SYSTEM PHYSIOLOGY PLANT

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A. Photosynthesis: Light harvesting complexes; mechanisms of electron transport; photoprotective

mechanisms; CO fixation-C , C and CAM pathways.
2 3 4

Photosynthesis is essentially the only mechanism of energy Photosynthetic Pigments: The photosynthetjc pigments
input in the living world. Photosynthesis (photos‐light, present in thylakoid membranes consist largely of two kinds
synthesis‐putting together) is an anabolic process of of green chlorophylls, Chlorophyll a (C55H70O5N4Mg) and
manufacture of organic compounds inside the chlorophyll Chlorophyll b (C55H72O6N4Mg). Also present are yellow to
containing cells from carbon dioxide and water with the help orange pigments classified as carotenoids. There are two
of sunlight as a source of energy. A simple equation of kinds of carotenoids, the pure hydrocarbon carotenes and
photosynthesis is as follows: the oxygen‐containing xanthophylls. Certain carotenoids,
Chlorophyll especially violaxanthin, a xanthophyll, also exist in the
6CO2 + 16H2O Æ C6H12O6 + 6O2 chloroplast envelope, giving it a yellowish colour. In most
Radiant Energy plants, including green algae, β‐carotene and lutein are the
most abundant carotenoids in the thylakoids.
However, the function of water is to provide hydrogen for
the synthesis of organic compounds.
All the liberated oxygen comes from it. Therefore, the
equation is modified as
Chlorophyll
6CO2 + 12H2O Æ C6H12O6 + 6H2O+ 6O2
Radiant Energy

The source of molecular oxygen was water and not carbon

dioxide as was believed earlier was experimentally proved
first by Robert Hill (1937) and later confirmed by M.D.
Kamen, and S. Ruben (1945), employing tracer technique in
which heavy isotopes of oxygen 18O were used. But this
experimental proof was based on the suggestions of C.B. Van
Niel's work on bacterial photosynthesis (1930). He
suggested that in green plants H2O is the source of reduction
and when split yields (H) and (OH), and O2 released by
plants is derived from water, not from CO2. This splitting of
water in light by green plants has come to be known as
photolysis of water and the theory, Van Niel's theory of
photolysis of water.
Events of Photosynthesis
Chloroplasts: Structures and Photosynthetic Pigments
Photosynthesis consists of two types of reactions: a light
Chloroplasts is the seat of photosynthesis and is best dependent one and a light independent one. The light‐
exemplified in the higher plants. A chloroplast is covered by dependent reaction is a photochemical reaction or light
an envelope of two membranes which are separated by reaction as it came to be called, culminating in the
periplastidial space of 10‐20 nm. Internally a chloroplast generation of NADPH2, ATP and evolution of molecular
contains a matrix or stroma in which are embedded a oxygen. The NADPH2 and ATP are energy‐rich, having
number of flattened membranous sacs called thylakoids or caught the electrons that became available when light
lamella. The external surface of thylakoids contains the impinged upon chlorophyll. They form the assimilatory
photosynthetic pigments and serves the ends of light re‐ power, utilized for CO2‐fixation. The events of CO2‐fixation is
action. The stroma, on the other hand, is concerned with the light independent reaction and is designated as dark
events of the dark reaction. In certain regions the thylakoids reaction.
are stacked to form grana. The longer thylakoids that
connect one granum to another extend through the stroma LIGHT REACTION
so these membranes are usually referred to as stroma
thylakoids. Light reaction consists of two phase:

Phase I­Energy absorption (Absorption and retention of

light by the photosynthetic pigments); and

Phase II­Energy transduction (conversion of light energy

absorbed in phase I into chemical energy‐ATP and NADPH2
by photophosphorylation).

Phase I. Energy absorption.

Photosynthetic Units­The events of light reaction are

mediated through photosynthetic units, a photosynthetic
unit being the smallest group of pigment molecules, together
with their lipo‐protein associate substances, able to bring
about a photochemical act (Photoact). The term,

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photochemical act, means absorption and migration of a purpose. Depending upon the pigment composition, the
light quantum by a trapping centre, as a result of which an various plant groups absorb and utilise light of different
electron is released. Emerson and Arnold thought that a spectral regions. Most green plants absorb light in the visible
photosynthetic unit (PSU) contained at least 2500 spectrum (390‐700 nm), whereas purple bacteria employ
chlorophyll molecules, but recent work by Bessel Kock wavelengths ranging from near ultraviolet to infrared (800‐
indicates that a photosynthetic unit contains only about 250 950 nm). This range of the spectrum through which
chlorophyll molecules. The occurrence of PSU as a distinct photosynthesis can take place is called photosynthetically
morphological entity was obtained by Park and his co‐ active radiation. But the entire range is not employable in
workers and they christened it quantasome. photosynthesis. The green plants, for example, absorb light
maximally in the red and blue regions of the spectrum. A
Absorption of light by pigments study of the absorption spectra shows the quantitative
relationship between the wavelength of light and its
All the light incident on photosynthesising surface is not absorption by the pigment in question. Thus, we see that
used for photosynthesis. Much of it is lost and quite some is chlorophyll‐a has its absorption peaks at 660 nm and 430
reflected back. Again, only a small fraction of the absorbed‐ nm chlorophyll‐b at 648 nm and 456 nm carotene at 478 nm
light is used to drive photosynthesis. It is estimated that in and 449 nm and xanthophyll same as carotene.
full sunlight, just about 3% is used for photosynthetic

Light Trap: Chlorophyll‐a utilises the light that it absorbs on

its own and also the light transferred to it by other pigments.
This funnelling of light from other pigments to chlorophyll a
has been called light trap or light sink. The light trap makes
for a much better light‐harvesting efficiency, for it ensures
funnelling of light quanta towards one acceptor molecule of
chlorophyll.

The light reaction actually consists of two photochemical

reactions which are separated both in time and space. They
are designated as Photoact I and Photoact II and the two
reactions are mediated by two different systems whose
composition differ in terms of pigments, electron carriers
and light trap mechanisms. The mediating agencies of the
two photoacts are respectively called Photosystem I and
Photosystem II.

Photosystems: The concept of two photosystems originated

in the work of Emerson and Lewis (1943). Working on the
action spectrum for the pigments of Chlorella, they found
that at wavelengths of light between 600 and 680 nm
(wavelengths corresponding to the 'red' region of the
spectrum) the evolution of oxygen was at its maximum. But
when light of wavelengths beyond 680 nm, a region of the
spectrum referred to as 'far‐red' was supplied there was a
drop in the evolution of oxygen, indicative of lessened
photosynthesis efficiency. This observation had been
christened the red drop. His research group found that if
light of shorter wave lengths was provided at the same time
as the longer red wavelengths, photosynthesis was even

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faster than when either of wavelengths alone was provided Photosystem I takes part in both cyclic and non‐cyclic
This synergism or enhancement became known as the photophosphorylations. PS‐I can carryon
Emerson enhancement effect. These two observations‐the cyclicphotosphosphorylation independently. Normally it
red drop effect and enhancement effect led to the first drives an electron from photosystem II to NADP+.
indication that the light reaction has two sites of action, one
in the red region of the spectrum and the other in the far‐ Photosystem II picks up electron released during photolysis
red. of water. The same is extruded on absorption of light energy.
As the extruded electron passes over cytochrome complex,
sufficient energy is released to take part in the synthesis of
ATP from ADP and inorganic phosphate. This
photophosphorylation is noncyclic. PS II can operate only in
conjunction with PS I.

Phase II : Energy Transduction

The excited molecules of P‐700. and P‐690 transduce their

energies to generate ATP and NADPH2. Molecular oxygen is
also produced but it escapes out of the photosynthetic
system. ATP and NADPH2 together constitute the
The explanation offered for these two effects is that the light assimilatory power and are employed in the fixation of CO 2
reaction actually consists of two photoacts, photoact I and in the dark reaction.
photoact II, mediated by two photosystems, photosystem I
and photosystem II. Photosystem I is driven by the far‐red Photophosphorylation
light and when it operates alone, it produces the red drop
effect. But when it operates along with photosystem II, Photophosphorylation is the light driven or light energized
which functions in the red region, enhancement effect is synthesis of ATP. It was discovered by Amon et al in 1954.
produced. Physical separation of the two photosystems had Photophosphorylation is of two main types, cyclic and
been sucessfully carried out and their functions clarified. noncyclic.
Photosystem I has been located in the thylakoid membranes. Cyclic Photophosphorylation: It is a process of
It is made up of three forms of chlorophyll‐a, one absorbing photophosphorylation in which an electron expelled by the
maximally at 683 nm, the second absorbing maximally at excited photocentre is returned to it after passing
695 nm and the third at 670 nm. The last of these has been through a series of electron carriers. Cyclic
called P‐700. Photosystem II had. been located in the stroma photophosphorylation is performed by photosystem I only.
thylakoids. It is made up of two forms of chlorophyll‐a with Its photocentre P 700 extrudes an electron with a gain of 23
maximum absorption at 670 and 690 nm. The second form is kcal/mole of energy after absorbing a photon of light (hv).
christened P‐690. Each photosystem has three components : After losing the electron the photocentre becomes oxidised.
(i) a reaction centre made up of a special chlorophyll The expelled electron passes through a series of carriers
molecule‐in photosystem I it is a protein‐bound chlorophyll‐ including X, ferredoxin, plastoquinone, cytochrome complex
a molecule, P‐700; in photosystem II it is P‐690. The reaction and plastocyanin before returning to photocentre.
centres are the actual sites where light energy is converted
to chemical energy. (ii) some electron carriers‐in While passing between ferredoxin and plastoquinone and/
photosystem I, X, plastocyanin, cytochrome‐f and ferrodoxin or over the cytochrome complex, the electron loses sufficient
as the electron carrier; photosystem II has plastoquinone energy to form ATP from ADP and inorganic phosphate.
and cytochrome b‐559. (iii) other chlorophyll and
carotenoids, which merely serve to transfer the light
absorbed by them to the active centres.

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Halobacteria or halophile bacteria also perform bacteriorhodopsin attached to plasma membrane. As light
photophosphorylation but ATP thus produced is not used in falls on the pigment, it creates a proton pump which is used
synthesis of food. These bacteria possess purple pigment in ATP synthesis.

Noncyclic Photophosphorylation: It is the normal process Oxygen evolution:

of photophosphorylation in which the electron expelled by
the excited photocentre does not return to it. Non‐cyclic The oxygen that is evolved during photosynthesis comes
photophosphorylation is carried out in collaboration of both from water and it is part of the photoact II mediated by PSII.
photosystems I and II. Electron released during photolysis of The light energy trapped by this system excites P‐690 and
water is picked up by photocentre of PS II called P 680 two electrons are ejected. The energy is used to remove two
electrons from the hydrogen of the water and boost them to
The same is extruded out when the photocentre absorbs a higher energy level. At this point, the molecular oxygen
light energy (hv). The extruded electron has an energy which escapes from the photosynthetic system is formed.
equivalent to 23 kcal/mole. It passes through a series of The electrons pass through the carriers plastoquinone (PQ),
electron carriers Q, PQ, cytochrome complex and plas‐ cytochrome b‐559, cytochrome‐F, plastocyanin and finally
tocyanin. While passing over cytochrome complex, the end up in P‐700 to take it back to the ground state. Thus in
electron loses sufficient energy for the syntheisis of ATP. The photosystem II, the electrons that brings the excited
electron is handed over to photocentre P 700 of PSI by chlorophyll molecule to the ground state comes from
plastocyanin P 700 extrudes the electron after absorbing photolysis of water. Another aspect of oxygen evolution
light energy. The extruded electron passes through X, Fe‐S during photosynthesis is its relationship to the presence of
centre A (ferredoxin), and NADP‐reductase which combines certain ions in the medium such as Cl‐, Mn2+ and
it with NADP+ for becoming reduced H+ released during bicarbonate.
photolysis to form NADPH. ATP synthesis is not direct. The 2H2OÆ O2 + 4H+ + 4e‐
energy released by electron is actually used for pumping H+
ions across the thylakoid membrane. It creates a proton Both the photosystems working in union produce, for every
gradient. The gradient triggers the coupling factor to two turns, two molecules of NADPH2, three ATPs, and a
syntl1esize ATP from ADP and inorganic phosphate. molecule of oxygen from two water molecules.

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Some Herbicides Block Electron Flow

The use of herbicides to kill unwanted plants is widespread

in modern agriculture. Many different classes of herbicides
have been developed, and they act by blocking amino acid,
carotenoid, or lipid biosynthesis or by disrupting cell
division. Other herbicides, such as DCMU
(dichlorophenyldimethylurea) and paraquat, block
photosynthetic electron flow (Figure). DCMU is also known
as diuron. Paraquat has acquired public notoriety because of
its use on marijuana crops.

Many herbicides, DCMU among them, act by blocking

electron flow at the quinone acceptors of photosystem II, by
competing for the binding site of plastoquinone that is
normally occupied by QB. Other herbicides, such as
paraquat, act by accepting electrons from the early acceptors
of photosystem I and then reacting with oxygen to form
superoxide, O2‐ , a species that is very damaging to
chloroplast components, especially lipids.

Carotenoids Serve as Photoprotective Agents

In addition to their role as accessory pigments, carotenoids

play an essential role in photoprotection. The
photosynthetic membrane can easily be damaged by the
large amounts of energy absorbed by the pigments if this
energy cannot be stored by photochemistry; this is why a
protection mechanism is needed. The photoprotection
mechanism can be thought of as a safety valve, venting
excess energy before it can damage the organism. When the
energy stored in chlorophylls in the excited state is rapidly
dissipated by excitation transfer or photochemistry, the
excited state is said to be quenched.

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If the excited state of chlorophyll is not rapidly quenched by Photoinhibition is reversible in early stages. Prolongued
excitation transfer or photochemistry, it can react with inhibition, however, results in damage to the system such
molecular oxygen to form an excited state of oxygen known that the PSII reaction center must be disassembled and
as singlet oxygen (1O2*). The extremely reactive singlet repaired. The main target of this damage is the D1 protein
oxygen goes on to react with and damage many cellular that makes up part of the PSII reaction center complex (see
components, especially lipids. Carotenoids exert their Figure). When D1 is damaged by excess light, it must be
photoprotective action by rapidly quenching the excited removed from the membrane and replaced with a newly
state of chlorophyll. The excited state of carotenoids does synthesized molecule. The other components of the PSII
not have sufficient energy to form singlet oxygen, so it reaction center are not damaged by excess excitation and
decays back to its ground state while losing its energy as are thought to be recycled, so the D1 protein is the only
heat. component that needs to be synthesized.

Mutant organisms that lack carotenoids cannot live in the

presence of both light and molecular oxygen—a rather
difficult situation for an O2‐evolving photosynthetic
organism.

For non‐O2‐evolving photosynthetic bacteria, mutants that

lack carotenoids can be maintained under laboratory
conditions if oxygen is excluded from the growth medium.

Recently carotenoids were found to play a role in

nonphotochemical quenching, which is a second protective
and regulatory mechanism.

Some Xanthophylls Also Participate in Energy

Dissipation

Nonphotochemical quenching, a major process regulating

the delivery of excitation energy to the reaction center, can
be thought of as a “volume knob” that adjusts the flow of
excitations to the PSII reaction center to a manageable level,
depending on the light intensity and other conditions. The
process appears to be an essential part of the regulation of
antenna systems in most algae and plants.

Nonphotochemical quenching is the quenching of

chlorophyll fluorescence by processes other than
photochemistry. As a result of nonphotochemical quenching, Photosystem I Is Protected from Active Oxygen Species
a large fraction of the excitations in the antenna system
caused by intense illumination are quenched by conversion Photosystem I is particularly vulnerable to damage from
into heat. Nonphotochemical quenching is thought to be active oxygen species. The ferredoxin acceptor of PSI is a
involved in protecting the photosynthetic machinery against very strong reductant that can easily reduce molecular
overexcitation and subsequent damage. oxygen to form superoxide (O2–). This reduction competes
with the normal channeling of electrons to the reduction of
The molecular mechanism of nonphotochemical quenching NADP+ and other processes. Superoxide is one of a series of
is not well understood, although it is clear that the pH of the active oxygen species that can be very damaging to
thylakoid lumen and the state of aggregation of the antenna biological membranes.
complexes are important factors. Three carotenoids, called
xanthophylls, are involved in nonphotochemical quenching: Superoxide formed in this way can be eliminated by the
violaxanthin, antheraxanthin, and zeaxanthin. action of a series of enzymes, including superoxide
dismutase and ascorbate peroxidase.
In high light, violaxanthin is converted into zeaxanthin, via
the intermediate antheraxanthin, by the enzyme Thylakoid Stacking Permits Energy Partitioning
violaxanthin de‐epoxidase. When light intensity decreases, between the Photosystems
the process is reversed. Binding of protons and zeaxanthin
to light‐harvesting antenna proteins is thought to cause The fact that photosynthesis in higher plants is driven by
conformational changes that lead to quenching and heat two photosystems with different light‐absorbing properties
dissipation. Nonphotochemical quenching appears to be poses a special problem. If the rate of delivery of energy to
preferentially associated with a peripheral antenna complex PSI and PSII is not precisely matched and conditions are
of photosystem II, the PsbS protein. such that the rate of photosynthesis is limited by the
available light (low light intensity), the rate of electron flow
The Photosystem II Reaction Center Is Easily Damaged will be limited by the photosystem that is receiving less
energy.
Another effect that appears to be a major factor in the
stability of the photosynthetic apparatus is photoinhibition, In the most efficient situation, the input of energy would be
which occurs when excess excitation arriving at the PSII the same to both photosystems. However, no single
reaction center leads to its inactivation and damage. arrangement of pigments would satisfy this requirement
Photoinhibition is a complex set of molecular processes,
defined as the inhibition of photosynthesis by excess light.

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because at different times of day the light intensity and Phase III: Energy Stabilization
spectral distribution tend to favor one photosystem or the
other. The production of carbon dioxide to a carbohydrate is the
essence of this phase and this is accomplished through the
This problem can be solved by a mechanism that shifts employment of the assimilatory power (ATP and NADPH2)
energy from one photosystem to the other in response to generated in the light reaction. This energy stabilization is a
different conditions. Such a regulating mechanism has been dark reaction. It does not require light, instead assimilatory
shown to operate in different experimental conditions. The power (ATP and NADPH2 produced during photochemical
observation that the overall quantum yield of phase is used here in fixation and reduction of CO2. The
photosynthesis is nearly independent of wavelength enzymes required for the process are present in the matrix
strongly suggests that such a mechanism exists. or stroma of the chloroplast. There are two mam pathways
for the biosynthetic or dark phase‐Calvin cycle and C4
Thylakoid membranes contain a protein kinase that can dicarboxylic acid cycle. The plants exhibiting the two are
phosphorylate a specific threonine residue on the surface of respectively called C3 and C4 plants.
LHCII, one of the membrane‐bound antenna pigment
proteins described earlier in the chapter. When LHCII is not Calvin cycle (the photosynthetic carbon reduction cycle
phosphorylated, it delivers more energy to photosystem II, or the C­3 photosynthetic pathway):
and when it is phosphorylated, it delivers more energy to
photosystem I. This cycle was discovered by Calvin, Benson and their
colleagues using unicellular algae Chlorella pyrenoidosa and
The kinase is activated when plastoquinone, one of the Scenedesmus obliques and radioactive isotope of 14C with a
electron carriers between PSI and PSII, accumulates in the half‐life of more than 5000 years.
reduced state. Reduced plastoquinone accumulates when
PSII is being activated more frequently than PSI. The Phases of Calvin Cycle: Calvin cycle is divided into the
phosphorylated LHCII then migrates out of the stacked following three phases‐carboxylation, glycolytic reversal and
regions of the membrane into the unstacked regions, regeneration of RuBP.
probably because of repulsive interactions with negative
charges on adjacent membranes. The lateral migration of 1. Carboxylation: It requires ribulose‐1, 5‐biphosphate or
LHCII shifts the energy balance toward photosystem I, which RuBP as acceptor of carbon dioxide and RuBP carboxylase or
is located in the stroma lamellae, and away from rubisco as enzyme. The enzyme was previously called
photosystem II, which is located in the stacked membranes carboxydismutase. Carbon dioxide combines with ribulose‐
of the grana. This situation is called state 2. If plastoquinone 1, 5‐biphosphate to produce a transient intermediate
becomes more oxidized because of excess excitation of compound called 2‐carboxy 3‐keto 1,5‐biphosphoribotol.
photosystem I, the kinase is deactivated and the level of The intermediate splits up immediately in the presence of
phosphorylation of LHCII is decreased by the action of a water to form the two molecules of 3‐phosphoglyceric acid
membrane‐bound phosphatase. or PGA. It is the first stable product of photosynthesis.

LHCII then moves back to the grana, and the system is in 2. Glycolytic Reversal: The processes involved in this step
state 1. The net result is a very precise control of the energy or phase are reversal of processes found during glycolysis
distribution between the photosystems, allowing the most part of respiration. Phosphoglyceric acid or PGA is further
efficient use of the available energy. phosphorylated by ATP with the help of enzyme triose
phosphate kinase. It give rise to 1, 3‐diphosphoglyceric acid.
BLACKMAN'S REACTION
Diphosphoglyceric acid is reduced by NADPH through the
agency of enzyme triose phosphate dehydrogenase. It
produces glyceraldehyde 3‐phosphate or 3‐
phosphoglyceraldehyde.

Glyceraldehyde‐3 phosphate is a key product which is used

in synthesis of both carbohydrates and fats. For forming
carbohydrates, say glucose, a part of it is changed into its
isomer called dihydroxyacetone‐3‐phosphate. The enzyme
that catalyses the reaction is phosphate isomerase.

The two isomers condense in the presence of enzyme

aldolase forming fructose 1, 6diphosphate.

Fructose 1,6‐diphosphate (FOP) loses one phosphate group,

forms fructose 6‐phosphate (F 6‐P) which is then changed to
glucose‐6‐phosphate (G 6 P). The latter can produce glucose
or become part of sucrose and polysaccharide.
As glucose is a six carbon compound, six turns of Calvin cycle
are required to synthesise its one molecule.

3. Regeneration of RuBP: Fructose 6‐phosphate (F 6‐P)

and glyceraldehyde 3‐phosphate (GAP) react to form
erythrose 4‐phosphate (E 4‐P) and xylulose 5‐phosphate (X
5‐P). Erythrose 4‐phosphate combines with dihydroxy
acetone 3‐phosphate to produce sedoheptulose 1 : 7‐
diphosphate (SDP) which loses a molecule of phosphate and

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gives rise to sedoheptulose 7‐phosphate (S 7‐P). their isomer ribulose 5‐phosphate (Ru 5‐P). Ribulose 5‐
Sedoheptulose 7‐phosphate reacts with glyceraldehyde 3‐ phosphate picks up a second phosphate from ATP to become
phosphate to produce xylulose 5‐phosphate (X 5‐P) and changed into ribulose 1, 5 biphosphate (RuBP).
ribose 5‐phosphate. (R 5‐P). Both of these are changed to

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REGULATION OF THE CALVIN CYCLE fructose‐1,6‐bisphosphatase), the thioredoxin‐linked

activation is enhanced by an effector (e.g., fructose‐1,6‐
The high energy efficiency of the Calvin cycle indicates that bisphosphate substrate). Inactivation of the target enzymes
some form of regulation ensures that all intermediates in the observed upon darkening appears to take place by a reversal
cycle are present at adequate concentrations and that the of the reduction (activation) pathway. That is, oxygen
cycle is turned off when it is not needed in the dark. In converts the thioredoxin and target enzyme from the
general, variation in the concentration or in the specific reduced state (—SH HS—) to the oxidized state (—S—S—)
activity of enzymes modulates catalytic rates, thereby and, in so doing, leads to inactivation of the enzyme. The last
adjusting the level of metabolites in the cycle. four of the enzymes listed here are regulated directly by
thioredoxin; the first, rubisco, is regulated indirectly by a
Changes in gene expression and protein biosynthesis thioredoxin accessory enzyme, rubisco activase.
regulate enzyme concentration. Posttranslational
modification of proteins contributes to the regulation of C2 cycle: Photorespiration (Respiration Associated with
enzyme activity. At the genetic level the amount of each Photosynthetic Tissues)
enzyme present in the chloroplast stroma is regulated by
mechanisms that control expression of the nuclear and It was discovered by Decker and Tio in 1959.
chloroplast genomes. Photorespiration is the light dependent utilization of oxygen
and release of carbon dioxide by the photosynthetic organs
Short‐term regulation of the Calvin cycle is achieved by of a plant. Normally photosynthetic organs do the reverse in
several mechanisms that optimize the concentration of the light i.e., uptake of CO2 and release of O2. Therefore,
intermediates. These mechanisms minimize reactions photorespiration is difficult to demonstrate. It is inferred
operating in opposing directions, which would waste from (i) Decrease in the rate of net photosynthesis when
resources. Two general mechanisms can change the kinetic oxygen concentration is increased from 2‐3% to 21% (ii)
properties of enzymes: Sudden increased evolution of O2 when an illuminated green
1. The transformation of covalent bonds such as the organ is transferred to dark.
reduction of disulfides and the carbamylation of amino
groups, which generate a chemically modified enzyme. The site for photorespiration is chloroplast. Peroxisome is
2. The modification of noncovalent interactions, such as the required for completing the process. RuBP carboxylase is
binding of metabolites or changes in the composition of the changed to RuBP oxygenase. This happens at high
cellular milieu (e.g., pH). In addition, the binding of the temperature and high oxygen concentration. At high
enzymes to the thylakoid membranes enhances the temperature and high oxygen concentration, the affinity of
efficiency of the Calvin cycle, thereby achieving a higher RuBP carboxylase for CO2 decreases and the affinity for O2
level of organization that favors the channeling and increases. High temperature occurs in tropical areas.
protection of substrates. Therefore, tropical plants are the major sufferers. At high
temperature, RuBP carboxylase functions as oxygenase and
Calvin cycle is not a Dark Reaction: Light­Dependent instead of fixing carbon dioxide, oxidises ribulose 1, 5‐
Enzyme Activation Regulates the Calvin Cycle biphosphate to produce phosphoglyceric acid and
phosphoglycolate.
Five light‐regulated enzymes operate in the Calvin cycle:
Phosphoglycolate is hydrolysed to form glycolate. Glycolate
1. Rubisco usually passes into peroxisome of the mesophyll cell and
2. NADP:glyceraldehyde­3­phosphate dehydrogenase forms glyoxylate. Glyoxylate is aminated and gives rise to
3. Fructose­1,6­bisphosphatase amino acid glycine. Inside mitochondrion and even
4. Sedoheptulose­1,7­bisphosphatase cytoplasm the two molecules of glycine condense to form a
5. Ribulose­5­phosphate kinase molecule of serine, C02 and ammonia are released in the
process. Serine can further be deaminated to form PGA. The
The last four enzymes contain one or more disulfide (—S— latter passes into chloroplasts for synthesis of pho‐
S—) groups. Light controls the activity of these four tosynthetic products as well as photorespiration. Since
enzymes via the ferredoxin–thioredoxin system, a photorespiration involves the synthesis of two‐carbon
covalent thiol‐based oxidation–reduction mechanism compounds, it is also called C2 cycle.
identified by Bob Buchanan and colleagues. In the dark these
residues exist in the oxidized state (—S—S—), which Photorespiration does not produce energy or reducing
renders the enzyme inactive or subactive. In the light the — power. Rather, it consumes energy. Further, it undoes the
S—S— group is reduced to the sulfhydryl state (—SH HS—). work of photosynthesis. It may reduce photosynthesis upto
This redox change leads to activation of the enzyme. The 50%. Therefore, photorespiration is a highly wasteful
resolution of the crystal structure of each member of the process. This happens only in case of C3 plants. C4 plants
ferredoxin– thioredoxin system and of the target enzymes have overcome the problem of photorespiration by
fructose‐1,6‐bisphosphatase and NADP : malate performing Calvin Cycle in the interior of leaves (bundle
dehydrogenase have provided valuable information about sheath cells) where both temperature and oxygen are lower.
the mechanisms involved. They have further ensured high CO2 supply to cells
performing Calvin cycle.
This sulfhydryl (also called dithiol) signal of the regulatory
protein thioredoxin is transmitted to specific target
enzymes, resulting in their activation In some cases (such as

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Hatch and Slack found it a regular mode of C02‐fixation in a

The Biological Function of Photorespiration Is Unknown number of tropical plants, both monocots and dicots, eg.,
Maize, Sugarcane, Sorghum, Panicum, Pennisetum, Atriplex,
Although the C2 oxidative photosynthetic carbon cycle Amaranthus, Salsola etc. These plants are called C4 plants
recovers 75% of the carbon originally lost from the Calvin because of the first stable photosynthetic product being a 4‐
cycle as 2‐phosphoglycolate, why does 2‐phosphoglycolate carbon‐compound. Other plants are C3 plants. C4 plants often
form at all? One possible explanation is that the formation of live in hot, arid and saline habitats. They have Kranz
2‐phosphoglycolate is a consequence of the chemistry of the anatomy. In Kranz anatomy, the mesophyll is undifferen‐
carboxylation reaction, which requires an intermediate that tiated and its cells occur in concentric layers around
can react with both CO2 and O2. vascular bundles having large bundle sheath cells. The
mesophyll and bundle sheath cells are connected by
Such a reaction would have had little consequence in early plasmodesmata or cytoplasmic bridges. The chloroplasts of
evolutionary times if the ratio of CO2 to O2 in air were higher the mesophyll cells are smaller. They have well developed
than it is today. However, the low CO2:O2 ratios prevalent in grana and a peripheral reticulum but no starch. The
modern times are conducive to photorespiration, with no chloroplasts of the bundle sheath cells are larger. They have
other function than the recovery of some of the carbon ill defined grana, a peripheral reticulum and starch grains.
present in 2‐phosphoglycolate.

Another possible explanation is that photorespiration is

important, especially under conditions of high light intensity
and low intercellular CO2 concentration (e.g., when stomata
are closed because of water stress), to dissipate excess ATP
and reducing power from the light reactions, thus
preventing damage to the photosynthetic apparatus.

Arabidopsis mutants that are unable to photorespire grow

normally under 2% CO2, but they die rapidly if transferred to
normal air. There is evidence from work with transgenic
plants that photorespiration protects C3 plants from
photooxidation and photoinhibition. Further work is needed
to improve our understanding of the function of
photorespiration.

C4­DICARBOXYLIC ACID PATHWAY (HATCH SLACK

PATHWAY, C4 PATHWAY)

It was worked out by Hatch and Slack (1965, 1967).

Koitschak et al (1965) found that labelled carbon dioxide
(l4C02) assimilated by Sugarcane leaves first appeared in a 4‐
carbon compound oxalo‐acetic acid (OAA or oxaloacetate).

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In C4 plants, initial fixation of carbon dioxide occurs in Malic acid + NADP+ ÆPyruvate + CO2 + NADPH
mesophyll cells. The primary acceptor of C02 is phosphoenol
pyruvate or PEP. CO2 is again fixed inside the bundle sheath cells through
Calvin cycle. RuBP of Calvin cycle is called secondary or final
It combines with carbon dioxide in the presence of PEP acceptor of CO2 in C4 plants. Pyruvate is sent back to
carboxylase or pepco to form oxalo‐acetic acid or mesophyll cells. Here, it is changed to phosphoenol pyruvate.
oxaloacetate. Energy is required for this. The same is provided by ATP.
PEP carboxylase The latter is changed into AMP (adenosine monophosphate).
PEP + CO2 + H2O Æ Oxalo acetic acid + H3PO4 Phosphopyruvate dikinase
Pyruvate + ATP Æ PEP + AMP + H3PO4
Oxalo‐acetic acid is reduced to malic acid or aminated to
form aspartic acid Conversion of AMP to ATP requires double the energy than
dehydrogenase conversion of ADP to ATP. Therefore, actual requirement of
Oxalo‐acetic Acid + NADPHÆ Malic Acid + NADP+ energy is equal to two molecules of ATP. This energy is in
dehydrogenase addition to 3 ATP required for fixation of one molecule of
Oxalo‐acetic Acid + NH3 + NADPHÆAspartic acid + NADP + CO2 through Calvin cycle. Therefore, C4 plants consume 5
H2O ATP molecules per molecule of CO2 fixed instead of 3 ATP
molecules for C3 plants. For the formation of a glucose
Malic acid or aspartic acid is translocated to bundle sheath molecule, C4 plants require 30 ATP while C3 plants utilize
cells through plasmodesmata. Inside the bundle sheath cells only 18 ATP.
they are decarboxylated (and demainated in case of aspartic
acid) to form pyruvate and CO2. 6PEP + 6RuBP + 6CO2 + 30 ATP + 12 NAPDH Æ 6 PEP +
Malic enzyme 6RuBP + C6H12O6 + 30 ADP + 30 H3P04 + 12 NADP+

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Importance

1. The C4‐plants are considered to possess greater 3. The chloroplasts of these plants seem to generate more
photosynthetic efficiency, for they can utilize CO2 until a ATP which of course makes for improved cellular work.
level of 5 ppm is reached but the Calvin cycle plants cannot
utilise CO2 if the level falls below 40‐50 ppm. 4. The presence of extensive peripheral reticulum in the
chloroplasts of these plants indirectly suggests quicker
2. C4‐plants can utilize greater light intensities and their transport of products and therefore greater utilisation of
temperature optima for photosynthesis exceeds those of C3 light and CO2.
plants.

CRASSULACEAN ACID METABOLISM occurs, the cell sap turns acidic. This process is called as
dark acidification. In the following daytime, the stomata
This pathway worked out by Ranson and Thomas (1960) remain closed, minimizing transpirational losses, but with
and Rouham, Vines and Black (1973) is found in succulents, the advent of light, the CO2 absorbed during the preceding
mostly members of Crassulaceae (Bryophylum and Sedum) night is utilized for photosynthetic purposes, the process , of
and a few members of Bromeliaceae, such as pineapple. It is light deacidification then occurs. Thus, there is a, time lag
a device designed to meet the pressures of heavy between absorption and reduction of CO2. This arrangement
transpiration, arising from their xerophytic environment. helps to lessen transpirational stress but is responsible for
These plants obtain their CO2 requirements during the night the extremely slow growth of these plants. Below is given a
time when they keep their stomata open and as CO2 build‐up short account of this pathway.

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Phase I : Dark acidification: At low light intensity the rate of photosynthesis is reduced.
In this phase, the reserve starch is broken to There is a point in light intensity where there is no gaseous
phosphoenoplyruvate (PEP) through a number of exchange in photosynthesis. It is called light compensation
respiratory reactions. PEP accept' the atmospheric C02 point. Light compensation point is defined as a point in light
absorbed by the plant and produces malic acid. This happens intensity when no gaseous exchange is observed between
in darkness when the stomata are open. the environment and the photosynthetic organ illuminated
Starch Æ PEP by that light intensity. A plant cannot survive for long at
PEP + NADPH2 + CO2 Æ Malic acid + NADP. compensation point because there is a net loss of organic
matter due to respiration of non‐green organs and
Phase II: Light deacidification respiration in dark.

During this phase, which occurs in light when the stomata The light intensity at which a plant can achieve maximum
are closed, malic acid is decarboxylated into pyruvic acid amount of photosynthesis is called saturation point. Its value
and CO2. The CO2 released is routed through Calvin cycle to is 800‐1000 ft candles (10% of full sunlight) in shade plants,
synthesise a hexose. The pyruvic acid is used to build up 50‐70% of full sunlight in C3 sun plants and upto 200% of
starch whose stocks are depleted earlier. full sunlight in C4 sun plants, (e.g., Sugarcane).
Malic acid Æ Pyruvic acid + NADPH2 + CO2 in plant

Calvin cycle events:

6 CO2 + 12 NADPH2 + 18 ATP + 11 H2O Æ Fructose ‐6‐

phosphate + 12 NADP + 18 ADP + 17 Pi

The assimilatory power needed for Calvin cycle is provided

by the events of light deacidification.

PRINCIPLE OR LAW OF LIMITING FACTORS

Optimum value of a factor is never constant. It depends

upon the magnitude of other factors. We may continue to
increase the magnitude of one or more factors without
influencing the rate of reaction. In such cases it is found that
a factor called limiting factor is holding the balance. A
limiting factor is defined as a factor which is deficient to
such an extent that increase in its magnitude directly
increases the rate of the process. The effect of limiting
factors was studied by Blac1anarm in 1905. He formulated
the principle of limiting factors which states that when a
process is conditioned as to its rapidity by a number of
separate factors, the rate of the process is limited by the
pace of the slowest factor. In other words the rate of a physi‐
ological process is limited at a given time by one and only
one factor which is deficient.

Factors Influencing Photosynthesis

External Factors

1. Carbon Dioxide: CO2 concentration of the atmosphere is

0.03% or 300 ppm. It is a limiting factor as the available CO 2
concentration is lower than the optimum for photosynthesis. Beyond saturation point (it is seldom realised in nature in
Increase in its concentration upto 0.1% increases the rate of C4 sun plants) the rate of photosynthesis begins to decline.
photosynthesis in most land plants. A decline is observed The phenomenon is called solarisation. It is due to (i) Photo‐
beyond it. When CO2 concentration is reduced, there comes a inhibition due to reduction in hydration and closure of
point at which illuminated plant parts stop absorbing carbon stomata (ii) Photo‐oxidation or oxidation of photosynthetic
dioxide from their environment. It is known as CO2 pigments, intermediates and enzymes.
compensation point or threshold value. At this value CO2
fixed by photosynthesis is equal to CO2 evolved in 3. Quality of Light: Maximum 'photosynthesis occurs in
respiration and photorespiration. The CO2 compensation blue‐violet and red regions of the light spectrum where most
point reflects the balance between photosynthesis and of the absorption is carried out by chlorophylls. Red light
respiration as a function of CO2 concentration, and the light favors carbohydrate accumulation while blue light
compensation point reflects that balance as a function of stimulates protein: synthesis. Minimum photosynthesis
photon flux. occurs in the green wavelengths. Plants 'growing under the
canopy of other trees receive very little red and blue‐violet
2. Light Intensity: Plants are broadly classified into two light because of its absorption by leaves of the canopy. They
groups depending upon their inability or ability to tolerate receive more of green light that is transmitted through the
high light intensity‐shade plants (sciophytes, e.g., Oxalis) and tree. leaves. As a result the rate of photosynthesis of herbs,
sun plants (heliophytes) shrubs and ot:4er undergrowths in a forest is comparatively
low. Ultraviolet rays‐ are harmful.

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4. Duration of Light: Continuous photosynthesis can occur photosynthesis begins to decline in all plants. The
in continuous illumination without any.harm to the plant phenomenon is called Warburg effect.
(Bohning, 1949) though the rate of photosynthesis may
slightly decline after six days. 7. Water: Water supplies H+ and electrons for carbon
dioxide fixation. However, .less than 1°/" of the total water
5. Temperature: It does not influence light reactions of absorbed is utilized in photosynthesis. The rest is lost in
photosynthesis but affects the enzyme controlled dark transpiration. Even a slight increase in transpiration reduces
reactions. The minimum temperature at which most plants the leaf hydration that cuts down photosynthesis by causing
start photosynthesis is 0°‐5°C but it can be as low as‐20°C stomatal closure and hence decreased CO2 absorption, loss
for lichens and ‐35°C for some gymnosperms. The maximum of leaf turgidity, reduced absorption of solar radiations and
temperature at which photosynthesis can occur is 55°C in decrease enzymatic activity. Thus photosynthesis is more
some desert plants and 75°C for hot spring algae. The sensitive to dehydration than any other metabolic process.
optimum temperature is 10°‐25°C for C3 plants and 30°‐
45°C for C4 plants. When temperature is increased from 8. Air Pollutants: Dust and smoke particles present in the
minimum to optimum, the rate of photosynthesis doubles atmosphere reduce photosynthesis by reducing light
for every l0 oC rise in temperature. Above the optimum penetration and forming a layer over the plants. Sulphur
temperature, the rate of photosynthesis shows an initial dioxide, nitrogen oxides, hydrogen fluorides and other air
increase for short duration but later declines. This decline pollutants also decrease photosynthesis.
with time is called time factor.
9. Minerals: Magnesium is a component of chlorophyll. Fe,
6. Oxygen: Small quantity of oxygen is essential for Cu and Mn are required for synthesis of chlorophyll. Mn and
photosynthesis except in some anaerobic bacteria. C3 plants Cl are linked to photolysis of water. P as phosphate is es‐
show optimum photosynthesis at low oxygen concentration. sential for ATP synthesis. Enzyme activators of
For example, Bjorkman et al (1968) found in beans twice the photosynthesis include potassium and sulphur. Lower
rate of photosynthesis at 2.5% oxygen as compared to availability of any of these minerals reduces rate of
normal atmospheric concentration. The possible reasons are photosynthesis.
(i) Oxygen takes part in oxidation of photosynthetic
pigments, intermediates and enzymes in the presence of Internal or Plant factors.
strong light (photo‐oxidation). (ii) Oxygen is a strong 1. Age: As a leaf develops, the rate of photosynthesis rises
quencher of excited state of chlorophyll. (iii) Oxygen with the age till it becomes maximum at full maturity.
competes with C02 for reducing power. However, this effect Afterwards the rate of photosynthesis begins to decline.
is not known in C4 plants. (iv) It converts RuBP‐carboxylase
to RuBP‐oxygenase. At a very high oxygen content the rate of

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B. Respiration: Citric acid cycle; plant mitochondrial electron transport and ATP synthesis; alternate oxidase

Respiration: the oxidative breakdown of organic breakdown of substrate, which yields CO2 and reduced
compounds coenzymes, NAD(P)H; and the stage of terminal oxidation,
which achieves oxidation of the reduced coenzymes. Both of
1 The overall process and respiratory substrates these stages yield ATP, but by far the greater proportion is
produced during terminal oxidation.
Earlier in this chapter it was discussed how respiration
counterbalances photosynthesis in the biosphere, oxidizing 2 Pathways of substrate breakdown
the C fixed in photosynthesis to CO2 and water again. Most of
the respiration is aerobic and utilizes the O2 produced in Glycolysis
photosynthesis, so that the O2 is recycled as well. The
primary function of respiration is to provide for the energy The word glycolysis means ‘sugar breakdown’; it brings
needs of living cells: some of the potential energy of the about the oxidative breakdown of glucose to pyruvate, as
oxidizable substrates is conserved in ATP. Respiration is illustrated in Fig. 1 . The glucose is first converted to
sometimes described as being ‘the reverse of fructose‐1,6‐bisphosphate by phosphorylation at the
photosynthesis’. Chemically, the overall result of respiration expense of ATP, catalysed by hexokinase, followed by
is the reverse of that of photosynthesis; taking carbohydrate isomerization and a second phosphorylation by
as the end product of photosynthesis or the substrate of phosphofructokinase. The fructose‐1,6‐bisphosphate is
respiration, one can write cleaved by aldolase to the two triose sugar phosphates, 3–
phosphoglyceraldehyde and dihydroxyacetone‐3 ‐
phosphate. Now comes the oxidative reaction: the 3‐
phosphoglyceraldehyde is oxidized by glyceraldehyde‐
phosphate dehydrogenase to 1,3 ‐phosphoglycerate (PGA)
With respect to the reaction mechanism, however, and the coenzyme NAD+ is reduced. The 1, 3 ‐PGA donates a
respiration as a whole is not the exact reverse of phosphate group to ADP to synthesize ATP, catalysed by
photosynthesis, although there are many common PGA kinase, a process known as substrate level
intermediates, and some reactions do run in the reverse phosphorylation.
direction in the two processes. Quantitatively, the amount of
photosynthesis by a plant must exceed its respiration or a After some molecular rearrangements,
positive mass balance would be impossible. It has been phosphoenolpyruvate, PEP, is formed and pyruvate kinase
estimated that a flowering plant respires daily 30 to 70% of catalyses a second substrate level phosphorylation to give
its photosynthate, not counting any photorespiratory losses the end product of glycolysis, pyruvate. Since all the
that may have occurred. reactions from the oxidation step onwards proceed twice
per molecule of glucose, 4 ATP per 1 glucose can be formed;
In most plant tissues, carbohydrate is the main respiratory but 2 ATP are used to prime the system, so that the net gain
substrate, entering the oxidative pathways as hexose sugars. is 2 ATP per 1 glucose:
Such sugars are metabolically reactive and are not stored in
cells in large amounts. Carbohydrate is stored as C6H12 O6 + 2ATP + 2NAD+Æ C3 H4 O3 + 4ATP + 2NADH2
polysaccharides of which starch, a glucose polymer, is the
most common; it is insoluble and forms starch grains in All the C of the glucose is still in organic combination, i.e. no
plastids. Some species store fructosans, soluble polymers of CO2 has been evolved and most of the potential energy of the
fructose, in vacuoles. The disaccharide sucrose is the main glucose is still present in the pyruvate and the NADH; the net
vacuolar store in yet other plants, and sucrose is also the gain of 2 ATP represents a very small percentage of the
most common form in which carbohydrate is transported in potential total energy. It may also be noted that no O2 has
plants. All these more complex carbohydrates have to be been used: glycolysis is an anaerobic pathway.
hydrolysed to their hexose monomers for respiration. Many
seeds store lipids as oil and this can serve as respiratory Substrates can enter the glycolytic pathway at several
substrate, although most of the lipid store is converted to points. Fructose‐6‐phosphate may come from the hydrolysis
carbohydrate before being respired. Protein is not of fructans or sucrose, or from the PPP. Triose sugar
commonly utilized as a respiratory substrate, but C phosphates are transported out of chloroplasts in the light
skeletons from amino acids can enter the respiratory and may also be derived from the PPP. Starch hydrolysis by
pathways. During starvation, or senescence, and during the starch phosphorylase enzyme produces glucose‐1‐
reserve mobilization in seed storage tissues, there is large‐ phosphate, which is easily isomerized to glucose‐ 6‐
scale hydrolysis of proteins and respiration of at least part of phosphate. When such phosphorylated sugars enter
the amino acid pool. glycolysis, one or both of the priming reactions with ATP
is/are bypassed and the ATP gain is correspondingly
Much of the basic biochemistry of respiration is common to greater. Intermediates can also be withdrawn into other
organisms from all the living kingdoms, though some details metabolic sequences from the glycolytic pathway at various
may be specific to plants. With reference to photosynthetic points before pyruvate is produced.
organisms, the respiration which passes through the
universal respiratory pathways, as described below, is often The complete glycolytic sequence is located in the cytosol. In
termed dark respiration, to distinguish it from the unique, plant cells, however, isozymes of all the glycolytic enzymes
photosynthesis‐linked photorespiration. are additionally found in plastids. (Isozymes are variants of
an enzyme, catalysing the same reaction, but differing
The so‐called dark respiration still proceeds in slightly in structure and properties such as substrate
photosynthetic tissues in the light. As in the case of affinity; isozymes may be coded by different genes, or their
photosynthesis, which proceeds in two stages, one can differences may result from post‐transcriptional or post‐
distinguish two stages in respiration: the stage of translational modifications.) The function of glycolysis in
plastids appears to be the production of pyruvate for fatty

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acid biosynthesis, a process confined to plastids in plant opposite direction to respiratory reactions. For instance, in
cells and particularly active in seed tissues synthesizing oil the C3 cycle chloroplast aldolase catalyses the condensation
as nutrient store. Photosynthetic CO2 metabolism involves of triose phosphates to fructose‐1,6‐bisphosphate; in
several reactions catalysed by glycolytic isozymes, but in the glycolysis, cytosolic aldolase cleaves the sugar.

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THE PENTOSE PHOSPHATE PATHWAY, PPP the aromatic amino acids and the precursors of lignin,
flavonoids, and phytoalexins
This reaction series is also known as the hexose • During the early stages of greening, before leaf tissues
monophosphate shunt, or as the oxidative pentose pathway, become fully photoautotrophic, the oxidative pentose
OPP, to distinguish it from an alternative name for the C3 phosphate pathway is thought to be involved in generating
cycle, which is sometimes termed the reductive pentose Calvin cycle intermediates.
phosphate pathway. The PPP is located in the cytosol. It is
outlined in Fig. 2. Control of the oxidative pathway.

The starting substrate for the PPP is glucose‐6‐phosphate The oxidative pentose phosphate pathway is controlled by
(or glucose followed by the hexokinase reaction). The PPP the initial reaction of the pathway catalyzed by glucose‐6‐
commences with the oxidation of glucose‐6‐phosphate to 6‐ phosphate dehydrogenase, the activity of which is markedly
phosphogluconate, followed by an oxidative decarboxylation inhibited by a high ratio of NADPH to NADP+.
of the gluconate to ribulose‐ 5‐phosphate with the release of
CO2 . The respective enzymes for these two reactions are In the light, however, little operation of the oxidative
glucose‐6‐phosphate dehydrogenase and phosphogluconate pathway is likely to occur in the chloroplast because the end
dehydrogenase; the coenzyme which receives the H products of the pathway, fructose‐6‐phosphate and
equivalents from both reactions is NADP+ . The ribulose‐5‐ glyceraldehyde‐3‐phosphate, are being synthesized by the
phosphate is recycled to glucose‐6‐phosphate: Calvin cycle. Thus, mass action will drive the nonoxidative
interconversions of the pathway in the direction of pentose
6 ribulose­5­phosphate Æ 5 glucose­6­phosphate synthesis. Moreover, glucose‐6‐phosphate dehydrogenase
will be inhibited during photosynthesis by the high ratio of
The recycling reactions here are largely a reversal of the C3 NADPH to NADP+ in the chloroplast, as well as by a
cycle reactionswhich regenerate the CO2 acceptor ribulose‐ reductive inactivation involving the ferredoxin–thioredoxin
1,5‐bisphosphate, but no ATP is expended and the System.
chloroplast and the cytosol each has a distinctive set of
isozymes, just as for glycolytic ones.

The balance sheet for the PPP is shown in Equations; to be

able to work with whole molecules, one must start with 6
hexose (C6) molecules. Phosphates have been omitted for
simplicity.

This reduces to:

The PPP thus does achieve the complete oxidation of glucose

to CO2 , but without ATP formation; there is no substrate‐
level phosphorylation involved. The major function of the
PPP is thought to be the provision of NADPH for reductive
biosyntheses, e.g. lipid formation, and for the production of
metabolic intermediates; the pentose sugars can be utilized
for nucleotide synthesis. (Some of the NADPH may be
oxidized by mitochondria with ATP formation.

The oxidative pentose phosphate pathway plays several

roles in plant metabolism:
• The product of the two oxidative steps is NADPH, and this
NADPH is thought to drive reductive steps associated with
various biosynthetic reactions that occur in the cytosol. In
nongreen plastids, such as amyloplasts, and in chloroplasts
functioning in the dark, the pathway may also supply
NADPH for biosynthetic reactions such as lipid biosynthesis
and nitrogen assimilation.
• Because plant mitochondria are able to oxidize cytosolic
NADPH via an NADPH dehydrogenase localized on the
external surface of the inner membrane, some of the
reducing power generated by this pathway may contribute
to cellular energy metabolism; that is, electrons from NADPH
may end up reducing O2 and generating ATP.
• The pathway produces ribose‐5‐phosphate, a precursor
of the ribose and deoxyribose needed in the synthesis
of RNA and DNA, respectively.
• Another intermediate in this pathway, the four‐carbon
erythrose‐4‐phosphate, combines with PEP in the initial
reaction that produces plant phenolic compounds, including

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The Krebs cycle has been regenerated. There is one substrate‐level

phosphorylation producing ATP, associated with the
The Krebs cycle is the reaction series which achieves the oxidation of 2‐oxoglutarate. At three steps, a molecule of
complete oxidation of pyruvate (coming mainly from water is added to the reactants. The overall final balance
glycolysis) to CO2 . It is located entirely and exclusively in the sheet for respiration shows water as a product, but water is
mitochondria. The reactions are summarized in Fig. 3 also a substrate in respiration. (Compare with
photosynthesis, where water is not only consumed as
The pyruvate (with three C atoms) first loses CO 2 by indicated by the overall equation, but is also a product.) The
oxidative decarboxylation catalysed by the enzyme pyruvate 5 pairs of H equivalents removed from the substrates in the
dehydrogenase, which also links the remaining 2 ‐C Krebs cycle reduce NAD+, except for the succinate oxidation
fragment, an acetyl moiety, to coenzyme A producing acetyl‐ step, where no coenzyme is involved. The enzymes are
CoA. This condenses with the 4 –C acid oxaloacetate to form present in the mitochondrial matrix, but succinate
the 6‐C acid citrate. Then, as indicated in Fig. 3, there follows dehydrogenase is again an exception, being bound to the
a series of molecular rearrangements, oxidation steps and crista membrane of the mitochondrion.
oxidative decarboxylation steps, until the equivalent of the
pyruvate has been converted to CO2 and the oxaloacetate

Between them the Krebs cycle and glycolysis can carry out the pairs of H equivalents removed from substrates, one can
the complete oxidation of glucose. With 2 [H] representing write

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CSIR NET LIFESCIENES NOTES‐UNIT 6

The H equivalents can be oxidized in the terminal oxidation

reactions using molecular O2 , and producing 12 H2O per 1
molecule glucose.

Like glycolysis and the PPP, the Krebs cycle can oxidize
intermediates fed in at any place in the sequence; its role in
this respect is discussed later. The Krebs cycle is also an
important source for metabolic intermediates. Malate,
oxaloacetate and 2‐ oxoglutarate are C skeletons for amino
acids. Running down of the cycle by removal of
intermediates is prevented by the occurrence of anaplerotic
reactions which replenish it. One such reaction is the
carboxylation of PEP by PEP carboxylase to give
oxaloacetate; this is of course the initial reaction in C 4 and
CAM photosynthesis, but it takes place also in non‐
photosynthetic cells, at a lower rate. Malate can be produced
by malic enzyme from pyruvate, CO2 and NADPH.

The Citric Acid Cycle of Plants Has Unique Features

The citric acid cycle reactions outlined in Figure are not all
identical with those carried out by animal mitochondria.

For example, the step catalyzed by succinyl‐CoA synthetase

produces ATP in plants and GTP in animals. A feature of
the plant citric acid cycle that is absent in many other
organisms is the significant activity of NAD+ malic enzyme,
which has been found in the matrix of all plant mitochondria
analyzed to date. This enzyme catalyzes the oxidative
decarboxylation of malate:

Malate + NAD+ Æ pyruvate + CO2 + NADH

The presence of NAD+ malic enzyme enables plant

mitochondria to operate alternative pathways for the
metabolism of PEP derived from glycolysis. As already
described, malate can be synthesized from PEP in the cytosol
via the enzymes PEP carboxylase and malate
dehydrogenase. Malate is then transported into the
mitochondrial matrix, where NAD+ malic enzyme can
oxidize it to pyruvate. This reaction makes possible the
complete net oxidation of citric acid cycle intermediates
such as malate or citrate.

Alternatively, the malate produced via the PEP carboxylase

can replace citric acid cycle intermediates used in
biosynthesis. Reactions that can replenish intermediates in a
metabolic cycle are known as anaplerotic. For example,
export of 2‐oxoglutarate for nitrogen assimilation in the
chloroplast will cause a shortage of malate needed in the
ELECTRON TRANSPORT AND ATP SYNTHESIS AT THE
citrate synthase reaction. This malate can be replaced
INNER MITOCHONDRIAL MEMBRANE
through the PEP carboxylase pathway.
ATP is the energy carrier used by cells to drive living
The presence of an alternative pathway for the oxidation of
processes, and chemical energy conserved during the citric
malate is consistent with the observation that many plants,
acid cycle in the form of NADH and FADH2 (redox
in addition to those that carry out crassulacean acid
equivalents with high‐energy electrons) must be converted
metabolism, store significant levels of malate in their central
to ATP to perform useful work in the cell. This O2‐
vacuole.
dependent process, called oxidative phosphorylation,
occurs in the inner mitochondrial membrane.

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Although fundamentally similar in all aerobic cells, the These reduced compounds must be reoxidized or the entire
electron transport chain of plants (and fungi) contains respiratory process will come to a halt. The electron
multiple NAD(P)H dehydrogenases and an alternative transport chain catalyzes an electron flow from NADH (and
oxidase not found in mammalian mitochondria. FADH2) to oxygen, the final electron acceptor of the
respiratory process. For the oxidation of NADH, the overall
The Electron Transport Chain Catalyzes a Flow of two‐electron transfer can be written as follows:
Electrons from NADH to O2
NADH + H+ + 1⁄2 O2 Æ NAD+ + H2O
For each molecule of sucrose oxidized through glycolysis
and the citric acid cycle pathways, 4 molecules of NADH are The electron transport chain of plants contains the same set
generated in the cytosol and 16 molecules of NADH plus 4 of electron carriers found in mitochondria from other
molecules of FADH2 (associated with succinate organisms. The individual electron transport proteins are
dehydrogenase) are generated in the mitochondrial matrix. organized into four multiprotein complexes (identified by
Roman numerals I through IV), all of which are localized in
the inner mitochondrial membrane:

Complex II (succinate dehydrogenase). Oxidation of

Complex I (NADH dehydrogenase). Electrons from NADH succinate in the citric acid cycle is catalyzed by this complex,
generated in the mitochondrial matrix during the citric acid and the reducing equivalents are transferred via the FADH2
cycle are oxidized by complex I (an NADH dehydrogenase). and a group of iron–sulfur proteins into the ubiquinone pool.
The electron carriers in complex I include a tightly bound This complex does not pump protons.
cofactor (flavin mononucleotide [FMN], which is chemically
similar to FAD) and several iron–sulfur centers. Complex I Complex III (cytochrome bc1 complex). This complex
then transfers these electrons to ubiquinone. Four protons oxidizes reduced ubiquinone (ubiquinol) and transfers the
are pumped from the matrix to the intermembrane space for electrons via an iron–sulfur center, two b‐type cytochromes
every electron pair passing through the complex. (b565 and b560), and a membrane‐bound cytochrome c1 to
cytochrome c. Four protons per electron pair are pumped by
Ubiquinone, a small lipid‐soluble electron and proton complex III.
carrier, is located within the inner membrane. It is not
tightly associated with any protein, and it can diffuse within Cytochrome c is a small protein loosely attached to the
the hydrophobic core of the membrane bilayer. outer surface of the inner membrane and serves as a mobile
carrier to transfer electrons between complexes III and IV.

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Complex IV (cytochrome c oxidase). This complex contains • 8 molecules of ATP by substrate‐level phosphorylation (4
two copper centers (CuA and CuB) and cytochromes a and during glycolysis and 4 in the citric acid cycle)
a3. Complex IV is the terminal oxidase and brings about the • 4 molecules of NADH in the cytosol
four‐electron reduction of O2 to two molecules of H2O. • 16 molecules of NADH plus 4 molecules of FADH2 (via
succinate dehydrogenase) in the mitochondrial matrix
Two protons are pumped per electon pair. Both structurally
and functionally, ubiquinone and the cytochrome bc1 On the basis of theoretical ADP:O values, a total of
complex are very similar to plastoquinone and the approximately 52 molecules of ATP will be generated per
cytochrome b6 f complex, respectively, in the photosynthetic sucrose by oxidative phosphorylation. The result is a total of
electron transport chain. about 60 ATPs synthesized per sucrose (Table).

Some Electron Transport Enzymes Are Unique to Plant

Mitochondria

In addition to the set of electron carriers described in the

previous section, plant mitochondria contain some
components not found in mammalian mitochondria. Note
that none of these additional enzymes pump protons and
that energy conservation is therefore lower whenever they
are used:
• Two NAD(P)H dehydrogenases, both Ca2+‐dependent,
attached to the outer surface of the inner membrane facing Using 50 kJ mol–1 (12 kcal mol–1) as the actual free energy of
the intermembrane space can oxidize cytosolic NADH and formation of ATP in vivo, we find that about 3010 kJ mol–1
NADPH. Electrons from these external NAD(P)H (720 kcal mol–1) of free energy is conserved in the form of
dehydrogenases—NDex(NADH) and NDex(NADPH)—enter ATP per mole of sucrose oxidized during aerobic respiration.
the main electron transport chain at the level of the This amount represents about 52% of the standard free
ubiquinone pool. energy available from the complete oxidation of sucrose; the
• Plant mitochondria have two pathways for oxidizing rest is lost as heat. This is a vast improvement over the
matrix NADH. Electron flow through complex I, described in conversion of only 4% of the energy available in sucrose to
the previous section, is sensitive to inhibition by several ATP that is associated with fermentative metabolism.
compounds, including rotenone and piericidin. In addition,
plant mitochondria have a rotenone‐resistant Plants Have Several Mechanisms That Lower the ATP
dehydrogenase, NDin(NADH), for the oxidation of NADH Yield
derived from citric acid cycle substrates. The role of this
pathway may well be as a bypass being engaged when As we have seen, a complex machinery is required for a high
complex I is overloaded, such as under photorespiratory efficiency of energy conservation in oxidative
conditions. phosphorylation. So it is perhaps surprising that plant
• An NADPH dehydrogenase, NDin(NADPH), is present on mitochondria have several functional proteins that reduce
the matrix surface. Very little is known about this enzyme. this efficiency. Probably plants are less limited by the energy
• Most, if not all, plants have an “alternative” respiratory supply (sunlight) than by other factors in the environment
pathway for the reduction of oxygen. This pathway involves (e.g., access to nitrogen or phosphate). As a consequence,
the so‐called alternative oxidase that, unlike cytochrome c adaptational flexibility may be more important than
oxidase, is insensitive to inhibition by cyanide, azide, or energetic efficiency.
carbon monoxide.
In the following subsections we will discuss the role of the
nonphosphorylating mechanisms and their possible
ATP Synthesis in the Mitochondrion Is Coupled to usefulness in the life of the plant.
Electron Transport
The alternative oxidase. If cyanide (1 mM) is added to
In oxidative phosphorylation, the transfer of electrons to actively respiring animal tissues, cytochrome c oxidase is
oxygen via complexes I to IV is coupled to the synthesis of inhibited and the respiration rate quickly drops to less than
ATP from ADP and Pi via the ATP synthase (complex V). The 1% of its initial level. However, most plant tissues display a
number of ATPs synthesized depends on the nature of the level of cyanide‐resistant respiration that can represent 10
electron donor. In experiments conducted with the use of to 25%, and in some tissues up to 100%, of the uninhibited
isolated mitochondria, electrons derived from internal control rate. The enzyme responsible for this oxygen uptake
(matrix) NADH give ADP:O ratios (the number of ATPs has been identified as a cyanide‐resistant oxidase
synthesized per two electrons transferred to oxygen) of 2.4 component of the plant mitochondrial electron transport
to 2.7. Succinate and externally added NADH each give chain called the alternative oxidase.
values in the range of 1.6 to 1.8, while ascorbate, which
serves as an artificial electron donor to cytochrome c, gives Electrons feed off the main electron transport chain into the
values of 0.8 to 0.9. Results such as these (for both plant and alternative pathway at the level of the ubiquinone pool. The
animal mitochondria) have led to the general concept that alternative oxidase, the only component of the alternative
there are three sites of energy conservation along the pathway, catalyzes a four‐electron reduction of oxygen to
electron transport chain, at complexes I, III, and IV. water and is specifically inhibited by several compounds,
most notably salicylhydroxamic acid (SHAM).
Aerobic Respiration Yields about 60 Molecules of
ATP per Molecule of Sucrose When electrons pass to the alternative pathway from the
ubiquinone pool, two sites of proton pumping (at complexes
The complete oxidation of a sucrose molecule leads to the III and IV) are bypassed. Because there is no energy
net formation of

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conservation site in the alternative pathway between It has been suggested that the alternative pathway can
ubiquinone and oxygen, the free energy that would normally function as an “energy overflow” pathway, oxidizing
be conserved as ATP is lost as heat when electrons are respiratory substrates that accumulate in excess of those
shunted through the alternative pathway. needed for growth, storage, or ATP synthesis. This view
suggests that electrons flow through the alternative pathway
How can a process as seemingly energetically wasteful as the only when the activity of the main pathway is saturated.
alternative pathway contribute to plant metabolism? Such saturation is reached in the test tube in state 4; in vivo,
saturation may occur if the respiration rate exceeds the cell’s
One example of the functional usefulness of the alternative demand for ATP (i.e., if ADP levels are very low). However, it
oxidase is its activity during floral development in certain is now clear that the alternative oxidase can be active before
members of the Araceae (the arum family)—for example, the cytochrome pathway is saturated. Thus the alternative
the voodoo lily (Sauromatum guttatum). Just before oxidase makes it possible for the mitochondrion to adjust
pollination, tissues of the clublike inflorescence, called the the relative rates of ATP production and synthesis of carbon
appendix, which bears male and female flowers, exhibit a skeletons for use in biosynthetic reactions.
dramatic increase in the rate of respiration via the
alternative pathway. As a result, the temperature of the Another possible function of the alternative pathway is in
upper appendix increases by as much as 25°C over the the response of plants to a variety of stresses (phosphate
ambient temperature for a period of about 7 hours. deficiency, chilling, drought, osmotic stress, and so on),
many of which can inhibit mitochondrial respiration. By
During this extraordinary burst of heat production, certain draining off electrons from the electron transport chain, the
amines, indoles, and terpenes are volatilized, and the plant alternative pathway prevents a potential overreduction of
therefore gives off a putrid odor that attracts insect the ubiquinone pool, which, if left unchecked, can lead to the
pollinators. Salicylic acid, a phenolic compound related generation of destructive reactive oxygen species such as
to aspirin, has been identified as the chemical signal superoxide anions and hydroxyl radicals. In this way the
responsible for initiating this thermogenic event in the alternative pathway may lessen the detrimental effects of
voodoo lily. stress on respiration.

In most plants, however, both the respiratory rates and the The uncoupling protein. A protein found in the inner
rate of cyanide‐resistant respiration are too low to generate membrane of mammalian mitochondria, the uncoupling
sufficient heat to raise the temperature significantly, so what protein, can dramatically increase the proton permeability
other role(s) does the alternative pathway play? of the membrane and thus act as an uncoupler. As a result,
less ATP and more heat is generated. Heat production
appears to be one of the uncoupling protein’s main functions
in mammalian cells.

It has long been thought that the alternative oxidase in

plants and the uncoupling protein in mammals were simply
two different means of achieving the same end. It was
therefore surprising when a protein similar to the ncoupling
protein was discovered in plant mitochondria. This protein
is stress induced and, like the alternative oxidase, may
function to prevent overreduction of the electron transport
chain. It remains unclear, however, why plant mitochondria
require both mechanisms.

The internal, rotenone­insensitive NADH dehydrogenase,

NDin(NADH).

This is one of the multiple NAD(P)H dehydrogenases found

in plant mitochondria. It has been suggested to work as a
nonproton‐pumping bypass when complex I is overloaded.
Complex I has a higher affinity for NADH (ten times lower
Km), than NDin(NADH). At lower NADH levels in the matrix,
typically when ADP is available (state 3), complex I will
dominate, whereas when ADP is rate limiting (state 4),
NADH levels will increase and NDin(NADH) will be more
active. The physiological importance of this enzyme is,
however, still unclear.

Mitochondrial Respiration Is Controlled by Key

Metabolites

The substrates of ATP synthesis—ADP and Pi—appear to be

key regulators of the rates of glycolysis in the cytosol, as well
as the citric acid cycle and oxidative phosphorylation in the
mitochondria. Control points exist at all three stages of
respiration; here we will give just a brief overview of some
major features.

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The best‐characterized site of regulation of the citric acid intermediates into glycolysis. The PPP accordingly should be
cycle is at the pyruvate dehydrogenase complex, which is considered an integral part of a glycolysis – Krebs cycle –
reversibly phosphorylated by a regulatory kinase and a PPP network. This network in turn interacts with the rest of
phosphatase. cellular metabolism via interchange of metabolites, at many
steps. The provision of intermediates and reduced
Pyruvate dehydrogenase is inactive in the phosphorylated coenzymes is just as much a function of respiration as
state, and the regulatory kinase is inhibited by pyruvate, provision of ATP; respiration, defined as a substrate
allowing the enzyme to be active when substrate is available. breakdown process, is also the starting point of many
In addition, several citric acid cycle enzymes, including biosyntheses.
pyruvate dehydrogenase and 2‐oxoglutarate dehydrogenase,
are directly inhibited by NADH. The ATP balance sheet and energy­conversion efficiency
of respiration
Lipid oxidation
The theoretical balance sheet for respiratory ATP
Lipids are frequently stored in seeds as oil bodies production is easily drawn up. If glucose is completely
(oleosomes, sphaerosomes), where they may account for oxidized via glycolysis and the Krebs cycle, with terminal
over 50% of the tissue mass. The storage oils are oxidation through cytochrome oxidase, one can add up the
triglycerides consisting of a glycerol molecule esterified with ATP molecules per 1 molecule glucose (Table 2). The NADH
three long‐chain fatty acids, mostly with 16 or 18 C atoms from glycolysis, reacting with mitochondria from the
per chain. The first step in lipid oxidation is hydrolysis by outside, is oxidized with the production of only 2 ATP per
lipase enzymes within the oil body to glycerol and fatty NADH, whilst mitochondrially produced coenzyme oxidation
acids. Glycerol, a 3 ‐C sugar alcohol, is closely related to yields 3 ATP per 1 NADH. The free energy of complete
triose sugars and is converted to dihydroxyacetone‐3 ‐ oxidation of glucose is 2880 kJ mol–1 . The free energy of
phosphate, which can then enter the glycolytic pathway. The hydrolysis of ATP is highly dependent on factors such as ATP
fatty acids are activated by linkage to coenzyme A and are concentration and pH, but under cellular conditions is at
metabolized in the glyoxysomes (specialized microbodies of least 42 kJ mol–1 . Assuming this value, a gain of 36 ATP per
oily seeds) by two metabolic sequences. Firstly, the process molecule of glucose is equivalent to an energy conservation
of β‐oxidation results in the cleavage of the fatty acid chain of [3 6 x 4 2 ], 1512 kJ mol–1 , or 52 % of the total available, a
into 2 ‐C fragments, acetyl‐CoA. Secondly, the glyoxysomes very high degree of energy conservation. (The value would
process the acetyl‐CoA via the glyoxylate cycle to succinate be pushed even higher if phosphorylated sugars enter the
and this can, through a series of reactions in the cytosol and glycolytic pathway.
the mitochondria, finally be synthesized to sucrose. Most of
the lipid stored in seeds is converted to sucrose and
transported to the growing parts of the seedling. Some of the
acetyl‐CoA is utilized in the mitochondria of the storage cells
as respiratory substrate in the Krebs cycle.

3 Interactions of pathways

In the cell, none of the pathways of respiratory substrate

oxidation functions in isolation. A summary overview of all
the pathways, and their interrelationships, is presented in
Fig. 2 .15. The first stage, ‘hydrolysis’ in Fig. 4, does not
involve oxidations and is not specific to respiration: the
monomers produced from the polymers serve as substrates
not only for respiration but for numerous other metabolic The calculation in Table 2 is, however, based on an
sequences. The free energy change associated with the assumption of complete coupling of substrate breakdown
hydrolytic reactions is minimal, less than 1% of the total and terminal oxidation to ATP synthesis. This is unlikely to
available, and cannot support ATP synthesis. The be the situation in a cell, at least not in all circumstances.
‘incomplete oxidation’ stage produces a small number of Any NADH which is used in reductive reactions does not
fairly simple organic acids and the free energy content of the yield ATP, and terminal oxidation by the alternative oxidase
substrate falls by about 33 %; some of the energy is produces only 1 ATP per molecule of NADH oxidized. The 12
conserved in ATP during substrate level phosphorylations, NADPH formed per molecule of glucose in the PPP could
and also in NADH. The organic acids feed into the ‘complete theoretically be oxidized by the mitochondria with the
oxidation’ stage, the Krebs cycle, which in association with production of 24 ATP, but, as stated, the PPP probably
the mitochondrial electron transport chain forms the final provides NADPH mainly for reductive reactions rather than
stage for most aerobic respiratory activity and where most for terminal oxidation. It is therefore not possible to say
of the ATP synthesis takes place. The PPP does not appear to exactly how much ATP is produced per glucose molecule in a
fit the overall pattern in that it is a direct oxidative pathway; particular situation.
sugars metabolized exclusively by the PPP do not pass
through the Krebs cycle. However, the PPP and glycolysis Anaerobic respiration
share common intermediates, fructose‐6‐phosphate and
triose phosphates. Since both reaction series take place in Occurrence and endurance of anaerobiosis in plants
the cytosol, it is almost inevitable that some exchange of
metabolites between the two pathways should occur. If, say, Flowering plants are obligate aerobes: no flowering plant
there is a high demand for NADPH, the flow of material can complete its life cycle without O2 . Most flowering plant
through the PPP might be boosted by intermediates from the organs, except dormant seeds, are killed by anoxia
glycolytic sequence. A large demand for pyruvate, on the (complete lack of O2 ) within hours or a few days at most.
other hand, could result in the channelling of PPP Nevertheless there are also many situations in which plant

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tissues regularly survive at least hypoxia, O2 concentrations intermittently damaged by anaerobiosis owing to flooding.
too low to support a normal level of aerobic respiration. Once the air spaces in the soil are filled with water, the
Anaerobic respiration (fermentation) is therefore not limited amount of O2 that is dissolved is rapidly used up by
unusual in plants. In hypoxia, both aerobic and anaerobic the respiration of plant roots and soil microorganisms and
pathways operate simultaneously. the roots then suffer an O2 shortage; this can have serious
consequences for an agricultural crop. An understanding of
Germinating seeds often undergo a period of O2 shortage anaerobic respiration is therefore of considerable practical
during imbibition, because the testa can be highly interest.Knowledge of how e.g. aquatic plants endure
impermeable towards O2 ; also before the cells are fully anaerobiosis might help the development of flooding‐
hydrated and expanded there are hardly any air spaces tolerant crop plants.
between them. Respiration during this period has a high RQ,
respiratory quotient, i.e. the ratio CO2 evolution : O2 uptake. Respiratory metabolism under anaerobiosis

For aerobic respiration this ratio is unity; in anaerobic The respiratory pathwaywhich is functional in anaerobic
respiration, CO2 is evolved without any O2 uptake, raising plant tissues is glycolysis, just as it is in anaerobic
the RQ. Analysis shows an accumulation of end products of microorganisms; this pathway is essentially an anaerobic
fermentation, such as ethanol, in the seed tissues. When the reaction sequence. On transfer to anoxic or hypoxic
emerging radicle splits the testa, the RQ falls. Complete conditions, the transcription and translation of most plant
anoxia is, however, tolerated by seeds of very few species. genes is inhibited, but there is enhanced transcription and
translation of some 20 genes, a major part of which code for
Meristematic tissues carry out partly anaerobic respiration. glycolytic enzymes: aldolase, glucose‐phosphate isomerase,
Cells in meristematic regions are closely packed, without air enolase and glyceraldehydes phosphate dehydrogenase,
spaces, making diffusion of O2 a problem. The vascular including some isozymes not synthesized aerobically. The
cambium lies quite deeply within plant organs and outside it pyruvate produced in glycolysis in the absence of O 2
lies the phloem, a living tissue without intercellular air becomes the oxidant which receives the H equivalents from
spaces and with a high demand for O2 . In apical meristems glycolytic NADH and recycles it to NAD+, which is vital for
the mitochondria are immature, with few cristae per unit the continuance of glycolysis; cells contain only very small
volume, so that their capacity for terminal oxidation is low. amounts of the coenzyme. Three pathways of fermentation
have been demonstrated in plant tissues:
All these factors favour anaerobic respiration. Inside bulky (1) Lactic fermentation: the enzyme lactate dehydrogenase
organs – large fruits, tubers, thick stems and thick roots – catalyses the reaction:
the level of O2 may be under 2 .5% and partially anaerobic pyruvate + NADH Æ lactate + NAD
respiration would be expected; ethanol accumulation has (2) Alcoholic fermentation: firstly pyruvate decarboxylase
been detected. However, some bulky organs are well decarboxylates the pyruvate to acetaldehyde, then this is
supplied with air spaces and then the problem of O 2 reduced with NADH by alcohol dehydrogenase (ADH):
diffusion is much reduced. PyruvateÆCO2 + acetaldehyde
acetaldehyde + NADHÆethanol
In seeds, large organs and meristems, hypoxia is the result of (3 ) Malic fermentation: malic enzyme carboxylates the
the structure of the plant itself. In aquatic plants hypoxia is pyruvate to malate:
imposed by the environment. The solubility of O2 in water is pyruvate + CO2 + NADH Æ malate + NAD+
low. Water at 10 0C in equilibrium with normal air, which is
21% O2 by volume, contains only about 0.80% O2 and the Increased transcription of mRNA for lactate dehydrogenase,
solubility falls with rising temperature. Roots and rhizomes pyruvate decarboxylase and ADH occurs as a response to
growing in mud at the bottom of a body of water, or in boggy anaerobiosis. Why should anaerobiosis be so harmful to
ground, will be in an essentially anoxic environment. By plants? There is still no clear answer to this, nor to what
daytime, green parts of submerged aquatics can respire confers tolerance on such plants as show it. One serious
aerobically on the O2 evolved in photosynthesis and O2 is drawback of anaerobic metabolism is the extremely low
then also conducted to the roots through the extensive air yield of ATP per unit of substrate respired. A switch from
spaces of these plants. By night the O2 levels in and around complete aerobic oxidation to complete anaerobiosis could
the plants will fall very low, especially for the parts in the mean a fall from 3 6 to 2 ATP produced per molecule of
muddy substratum. These parts are highly tolerant of glucose oxidized, a decrease to 5.5%, insufficient to keep a
anoxia. The marsh plant Acorus calamus grows from a cell alive. Even if aerobic respiration were producing only
rhizome and normally overwinters in a submerged resting half the theoretical maximal ATP, the decrease induced by
state, with a low metabolic rate. anaerobiosis would still be drastic. The low ATP yield can be
offset to some degree by the increases in the rate of
Explants from the resting plants, consisting of segments of glycolysis which typically occur under anaerobiosis (the
rhizome with attached roots and small leaves, have survived Pasteur effect). But increases in the rate of glycolysis
laboratory incubation under total anoxia, in the dark, for two induced in plant tissues by anaerobiosis are only 1.5‐ to 6‐
months. Seeds of a limited number of aquatic species can fold. With high rates of glycolysis there is a danger of
germinate in a totally anaerobic environment. Examples are substrate exhaustion and starvation, and also much
rice, a few species of the grass Echinochloa found as a weed accumulation of potentially harmful products of
in rice fields, and the yellow water lily (Nuphar luteum). This fermentation. Lactate and malate are acids and acidification
is obviously an adaptation to their natural environment, of the cytoplasm has been cited as the main cause of cell
where these seeds germinate at the bottom of the water. death in some instances. Acetaldehyde, the intermediate in
Rice and Echinochloa seedlings can survive for at least four alcoholic fermentation, is highly toxic; ethanol is, however,
days anaerobically. tolerated comparatively well. Maize mutants deficient in an
ADH gene succumb rapidly to anaerobiosis, presumably
As the above examples demonstrate, varying degrees of because they cannot remove the acetaldehyde. There is also,
anaerobiosis are normal for, and tolerated by, numerous strange as it may seem, evidence of oxidative stress, with
plant species or tissues. On the other hand, many species are formation of hydrogen peroxide, a dangerous oxidant, from

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trace amounts of O2 . Tissue damage and death could result metabolically more inert tissues have larger proportions of
fromsubstrate exhaustion, acidification, lipid peroxidation cell wall and/or vacuoles and storage materials per unit
and toxin accumulation, singly or in combination. mass, and these tissue compartments do not contribute to
respiratory activity. On a unit nitrogen basis, which reflects
In plant tissues tolerant to anoxia or hypoxia, no basic more truly the ‘living’ mass, differences between tissues of
differences in anaerobic metabolism have been detected, in varying maturity and metabolic activity become less marked.
comparison with sensitive material. Differences seem to be But in the same tissue, respiration rate can be shown to
quantitative rather than qualitative; alcoholic fermentation increase with increasing activity. For instance, when roots
predominates. In submerged plants, at least a sizeable are washed in distilled water and then transferred to a
proportion of the ethanol leaches out into the surrounding nutrient solution from which they proceed to take up ions,
water, the permeability of cellular membranes towards their respiration rate increases concomitantly with ion
ethanol being high. This helps to keep down the absorption. In nectaries, the period of rapid sugar secretion
concentration of alcohol in the tissues. The Acorus calamus coincides with a period of rapid respiration.
rhizome which overwinters in mud, lays down an abundant
store of starch during the summer. Rice grains, which are The relationship between respiration and growth has been
very tolerant to anaerobiosis, are able to synthesize a‐ the subject of much study and speculation. As already noted,
amylase in anoxic conditions and hence can mobilize their growing tissues are characterized by high rates of
starch reserves. The sensitive wheat grain is unable to respiration. The quantitative relationship between growth
synthesize the enzyme and cannot mobilize starch in the rate and respiration rate is, however, not a simple one. On
absence of O2 , but can be induced to germinate the whole, plants with higher growth rates tend to have the
anaerobically by feeding with sucrose or glucose. These higher respiration rates, although there are also reports to
examples show that an ability to maintain an adequate the contrary, e.g. of faster‐growing genotypes of a grass
supply of respiratory substrate can be a factor in survival (Lolium perenne) having lower respiration rates. When plant
under anaerobiosis. systems are compared, the differences in their rates of
respiration are typically less than the differences in their
Tolerance towards anaerobiosis may need induction. Rice growth rates. In a study with nine species of grasses, for a 2
seedlings (as distinct from the ungerminated grains), which –3 ‐fold higher relative growth rate, RGR (for definition of
are killed within 24 hours when suddenly exposed to RGR), the increase in respiration was only 1.4 –1.7‐fold.
complete anoxia, can survive anoxia for several days when Explanations must be sought in differences in metabolism,
first pretreated at low O2 levels. During the pretreatment resulting in a greater efficiency of respiratory energy
period, there are increases in the activities of ADH and production (or utilization) in the faster‐growing species. One
pyruvate decarboxylase, giving the plants a greater potential such difference might be in the degree to which the
for alcoholicfermentation. In maize, development of flooding alternative oxidase participates in the respiration of
tolerance involves increases in activity of several genes different tissues. In a study of root respiration and growth in
coding for ADH and other enzymes of anaerobic respiration. four inbred lines of the greater plantain (Plantago major) it
was found that higher RGR were associated with a higher
Correlation of respiration rate with physiological proportion of respiration passing through the more energy‐
activity efficient cytochrome pathway as opposed to the alternative
oxidase.
Respiration proceeds unceasingly in all active (i.e. non‐
dormant) living cells. Even when a cell is not performing any An understanding of relationships between growth and
net metabolic work and is simply subsisting unchanged – say respiration is of great relevance to considerations of plant
a mature pith parenchyma cell – it still requires repair and productivity. Respiration results in a loss of biomass, even
resynthesis of protoplasmic components, which are labile while it is ecessary to support growth. There have been
and in a constant state of turnover. Membrane potentials are arguments as to whether it is more favourable to breed a
sustained only by continued pumping of ions across crop with a low respiration rate, for minimal loss of biomass,
membranes, requiring ATP. This aspect of cellular activity or with a high rate, associated with a high RGR. It appears
has led to the concept of maintenance respiration, required nowthat it is not just overall respiration rate that is relevant
for such processes. The proportion of respiration that to productivity. To maximize productivity, one would need
supports net cellular work is then termed growth to maximize the efficiency with which the potential energy
respiration (synthetic respiration). More precise definitions of the respiratory substrates is converted to usable form –
of the terms have been attempted, while some plant ATP and reduced coenzymes. We are not yet certain wherein
physiologists have disputed the validity of any division of this efficiency lies.
respiration into these components. The general concept of
maintenance respiration is, however, useful as a reminder Metabolic control of rates: feedback mechanisms
that cells must spend energy for their survival, without
cessation. It is not implied that there is a biochemical Respiration rate is geared to the requirements of tissues by
distinction between growth respiration and maintenance numerous mechanisms. One of these is the cellular
respiration. Measurement of the proportion of maintenance concentration of ADP, and the ATP : ADP ratio. Respiration
respiration is even more controversial than its definition, produces ATP, growth and maintenance processes consume
but for plant cells, its magnitude has been estimated at up to it and produce ADP, the total cellular amount of [ATP + ADP]
50% of the total. remaining constant for prolonged periods. ADP is an
essential reactant in respiration. It is directly used in
Respiration rates are positively correlated with substrate‐level phosphorylations, and mitochondrial
physiological activity. On a unit mass basis (fresh or dry electron transport through cytochrome oxidase is normally
mass), high rates are found in young, actively growing coupled to oxidative phosphorylation, which requires ADP.
regions, such as growing apices, or in tissues performing When there is an increase in an ATP‐utilizing cellular
metabolic work at a high rate, such as glands. To some activity, more ATP per unit time is converted to ADP. The
extent this is the result of the higher ratio of living higher ADP concentration stimulates a faster rate of
protoplasm per unit mass in such tissues; mature and substrate breakdown and mitochondrial electron transport

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– with a higher rate of ATP synthesis. The result is a higher and demand was established. For the PPP, the ratio of NADP+
rate of respiration and a higher turnover of ATP and ADP. : NADPH is a regulatory factor.
The ratio ATP : ADP seems to be fairly constant in cells.
When metabolic pathways were first elucidated, rate control
The effects of ATP and ADP are not only simple was postulated to be concentrated at a few key steps
concentration effects. The glycolytic enzyme catalysed by ‘pacemaker’ enzymes. For glycolysis,
phosphofructokinase is inhibited by ATP, which is one of its phosphofructokinase and pyruvate kinase were considered
substrates; pyruvate kinase is also inhibited by ATP. On the to be pacemakers, these being the two glycolytic enzymes
other hand, ADP activates pyruvate kinase. These effects for which the reactions are irreversible under physiological
would result in slowing glycolysis by an increase in ATP conditions. Several metabolites are moreover known to
concentration and a speeding up by a rise in the regulate the activities of these enzymes, as noted in the
concentration of ADP. previous paragraph. But changing the amounts of individual
enzymes in cells by genetic manipulation has shown that
Accumulation of intermediates of the respiratory pathways large changes in the activities of phosphofructokinase and
causes inhibition of enzymes acting earlier in the pathways – pyruvate kinase have little effect on respiration rate. It now
the well‐known phenomenon of feedback inhibition. appears that all steps in glycolysis and the Krebs cycle
Phosphofructokinase, one ofthe first enzymes of the contribute to regulation of respiration rate, though not all to
glycolytic pathway, is inhibited by phosphoglycerate and by the same degree. From the viewpoint of the cell, the larger
phosphoenolpyruvate. Citrate (from the Krebs cycle) the number of control points, the more opportunities there
inhibits both phosphofructokinase and pyruvate kinase. A are for finetuning and interactions between pathways. From
fall in the demand for ATP would slow down the the viewpoint of the investigator, it makes the study of rate
mitochondrial electron transport; citrate metabolism would control exceedingly complex. There is nevertheless no doubt
slow down correspondingly, being dependent on the regarding the basic principle: respiration rate is integrated
mitochondrial terminal oxidation. The accumulating citrate with cellular activity so that a depletion of respiratory
would cause slowing down of glycolysis. Similarly, a fall in products (ATP and metabolites) leads to an increase in the
the demand for biosynthetic intermediates would result in a rate of respiration. An accumulation of the same leads to a
build‐up of these intermediates, and in an inhibition of decrease in the respiration rate and in each case a new
preceding steps. Any change in requirement for ATP or/and steady state is achieved.
metabolic intermediates would result in an adjustment in
respiration rate, until a new equilibrium between supply

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C. NITROGEN METABOLISM: NITRATE AND AMMONIUM ASSIMILATION; BIOLOGICAL NITROGEN FIXATION

Higher plants are autotrophic organisms that can synthesize NITROGEN IN THE ENVIRONMENT
their organic molecular components out of inorganic
nutrients obtained from their surroundings. For many Many biochemical compounds present in plant cells contain
mineral nutrients, this process involves absorption from the nitrogen. For example, nitrogen is found in the nucleoside
soil by the roots and incorporation into the organic phosphates and amino acids that form the building blocks of
compounds that are essential for growth and development. nucleic acids and proteins, respectively. Only the elements
This incorporation of mineral nutrients into organic oxygen, carbon, and hydrogen are more abundant in plants
substances such as pigments, enzyme cofactors, lipids, than nitrogen. Most natural and agricultural ecosystems
nucleic acids, and amino acids is termed nutrient show dramatic gains in productivity after fertilization with
assimilation. inorganic nitrogen, attesting to the importance of this
element. In this section we will discuss the biogeochemical
Assimilation of some nutrients—particularly nitrogen cycle of nitrogen, the crucial role of nitrogen fixation in the
requires a complex series of biochemical reactions that are conversion of molecular nitrogen into ammonium and
among the most energy‐requiring reactions in living nitrate, and the fate of nitrate and ammonium in plant
organisms: tissues.
• In nitrate (NO3 –) assimilation, the nitrogen in NO3– is
converted to a higher‐energy form in nitrite (NO2 –), then to Nitrogen Passes through Several Forms in a
a yet higher‐energy form in ammonium (NH4 +), and finally Biogeochemical Cycle
into the amide nitrogen of glutamine. This process
consumes the equivalent of 12 ATPs per nitrogen. Nitrogen is present in many forms in the biosphere. The
• Plants such as legumes form symbiotic relationships with atmosphere contains vast quantities (about 78% by volume)
nitrogen‐ fixing bacteria to convert molecular nitrogen (N2) of molecular nitrogen (N2). For the most part, this large
into ammonia (NH3). Ammonia (NH3) is the first stable reservoir of nitrogen is not directly available to living
product of natural fixation; at physiological pH, however, organisms. Acquisition of nitrogen from the atmosphere
ammonia is protonated to form the ammonium ion (NH4 +). requires the breaking of an exceptionally stable triple
The process of biological nitrogen fixation, together with the covalent bond between two nitrogen atoms (N—N) to
subsequent assimilation of NH3 into an amino acid, produce ammonia (NH3) or nitrate (NO3–).
consumes about 16 ATPs per nitrogen.
These reactions, known as nitrogen fixation, can be
For some perspective on the enormous energies involved, accomplished by both industrial and natural processes.
consider that if these reactions run rapidly in reverse—say, Under elevated temperature (about 200°C) and high
from NH4NO3 (ammonium nitrate) to N2—they become pressure (about 200 atmospheres), N2 combines with
explosive, liberating vast amounts of energy as motion, heat, hydrogen to form ammonia. The extreme conditions are
and light. Nearly all explosives are based on the rapid required to overcome the high activation energy of the
oxidation of nitrogen or sulfur compounds. reaction. This nitrogen fixation reaction, called the Haber–
Bosch process, is a starting point for the manufacture of
many industrial and agricultural products. Worldwide
industrial production of nitrogen fertilizers amounts to
more than 80 x 1012 g yr–1. Natural processes fix about 190 x
1012 g yr–1 of nitrogen (Table 1) through the following
processes:

• Lightning. Lightning is responsible for about 8% of the

nitrogen fixed. Lightning converts water vapor and oxygen

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into highly reactive hydroxyl free radicals, free hydrogen in their vacuoles, thus avoiding toxic effects on membranes
atoms, and free oxygen atoms that attack molecular nitrogen and the cytosol.
(N2) to form nitric acid (HNO3). This nitric acid subsequently
falls to Earth with rain.
• Photochemical reactions. Approximately 2% of the nitrogen
fixed derives from photochemical reactions between
gaseous nitric oxide (NO) and ozone (O3) that produce nitric
acid (HNO3).
• Biological nitrogen fixation. The remaining 90% results
from biological nitrogen fixation, in which bacteria or blue‐
green algae (cyanobacteria) fix N2 into ammonium (NH4+).
From an agricultural standpoint, biological nitrogen fixation
is critical because industrial production of nitrogen
fertilizers seldom meets agricultural demand.

Once fixed in ammonium or nitrate, nitrogen enters a

biogeochemical cycle and passes through several organic or
inorganic forms before it eventually returns to molecular NITRATE ASSIMILATION
nitrogen (Figure 1; see also Table 1). The ammonium (NH4+)
and nitrate (NO3–) ions that are generated through fixation Plants assimilate most of the nitrate absorbed by their roots
or released through decomposition of soil organic matter into organic nitrogen compounds. The first step of this
become the object of intense competition among plants and process is the reduction of nitrate to nitrite in the cytosol.
microorganisms. To remain competitive, plants have The enzyme nitrate reductase catalyzes this reaction:
developed mechanisms for scavenging these ions from the NO3‐+NAD(P)H + H++2e‐ Æ NO2‐ + NAD(P)++H2O (eq.1)
soil solution as quickly as possible. Under the elevated soil where NAD(P)H indicates NADH or NADPH. The most
concentrations that occur after fertilization, the absorption common form of nitrate reductase uses only NADH as an
of ammonium and nitrate by the roots may exceed the electron donor; another form of the enzyme that is found
capacity of a plant to assimilate these ions, leading to their predominantly in nongreen tissues such as roots can use
accumulation within the plant’s tissues. either NADH or NADPH.

Stored Ammonium or Nitrate Can Be Toxic The nitrate reductases of higher plants are composed of two
identical subunits, each containing three prosthetic groups:
Plants can store high levels of nitrate, or they can translocate FAD (flavin adenine dinucleotide), heme, and a molybdenum
it from tissue to tissue without deleterious effect. However, complexed to an organic molecule called a pterin.
if livestock and humans consume plant material that is high
in nitrate, they may suffer methemoglobinemia, a disease in
which the liver reduces nitrate to nitrite, which combines
with hemoglobin and renders the hemoglobin unable to bind
oxygen. Humans and other animals may also convert nitrate
into nitrosamines, which are potent carcinogens. Some
countries limit the nitrate content in plant materials sold for
human consumption.
Nitrate reductase is the main molybdenum‐containing
In contrast to nitrate, high levels of ammonium are toxic to protein in vegetative tissues, and one symptom of
both plants and animals. Ammonium dissipates molybdenum deficiency is the accumulation of nitrate that
transmembrane proton gradients (Figure 2) that are results from diminished nitrate reductase activity.
required for both photosynthetic and respiratory electron Comparison of the amino acid sequences for nitrate
transport and for sequestering metabolites in the vacuole. reductase from several species with those of other
Because high levels of ammonium are dangerous, animals wellcharacterized proteins that bind FAD, heme, or
have developed a strong aversion to its smell. The active molybdenum has led to the three‐domain model for nitrate
ingredient in smelling salts, a medicinal vapor released reductase shown in Figure 3. The FAD‐binding domain
under the nose to revive a person who has fainted, is accepts two electrons from NADH or NADPH. The electrons
ammonium carbonate. Plants assimilate ammonium near the then pass through the heme domain to the molybdenum
site of absorption or generation and rapidly store any excess complex, where they are transferred to nitrate.

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NO2– + 6 Fdred + 8 H+ + 6 e–→ NH4+ + 6 Fdox + 2H2O (eq. 2)

where Fd is ferredoxin, and the subscripts red and ox stand

for reduced and oxidized, respectively. Reduced ferredoxin
derives from photosynthetic electron transport in the
chloroplasts and from NADPH generated by the oxidative
pentose phosphate pathway in nongreen tissues.
Chloroplasts and root plastids contain different forms of the
enzyme, but both forms consist of a single polypeptide
containing two prosthetic groups: an iron–sulfur cluster
(Fe4S4) and a specialized heme.

These groups acting together bind nitrite and reduce it

directly to ammonium, without accumulation of nitrogen
compounds of intermediate redox states. The electron flow
through ferredoxin (Fe4S4) and heme can be represented as
in Figure 5.

Nitrate, Light, and Carbohydrates Regulate Nitrate

Reductase

Nitrate, light, and carbohydrates influence nitrate reductase

at the transcription and translation levels. In barley
seedlings, nitrate reductase mRNA was detected
approximately 40 minutes after addition of nitrate, and
maximum levels were attained within 3 hours (Figure 4). In
contrast to the rapid mRNA accumulation, here was a
gradual linear increase in nitrate reductase activity,
reflecting the slower synthesis of the protein. In addition,
the protein is subject to posttranslational modulation Nitrite reductase is encoded in the nucleus and synthesized
(involving a reversible phosphorylation) that is analogous to in the cytoplasm with an N‐terminal transit peptide that
the regulation of sucrose phosphate synthase. Light, targets it to the plastids. Whereas NO3– and light induce the
carbohydrate levels, and other environmental factors transcription of nitrite reductase mRNA, the end products of
stimulate a protein phosphatase that dephosphorylates the process—asparagine and glutamine—repress this
several serine residues on the nitrate reductase protein and induction.
thereby activates the enzyme.
Plants Can Assimilate Nitrate in Both Roots and Shoots

In many plants, when the roots receive small amounts of

nitrate, nitrate is reduced primarily in the roots. As the
supply of nitrate increases, a greater proportion of the
absorbed nitrate is translocated to the shoot and assimilated
there. Even under similar conditions of nitrate supply, the
balance between root and shoot nitrate metabolism—as
indicated by the proportion of nitrate reductase activity in
each of the two organs or by the relative concentrations of
nitrate and reduced nitrogen in the xylem sap—varies from
species to species.

In plants such as the cocklebur (Xanthium strumarium),

nitrate metabolism is restricted to the shoot; in other plants,
Operating in the reverse direction, darkness and Mg2+ such as white lupine (Lupinus albus), most nitrate is
stimulate a protein kinase that phosphorylates the same metabolized in the roots. Generally, species native to
serine residues, which then interact with a 14‐3‐3 inhibitor temperate regions rely more heavily on nitrate assimilation
protein, and thereby inactivate nitrate reductase. Regulation by the roots than do species of tropical or subtropical
of nitrate reductase activity through phosphorylationand origins.
dephosphorylation provides more rapid control than can be
achieved through synthesis or degradation of theenzyme AMMONIUM ASSIMILATION
(minutes versus hours).
Plant cells avoid ammonium toxicity by rapidly converting
Nitrite Reductase Converts Nitrite to Ammonium the ammonium generated from nitrate assimilation or
photorespiration into amino acids. The primary pathway for
Nitrite (NO2–) is a highly reactive, potentially toxic ion. Plant this conversion involves the sequential actions of glutamine
cells immediately transport the nitrite generated by nitrate synthetase and glutamate synthase. In this section we will
reduction from the cytosol into chloroplasts in leaves and discuss the enzymatic processes that mediate the
plastids in roots. In these organelles, the enzyme nitrite assimilation of ammonium into essential amino acids, and
reductase reduces nitrite to ammonium according to the the role of amides in the regulation of nitrogen and carbon
following overall reaction: metabolism.

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Conversion of Ammonium to Amino Acids Requires Two contain two types of GOGAT: One accepts electrons from
Enzymes NADH; the other accepts electrons from ferredoxin (Fd):

Glutamine synthetase (GS) combines ammonium with Glutamine + 2‐oxoglutarate + NADH + H+ →

glutamate to form glutamine (Figure 6A): 2 glutamate + NAD+ (eq. 4)
Glutamate + NH4+ + ATP → glutamine + ADP + Pi (eq.3) Glutamine + 2‐oxoglutarate + Fdred →
This reaction requires the hydrolysis of one ATP and 2 glutamate + Fdox (eq.5)
involves a divalent cation such as Mg2+, Mn2+, or Co2+ as a
cofactor. Plants contain two classes of GS, one in the cytosol The NADH type of the enzyme (NADH‐GOGAT) is located in
and the other in root plastids or shoot chloroplasts. The plastids of nonphotosynthetic tissues such as roots or
cytosolic forms are expressed in germinating seeds or in the vascular bundles of developing leaves. In roots, NADH‐
vascular bundles of roots and shoots and produce glutamine GOGAT is involved in the assimilation of NH4+ absorbed from
for intracellular nitrogen transport. The GS in root plastids the rhizosphere (the soil near the surface of the roots); in
generates amide nitrogen for local consumption; the GS in vascular bundles of developing leaves, NADH‐GOGAT
shoot chloroplasts reassimilates photorespiratory NH4+. assimilates glutamine translocated from roots or senescing
Light and carbohydrate levels alter leaves.
the expression of the plastid forms of the enzyme, but they
have little effect on the cytosolic forms. The ferredoxin‐dependent type of glutamate synthase (Fd‐
GOGAT) is found in chloroplasts and serves in
Elevated plastid levels of glutamine stimulate the activity of photorespiratory nitrogen metabolism. Both the amount of
glutamate synthase (also known as glutamine:2­ protein and its activity increase with light levels. Roots,
oxoglutarate aminotransferase, or GOGAT). This enzyme particularly those under nitrate nutrition, have Fd‐GOGAT in
transfers the amide group of glutamine to 2‐oxoglutarate, plastids. Fd‐GOGAT in the roots presumably functions to
yielding two molecules of glutamate (see Figure 6A). Plants incorporate the glutamine generated during nitrate
assimilation.

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Ammonium Can Be Assimilated via an Alternative carbohydrates) stimulate GS and GOGAT, inhibit AS, and
Pathway thus favor nitrogen assimilation into glutamine and
glutamate, compounds that are rich in carbon and
Glutamate dehydrogenase (GDH) catalyzes a reversible participate in the synthesis of new plant materials.
reaction that synthesizes or deaminates glutamate (Figure
6B): By contrast, energy‐limited conditions inhibit GS and
2‐Oxoglutarate + NH4+ + NAD(P)H ↔ GOGAT, stimulate AS, and thus favor nitrogen assimilation
glutamate + H2O + NAD(P)+ (eq.6) into asparagine, a compound that is rich in nitrogen and
sufficiently stable for long‐distance transport or long‐term
An NADH‐dependent form of GDH is found in mitochondria, storage.
and an NADPH‐dependent form is localized in the
chloroplasts of photosynthetic organs. Although both forms BIOLOGICAL NITROGEN FIXATION
are relatively abundant, they cannot substitute for the GS–
GOGAT pathway for assimilation of ammonium, and their Biological nitrogen fixation accounts for most of the fixation
primary function is to deaminate glutamate (see Figure 6B). of atmospheric N2 into ammonium, thus representing the
key entry point of molecular nitrogen into the
Transamination Reactions Transfer Nitrogen biogeochemical cycle of nitrogen (see Figure 1). This section
describes the properties of the nitrogenase enzymes that fix
Once assimilated into glutamine and glutamate, nitrogen is nitrogen, the symbiotic relations between nitrogen‐fixing
incorporated into other amino acids via transamination organisms and higher plants, the specialized structures that
reactions. The enzymes that catalyze these reactions are form in roots when infected by nitrogen‐fixing bacteria, and
known as aminotransferases. An example is aspartate the genetic and signaling interactions that regulate nitrogen
aminotransferase (Asp­AT), which catalyzes the following fixation by symbiotic prokaryotes and their hosts.
reaction (Figure C):
Glutamate + oxaloacetate → Free­Living and Symbiotic Bacteria Fix Nitrogen
aspartate + 2‐oxoglutarate (eq.7)
Some bacteria, as stated earlier, can convert atmospheric
in which the amino group of glutamate is transferred to the nitrogen into ammonium (Table 2). Most of these nitrogen‐
carboxyl atom of aspartate. Aspartate is an amino acid that fixing prokaryotes are free‐living in the soil. A few form
participates in the malate–aspartate shuttle to transfer symbiotic associations with higher plants in which the
reducing equivalents from the mitochondrion and prokaryote directly provides the host plant with fixed
chloroplast into the cytosol and in the transport of carbon nitrogen in exchange for other nutrients and carbohydrates
from mesophyll to bundle sheath for C4 carbon fixation. All (top portion of Table 2). Such symbioses occur in nodules
transamination reactions require pyridoxal phosphate that form on the roots of the plant and contain the nitrogen‐
(vitamin B6) as a cofactor. Aminotransferases are found in fixing bacteria. The most common type of symbiosis occurs
the cytoplasm, chloroplasts, mitochondria, glyoxysomes, and between members of the plant family Leguminosae and soil
peroxisomes. The aminotransferases localized in the bacteria of the genera Azorhizobium, Bradyrhizobium,
chloroplasts may have a significant role in amino acid Photorhizobium, Rhizobium, and Sinorhizobium (collectively
biosynthesis because plant leaves or isolated chloroplasts called rhizobia; Table 3). Another common type of
exposed to radioactively labeled carbon dioxide rapidly symbiosis occurs between several woody plant species, such
incorporate the label into glutamate, aspartate, alanine, as alder trees, and soil bacteria of the genus Frankia. Still
serine, and glycine. other types involve the South American herb Gunnera and
the tiny water fern Azolla, which form associations with the
Asparagine and Glutamine Link Carbon and Nitrogen cyanobacteria Nostoc and Anabaena, respectively (see Table
Metabolism 2).

Asparagine, isolated from asparagus as early as 1806, was

the first amide to be identified. It serves not only as a protein
precursor, but as a key compound for nitrogen transport and
storage because of its stability and high nitrogen‐to‐carbon
ratio (2 N to 4 C for asparagine, versus 2 N to 5 C for
glutamine or 1 N to 5 C for glutamate). The major pathway
for asparagine synthesis involves the transfer of the amide
nitrogen from glutamine to asparagine (Figure 6D):
Glutamine + aspartate + ATP →
asparagine + glutamate + AMP + PPi (eq.8)

Asparagine synthetase (AS), the enzyme that catalyzes this

reaction, is found in the cytosol of leaves and roots and in
nitrogen‐fixing nodules (see the next . In maize roots,
particularly those under potentially toxic levels of ammonia,
ammonium may replace glutamine as the source of the
amide group.

High levels of light and carbohydrate—conditions that

stimulate plastid GS and Fd‐GOGAT—inhibit the expression
of genes coding for AS and the activity of the enzyme. The
opposing regulation of these competing pathways helps
balance the metabolism of carbon and nitrogen in plants.
Conditions of ample energy (i.e., high levels of light and

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Nitrogen Fixation Requires Anaerobic Conditions nitrogen to grant independence from nitrogen fertilization.
The potential for applying Azospirillum to corn and other
Because oxygen irreversibly inactivates the nitrogenase grains has been explored, but Azospirillum seems to fix little
enzymes involved in nitrogen fixation, nitrogen must be nitrogen when associated with plants.
fixed under anaerobic conditions. Thus each of the nitrogen‐
fixing organisms listed in Table 2 either functions under Legumes and actinorhizal plants regulate gas permeability in
natural anaerobic conditions or can create an internal their nodules, maintaining a level of oxygen within the
anaerobic environment in the presence of oxygen. In nodule that can support respiration but is sufficiently low to
cyanobacteria, anaerobic conditions are created in avoid inactivation of the nitrogenase. Gas permeability
specialized cells called heterocysts. Heterocysts are thick‐ increases in the light and decreases under drought or upon
walled cells that differentiate when filamentous exposure to nitrate. The mechanism for regulating gas
cyanobacteria are deprived of NH4+. These cells lack permeability is not yet known.
photosystem II, the oxygen‐producing photosystem of
chloroplasts, so they do not generate oxygen. Heterocysts
appear to represent an adaptation for nitrogen fixation, in
that they are widespread among aerobic cyanobacteria that
fix nitrogen.

Cyanobacteria that lack heterocysts can fix nitrogen only

under anaerobic conditions such as those that occur in
flooded fields. In Asian countries, nitrogen‐fixing
cyanobacteria of both the heterocyst and nonheterocyst
types are a major means for maintaining an adequate
nitrogen supply in the soil of rice fields. These
microorganisms fix nitrogen when the fields are flooded and
die as the fields dry, releasing the fixed nitrogen to the soil.
Another important ource of available nitrogen in flooded
rice fields is the water fern Azolla, which associates with the
cyanobacterium Anabaena. The Azolla–Anabaena association
can fix as much as 0.5 kg of atmospheric nitrogen per
hectare per day, a rate of fertilization that is sufficient to
attain moderate rice yields. Nodules contain an oxygen‐binding heme protein called
leghemoglobin. Leghemoglobin is present in the cytoplasm
Free‐living bacteria that are capable of fixing nitrogen are of infected nodule cells at high concentrations (700 µM in
aerobic, facultative, or anaerobic (see Table 2, bottom): soybean nodules) and gives the nodules a pink color.
• Aerobic nitrogen‐fixing bacteria such as Azotobacter are
thought to maintain reduced oxygen conditions The host plant produces the globin portion of leghemoglobin
(microaerobic conditions) through their high levels of in response to infection by the bacteria; the bacterial
respiration. Others, such as Gloeothece, evolve O2 symbiont produces the heme portion. Leghemoglobin has a
photosynthetically during the day and fix nitrogen during high affinity for oxygen (a Km of about 0.01 µM), about ten
the night. times higher than the β chain of human hemoglobin.
• Facultative organisms, which are able to grow under both
aerobic and anaerobic conditions, generally fix nitrogen only Although leghemoglobin was once thought to provide a
under anaerobic conditions. buffer for nodule oxygen, recent studies indicate that it
• For anaerobic nitrogen‐fixing bacteria, oxygen does not stores only enough oxygen to support nodule respiration for
pose a problem, because it is absent in their habitat. a few seconds. Its function is to help transport oxygen to the
respiring symbiotic bacterial cells in a manner analogous to
These anaerobic organisms can be either photosynthetic hemoglobin transporting oxygen to respiring tissues in
(e.g., Rhodospirillum), or nonphotosynthetic (e.g., animals.
Clostridium).
Establishing Symbiosis Requires an Exchange of Signals
Symbiotic Nitrogen Fixation Occurs in Specialized
Structures The symbiosis between legumes and rhizobia is not
obligatory. Legume seedlings germinate without any
Symbiotic nitrogen‐fixing prokaryotes dwell within nodules, association with rhizobia, and they may remain
the special organs of the plant host that enclose the unassociated throughout their life cycle. Rhizobia also occur
nitrogen‐fixing bacteria. In the case of Gunnera, these organs as free‐living organisms in the soil. Under nitrogen‐limited
are existing stem glands that develop independently of the conditions, however, the symbionts seek out one another
symbiont. In the case of legumes and actinorhizal plants, the through an elaborate exchange of signals. This signaling, the
nitrogen‐fixing bacteria induce the plant to form root subsequent infection process, and the development of
nodules. nitrogenfixing nodules involve specific genes in both the
host and the symbionts.
Grasses can also develop symbiotic relationships with
nitrogen‐fixing organisms, but in these associations root Plant genes specific to nodules are called nodulin (Nod)
nodules are not produced. Instead, the nitrogen‐fixing genes; rhizobial genes that participate in nodule formation
bacteria seem to colonize plant tissues or anchor to the root are called nodulation (nod) genes. The nod genes are
surfaces, mainly around the elongation zone and the root classified as common nod genes or host‐specific nod genes.
hairs. For example, the nitrogen‐fixing bacterium The common nod genes—nodA, nodB, and nodC—are found
Acetobacter diazotrophicus lives in the apoplast of stem in all rhizobial strains; the host‐specific nod genes—such as
tissues in sugarcane and may provide its host with sufficient nodP, nodQ, and nodH; or nodF, nodE, and nodL—differ

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among rhizobial species and determine the host range. Only A particular legume host responds to a specific Nod
one of the nod genes, the regulatory nodD, is constitutively factor.The legume receptors for Nod factors appear to be
expressed, and as we will explain in detail, its protein special lectins (sugar‐binding proteins) produced in the root
product (NodD) regulates the transcription of the other nod hairs. Nod factors activate these lectins, increasing their
genes. hydrolysis of phosphoanhydride bonds of nucleoside di‐ and
triphosphates. This lectin activation directs particular
The first stage in the formation of the symbiotic relationship rhizobia to appropriate hosts and facilitates attachment of
between the nitrogen‐fixing bacteria and their host is the rhizobia to the cell walls of a root hair.
migration of the bacteria toward the roots of the host plant.
This migration is a chemotactic response mediated by Nodule Formation Involves Several Phytohormones
chemical attractants, especially (iso)flavonoids and betaines,
secreted by the roots. These attractants activate the Two processes—infection and nodule organogenesis—
rhizobial NodD protein, which then induces transcription of occur simultaneously during root nodule formation. During
the other nod genes. the infection process, rhizobia that are attached to the root
hairs release Nod factors that induce a pronounced curling
The promoter region of all nod operons, except that of nodD, of the root hair cells (Figure 8A and B). The rhizobia become
contains a highly conserved sequence called the nod box. enclosed in the small compartment formed by the curling.
Binding of the activated NodD to the nod box induces The cell wall of the root hair degrades in these regions, also
transcription of the other nod genes. in response to Nod factors, allowing the bacterial cells direct
access to the outer surface of the plant plasma membrane.
Nod Factors Produced by Bacteria Act as Signals for
Symbiosis The next step is formation of the infection thread (Figure
8C), an internal tubular extension of the plasma membrane
The nod genes activated by NodD code for nodulation that is produced by the fusion of Golgi‐derived membrane
proteins, most of which are involved in the biosynthesis of vesicles at the site of infection. The thread grows at its tip by
Nod factors. Nod factors are lipochitin oligosaccharide the fusion of secretory vesicles to the end of the tube. Deeper
signal molecules, all of which have a chitin β‐1→4‐linked into the root cortex, near the xylem, cortical cells
Nacetyl‐ D‐glucosamine backbone (varying in length from dedifferentiate and start dividing, forming a distinct area
three to six sugar units) and a fatty acyl chain on the C‐2 within the cortex, called a nodule primordium, from which
position of the nonreducing sugar (Figure 7). the nodule will develop. The nodule primordia form
opposite the protoxylem poles of the root vascular bundle.

Different signaling compounds, acting either positively or

negatively, control the position of nodule primordia. The
nucleoside uridine diffuses from the stele into the cortex in
the protoxylem zones of the root and stimulates cell division.
Ethylene is synthesized in the region of the pericycle,
diffuses into the cortex, and blocks cell division opposite the
phloem poles of the root. The infection thread filled with
proliferating rhizobia elongates through the root hair and
cortical cell layers, in the direction of the nodule
primordium. When the infection thread reaches specialized
cells within the nodule, its tip fuses with the plasma
membrane of the host cell, releasing bacterial cells that are
packaged in a membrane derived from the host cell plasma
membrane (see Figure 8D).

Three of the nod genes (nodA, nodB, and nodC) encode Branching of the infection thread inside the nodule enables
enzymes (NodA, NodB, and NodC, respectively) that are the bacteria to infect many cells (see Figure 8E and F). At
required for synthesizing this basic structure: first the bacteria continue to divide, and the surrounding
1. NodA is an N‐acyltransferase that catalyzes the addition of membrane increases in surface area to accommodate this
a fatty acyl chain. growth by fusing with smaller vesicles. Soon thereafter,
2. NodB is a chitin‐oligosaccharide deacetylase that removes upon an undetermined signal from the plant, the bacteria
the acetyl group from the terminal nonreducing sugar. stop dividing and begin to enlarge and to differentiate into
3. NodC is a chitin‐oligosaccharide synthase that links N‐ nitrogen‐fixing endosymbiotic organelles called bacteroids.
acetyl‐D‐glucosamine monomers. The membrane surrounding the bacteroids is called the
peribacteroid membrane.
Host‐specific nod genes that vary among rhizobial species
are involved in the modification of the fatty acyl chain or the The nodule as a whole develops such features as a vascular
addition of groups important in determining host specificity: system (which facilitates the exchange of fixed nitrogen
• NodE and NodF determine the length and degree of produced by the bacteroids for nutrients contributed by the
saturation of the fatty acyl chain; those of Rhizobium plant) and a layer of cells to exclude O2 from the root nodule
leguminosarum bv. viciae and R. meliloti result in the interior. In some temperate legumes (e.g., peas), the nodules
synthesis of an 18:4 and a 16:2 fatty acyl group, respectively. are elongated and cylindrical because of the presence of a
• Other enzymes, such as NodL, influence the host specificity nodule meristem. The nodules of tropical legumes, such as
of Nod factors through the addition of specific substitutions soybeans and peanuts, lack a persistent meristem and are
at the reducing or nonreducing sugar moieties of the chitin spherical.
backbone.

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The Nitrogenase Enzyme Complex Fixes N2 In the overall nitrogen reduction reaction (see Figure 9),
ferredoxin serves as an electron donor to the Fe protein,
Biological nitrogen fixation, like industrial nitrogen fixation, which in turn hydrolyzes ATP and reduces the MoFe protein.
produces ammonia from molecular nitrogen. The overall The MoFe protein then can reduce numerous substrates
reaction is (Table 4), although under natural conditions it reacts only
N2 + 8 e– + 8 H+ + 16 ATP → with N2 and H+. One of the reactions catalyzed by
2 NH3 + H2 + 16 ADP + 16 Pi (eq.9) nitrogenase, the reduction of acetylene to ethylene, is used
in estimating nitrogenase activity.
Note that the reduction of N2 to 2 NH3, a six‐electron
transfer, is coupled to the reduction of two protons to evolve
H2. The nitrogenase enzyme complex catalyzes this
reaction. The nitrogenase enzyme complex can be separated
into two components—the Fe protein and the MoFe
protein— neither of which has catalytic activity by itself
(Figure 9):
• The Fe protein is the smaller of the two components and
has two identical subunits of 30 to 72 kDa each, depending
on the organism. Each subunit contains an iron–sulfur
cluster (4 Fe and 4 S2– ) that participates in the redox
reactions involved in the conversion of N2 to NH3. The Fe
protein is irreversibly inactivated by O2 with typical half‐
decay times of 30 to 45 seconds.
• The MoFe protein has four subunits, with a total molecular
mass of 180 to 235 kDa, depending on the species. Each
subunit has two Mo–Fe–S clusters. The MoFe protein is also
inactivated by oxygen, with a half‐decay time in air of 10
minutes.

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The energetics of nitrogen fixation is complex. The to the shoot via the xylem. Nitrogen‐fixing legumes can be
production of NH3 from N2 and H2 is an exergonic reaction, divided into amide exporters or ureide exporters on the
with a ∆G0`(change in free energy) of –27 kJ mol–1. However, basis of the composition of the xylem sap. Amides
industrial production of NH3 from N2 and H2 is endergonic, (principally the amino acids asparagine or glutamine) are
requiring a very large energy input because of the activation exported by temperate‐region legumes, such as pea (Pisum),
energy needed to break the triple bond in N2. For the same clover (Trifolium), broad bean (Vicia), and lentil (Lens).
reason, the enzymatic reduction of N2 by nitrogenase also Ureides are exported by legumes of tropical origin, such as
requires a large investment of energy (see Equation 9), soybean (Glycine), kidney bean (Phaseolus), peanut
although the exact changes in free energy are not yet known. (Arachis), and southern pea (Vigna). The three major ureides
are allantoin, allantoic acid, and citrulline.
Calculations based on the carbohydrate metabolism of
legumes show that a plant consumes 12 g of organic carbon
per gram of N2 fixed. On the basis of Equation, the ∆G0` for
the overall reaction of biological nitrogen fixation is about –
200 kJ mol–1. Because the overall reaction is highly
exergonic, ammonium production is limited by the slow
operation (number of N2 molecules reduced per unit time) of
the nitrogenase complex.

Under natural conditions, substantial amounts of H+ are

reduced to H2 gas, and this process can compete with N2
reduction for electrons from nitrogenase. In rhizobia, 30 to
60% of the energy supplied to nitrogenase may be lost as H 2,
diminishing the efficiency of nitrogen fixation. Some
rhizobia, however, contain hydrogenase, an enzyme that can
split the H2 formed and generate electrons for N2 reduction,
thus improving the efficiency of nitrogen fixation.

Allantoin is synthesized in peroxisomes from uric acid, and

allantoic acid is synthesized from allantoin in the
endoplasmic reticulum. The site of citrulline synthesis from
the amino acid ornithine has not yet been determined. All
three compounds are ultimately released into the xylem and
Amides and Ureides Are the Transported Forms of transported to the shoot, where they are rapidly catabolized
Nitrogen to ammonium. This ammonium enters the assimilation
pathway described earlier.
The symbiotic nitrogen‐fixing prokaryotes release ammonia
that, to avoid toxicity, must be rapidly converted into
organic forms in the root nodules before being transported

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D. PLANT HORMONES: BIOSYNTHESIS, STORAGE, BREAKDOWN AND TRANSPORT; PHYSIOLOGICAL EFFECTS AND MECHANISMS OF
ACTION.

1 Introduction these compounds. They are transported, perhaps through

only a few cells, but often throughout the plant, giving rise to
A constant theme underlying the study of plant physiology is a response in target tissues. It is possible, and has been
that plant growth and development are controlled by the proposed, that volatile chemical signals might be exchanged
environment. Plants being sessile organisms, it is not between plants although the importance of this in field
surprising that their development is exquisitely sensitive to conditions remains controversial. Thirdly, these compounds
a wide range of environmental factors and is extremely cause a response. Different cells will respond in different
plastic, i.e. very flexible. There are underlying basic patterns ways (or not at all) to a particular hormone, giving rise to
in plant development, but there is considerable regulation the concept of ‘competence to respond’ – that is whether a
by environmental signals of how and when these patterns particular cell type will respond to a given concentration of a
are expressed. In addition, there are internal signals within plant growth regulator or not.
the plant. One of the most important factors influencing the
development of a cell is its position within the plant. A plant Fourthly, the hormones are effective at low concentrations.
cell develops depending on its location in relation to This would rule out compounds such as sucrose which is
neighbouring cells, and this in turn will determine its produced within the leaves by photosynthesis, is
response to environmental signals. For example, the transported throughout the plant in the phloem, and has a
response to drought of a cell within the leaf will differ in profound effect on plant development, but is found at high
many ways from that of a cell within the root. The key concentrations. Sucrose has been considered by some to be a
question arises of how a complex set of environmental plant growth hormone. However, in this chapter the use of
factors can interact with cells to elicit an appropriate the term is limited to compounds which have traditionally
response within a given cell type: what are the internal been classed as plant growth hormones. These have the
signals that communicate between cells, and mediate following characteristics which can be added to the
between environmental factors and the plant tissues? definition above.

It has been known for decades (if not centuries) that plants In general :
contain a range of compounds which have profound effects • Plant growth hormones are small, relatively
on many aspects of growth and developmental physiology, simple compounds.
and act as a means of communication within the plant. These • Specific receptors exist which bind these
plant growth hormones, sometimes referred to as plant compounds.
growth regulators, are still being discovered. They are of • Often the presence of one plant growth hormone
supreme importance in controlling cell division, growth and will affect the synthesis or action of other plant
differentiation, which in turn determine the morphology and growth hormones.
ultimately the physiology of the whole plant. The
concentrations of the hormones are greatly influenced by The concentration of a plant growth hormone at a particular
environmental factors, and the movement of hormones in site will depend upon many different factors including the
the plant can also be under environmental control. Although rate of synthesis, degradation and transport to and from the
the mechanisms by which they act cannot be considered to target cell. In addition, plant growth hormones are often
be well understood, we are beginning to unravel their modes chemically modified, which may inactivate them, although –
of action and the ways in which they mutually interact to as this process is often reversible – it can also increase the
regulate plant functions. effective concentration of a plant growth hormone in a cell.
Finally, as the activity of plant hormones is thought to
2 Plant growth hormones require binding to specific receptors, transport in and out of
subcellular compartments also controls the concentration
2 .1 Concepts and definitions perceived by the cell. As all of these processes have the
potential to be regulated by the environment, it can be seen
In any area of biology, attempting to formulate definitions is that plant growth hormones act as a means of integrating
difficult and this is especially true of plant growth hormones. environmental signals and distributing them around the
One reasonable definition might be: A plant growth hormone plant.
is a substance produced within the plant in very low
concentrations and transported to another part of the plant Classically there are five major groups of plant growth
where it causes a response. hormones. These are the auxins, gibberellins, cytokinins,
abscisins and ethylene However, many more compounds
There are a number of key concepts in this definition. Firstly, exist in plants which have all of the characteristics of the
plant growth hormones are naturally occurring compounds more established plant growth hormones, and their role in
synthesized within the plant. However, similar if not regulating plant development is becoming clearer. These
identical compounds can be synthesized by other organisms. compounds include the jasmonates, salicylates,
For example, many plant pathogens produce substances brassinosteroids and peptide hormones. Recently, small
similar to plant hormones which are important in the RNA molecules have been identified which regulate gene
progression of the disease. Man‐made plant hormones are expression in different parts of the plants; these too might
commercially extremely important as they have such a be considered to fulfil many of the criteria of being a plant
diverse range of effects on plant development from acting as growth hormone.
weedkillers to stimulating fruit development.

The second key concept is that of transport. Plant growth

hormones are synthesized throughout the plant but apical
meristems and young, developing tissues are rich sources of

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THE EMERGENCE OF THE AUXIN CONCEPT side to grow faster than the illuminated side. The results of
their experiments were published in 1881 in a remarkable
During the latter part of the nineteenth century, Charles book entitled The Power of Movement in Plants. There
Darwin and his son Francis studied plant growth followed a long period of experimentation by many
phenomena involving tropisms. One of their interests was investigators on the nature of the growth stimulus in
the bending of plants toward light. This phenomenon, which coleoptiles. This research culminated in the demonstration
is caused by differential growth, is called phototropism. In in 1926 by Frits Went of the presence of a growth‐
some experiments the Darwins used seedlings of canary promoting chemical in the tip of oat (Avena sativa)
grass (Phalaris canariensis), in which, as in many other coleoptiles. It was known that if the tip of a coleoptile was
grasses, the youngest leaves are sheathed in a protective removed, coleoptile growth ceased. Previous workers had
organ called the coleoptile (Figure). attempted to isolate and identify the growth‐promoting
chemical by grinding up coleoptile tips and testing the
Coleoptiles are very sensitive to light, especially to blue light activity of the extracts. This approach failed because
(see Chapter 18). If illuminated on one side with a short grinding up the tissue released into the extract inhibitory
pulse of dim blue light, they will bend (grow) toward the substances that normally were compartmentalized in the
source of the light pulse within an hour. The Darwins found cell. Went’s major breakthrough was to avoid grinding by
that the tip of the coleoptile perceived the light, for if they allowing the material to diffuse out of excised coleoptiles
covered the tip with foil, the coleoptile would not bend. But tips directly into gelatin blocks. If placed asymmetrically on
the region of the coleoptile that is responsible for the top of a decapitated coleoptile, these blocks could be tested
bending toward the light, called the growth zone, is several for their ability to cause bending in the absence of a
millimeters below the tip. unilateral light source (see Figure). Because the substance
promoted the elongation of the coleoptile sections, it was
Thus they concluded that some sort of signal is produced in eventually named auxin from the Greek auxein, meaning “to
the tip, travels to the growth zone, and causes the shaded increase” or “to grow.”

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BIOSYNTHESIS AND METABOLISM OF AUXIN Auxin bioassays are still used today to detect the presence of
auxin activity in a sample. The Avena coleoptile curvature
Went’s studies with agar blocks demonstrated unequivocally assay is a sensitive measure of auxin activity (it is effective
that the growth‐promoting “influence” diffusing from the for IAA concentrations of about 0.02 to 0.2 mg L–1). Another
coleoptile tip was a chemical substance. The fact that it was bioassay measures auxin‐induced changes in the straight
produced at one location and transported in minute growth of Avena coleoptiles floating in solution. Both of
amounts to its site of action qualified it as an authentic plant these bioassays can establish the presence of an auxin in a
hormone. sample, but they cannot be used for precise quantification or
identification of the specific compound.
The Principal Auxin in Higher Plants Is Indole­3­Acetic
Acid Mass spectrometry is the method of choice when
information about both the chemical structure and the
In the mid‐1930s it was determined that auxin is indole­3­ amount of IAA is needed. This method is used in conjunction
acetic acid (IAA). Several other auxins in higher plants were with separation protocols involving gas chromatography. It
discovered later (Figure), but IAA is by far the most allows the precise quantification and identification of auxins,
abundant and physiologically relevant. Because the and can detect as little as 10–12 g (1 picogram, or pg) of IAA,
structure of IAA is relatively simple, academic and industrial which is well within the range of auxin found in a single pea
laboratories were quickly able to synthesize a wide array of stem section or a corn kernel. These sophisticated
molecules with auxin activity. Some of these are used as techniques have enabled researchers to accurately analyze
herbicides in horticulture and agriculture. auxin precursors, auxin turnover, and auxin distribution
within the plant.

IAA Is Synthesized in Meristems,Young Leaves, and

Developing Fruits and Seeds

IAA biosynthesis is associated with rapidly dividing and

rapidly growing tissues, especially in shoots. Although
virtually all plant tissues appear to be capable of producing
low levels of IAA, shoot apical meristems, young leaves, and
developing fruits and seeds are the primary sites of IAA
synthesis.

In very young leaf primordia of Arabidopsis, auxin is

An early definition of auxins included all natural and synthesized at the tip. During leaf development there is a
synthetic chemical substances that stimulate elongation in gradual shift in the site of auxin production basipetally along
coleoptiles and stem sections. However, auxins affect many the margins, and later, in the central region of the lamina.
developmental processes besides cell elongation. Thus The basipetal shift in auxin production correlates closely
auxins can be defined as compounds with biological with, and is probably causally related to, the basipetal
activities similar to those of IAA, including the ability to maturation sequence of leaf development and vascular
promote cell elongation in coleoptile and stem sections, cell differentiation.
division in callus cultures in the presence of cytokinins,
formation of adventitious roots on detached leaves and By fusing the GUS (β‐glucuronidase) reporter gene to a
stems, and other developmental phenomena associated with promoter containing an auxin response element, and
IAA action. transforming Arabidopsis leaves with this construct in a Ti
plasmid using Agrobacterium, it is possible to visualize the
Although they are chemically diverse, a common feature of distribution of free auxin in young, developing leaves.
all active auxins is a molecular distance of about 0.5 nm Wherever free auxin is produced, GUS expression occurs—
between a fractional positive charge on the aromatic ring and can be detected histochemically. By use of this
and a negatively charged carboxyl group. technique, it has recently been demonstrated that auxin is
produced by a cluster of cells located at sites where
Auxins in Biological Samples Can Be Quantified hydathodes will develop.

Depending on the information that a researcher needs, the Hydathodes are glandlike modifications of the ground and
amounts and/or identity of auxins in biological samples can vascular tissues, typically at the margins of leaves, that allow
be determined by bioassay, mass spectrometry, or enzyme‐ the release of liquid water (guttation fluid) through pores in
linked immunosorbent assay, which is abbreviated as ELISA. the epidermis in the presence of root pressure.

A bioassay is a measurement of the effect of a known or Multiple Pathways Exist for the Biosynthesis of IAA
suspected biologically active substance on living material. In
his pioneering work more than 60 years ago, Went used IAA is structurally related to the amino acid tryptophan, and
Avena sativa (oat) coleoptiles in a technique called the early studies on auxin biosynthesis focused on tryptophan as
Avena coleoptile curvature test. The coleoptiles curved the probable precursor. However, the incorporation of
because the increase in auxin on one side stimulated cell exogenous labeled tryptophan (e.g., [3H]tryptophan) into
elongation, and the decrease in auxin on the other side (due IAA by plant tissues has proved difficult to demonstrate.
to the absence of the coleoptile tip) caused a decrease in the Nevertheless, an enormous body of evidence has now
growth rate—a phenomenon called differential growth. accumulated showing that plants convert tryptophan to IAA
by several pathways, which are described in the paragraphs
Went found that he could estimate the amount of auxin in a that follow.
sample by measuring the resulting coleoptile curvature.

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The IPA pathway. The indole­3­pyruvic acid (IPA) Another tryptophan‐dependent biosynthetic pathway— one
pathway (see Figure C), is probably the most common of the that uses indole­3­acetamide (IAM) as an intermediate
tryptophan‐dependent pathways. It involves a deamination (see Figure A)—is used by various pathogenic bacteria, such
reaction to form IPA, followed by a decarboxylation reaction as Pseudomonas savastanoi and Agrobacterium tumefaciens.
to form indole‐3‐acetaldehyde (IAld). Indole‐3‐ acetaldehyde This pathway involves the two enzymes tryptophan
is then oxidized to IAA by a specific dehydrogenase. monooxygenase and IAM hydrolase. The auxins produced by
these bacteria often elicit morphological changes in their
The TAM pathway. The tryptamine (TAM) pathway (see plant hosts.
Figure D) is similar to the IPA pathway, except that the order
of the deamination and decarboxylation reactions is In addition to the tryptophan‐dependent pathways, recent
reversed, and different enzymes are involved. Species that genetic studies have provided evidence that plants can
do not utilize the IPApathway possess the TAM pathway. In synthesize IAA via one or more tryptophan‐independent
at least one case (tomato), there is evidence for both the pathways. The existence of multiple pathways for IAA
IPAand the TAM pathways. biosynthesis makes it nearly impossible for plants to run out
of auxin and is probably a reflection of the essential role of
The IAN pathway. In the indole­3­acetonitrile (IAN) this hormone in plant development.
pathway (see Figure B), tryptophan is first converted to
indole‐3‐acetaldoxime and then to indole‐3‐acetonitrile. The IAA Is Also Synthesized from Indole or from Indole­3­
enzyme that converts IAN to IAA is called nitrilase. Glycerol Phosphate

The IAN pathway may be important in only three plant Although a tryptophan‐independent pathway of IAA
families: the Brassicaceae (mustard family), Poaceae (grass biosynthesis had long been suspected because of the low
family), and Musaceae (banana family). Nevertheless, levels of conversion of radiolabeled tryptophan to IAA, not
nitrilase‐like genes or activities have recently been until genetic approaches were available could the existence
identified in the Cucurbitaceae (squash family), Solanaceae of such pathways be confirmed and defined. Perhaps the
(tobacco family), Fabaceae (legumes), and Rosaceae (rose most striking of these studies in maize involves the orange
family). Four genes (NIT1 through NIT4) that encode pericarp (orp) mutant, in which both subunits of the enzyme
nitrilase enzymes have now been cloned from Arabidopsis. tryptophan synthase are inactive (Figure). The orp mutant is
a true tryptophan auxotroph, requiring exogenous
When NIT2 was expressed in transgenic tobacco, the tryptophan to survive. However, neither the orp seedlings
resultant plants acquired the ability to respond to IAN as an nor the wild‐type seedlings can convert tryptophan to IAA,
auxin by hydrolyzing it to IAA. even when the mutant seedlings are given enough
tryptophan to reverse the lethal effects of the mutation.

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Despite the block in tryptophan biosynthesis, the orp mutant have been identified in all higher plants and are considered
contains amounts of IAA 50‐fold higher than those of a wild‐ hormonally inactive.
type plant. Signficantly, when orp seedlings were fed
[15N]anthranilate (see Figure), the label subsequently IAA has been found to be conjugated to both high‐ and low‐
appeared in IAA, but not in tryptophan. These results molecular‐weight compounds.
provided the best experimental evidence for a tryptophan‐ • Low‐molecular‐weight conjugated auxins include esters of
independent pathway of IAA biosynthesis. IAA with glucose or myo‐inositol and amide conjugates such
as IAA‐N‐aspartate.
Further studies established that the branch point for IAA • High‐molecular‐weight IAA conjugates include IAA glucan
biosynthesis is either indole or its precursor, indole‐3‐ (7–50 glucose units per IAA) and IAA‐glycoproteins found in
glycerol phosphate (see Figure). IAN and IPA are possible cereal seeds.
intermediates, but the immediate precursor of IAA in the
tryptophan‐independent pathway has not yet been The compound to which IAA is conjugated and the extent of
identified. the conjugation depend on the specific conjugating enzymes.
The best‐studied reaction is the conjugation of IAA to
The discovery of the tryptophan‐independent pathway has glucose in Zea mays.
drastically altered our view of IAA biosynthesis, but the
relative importance of the two pathways (tryptophan The highest concentrations of free auxin in the living plant
dependent versus tryptophan‐independent) is poorly are in the apical meristems of shoots and in young leaves
understood. In several plants it has been found that the type because these are the primary sites of auxin synthesis.
of IAA biosynthesis pathway varies between different However, auxins are widely distributed in the plant.
tissues, and between different times of development. For
example, during embryogenesis in carrot, the tryptophan Metabolism of conjugated auxin may be a major contributing
dependent pathway is important very early in development, factor in the regulation of the levels of free auxin. For
whereas the tryptophan‐independent pathway takes over example, during the germination of seeds of Zea mays, IAA‐
soon after the root–shoot axis is established. myo‐inositol is translocated from the endosperm to the
coleoptile via the phloem. At least a portion of the free IAA
Most IAA in the Plant Is in a Covalently Bound Form produced in coleoptile tips of Zea mays is believed to be
derived from the hydrolysis of IAA‐myo‐inositol.
Although free IAA is the biologically active form of the
hormone, the vast majority of auxin in plants is found in a In addition, environmental stimuli such as light and gravity
covalently bound state. These conjugated, or “bound,” auxins have been shown to influence both the rate of auxin
conjugation (removal of free auxin) and the rate of release of

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free auxin (hydrolysis of conjugated auxin). The formation of Polar Transport Requires Energy and Is Gravity
conjugated auxins may serve other functions as well, Independent
including storage and protection against oxidative
degradation.

IAA Is Degraded by Multiple Pathways

Like IAA biosynthesis, the enzymatic breakdown (oxidation)

of IAA may involve more than one pathway. For some time it
has been thought that peroxidative enzymes are chiefly
responsible for IAA oxidation, primarily because these
enzymes are ubiquitous in higher plants and their ability to
degrade IAA can be demonstrated in vitro. However, the
physiological significance of the peroxidase pathway is
unclear. For example, no change in the IAA levels of
transgenic plants was observed with either a tenfold
increase in peroxidase expression or a tenfold repression of
peroxidase activity.

On the basis of isotopic labeling and metabolite To study polar transport, researchers have employed the
identification, two other oxidative pathways are more likely donor–receiver agar block method (Figure): An agar block
to be involved in the controlled degradation of IAA. The end containing radioisotope‐labeled auxin (donor block) is
product of this pathway is oxindole‐3‐acetic acid (OxIAA), a placed on one end of a tissue segment, and a receiver block
naturally occurring compound in the endosperm and shoot is placed on the other end. The movement of auxin through
tissues of Zea mays. In one pathway, IAA is oxidized without the tissue into the receiver block can be determined over
decarboxylation to OxIAA. time by measurement of the radioactivity in the receiver
block.
In another pathway, the IAA‐aspartate conjugate is oxidized
first to the intermediate dioxindole‐3‐acetylaspartate, and From a multitude of such studies, the general properties of
then to OxIAA. polar IAA transport have emerged. Tissues differ in the
degree of polarity of IAA transport. In coleoptiles, vegetative
In vitro, IAA can be oxidized nonenzymatically when stems, and leaf petioles, basipetal transport predominates.
exposed to high‐intensity light, and its photodestruction in Polar transport is not affected by the orientation of the
vitro can be promoted by plant pigments such as riboflavin. tissue (at least over short periods of time), so it is
Although the products of auxin photooxidation have been independent of gravity.
isolated from plants, the role, if any, of the photooxidation
pathway in vivo is presumed to be minor. When stem cuttings (in this case bamboo) are placed in a
moist chamber, adventitious roots always form at the basal
Two Subcellular Pools of IAA Exist: The Cytosol and the end of the cuttings, even when the cuttings are inverted.
Chloroplasts Because root differentiation is stimulated by an increase in
auxin concentration, auxin must be transported basipetally
The distribution of IAA in the cell appears to be regulated in the stem even when the cutting is oriented upside down.
largely by pH. Because IAA− does not cross membranes
unaided, whereas IAAH readily diffuses across membranes, Polar transport proceeds in a cell‐to‐cell fashion, rather than
auxin tends to accumulate in the more alkaline via the symplast. That is, auxin exits the cell through the
compartments of the cell. plasma membrane, diffuses across the compound middle
lamella, and enters the cell below through its plasma
The distribution of IAA and its metabolites has been studied membrane. The loss of auxin from cells is termed auxin
in tobacco cells. About one‐third of the IAA is found in the efflux; the entry of auxin into cells is called auxin uptake or
chloroplast, and the remainder is located in the cytosol. IAA influx. The overall process requires metabolic energy, as
conjugates are located exclusively in the cytosol. IAA in the evidenced by the sensitivity of polar transport to O2
cytosol is metabolized either by conjugation or by deprivation and metabolic inhibitors.
nondecarboxylative catabolism.
The velocity of polar auxin transport is 5 to 20 cm h–1—
The IAA in the chloroplast is protected from these processes, faster than the rate of diffusion, but slower than phloem
but it is regulated by the amount of IAA in the cytosol, with translocation rates.
which it is in equilibrium.
Polar transport is also specific for active auxins, both natural
AUXIN TRANSPORT and synthetic. Neither inactive auxin analogs nor auxin
metabolites are transported polarly, suggesting that polar
The main axes of shoots and roots, along with their transport involves specific protein carriers on the plasma
branches, exhibit apex–base structural polarity, and this membrane that can recognize the hormone and its active
structural polarity has its origin in the polarity of auxin analogs.
transport. Soon after Went developed the coleoptile
curvature test for auxin, it was discovered that IAA moves The major site of basipetal polar auxin transport in stems
mainly from the apical to the basal end (basipetally) in and leaves is the vascular parenchyma tissue. Coleoptiles
excised oat coleoptile sections. This type of unidirectional appear to be the exception in that basipetal polar transport
transport is termed polar transport. Auxin is the only plant
growth hormone known to be transported polarly.

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occurs mainly in the nonvascular tissues. Acropetal polar because it is driven across the membrane by the proton
transport in the root is specifically associated with the xylem motive force.
parenchyma of the stele.
A permease‐type auxin uptake carrier, AUX1, related to
However, most of the auxin that reaches the root tip is bacterial amino acid carriers, has been identified in
translocated via the phloem. Arabidopsis roots. The roots of aux1 mutants are
agravitropic, suggesting that auxin influx is a limiting factor
A small amount of basipetal auxin transport from the root for gravitropism in roots. As predicted by the chemiosmotic
tip has also been demonstrated. In maize roots, for example, model, AUX1 appears to be uniformly distributed around
radiolabeled IAA applied to the root tip is transported cells in the polar transport pathway. Thus in general, the
basipetally about 2 to 8 mm. Basipetal auxin transport in the polarity of auxin transport is governed by the efflux step
root occurs in the epidermal and cortical tissues, and as we rather than the influx step.
shall see, it plays a central role in gravitropism.

A Chemiosmotic Model Has Been Proposed to Explain

Polar Transport

The discovery of the chemiosmotic mechanism of solute

transport in the late 1960s led to the application of this
model to polar auxin transport. According to the now
generally accepted chemiosmotic model for polar auxin
transport, auxin uptake is driven by the proton motive force
(∆E + ∆pH) across the plasma membrane, while auxin efflux
is driven by the membrane potential, ∆E.

Acrucial feature of the polar transport model is that the

auxin efflux carriers are localized at the basal ends of the
conducting cells. The evidence for each step in this model is
considered separately in the discussion that follows.

Auxin influx. The first step in polar transport is auxin influx.

According to the model, auxin can enter plant cells from any
direction by either of two mechanisms:
1. Passive diffusion of the protonated (IAAH) form across the
phospholipid bilayer
2. Secondary active transport of the dissociated (IAA–) form
via a 2H+–IAA– symporter. The dual pathway of auxin uptake Auxin efflux. Once IAA enters the cytosol, which has a pH of
arises because the passive permeability of the membrane to approximately 7.2, nearly all of it will dissociate to the
auxin depends strongly on the apoplastic pH. anionic form. Because the membrane is less permeable to
IAA– than to IAAH, IAA– will tend to accumulate in the
The undissociated form of indole‐3‐acetic acid, in which the cytosol. However, much of the auxin that enters the cell
carboxyl group is protonated, is lipophilic and readily escapes via an auxin anion efflux carrier.
diffuses across lipid bilayer membranes. In contrast, the
dissociated form of auxin is negatively charged and According to the chemiosmotic model, transport of IAA– out
therefore does not cross membranes unaided. Because the of the cell is driven by the inside negative membrane
plasma membrane H+‐ATPase normally maintains the cell potential.
wall solution at about pH 5, about half of the auxin (pKa =
4.75) in the apoplast will be in the undissociated form and As noted earlier, the central feature of the chemiosmotic
will diffuse passively across the plasma membrane down a model for polar transport is that IAA– efflux takes place
concentration gradient. Experimental support for pH‐ preferentially at the basal end of each cell. The repetition of
dependent, passive auxin uptake was first provided by the auxin uptake at the apical end of the cell and preferential
demonstration that IAA uptake by plant cells increases as release from the base of each cell in the pathway gives rise
the extracellular pH is lowered from a neutral to a more to the total polar transport effect. A family of putative auxin
acidic value. efflux carriers known as PIN proteins (named after the pin‐
shaped inflorescences formed by the pin1 mutant of
A carrier‐mediated, secondary active uptake mechanism was Arabidopsis) are localized precisely as the model would
shown to be saturable and specific for active auxins. In predict—that is, at the basal ends of the conducting cells.
experiments in which the ∆pH and ∆E values of isolated
membrane vesicles from zucchini (Cucurbita pepo) Inhibitors of Auxin Transport Block Auxin Efflux
hypocotyls were manipulated artificially, the uptake of
radiolabeled auxin was shown to be stimulated in the Several compounds have been synthesized that can act as
presence of a pH gradient, as in passive uptake, but also auxin transport inhibitors (ATIs), including NPA (1­
when the inside of the vesicle was negatively charged Nnaphthylphthalamic acid) and TIBA (2,3,5­
relative to the outside. triiodobenzoic acid). These inhibitors block polar
transport by preventing auxin efflux. We can demonstrate
These and other experiments suggested that an H+–IAA– this phenomenon by incorporating NPA or TIBA into either
symporter cotransports two protons along with the auxin the donor or the receiver block in an auxin transport
anion. This secondary active transport of auxin allows for experiment.
greater auxin accumulation than simple diffusion does

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Both compounds inhibit auxin efflux into the receiver block,

but they do not affect auxin uptake from the donor block.
Some ATIs, such as TIBA, that have weak auxin activity and
are transported polarly, may inhibit polar transport in part
by competing with auxin for its binding site on the efflux
carrier. Others, such as NPA, are not transported polarly and
are believed to interfere with auxin transport by binding to
proteins associated in a complex with the efflux carrier. Such
NPA‐binding proteins are also found at the basal ends of the
conducting cells, consistent with the localization of PIN
proteins.

Recently another class of ATIs has been identified that

inhibits the AUX1 uptake carrier. For example, 1‐
naphthoxyacetic acid (1‐NOA) blocks auxin uptake into cells,
and when applied to Arabidopsis plants it causes root
agravitropism similar to that of the aux1 mutant. Like the
aux1 mutation, neither 1‐ NOA nor any of the other AUX1‐
specific inhibitors block polar auxin transport.

PIN Proteins Are Rapidly Cycled to and from the Plasma

Membrane

The basal localization of the auxin efflux carriers involves

targeted vesicle secretion to the basal ends of the conducting
cells. Recently it has been demonstrated that PIN proteins,
although stable, do not remain on the plasma membrane
permanently, but are rapidly cycled to an unidentified
endosomal compartment via endocytotic vesicles, and then
recycled back to the plasma membrane.

Prior to treatment, the PIN1 protein is localized at the basal

ends (top) of root cortical parenchyma cells. Treatment of Flavonoids Serve as Endogenous ATIs
Arabidopsis seedlings with brefeldin A(BFA), which causes
Golgi vesicles and other endosomal compartments to There is mounting evidence that flavonoids can function as
aggregate near the nucleus, causes PIN to accumulate in endogenous regulators of polar auxin transport. Indeed,
these abnormal intracellular compartments. When the BFA naturally occurring aglycone flavonoid compounds
is washed out with buffer, the normal localization on the (flavonoids without attached sugars) are able to compete
plasma membrane at the base of the cell is restored. with NPA for its binding site on membranes and are typically
localized on the plasma membrane at the basal ends of cells
But when cytochalasin D, an inhibitor of actin where the efflux carrier is concentrated.
polymerization, is included in the buffer washout solution,
normal relocalization of PIN to the plasma membrane is Auxin Is Also Transported Nonpolarly in the Phloem
prevented. These results indicate that PIN is rapidly cycled
between the plasma membrane at the base of the cell and an Most of the IAA that is synthesized in mature leaves appears
unidentified endosomal compartment by an actin‐dependent to be transported to the rest of the plant nonpolarly via the
mechanism. phloem. Auxin, along with other components of phloem sap,
can move from these leaves up or down the plant at
Although they bind different targets, both TIBA and NPA velocities much higher than those of polar transport. Auxin
interfere with vesicle traffic to and from the plasma translocation in the phloem is largely passive, not requiring
membrane. energy directly.

The best way to demonstrate this phenomenon is to include Although the overall importance of the phloem pathway
TIBA in the washout solution after BFA treatment. Under versus the polar transport system for the long‐distance
these conditions, TIBA prevents the normal relocalization of movement of IAA in plants is still unresolved, the evidence
PIN on the plasma membrane following the washout suggests that long‐distance auxin transport in the phloem is
treatment. important for controlling such processes as cambial cell
divisions, callose accumulation or removal from sieve tube
The effects of TIBA and NPA on cycling are not specific for elements, and branch root formation. Indeed, the phloem
PIN proteins, and it has been proposed that ATIs may appears to represent the principal pathway for long‐distance
actually represent general inhibitors of membrane cycling. auxin translocation to the root.
On the other hand, neither TIBA nor NPA alone causes PIN
delocalization, even though they block auxin efflux. Polar transport and phloem transport are not independent
Therefore, TIBA and NPA must also be able to directly of each other. Recent studies with radiolabeled IAA suggest
inhibit the transport activity of PIN complexes on the plasma that in pea, auxin can be transferred from the nonpolar
membrane—by binding either to PIN (as TIBA does) or to phloem pathway to the polar transport pathway. This
one or more regulatory proteins (as NPA does). transfer takes place mainly in the immature tissues of the
shoot apex.
A simplified model of the effects of TIBA and NPA on PIN
cycling and auxin efflux is shown in Figure.

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PHYSIOLOGICAL EFFECTS OF AUXIN: This result indicates that, in the absence of auxin the central
tissues, including the pith, vascular tissues, and inner cortex,
Auxins Promote Growth in Stems and Coleoptiles, While elongate at a faster rate than the outer tissues, consisting of
Inhibiting Growth in Roots the outer cortex and epidermis. Thus the outer tissues must
be limiting the extension rate of the stem in the absence of
As we have seen, auxin is synthesized in the shoot apex and auxin. However, when the split sections are incubated in
transported basipetally to the tissues below. The steady buffer plus auxin, the two halves now curve inward,
supply of auxin arriving at the subapical region of the stem demonstrating that the outer tissues of dicot stems are the
or coleoptile is required for the continued elongation of primary targets of auxin action during cell elongation.
these cells. Because the level of endogenous auxin in the
elongation region of a normal healthy plant is nearly optimal The observation that the outer cell layers are the targets of
for growth, spraying the plant with exogenous auxin causes auxin seems to conflict with the localization of polar
only a modest and short‐lived stimulation in growth, and transport in the parenchyma cells of the vascular bundles.
may even be inhibitory in the case of darkgrown seedlings,
which are more sensitive to supraoptimal auxin However, auxin can move laterally from the vascular tissues
concentrations than light‐grown plants are. of dicot stems to the outer tissues of the elongation zone. In
coleoptiles, on the other hand, all of the nonvascular tissues
However, when the endogenous source of auxin is removed (epidermis plus mesophyll) are capable of transporting
by excision of sections containing the elongation zones, the auxin, as well as responding to it.
growth rate rapidly decreases to a low basal rate. Such
excised sections will often respond dramatically to The Minimum Lag Time for Auxin­Induced Growth Is
exogenous auxin by rapidly increasing their growth rate Ten Minutes
back to the level in the intact plant.
When a stem or coleoptile section is excised and inserted
In long‐term experiments, treatment of excised sections of into a sensitive growth‐measuring device, the growth
coleoptiles or dicot stems with auxin stimulates the rate of response to auxin can be monitored at very high resolution.
elongation of the section for up to 20 hours. The optimal Without auxin in the medium, the growth rate declines
auxin concentration for elongation growth is typically 10 –6 rapidly. Addition of auxin markedly stimulates the growth
to 10–5 M(Figure). rate after a lag period of only 10 to 12 minutes.

Both Avena (oat) coleoptiles and Glycine max (soybean)

hypocotyls (dicot stem) reach a maximum growth rate after
30 to 60 minutes of auxin treatment (Figure).

This maximum represents a five‐ to tenfold increase over the

The inhibition beyond the optimal concentration is generally basal rate. Oat coleoptile sections can maintain this
attributed to auxin‐induced ethylene biosynthesis. Auxin maximum rate for up to 18 hours in the presence of
control of root elongation growth has been more difficult to osmotically active solutes such as sucrose or KCl.
demonstrate, perhaps because auxin induces the production
of ethylene, a root growth inhibitor. However, even if As might be expected, the stimulation of growth by auxin
ethylene biosynthesis is specifically blocked, low requires energy, and metabolic inhibitors inhibit the
concentrations (10–10 to 10–9 M) of auxin promote the response within minutes. Auxin‐induced growth is also
growth of intact roots, whereas higher concentrations (10–6 sensitive to inhibitors of protein synthesis such as
M) inhibit growth. Thus, roots may require a minimum cycloheximide, suggesting that proteins with high turnover
concentration of auxin to grow, but root growth is strongly rates are involved. Inhibitors of RNA synthesis also inhibit
inhibited by auxin concentrations that promote elongation auxininduced growth, after a slightly longer delay.
in stems and coleoptiles.
Although the length of the lag time for auxin‐stimulated
The Outer Tissues of Dicot Stems Are the Targets of growth can be increased by lowering of the temperature or
Auxin Action by the use of suboptimal auxin concentrations, the lag time
cannot be shortened by raising of the temperature, by the
Dicot stems are composed of many types of tissues and cells, use of supraoptimal auxin concentrations, or by abrasion of
only some of which may limit the growth rate. This point is the waxy cuticle to allow auxin to penetrate the tissue more
illustrated by a simple experiment. When stem sections from rapidly. Thus the minimum lag time of 10 minutes is not
growing regions of an etiolated dicot stem, such as pea, are determined by the time required for auxin to reach its site of
split lengthwise and incubated in buffer, the two halves bend action. Rather, the lag time reflects the time needed for the
outward. biochemical machinery of the cell to bring about the increase
in the growth rate.

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Auxin Rapidly Increases the Extensibility of the Cell Wall Auxin‐induced growth has also been shown to be inhibited
by neutral buffers, as long as the cuticle has been abraided.
How does auxin cause a five‐ to tenfold increase in the Fusicoccin, a fungal phytotoxin, stimulates both rapid
growth rate in only 10 minutes? To understand the proton extrusion and transient growth in stem and
mechanism, we must first review the process of cell coleoptile sections. And finally, wall loosening proteins
enlargement in plants. Plant cells expand in three steps: called expansins have been identified in the cell walls of a
1. Osmotic uptake of water across the plasma membrane is wide range of plant species. At acidic pH values, expansins
driven by the gradient in water potential (∆Yw). loosen cell walls by weakening the hydrogen bonds between
2. Turgor pressure builds up because of the rigidity of the the polysaccharide components of the wall.
cell wall.
3. Biochemical wall loosening occurs, allowing the cell to Auxin­Induced Proton Extrusion May Involve Both
expand in response to turgor pressure. Activation and Synthesis

The effects of these parameters on the growth rate are In theory, auxin could increase the rate of proton extrusion
encapsulated in the growth rate equation: by two possible mechanisms:
GR = m (Yp – Y) 1. Activation of preexisting plasma membrane H+‐ ATPases
where GR is the growth rate, Yp is the turgor pressure, Y is 2. Synthesis of new H+‐ATPases on the plasma membrane
the yield threshold, and m is the coefficient (wall
extensibility) that relates the growth rate to the difference H+­ATPase activation. When auxin was added directly to
between Yp and Y. isolated plasma membrane vesicles from tobacco cells, a
small stimulation (about 20%) of the ATP‐driven proton‐
In principle, auxin could increase the growth rate by pumping activity was observed, suggesting that auxin
increasing m, increasing Yp, or decreasing Y. Although directly activates the H+‐ATPase. A greater stimulation
extensive experiments have shown that auxin does not (about 40%) was observed if the living cells were treated
increase turgor pressure when it stimulates growth, with IAA just before the membranes were isolated,
conflicting results have been obtained regarding auxin suggesting that a cellular factor is also required.
induced decreases in Y. However, there is general agreement
that auxin causes an increase in the wall extensibility Although an auxin receptor has not yet been unequivocally
parameter, m. identified, various auxin‐binding proteins (ABPs) have been
isolated and appear to be able to activate the plasma
Auxin­Induced Proton Extrusion Acidifies the Cell Wall membrane H+‐ATPase in the presence of auxin.
and Increases Cell Extension
Recently an ABP from rice, ABP57, was shown to bind
According to the widely accepted acid growth hypothesis, directly to plasma membrane H+‐ATPases and stimulate
hydrogen ions act as the intermediate between auxin and proton extrusion—but only in the presence of IAA. When
cell wall loosening. The source of the hydrogen ions is the IAA is absent, the activity of the H+‐ ATPase is repressed by
plasma membrane H+‐ATPase, whose activity is thought to the C‐terminal domain of the enzyme, which can block the
increase in response to auxin. The acid growth hypothesis catalytic site. ABP57 (with bound IAA) interacts with the H+‐
allows five main predictions: ATPase, activating the enzyme. A second auxin‐binding site
1. Acid buffers alone should promote short‐term growth, interferes with the action of the first, possibly explaining the
provided the cuticle has been abraded to allow the protons bell‐shaped curve of auxin action. This hypothetical model
access to the cell wall. for the action of ABP57 is shown in Figure.
2. Auxin should increase the rate of proton extrusion (wall
acidification), and the kinetics of proton extrusion should
closely match those of auxin‐induced growth.
3. Neutral buffers should inhibit auxin‐induced growth.
4. Compounds (other than auxin) that promote proton
extrusion should stimulate growth.
5. Cell walls should contain a “wall loosening factor” with an
acidic pH optimum.

All five of these predictions have been confirmed. Acidic

buffers cause a rapid and immediate increase in the growth
rate, provided the cuticle has been abraded. Auxin
stimulates proton extrusion into the cell wall after 10 to 15
minutes of lag time, consistent with the growth kinetics
(Figure).

H+­ATPase synthesis. The ability of protein synthesis

inhibitors, such as cycloheximide, to rapidly inhibit auxin
induced proton extrusion and growth suggests that auxin
might also stimulate proton pumping by increasing the
synthesis of the H+‐ATPase. An increase in the amount of
plasma membrane ATPase in corn coleoptiles was detected
immunologically after only 5 minutes of auxin treatment,
and a doubling of the H+‐ATPase was observed after 40
minutes of treatment. A threefold stimulation by auxin of an
mRNA for the H+‐ATPase was demonstrated specifically in
the nonvascular tissues of the coleoptiles.

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In summary, the question of activation versus synthesis is tip to base is developmentally determined and is
still unresolved, and it is possible that auxin stimulates independent of orientation with respect to gravity. However,
proton extrusion by both activation and stimulation of auxin can also be transported laterally, and this lateral
synthesis of the H+‐ATPase. Figure summarizes the proposed movement of auxin lies at the heart of a model for tropisms
mechanisms of auxin‐induced cell wall loosening via proton originally proposed separately by the Russian plant
extrusion. physiologist, Nicolai Cholodny and Frits Went from the
Netherlands in the 1920s.
PHYSIOLOGICAL EFFECTS OF AUXIN: PHOTOTROPISM
AND GRAVITROPISM According to the Cholodny–Went model of phototropism,
the tips of grass coleoptiles have three specialized functions:
Three main guidance systems control the orientation of 1. The production of auxin
plant growth: 2. The perception of a unilateral light stimulus
1. Phototropism, or growth with respect to light, is 3. The lateral transport of IAA in response to the phototropic
expressed in all shoots and some roots; it ensures that leaves stimulus
will receive optimal sunlight for photosynthesis.
2. Gravitropism, growth in response to gravity, enables Thus, in response to a directional light stimulus, the auxin
roots to grow downward into the soil and shoots to grow produced at the tip, instead of being transported basipetally,
upward away from the soil, which is especially critical is transported laterally toward the shaded side. The precise
during the early stages of germination. sites of auxin production, light perception, and lateral
3. Thigmotropism, or growth with respect to touch, enables transport have been difficult to define. In maize coleoptiles,
roots to grow around rocks and is responsible for the ability auxin is produced in the upper 1 to 2 mm of the tip. The
of the shoots of climbing plants to wrap around other zones of photosensing and lateral transport extend farther,
structures for support. within the upper 5 mm of the tip. The response is also
strongly dependent on the light fluence.
Phototropism Is Mediated by the Lateral Redistribution
of Auxin Two flavoproteins, phototropins 1 and 2, are the
photoreceptors for the blue‐light signaling pathway that
As we saw earlier, Charles and Francis Darwin provided the induces phototropic bending in Arabidopsis hypocotyls and
first clue concerning the mechanism of phototropism by oat coleoptiles under both high‐ and low‐fluence conditions.
demonstrating that the sites of perception and differential
growth (bending) are separate: Light is perceived at the tip, Phototropins are autophosphorylating protein kinases
but bending occurs below the tip. The Darwins proposed whose activity is stimulated by blue light. The action
that some “influence” that was transported from the tip to spectrum for blue­light activation of the kinase activity
the growing region brought about the observed asymmetric closely matches the action spectrum for phototropism,
growth response. This influence was later shown to be including the multiple peaks in the blue region. Phototropin
indole‐3‐acetic acid—auxin. When a shoot is growing 1 displays a lateral gradient in phosphorylation during
vertically, auxin is transported polarly from the growing tip exposure to low‐fluence unilateral blue light.
to the elongation zone. The polarity of auxin transport from

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According to the current hypothesis, the gradient in greater amount of auxin diffuses from the lower half than
phototropin phosphorylation induces the movement of the upper half.
auxin to the shaded side of the coleoptile. Once the auxin
reaches the shaded side of the tip, it is transported Tissues below the tip are able to respond to gravity as well.
basipetally to the elongation zone, where it stimulates cell For example, when vertically oriented maize coleoptiles are
elongation. The acceleration of growth on the shaded side decapitated by removal of the upper 2 mm of the tip and
and the slowing of growth on the illuminated side oriented horizontally, gravitropic bending occurs at a slow
(differential growth) give rise to the curvature toward light rate for several hours even without the tip. Application of
(Figure). IAA to the cut surface restores the rate of bending to normal
levels. This finding indicates that both the perception of the
gravitational stimulus and the lateral redistribution of auxin
can occur in the tissues below the tip, although the tip is still
required for auxin production. Lateral redistribution of
auxin in shoot apical meristems is more difficult to
demonstrate than in coleoptiles because of the presence of
leaves. In recent years, molecular markers have been widely
used as reporter genes to detect lateral auxin gradients in
horizontally placed stems and roots.

In soybean hypocotyls, gravitropism leads to a rapid

asymmetry in the accumulation of a group of auxin‐
stimulated mRNAs called SAURs (small auxin up‐regulated
RNAs). In vertical seedlings, SAUR gene expression is
symmetrically distributed. Within 20 minutes after the
seedling is oriented horizontally, SAURs begin to accumulate
on the lower half of the hypocotyl. Under these conditions,
gravitropic bending first becomes evident after 45 minutes,
well after the induction of the SAURs. The existence of a
lateral gradient in SAUR gene expression is indirect evidence
for the existence of a lateral gradient in auxin detectable
within 20 minutes of the gravitropic stimulus. The GH3 gene
Direct tests of the Cholodny–Went model using the agar family is also up‐regulated within 5 minutes of auxin
block/coleoptile curvature bioassay have supported the treatment and has been used as a molecular marker for the
model’s prediction that auxin in coleoptile tips is presence of auxin. By fusing an artificial promoter sequence
transported laterally in response to unilateral light. The total based on the GH3 promoter to the GUS reporter gene, it is
amount of auxin diffusing out of the tip (here expressed as possible to visualize the lateral gradient in auxin
the angle of curvature) is the same in the presence of concentration that occurs during both photo‐ and
unilateral light as in darkness (compare Figure A and B). gravitropism.
This result indicates that light does not cause the
photodestruction of auxin on the illuminated side, as had Statoliths Serve as Gravity Sensors in Shoots and Roots
been proposed by some investigators.
Unlike unilateral light, gravity does not form a gradient
Consistent with both the Cholodny–Went hypothesis and the between the upper and lower sides of an organ. All parts of
acid growth hypothesis, the apoplastic pH on the shaded the plant experience the gravitational stimulus equally. How
side of a phototropically bending stem or coleoptiles is more do plant cells detect gravity? The only way that gravity can
acidic than the side facing the light. be sensed is through the motion of a falling or sedimenting
body.

Obvious candidates for intracellular gravity sensors in plants

are the large, dense amyloplasts that are present in many
plant cells. These specialized amyloplasts are of sufficiently
high density relative to the cytosol that they readily
sediment to the bottom of the cell. Amyloplasts that function
as gravity sensors are called statoliths, and the specialized
gravity‐sensing cells in which they occur are called
statocytes. Whether the statocyte is able to detect the
downward motion of the statolith as it passes through the
Gravitropism Also Involves Lateral Redistribution of cytoskeleton or whether the stimulus is perceived only
Auxin when the statolith comes to rest at the bottom of the cell has
not yet been resolved.
When dark‐grown Avena seedlings are oriented horizontally,
the coleoptiles bend upward in response to gravity. Shoots and Coleoptiles. In shoots and coleoptiles, gravity is
According to the Cholodny–Went model, auxin in a perceived in the starch sheath, a layer of cells that
horizontally oriented coleoptile tip is transported laterally surrounds the vascular tissues of the shoot. The starch
to the lower side, causing the lower side of the coleoptile to sheath is continuous with the endodermis of the root, but
grow faster than the upper side. Early experimental unlike the endodermis it contains amyloplasts. Arabidopsis
evidence indicated that the tip of the coleoptile can perceive mutants lacking amyloplasts in the starch sheath display
gravity and redistribute auxin to the lower side. For agravitropic shoot growth but normal gravitropic root
example, if coleoptile tips are oriented horizontally, a growth.

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Auxin Regulates Apical Dominance senescence, the walls of the cells in the abscission layer are
digested, which causes them to become soft and weak. The
In most higher plants, the growing apical bud inhibits the leaf eventually breaks off at the abscission layer as a result
growth of lateral (axillary) buds—a phenomenon called of stress on the weakened cell walls. Auxin levels are high in
apical dominance. Removal of the shoot apex young leaves, progressively decrease in maturing leaves, and
(decapitation) usually results in the growth of one or more are relatively low in senescing leaves when the abscission
of the lateral buds. Not long after the discovery of auxin, it process begins. The role of auxin in leaf abscission can be
was found that IAA could substitute for the apical bud in readily demonstrated by excision of the blade from a mature
maintaining the inhibition of lateral buds of bean (Phaseolus leaf, leaving the petiole intact on the stem. Whereas removal
vulgaris) plants. of the leaf blade accelerates the formation of the abscission
layer in the petiole, application of IAA in lanolin paste to the
How does auxin from the shoot apex inhibit the growth of cut surface of the petiole prevents the formation of the
lateral buds? Kenneth V. Thimann and Folke Skoog originally abscission layer. (Lanolin paste alone does not prevent
proposed that auxin from the shoot apex inhibits the growth abscission.). These results suggest the following:
of the axillary bud directly—the so‐called direct inhibition • Auxin transported from the blade normally prevents
model. According to the model, the optimal auxin abscission.
concentration for bud growth is low, much lower than the • Abscission is triggered during leaf senescence, when auxin
auxin concentration normally found in the stem. The level of is no longer being produced.
auxin normally present in the stem was thought to inhibit
the growth of lateral buds. Auxin Transport Regulates Floral Bud Development

Other hormones, such as cytokinins and ABA, may be Treating Arabidopsis plants with the auxin transport
involved. Direct application of cytokinins to axillary buds inhibitor NPA causes abnormal floral development,
stimulates bud growth in many species, overriding the suggesting that polar auxin transport in the inflorescence
inhibitory effect of the shoot apex. Auxin makes the shoot meristem is required for normal floral development.
apex a sink for cytokinin synthesized in the root, and this
may be one of the factors involved in apical dominance. Auxin Promotes Fruit Development

Finally, ABA has been found in dormant lateral buds in intact Much evidence suggests that auxin is involved in the
plants. When the shoot apex is removed, the ABA levels in regulation of fruit development. Auxin is produced in pollen
the lateral buds decrease. High levels of IAA in the shoot may and in the endosperm and the embryo of developing seeds,
help keep ABA levels high in the lateral buds. Removing the and the initial stimulus for fruit growth may result from
apex removes a major source of IAA, which may allow the pollination. Successful pollination initiates ovule growth,
levels of bud growth inhibitor to fall. which is known as fruit set. After fertilization, fruit growth
may depend on auxin produced in developing seeds. The
Auxin Promotes the Formation of Lateral and endosperm may contribute auxin during the initial stage of
Adventitious Roots fruit growth, and the developing embryo may take over as
the main auxin source during the later stages.
Although elongation of the primary root is inhibited by auxin
concentrations greater than 10–8 M, initiation of lateral Auxin Induces Vascular Differentiation
(branch) roots and adventitious roots is stimulated by high
auxin levels. Lateral roots are commonly found above the New vascular tissues differentiate directly below developing
elongation and root hair zone and originate from small buds and young growing leaves, and removal of the young
groups of cells in the pericycle. Auxin stimulates these leaves prevents vascular differentiation. The ability of an
pericycle cells to divide. The dividing cells gradually form apical bud to stimulate vascular differentiation can be
into a root apex, and the lateral root grows through the root demonstrated in tissue culture. When the apical bud is
cortex and epidermis. Adventitious roots (roots originating grafted onto a clump of undifferentiated cells, or callus,
from nonroot tissue) can arise in a variety of tissue locations xylem and phloem differentiate beneath the graft.
from clusters of mature cells that renew their cell division
activity. These dividing cells develop into a root apical The relative amounts of xylem and phloem formed are
meristem in a manner somewhat analogous to the formation regulated by the auxin concentration: High auxin
of lateral roots. In horticulture, the stimulatory effect of concentrations induce the differentiation of xylem and
auxin on the formation of adventitious roots has been very phloem, but only phloem differentiates at low auxin
useful for the vegetative propagation of plants by cuttings. concentrations. Similarly, experiments on stem tissues have
shown that low auxin concentrations induce phloem
IAA is required for at least two steps in the formation of differentiation, whereas higher IAA levels induce xylem.
lateral roots:
1. IAA transported acropetally (toward the tip) in the stele is The regeneration of vascular tissue following wounding is
required to initiate cell division in the pericycle. also controlled by auxin produced by the young leaf directly
2. IAA is required to promote cell division and maintain cell above the wound site. Removal of the leaf prevents the
viability in the developing lateral root. regeneration of vascular tissue, and applied auxin can
substitute for the leaf in stimulating regeneration.
Auxin Delays the Onset of Leaf Abscission
Vascular differentiation is polar and occurs from leaves to
The shedding of leaves, flowers, and fruits from the living roots. In woody perennials, auxin produced by growing buds
plant is known as abscission. These parts abscise in a region in the spring stimulates activation of the cambium in a
called the abscission zone, which is located near the base of basipetal direction. The new round of secondary growth
the petiole of leaves. In most plants, leaf abscission is begins at the smallest twigs and progresses downward
preceded by the differentiation of a distinct layer of cells, the toward the root tip.
abscission layer, within the abscission zone. During leaf

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Synthetic Auxins Have a Variety of Commercial Uses activates the H+‐ATPase (see Figure), is involved in a signal
transduction pathway.
Auxins have been used commercially in agriculture and
horticulture for more than 50 years. The early commercial Calcium and Intracellular pH Are Possible Signaling
uses included prevention of fruit and leaf drop, promotion of Intermediates
flowering in pineapple, induction of parthenocarpic fruit,
thinning of fruit, and rooting of cuttings for plant Calcium plays an important role in signal transduction in
propagation. Rooting is enhanced if the excised leaf or stem animals and is thought to be involved in the action of certain
cutting is dipped in an auxin solution, which increases the plant hormones as well. The role of calcium in auxin action
initiation of adventitious roots at the cut end. This is the seems very complex and, at this point in time, very
basis of commercial rooting compounds, which consist uncertain. Nevertheless, some experimental evidence shows
mainly of a synthetic auxin mixed with talcum powder. that auxin increases the level of free calcium in the cell.
Changes in cytoplasmic pH can also serve as a second
In some plant species, seedless fruits may be produced messenger in animals and plants. In plants, auxin induces a
naturally, or they may be induced by treatment of the decrease in cytosolic pH of about 0.2 units within 4 minutes
unpollinated flowers with auxin. The production of such of application. The cause of this pH drop is not known. Since
seedless fruits is called parthenocarpy. In stimulating the the cytosolic pH is normally around 7.4, and the pH optimum
formation of parthenocarpic fruits, auxin may act primarily of the plasma membrane H+‐ATPase is 6.5, a decrease in the
to induce fruit set, which in turn may trigger the endogenous cytosolic pH of 0.2 units could cause a marked increase in
production of auxin by certain fruit tissues to complete the the activity of the plasma membrane H+‐ATPase.
developmental process.
The decrease in cytosolic pH might also account for the
Ethylene is also involved in fruit development, and some of auxin‐induced increase in free intracellular calcium, by
the effects of auxin on fruiting may result from the promoting the dissociation of bound forms. MAP kinases
promotion of ethylene synthesis. In addition to these that play a role in signal transduction by phosphorylating
applications, today auxins are widely used as herbicides. The proteins in a cascade that ultimately activates transcription
chemicals 2,4‐D and dicamba are probably the most widely factors have also been implicated in auxin responses. When
used synthetic auxins. Synthetic auxins are very effective tobacco cells are deprived of auxin, they arrest at the end of
because they are not metabolized by the plant as quickly as either the G1 or the G2 phase and cease dividing; if auxin is
IAA is. Because maize and other monocotyledons can rapidly added back into the culture medium, the cell cycle resumes.
inactivate synthetic auxins by conjugation, these auxins are Auxin appears to exert its effect on the cell cycle primarily
used by farmers for the control of dicot weeds, also called by stimulating the synthesis of the major cyclin‐dependent
broad­leaved weeds, in commercial cereal fields, and by protein kinase (CDK): Cdc2 (cell division cycle 2).
home gardeners for the control of weeds such as dandelions
and daisies in lawns. Auxin­Induced Genes Fall into Two Classes: Early and
Late
AUXIN SIGNAL TRANSDUCTION PATHWAYS
One of the important functions of the signal transduction
The ultimate goal of research on the molecular mechanism pathway(s) initiated when auxin binds to its receptor is the
of hormone action is to reconstruct each step in the signal activation of a select group of transcription factors. The
transduction pathway, from receptor binding to the activated transcription factors enter the nucleus and
physiological response. In this last section, we will examine promote the expression of specific genes. Genes whose
candidates for the auxin receptor and then discuss the expression is stimulated by the activation of preexisting
various signaling pathways that have been implicated in transcription factors are called primary response genes or
auxin action. Finally we will turn our attention to early genes.
auxinregulated gene expression.
This definition implies that all of the proteins required for
ABP1 Functions as an Auxin Receptor: auxin‐induced expression of the early genes are present in
the cell at the time of exposure to the hormone; thus, early‐
In addition to its possible direct role in plasma membrane gene expression cannot be blocked by inhibitors of protein
H+‐ATPase activation (discussed earlier), the auxin‐binding synthesis such as cycloheximide. As a consequence, the time
protein ABP1 appears to function as an auxin receptor in required for the expression of the early genes can be quite
other signal transduction pathways. ABP1 homologs have short, ranging from a few minutes to several hours.
been identified in a variety of monocot and dicot species.
Knockouts of the ABP1 gene in Arabidopsis are lethal, and In general, primary response genes have three main
less severe mutations result in altered development. Recent functions:
studies indicate that, despite being localized primarily on the
endoplasmic reticulum (ER), a small amount of ABP1 is (1) Some of the early genes encode proteins that regulate
secreted to the plasma membrane outer surface where it the transcription of secondary response genes, or late
interacts with auxin to cause protoplast swelling and H+‐ genes, that are required for the long‐term responses to the
pumping . hormone. Because late genes require de novo protein
synthesis, their expression can be blocked by protein
However, it is unlikely that ABP1 mediates all auxin synthesis inhibitors.
response pathways because expression of a number of (2) Other early genes are involved in intercellular
auxin‐responsive genes is not affected when protoplasts are communication, or cell‐to‐cell signaling.
incubated with anti‐ABP1 antibodies. It is also unclear what (3) Another group of early genes is involved in adaptation to
role the ABP1 in the ER plays in auxin‐responsive signal stress.
transduction. Finally, it remains to be determined whether
ABP57, the soluble and unrelated ABP from rice that

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Five major classes of early auxin‐responsive genes have Auxin­Responsive Domains Are Composite Structures
been identified:
Aconserved auxin response element (AuxRE) within the
• Genes involved in auxin­regulated growth and promoters of the early auxin genes, like GH3, is usually
development: combined with other response elements to form auxin
1. The AUX/IAA gene family response domains (AuxRDs). For example, the GH3 gene
2. The SAUR gene family promoter of soybean is composed of three independently
3. The GH3 gene family acting AuxRDs (each containing multiple AuxREs) that
contribute incrementally to the strong auxin inducibility of
• Stress response genes: the promoter.
1. Genes encoding glutathione S‐transferases
2. Genes encoding 1‐aminocyclopropane‐1‐carboxylic Early Auxin Genes Are Regulated by Auxin Response
acid (ACC) synthase, the key enzyme in the ethylene Factors
biosynthetic pathway
As noted previously, early auxin genes are by definition
Early genes for growth and development. insensitive to protein synthesis inhibitors such as
cycloheximide. Instead of being inhibited, the expression of
Members of the AUX/IAA gene family encode short‐lived many of the early auxin genes has been found to be
transcription factors that function as repressors or stimulated by cycloheximide.
activators of the expression of late auxin‐inducible genes.
The expression of most of the AUX/IAA family of genes is Cycloheximide stimulation of gene expression is
stimulated by auxin within 5 to 60 minutes of hormone accomplished both by transcriptional activation and by
addition All the genes encode small hydrophilic polypeptides mRNA stabilization. Transcriptional activation of a gene by
that have putative DNA‐binding motifs similar to those of inhibitors of protein synthesis usually indicates that the
bacterial repressors. They also have short half‐lives (about 7 gene is being repressed by a short‐lived repressor protein or
minutes), indicating that they are turning over rapidly. by a regulatory pathway that involves a protein with a high
turnover rate.
The SAUR gene family was mentioned earlier in the chapter
in relation to tropisms. Auxin stimulates the expression of A family of auxin response factors (ARFs) function as
SAUR genes within 2 to 5 minutes of treatment, and the transcriptional activators by binding to the auxin response
response is insensitive to cycloheximide. The five SAUR element TGTCTC, which is present in the promoters of GH3
genes of soybean are clustered together, contain no introns, and other early auxin response genes. Mutations in ARF
and encode highly similar polypeptides of unknown genes result in severe developmental defects. To bind the
function. Because of the rapidity of the response, expression AuxRE stably, ARFs must form dimers. It has been proposed
of SAUR genes has proven to be a convenient probe for the that ARF dimers promote transcription by binding to two
lateral transport of auxin during photo‐ and gravitropism. AuxREs arranged in a palindrome.

GH3 early‐gene family members, identified in both soybean Recent studies also indicate that proteins encoded by the
and Arabidopsis, are stimulated by auxin within 5 minutes. AUX/IAA gene family (itself one of the early auxin response
Mutations in Arabidopsis GH3‐like genes result in dwarfism gene families) can inhibit the transcription of early auxin
and appear to function in light‐regulated auxin responses. response genes by forming inactive heterodimers with ARFs.
Because GH3 expression is a good reflection of the presence
of endogenous auxin, a synthetic GH3‐based reporter gene These inactive heterodimers may act to inhibit ARF–AuxRE
known as DR5 is widely used in auxin bioassays. binding, thereby blocking either gene activation or
repression. AUX/IAA proteins may thus function as ARF
Early genes for stress adaptations. As mentioned earlier in inhibitors.
the chapter, auxin is involved in stress responses, such as
wounding. Several genes encoding glutathione‐S‐ It is now believed that auxin induces the transcription of the
transferases (GSTs), a class of proteins stimulated by various early response genes by promoting the proteolytic
stress conditions, are induced by elevated auxin degradation of the inhibitory AUX/IAA proteins so that
concentrations. active ARF dimers can form. The precise mechanism by
which auxin causes AUX/IAA turnover is unknown, although
Likewise, ACC synthase, which is also induced by stress and it is known to involve ubiquitination by a ubiquitin ligase
is the rate‐limiting step in ethylene biosynthesis, is induced and proteolysis by the massive 26S proteasome complex.
by high levels of auxin. Note that a negative feedback loop is introduced into the
pathway by virtue of the fact that one of the gene families
To be induced, the promoters of the early auxin genes must turned on by auxin is AUX/IAA, which inhibits the response.
contain response elements that bind to the transcription
factors that become activated in the presence of auxin. A model for auxin regulation of the early response genes
based on the findings described here is shown in Figure.
A limited number of these response elements appear to be
arranged combinatorily within the promoters of a variety of
auxin‐induced genes.

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CYTOKININS
Differentiated Plant Cells Can Resume Division
The cytokinins were discovered in the search for factors that
stimulate plant cells to divide (i.e., undergo cytokinesis). Under some circumstances, mature, differentiated plant cells
Since their discovery, cytokinins have been shown to have may resume cell division in the intact plant. In many species,
effects on many other physiological and developmental mature cells of the cortex and/or phloem resume division to
processes, including leaf senescence, nutrient mobilization, form secondary meristems, such as the vascular cambium or
apical dominance, the formation and activity of shoot apical the cork cambium. The abscission zone at the base of a leaf
meristems, floral development, the breaking of bud petiole is a region where mature parenchyma cells begin to
dormancy, and seed germination. Cytokinins also appear to divide again after a period of mitotic inactivity, forming a
mediate many aspects of light‐regulated development, layer of cells with relatively weak cell walls where
including chloroplast differentiation, the development of abscission can occur.
autotrophic metabolism, and leaf and cotyledon expansion.
Wounding of plant tissues induces cell divisions at the
Although cytokinins regulate many cellular processes, the wound site. Even highly specialized cells, such as phloem
control of cell division is central in plant growth and fibers and guard cells, may be stimulated by wounding to
development and is considered diagnostic for this class of divide at least once. Wound‐induced mitotic activity
plant growth regulators. typically is self‐limiting; after a few divisions the derivative
cells stop dividing and redifferentiate. However, when the
CELL DIVISION AND PLANT DEVELOPMENT soildwelling bacterium Agrobacterium tumefaciens invades a
wound, it can cause the neoplastic (tumor‐forming) disease
Plant cells form as the result of cell divisions in a primary or known as crown gall. This phenomenon is dramatic natural
secondary meristem. Newly formed plant cells typically evidence of the mitotic potential of mature plant cells.
enlarge and differentiate, but once they have assumed their
function—whether transport, photosynthesis, support, Without Agrobacterium infection, the wound‐induced cell
storage, or protection—usually they do not divide again division would subside after a few days and some of the new
during the life of the plant. In this respect they appear to be cells would differentiate as a protective layer of cork cells or
similar to animal cells, which are considered to be terminally vascular tissue. However, Agrobacterium changes the
differentiated. character of the cells that divide in response to the wound,
making them tumorlike. They do not stop dividing; rather
However, this similarity to the behavior of animal cells is they continue to divide throughout the life of the plant to
only superficial. Almost every type of plant cell that retains produce an unorganized mass of tumorlike tissue called a
its nucleus at maturity gall.

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Diffusible Factors May Control Cell Division Kinetin is not a naturally occurring plant growth regulator,
and it does not occur as a base in the DNA of any species. It is
The considerations addressed in the previous section a by‐product of the heat‐induced degradation of DNA, in
suggest that mature plant cells stop dividing because they no which the deoxyribose sugar of adenosine is converted to a
longer receive a particular signal, possibly a hormone, that is furfuryl ring and shifted from the 9 position to the 6 position
necessary for the initiation of cell division. The idea that cell on the adenine ring.
division may be initiated by a diffusible factor originated
with the Austrian plant physiologist G. Haberlandt, who, in The discovery of kinetin was important because it
about 1913, demonstrated that vascular tissue contains a demonstrated that cell division could be induced by a simple
water‐soluble substance or substances that will stimulate chemical substance. Of greater importance, the discovery of
the division of wounded potato tuber tissue. The effort to kinetin suggested that naturally occurring molecules with
determine the nature of this factor (or factors) led to the structures similar to that of kinetin regulate cell division
discovery of the cytokinins in the 1950s. activity within the plant. This hypothesis proved to be
correct.
THE DISCOVERY, IDENTIFICATION, AND PROPERTIES OF
CYTOKININS Zeatin Is the Most Abundant Natural Cytokinin

A great many substances were tested in an effort to initiate Several years after the discovery of kinetin, extracts of the
and sustain the proliferation of normal stem tissues in immature endosperm of corn (Zea mays) were found to
culture. Materials ranging from yeast extract to tomato juice contain a substance that has the same biological effect as
were found to have a positive effect, at least with some kinetin. This substance stimulates mature plant cells to
tissues. However, culture growth was stimulated most divide when added to a culture medium along with an auxin.
dramatically when the liquid endosperm of coconut, also Letham (1973) isolated the molecule responsible for this
known as coconut milk, was added to the culture medium. activity and identified it as trans‐6‐(4‐hydroxy‐3‐ methylbut‐
2‐enylamino)purine, which he called zeatin:
Philip White’s nutrient medium, supplemented with an
auxin and 10 to 20% coconut milk, will support the
continued cell division of mature, differentiated cells from a
wide variety of tissues and species, leading to the formation
of callus tissue . This finding indicated that coconut milk
contains a substance or substances that stimulate mature
cells to enter and remain in the cell division cycle.

Eventually coconut milk was shown to contain the cytokinin

zeatin, but this finding was not obtained until several years
after the discovery of the cytokinins. The first cytokinin to be
discovered was the synthetic analog kinetin. The molecular structure of zeatin is similar to that of kinetin.
Both molecules are adenine or aminopurine derivatives.
Kinetin Was Discovered as a Breakdown Product of DNA Although they have different side chains, in both cases the
side chain is attached to the 6 nitrogen of the aminopurine.
In the 1940s and 1950s, Folke Skoog and coworkers at the Because the side chain of zeatin has a double bond, it can
University of Wisconsin tested many substances for their exist in either the cis or the trans configuration.
ability to initiate and sustain the proliferation of cultured
tobacco pith tissue. They had observed that the nucleic acid In higher plants, zeatin occurs in both the cis and the trans
base adenine had a slight promotive effect, so they tested the configurations, and these forms can be interconverted by an
possibility that nucleic acids would stimulate division in this enzyme known as zeatin isomerase. Although the trans form
tissue. Surprisingly, autoclaved herring sperm DNA had a of zeatin is much more active in biological assays, the cis
powerful cell division–promoting effect. form may also play important roles, as suggested by the fact
that it has been found in high levels in a number of plant
After much work, a small molecule was identified from the species and particular tissues. Agene encoding a glucosyl
autoclaved DNA and named kinetin. It was shown to be an transferase enzyme specific to cis‐zeatin has recently been
adenine (or aminopurine) derivative, 6‐furfurylaminopurine cloned, which further supports a biological role for this
isoform of zeatin.

Since its discovery in immature maize endosperm, zeatin has

been found in many plants and in some bacteria. It is the
most prevalent cytokinin in higher plants, but other
substituted aminopurines that are active as cytokinins have
been isolated from many plant and bacterial species. These
aminopurines differ from zeatin in the nature of the side
chain attached to the 6 nitrogen or in the attachment of a
side chain to carbon 2:

In the presence of an auxin, kinetin would stimulate tobacco

pith parenchyma tissue to proliferate in culture. No kinetin‐
induced cell division occurs without auxin in the culture
medium.

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In addition, these cytokinins can be present in the plant as a group of plants. They have also been found in algae, diatoms,
riboside (in which a ribose sugar is attached to the 9 mosses, ferns, and conifers.
nitrogen of the purine ring), a ribotide (in which the ribose
sugar moiety contains a phosphate group), or a glycoside The regulatory role of cytokinins has been demonstrated
(in which a sugar molecule is attached to the 3, 7, or 9 only in angiosperms, conifers, and mosses, but they may
nitrogen of the purine ring, or to the oxygen of the zeatin or function to regulate the growth, development, and
dihydrozeatin side chain). metabolism of all plants. Usually zeatin is the most abundant
naturally occurring free cytokinin, but dihydrozeatin (DZ)
Some Synthetic Compounds Can Mimic or Antagonize and isopentenyl adenine (iP) also are commonly found in
Cytokinin Action higher plants and bacteria. Numerous derivatives of these
three cytokinins have been identified in plant extract.
Cytokinins are defined as compounds that have biological
activities similar to those of trans‐zeatin. These activities Transfer RNA (tRNA) contains not only the four nucleotides
include the ability to do the following: used to construct all other forms of RNA, but also some
• Induce cell division in callus cells in the presence of an unusual nucleotides in which the base has been modified.
auxin Some of these “hypermodified” bases act as cytokinins when
• Promote bud or root formation from callus cultures when the tRNA is hydrolyzed and tested in one of the cytokinin
in the appropriate molar ratios to auxin bioassays. Some plant tRNAs contain cis‐zeatin as a
• Delay senescence of leaves hypermodified base. However, cytokinins are not confined to
• Promote expansion of dicot cotyledons plant tRNAs. They are part of certain tRNAs from all
organisms, from bacteria to humans.
Many chemical compounds have been synthesized and
tested for cytokinin activity. Analysis of these compounds The Hormonally Active Cytokinin Is the Free Base
provides insight into the structural requirements for
activity. Nearly all compounds active as cytokinins are N6‐ It has been difficult to determine which species of cytokinin
substituted aminopurines, such as benzyladenine (BA): represents the active form of the hormone, but the recent
identification of the cytokinin receptor CRE1 has allowed
this question to be addressed. The relevant experiments
have shown that the free‐base form of trans‐zeatin, but not
its riboside or ribotide derivatives, binds directly to CRE1,
indicating that the free base is the active form.

Although the free‐base form of trans‐zeatin is thought to be

the hormonally active cytokinin, some other compounds
have cytokinin activity, either because they are readily
converted to zeatin, dihydrozeatin, or isopentenyl adenine,
and all the naturally occurring cytokinins are aminopurine
or because they release these compounds from other
derivatives. There are also synthetic cytokinin compounds
molecules, such as cytokinin glucosides. For example,
that have not been identified in plants, most notable of
tobacco cells in culture do not grow unless cytokinin
which are the diphenylurea‐type cytokinins, such as
ribosides supplied in the culture medium are converted to
thidiazuron, which is used commercially as a defoliant and
the free base.
an herbicide:
In another example, excised radish cotyledons grow when
they are cultured in a solution containing the cytokinin base
benzyladenine (BA, an N6‐substituted aminopurine
cytokinin). The cultured cotyledons readily take up the
hormone and convert it to various BA glucosides, BA
ribonucleoside, and BA ribonucleotide. When the cotyledons
are transferred back to a medium lacking a cytokinin, their
growth rate declines, as do the concentrations of BA, BA
ribonucleoside, and BA ribonucleotide in the tissues.

In the course of determining the structural requirements for However, the level of the BA glucosides remains constant.
cytokinin activity, investigators found that some molecules This finding suggests that the glucosides cannot be the active
act as cytokinin antagonists: form of the hormone.

Some Plant Pathogenic Bacteria, Insects, and Nematodes

Secrete Free Cytokinins

Some bacteria and fungi are intimately associated with

higher plants. Many of these microorganisms produce and
secrete substantial amounts of cytokinins and/or cause the
plant cells to synthesize plant hormones, including
Cytokinins Occur in Both Free and Bound Forms cytokinins. The cytokinins produced by microorganisms
include trans‐zeatin, [9R]iP, cis‐zeatin, and their ribosides.
Hormonal cytokinins are present as free molecules (not Infection of plant tissues with these microorganisms can
covalently attached to any macromolecule) in plants and induce the tissues to divide and, in some cases, to form
certain bacteria. Free cytokinins have been found in a wide special structures, such as mycorrhizae, in which the
spectrum of angiosperms and probably are universal in this microorganism can reside in a mutualistic relationship with
the plant.

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In addition to the crown gall bacterium, Agrobacterium

tumefaciens, other pathogenic bacteria may stimulate plant During infection by Agrobacterium tumefaciens, plant cells
cells to divide. For example, Corynebacterium fascians is a incorporate bacterial DNA into their chromosomes. The
major cause of the growth abnormality known as witches’­ virulent strains of Agrobacterium contain a large plasmid
broom. The shoots of plants infected by C. fascians resemble known as the Ti plasmid. Plasmids are circular pieces of
an old‐fashioned straw broom because the lateral buds, extrachromosomal DNA that are not essential for the life of
which normally remain dormant, are stimulated by the the bacterium. However, plasmids frequently contain genes
bacterial cytokinin to grow. that enhance the ability of the bacterium to survive in
special environments.

A small portion of the Ti plasmid, known as the TDNA, is

incorporated into the nuclear DNA of the host plant cell. T‐
DNA carries genes necessary for the biosynthesis of trans‐
zeatin and auxin, as well as a member of a class of unusual
nitrogencontaining compounds called opines . Opines are not
synthesized by plants except after crown gall
transformation. The T‐DNA gene involved in cytokinin
biosynthesis—known as the ipt1 gene—encodes an
isopentenyl transferase

Infection with a close relative of the crown gall organism,

Agrobacterium rhizogenes, causes masses of roots instead of
callus tissue to develop from the site of infection. A. (IPT) enzyme that transfers the isopentenyl group from
rhizogenes is able to modify cytokinin metabolism in DMAPP to AMP (adenosine monophosphate) to form
infected plant tissues. isopentenyl adenine ribotide. The ipt gene has been called
the tmr locus because, when inactivated by mutation, it
Certain insects secrete cytokinins, which may play a role in results in “rooty” tumors. Isopentenyl adenine ribotide can
the formation of galls utilized by these insects as feeding be converted to the active cytokinins isopentenyl adenine,
sites. Root‐knot nematodes also produce cytokinins, which transzeatin, and dihydrozeatin by endogenous enzymes in
may be involved in manipulating host development to plant cells. This conversion route is similar to the pathway
produce the giant cells from which the nematode feeds. for cytokinin synthesis that has been postulated for normal
tissue.
BIOSYNTHESIS, METABOLISM, AND TRANSPORT OF
CYTOKININS The T‐DNA also contains two genes encoding enzymes that
convert tryptophan to the auxin indole‐3‐acetic acid (IAA).
The side chains of naturally occurring cytokinins are This pathway of auxin biosynthesis differs from the one in
chemically related to rubber, carotenoid pigments, the plant nontransformed cells and involves indoleacetamide as an
hormones gibberellin and abscisic acid, and some of the intermediate. The ipt gene and the two auxin biosynthetic
plant defense compounds known as phytoalexins. All of enes of T‐DNA are phyto­oncogenes, since they can induce
these compounds are constructed, at least in part, from tumors in plants..
isoprene units.
Because their promoters are plant eukaryotic promoters,
Isoprene is similar in structure to the side chains of zeatin none of the T‐DNAgenes are expressed in the bacterium;
and iP. These cytokinin side chains are synthesized from an rather they are transcribed after they are inserted into the
isoprene derivative. Large molecules of rubber and the plant chromosomes. Transcription of the genes leads to
carotenoids are constructed by the polymerization of many synthesis of the enzymes they encode, resulting in the
isoprene units; cytokinins contain just one of these units. production of zeatin, auxin, and an opine. The bacterium can
The precursor(s) for the formation of these isoprene utilize the opine as a nitrogen source, but cells of higher
structures are either mevalonic acid or pyruvate plus 3‐ plants cannot. Thus, by transforming the plant cells, the
phosphoglycerate, depending on which pathway is involved. bacterium provides itself with an expanding environment
These precursors are converted to the biological isoprene (the gall tissue) in which the host cells are directed to
unit dimethylallyl diphosphate (DMAPP). produce a substance (the opine) that only the bacterium can
utilize for its nutrition .
Crown Gall Cells Have Acquired a Gene for Cytokinin
Synthesis An important difference between the control of cytokinin
biosynthesis in crown gall tissues and in normal tissues is
Bacteria‐free tissues from crown gall tumors proliferate in that the T‐DNA genes for cytokinin synthesis are expressed
culture without the addition of any hormones to the culture in all infected cells, even those in which the native plant
medium. Crown gall tissues contain substantial amounts of genes for biosynthesis of the hormone are normally
both auxin and free cytokinins. Furthermore, when repressed.
radioactively labeled adenine is fed to periwinkle (Vinca
rosea) crown gall tissues, it is incorporated into both zeatin IPT Catalyzes the First Step in Cytokinin Biosynthesis
and zeatin riboside, demonstrating that gall tissues contain
the cytokinin biosynthetic pathway. Control stem tissue, The first committed step in cytokinin biosynthesis is the
which has not been transformed by Agrobacterium, does not transfer of the isopentenyl group of dimethylallyl
incorporate labeled adenine into cytokinins. diphosphate (DMAPP ) to an adenosine moiety. An enzyme

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that catalyzes such an activity was first identified in the tRNA cytokinins are synthesized by modification of specific
cellular slime mold Dictyostelium discoideum, and adenine residues within the fully transcribed tRNA. As with
subsequently the ipt gene from Agrobacterium was found to the free cytokinins, isopentenyl groups are transferred to
encode such an enzyme. In both cases, DMAPP and AMP are the adenine molecules from DMAPP by an enzyme call tRNA‐
converted to isopentenyladenosine‐5’‐monophosphate IPT. The genes for tRNA‐IPT have been cloned from many
(iPMP). As noted earlier, cytokinins are also present in the species.
tRNAs of most cells, including plant and animal cells. The

Cytokinins from the Root Are Transported to the Shoot

The possibility that free cytokinins are derived from tRNA via the Xylem
has been explored extensively. Although the tRNA bound
cytokinins can act as hormonal signals for plant cells if the Root apical meristems are major sites of synthesis of the free
tRNA is degraded and fed back to the cells, it is unlikely that cytokinins in whole plants. The cytokinins synthesized in
any significant amount of the free hormonal cytokinin in roots appear to move through the xylem into the shoot,
plants is derived from the turnover of tRNA. along with the water and minerals taken up by the roots.
This pathway of cytokinin movement has been inferred from
An enzyme with IPT activity was identified from crude the analysis of xylem exudate.
extracts of various plant tissues, but researchers were Roots are not the only parts of the plant capable of
unable to purify the protein to homogeneity. Recently, plant synthesizing cytokinins. For example, young maize embryos
IPT genes were cloned after the Arabidopsis genome was synthesize cytokinins, as do young developing leaves, young
analyzed for potential ipt‐like sequence. Nine different IPT fruits, and possibly many other tissues. Clearly, further
genes were identified studies will be needed to resolve the roles of cytokinins
transported from the root versus cytokinins synthesized in
the shoot.

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A Signal from the Shoot Regulates the Transport of Dormant seeds often have high levels of cytokinin glucosides
Zeatin Ribosides from the Root but very low levels of hormonally active free cytokinins.
Levels of free cytokinins increase rapidly, however, as
The cytokinins in the xylem exudate are mainly in the form germination is initiated, and this increase in free cytokinins
of zeatin ribosides. Once they reach the leaves, some of these is accompanied by a corresponding decrease in cytokinin
nucleosides are converted to the free‐base form or to glucosides.
glucosides. Cytokinin glucosides may accumulate to high
levels in seeds and in leaves, and substantial amounts may THE BIOLOGICAL ROLES OF CYTOKININS
be present even in senescing leaves. Although the glucosides
are active as cytokinins in bioassays, often they lack Although discovered as a cell division factor, cytokinins can
hormonal activity after they form within cells, possibly stimulate or inhibit a variety of physiological, metabolic,
because they are compartmentalized in such a way that they biochemical, and developmental processes when they are
are unavailable. Evidence from grafting experiments with applied to higher plants, and it is increasingly clear that
mutants suggests that the transport of zeatin riboside from endogenous cytokinins play an important role in the
the root to the shoot is regulated by signals from the shoot. regulation of these events in the intact plant.

Cytokinins Are Rapidly Metabolized by Plant Tissues In this section we will survey some of the diverse effects of
cytokinin on plant growth and development, including a
Free cytokinins are readily converted to their respective discussion of its role in regulating cell division. The
nucleoside and nucleotide forms. Such interconversions discovery of the tumor‐inducing Ti plasmid in the plant‐
likely involve enzymes common to purine metabolism. Many pathogenic bacterium Agrobacterium tumefaciens provided
plant tissues contain the enzyme cytokinin oxidase, which plant scientists with a powerful new tool for introducing
cleaves the side chain from zeatin (both cis and trans), zeatin foreign genes into plants, and for studying the role of
riboside, iP, and their N‐glucosides, but not their O‐glucoside cytokinin in development. In addition to its role in cell
derivatives (Figure). proliferation, cytokinin affects many other processes,
including differentiation, apical dominance, and senescence.

Cytokinins Regulate Cell Division in Shoots and Roots

As discussed earlier, cytokinins are generally required for

cell division of plant cells in vitro. Several lines of evidence
suggest that cytokinins also play key roles in the regulation
of cell division in vivo.

Much of the cell division in an adult plant occurs in the

However, dihydrozeatin and its conjugates are resistant to meristems. Localized expression of the ipt gene of
cleavage. Cytokinin oxidase irreversibly inactivates Agrobacterium in somatic sectors of tobacco leaves causes
cytokinins, and it could be important in regulating or the formation of ectopic (abnormally located) meristems,
limiting cytokinin effects. The activity of the enzyme is indicating that elevated levels of cytokinin are sufficient to
induced by high cytokinin concentrations, due at least in initiate cell divisions in these leaves Overexpression of
part to an elevation of the RNA levels for a subset of the several of the Arabidopsis cytokinin oxidase genes in tobacco
genes. results in a reduction of endogenous cytokinin levels and a
consequent strong retardation of shoot development due to
Cytokinin levels can also be regulated by conjugation of the a reduction in the rate of cell proliferation in the shoot apical
hormone at various positions. The nitrogens at the 3, 7, and meristem. This finding strongly supports the notion that
9 positions of the adenine ring of cytokinins can be endogenous cytokinins regulate cell division in vivo.
conjugated to glucose residues. Alanine can also be
conjugated to the nitrogen at the 9 positon, forming lupinic Surprisingly, the same overexpression of cytokinin oxidase
acid. These modifications are generally irreversible, and in tobacco led to an enhancement of root growth, primarily
such conjugated forms of cytokinin are inactive in bioassays, by increasing the size of the root apical meristem. Since the
with the exception of the N3‐glucosides. root is a major source of cytokinin, this result may indicate
that cytokinins play opposite roles in regulating cell
The hydroxyl group of the side chain of cytokinins is also the proliferation in root and shoot meristems.
target for conjugation to glucose residues, or in some cases
xylose residues, yielding O‐glucoside and Oxyloside Cytokinins Regulate Specific Components of the Cell
cytokinins. O‐glucosides are resistant to cleavage by Cycle
cytokinin oxidases, which may explain why these derivatives
have higher biological activity in some assays than their Cytokinins regulate cell division by affecting the controls
corresponding free bases have. that govern the passage of the cell through the cell division
cycle. Zeatin levels were found to peak in synchronized
The conjugations at the side chain can be removed by culture tobacco cells at the end of S phase, mitosis, and G1
glucosidase enzymes to yield free cytokinins, which, as phase. Cytokinins were discovered in relation to their ability
discussed earlier, are the active forms. Thus, cytokinin to stimulate cell division in tissues supplied with an optimal
glucosides may be a storage form, or metabolically inactive level of auxin. Evidence suggests that both auxin and
state, of these compounds. Agene encoding a glucosidase cytokinins participate in regulation of the cell cycle and that
that can release cytokinins from sugar conjugates has been they do so by controlling the activity of cyclin‐dependent
cloned from maize, and its expression could play an kinases. Cyclin­dependent protein kinases (CDKs), in concert
important role in the germination of maize seeds. with their regulatory subunits, the cyclins, are enzymes that
regulate the eukaryotic cell cycle. The expression of the gene
that encodes the major CDK, Cdc2 (cell division cycle 2), is

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regulated by auxin. In pea root tissues, CDC2 mRNA was Cytokinins Induce Bud Formation in a Moss
induced within 10 minutes after treatment with auxin, and
high levels of CDK are induced in tobacco pith when it is Thus far we have restricted our discussion of plant
cultured on medium containing auxin . However, the CDK hormones to the angiosperms. However, many plant
induced by auxin is enzymatically inactive, and high levels of hormones are present and developmentally active in
CDK alone are not sufficient to permit cells to divide. representative species throughout the plant kingdom. The
moss Funaria hygrometrica is a well‐studied example. The
Cytokinin has been linked to the activation of a Cdc25‐ like germination of moss spores gives rise to a filament of cells
phosphatase, whose role is to remove an inhibitory called a protonema (plural protonemata). The protonema
phosphate group from the Cdc2 kinase . elongates and undergoes cell divisions at the tip, and it
forms branches some distance back from the tip.
This action of cytokinin provides one potential link between
cytokinin and auxin in regulating the cell cycle. Recently, a The transition from filamentous growth to leafy growth
second major input for cytokinin in regulating the cell cycle begins with the formation of a swelling or protuberance
has emerged. Cytokinins elevate the expression of the CYCD3 near the apical ends of specific cells. An asymmetric cell
gene, which encodes a D­type cyclin. In animal cells, D‐type division follows, creating the initial cell. The initial cell then
cyclins are regulated by a wide variety of growth factors and divides mitotically to produce the bud, the structure that
play a key role in regulating the passage through the gives rise to the leafy gametophyte. During normal growth,
restriction point of the cell cycle in G1. D‐type cyclins are buds and branches are regularly initiated, usually beginning
thus key players in the regulation of cell proliferation. at the third cell from the tip of the filament. Light, especially
red light, is required for bud formation in Funaria. In the
The Auxin: Cytokinin Ratio Regulates Morphogenesis in dark, buds fail to develop, but cytokinin added to the
Cultured Tissues medium can substitute for the light requirement.

Shortly after the discovery of kinetin, it was observed that Cytokinin not only stimulates normal bud development; it
the differentiation of cultured callus tissue derived from also increases the total number of buds. Even very low levels
tobacco pith segments into either roots or shoots depends of cytokinin (picomolar, or 10–12 M) can stimulate the first
on the ratio of auxin to cytokinin in the culture medium. step in bud formation: the swelling at the apical end of the
specific protonemal cell.
Whereas high auxin:cytokinin ratios stimulated the
formation of roots, low auxin:cytokinin ratios led to the Cytokinin Overproduction Has Been Implicated in
formation of shoots. At intermediate levels the tissue grew Genetic Tumors
as an undifferentiated callus.
Many species in the genus Nicotiana can be crossed to
The effect of auxin: cytokinin ratios on morphogenesis can generate interspecific hybrids. More than 300 such
also be seen in crown gall tumors by mutation of the T‐DNA interspecific hybrids have been produced; 90% of these
of the Agrobacterium Ti plasmid. Mutating the ipt gene (the hybrids are normal, exhibiting phenotypic characteristics
tmr locus) of the Ti plasmid blocks zeatin biosynthesis in the intermediate between those of both parents. The plant used
infected cells. The resulting high auxin:cytokinin ratio in the for cigarette tobacco, Nicotiana tabacum, for example, is an
tumor cells causes the proliferation of roots instead of interspecific hybrid. However, about 10% of these
undifferentiated callus tissue. In contrast, mutating either of interspecific crosses result in progeny that tend to form
the genes for auxin biosynthesis (tms locus) lowers the spontaneous tumors called genetic tumors.
auxin:cytokinin ratio and stimulates the proliferation of
shoots. These partially differentiated tumors are known as Genetic tumors are similar morphologically to those induced
teratomas. by Agrobacterium tumefaciens, discussed at the beginning of
this chapter, but genetic tumors form spontaneously in the
Cytokinins Modify Apical Dominance and Promote absence of any external inducing agent. The tumors are
Lateral Bud Growth composed of masses of rapidly proliferating cells in regions
of the plant that ordinarily would contain few dividing cells.
One of the primary determinants of plant form is the degree Furthermore, the cells divide without differentiating into the
of apical dominance. Plants with strong apical dominance, cell types normally associated with the tissues giving rise to
such as maize, have a single growing axis with few lateral the tumor. Nicotiana hybrids that produce genetic tumors
branches. In contrast, many lateral buds initiate growth in have abnormally high levels of both auxin and cytokinins.
shrubby plants. Typically, the cytokinin levels in tumor‐prone hybrids are
five to six times higher than those found in either parent.
Although apical dominance may be determined primarily by
auxin, physiological studies indicate that cytokinins play a Cytokinins Delay Leaf Senescence
role in initiating the growth of lateral buds. For example,
direct applications of cytokinins to the axillary buds of many Leaves detached from the plant slowly lose chlorophyll,
species stimulate cell division activity and growth of the RNA, lipids, and protein, even if they are kept moist and
buds. provided with minerals. This programmed aging process
The phenotypes of cytokinin‐overproducing mutants are leading to death is termed senescence. Leaf senescence is
consistent with this result. Wild‐type tobacco shows strong more rapid in the dark than in the light. Treating isolated
apical dominance during vegetative development, and the leaves of many species with cytokinins will delay their
lateral buds of cytokinin overproducers grow vigorously, senescence.
developing into shoots that compete with the main shoot.
Consequently, cytokinin‐overproducing plants tend to be Although applied cytokinins do not prevent senescence
bushy. completely, their effects can be dramatic, particularly when
the cytokinin is sprayed directly on the intact plant. If only
one leaf is treated, it remains green after other leaves of

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similar developmental age have yellowed and dropped off The ability of exogenous cytokinin to enhance de‐etiolation
the plant. Even a small spot on a leaf will remain green if of dark‐grown seedlings is mimicked by certain mutations
treated with a cytokinin, after the surrounding tissues on the that lead to cytokinin overproduction.
same leaf begin to senesce.
Cytokinins Promote Cell Expansion in Leaves and
Unlike young leaves, mature leaves produce little if any Cotyledons
cytokinin. Mature leaves may depend on root‐derived
cytokinins to postpone their senescence. Senescence is The promotion of cell enlargement by cytokinins is most
initiated in soybean leaves by seed maturation—a clearly demonstrated in the cotyledons of dicots with leafy
phenomenon known as monocarpic senescence—and can be cotyledons, such as mustard, cucumber, and sunflower. The
delayed by seed removal. Although the seedpods control the cotyledons of these species expand as a result of cell
onset of senescence, they do so by controlling the delivery of enlargement during seedling growth. Cytokinin treatment
root‐derived cytokinins to the leaves. promotes additional cell expansion, with no increase in the
dry weight of the treated cotyledons.
The cytokinins involved in delaying senescence are
primarily zeatin riboside and dihydrozeatin riboside, which Leafy cotyledons expand to a much greater extent when the
may be transported into the leaves from the roots through seedlings are grown in the light than in the dark, and
the xylem, along with the transpiration stream. cytokinins promote cotyledon growth in both light‐ and
dark‐grown seedlings. As with auxin‐ induced growth,
Cytokinins Promote Movement of Nutrients cytokinin‐stimulated expansion of radish cotyledons is
associated with an increase in the mechanical extensibility
Cytokinins influence the movement of nutrients into leaves of the cell walls. However, cytokinin‐induced wall loosening
from other parts of the plant, a phenomenon known as is not accompanied by proton extrusion. Neither auxin nor
cytokinin­induced nutrient mobilization. This process is gibberellin promotes cell expansion in cotyledons.
revealed when nutrients (sugars, amino acids, and so on)
radiolabeled with 14C or 3H are fed to plants after one leaf or Cytokinins Regulate Growth of Stems and Roots
part of a leaf is treated with a cytokinin. Later the whole
plant is subjected to autoradiography to reveal the pattern Although endogenous cytokinins are clearly required for
of movement and the sites at which the labeled nutrients normal cell proliferation in the apical meristem, and
accumulate. therefore normal shoot growth, applied cytokinins typically
inhibit the process of cell elongation in both stems and roots.
Experiments of this nature have demonstrated that For example, exogenous cytokinin inhibits hypocotyl
nutrients are preferentially transported to, and accumulated elongation at concentrations that promote leaf and
in, the cytokinin‐treated tissues. It has been postulated that cotyledon expansion in the dark‐grown seedlings.
the hormone causes nutrient mobilization by creating a new
source–sink relationship. As discussed in Chapter 10, It is likely that the inhibition of hypocotyl and internode
nutrients translocated in the phloem move from a site of elongation induced by excess cytokinin is due to the
production or storage (the source) to a site of utilization (the production of ethylene, and this inhibition thus may
sink). The metabolism of the treated area may be stimulated represent another example of the interdependence of
by the hormone so that nutrients move toward it. However, hormonal regulatory pathways.
it is not necessary for the nutrient itself to be metabolized in
the sink cells because even nonmetabolizable substrate On the other hand, other experiments suggest that
analogs are mobilized by cytokinins. endogenous cytokinins at normal physiological
concentrations inhibit root growth.
Cytokinins Promote Chloroplast Development
Cytokinin­Regulated Processes Are Revealed in Plants
Although seeds can germinate in the dark, the morphology That Overproduce Cytokinin
of dark‐grown seedlings is very different from that of
lightgrown seedlings: Dark‐grown seedlings are said to be The ipt gene from the Agrobacterium Ti plasmid has been
etiolated. The hypocotyl and internodes of etiolated introduced into many species of plants, resulting in
seedlings are more elongated, cotyledons and leaves do not cytokinin overproduction. These transgenic plants exhibit an
expand, and chloroplasts do not mature. Instead of maturing array of developmental abnormalities that tell us a great
as chloroplasts, the proplastids of dark‐grown seedlings deal about the biological role of cytokinins.
develop into etioplasts, which do not synthesize chlorophyll
or most of the enzymes and structural proteins required for As discussed earlier, plant tissues transformed by
the formation of the chloroplast thylakoid system and Agrobacterium carrying a wild‐type Ti plasmid proliferate as
photosynthesis machinery. tumors as a result of the overproduction of both auxin and
cytokinin. And as mentioned already, if all of the other genes
When seedlings germinate in the light, chloroplasts mature in the T‐DNA are deleted and plant tissues are transformed
directly from the proplastids present in the embryo, but with T‐DNA containing only a selective antibiotic resistance
etioplasts also can mature into chloroplasts when etiolated marker gene and the ipt gene, shoots proliferate instead of
seedlings are illuminated. callus.
If the etiolated leaves are treated with cytokinin before The shoot teratomas formed by ipt‐transformed tissues are
being illuminated, they form chloroplasts with more difficult to root, and when roots are formed, they tend to be
extensive grana, and chlorophyll and photosynthetic stunted in their growth. As a result, it is difficult to obtain
enzymes are synthesized at a greater rate upon illumination. plants from shoots expressing the ipt gene under the control
These results suggest that cytokinins—along with other of its own promoter because the promoter is a constitutive
factors, such as light, nutrition, and development— regulate promoter and the gene is continuously expressed. To
the synthesis of photosynthetic pigments and proteins. circumvent this problem, a variety of promoters whose

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expression can be regulated have been used to drive the CELLULAR AND MOLECULAR MODES OF CYTOKININ
expression of the ipt gene in the transformed tissues. ACTION

For example, several studies have employed a heat shock The diversity of the effects of cytokinin on plant growth and
promoter, which is induced in response to elevated development is consistent with the involvement of signal
temperature, to drive inducible expression of the ipt gene in transduction pathways with branches leading to specific
transgenic tobacco and Arabidopsis. In these plants, heat responses. Although our knowledge of how cytokinin works
induction substantially increased the level of zeatin, zeatin at the cellular and molecular levels is still quite fragmentary,
riboside and ribotide, and N‐conjugated zeatin. significant progress has been achieved. In this section we
will discuss the nature of the cytokinin receptor and various
These cytokinin‐overproducing plants exhibit several cytokinin‐regulated genes, as well as a model for cytokinin
characteristics that point to roles played by cytokinin in signaling based on current information.
plant physiology and development:
• The shoot apical meristems of cytokinin‐ A Cytokinin Receptor Related to Bacterial Two­
overproducing plants produce more leaves. Component Receptors Has Been Identified
• The leaves have higher chlorophyll levels and are
much greener. The first clue to the nature of the cytokinin receptor came
• Adventitious shoots may form from unwounded from the discovery of the CKI1 gene. CKI1 was identified in a
leaf veins and petioles. screen for genes that, when overexpressed, conferred
• Leaf senescence is retarded. cytokinin‐independent growth on Arabidopsis cells in
• Apical dominance is greatly reduced. culture.
• The more extreme cytokinin‐overproducing As discussed already, plant cells generally require cytokinin
plants are stunted, with greatly shortened in order to divide in culture. However, a cell line that
internodes. overexpresses CKI1 is capable of growing in culture in the
• Rooting of stem cuttings is reduced, as is the root absence of added cytokinin.
growth rate.
CKI1 encodes a protein similar in sequence to bacterial two‐
Some of the consequences of cytokinin overproduction could component sensor histidine kinases, which are ubiquitous
be highly beneficial for agriculture if synthesis of the receptors in prokaryotes. Bacterial two‐component
hormone can be controlled. Because leaf senescence is regulatory systems mediate a range of responses to
delayed in the cytokinin‐overproducing plants, it should be environmental stimuli, such as osmoregulation and
possible to extend their photosynthetic productivity. chemotaxis. Typically these systems are composed of two
functional elements: a sensor histidine kinase, to which a
In addition, cytokinin production could be linked to damage signal binds, and a downstream response regulator, whose
caused by predators. For example, tobacco plants activity is regulated via phosphorylation by the sensor
transformed with an ipt gene under the control of the histidine kinase. The sensor histidine kinase is usually a
promoter from a wound‐inducible protease inhibitor II gene membrane‐bound protein that contains two distinct
were more resistant to insect damage. The tobacco domains, called the input and histidine kinase, or
hornworm consumed up to 70% fewer tobacco leaves in “transmitter,” domains (Figure ).
plants that expressed the ipt gene driven by the protease
inhibit or promoter.

Detection of a signal by the input domain alters the activity aspartate residue in the receiver domain of a cognate
of the histidine kinase domain. Active sensor kinases are response regulator, and this phosphorylation alters the
dimers that transphosphorylate a conserved histidine activity of the kinases. Most response regulators also contain
residue. This phosphate is then transferred to a conserved output domains that act as transcription factors.

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The phenotype resulting from CKI1 overexpression, The rapid induction of the type‐A genes, coupled with their
combined with its similarity to bacterial receptors, similarity to signaling elements predicted to act downstream
suggested that the CKI1 and/or similar histidine kinases are of sensor histidine kinases, suggests that these elements act
cytokinin receptors. Support for this model came from downstream of the CRE1 cytokinin receptor family to
identification of the CRE1 gene. mediate the primary cytokinin response. Interestingly, one
of these type‐A genes, ARR5, is expressed primarily in the
Like CKI1, CRE1 encodes a protein similar to bacterial apical meristems of both shoots and roots, consistent with a
histidine kinases. Loss‐of‐function cre1 mutations were role in regulating cell proliferation, a key aspect of cytokinin
identified in a genetic screen for mutants that failed to action.
develop shoots from undifferentiated tissue culture cells in
response to cytokinin. This is essentially the opposite screen The expression of a wide variety of other genes is altered in
from the one just described, from which the CKI1 gene was response to cytokinin, but generally with slower kinetics
identified by a gain‐of‐function (ability to divide in the than the type‐A genes. These include the gene that encodes
absence of cytokinin) mutation. The cre1 mutants are also nitrate reductase, light‐regulated genes such as LHCB and
resistant to the inhibition of root elongation observed in SSU, and defense‐related genes such as PR1, as well as genes
response to cytokinin. that encode an extensin (cell wall protein rich in
hydroxyproline), rRNAs, cytochrome P450s, and peroxidase.
Convincing evidence that CRE1 encodes a cytokinin receptor Cytokinin elevates the expression of these genes both by
came from analysis of the expression of the protein in yeast. increasing the rate of transcription (as in the case of the
Yeast cells also contain a sensor histidine kinase, and type‐A ARRs) and/or by a stabilization of the RNA transcript
deletion of the gene that encodes this kinase— SLN1—is (e.g., the extensin gene).
lethal. Expression of CRE1 in SLN1‐deficient yeast can
restore viability, but only if cytokinins are present in the Histidine Phosphotransferases May Mediate the
medium. Thus the activity of CRE1 (i.e., its ability to replace Cytokinin Signaling Cascade
SLN1) is dependent on cytokinin, which, coupled with the
cytokinin‐insensitive phenotype of the cre1 mutants in From the preceding discussions we have seen that cytokinin
Arabidopsis, unequivocally demonstrates that CRE1 is a binds to the CRE1 receptors to initiate a response that
cytokinin receptor. It remains to be determined if CKI1 is culminates in the elevation of transcription of the type‐ A
also a cytokinin receptor. ARRs. The type‐A ARR proteins, in turn, may regulate the
expression of numerous other genes, as well as the activities
Two other genes in the Arabidopsis genome (AHK2 and of various target proteins that ultimately alter cellular
AHK3) are closely related to CRE1, suggesting that, like the function. How is the signal propagated from CRE1 (which is
ethylene receptors, the cytokinin receptors are encoded by a at the plasma membrane) to the nucleus to alter type‐A ARR
multigene family. Indeed, it has been demonstrated that transcription?
cytokinins bind to the predicted extracellular domains of
CRE1, AHK2, and AHK3 with high affinity, confirming that One set of genes that are likely to be involved in this
they are indeed cytokinin receptors. This raises the signaling cascade encode the AHP (Arabidopsis histidine
possibility that these genes are at least partially genetically phosphotransfer) proteins. In two‐component systems that
redundant (as are the ethylene receptors), which may involve a sensor kinase fused to a receiver domain (the
explain the relatively mild phenotypes that result from loss‐ structure of most eukaryotic sensor histidine kinases,
of‐function cre1 mutations. including those of the CRE1 family), there is an additional set
of phosphotransfers that are mediated by a histidine
Cytokinins Cause a Rapid Increase in the Expression of phosphotransfer protein (Hpt).
Response Regulator Genes
Phosphate is first transferred from ATP to a histidine within
One of the primary effects of cytokinin is to alter the the histidine kinase domain, and then transferred to an
expression of various genes. The first set of genes to be aspartate residue on the fused receiver. From the aspartate
upregulated in response to cytokinin are the ARR residue the phosphate group is then transferred to a
(Arabidopsis response regulator) genes. These genes are histidine on the Hpt protein and then finally to an aspartate
homologous to the receiver domain of bacterial two‐ on the receiver domain of the response regulator. This
component response regulators, the downstream target of phosphorylation of the receiver domain of the response
sensor histidine kinases. regulator alters its activity. Thus, Hpt proteins are predicted
to mediate the phosphotransfer between sensor kinases and
In Arabidopsis, response regulators are encoded by a response regulators.
multigene family. They fall into two basic classes: the type­
A ARR genes, which are made up solely of a receiver domain, In Arabidopsis there are 5 Hpt genes, called AHPs. The AHP
and the type­B ARR genes, which contain a transcription proteins have been shown to physically associate with
factor domain in addition to the receiver domain. The rate of receiver domains from several Arabidopsis histidine kinases,
transcription of the type‐A gene is increased within 10 including CRE1, and a subset of the AHPs have been
minutes in response to applied cytokinin. This rapid demonstrated to transiently translocate from the cytoplasm
induction is specific for cytokinin and does not require new to the nucleus in response to cytokinin. This finding suggests
protein synthesis. Both of these features are hallmarks of that the AHPs are the immediate downstream targets of the
primary response genes. activated CRE receptors, and that these proteins transduce
the cytokinin signal into the nucleus.

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This conclusion is supported by the findings that type‐ B

Cytokinin­Induced Phosphorylation Activates ARRs operate as transcriptional activators and that there are
Transcription Factors multiple binding sites for ARR1, a type‐B ARR, in the 5
DNAregulatory sequences of the type‐A ARR genes. A model
The question now becomes, How do the activated AHPs, of cytokinin signaling is presented in Figure. Cytokinin binds
once in the nucleus, act to regulate gene transcription? to the CRE1 receptor and initiates a phosphorylation
Genetic studies in intact Arabidopsis plants and cascade that results in the phosphorylation and activation of
overexpression studies in isolated Arabidopsis protoplasts a subset of the type‐B ARR proteins. Activation of the type‐B
using a cytokinin responsive reporter have provided a likely proteins (transcription factors) leads to the transcriptional
answer. activation of the type‐A genes. The type‐AARR proteins are
likely also phosphorylated in response to cytokinin, and
perhaps together with the type‐ B proteins, they interact
Disruption of ARR1, one of the type‐B ARR genes, reduces the with various targets to mediate the changes in cellular
induction of the type‐AARR genes in response to cytokinin. function, such as an activation of the cell cycle. Type‐A ARRs
Conversely, an increase in ARR1 function increases the are also able to inhibit their own expression by an unknown
response of the type‐A genes to cytokinin. This suggests that mechanism, providing a negative feedback loop (see Figure).
ARR1, which is a transcription factor, directly regulates Much work needs to be done to confirm and refine this
transcription of the type‐A ARRs, and that by analogy other model, but we are beginning to glimpse for the first time the
members of the type‐B ARR family also mediate cytokinin‐ molecular basis for cytokinin action in plants.
regulated gene expression.

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3. GIBBERLIC ACID
It became evident that an entire family of gibberellins exists
For nearly 30 years after the discovery of auxin in 1927, and and that in each fungal culture different gibberellins
more than 20 years after its structural elucidation as indole‐ predominate, though gibberellic acid is always a principal
3‐acetic acid, Western plant scientists tried to ascribe the component. As we will see, the structural feature that all
regulation of all developmental phenomena in plants to gibberellins have in common, and that defines them as a
auxin. However, plant growth and development are family of molecules, is that they are derived from the ent
regulated by several different types of hormones acting kaurene ring structure:
individually and in concert.

In the 1950s the second group of hormones, the gibberellins

(GAs), was characterized. The gibberellins are a large group
of related compounds (more than 125 are known) that,
unlike the auxins, are defined by their chemical structure
rather than by their biological activity. Gibberellins are most
often associated with the promotion of stem growth, and the
application of gibberellin to intact plants can induce large
increases in plant height. As we will see, however, As gibberellic acid became available, physiologists began
gibberellins play important roles in a variety of physiological testing it on a wide variety of plants. Spectacular responses
phenomena. were obtained in the elongation growth of dwarf and rosette
plants, particularly in genetically dwarf peas (Pisum
The biosynthesis of gibberellins is under strict genetic, sativum), dwarf maize (Zea mays), and many rosette plants.
developmental, and environmental control, and numerous In contrast, plants that were genetically very tall showed no
gibberellin‐deficient mutants have been isolated. Mendel’s further response to applied gibberellins. More recently,
tall/dwarf alleles in peas are a famous example. Such experiments with dwarf peas and dwarf corn have
mutants have been useful in elucidating the complex confirmed that the natural elongation growth of plants is
pathways of gibberellin biosynthesis. regulated by gibberellins, as we will describe later.

THE DISCOVERY OF THE GIBBERELLINS Because applications of gibberellins could increase the
height of dwarf plants, it was natural to ask whether plants
Although gibberellins did not become known to American contain their own gibberellins. Shortly after the discovery of
and British scientists until the 1950s, they had been the growth effects of gibberellic acid, gibberellin‐like
discovered much earlier by Japanese scientists. Rice farmers substances were isolated from several species of plants.
in Asia had long known of a disease that makes the rice Gibberellin­like substance refers to a compound or an extract
plants grow tall but eliminates seed production. In Japan this that has gibberellin‐like biological activity, but whose
disease was called the “foolish seedling,” or bakanae, chemical structure has not yet been defined. Such a response
disease. indicates, but does not prove, that the tested substance is a
gibberellin.
Plant pathologists investigating the disease found that the
tallness of these plants was induced by a chemical secreted In 1958 a gibberellin (gibberellin A1) was conclusively
by a fungus that had infected the tall plants. This chemical identified from a higher plant (runner bean seeds, Phaseolus
was isolated from filtrates of the cultured fungus and called coccineus):
gibberellin after Gibberella fujikuroi, the name of the fungus.

In the 1930s Japanese scientists succeeded in obtaining

impure crystals of two fungal growth‐active compounds,
which they termed gibberellin A and B, but because of
communication barriers and World War II, the information
did not reach the West. Not until the mid‐1950s did two
groups—one at the Imperial Chemical Industries (ICI)
research station at Welyn in Britain, the other at the U.S.
Department of Agriculture (USDA) in Peoria, Illinois—
succeed in elucidating the structure of the material that they Because the concentration of gibberellins in immature seeds
had purified from fungal culture filtrates, which they named far exceeds that in vegetative tissue, immature seeds were
gibberellic acid: the tissue of choice for gibberellin extraction. However,
because the concentration of gibberellins in plants is very
low (usually 1–10 parts per billion for the active gibberellin
in vegetative tissue and up to 1 part per million of total
gibberellins in seeds), chemists had to use truckloads of
seeds. As more and more gibberellins from fungal and plant
sources were characterized, they were numbered as
gibberellin AX (or GAX), where X is a number, in the order of
their discovery. This scheme was adopted for all gibberellins
in 1968. However, the number of a gibberellin is simply a
cataloging convenience, designed to prevent chaos in the
At about the same time scientists at Tokyo University
naming of the gibberellins. The system implies no close
isolated three gibberellins from the original gibberellin A
chemical similarity or metabolic relationship between
and named them gibberellin A1, gibberellin A2, and
gibberellins with adjacent numbers.
gibberellin A3. Gibberellin A3 and gibberellic acid proved to
be identical.

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All gibberellins are based on the ent‐gibberellane skeleton: near the base of the internode that produces derivatives
above and below. Deep water rice is a particularly striking
example.

Although stem growth may be dramatically enhanced by

GAs, gibberellins have little direct effect on root growth.
However, the root growth of extreme dwarfs is less than that
of wild‐type plants, and gibberellin application to the shoot
enhances both shoot and root growth. Whether the effect of
gibberellin on root growth is direct or indirect is currently
unresolved.
Some gibberellins have the full complement of 20 carbons
(C20‐GAs): Gibberellins Regulate the Transition from Juvenile to
Adult Phases

Many woody perennials do not flower until they reach a

certain stage of maturity; up to that stage they are said to be
juvenile. The juvenile and mature stages often have different
leaf forms, as in English ivy (Hedera helix). Applied
gibberellins can regulate this juvenility in both directions,
depending on the species. Thus, in English ivy GA3 can cause
a reversion from a mature to a juvenile state, and many
juvenile conifers can be induced to enter the reproductive
Others have only 19 (C19‐GAs), having lost one carbon to phase by applications of non polar gibberellins such as GA4
metabolism. There are other variations in the basic + GA7. (The latter example is one instance in which GA3 is
structure, especially the oxidation state of carbon 20 (in not effective.)
C20‐GAs) and the number and position of hydroxyl groups
on the molecule. Despite the plethora of gibberellins present Gibberellins Influence Floral Initiation and Sex
in plants, genetic analyses have demonstrated that only a Determination
few are biologically active as hormones. All the others serve
as precursors or represent inactivated forms. As already noted, gibberellin can substitute for the long day
or cold requirement for flowering in many plants, especially
EFFECTS OF GIBBERELLIN ON GROWTH AND rosette species. Gibberellin is thus a component of the
DEVELOPMENT flowering stimulus in some plants, but apparently not in
others.
Though they were originally discovered as the cause of a
disease of rice that stimulated internode elongation, In plants where flowers are unisexual rather than
endogenous gibberellins influence a wide variety of hermaphroditic, floral sex determination is genetically
developmental processes. In addition to stem elongation, regulated. However, it is also influenced by environmental
gibberellins control various aspects of seed germination, factors, such as photoperiod and nutritional status, and
including the loss of dormancy and the mobilization of these environmental effects may be mediated by gibberellin.
endosperm reserves. In reproductive development, In maize, for example, the staminate flowers (male) are
gibberellin can affect the transition from the juvenile to the restricted to the tassel, and the pistillate flowers (female)
mature stage, as well as floral initiation, sex determination, are contained in the ear. Exposure to short days and cool
and fruit set. In this section we will review some of these nights increases the endogenous gibberellin levels in the
gibberellin‐regulated phenomena. tassels 100‐fold and simultaneously causes feminization of
the tassel flowers. Application of exogenous gibberellic acid
Gibberellins Stimulate Stem Growth in Dwarf and to the tassels can also induce pistillate flowers. For studies
Rosette Plants on genetic regulation, a large collection of maize mutants
that have altered patterns of sex determination have been
Applied gibberellin promotes internodal elongation in a isolated. Mutations in genes that affect either gibberellin
wide range of species. However, the most dramatic biosynthesis or gibberellin signal transduction result in a
stimulations are seen in dwarf and rosette species, as well as failure to suppress stamen development in the flowers of the
members of the grass family. Exogenous GA3 causes such ear. Thus the primary role of gibberellin in sex
extreme stem elongation in dwarf plants that they resemble determination in maize seems to be to suppress stamen
the tallest varieties of the same species. development.

Accompanying this effect are a decrease in stem thickness, a In dicots such as cucumber, hemp, and spinach, gibberellin
decrease in leaf size, and a pale green color of the leaves. seems to have the opposite effect. In these species,
Some plants assume a rosette form in short days and application of gibberellin promotes the formation of
undergo shoot elongation and flowering only in long days. staminate flowers, and inhibitors of gibberellin biosynthesis
Gibberellin application results in bolting (stem growth) in promote the formation of pistillate flowers.
plants kept in short days, and normal bolting is regulated by
endogenous gibberellin. In addition, as noted earlier, many Gibberellins Promote Fruit Set
long‐day rosette plants have a cold requirement for stem
elongation and flowering, and this requirement is overcome Applications of gibberellins can cause fruit set (the initiation
by applied gibberellin. GA also promotes internodal of fruit growth following pollination) and growth of some
elongation in members of the grass family. The target of fruits, in cases where auxin may have no effect. For example,
gibberellin action is the intercalary meristem—a meristem stimulation of fruit set by gibberellin has been observed in
apple (Malus sylvestris).

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Gibberellins Promote Seed Germination crop with gibberellin can increase the yield of raw cane by
up to 20 tons per acre, and the sugar yield by 2 tons per acre.
Seed germination may require gibberellins for one of several This increase is a result of the stimulation of internode
possible steps: the activation of vegetative growth of the elongation during the winter season.
embryo, the weakening of a growth‐constraining endosperm
layer surrounding the embryo, and the mobilization of Uses in plant breeding. The long juvenility period in
stored food reserves of the endosperm. Some seeds, conifers can be detrimental to a breeding program by
particularly those of wild plants, require light or cold to preventing the reproduction of desirable trees for many
induce germination. In such seeds this dormancy can often years. Spraying with GA4 + GA7 can considerably reduce the
be overcome by application of gibberellin. time to seed production by inducing cones to form on very
young trees. In addition, the promotion of male flowers in
Since changes in gibberellin levels are often, but not always, cucurbits, and the stimulation of bolting in biennial rosette
seen in response to chilling of seeds, gibberellins may crops such as beet (Beta vulgaris) and cabbage (Brassica
represent a natural regulator of one or more of the oleracea), are beneficial effects of gibberellins that are
processes involved in germination. Gibberellin application occasionally used commercially in seed production.
also stimulates the production of numerous hydrolases,
notably α‐amylase, by the aleurone layers of germinating Gibberellin biosynthesis inhibitors. Bigger is not always
cereal grains. This aspect of gibberellin action has led to its better. Thus, gibberellin biosynthesis inhibitors are used
use in the brewing industry in the production of malt. commercially to prevent elongation growth in some plants.
In floral crops, short, stocky plants such as lilies,
Gibberellins Have Commercial Applications chrysanthemums, and poinsettias are desirable, and
restrictions on elongation growth can be achieved by
The major uses of gibberellins (GA3, unless noted applications of gibberellin synthesis inhibitors such as
otherwise), applied as a spray or dip, are to manage fruit ancymidol (known commercially as A‐Rest) or paclobutrazol
crops, to malt barley, and to increase sugar yield in (known as Bonzi). Tallness is also a disadvantage for cereal
sugarcane. In some crops a reduction in height is desirable, crops grown in cool, damp climates, as occur in Europe,
and this can be accomplished by the use of gibberellin where lodging can be a problem. Lodging—the bending of
synthesis inhibitors. stems to the ground caused by the weight of water collecting
on the ripened heads—makes it difficult to harvest the grain
Fruit production. A major use of gibberellins is to increase with a combine harvester. Shorter internodes reduce the
the stalk length of seedless grapes. Because of the shortness tendency of the plants to lodge, increasing the yield of the
of the individual fruit stalks, bunches of seedless grapes are crop. Even genetically dwarf wheats grown in Europe are
too compact and the growth of the berries is restricted. sprayed with gibberellin biosynthesis inhibitors to further
Gibberellin stimulates the stalks to grow longer, thereby reduce stem length and lodging. Yet another application of
allowing the grapes to grow larger by alleviating gibberellin biosynthesis inhibitors is the restriction of
compaction, and it promotes elongation of the fruit. growth in roadside shrub plantings.

A mixture of benzyladenine (a cytokinin) and GA4 + GA7 can BIOSYNTHESIS AND METABOLISM OF GIBBERELLIN
cause apple fruit to elongate and is used to improve the
shape of Delicious‐type apples under certain conditions. Gibberellins constitute a large family of diterpene acids and
Although this treatment does not affect yield or taste, it is are synthesized by a branch of the terpenoid pathway. The
considered commercially desirable. In citrus fruits, elucidation of the gibberellin biosynthetic pathway would
gibberellins delay senescence, allowing the fruits to be left not have been possible without the development of sensitive
on the tree longer to extend the market period. methods of detection. As noted earlier, plants contain a
bewildering array of gibberellins, many of which are
Malting of barley. Malting is the first step in the brewing biologically inactive. In this section we will discuss the
process. During malting, barley seeds (Hordeum vulgare) are biosynthesis of GAs, as well as other factors that regulate the
allowed to germinate at temperatures that maximize the steady‐state levels of the biologically active form of the
production of hydrolytic enzymes by the aleurone layer. hormone in different plant tissues.
Gibberellin is sometimes used to speed up the malting
process. The germinated seeds are then dried and Gibberellins Are Measured via Highly Sensitive Physical
pulverized to produce “malt,” consisting mainly of a mixture Techniques
of amylolytic (starch‐degrading) enzymes and partly
digested starch. Systems of measurement using a biological response, called
bioassays, were originally important for detecting
During the subsequent “mashing” step, water is added and gibberellin‐ like activity in partly purified extracts and for
the amylases in the malt convert the residual starch, as well assessing the biological activity of known gibberellins. The
as added starch, to the disaccharide maltose, which is use of bioassays, however, has declined with the
converted to glucose by the enzyme maltase. The resulting development of highly sensitive physical techniques that
“wort” is then boiled to stop the reaction. In the final step, allow precise identification and quantification of specific
yeast converts the glucose in the wort to ethanol by gibberellins from small amounts of tissue.
fermentation.
High‐performance liquid chromatography (HPLC) of plant
Increasing sugarcane yields. Sugarcane (Saccharum extracts, followed by the highly sensitive and selective
officinarum) is one of relatively few plants that store their analytical method of gas chromatography combined with
carbohydrate as sugar (sucrose) instead of starch (the other mass spectrometry (GC‐MS), has now become the method of
important sugar‐storing crop is sugar beet). Originally from choice. With the availability of published mass spectra,
New Guinea, sugarcane is a giant perennial grass that can researchers can now identify gibberellins without
grow from 4 to 6 m tall. The sucrose is stored in the central possessing pure standards. The availability of heavy‐
vacuoles of the internode parenchyma cells. Spraying the isotope‐ labeled standards of common gibberellins, which

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can themselves be separately detected on a mass

spectrometer, allows the accurate measurement of levels in
plant tissues by mass spectrometry with these heavy‐
isotope‐labeled gibberellins as internal standards for
quantification.
joined head to tail. Researchers have determined the entire
Gibberellins Are Synthesized via the Terpenoid Pathway gibberellin biosynthetic pathway in seed and vegetative
in Three Stages tissues of several species by feeding various radioactive
precursors and intermediates and examining the production
Gibberellins are tetracyclic diterpenoids made up of four of the other compounds of the pathway. The gibberellin
isoprenoid units. Terpenoids are compounds made up of biosynthetic pathway can be divided into three stages, each
five‐carbon (isoprene) building blocks: residing in a different cellular compartment (Figure).

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Stage 1: Production of terpenoid precursors and ent­ 2. A successive oxidation at carbon 20 (CH2 →CH2OH
kaurene in plastids. The basic biological isoprene unit is →CHO). The final step of this oxidation is the loss of carbon
isopentenyl diphosphate (IPP).2 IPP used in gibberellin 20 as CO2.
biosynthesis in green tissues is synthesized in plastids from
glyceraldehyde‐3‐phosphate and pyruvate. However, in the When these reactions involve gibberellins initially
endosperm of pumpkin seeds, which are very rich in hydroxylated at C‐13, the resulting gibberellin is GA20. GA20
gibberellin, IPP is formed in the cytosol from mevalonic acid, is then converted to the biologically active form, GA1, by
which is itself derived from acetyl‐CoA. hydroxylation of carbon 3. (Because this is in the beta
configuration [drawn as if the bond to the hydroxyl group
Thus the IPP used to make gibberellins may arise from were toward the viewer], it is referred to as 3β‐
different cellular compartments in different tissues. Once hydroxylation.)
synthesized, the IPP isoprene units are added successively to
produce intermediates of 10 carbons (geranyl diphosphate), Finally, GA1 is inactivated by its conversion to GA8 by a
15 carbons (farnesyl diphosphate), and 20 carbons hydroxylation on carbon 2. This hydroxylation can also
(geranylgeranyl diphosphate, GGPP). GGPP is a precursor of remove GA20 from the biosynthetic pathway by converting
many terpenoid compounds, including carotenoids and it to GA29.
many essential oils, and it is only after GGPP that the
pathway becomes specific for gibberellins. Inhibitors of the third stage of the gibberellin biosynthetic
pathway interfere with enzymes that utilize 2‐oxoglutarate
The cyclization reactions that convert GGPP to ent‐kaurene as cosubstrates. Among these, the compound prohexadione
represent the first step that is specific for the gibberellins. (BX‐112), is especially useful because it specifically inhibits
The two enzymes that catalyze the reactions are localized in GA 3‐oxidase, the enzyme that converts inactive GA20 to
the proplastids of meristematic shoot tissues, and they are growth‐active GA1.
not present in mature chloroplasts. Thus, leaves lose their
ability to synthesize gibberellins from IPP once their
chloroplasts mature.

Compounds such as AMO‐1618, Cycocel, and Phosphon D

are specific inhibitors of the first stage of gibberellin
biosynthesis, and they are used as growth height reducers.

Stage 2: Oxidation reactions on the ER form GA12 and

GA53. In the second stage of gibberellin biosynthesis, a
methyl group on ent‐kaurene is oxidized to a carboxylic acid,
followed by contraction of the B ring from a six‐ to a five‐
carbon ring to give GA12‐aldehyde. GA12‐aldehyde is then
oxidized to GA12, the first gibberellin in the pathway in all
plants and thus the precursor of all the other gibberellins.

Many gibberellins in plants are also hydroxylated on carbon

13. The hydroxylation of carbon 13 occurs next, forming
GA53 from GA12. All the enzymes involved are
monooxygenases that utilize cytochrome P450 in their
reactions.

These P450 monooxygenases are localized on the

endoplasmic reticulum. Kaurene is transported from the
plastid to the endoplasmic reticulum, and is oxidized en
route to kaurenoic acid by kaurene oxidase, which is
associated with the plastid envelope.

Further conversions to GA12 take place on the endoplasmic

reticulum. Paclobutrazol and other inhibitors of P450
monooxygenases specifically inhibit this stage of gibberellin
biosynthesis before GA12‐aldehyde, and they are also
growth retardants.

Stage 3: Formation in the cytosol of all other gibberellins

from GA12 or GA53. All subsequent steps in the pathway The Enzymes and Genes of the Gibberellin Biosynthetic
are carried out by a group of soluble dioxygenases in the Pathway Have Been Characterized
cytosol. These enzymes require 2‐ oxoglutarate and
molecular oxygen as cosubstrates, and they use Fe2+ and The enzymes of the gibberellin biosynthetic pathway are
ascorbate as cofactors. The specific steps in the modification now known, and the genes for many of these enzymes have
of GA12 vary from species to species, and between organs of been isolated and characterized. Most notable from a
the same species. regulatory standpoint are two biosynthetic enzymes—GA
20‐oxidase (GA20ox)3 and GA 3‐oxidase (GA3ox)—and an
Two basic chemical changes occur in most plants: enzyme involved in gibberellin metabolism, GA 2‐oxidase
1. Hydroxylation at carbon 13 (on the endoplasmic (GA2ox):
reticulum) or carbon 3, or both.

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• GA 20­oxidase catalyzes all the reactions involving the conclusively that the Le gene encodes an enzyme that 3β‐
successive oxidation steps of carbon 20 between GA53 and hydroxylates GA20 to produce GA1 (Figure).
GA20, including the removal of C‐20 as CO2.
• GA 3­oxidase functions as a β‐hydroxylase, adding a Mendel’s Le(T) gene was isolated, and the recessive le (t)
hydroxyl group to C‐3 to form the active gibberellin, GA1. allele was shown to have a single base change leading to a
(The evidence demonstrating that GA1 is the active defective enzyme only one‐twentieth as active as the wild‐
gibberellin will be discussed shortly.) type enzyme, so much less GA1 is produced and the plants
• GA 2­oxidase inactivates GA1 by catalyzing the addition of are dwarf.
a hydroxyl group to C‐2.
Endogenous GA1 Levels Are Correlated with Tallness
The transcription of the genes for the two gibberellin
biosynthetic enzymes, as well as for GA 2‐oxidase, is highly Although the shoots of gibberellin‐deficient le dwarf peas
regulated. All three of these genes have sequences in are much shorter than those of normal plants (internodes of
common with each other and with other enzymes utilizing 2‐ 3 cm in mature dwarf plants versus 15 cm in mature normal
oxoglutarate and Fe2+ as cofactors. The common sequences plants), the mutation is “leaky” (i.e., the mutated gene
represent the binding sites for 2‐oxoglutarate and Fe2+. produces a partially active enzyme) and some endogenous
GA1 remains to cause growth. Different le alleles give rise to
Gibberellins May Be Covalently Linked to Sugars peas differing in their height, and the height of the plant has
been correlated with the amount of endogenous GA1.
Although active gibberellins are free, a variety of gibberellin
glycosides are formed by a covalent linkage between There is also an extreme dwarf mutant of pea that has even
gibberellin and a sugar. These gibberellin conjugates are fewer gibberellins. This dwarf has the allele na (the wild‐
particularly prevalent in some seeds. The conjugating sugar type allele is Na), which completely blocks gibberellin
is usually glucose, and it may be attached to the gibberellin biosynthesis between ent‐kaurene and GA12‐aldehyde. As a
via a carboxyl group forming a gibberellin glycoside, or via a result, homozygous (nana) mutants, which are almost
hydroxyl group forming a gibberellin glycosyl ether. completely free of gibberellins, achieve a stature of only
about 1 cm at maturity. However, nana plants may still
When gibberellins are applied to a plant, a certain possess an active GA 3β‐ hydroxylase encoded by Le, and
proportion usually becomes glycosylated. Glycosylation thus can convert GA20 to GA1. If a nana naLe shoot is grafted
therefore represents another form of inactivation. In some onto a dwarf le plant, the resulting plant is tall because the
cases, applied glucosides are metabolized back to free GAs, nana shoot tip can convert the GA20 from the dwarf into
so glucosides may also be a storage form of gibberellins. GA1.

GA1 Is the Biologically Active Gibberellin Controlling Such observations have led to the conclusion that GA1 is the
Stem Growth biologically active gibberellin that regulates tallness in peas.
The same result has been obtained for maize, a monocot, in
Knowledge of biosynthetic pathways for gibberellins reveals parallel studies using genotypes that have blocks in the
where and how dwarf mutations act. Although it had long gibberellin biosynthetic pathway. Thus the control of stem
been assumed that gibberellins were natural growth elongation by GA1 appears to be universal.
regulators because gibberellin application caused dwarf
plants to grow tall, direct evidence was initially lacking. In Although GA1 appears to be the primary active gibberellin in
the early 1980s it was demonstrated that tall stems do stem growth for most species, a few other gibberellins have
contain more bioactive gibberellin than dwarf stems have, biological activity in other species or tissues. For example,
and that the level of the endogenous bioactive gibberellin GA3, which differs from GA1 only in having one double bond,
mediates the genetic control of tallness. is relatively rare in higher plants but is able to substitute for
GA1 in most bioassays:

The gibberellins of tall pea plants containing the

homozygous Le allele (wild type) were compared with dwarf
plants having the same genetic makeup, except containing
the le allele (mutant). Le and le are the two alleles of the GA4, which lacks an OH group at C‐13, is present in both
gene that regulates tallness in peas, the genetic trait first Arabidopsis and members of the squash family
investigated by Gregor Mendel in his pioneering study in (Cucurbitaceae). It is as active as GA1, or even more active,
1866. We now know that tall peas contain much more in some bioassays, indicating that GA4 is a bioactive
bioactive GA1 than dwarf peas have (Ingram et al. 1983). As gibberellin in the species where it occurs. The structure of
we have seen, the precursor of GA1 in higher plants is GA20 GA4 looks like this:
(GA1 is 3β‐OH GA20). If GA20 is applied to homozygous
dwarf (le) pea plants, they fail to respond, although they do
respond to applied GA1. The implication is that the Le gene
enables the plants to convert GA20 to GA1. Metabolic studies
using both stable and radioactive isotopes demonstrated

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can alter the levels of active gibberellins by affecting gene

transcription for specific steps in the biosynthetic pathway.

Light regulation of GA1 biosynthesis. The presence of light

has many profound effects. Some seeds germinate only in
the light, and in such cases gibberellin application can
stimulate germination in darkness. The promotion of
germination by light has been shown to be due to increases
in GA1 levels resulting from a light‐induced increase in the
transcription of the gene for GA3ox, which converts GA20 to
Gibberellins Are Biosynthesized in Apical Tissues
GA1. This effect shows red/far‐red photoreversibility and is
mediated by phytochrome.
The highest levels of gibberellins are found in immature
seeds and developing fruits. However, because the
When a seedling becomes exposed to light as it emerges
gibberellin level normally decreases to zero in mature seeds,
from the soil, it changes its form—a process referred to as
there is no evidence that seedlings obtain any active
de‐etiolation. One of the most striking changes is a decrease
gibberellins from their seeds.
in the rate of stem elongation such that the stem in the light
is shorter than the one in the dark. Initially it was assumed
Work with pea seedlings indicates that the gibberellin
that the light‐grown plants would contain less GA1 than
biosynthetic enzymes and GA3ox are specifically localized in
dark‐grown plants. However, light‐grown plants turned out
young, actively growing buds, leaves, and upper internodes.
to contain more GA1 than dark‐grown plants—indicating
In Arabidopsis, GA20ox is expressed primarily in the apical
that de‐etiolation is a complex process involving changes in
bud and young leaves, which thus appear to be the principal
the level of GA1, as well as changes in the responsiveness of
sites of gibberellin synthesis.
the plant to GA1. In peas, for example, the level of GA1
initially falls within 4 hours of exposure to light because of
The gibberellins that are synthesized in the shoot can be
an increase in transcription of the gene for GA2ox, leading to
transported to the rest of the plant via the phloem.
an increase in GA1 breakdown. The level of GA1 remains low
Intermediates of gibberellin biosynthesis may also be
for a day but then increases, so that by 5 days there is a
translocated in the phloem. Indeed, the initial steps of
fivefold increase in the GA1 content of the stems, even
gibberellin biosynthesis may occur in one tissue, and
though the stem elongation rate is lower. The reason that
metabolism to active gibberellins in another.
growth slows down despite the increase in GA1 level is that
the plants are now severalfold less sensitive to the GA1
Gibberellins also have been identified in root exudates and
present. As will be discussed later in the chapter, sensitivity
root extracts, suggesting that roots can also synthesize
to active gibberellin is governed by components of the
gibberellins and transport them to the shoot via the xylem.
gibberellin signal transduction pathway.
Gibberellin Regulates Its Own Metabolism
Photoperiod regulation of GA1 biosynthesis. When plants
that require long days to flower are shifted from short days
Endogenous gibberellin regulates its own metabolism by
to long days, gibberellin metabolism is altered. In spinach
either switching on or inhibiting the transcription of the
(Spinacia oleracea), in short days, when the plants maintain
genes that encode enzymes of gibberellin biosynthesis and
a rosette form, the level of gibberellins hydroxylated at
degradation (feedback and feed‐forward regulation,
carbon 13 is relatively low. In response to increasing day
respectively). In this way the level of active gibberellins is
length, the shoots of spinach plants begin to elongate after
kept within a narrow range, provided that precursors are
about 14 long days. The levels of all the gibberellins of the
available and the enzymes of gibberellin biosynthesis and
carbon 13–hydroxylated gibberellin pathway (GA53
degradation are functional.
→ GA44 → GA19→GA20→GA1→GA8) start to increase
after about 4 days. Although the level of GA20 increases 16‐
For example, the application of gibberellin causes a down‐
fold during the first 12 days, it is the fivefold increase in GA1
regulation of the biosynthetic genes—GA20ox and GA3ox—
that induces stem growth.
and an elevation in transcription of the degradative gene—
GA2ox. A mutation in the GA 2‐oxidase gene, which prevents
The dependence of stem growth on GA1 has been shown
GA1 from being degraded, is functionally equivalent to
through the use of different inhibitors of gibberellin
applying exogenous gibberellin to the plant, and produces
synthesis and metabolism. The inhibitors AMO‐1618 and
the same effect on the biosynthetic gene transcription.
BX‐112 both prevent internode elongation (bolting). The
Conversely, a mutation that lowers the level of active
effect of AMO‐1618, which blocks gibberellin biosynthesis
gibberellin, such as GA1, in the plant stimulates the
prior to GA12‐aldehyde, can be overcome by applications of
transcription of the biosynthetic genes—GA20ox and
GA20. However, the effect of another inhibitor, BX‐112,
GA3ox— and down‐regulates the degradative enzyme—
which blocks the production of GA1 from GA20, can be
GA2ox. In peas this is particularly evident in very dwarf
overcome only by GA1. This result demonstrates that the
plants, such as those with a mutation in the LS gene (CPP
rise in GA1 is the crucial factor in regulating spinach stem
synthase) or even more severely dwarf na plants (defective
growth.
GA12‐aldehyde synthase).
The level of GA20‐oxidase mRNA in spinach tissues, which
Environmental Conditions Can Alter the Transcription
occurs in the highest amount in shoot tips and elongating
of Gibberellin Biosynthesis Genes
stems, is increased under long‐day conditions. The fact that
GA 20‐ oxidase is the enzyme that converts GA53 to GA20
Gibberellins play an important role in mediating the effects
explains why the concentration of GA20 was found to be
of environmental stimuli on plant development.
higher in spinach under long‐day conditions.
Environmental factors such as photoperiod and temperature

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Photoperiod control of tuber formation. Potato

tuberization is another process regulated by photoperiod.
Tubers form on wild potatoes only in short days (although
the requirement for short days has been bred out of many
cultivated varieties), and this tuberization can be blocked by
applications of gibberellin. The transcription of GA20ox was
found to fluctuate during the light–dark cycle, leading to
lower levels of GA1 in short days. Potato plants
overexpressing the GA20ox gene showed delayed
tuberization, whereas trans‐formation with the antisense
gene for GA20ox promoted tuberization, demonstrating the
importance of the transcription of this gene in the regulation
of potato tuberization.

In general, de‐etiolation, light‐dependent seed germination,

and the photoperiodic control of stem growth in rosette
plants and tuberization in potato are all mediated by
phytochromes. There is mounting evidence that many
phytochrome effects are in part due to modulation of the
levels of gibberellins through changes in the transcription of
the genes for gibberellin biosynthesis and degradation.
Figure summarizes some of the factors that modulate the
Temperature effects. Cold temperatures are required for active gibberellin level through regulation of the
the germination of certain seeds (stratification) and for transcription of the genes for gibberellin biosynthesis or
flowering in certain species (vernalization). For example, a metabolism.
prolonged cold treatment is required for both the stem
elongation and the flowering of Thlaspi arvense (field Dwarfness Can Now Be Genetically Engineered
pennycress), and gibberellins can substitute for the cold
treatment. The characterization of the gibberellin biosynthesis and
metabolism genes—GA20ox, GA3ox, and GA2ox—has
In the absence of the cold treatment, ent‐kaurenoic acid enabled genetic engineers to modify the transcription of
accumulates to high levels in the shoot tip, which is also the these genes to alter the gibberellin level in plants, and thus
site of perception of the cold stimulus. After cold treatment affect their height. The desired effect is usually to increase
and a return to high temperatures, the ent‐kaurenoic acid is dwarfness because plants grown in dense crop communities,
converted to GA9, the most active gibberellin for stimulating such as cereals, often grow too tall and thus are prone to
the flowering response. These results are consistent with a lodging. In addition, because gibberellin regulates bolting,
cold‐induced increase in the activity of ent‐kaurenoic acid one can prevent bolting by inhibiting the rise in gibberellin.
hydroxylase in the shoot tip. An example of the latter is the inhibition of bolting in sugar
beet.
Auxin Promotes Gibberellin Biosynthesis
Sugar beet is a biennial, forming a swollen storage root in
Although we often discuss the action of hormones as if they the first season and a flower and seed stalk in the second. To
act singly, the net growth and development of the plant are extend the growing season and obtain bigger beets, farmers
the results of many combined signals. In addition, hormones sow the beets as early as possible in the spring, but sowing
can influence each other’s biosynthesis so that the effects too early leads to bolting in the first year, with the result that
produced by one hormone may in fact be mediated by no storage roots form. Areduction in the capacity to make
others. gibberellin inhibits bolting, allowing earlier sowing of the
seeds and thus the growth of larger beets. Reductions in GA1
For example, it has long been known that auxin induces levels have recently been achieved in such crops as sugar
ethylene biosynthesis. It is now evident that gibberellin can beet and wheat, either by the transformation of plants with
induce auxin biosynthesis and that auxin can induce antisense constructs of the GA20ox or GA3ox genes, which
gibberellin biosynthesis. If pea plants are decapitated, encode the enzymes leading to the synthesis of GA1, or by
leading to a cessation in stem elongation, not only is the level overexpressing the gene responsible for GA1 metabolism:
of auxin lowered because its source has been removed, but GA2ox. Either approach results in dwarfing in wheat or an
the level of GA1 in the upper stem drops sharply. This inhibition of bolting in rosette plants such as beet.
change can be shown to be an auxin effect because replacing
the bud with a supply of auxin restores the GA1 level. The inhibition of seed production in such transgenic plants
can be overcome by sprays of gibberellin solution, provided
The presence of auxin has been shown to promote the that the reduction in gibberellin has been achieved by
transcription of GA3ox and to repress the transcription of blocking the genes for GA20ox or GA3ox, the gibberellin
GA2ox. In the absence of auxin the reverse occurs. Thus the biosynthetic enzymes. A similar strategy has recently been
apical bud promotes growth not only through the direct applied to turf grass, keeping the grass short with no
biosynthesis of auxin, but also through the auxin‐induced seedheads, so that mowing can be virtually eliminated—a
biosynthesis of GA1. boon for homeowners!

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PHYSIOLOGICAL MECHANISMS OF GIBBERELLIN­ Gibberellin has no effect on the osmotic parameters but has
INDUCED GROWTH consistently been observed to cause an increase in both the
mechanical extensibility of cell walls and the stress
As we have seen, the growth‐promoting effects of gibberellin relaxation of the walls of living cells. An analysis of pea
are most evident in dwarf and rosette plants. When dwarf genotypes differing in gibberellin content or sensitivity
plants are treated with gibberellin, they resemble the tallest showed that gibberellin decreases the minimum force that
varieties of the same species. Other examples of gibberellin will cause wall extension (the wall yield threshold). Thus,
action include the elongation of hypocotyls and of grass both gibberellin and auxin seem to exert their effects by
internodes. modifying cell wall properties.

A particularly striking example of internode elongation is In the case of auxin, cell wall loosening appears to be
found in deep‐water rice (Oryza sativa). In general, rice mediated in part by cell wall acidification. However, this
plants are adapted to conditions of partial submergence. To does not appear to be the mechanism of gibberellin action.
enable the upper foliage of the plant to stay above water, the In no case has a gibberellin‐stimulated increase in proton
internodes elongate as the water level rises. Deep‐water rice extrusion been demonstrated. On the other hand, gibberellin
has the greatest potential for rapid internode elongation. is never present in tissues in the complete absence of auxin,
Under field conditions, growth rates of up to 25 cm per day and the effects of gibberellin on growth may depend on
have been measured. auxin‐induced wall acidification. The typical lag time before
gibberellin‐stimulated growth begins is longer than for
The initial signal is the reduced partial pressure of O2 auxin; as noted already, in deepwater rice it is about 40
resulting from submergence, which induces ethylene minutes, and in peas it is 2 to 3 hours. These longer lag times
biosynthesis. The ethylene trapped in the submerged point to a growth‐promoting mechanism distinct from that
tissues, in turn, reduces the level of abscisic acid, which acts of auxin. Consistent with the existence of a separate
as an antagonist of gibberellin. The end result is that the gibberellin‐
tissue becomes more responsive to its endogenous specific wall‐loosening mechanism, the growth responses to
gibberellin. Because inhibitors of gibberellin biosynthesis applied gibberellin and auxin are additive. Various
block the stimulatory effect of both submergence and suggestions have been made regarding the mechanism of
ethylene on growth, and exogenous gibberellin can stimulate gibberellin‐stimulated stem elongation, and all have some
growth in the absence of submergence, gibberellin appears experimental support, but as yet none provide a clear‐cut
to be the hormone directly responsible for growth answer. For example, there is evidence that the enzyme
stimulation. xyloglucan endotransglycosylase (XET) is involved in
gibberellin‐promoted wall extension. The function of XET
GA‐stimulated growth in deep‐water rice can be studied in may be to facilitate the penetration of expansins into the cell
an excised stem system. The addition of gibberellin causes a wall. (Recall that expansins are cell wall proteins that cause
marked increase in the growth rate after a lag period of wall loosening in acidic conditions by weakening hydrogen
about 40 minutes. Cell elongation accounts for about 90% of bonds between wall polysaccharides) Both expansins and
the length increase during the first 2 hours of gibberellin XET may be required for gibberellin‐stimulated cell
treatment. elongation.

Gibberellins Stimulate Cell Elongation and Cell Division Gibberellins Regulate the Transcription of Cell Cycle
Kinases in Intercalary Meristems
The effect of gibberellins applied to intact dwarf plants is so
dramatic that it would seem to be a simple task to determine As noted earlier, the growth rate of the internodes of
how they act. Unfortunately, this is not the case because, as deepwater rice dramatically increases in response to
we have seen with auxin, so much about plant cell growth is submergence, and part of this response is due to increased
not understood. However, we do know some characteristics cell divisions in the intercalary meristem. To study the effect
of gibberellin‐induced stem elongation. Gibberellin increases of gibberellin on the cell cycle, researchers isolated nuclei
both cell elongation and cell division, as evidenced by from the intercalary meristem and quantified the amount of
increases in cell length and cell number in response to DNA per nucleus.
applications of gibberellin. For example, internodes of tall
peas have more cells than those of dwarf peas, and the cells In submergence‐induced plants, gibberellin activates the cell
are longer. Mitosis increases markedly in the subapical division cycle first at the transition from G1 to S phase,
region of the meristem of rosette long‐day plants after leading to an increase in mitotic activity. To do this,
treatment with gibberellin. The dramatic stimulation of gibberellin induces the expression of the genes for several
internode elongation in deep‐water rice is due in part to cyclin­dependent protein kinases (CDKs), which are
increased cell division activity in the intercalary meristem. involved in regulation of the cell cycle. The transcription of
Moreover, only the cells of the intercalary meristem whose these genes—first those regulating the transition from G1 to
division is increased by gibberellin exhibit gibberellin‐ S phase, followed by those regulating the transition from G2
stimulated cell elongation. to M phase—is induced in the intercalary meristem by
gibberellin. The result is a gibberellin induced increase in
Because gibberellin‐induced cell elongation appears to the progression from the G1 to the S phase through to
precede gibberellin‐induced cell division, we begin our mitosis and cell division.
discussion with the role of gibberellin in regulating cell
elongation. Gibberellin Response Mutants Have Defects in Signal
Transduction
Gibberellins Enhance Cell Wall Extensibility without
Acidification Single‐gene mutants impaired in their response to
gibberellin provide valuable tools for identifying genes that
The elongation rate can be influenced by both cell wall encode possible gibberellin receptors or components of
extensibility and the osmotically driven rate of water uptake. signal transduction pathways. In screenings for such

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mutants, three main classes of mutations affecting plant

height have been selected: Different Genetic Screens Have Identified the Related
1. Gibberellin‐insensitive dwarfs Repressors GAI and RGA
2. Gibberellin‐deficient mutants in which the gibberellin
deficiency has been overcome by a second “suppressor” Several gibberellin‐insensitive dwarf mutants have been
mutation, so the plants look closer to normal isolated from various species. The first to be isolated in
3. Mutants with a constitutive gibberellin response Arabidopsis was the gai­1 mutant. The gai­1 mutants
(“slender” mutants) resemble gibberellin‐deficient mutants, except that they do
not respond to exogenous gibberellin. Another mutant was
All three types of gibberellin response mutants have been obtained by screening for a second mutation in a gibberellin‐
generated in Arabidopsis, but equivalent mutations have also deficient Arabidopsis mutant that restores, or partially
been found in several other species; in fact, some have been restores, wild‐type growth. The original gibberellin‐deficient
in agricultural use for many years. The three types of mutant mutant was ga1­3, and the second mutation that partially
screens have sometimes identified genes encoding the same “rescued” the phenotype (i.e., restored normal growth) was
signal transduction components, even though the called rga (for repressor of ga1­3).4 The rga mutation is a
phenotypes being selected are completely different. This is recessive mutation that, when present in double copy, gives
possible because mutations at different sites in the same a plant of intermediate height.
protein can produce vastly different phenotypes, depending
on whether the mutation is in a regulatory domain or in an Despite the contrasting phenotypes of the mutants, the wild‐
activity, or functional, domain. Some examples of the type GAI and RGA genes turned out to be closely related,
different phenotypes that can result from changes at with a very high (82%) sequence identity. The gai­1
different sites in the same protein are described in the mutation is semidominant, as are similar gibberellin‐
sections that follow. insensitive dwarf mutations in other species.

Functional domain (repression). The principal gibberellin Genetic analyses have indicated that both the GAI and RGA
signal transduction components that have been identified so proteins normally act as repressors of gibberellin responses.
far are repressors of gibberellin signaling; that is, they Gibberellin acts indirectly through an unidentified signaling
repress what we regard as gibberellin‐induced tall growth intermediate, which is thought to bind to the regulatory
and make the plant dwarf. The repressor proteins are domains of the GAI and RGA proteins.
negated or turned off by gibberellin so that the default type
growth—namely, tall—is allowed to proceed. The loss of
function resulting from a mutation in the functional domain
of such a negative regulator results in the mutant appearing
as if it has been treated with gibberellin; that is, it has a tall
phenotype. Thus a loss‐of‐function mutation of
a negative regulator is like a double negative in English
grammar: It translates into a positive. Because the effects of
these loss‐of‐function mutations are pleiotropic—that is,
they also affect developmental processes other than stem
elongation—the steps in the pathway involved in the growth
response are probably common to all gibberellin responses.

Regulatory domain. If a mutation in the gene for the same

negative regulator causes a change in the regulatory domain
(i.e., that part of the protein that receives a signal from the
gibberellin receptor indicating the presence of gibberellin),
the protein is unable to receive the signal, and it retains its
growth‐repressing activity. The phenotype of such a mutant
will be that of a gibberellin‐insensitive dwarf. Thus, different
mutations in the same gene can give opposite phenotypes
(tall versus dwarf), depending on whether the mutation is
located in the repression domain or the regulatory domain.

The regulatory domain mutations that confer loss of

gibberellin sensitivity result in the synthesis of a
constitutively active form of the repressor than cannot be
turned off by gibberellin. The more of this type of mutant
repressor that is present in the cell, the more dwarf the plant
will be. Hence, such regulatory domain mutations are
semidominant. In contrast, mutations in the repression The repressor is no longer able to inhibit growth, and the
domain inactivate the negative regulator (i.e., they act as resulting plant is tall. The reason that gai­1 is dwarf, while
“knockout” alleles) so that it no longer represses growth; rga is tall, is that the mutations are in different parts of the
such mutations are recessive because in a heterozygote half protein. Whereas the gai­1 mutation (which negates
the proteins will still be able to repress growth in the sensitivity of the repressor to gibberellin) is in the
absence of gibberellin. All of the negative regulators have to regulatory domain, the rga mutation (which prevents the
be nonfunctional for the plant to grow tall without action of the repressor in blocking growth) is located in the
gibberellin. With this as background, we now examine repression domain, as illustrated in figure.
specific examples of mutations in the genes that encode
proteins in the gibberellin signal transduction pathway.

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The mutant gai­1 gene has been shown to encode a mutant pathway. However, RGA appears to play a more dominant
protein with a deletion of 17 amino acids, which role than GAI because in a gibberellin‐deficient mutant, a
corresponds to the regulatory domain of the repressor. A second mutation in the repression domain of gai (gai­t6)
similar mutation in the receptor domain of the RGA gene also does not restore growth, whereas a comparable mutation in
produces a gibberellin‐insensitive dwarf, demonstrating that rga does. On the other hand, the existence of repression
the two related proteins have overlapping functions. domain mutations in both of these genes allows for complete
Because of this deletion in the gai­1 mutant, the action of the expression of many characteristics induced by GA, including
repressor cannot be alleviated by gibberellin, and growth is plant height, in the absence of gibberellin.
constitutively inhibited.
DELLA Repressors Have Been Identified in Crop Plants
Gibberellins Cause the Degradation of RGA
Transcriptional Repressors Functional DELLA repressors have been found in several
crop plants that have dwarfing mutations, analogous to gai­
The Arabidopsis wild‐type GAI and RGA genes are members 1, in the genes encoding these proteins. Most notable are the
of a large gene family encoding transcriptional repressors rht (reduced height) mutations of wheat that have been in
that have highly conserved regions with nuclear localization use in agriculture for 30 years. These alleles encode
signals. To demonstrate the nuclear localization and gibberellin response modulators that lack gibberellin
repressor nature of the RGA product, the RGA promoter was responsiveness, leading to dwarfness.
fused to the gene for a green fluorescent protein whose
product can be visualized under the microscope. The green Cereal dwarfs such as these are very important as the
color could be seen in cell nuclei. When the plants were foundations of the green revolution that enabled large
treated with gibberellin, there was no green color, showing increases in yield to be obtained. Normal cereals grow too
that the RGA protein was not present following gibberellin tall when close together in a field, especially with high levels
treatment. However, when the gibberellin content was of fertilizer. The result is that plants fall down (lodge), and
severely lowered by treatment with the gibberellin the yield decreases concomitantly. The use of these stiff‐
biosynthesis inhibitor paclobutrazol, the nuclei acquired a strawed dwarf varieties that resist lodging enables high
very intense green fluorescence, demonstrating both the yields.
presence and nuclear localization of the RGA protein only
when gibberellin was absent or low. The Negative Regulator SPINDLY Is an Enzyme That
Alters Protein Activity
Both GAI and RGA also have a conserved region at the amino
terminus of the protein referred to as DELLA, after the code “Slender mutants” resemble wild‐type plants that have been
letters for the amino acids in that sequence. This region is treated with gibberellin repeatedly. They exhibit elongated
involved in the gibberellin response because it is the internodes, parthenocarpic (seed‐free) fruit growth (in
location of the mutation in gai­1 that renders it non‐ dicots), and poor pollen production. Slender mutants are
responsive to gibberellin. It turns out that the RGA protein is rare compared to dwarf mutants. One possible explanation
synthesized all the time; in the presence of gibberellin this of the slender phenotype could be simply that the mutants
protein is targeted for destruction, and the DELLA region is have higher‐than‐normal levels of endogenous gibberellins.
required for this response. For example, in the sln mutation of peas, a gibberellin
It is likely that gibberellin also brings about the turnover of deactivation step is blocked in the seed. As a result, the
GAI. RGA and GAI have partially redundant functions in mature seed, which in the wild type contains little or no GA,
maintaining the repressed state of the gibberellin signaling has abnormally high levels of GA20. The GA20 from the seed

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is then taken up by the germinating seedling and converted occur, but the default pathway is prevented by the presence
to the bioactive GA1, giving rise to the slender phenotype. of various negative regulators.
However, once the seedling runs out of GA20 from the seed,
its phenotype returns to normal.

If, on the other hand, the slender phenotype is not due to an

overproduction of endogenous gibberellin, the mutant is
considered to be a constitutive response mutant. The best
characterized of such mutants are the ultratall mutants: la
crys in pea, (representing mutations at two loci: La and
Crys); procera (pro) in tomato; slender (sln) in barley; and
spindly (spy) in Arabidopsis. All of these mutations are
recessive and appear to be loss‐of‐function mutations in
negative regulators of the gibberellin response pathway, as
in the case of the DELLA regulators.

SPINDLY (SPY) in Arabidopsis and related genes in other

species are similar in sequence to genes that encode
glucosamine transferases in animals. These enzymes modify
target proteins by the glycosylation of serine or threonine
residues. Glycosylation can modify protein activity either
directly or indirectly by interfering with or blocking sites of
phosphorylation by protein kinases. The target protein for
spindly proteins has not yet been identified.

Rather than directly promoting an effect, the arrival of the

developmental signal—in this case gibberellin—negates the
growth repressor, enabling the default condition.

GIBBERELLIN SIGNAL TRANSDUCTION: CEREAL

ALEURONE LAYERS

Genetic analyses of gibberellin‐regulated growth, such as the

studies described in the previous section, have identified
some of the genes and their gene products, but not the
biochemical pathways involved in gibberellin signal
SPY Acts Upstream of GAI and RGA in the Gibberellin transduction. The biochemical and molecular mechanisms,
Signal Transduction Chain which are probably common to all gibberellin responses,
have been studied most extensively in relation to the
On the basis of the evidence presented in the preceding gibberellin‐ stimulated synthesis and secretion of α‐amylase
sections and other studies on the expression of SPY, GAI, and in cereal aleurone layers.
RGA (Sun 2000; Dill et al. 2001), we can begin to sketch out
the following elements of the gibberellin signal transduction In this section we will describe how such studies have shed
chain (Figures): light on the location of the gibberellin receptor, the
• Two or more transcriptional regulators encoded by GAI transcriptional regulation of the genes for α‐amylase and
and RGA act as inhibitors of the transcription of genes that other proteins, and the possible signal transduction
directly or indirectly promote growth. pathways involved in the control of α‐amylase synthesis and
• SPY appears to be a signal transduction intermediate secretion by gibberellin.
acting upstream of GAI and RGA that, itself, turns on or
enhances the transcription or action of GAI and RGA, or Gibberellin from the Embryo Induces α­Amylase
another negative regulator. Production by Aleurone Layers
• In the presence of gibberellin, SPY, GAI, and RGA are all
negated or turned off. Cereal grains (caryopses; singular caryopsis) can be divided
• The RGA protein is degraded, and it is likely that GAI is into three parts: the diploid embryo, the triploid endosperm,
similarly destroyed. and the fused testa–pericarp (seed coat–fruit wall). The
embryo part consists of the plant embryo proper, along with
Whether gibberellin negates GAI and RGA through SPY, or its specialized absorptive organ, the scutellum (plural
independently, or both, is currently under investigation. scutella), which functions in absorbing the solubilized food
However, the basic message in this case and in the cases of reserves from the endosperm and transmitting them to the
other plant hormones, such as ethylene and the growing embryo. The endosperm is composed of two
photoreceptor phytochrome, is that the default tissues: the centrally located starchy endosperm and the
developmental program is for the induced type of growth to aleurone layer. The starchy endosperm, typically nonliving
at maturity, consists of thin‐walled cells filled with starch

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grains. The aleurone layer surrounds the starchy endosperm 3. What signal transduction pathways operate between the
and is cytologically and biochemically distinct from it. gibberellin receptor and a‐amylase production?
Aleurone cells are enclosed in thick primary cell walls and
contain large numbers of protein‐storing vacuoles called
protein bodies, enclosed by a single membrane. The protein
bodies also contain phytin, a mixed cation salt (mainly Mg 2+
and K+) of myo‐inositol hexaphosphoric acid (phytic acid).

During germination and early seedling growth, the stored

food reserves of the endosperm—chiefly starch and
protein—are broken down by a variety of hydrolytic
enzymes, and the solubilized sugars, amino acids, and other
products are transported to the growing embryo. The two
enzymes responsible for starch degradation are α‐ and β‐
amylase. α‐Amylase hydrolyzes starch chains internally to
produce oligosaccharides consisting of α‐1,4‐linked glucose
residues. β‐Amylase degrades these oligosaccharides from
the ends to produce maltose, a disaccharide. Maltase then
converts maltose to glucose.

α‐Amylase is secreted into the starchy endosperm of cereal

seeds by both the scutellum and the aleurone layer. The sole Gibberellic Acid Enhances the Transcription of α­
function of the aleurone layer of the seeds of graminaceous Amylase mRNA
monocots (e.g., barley, wheat, rice, rye, and oats) appears to
be the synthesis and release of hydrolytic enzymes. After Before molecular biological approaches were developed,
completing this function, aleurone cells undergo there was already physiological and biochemical evidence
programmed cell death. Experiments carried out in the that gibberellic acid might enhance α‐amylase production at
1960s confirmed Gottlieb Haberlandt’s original observation the level of gene transcription. The two main lines of
of 1890 that the secretion of starch‐degrading enzymes by evidence were as follows:
barley aleurone layers depends on the presence of the 1. GA3‐stimulated α‐amylase production was shown to be
embryo. When the embryo was removed (i.e., the seed was blocked by inhibitors of transcription and translation.
de‐embryonated), no starch was degraded. However, when 2. Heavy‐isotope‐ and radioactive‐isotope‐labeling studies
the de‐embryonated “half‐seed” was incubated in close demonstrated that the stimulation of α‐amylase activity by
proximity to the excised embryo, starch was digested, gibberellin involved de novo synthesis of the enzyme from
demonstrating that the embryo produced a diffusible amino acids, rather than activation of preexisting enzyme.
substance that triggered α‐ amylase release by the aleurone
layer. Definitive molecular evidence now shows that gibberellin
acts primarily by inducing the expression of the gene for α‐
It was soon discovered that gibberellic acid (GA3) could amylase. It has been shown that GA3 enhances the level of
substitute for the embryo in stimulating starch degradation. translatable mRNA for α‐amylase in aleurone layers.
When de‐embryonated half‐seeds were incubated in Furthermore, by using isolated nuclei, investigators also
buffered solutions containing gibberellic acid, secretion of α‐ demonstrated that there was an enhanced transcription of
amylase into the medium was greatly stimulated after an 8‐ the α‐amylase gene rather than a decrease in mRNA
hour lag period (relative to the control half‐seeds incubated turnover.
in the absence of gibberellic acid).
The purification of α‐amylase mRNA, which is produced in
The significance of the gibberellin effect became clear when relatively large amounts in aleurone cells, enabled the
it was shown that the embryo synthesizes and releases isolation of genomic clones containing both the structural
gibberellins (chiefly GA1) into the endosperm during gene for α‐amylase and its upstream promoter sequences.
germination. Thus the cereal embryo efficiently regulates These promoter sequences were then fused to the reporter
the mobilization of its own food reserves through the gene that encodes the enzyme α‐glucuronidase (GUS), which
secretion of gibberellins, which stimulate the digestive yields a blue color in the presence of an artificial substrate
function of the aleurone layer (see Figure A). when the gene is expressed. The regulation of transcription
by gibberellin was proved when such chimeric genes
Gibberellin has been found to promote the production containing α‐amylase promoters that were fused to reporter
and/or secretion of a variety of hydrolytic enzymes that are genes were introduced into aleurone protoplasts and the
involved in the solubilization of endosperm reserves; production of the blue color was shown to be stimulated by
principal among these is α‐amylase. Since the 1960s, gibberellin.
investigators have utilized isolated aleurone layers, or even
aleurone cell protoplasts, rather than half seeds. The isolated The partial deletion of known sequences of bases from α‐
aleurone layer, consisting of a homogeneous population of amylase promoters from several cereals indicates that the
target cells, provides a unique opportunity to study the sequences conferring gibberellin responsiveness, termed
molecular aspects of gibberellin action in the absence of gibberellin response elements, are located 200 to 300 base
nonresponding cell types. pairs upstream of the transcription start site.
In the following discussion of gibberellin‐induced α‐ amylase
production we focus on three questions:
1. How does gibberellin regulate the increase in a‐amylase?
2. Where is the gibberellin receptor located in the cell?

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A GA­MYB Transcription Factor Regulates α­ Amylase In animal cells, heterotrimeric GTP‐binding proteins (G
Gene Expression proteins) in the cell membrane are often involved as first
steps in a pathway between a hormone receptor and
The stimulation of α‐amylase gene expression by gibberellin subsequent cytosolic signals. Evidence has been obtained
is mediated by a specific transcription factor that binds to that G‐proteins are also involved in the early gibberellin
the promoter of the α‐amylase gene. To demonstrate such signaling events in aleurone cells.
DNA‐binding proteins in rice, a technique called a mobility
shift assay was used. This assay detects the increase in size Treatment of oat aleurone protoplasts with a peptide called
that occurs when the α‐amylase promoter binds to a protein Mas7, which stimulates GTP/GDP exchange by G proteins,
isolated from gibberellin‐treated aleurone cells. was found to induce α‐amylase gene expression and to
stimulate α‐amylase secretion, suggesting that such a
The mobility shift assay also allowed identification of the GTP/GDP exchange on the cell membrane is a reaction en
regulatory DNA sequences (gibberellin response route to the induction of α‐amylase biosynthesis by
elements) in the promoter that are involved in binding the gibberellin. In addition, gibberellin‐induced α‐amylase gene
protein. Identical gibberellin response elements were found expression and secretion were inhibited by a guanine
to occur in all cereal α‐amylase promoters, and their nucleotide analog that binds to the α subunit of
presence was shown to be essential for the induction of α‐ heterotrimeric G‐proteins and inhibits GTP/GDP exchange,
amylase gene transcription by gibberellin. These studies further supporting the preceding conclusion.
demonstrated that gibberellin increases either the level or
the activity of a transcription factor protein that switches on Recent genetic studies have provided further support for the
the production of α‐amylase mRNA by binding to an role of G‐proteins as intermediates in the gibberellin signal
upstream regulatory element in the α‐amylase gene transduction pathway. The rice dwarf mutant dwarf 1 (d1)
promoter. The sequence of the gibberellin response element has a defective gene encoding the α subunit. Besides being
in the α‐amylase gene promoter turned out to be similar to dwarf, the aleurone layers of the d1 mutant synthesize less
that of the binding sites for MYB transcription factors that α‐amylase in response to gibberellin than wildtype aleurone
are known to regulate growth and development in layers do. This reduction in α‐amylase production by the d1
phytochrome responses. This knowledge enabled the mutant demonstrates that G‐proteins are one of the
isolation of mRNA for a MYB transcription factor, named GA‐ components of the gibberellin signal transduction pathway
MYB, associated with the gibberellin induction of α‐amylase involved in both the growth response and the production of
gene expression. α‐amylase. However, the difference in α‐
amylase production between the mutant and the wild type
The synthesis of GA­MYB mRNA in aleurone cells increases goes away with increasing gibberellin concentration,
within 3 hours of gibberellin application, several hours suggesting that gibberellin can also stimulate α‐amylase
before the increase in α‐amylase mRNA. The inhibitor of production by a G‐protein‐independent pathway.
translation, cycloheximide, has no effect on the production
of MYB mRNA, indicating that GA­MYB is a primary response Cyclic GMP, Ca2+, and Protein Kinases Are Possible
gene, or early gene. In contrast, the α‐amylase gene is a Signaling Intermediates
secondary response gene, or late gene, as indicated by the fact
that its transcription is blocked by cycloheximide. In animal cells, G‐proteins can activate the enzyme guanylyl
cyclase, the enzyme that synthesizes cGMP from GTP,
How does gibberellin cause the MYB gene to be expressed? leading to an increase in cGMP concetration. Cyclic GMP, in
Because protein synthesis is not involved, gibberellin may turn, can regulate ion channels, Ca2+ levels, protein kinase
bring about the activation of one or more preexisting activity, and gene transcription. Gibberellin has been
transcription factors. The activation of transcription factors reported to cause a transient rise in cGMP levels in barley
is typically mediated by protein phosphorylation events aleurone layers, suggesting a possible role for cGMP in α‐
occurring at the end of a signal transduction pathway. We amylase production.
will now examine what is known about the signaling
pathways involved in gibberellin‐induced α‐amylase Calcium and the calcium‐binding protein calmodulin act as
production up to the point of GA‐MYB production. second messengers for many hormonal responses in animal
cells, and they have been implicated in various plant
Gibberellin Receptors May Interact with G Proteins on responses to environmental and hormonal stimuli. The
the Plasma Membrane earliest event in aleurone protoplasts after the application of
gibberellin is a rise in the cytoplasmic calcium concentration
A cell surface localization of the gibberellin receptor is that occurs well before the onset of α‐amylase synthesis.
suggested from the fact that gibberellin that has been bound Without calcium, α‐amylase secretion does not occur,
to microbeads that are unable to cross the plasma though in barley aleurone protoplasts its synthesis goes
membrane is still active in inducing α‐amylase production in ahead normally, so we have to conclude that, in barley,
aleurone protoplasts. In addition, microinjection of GA3 into calcium is not on the signaling pathway to α‐amylase gene
aleurone protoplasts had no effect, but when the protoplasts transcription, though it does play a role in enzyme secretion.
were immersed in GA3 solution, they produced α‐amylase.
These results suggest that gibberellin acts on the outer face Protein phosphorylation by protein kinases is another
of the plasma membrane. Two gibberellin‐binding plasma component in many signaling pathways, and gibberellin
membrane proteins have been isolated through the use of appears to be no exception. The injection of a protein kinase
purified plasma membrane and a radioactively labeled substrate into barley aleurone protoplasts to compete with
gibberellin that was chemically modified to permanently endogenous protein phosphorylation inhibited α‐amylase
attach to protein to which it was weakly bound. Because the secretion, suggesting the involvement of protein
presence of excess gibberellin reduces binding, and these phosphorylation in the α‐amylase secretion pathway. This
proteins from a semidwarf, gibberellin‐insensitive sweet pea did not affect the gibberellin‐ stimulated increase in calcium,
bind gibberellin less strongly, they may represent the indicating that the protein kinase step is downstream of the
gibberellin receptors. calcium signaling event.

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In conclusion, gibberellin signal transduction in aleurone increase in cytoplasmic calcium and protein
cells seems to involve G‐proteins as well as cyclic GMP, phosphorylation. The detailed signaling pathways remain to
leading to production of the transcription factor GAMYB, be worked out. A model of the known biochemical
which induces α‐amylase gene transcription α‐Amylase components of the gibberellin signal transduction pathways
secretion has similar initial components but also involves an in aleurone cells is illustrated in Figure.

The Gibberellin Signal Transduction Pathway Is Similar SPY‐type gene associated with α‐amylase production has
for Stem Growth and α­Amylase Production been isolated from barley (HvSPY), and its expression is able
to inhibit gibberellin‐induced α‐amylase synthesis, while GA‐
It is widely believed that gibberellin initially acts through a MYB‐type factors are also implicated in the gibberellin
common pathway or pathways in all of its effects on transduction chain regulating stem growth.
development. As we have seen, the genetic approaches
applied to the study of gibberellin‐stimulated growth led to Rice with the dwarf 1 mutation also produces little α‐
the identification of the SPY/GAI/RGA negative regulatory amylase in response to gibberellin. As noted earlier, the
pathway. The proteins SPY, GAI, and RGA act as repressors mutation causing dwarf 1 is known to be in the α subunit of
of gibberellin responses. Gibberellin deactivates these the G‐protein complex, providing evidence that the action of
repressors. Because the aleurone layers of gibberellin‐ gibberellin in both stem elongation and the production of α‐
insensitive dwarf wheat are also insensitive to GA, the same amylase are regulated by plasma membrane heterotrimeric
signal transduction pathways that regulate growth appear to G‐proteins.
regulate gibberellin‐induced α‐amylase production. Indeed a

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4. ETHYLENE we will see, the complete pathway is a cycle, taking its place
among the many metabolic cycles that operate in plant cells.
DURING THE NINETEENTH CENTURY, when coal gas was
used for street illumination, it was observed that trees in the The Properties of Ethylene Are Deceptively Simple
vicinity of streetlamps defoliated more extensively than
other trees. Eventually it became apparent that coal gas and Ethylene is the simplest known olefin (its molecular weight
air pollutants affect plant growth and development, and is 28), and it is lighter than air under physiological
ethylene was identified as the active component of coal gas. conditions:

In 1901, Dimitry Neljubov, a graduate student at the

Botanical Institute of St. Petersburg in Russia, observed that
dark‐grown pea seedlings growing in the laboratory
exhibited symptoms that were later termed the triple
response: reduced stem elongation, increased lateral growth
(swelling), and abnormal, horizontal growth. When the It is flammable and readily undergoes oxidation. Ethylene
plants were allowed to grow in fresh air, they regained their can be oxidized to ethylene oxide:
normal morphology and rate of growth. Neljubov identified
ethylene, which was present in the laboratory air from coal
gas, as the molecule causing the response.

The first indication that ethylene is a natural product of

plant tissues was published by H. H. Cousins in 1910.
Cousins reported that “emanations” from oranges stored in a and ethylene oxide can be hydrolyzed to ethylene glycol:
chamber caused the premature ripening of bananas when
these gases were passed through a chamber containing the In most plant tissues, ethylene can be completely oxidized to
fruit. However, given that oranges synthesize relatively little CO2, in the following reaction:
ethylene compared to other fruits, such as apples, it is likely
that the oranges used by Cousins were infected with the
fungus Penicillium, which produces copious amounts of
ethylene. In 1934, R. Gane and others identified ethylene
chemically as a natural product of plant metabolism, and
because of its dramatic effects on the plant it was classified
as a hormone.

For 25 years ethylene was not recognized as an important Ethylene is released easily from the tissue and diffuses in the
plant hormone, mainly because many physiologists believed gas phase through the intercellular spaces and outside the
that the effects of ethylene were due to auxin, the first plant tissue. At an ethylene concentration of 1 μL L–1 in the gas
hormone to be discovered. Auxin was thought to be the main phase at 25°C, the concentration of ethylene in water is 4.4 ×
plant hormone, and ethylene was considered to play only an 10–9 M. Because they are easier to measure, gas phase
insignificant and indirect physiological role. Work on concentrations are normally given for ethylene. Because
ethylene was also hampered by the lack of chemical ethylene gas is easily lost from the tissue and may affect
techniques for its quantification. However, after gas other tissues or organs, ethylene‐trapping systems are used
chromatography was introduced in ethylene research in during the storage of fruits, vegetables, and flowers.
1959, the importance of ethylene was rediscovered and its Potassium permanganate (KMnO4) is an effective absorbent
physiological significance as a plant growth regulator was of ethylene and can reduce the concentration of ethylene in
recognized. apple storage areas from 250 to 10 μLL–1, markedly
extending the storage life of the fruit.
In this chapter we will describe the discovery of the ethylene
biosynthetic pathway and outline some of the important Bacteria, Fungi, and Plant Organs Produce Ethylene
effects of ethylene on plant growth and development. At the
end of the chapter we will consider how ethylene acts at the Even away from cities and industrial air pollutants, the
cellular and molecular levels. environment is seldom free of ethylene because of its
production by plants and microorganisms. The production
STRUCTURE, BIOSYNTHESIS, AND MEASUREMENT OF of ethylene in plants is highest in senescing tissues and
ETHYLENE ripening fruits (>1.0 nL g‐fresh‐weight–1 h–1), but all organs
of higher plants can synthesize ethylene. Ethylene is
Ethylene can be produced by almost all parts of higher biologically active at very low concentrations—less than 1
plants, although the rate of production depends on the type part per million (1 μL L–1). The internal ethylene
of tissue and the stage of development. In general, concentration in a ripe apple has been reported to be as high
meristematic regions and nodal regions are the most active as 2500 μL L–1.
in ethylene biosynthesis. However, ethylene production also
increases during leaf abscission and flower senescence, as Young developing leaves produce more ethylene than do
well as during fruit ripening. Any type of wounding can fully expanded leaves. In bean (Phaseolus vulgaris), young
induce ethylene biosynthesis, as can physiological stresses leaves produce 0.4 nL g–1 h–1, compared with 0.04 nL g–1
such as flooding, chilling, disease, and temperature or h–1 for older leaves. With few exceptions, nonsenescent
drought stress. tissues that are wounded or mechanically perturbed will
temporarily increase their ethylene production several fold
The amino acid methionine is the precursor of ethylene, and within 30 minutes. Ethylene levels later return to normal.
ACC (1‐aminocyclopropane‐1‐carboxylic acid) serves as an
intermediate in the conversion of methionine to ethylene. As

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Gymnosperms and lower plants, including ferns, mosses, immediate precursor of ethylene is 1­aminocyclopropane­
liverworts, and certain cyanobacteria, all have shown the 1­carboxylic acid (ACC) (see Figure ).
ability to produce ethylene. Ethylene production by fungi
and bacteria contributes significantly to the ethylene content The role of ACC became evident in experiments in which
of soil. Certain strains of the common enteric bacterium plants were treated with [14C]methionine. Under anaerobic
Escherichia coli and of yeast (a fungus) produce large conditions, ethylene was not produced from the
amounts of ethylene from methionine. There is no evidence [14C]methionine, and labeled ACC accumulated in the tissue.
that healthy mammalian tissues produce ethylene, nor does On exposure to oxygen, however, ethylene production
ethylene appear to be a metabolic product of invertebrates. surged. The labeled ACC was rapidly converted to ethylene
in the presence of oxygen by various plant tissues,
However, recently it was found that both a marine sponge suggesting that ACC is the immediate precursor of ethylene
and cultured mammalian cells can respond to ethylene, in higher plants and that oxygen is required for the
raising the possibility that this gaseous molecule acts as a conversion. In general, when ACC is supplied exogenously to
signaling molecule in animal cells. plant tissues, ethylene production increases substantially.
This observation indicates that the synthesis of ACC is
Regulated Biosynthesis Determines the Physiological usually the biosynthetic step that limits ethylene production
Activity of Ethylene in plant tissues.

In vivo experiments showed that plant tissues convert l‐ ACC synthase, the enzyme that catalyzes the conversion of
[14C]methionine to [14C]ethylene, and that the ethylene is AdoMet to ACC (see Figure ), has been characterized in many
derived from carbons 3 and 4 of methionine (Figure ). types of tissues of various plants. ACC synthase is an
unstable, cytosolic enzyme. Its level is regulated by
The CH3—S group of methionine is recycled via the Yang environmental and internal factors, such as wounding,
cycle. Without this recycling, the amount of reduced sulfur drought stress, flooding, and auxin. Because ACC synthase is
present would limit the available methionine and the present in such low amounts in plant tissues (0.0001% of
synthesis of ethylene. S‐adenosylmethionine (AdoMet), the total protein of ripe tomato) and is very unstable, it is
which is synthesized from methionine and ATP, is an difficult to purify the enzyme for biochemical analysis.
intermediate in the ethylene biosynthetic pathway, and the

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ACC synthase is encoded by members of a divergent pathway, and the increased ethylene production has been
multigene family that are differentially regulated by various shown to result at least in part from an increase in
inducers of ethylene biosynthesis. In tomato, for example, transcription of ACC synthase mRNA. This “stress ethylene”
there are at least nine ACC synthase genes, different subsets is involved in the onset of stress responses such as
of which are induced by auxin, wounding, and/or fruit abscission, senescence, wound healing, and increased
ripening. disease resistance.

ACC oxidase catalyzes the last step in ethylene biosynthesis: Auxin­induced ethylene production. In some instances,
the conversion of ACC to ethylene. In tissues that show high auxins and ethylene can cause similar plant responses, such
rates of ethylene production, such as ripening fruit, ACC as induction of flowering in pineapple and inhibition of stem
oxidase activity can be the rate‐limiting step in ethylene elongation. These responses might be due to the ability of
biosynthesis. The gene that encodes ACC oxidase has been auxins to promote ethylene synthesis by enhancing ACC
cloned. Like ACC synthase, ACC oxidase is encoded by a synthase activity. These observations suggest that some
multigene family that is differentially regulated. For responses previously attributed to auxin (indole‐3‐ acetic
example, in ripening tomato fruits and senescing petunia acid, or IAA) are in fact mediated by the ethylene produced
flowers, the mRNA levels of a subset of ACC oxidase genes in response to auxin.
are highly elevated. The deduced amino acid sequences of
ACC oxidases revealed that these enzymes belong to the Inhibitors of protein synthesis block both ACC and IAA‐
Fe2+/ascorbate oxidase superfamily. This similarity induced ethylene synthesis, indicating that the synthesis of
suggested that ACC oxidase might require Fe2+ and new ACC synthase protein caused by auxins brings about the
ascorbate for activity—a requirement that has been marked increase in ethylene production. Several ACC
confirmed by biochemical analysis of the protein. The low synthase genes have been identified whose transcription is
abundance of ACC oxidase and its requirement for cofactors elevated following application of exogenous IAA, suggesting
presumably explain why the purification of this enzyme that increased transcription is at least partly responsible for
eluded researchers for so many years. the increased ethylene production observed in response to
auxin.
Catabolism. Researchers have studied the catabolism of
ethylene by supplying 14C2H4 to plant tissues and tracing the Posttranscriptional regulation of ethylene production.
radioactive compounds produced. Carbon dioxide, ethylene
oxide, ethylene glycol, and the glucose conjugate of ethylene Ethylene production can also be regulated
glycol have been identified as metabolic breakdown posttranscriptionally. Cytokinins also promote ethylene
products. However, because certain cyclic olefin compounds, biosynthesis in some plant tissues. For example, in etiolated
such as 1,4‐cyclohexadiene, have been shown to block Arabidopsis seedlings, application of exogenous cytokinins
ethylene breakdown without inhibiting ethylene action, causes a rise in ethylene production, resulting in the
ethylene catabolism does not appear to play a significant tripleresponse phenotype.
role in regulating the level of the hormone.
Molecular genetic studies in Arabidopsis have shown that
Conjugation. Not all the ACC found in the tissue is converted cytokinins elevate ethylene biosynthesis by increasing the
to ethylene. ACC can also be converted to a conjugated form, stability and/or activity of one isoform of ACC synthase. The
N‐malonyl ACC, which does not appear to break down and carboxy‐terminal domain of this ACC synthase isoform
accumulates in the tissue. appears to be the target for this posttranscriptional
regulation. Consistent with this, the carboxy‐terminal
A second conjugated form of ACC, 1‐(γ‐L‐glutamylamino) domain of an ACC synthase isoform from tomato has been
cyclopropane‐1‐carboxylic acid (GACC), has also been shown to be the target for a calciumdependent
identified. The conjugation of ACC may play an important phosphorylation.
role in the control of ethylene biosynthesis, in a manner
analogous to the conjugation of auxin and cytokinin. Ethylene Production and Action Can Be Inhibited

Environmental Stresses and Auxins Promote Ethylene Inhibitors of hormone synthesis or action are valuable for
Biosynthesis the study of the biosynthetic pathways and physiological
roles of hormones. Inhibitors are particularly helpful when it
Ethylene biosynthesis is stimulated by several factors, is difficult to distinguish between different hormones that
including developmental state, environmental conditions, have identical effects in plant tissue or when a hormone
other plant hormones, and physical and chemical injury. affects the synthesis or the action of another hormone.
Ethylene biosynthesis also varies in a circadian manner,
peaking during the day and reaching a minimum at night. For example, ethylene mimics high concentrations of auxins
by inhibiting stem growth and causing epinasty (a
Fruit ripening. As fruits mature, the rate of ACC and downward curvature of leaves). Use of specific inhibitors of
ethylene biosynthesis increases. Enzyme activities for both ethylene biosynthesis and action made it possible to
ACC oxidase and ACC synthase increase, as do the mRNA discriminate between the actions of auxin and ethylene.
levels for subsets of the genes encoding each enzyme. Studies using inhibitors showed that ethylene is the primary
However, application of ACC to unripe fruits only slightly effector of epinasty and that auxin acts indirectly by causing
enhances ethylene production, indicating that an increase in a substantial increase in ethylene production.
the activity of ACC oxidase is the rate‐limiting step in
ripening. Inhibitors of ethylene synthesis. Aminoethoxy­
vinylglycine (AVG) and aminooxyacetic acid (AOA) block
Stress­induced ethylene production. Ethylene biosynthesis the conversion of AdoMet to ACC. AVG and AOA are known
is increased by stress conditions such as drought, flooding, to inhibit enzymes that use the cofactor pyridoxal
chilling, exposure to ozone, or mechanical wounding. In all phosphate. The cobalt ion (Co2+) is also an inhibitor of the
these cases ethylene is produced by the usual biosynthetic ethylene biosynthetic pathway, blocking the conversion of

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ACC to ethylene by ACC oxidase, the last step in ethylene Ethylene Promotes the Ripening of Some Fruits
biosynthesis.
In everyday usage, the term fruit ripening refers to the
Inhibitors of ethylene action. Most of the effects of ethylene changes in fruit that make it ready to eat. Such changes
can be antagonized by specific ethylene inhibitors. Silver typically include softening due to the enzymatic breakdown
ions (Ag+) applied as silver nitrate (AgNO3) or as silver of the cell walls, starch hydrolysis, sugar accumulation, and
thiosulfate (Ag(S2O3)2 3–) are potent inhibitors of ethylene the disappearance of organic acids and phenolic compounds,
action. Silver is very specific; the inhibition it causes cannot including tannins. From the perspective of the plant, fruit
be induced by any other metal ion. Carbon dioxide at high ripening means that the seeds are ready for dispersal. For
concentrations (in the range of 5 to 10%) also inhibits many seeds whose dispersal depends on animal ingestion, ripeness
effects of ethylene, such as the induction of fruit ripening, and edibility are synonymous. Brightly colored anthocyanins
although CO2 is less efficient than Ag+. This effect of CO2 has and carotenoids often accumulate in the epidermis of such
often been exploited in the storage of fruits, whose ripening fruits, enhancing their visibility. However, for seeds that rely
is delayed at elevated CO2 concentrations. The high on mechanical or other means for dispersal, fruit ripening
concentrations of CO2 required for inhibition make it may mean drying followed by splitting.
unlikely that CO2 acts as an ethylene antagonist under
natural conditions. Because of their importance in agriculture, the vast majority
of studies on fruit ripening have focused on edible fruits.
The volatile compound trans‐cyclooctene, but not its isomer
cis‐cyclooctene, is a strong competitive inhibitor of ethylene Ethylene has long been recognized as the hormone that
binding; trans‐cyclooctene is thought to act by competing accelerates the ripening of edible fruits. Exposure of such
with ethylene for binding to the receptor. A novel inhibitor, fruits to ethylene hastens the processes associated with
1­methylcyclopropene (MCP), was recently found that ripening, and a dramatic increase in ethylene production
binds almost irreversibly to the ethylene receptor. MCP accompanies the initiation of ripening. However, surveys of
shows tremendous promise in commercial applications. a wide range of fruits have shown that not all of them
respond to ethylene.

All fruits that ripen in response to ethylene exhibit a

characteristic respiratory rise before the ripening phase
called a climacteric. Such fruits also show a spike of
ethylene production immediately before the respiratory rise.
In a smuch as treatment with ethylene induces the fruit to
produce additional ethylene, its action can be described as
autocatalytic. Apples, bananas, avocados, and tomatoes are
examples of climacteric fruits. In contrast, fruits such as
citrus fruits and grapes do not exhibit the respiration and
ethylene production rise and are called nonclimacteric
fruits. Other examples of climacteric and nonclimacteric
fruits are given in Table 22.1.
Ethylene Can Be Measured by Gas Chromatography

Historically, bioassays based on the seedling triple response

were used to measure ethylene levels, but they have been
replaced by gas chromatography. As little as 5 parts per
billion (ppb) (5 pL per liter)1 of ethylene can be detected,
and the analysis time is only 1 to 5 minutes.

Usually the ethylene produced by a plant tissue is allowed to

accumulate in a sealed vial, and a sample is withdrawn with
a syringe. The sample is injected into a gas chromatograph
column in which the different gases are separated and
detected by a flame ionization detector. Quantification of
ethylene by this method is very accurate. Recently a novel
method to measure ethylene was developed that uses a
laser‐driven photoacoustic detector that can detect as little
as 50 parts per trillion (50 ppt = 0.05 pL L–1) ethylene .

DEVELOPMENTAL AND PHYSIOLOGICAL EFFECTS OF When unripe climacteric fruits are treated with ethylene, the
ETHYLENE onset of the climacteric rise is hastened. When
nonclimacteric fruits are treated in the same way, the
As we have seen, ethylene was discovered in connection magnitude of the respiratory rise increases as a function of
with its effects on seedling growth and fruit ripening. It has the ethylene concentration, but the treatment does not
since been shown to regulate a wide range of responses in trigger production of endogenous ethylene and does not
plants, including seed germination, cell expansion, cell accelerate ripening.
differentiation, flowering, senescence, and abscission. In this
section we will consider the phenotypic effects of ethylene in Elucidation of the role of ethylene in the ripening of
more detail. climacteric fruits has resulted in many practical applications
aimed at either uniform ripening or the delay of ripening.
Although the effects of exogenous ethylene on fruit ripening
are straightforward and clear, establishing a causal relation

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between the level of endogenous ethylene and fruit ripening Because water fills the air spaces in waterlogged soil and O2
is more difficult. Inhibitors of ethylene biosynthesis (such as diffuses slowly through water, the concentration of oxygen
AVG) or of ethylene action (such as CO2, MCP, or Ag+) have around flooded roots decreases dramatically. The elevated
been shown to delay or even prevent ripening. production of ethylene appears to be caused by the
accumulation of ACC in the roots under anaerobic
However, the definitive demonstration that ethylene is conditions, since the conversion of ACC to ethylene requires
required for fruit ripening was provided by experiments in oxygen. The ACC accumulated in the anaerobic roots is then
which ethylene biosynthesis was blocked by expression of transported to shoots via the transpiration stream, where it
an antisense version of either ACC synthase or ACC oxidase is readily converted to ethylene.
in transgenic tomatoes. Elimination of ethylene biosynthesis
in these transgenic tomatoes completely blocked fruit Ethylene Induces Lateral Cell Expansion
ripening, and ripening was restored by application of
exogenous ethylene.. At concentrations above 0.1 μL L–1, ethylene changes the
growth pattern of seedlings by reducing the rate of
Further demonstration of the requirement for ethylene in elongation and increasing lateral expansion, leading to
fruit ripening came from the analysis of the never­ripe swelling of the region below the hook. These effects of
mutation in tomato. As the name implies, this mutation ethylene are common to growing shoots of most dicots,
completely blocks the ripening of tomato fruit. Molecular forming part of the triple response. In Arabidopsis, the
analysis revealed that never­ripe was due to a mutation in an triple response consists of inhibition and swelling of the
ethylene receptor that rendered it unable to bind ethylene. hypocotyl, inhibition of root elongation, and exaggeration of
These experiments provided unequivocal proof of the role of the apical hook.
ethylene in fruit ripening, and they opened the door to the
manipulation of fruit ripening through biotechnology. The directionality of plant cell expansion is determined by
the orientation of the cellulose microfibrils in the cell wall.
In tomatoes several genes have been identified that are Transverse microfibrils reinforce the cell wall in the lateral
highly regulated during ripening. During tomato fruit direction, so that turgor pressure is channeled into cell
ripening, the fruit softens as the result of cell wall hydrolysis elongation. The orientation of the microfibrils in turn is
and changes from green to red as a consequence of determined by the orientation of the cortical array of
chlorophyll loss and the synthesis of the carotenoid pigment microtubules in the cortical (peripheral) cytoplasm. In
lycopene. At the same time, aroma and flavor components typical elongating plant cells, the cortical microtubules are
are produced. arranged transversely, giving rise to transversely arranged
cellulose microfibrils. During the seedling triple response to
Analysis of mRNA from tomato fruits from wild‐type and ethylene, the transverse pattern of microtubule alignment is
transgenic tomato plants genetically engineered to lack disrupted, and the microtubules switch over to a
ethylene has revealed that gene expression during ripening longitudinal orientation. This 90° shift in microtubule
is regulated by at least two independent pathways: orientation leads to a parallel shift in cellulose microfibril
1. An ethylene­dependent pathway includes genes involved in deposition. The newly deposited wall is reinforced in the
lycopene and aroma biosynthesis, respiratory longitudinal direction rather than the transverse direction,
metabolism, and ACC synthase. which promotes lateral expansion instead of elongation.
2. A developmental, ethylene­independent pathway includes
genes encoding ACC oxidase and chlorophyllase. How do microtubules shift from one orientation to another?
To study this phenomenon, pea (Pisum sativum) epidermal
Thus, not all of the processes associated with ripening in cells were injected with the microtubule protein tubulin, to
tomato are ethylene dependent. which a fluorescent dye was covalently attached. The
fluorescent “tag” did not interfere with the assembly of
Leaf Epinasty Results When ACC from the Root Is microtubules. This procedure allowed researchers to
Transported to the Shoot monitor the assembly of microtubules in living cells using a
confocal laser scanning microscope, which can focus in many
The downward curvature of leaves that occurs when the planes throughout the cell. It was found that microtubules
upper (adaxial) side of the petiole grows faster than the do not reorient from the transverse to the longitudinal
lower (abaxial) side is termed epinasty. Ethylene and high direction by complete depolymerization of the transverse
concentrations of auxin induce epinasty, and it has now been microtubules followed by repolymerization of a new
established that auxin acts indirectly by inducing ethylene longitudinal array of microtubules.
production.
Instead, increasing numbers of nontransversely aligned
As will be discussed later in the chapter, a variety of stress microtubules appear in particular locations. Neighboring
conditions, such as salt stress or pathogen infection, increase microtubules then adopt the new alignment, so at one stage
ethylene production and also induce epinasty. There is no different alignments coexist before they adopt a uniformly
known physiological function for the response. longitudinal orientation. Although the reorientations
observed in this study were spontaneous rather than
In tomato and other dicots, flooding (waterlogging) or induced by ethylene, it is presumed that ethylene‐induced
anaerobic conditions around the roots enhances the microtubule reorientation operates by a similar mechanism.
synthesis of ethylene in the shoot, leading to the epinastic
response. Because these environmental stresses are sensed The Hooks of Dark­Grown Seedlings Are Maintained by
by the roots and the response is displayed by the shoots, a Ethylene Production
signal from the roots must be transported to the shoots. This
signal is ACC, the immediate precursor of ethylene. ACC Etiolated dicot seedlings are usually characterized by a
levels were found to be significantly higher in the xylem sap pronounced hook located just behind the shoot apex. This
after flooding of tomato roots for 1 to 2 days. hook shape facilitates penetration of the seedling through
the soil, protecting the tender apical meristem.

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Like epinasty, hook formation and maintenance result from synthesis in the underwater parts is usually provided by
ethylene‐induced asymmetric growth. The closed shape of aerenchyma tissue.
the hook is a consequence of the more rapid elongation of
the outer side of the stem compared with the inner side. In deepwater rice it has been shown that ethylene stimulates
When the hook is exposed to white light, it opens because internode elongation by increasing the amount of, and the
the elongation rate of the inner side increases, equalizing the sensitivity to, gibberellin in the cells of the intercalary
growth rates on both sides. Red light induces hook opening, meristem. The increased sensitivity to GA (gibberellic acid)
and far‐red light reverses the effect of red, indicating that in these cells in response to ethylene is brought about by a
phytochrome is the photoreceptor involved in this process. decrease in the level of abscisic acid (ABA), a potent
A close interaction between phytochrome and ethylene antagonist of GA.
controls hook opening. As long as ethylene is produced by
the hook tissue in the dark, elongation of the cells on the Ethylene Induces the Formation of Roots and Root Hairs
inner side is inhibited. Red light inhibits ethylene formation,
promoting growth on the inner side, thereby causing the Ethylene is capable of inducing adventitious root formation
hook to open. in leaves, stems, flower stems, and even other roots.
Ethylene has also been shown to act as a positive regulator
The auxin‐insensitive mutation axr1 and treatment of wild‐ of root hair formation in several species. This relationship
type seedlings with NPA (1‐N‐naphthylphthalamic acid), an has been best studied in Arabidopsis, in which root hairs
inhibitor of polar auxin transport, both block the formation normally are located in the epidermal cells that overlie a
of the apical hook in Arabidopsis. These and other results junction between the underlying cortical cells.
indicate a role for auxin in maintaining hook structure. The
more rapid growth rate of the outer tissues relative to the In ethylene‐treated roots, extra hairs form in abnormal
inner tissues could reflect an ethylene‐dependent auxin locations in the epidermis; that is, cells not overlying a
gradient, analogous to the lateral auxin gradient that cortical cell junction differentiate into hair cells. Seedlings
develops during phototropic curvature. grown in the presence of ethylene inhibitors (such as Ag+),
as well as ethylene‐insensitive mutants, display a reduction
A gene required for formation of the apical hook, in root hair formation in response to ethylene. These
HOOKLESS1 (so called because mutations in this gene result observations suggest that ethylene acts as a positive
in seedlings lacking an apical hook), was identified in regulator in the differentiation of root hairs.
Arabidopsis. Disruption of this gene severely alters the
pattern of expression of auxin‐responsive genes. When the Ethylene Induces Flowering in the Pineapple Family
gene is overexpressed in Arabidopsis, it causes constitutive
hook formation even in the light. HOOKLESS1 encodes a Although ethylene inhibits flowering in many species, it
putative N‐acetyltransferase that is hypothesized to induces flowering in pineapple and its relatives, and it is
regulate—by an unknown mechanism— differential auxin used commercially in pineapple for synchronization of fruit
distribution in the apical hook induced by ethylene. set. Flowering of other species, such as mango, is also
initiated by ethylene. On plants that have separate male and
Ethylene Breaks Seed and Bud Dormancy in Some female flowers (monoecious species), ethylene may change
Species the sex of developing flowers. The promotion of female
flower formation in cucumber is one example of this effect.
Seeds that fail to germinate under normal conditions (water,
oxygen, temperature suitable for growth) are said to be Ethylene Enhances the Rate of Leaf Senescence
dormant. Ethylene has the ability to break dormancy and
initiate germination in certain seeds, such as cereals. In Senescence is a genetically programmed developmental
addition to its effect on dormancy, ethylene increases the process that affects all tissues of the plant. Several lines of
rate of seed germination of several species. In peanuts physiological evidence support roles for ethylene and
(Arachis hypogaea), ethylene production and seed cytokinins in the control of leaf senescence:
germination are closely correlated. Ethylene can also break • Exogenous applications of ethylene or ACC (the precursor
bud dormancy, and ethylene treatment is sometimes used to of ethylene) accelerate leaf senescence, and
promote bud sprouting in potato and other tubers. treatment with exogenous cytokinins delays leaf senescence.
• Enhanced ethylene production is associated with
Ethylene Promotes the Elongation Growth of Submerged chlorophyll loss and color fading, which are characteristic
Aquatic Species features of leaf and flower senescence; an inverse
correlation has been found between cytokinin levels in
Although usually thought of as an inhibitor of stem leaves and the onset of senescence.
elongation, ethylene is able to promote stem and petiole • Inhibitors of ethylene synthesis (e.g., AVG or Co2+) and
elongation in various submerged or partially submerged action (e.g., Ag+ or CO2) retard leaf senescence.
aquatic plants. These include the dicots Ranunculus
sceleratus, Nymphoides peltata, and Callitriche platycarpa, Taken together, the physiological studies suggest that
and the fern Regnellidium diphyllum. Another agriculturally senescence is regulated by the balance of ethylene and
important example is the cereal deepwater rice. cytokinin. In addition, abscisic acid (ABA) has been
implicated in the control of leaf senescence.
In these species, submergence induces rapid internode or
petiole elongation, which allows the leaves or upper parts of Senescence in ethylene mutants. Direct evidence for the
the shoot to remain above water. Treatment with ethylene involvement of ethylene in the regulation of leaf senescence
mimics the effects of submergence. Growth is stimulated in has come from molecular genetic studies on Arabidopsis. As
the submerged plants because ethylene builds up in the will be discussed later in the chapter, several mutants
tissues. In the absence of O2, ethylene synthesis is affecting the response to ethylene have been identified. The
diminished, but the loss of ethylene by diffusion is retarded specific bioassay employed was the tripleresponse assay in
under water. Sufficient oxygen for growth and ethylene

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which ethylene significantly inhibits seedling hypocotyl Arabidopsis mutants become susceptible to several
elongation and promotes lateral expansion. necrotrophic (cell‐killing) soil fungal pathogens that are
normally not plant pathogens. Thus, ethylene appears to be
Ethylene‐insensitive mutants, such as etr1 involved in the resistance response to some pathogens, but
(ethyleneresistant 1) and ein2 (ethylene‐insensitive 2), were not others.
identified by their failure to respond to ethylene (as will be
described later in the chapter). The etr1 mutant is unable to
perceive the ethylene signal because of a mutation in the Ethylene Biosynthesis in the Abscission Zone Is
gene that codes for the ethylene receptor protein; the ein2 Regulated by Auxin
mutant is blocked at a later step in the signal transduction
pathway. The shedding of leaves, fruits, flowers, and other plant
organs is termed abscission. Abscission takes place in
Consistent with a role for ethylene in leaf senescence, both specific layers of cells, called abscission layers, which
etr1 and ein2 were found to be affected not only during the become morphologically and biochemically differentiated
early stages of germination, but throughout the life cycle, during organ development. Weakening of the cell walls at
including senescence. The ethylene mutants retained their the abscission layer depends on cell wall–degrading
chlorophyll and other chloroplast components for a longer enzymes such as cellulase and polygalacturonase.
period of time compared to the wild type. However, because
the total life spans of these mutants were increased by only The ability of ethylene gas to cause defoliation in birch trees.
30% over that of the wild type, ethylene appears to increase The wild‐type tree on the left has lost all its leaves. The tree
the rate of senescence, rather than acting as a developmental on the right has been transformed with a gene for the
switch that initiates the senescence process. Arabidopsis ethylene receptor ETR1‐1 carrying a dominant
mutation (discussed in the next section). This tree is unable
Use of genetic engineering to probe senescence. Another to respond to ethylene and does not shed its leaves after
very useful genetic approach that offers direct evidence for ethylene treatment.
the function of specific gene(s) is based on transgenic plants.
Through genetic engineering technology, the roles of both Ethylene appears to be the primary regulator of the
ethylene and cytokinins in the regulation of leaf senescence abscission process, with auxin acting as a suppressor of the
have been confirmed. ethylene effect. However, supraoptimal auxin concentrations
stimulate ethylene production, which has led to the use of
One way to suppress the expression of a gene is to transform auxin analogs as defoliants. For example, 2,4,5‐T, the active
the plant with antisense DNA, which consists of the gene of ingredient in Agent Orange, was widely used as a defoliant
interest in the reverse orientation with respect to the during the Vietnam War. Its action is based on its ability to
promoter. When the antisense gene is transcribed, the increase ethylene biosynthesis, thereby stimulating leaf
resulting antisense mRNA is complementary to the sense abscission.
mRNAand will hybridize to it. Because double‐stranded RNA
is rapidly degraded in the cell, the effect of the antisense A model of the hormonal control of leaf abscission describes
gene is to deplete the cell of the sense mRNA. the process in three distinct sequential phases:
1. Leaf maintenance phase. Prior to the perception of any
Transgenic plants expressing antisense versions of genes signal (internal or external) that initiates the abscission
that encode enzymes involved in the ethylene biosynthetic process, the leaf remains healthy and fully functional in the
pathway, such as ACC synthase and ACC oxidase, can plant. A gradient of auxin from the blade to the stem
synthesize ethylene only at very low levels. Consistent with maintains the abscission zone in a nonsensitive state.
a role for ethylene in senescence, such antisense mutants 2. Shedding induction phase. A reduction or reversal in the
have been shown to exhibit delayed leaf senescence, as well auxin gradient from the leaf, normally associated with leaf
as fruit ripening, in tomato. senescence, causes the abscission zone to become sensitive
to ethylene. Treatments that enhance leaf senescence may
The Role of Ethylene in Defense Responses Is Complex promote abscission by interfering with auxin synthesis
and/or transport in the leaf.
Pathogen infection and disease will occur only if the 3. Shedding phase. The sensitized cells of the abscission zone
interactions between host and pathogen are genetically respond to low concentrations of endogenous ethylene by
compatible. However, ethylene production generally synthesizing and secreting cellulase and other cell wall–
increases in response to pathogen attack in both compatible degrading enzymes, resulting in shedding.
(i.e., pathogenic) and noncompatible (nonpathogenic)
interactions. The discovery of ethylene‐insensitive mutants During the early phase of leaf maintenance, auxin from the
has allowed the role of ethylene in the response to various leaf prevents abscission by maintaining the cells of the
pathogens to be assessed. The emerging picture is that the abscission zone in an ethylene‐insensitive state. It has long
involvement of ethylene in pathogenesis is complex and been known that removal of the leaf blade (the site of auxin
depends on the particular host–pathogen interaction. For production) promotes petiole abscission. Application of
example, blocking the ethylene response does not affect the exogenous auxin to petioles from which the leaf blade has
resistance response to Pseudomonas bacteria in Arabidopsis been removed delays the abscission process. However,
or to tobacco mosaic virus in tobacco. In compatible application of auxin to the proximal side of the abscission
interactions of these pathogens and hosts, however, zone (i.e., the side closest to the stem) actually accelerates
elimination of ethylene responsiveness prevents the the abscission process. These results indicate that it is not
development of disease symptoms, even though the growth the absolute amount of auxin at the abscission zone, but
of the pathogen appears to be unaffected. rather the auxin gradient, that controls the ethylene
sensitivity of these cells.
On the other hand, ethylene, in combination with jasmonic
acid, is required for the activation of several plant defense In the shedding induction phase, the amount of auxin from
genes. In addition, ethylene‐insensitive tobacco and the leaf decreases and the ethylene level rises. Ethylene

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appears to decrease the activity of auxin both by reducing its of ACC synthase and ACC oxidase has already been
synthesis and transport and by increasing its destruction. mentioned. Another example of this technology is in petunia,
The reduction in the concentration of free auxin increases in which ethylene biosynthesis has been blocked by
the response of specific target cells to ethylene. transformation of an antisense version of ACC oxidase.
Senescence and petal wilting of cut flowers are delayed for
The shedding phase is characterized by the induction of weeks in these transgenic plants.
genes encoding specific hydrolytic enzymes of cell wall
polysaccharides and proteins. The target cells, located in the CELLULAR AND MOLECULAR MODES OF ETHYLENE
abscission zone, synthesize cellulase and other ACTION
polysaccharide‐degrading enzymes, and secrete them into
the cell wall via secretory vesicles derived from the Golgi. Despite the broad range of ethylene’s effects on
The action of these enzymes leads to cell wall loosening, cell development, the primary steps in ethylene action are
separation, and abscission. assumed to be similar in all cases: They all involve binding to
a receptor, followed by activation of one or more signal
Ethylene Has Important Commercial Uses transduction pathways leading to the cellular response.
Ultimately, ethylene exerts its effects primarily by altering
Because ethylene regulates so many physiological processes the pattern of gene expression. In recent years, remarkable
in plant development, it is one of the most widely used plant progress has been made in our understanding of ethylene
hormones in agriculture. Auxins and ACC can trigger the perception, as the result of molecular genetic studies of
natural biosynthesis of ethylene and in several cases are Arabidopsis thaliana.
used in agricultural practice. Because of its high diffusion
rate, ethylene is very difficult to apply in the field as a gas, One key to the elucidation of ethylene signaling components
but this limitation can be overcome if an ethylene‐releasing has been the use of the triple‐response morphology of
compound is used. The most widely used such compound is etiolated Arabidopsis seedlings to isolate mutants affected in
ethephon, or 2‐ chloroethylphosphonic acid, which was their response to ethylene. Two classes of mutants have
discovered in the 1960s and is known by various trade been identified by experiments in which mutagenized
names, such as Ethrel. Arabidopsis seeds were grown on an agar medium in the
presence or absence of ethylene for 3 days in the dark:
Ethephon is sprayed in aqueous solution and is readily 1. Mutants that fail to respond to exogenous ethylene
absorbed and transported within the plant. It releases (ethylene‐resistant or ethylene‐insensitive mutants)
ethylene slowly by a chemical reaction, allowing the 2. Mutants that display the response even in the absence of
hormone to exert its effects: Ethephon hastens fruit ripening ethylene (constitutive mutants)
of apple and tomato and degreening of citrus, synchronizes
flowering and fruit set in pineapple, and accelerates Ethylene‐insensitive mutants are identified as tall seedlings
abscission of flowers and fruits. It can be used to induce fruit extending above the lawn of short, tripleresponding
thinning or fruit drop in cotton, cherry, and walnut. It is also seedlings when grown in the presence of ethylene.
used to promote female sex expression in cucumber, to Conversely, constitutive ethylene response mutants are
prevent self‐ pollination and increase yield, and to inhibit identified as seedlings displaying the triple response in the
terminal growth of some plants in order to promote lateral absence of exogenous ethylene.
growth and compact flowering stems.
Ethylene Receptors Are Related to Bacterial Two­
Storage facilities developed to inhibit ethylene production Component System Histidine Kinases
and promote preservation of fruits have a controlled
atmosphere of low O2 concentration and low temperature The first ethylene‐insensitive mutant isolated was etr1
that inhibits ethylene biosynthesis. A relatively high (ethylene‐resistant 1). The etr1 mutant was identified in a
concentration of CO2 (3 to 5%) prevents ethylene’s action as screen for mutations that block the response of Arabidopsis
a ripening promoter. Low pressure (vacuum) is used to seedlings to ethylene. The amino acid sequence of the
remove ethylene and oxygen from the storage chambers, carboxy‐terminal half of ETR1 is similar to bacterial two‐
reducing the rate of ripening and preventing overripening. component histidine kinases—receptors used by bacteria to
Specific inhibitors of ethylene biosynthesis and action are perceive various environmental cues, such as chemo‐
also useful in postharvest preservation. Silver (Ag+) is used sensory stimuli, phosphate availability, and osmolarity.
extensively to increase the longevity of cut carnations and
several other flowers. The potent inhibitor AVG retards fruit Bacterial two‐component systems consist of a sensor
ripening and flower fading, but its commercial use has not histidine kinase and a response regulator, which often acts
yet been approved by regulatory agencies. The strong, as a transcription factor. ETR1 was the first example of a
eukaryotic histidine kinase, but others have since been
found in yeast, mammals, and plants. Both phytochrome and
the cytokinin receptor also share sequence similarity to
bacterial two‐component histidine kinases.

The similarity to bacterial receptors and the ethylene

insensitivity of the etr1 mutants suggested that ETR1 might
offensive odor of trans‐cyclooctene precludes its use in be an ethylene receptor. Consistent with this hypothesis,
agriculture. Currently, 1‐methylcyclopropene (MCP) is being ETR1 expression in yeast conferred the ability to bind
developed for use in a variety of postharvest applications. radiolabeled ethylene with an affinity that closely parallels
The near future may see a variety of agriculturally important the dose‐response curve of Arabidopsis seedlings to
species that have been genetically modified to manipulate ethylene.
the biosynthesis of ethylene or its perception. The inhibition
of ripening in tomato by expression of an antisense version The Arabidopsis genome encodes four additional proteins
similar to ETR1 that also function as ethylene receptors:

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ETR2, ERS1 (ETR1‐related sequence 1), ERS2, and EIN4. Like

ETR1, these receptors have been shown to bind ethylene,
and missense mutations in the genes that encode these
proteins, analogous to the original etr1 mutation, prevent
ethylene binding to the receptor while allowing the receptor
to function normally as a regulator of the ethylene response
pathway in the absence of ethylene.

All of these proteins share at least two domains:

1. The amino‐terminal domain spans the membrane at least
three times and contains the ethylene‐binding
site. Ethylene can readily access this site because of its
hydrophobicity.
2. The middle portion of the ethylene receptors contains a
histidine kinase catalytic domain.

A subset of the ethylene receptors also have a

carboxyterminal domain that is similar to bacterial two‐
component receiver domains. In other two‐component
systems, binding of ligand regulates the activity of the
histidine kinase domain, which autophosphorylates a Unbound Ethylene Receptors Are Negative Regulators of
conserved histidine residue. The phosphate is then the Response Pathway
transferred to an aspartic acid residue located within the
fused receiver domain. Although histidine kinase activity has In Arabidopsis, tomato, and probably most other plant
been demonstrated for one of the ethylene receptors— species, the ethylene receptors are encoded by multigene
ETR1—several others are missing critical amino acids, families. Targeted disruption (complete inactivation) of the
making it unlikely that they possess histidine kinase activity. five Arabidopsis ethylene receptors (ETR1, ETR2, ERS1,
Thus the biochemical mechanism of these ethylene ERS2, and EIN4) has revealed that they are functionally
receptors is not known. redundant. That is, disruption of any single gene encoding
one of these proteins has no effect, but a plant with
Recent studies indicate that ETR1 is located on the disruptions in all five receptor genes exhibits a constitutive
endoplasmic reticulum, rather than on the plasma membrane ethylene response phenotype.
as originally assumed. Such an intracellular location for the
ethylene receptor is consistent with the hydrophobic nature The observation that ethylene responses, such as the triple
of ethylene, which enables it to pass freely through the response, become constitutive when the receptors are
plasma membrane into the cell. In this respect ethylene is disrupted indicates that the receptors are normally “on” (i.e.,
similar to the hydrophobic signaling molecules of animals, in the active state) in the absence of ethylene, and that the
such as steroids and the gas nitric oxide, which also bind to function of the receptor minus its ligand (ethylene), is to shut
intracellular receptors. off the signaling pathway that leads to the response. Binding
of ethylene turns off the receptors, thus allowing the
High­Affinity Binding of Ethylene to Its Receptor response pathway to proceed.
Requires a Copper Cofactor
This somewhat counterintuitive model for ethylene
Even prior to the identification of its receptor, scientists had receptors as negative regulators of a signaling pathway is
predicted that ethylene would bind to its receptor via a unlike the mechanism of most animal receptors, which, after
transition metal cofactor, most likely copper or zinc. This binding their ligands, serve as positive regulators of their
prediction was based on the high affinity of olefins, such as respective signal transduction pathways. In contrast to the
ethylene, for these transition metals. Recent genetic and disrupted receptors, receptors with missense mutations at
biochemical studies have borne out these predictions. the ethylene binding site (as occurs in the original etr1
mutant) are unable to bind ethylene, but are still active as
Analysis of the ETR1 ethylene receptor expressed in yeast negative regulators of the ethylene response pathway. Such
demonstrated that a copper ion was coordinated to the missense mutations result in a plant that expresses a subset
protein and that this copper was necessary for highaffinity of receptors that can no longer be turned off by ethylene,
ethylene binding. Silver ion could substitute for copper to and thus confer a dominant ethylene­insensitive phenotype.
yield high‐affinity binding, which indicates that silver blocks Even though the normal receptors can all be turned off by
the action of ethylene not by interfering with ethylene ethylene, the mutant receptors continue to signal the cell to
binding, but by preventing the changes in the protein that suppress ethylene responses whether ethylene is present or
normally occur when ethylene binds to the receptor. not.
Evidence that copper binding is required for ethylene A Serine/Threonine Protein Kinase Is Also Involved in
receptor function in vivo came from identification of the Ethylene Signaling
RAN1 gene in Arabidopsis. Strong ran1 mutations block the
formation of functional ethylene receptors. Cloning of RAN1 The recessive ctr1 (constitutive triple response 1 = triple
revealed that it encodes a protein similar to a yeast protein response in the absence of ethylene) mutation was identified
required for the transfer of a copper ion cofactor to an iron in screens for mutations that constitutively activated
transport protein. In an analogous manner, RAN1 is likely to ethylene responses. The fact that the mutation caused an
be involved in the addition of a copper ion cofactor activation of the ethylene response suggests that the wild‐
necessary for the function of the ethylene receptors. type protein also acts as a negative regulator of the response
pathway, similar to the ethylene receptors.

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CTR1 appears to be related to RAF‐1, a MAPKKK serine/

threonine protein kinase (mitogen‐activated protein kinase Ethylene Regulates Gene Expression
kinase kinase) that is involved in the transduction of various
external regulatory signals and developmental signaling One of the primary effects of ethylene signaling is an
pathways in organisms ranging from yeast to humans. In alteration in the expression of various target genes. Ethylene
animal cells, the final product in the MAP kinase cascade is a affects the mRNA transcript levels of numerous genes,
phosphorylated transcription factor that regulates gene including the genes that encode cellulase, as well as
expression in the nucleus. ripening‐ related genes and ethylene biosynthesis genes.
Regulatory sequences called ethylene response elements,
EIN2 Encodes a Transmembrane Protein or EREs, have been identified from the ethylene‐regulated
genes. Key components mediating ethylene’s effects on gene
The ein2 (ethylene‐insensitive 2) mutation blocks all expression are the EIN3 family of transcription factors.
ethylene responses in both seedling and adult Arabidopsis There are at least four EIN3‐like genes in Arabidopsis, and
plants. The EIN2 gene encodes a protein containing 12 homologs have been identified in both tomato and tobacco.
membranespanning domains that is most similar to the N‐ In response to an ethylene signal, homodimers of EIN3 or its
RAMP (natural resistance–associated macrophage protein) paralogs (closely related proteins), bind to the promoter of a
family of cation transporters in animals, suggesting that it gene called ERF1 (ethylene response factor 1) and activate
may act as a channel or pore. To date, however, researchers its transcription.
have failed to demonstrate a transport activity for this
protein, and the intracellular location of the protein is not ERF1 encodes a protein that belongs to the ERE­binding
known. protein (EREBP) family of transcription factors, which were
first identified in tobacco as proteins that bind to ERE
Interestingly, mutations in the EIN2 gene have also been sequences. Several EREBPs are rapidly up‐regulated in
identified in genetic screens for resistance to other response to ethylene. The EREBP genes exist in Arabidopsis
hormones, such as jasmonic acid and ABA, suggesting that as a very large gene family, but only a few of the genes are
EIN2 may be a common intermediate in the signal inducible by ethylene.
transduction pathways of various hormones and other
chemical signals.

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Genetic Epistasis Reveals the Order of the Ethylene From this it can be inferred that CTR1 acts downstream of
Signaling Components ETR1. In this way, the order of action of ETR1, EIN2, and
EIN3 were determined relative to CTR1.
The order of action of the genes ETR1, EIN2, EIN3, and CTR1
has been determined by the analysis of how the mutations The ETR1 protein has been shown to interact physically
interact with each other (i.e., their epistatic order). Two with the predicted downstream protein, CTR1, suggesting
mutants with opposite phenotypes are crossed, and a line that the ethylene receptors may directly regulate the kinase
harboring both mutations (the double mutant) is identified activity of CTR1. The model in Figure summarizes these and
in the F2 generation. In the case of the ethylene response other data. Genes that are similar to several of these
mutants, researchers constructed a line doubly mutant for Arabidopsis signaling genes have been found in other species
ctr1, a constitutive ethylene response mutant, and one of the .
ethylene‐insensitive mutations.
This model is still incomplete because other ethylene
The phenotype that the double mutant displays reveals response mutations have been identified that act in this
which of the mutations is epistatic to the other. For example, pathway. In addition, we are only beginning to understand
if an etr1/ctr1 double mutant displays a ctr1 mutant the biochemical properties of these proteins and how they
phenotype, the ctr1 mutation is said to be epistatic to etr1. interact. However, we are beginning to glimpse the outline
of the molecular basis for the perception and transduction of
this hormonal signal.

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5. ABSCISSIC ACID The Chemical Structure of ABA Determines Its

Physiological Activity
THE EXTENT AND TIMING OF PLANT GROWTH are
controlled by the coordinated actions of positive and ABA is a 15‐carbon compound that resembles the terminal
negative regulators. Some of the most obvious examples of portion of some carotenoid molecules (Figure). The
regulated nongrowth are seed and bud dormancy, adaptive orientation of the carboxyl group at carbon 2 determines the
features that delay growth until environmental conditions cis and trans isomers of ABA. Nearly all the naturally
are favorable. For many years, plant physiologists suspected occurring ABA is in the cis form, and by convention the name
that the phenomena of seed and bud dormancy were caused abscisic acid refers to that isomer.
by inhibitory compounds, and they attempted to extract and
isolate such compounds from a variety of plant tissues, ABA also has an asymmetric carbon atom at position 1′in the
especially dormant buds. ring, resulting in the S and R (or + and –, respectively)
enantiomers. The S enantiomer is the natural form;
Early experiments used paper chromatography for the commercially available synthetic ABA is a mixture of
separation of plant extracts, as well as bioassays based on approximately equal amounts of the S and R forms. The S
oat coleoptile growth. These early experiments led to the enantiomer is the only one that is active in fast responses to
identification of a group of growth‐inhibiting compounds, ABA, such as stomatal closure. In long‐term responses, such
including a substance known as dormin purified from as seed maturation, both enantiomers are active. In contrast
sycamore leaves collected in early autumn, when the trees to the cis and trans isomers, the S and R forms cannot be
were entering dormancy. Upon discovery that dormin was interconverted in the plant tissue.
chemically identical to a substance that promotes the
abscission of cotton fruits, abscisin II, the compound was Studies of the structural requirements for biological activity
renamed abscisic acid (ABA), to reflect its supposed of ABA have shown that almost any change in the molecule
involvement in the abscission process. results in loss of activity.

It is now known that ethylene is the hormone that triggers ABA Is Assayed by Biological, Physical, and Chemical
abscission and that ABA‐induced abscission of cotton fruits Methods
is due to ABA’s ability to stimulate ethylene production. As
will be discussed in this chapter, ABAis now recognized as Avariety of bioassays have been used for ABA, including
an important plant hormone in its own right. It inhibits inhibition of coleoptile growth, germination, or GAinduced
growth and stomatal opening, particularly when the plant is ‐amylase synthesis. Alternatively, promotion of stomatal
under environmental stress. Another important function is closure and gene expression are examples of rapid inductive
its regulation of seed maturation and dormancy. In responses.
retrospect, dormin would have been a more appropriate
name for this hormone, but the name abscisic acid is firmly Physical methods of detection are much more reliable than
entrenched in the literature. bioassays because of their specificity and suitability for
quantitative analysis. The most widely used techniques are
OCCURRENCE, CHEMICAL STRUCTURE, AND those based on gas chromatography or high‐performance
MEASUREMENT OF ABA liquid chromatography (HPLC). Gas chromatography allows
detection of as little as 10–13 g ABA, but it requires several
Abscisic acid has been found to be a ubiquitous plant preliminary purification steps, including thin‐layer
hormone in vascular plants. It has been detected in mosses chromatography. Immunoassays are also highly sensitive
but appears to be absent in liverworts. Several genera of and specific.
fungi make ABA as a secondary metabolite. Within the plant,
ABA has been detected in every major organ or living tissue BIOSYNTHESIS, METABOLISM, AND TRANSPORT OF ABA
from the root cap to the apical bud. ABA is synthesized in
almost all cells that contain chloroplasts or amyloplasts. As with the other hormones, the response to ABA depends
on its concentration within the tissue and on the sensitivity
of the tissue to the hormone. The processes of biosynthesis,
catabolism, compartmentation, and transport all contribute
to the concentration of active hormone in the tissue at any
given stage of development. The complete biosynthetic
pathway of ABA was elucidated with the aid of ABA‐deficient
mutants blocked at specific steps in the pathway.

ABA Is Synthesized from a Carotenoid Intermediate

ABA biosynthesis takes place in chloroplasts and other

plastids via the pathway depicted in Figure. Several ABA‐
deficient mutants have been identified with lesions at
specific steps of the pathway. These mutants exhibit
abnormal phenotypes that can be corrected by the
application of exogenous ABA. For example, flacca (flc) and
sitiens (sit) are “wilty mutants” of tomato in which the
tendency of the leaves to wilt (due to an inability to close
their stomata) can be prevented by the application of
exogenous ABA. The aba mutants of Arabidopsis also exhibit
a wilty phenotype. These and other mutants have been
useful in elucidating the details of the pathway.

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pathway also have reduced levels of ABA and exhibit

The pathway begins with isopentenyl diphosphate (IPP), the vivipary—the precocious germination of seeds in the fruit
biological isoprene unit, and leads to the synthesis of the while still attached to the plant. Vivipary is a feature of many
C40 xanthophyll (i.e., oxygenated carotenoid) violaxanthin. ABA‐deficient seeds.
Synthesis of violaxanthin is catalyzed by zeaxanthin
epoxidase (ZEP), the enzyme encoded by the ABA1 locus of Violaxanthin is converted to the C40 compound 9′­
Arabidopsis. This discovery provided conclusive evidence cisneoxanthin, which is then cleaved to form the C15
that ABA synthesis occurs via the “indirect” or carotenoid compound xanthoxal, previously called xanthoxin, a neutral
pathway, rather than as a small molecule. Maize mutants growth inhibitor that has physiological properties similar to
(vp) that are blocked at other steps in the carotenoid those of ABA. The cleavage is catalyzed by 9­cis­

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epoxycarotenoid dioxygenase (NCED), so named because radioactive ABAis applied to a leaf, it is transported both up
it can cleave both 9‐cis‐violaxanthin and 9′‐cis‐neoxanthin. the stem and down toward the roots. Most of the radioactive
Synthesis of NCED is rapidly induced by water stress, ABA is found in the roots within 24 hours. Destruction of the
suggesting that the reaction it catalyzes is a key regulatory phloem by a stem girdle prevents ABA accumulation in the
step for ABA synthesis. The enzyme is localized on the roots, indicating that the hormone is transported in the
thylakoids, where the carotenoid substrate is located. phloem sap.
Finally, xanthoxal is converted to ABA via oxidative steps
involving the intermediate(s) ABA­aldehyde and/or ABAsynthesized in the roots can also be transported to the
possibly xanthoxic acid. This final step is catalyzed by a shoot via the xylem. Whereas the concentration of ABA in
family of aldehyde oxidases that all require a molybdenum the xylem sap of well‐watered sunflower plants is between
cofactor; the aba3 mutants of Arabidopsis lack a functional 1.0 and 15.0 nM, the ABAconcentration in waterstressed
molybdenum cofactor and are therefore unable to sunflower plants increases to as much as 3000 nM (3.0 µM ).
synthesize ABA. The magnitude of the stress induced change in xylem ABA
content varies widely among species, and it has been
ABA Concentrations in Tissues Are Highly Variable suggested that ABA also is transported in a conjugated form,
then released by hydrolysis in leaves. However, the
ABA biosynthesis and concentrations can fluctuate postulated hydrolases have yet to be identified.
dramatically in specific tissues during development or in
response to changing environmental conditions. In As water stress begins, some of the ABA carried by the xylem
developing seeds, for example, ABAlevels can increase 100‐ stream is synthesized in roots that are in direct contact with
fold within a few days and then decline to vanishingly low the drying soil. Because this transport can occur before the
levels as maturation proceeds. Under conditions of water low water potential of the soil causes any measurable
stress, ABA in the leaves can increase 50‐fold within 4 to 8 change in the water status of the leaves, ABA is believed to
hours. Upon rewatering, the ABAlevel declines to normal in be a root signal that helps reduce the transpiration rate by
the same amount of time. Biosynthesis is not the only factor closing stomata in leaves.
that regulates ABA concentrations in the tissue. As with
other plant hormones, the concentration of free ABAin the Although a concentration of 3.0 µM ABA in the apoplast is
cytosol is also regulated by degradation, compartmentation, sufficient to close stomata, not all of the ABA in the xylem
conjugation, and transport. For example, cytosolic ABA stream reaches the guard cells. Much of the ABA in the
increases during water stress as a result of synthesis in the transpiration stream is taken up and metabolized by the
leaf, redistribution within the mesophyll cell, import from mesophyll cells. During the early stages of water stress,
the roots, and recirculation from other leaves. The however, the pH of the xylem sap becomes more alkaline,
concentration of ABA declines after rewatering because of increasing from about pH 6.3 to about pH 7.2.
degradation and export from the leaf, as well as a decrease
in the rate of synthesis. The major control of ABA distribution among plant cell
compartments follows the “anion trap” concept: The
ABA Can Be Inactivated by Oxidation or Conjugation dissociated (anion) form of this weak acid accumulates in
alkaline compartments and may be redistributed according
A major cause of the inactivation of free ABA is oxidation, to the steepness of the pH gradients across membranes. In
yielding the unstable intermediate 6‐hydroxymethyl ABA, addition to partitioning according to the relative pH of
which is rapidly converted to phaseic acid (PA) and compartments, specific uptake carriers contribute to
dihydrophaseic acid (DPA). PAis usually inactive, or it maintaining a low apoplastic ABA concentration in
exhibits greatly reduced activity, in bioassays. However, unstressed plants.
PAcan induce stomatal closure in some species, and it is as
active as ABA in inhibiting gibberellic acid–induced ‐
amylase production in barley aleurone layers. These effects
suggest that PA may be able to bind to ABA receptors. In
contrast to PA, DPA has no detectable activity in any of the
bioassays tested.

Free ABA is also inactivated by covalent conjugation to

another molecule, such as a monosaccharide. A common
example of an ABA conjugate is ABA­b­D­glucosyl ester
(ABA­GE). Conjugation not only renders ABA inactive as a
hormone; it also alters its polarity and cellular distribution.
Whereas free ABAis localized in the cytosol, ABA‐GE
accumulates in vacuoles and thus could theoretically serve
as a storage form of the hormone. Esterase enzymes in plant
cells could release free ABA from the conjugated form.
However, there is no evidence that ABA‐GE hydrolysis
contributes to the rapid increase in ABA in the leaf during
water stress. When plants were subjected to a series of Stress‐induced alkalinization of the apoplast favors
stress and rewatering cycles, the ABA GE concentration formation of the dissociated form of abscisic acid, ABA–,
increased steadily, suggesting that the conjugated form is which does not readily cross membranes. Hence, less ABA
not broken down during water stress. enters the mesophyll cells, and more reaches the guard cells
via the transpiration stream. Note that ABA is redistributed
ABA Is Translocated in Vascular Tissue in the leaf in this way without any increase in the total ABA
level. This increase in xylem sap pH may function as a root
ABA is transported by both the xylem and the phloem, but it signal that promotes early closure of the stomata.
is normally much more abundant in the phloem sap. When

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DEVELOPMENTAL AND PHYSIOLOGICAL EFFECTS OF be affecting the translocation of sugars and amino acids, the
ABA synthesis of the reserve materials, or both. Studies in
mutants impaired in both ABA synthesis and response
Abscisic acid plays primary regulatory roles in the initiation showed no effect of ABA on sugar translocation. In contrast,
and maintenance of seed and bud dormancy and in the ABA has been shown to affect the amounts and composition
plant’s response to stress, particularly water stress. In of storage proteins. For example, exogenous ABA promotes
addition, ABA influences many other aspects of plant accumulation of storage proteins in cultured embryos of
development by interacting, usually as an antagonist, with many species, and some ABA‐deficient or ‐insensitive
auxin, cytokinin, gibberellin, ethylene, and brassinosteroids. mutants have reduced storage protein accumulation.
In this section we will explore the diverse physiological However, storage protein synthesis is also reduced in other
effects of ABA, beginning with its role in seed development. seed developmental mutants with normal ABA levels and
responses, indicating that ABA is only one of several signals
ABA Levels in Seeds Peak during Embryogenesis controlling the expression of storage protein genes during
embryogenesis. ABA not only regulates the accumulation of
Seed development can be divided into three phases of storage proteins during embryogenesis; it can also maintain
approximately equal duration: the mature embryo in a dormant state until the
1. During the first phase, which is characterized by cell environmental conditions are optimal for growth. Seed
divisions and tissue differentiation, the zygote undergoes dormancy is an important factor in the adaptation of plants
embryogenesis and the endosperm tissue proliferates. to unfavorable environments. As we will discuss in the next
2. During the second phase, cell divisions cease and storage few sections, plants have evolved a variety of mechanisms,
compounds accumulate. some of them involving ABA, that enable them to maintain
3. In the final phase, the embryo becomes tolerant to their seeds in a dormant state.
desiccation, and the seed dehydrates, losing up to 90% of its
water. As a consequence of dehydration, metabolism comes Seed Dormancy May Be Imposed by the Coat or the
to a halt and the seed enters a quiescent (“resting”) state. In Embryo
contrast to dormant seeds, quiescent seeds will germinate
upon rehydration. During seed maturation, the embryo enters a quiescent
phase in response to desiccation. Seed germination can be
The latter two phases result in the production of viable defined as the resumption of growth of the embryo of the
seeds with adequate resources to support germination and mature seed; it depends on the same environmental
the capacity to wait weeks to years before resuming growth. conditions as vegetative growth does. Water and oxygen
Typically, the ABA content of seeds is very low early in must be available, the temperature must be suitable, and
embryogenesis, reaches a maximum at about the halfway there must be no inhibitory substances present.
point, and then gradually falls to low levels as the seed
reaches maturity. Thus there is a broad peak of ABA In many cases a viable (living) seed will not germinate even
accumulation in the seed corresponding to mid‐ to late if all the necessary environmental conditions for growth are
embryogenesis. satisfied. This phenomenon is termed seed dormancy. Seed
dormancy introduces a temporal delay in the germination
The hormonal balance of seeds is complicated by the fact process that provides additional time for seed dispersal over
that not all the tissues have the same genotype. The seed greater geographic distances. It also maximizes seedling
coat is derived from maternal tissues; the zygote and survival by preventing germination under unfavorable
endosperm are derived from both parents. conditions. Two types of seed dormancy have been
recognized: coat‐imposed dormancy and embryo dormancy.
Genetic studies with ABA‐deficient mutants of Arabidopsis
have shown that the zygotic genotype controls ABA Coat­imposed dormancy. Dormancy imposed on the
synthesis in the embryo and endosperm and is essential to embryo by the seed coat and other enclosing tissues, such as
dormancy induction, whereas the maternal genotype endosperm, pericarp, or extrafloral organs, is known as
controls the major, early peak of ABA accumulation and coat­imposed dormancy. The embryos of such seeds will
helps suppress vivipary in mid‐embryogenesis. germinate readily in the presence of water and oxygen once
the seed coat and other surrounding tissues have been
ABA Promotes Desiccation Tolerance in the Embryo either removed or damaged. There are five basic
mechanisms of coat‐imposed dormancy:
An important function of ABAin the developing seed is to
promote the acquisition of desiccation tolerance. Desiccation 1. Prevention of water uptake.
can severely damage membranes and other cellular 2. Mechanical constraint. The first visible sign of germination
constituents. During the mid‐ to late stages of seed is typically the radicle breaking through the seed coat. In
development, specific mRNAs accumulate in embryos at the some cases, however, the seed coat may be too rigid for the
time of high levels of endogenous ABA. These mRNAs encode radicle to penetrate. For the seeds to germinate, the
so‐called late­embryogenesis­abundant (LEA) proteins endosperm cell walls must be weakened by the production
thought to be involved in desiccation tolerance. Synthesis of of cell wall–degrading enzymes.
many LEA proteins, or related family members, can be 3. Interference with gas exchange. Lowered permeability of
induced by ABA treatment of either young embryos or seed coats to oxygen suggests that the seed coat inhibits
vegetative tissues. Thus the synthesis of most LEA proteins germination by limiting the oxygen supply to the embryo.
is under ABA control. 4. Retention of inhibitors. The seed coat may prevent the
escape of inhibitors from the seed.
ABA Promotes the Accumulation of Seed Storage Protein 5. Inhibitor production. Seed coats and pericarps may contain
during Embryogenesis relatively high concentrations of growth inhibitors,
including ABA, that can suppress germination of the embryo
Storage compounds accumulate during mid‐ to late
embryogenesis. Because ABA levels are still high, ABA could

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Embryo dormancy. The second type of seed dormancy is seeds that require afterripening, chilling, or light to
embryo dormancy, a dormancy that is intrinsic to the germinate.
embryo and is not due to any influence of the seed coat or
other surrounding tissues. In some cases, embryo dormancy ABA mutants have been extremely useful in demonstrating
can be relieved by amputation of the cotyledons. Species in the role of ABA in seed dormancy. Dormancy of Arabidopsis
which the cotyledons exert an inhibitory effect include seeds can be overcome with a period of after ripening
European hazel (Corylus avellana) and European ash and/or cold treatment. ABA‐deficient (aba) mutants of
(Fraxinus excelsior). Arabidopsis have been shown to be non‐dormant at
maturity. When reciprocal crosses between aba and wild
A fascinating demonstration of the cotyledon’s ability to type plants were carried out, the seeds exhibited dormancy
inhibit growth is found in species (e.g., peach) in which the only when the embryo itself produced the ABA. Neither
isolated dormant embryos germinate but grow extremely maternal nor exogenously applied ABA was effective in
slowly to form a dwarf plant. If the cotyledons are removed inducing dormancy in an aba embryo. On the other hand,
at an early stage of development, however, the plant maternally derived ABA constitutes the major peak present
abruptly shifts to normal growth. in seeds and is required for other aspects of seed
development—for example, helping suppress vivipary in
Embryo dormancy is thought to be due to the presence of midembryogenesis. Thus the two sources of ABA function in
inhibitors, especially ABA, as well as the absence of growth different developmental pathways. Dormancy is also greatly
promoters, such as GA (gibberellic acid). The loss of embryo reduced in seeds from the ABA insensitive mutants abi1
dormancy is often associated with a sharp drop in the ratio (ABA­insensitive1), abi2, and abi3, even though these seeds
of ABA to GA. contain higher ABA concentrations than those of the wild
type throughout development, possibly reflecting feedback
Primary versus secondary seed dormancy. Different types regulation of ABA metabolism.
of seed dormancy also can be distinguished on the basis of
the timing of dormancy onset rather than the cause of ABA‐deficient tomato mutants seem to function in the same
dormancy: way, indicating that the phenomenon is probably a general
• Seeds that are released from the plant in a dormant state one. However, other mutants with reduced dormancy, but
are said to exhibit primary dormancy. normal ABA levels and sensitivity, point to additional
• Seeds that are released from the plant in a non dormant regulators of dormancy. Although the role of ABA in
state, but that become dormant if the conditions for initiating and maintaining seed dormancy is well
germination are unfavorable, exhibit secondary dormancy. established, other hormones contribute to the overall effect.
For example, in most plants the peak of ABA production in
For example, seeds of Avena sativa (oat) can become the seed coincides with a decline in the levels of IAA and GA.
dormant in the presence of temperatures higher than the
maximum for germination, whereas seeds of Phacelia dubia An elegant demonstration of the importance of the ratio of
(small‐flower scorpion weed) become dormant at ABA to GA in seeds was provided by the genetic screen that
temperatures below the minimum for germination. The led to isolation of the first ABA‐deficient mutants of
mechanisms of secondary dormancy are poorly understood. Arabidopsis. Seeds of a GA‐deficient mutant that could not
germinate in the absence of exogenous GA were
Environmental Factors Control the Release from Seed mutagenized and then grown in the greenhouse.
Dormancy
The seeds produced by these mutagenized plants were then
Various external factors release the seed from embryo screened for revertants—that is, seeds that had regained
dormancy, and dormant seeds typically respond to more their ability to germinate. Revertants were isolated, and they
than one of three factors: turned out to be mutants of abscisic acid synthesis. The
1. After ripening. Many seeds lose their dormancy when their revertants germinated because dormancy had not been
moisture content is reduced to a certain level by drying—a induced, so subsequent synthesis of GA was no longer
phenomenon known as afterripening. required to overcome it. This study elegantly illustrates the
2. Chilling. Low temperature, or chilling, can release seeds general principle that the balance of plant hormones is often
from dormancy. Many seeds require a period of cold (0– more critical than are their absolute concentrations in
10°C) while in a fully hydrated (imbibed) state in order to regulating development. However, ABA and GA exert their
germinate. effects on seed dormancy at different times, so their
3. Light. Many seeds have a light requirement for antagonistic effects on dormancy do not necessarily reflect a
germination, which may involve only a brief exposure, as in direct interaction.
the case of lettuce, an intermittent treatment (e.g.,
succulents of the genus Kalanchoe), or even a specific Recent genetic screens for suppressors of ABA insensitivity
photoperiod involving short or long days. have identified additional antagonistic interactions between
ABA and ethylene or brassinosteroid effects on germination.
Seed Dormancy Is Controlled by the Ratio of ABA to GA In addition, many new alleles of ABA‐deficient or ABA­
insensitive4 (abi4) mutants have been identified in screens
Mature seeds may be either dormant or nondormant, for altered sensitivity to sugar. These studies show that a
depending on the species. Nondormant seeds, such as pea, complex regulatory web integrates hormonal and nutrient
will germinate readily if provided with water only. Dormant signaling.
seeds, on the other hand, fail to germinate in the presence of
water, and instead require some additional treatment or ABA Inhibits Precocious Germination and Vivipary
condition. As we have seen, dormancy may arise from the
rigidity or impermeability of the seed coat (coat imposed When immature embryos are removed from their seeds and
dormancy) or from the persistence of the state of arrested placed in culture midway through development before the
development of the embryo. Examples of the latter include onset of dormancy, they germinate precociously—that is,
without passing through the normal quiescent and/or

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dormant stage of development. ABA added to the culture underscores the need for extending such approaches to
medium inhibits precocious germination. This result, in woody species.
combination with the fact that the level of endogenous ABA
is high during mid‐ to late seed development, suggests that Analyses of traits such as dormancy are complicated by the
ABA is the natural constraint that keeps developing embryos fact that they are often controlled by the combined action of
in their embryogenic state. several genes, resulting in a gradation of phenotypes
referred to as quantitative traits. Recent genetic mapping
Further evidence for the role of ABA in preventing studies suggest that homologs of ABI1 may regulate bud
precocious germination has been provided by genetic dormancy in poplar trees. For a description of such studies
studies of vivipary. The tendency toward vivipary, also
known as preharvest sprouting, is a varietal characteristic in ABA Inhibits GA­Induced Enzyme Production
grain crops that is favored by wet weather. In maize, several
viviparous (vp) mutants have been selected in which the ABA inhibits the synthesis of hydrolytic enzymes that are
embryos germinate directly on the cob while still attached to essential for the breakdown of storage reserves in seeds. For
the plant. example, GA stimulates the aleurone layer of cereal grains to
produce α‐amylase and other hydrolytic enzymes that break
Several of these mutants are ABA deficient (vp2, vp5, vp7, down stored resources in the endosperm during
and vp14); one is ABA insensitive (vp1). Vivipary in the ABA‐ germination. ABAinhibits this GA‐dependent enzyme
deficient mutants can be partially prevented by treatment synthesis by inhibiting the transcription of ‐ amylase
with exogenous ABA. Vivipary in maize also requires mRNA. ABA exerts this inhibitory effect via at least two
synthesis of GA early in embryogenesis as a positive signal; mechanisms:
double mutants deficient in both GAand ABA do not exhibit 1. VP1, a protein originally identified as an activator of ABA‐
vivipary. induced gene expression, acts as a transcriptional repressor
of some GA‐regulated genes.
In contrast to the maize mutants, single‐gene mutants of 2. ABA represses the GA‐induced expression of GAMYB, a
Arabidopsis (aba1, aba3, abi1, and abi3) fail to exhibit transcription factor that mediates the GA induction of α‐
vivipary, although they are nondormant. The lack of vivipary amylase expression.
might reflect a lack of moisture because such seeds will
germinate within the fruits under conditions of high relative ABA Closes Stomata in Response to Water Stress
humidity. However, other Arabidopsis mutants with a
normal ABA response and only moderately reduced ABA Elucidation of the roles of ABA in freezing, salt, and water
levels (e.g., fusca3, which belongs to a class of mutants1 stress led to the characterization of ABA as a stress
defective in regulating the transition from embryogenesis to hormone. As noted earlier, ABA concentrations in leaves can
germination) exhibit some vivipary even at low humidities. increase up to 50 times under drought conditions—the most
Furthermore, double mutants combining either defects in dramatic change in concentration reported for any hormone
ABA biosynthesis or ABA response with the fusca3 mutation in response to an environmental signal. Redistribution or
have a high frequency of vivipary, suggesting that redundant biosynthesis of ABA is very effective in causing stomatal
control mechanisms suppress vivipary in Arabidopsis. closure, and its accumulation in stressed leaves plays an
important role in the reduction of water loss by
ABA Accumulates in Dormant Buds transpiration under water stress conditions.

In woody species, dormancy is an important adaptive Stomatal closing can also be caused by ABA synthesized in
feature in cold climates. When a tree is exposed to very low the roots and exported to the shoot. Mutants that lack the
temperatures in winter, it protects its meristems with bud ability to produce ABA exhibit permanent wilting and are
scales and temporarily stops bud growth. This response to called wilty mutants because of their inability to close their
low temperatures requires a sensory mechanism that stomata. Application of exogenous ABA to such mutants
detects the environmental changes (sensory signals), and a causes stomatal closure and a restoration of turgor pressure.
control system that transduces the sensory signals and
triggers the developmental processes leading to bud ABA Promotes Root Growth and Inhibits Shoot Growth
dormancy. ABA was originally suggested as the dormancy‐ at Low Water Potentials
inducing hormone because it accumulates in dormant buds
and decreases after the tissue is exposed to low ABA has different effects on the growth of roots and shoots,
temperatures. However, later studies showed that the ABA and the effects are strongly dependent on the water status of
content of buds does not always correlate with the degree of the plant. Endogenous ABA promotes shoot growth in well‐
dormancy. watered plants by suppressing ethylene production. When
water is limiting (i.e., at low water potentials), the opposite
As we saw in the case of seed dormancy, this apparent occurs: Shoot growth is greater in the ABA‐deficient mutant
discrepancy could reflect interactions between ABA and than in the wild type. Thus, endogenous ABA acts as a signal
other hormones as part of a process in which bud dormancy to reduce shoot growth only under water stress conditions.
and growth are regulated by the balance between bud
growth inhibitors, such as ABA, and growth‐inducing Now let’s examine how ABA affects roots. When water is
substances, such as cytokinins and gibberellins. abundant, root growth is slightly greater in the wild type
(normal endogenous ABA) than in the ABA‐deficient mutant,
Although much progress has been achieved in elucidating similar to growth in shoots. Therefore, at high water
the role of ABA in seed dormancy by the use of ABA‐deficient potentials (when the total ABA levels are low), endogenous
mutants, progress on the role of ABAin bud dormancy, which ABA exerts a slight positive effect on the growth of both
applies mainly to woody perennials, has lagged because of roots and shoots.
the lack of a convenient genetic system. This discrepancy
illustrates the tremendous contribution that genetics and Under dehydrating conditions, however, the growth of the
molecular biology have made to plant physiology, and it roots is much higher in the wild type than in the ABA

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deficient mutant, although growth is still inhibited relative protein conjugates have been shown to activate both ion
to root growth of either genotype when water is abundant. channel activity and gene expression.
In this case, endogenous ABA promotes root growth,
apparently by inhibiting ethylene production during water Other experiments, however, support an intracellular
stress. location for the ABA receptor: Extracellular application of
ABA was nearly twice as effective at inhibiting stomatal
To summarize, under dehydrating conditons, when ABA opening at pH 6.15, when it is fully protonated and readily
levels are high, the endogenous hormone exerts a strong taken up by guard cells, versus at pH 8, when it is largely
positive effect on root growth by suppressing ethylene dissociated to the anionic form that does not readily cross
production, and a slight negative effect on shoot growth. The membranes.
overall effect is a dramatic increase in the root:shoot ratio at • ABA supplied directly and continuously to the cytosol via a
low water potentials, which, along with the effect of ABA on patch pipette inhibited K+ in channels, which are required
stomatal closure, helps the plant cope with water stress. For for stomatal opening .
another example of the role of ABA in the response to • Microinjection of an inactive “caged” form of ABA into
dehydration guard cells of Commelina resulted in stomatal closure after
the stomata were treated briefly with UV irradiation to
ABA Promotes Leaf Senescence Independently of activate the hormone—that is, release it from its molecular
Ethylene cage. Control guard cells injected with a nonphotolyzable
form of the caged ABA did not close after UV irradiation.
Abscisic acid was originally isolated as an abscission‐causing
factor. However, it has since become evident that ABA Taken together, these results indicate that extracellular
stimulates abscission of organs in only a few species and perception of ABA can prevent stomatal opening and
that the primary hormone causing abscission is ethylene. On regulate gene expression, and intracellular ABA can both
the other hand, ABA is clearly involved in leaf senescence, induce stomatal closure and inhibit the K+ in current
and through its promotion of senescence it might indirectly required for opening. Thus there appear to be both
increase ethylene formation and stimulate abscission. extracellular and intracellular ABA receptors. However, they
have yet to be identified or localized.
Leaf segments senesce faster in darkness than in light, and
they turn yellow as a result of chlorophyll breakdown. In ABA Increases Cytosolic Ca2+, Raises Cytosolic pH, and
addition, the breakdown of proteins and nucleic acids is Depolarizes the Membrane
increased by the stimulation of several hydrolases. ABA
greatly accelerates the senescence of both leaf segments and Stomatal closure is driven by a reduction in guard cell turgor
attached leaves. pressure caused by a massive long‐term efflux of K+ and
anions from the cell. During the subsequent shrinkage of the
CELLULAR AND MOLECULAR MODES OF ABA ACTION cell due to water loss, the surface area of the plasma
membrane may contract by as much as 50%. Where does the
ABA is involved in short‐term physiological effects (e.g., extra membrane go? The answer seems to be that it is taken
stomatal closure), as well as long‐term developmental up as small vesicles by endocytosis—a process that also
processes (e.g., seed maturation). Rapid physiological involves reorganization of the actin cytoskeleton. However,
responses frequently involve alterations in the fluxes of ions the first changes detected after exposure of guard cells to
across membranes and may involve some gene regulation as ABA are transient membrane depolarization caused by the
well, and long‐term processes inevitably involve major net influx of positive charge, and transient increases in the
changes in the pattern of gene expression. Signal cytosolic calcium concentration.
transduction pathways, which amplify the primary signal
generated when the hormone binds to its receptor, are ABA stimulates elevations in the concentration of cytosolic
required for both the short‐term and the long term effects of Ca2+ by inducing both influx through plasma membrane
ABA. Genetic studies have shown that many conserved channels and release of calcium into the cytosol from
signaling components regulate both short‐ and long‐term internal compartments, such as the central vacuole.
responses, indicating that they share common signaling Stimulation of influx occurs via a pathway that uses reactive
mechanisms. In this section we will describe what is known oxygen species (ROS), such as hydrogen peroxide (H2O2) or
about the mechanism of ABA action at the cellular and superoxide (O2–), as secondary messengers leading to
molecular levels. plasma membrane channel activation. Calcium release from
intracellular stores can be induced by a variety of second
ABA Is Perceived Both Extracellularly and messengers, including inositol 1,4,5‐ trisphosphate (IP3),
Intracellularly cyclic ADP‐ribose (cADPR), and self‐ amplifying (calcium‐
induced) Ca2+ release. Recent studies have shown that ABA
Although ABA has been shown to interact directly with stimulates nitric oxide (NO) synthesis in guard cells, which
phospholipids, it is widely assumed that the ABA receptor is induces stomatal closure in a cADPR‐dependent manner,
a protein. To date, however, the protein receptor for ABA indicating that NO is an even earlier secondary messenger in
has not been identified. Experiments have been performed this response pathway.
to determine whether the hormone must enter the cell to be
effective, or whether it can act externally by binding to a The combination of calcium influx and the release of calcium
receptor located on the outer surface of the plasma from internal stores raises the cytosolic calcium
membrane. The results so far suggest multiple sites of concentration from 50 to 350 nM to as high as 1100 nM (1.1
perception. Some experiments point to a receptor on the mM). This increase is sufficient to cause stomatal closure, as
outer surface of the cell. For example, microinjected ABA demonstrated by the following experiment.
fails to alter stomatal opening in the spiderwort Commelina,
or to inhibit GA‐induced α‐amylase synthesis in barley As in the experiment described earlier, calcium was
aleurone protoplasts. Furthermore, impermeant ABA– microinjected into guard cells in a caged form that could be
hydrolyzed by a pulse of UV light. This method allowed the

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investigators to control both the concentration of free Another factor that can contribute to membrane
calcium and the time of release to the cytosol. At cytosolic depolarization is inhibition of the plasma membrane H+‐
concentrations of 600 nM or more, release of calcium from ATPase. ABAinhibits blue light–stimulated proton pumping
its cage triggered stomatal closure. This level of intracellular by guard cell protoplasts, consistent with the model that the
calcium is well within the concentration range observed depolarization of the plasma membrane by ABA is partially
after ABA treatment. caused by a decrease in the activity of the plasma membrane
H+‐ATPase. However, ABAdoes not inhibit the proton pump
In the preceding studies, intracellular free calcium was directly.
measured by the use of microinjected calcium‐sensitive
ratiometric fluorescent dyes2, such as fura‐2 or indo‐1. In Vicia faba (broad bean), at least, the plasma membrane
However, microinjections of fluorescent dyes into single H+‐ATPase of the leaves is strongly inhibited by calcium. A
plant cells are difficult and often result in cell death. Success calcium concentration of 0.3 µMblocks 50% of the activity of
rates of viable injections into Arabidopsis guard cells can be H+‐ATPase, and 1 µM calcium blocks the enzyme completely.
less than 3%. In contrast, transgenic plants expressing the It appears that two factors contribute to ABAinhibition of
gene for the calcium indicator protein yellow cameleon the plasma membrane proton pump: an increase in the
make it possible to monitor several fluorescing cells in cytosolic Ca2+ concentration, and alkalinization of the
parallel, without the need for invasive injections. Such cytosol.
studies have demonstrated that the cytosolic Ca2+
concentration oscillates with distinct periodicities, In addition to causing stomatal closure, ABA prevents light‐
depending on the signals received. induced stomatal opening. In this case ABA acts by inhibiting
the inward K+ channels, which are open when the membrane
These results support the hypothesis that an increase in is hyperpolarized by the proton pump. Inhibition of the
cytosolic calcium, partly derived from intracellular stores, is inward K+ channels is mediated by the ABA‐induced
responsible for ABA‐induced stomatal closure. However, the increase in cytosolic calcium concentration. Thus calcium
growth hormone auxin can induce stomatal opening, and and pH affect guard cell plasma membrane channels in two
this auxin‐induced stomatal opening, like ABA‐induced ways:
stomatal closure, is accompanied by increases in cytosolic 1. They prevent stomatal opening by inhibiting inward K +
calcium. This finding suggests that the detailed channels and plasma membrane proton pumps.
characteristics of the location and periodicity of Ca2+ 2. They promote stomatal closing by activating outward
oscillations (the “Ca2+ signature”), rather than the overall anion channels, thus leading to activation of K+ efflux
concentration of cytosolic calcium, determine the cellular channels.
response. In addition to increasing the cytosolic calcium
concentration, ABA causes an alkalinization of the cytosol ABA Stimulates Phospholipid Metabolism
from about pH 7.67 to pH 7.94. The increase in cytosolic pH
has been shown to activate the K+ efflux channels on the As discussed previously, much evidence supports a role for
plasma membrane apparently by increasing the number of calcium both in the promotion of stomatal closing and in the
channels available for activation. inhibition of stomatal opening. According to the classic
calcium‐dependent signal transduction pathway of animal
ABA Activation of Slow Anion Channels Causes Long­ cells, IP3 is released, along with diacylglycerol (DAG), when
Term Membrane Depolarization phospholipase C is activated by a G‐protein in the plasma
membrane . Does ABAuse the same pathway when it induces
The rapid, transient depolarizations induced by ABA are stomatal closure? In agreement with this model, ABA has
insufficient to open the K+ efflux channels, which require been shown to stimulate phosphoinositide metabolism in
long‐term membrane depolarization in order to open. Vicia faba (broad bean) guard cells. To detect the effect of
However, long‐term depolarizations in response to ABA ABA on IP3 release, it was necessary to include Li+ in the
have been demonstrated. According to a widely accepted incubation medium as an inhibitor of inositol phosphatase,
model, long‐term membrane depolarization is triggered by which rapidly removes phosphate groups from IP3. Under
two factors: (1) an ABA‐induced transient depolarization of these conditions, a 90% ABA‐induced increase in the level of
the plasma membrane, coupled with (2) an increase in IP3 was measured within 10 seconds of hormone treatment.
cytosolic calcium. Both of these conditions are required to Recent studies in Arabidopsis using antisense DNA to block
open calcium‐activated slow (S‐type) anion channels on the expression of an ABAinduced phospholipase C have shown
plasma membrane. ABAhas been shown to activate slow that this enzyme is required for ABA effects on germination,
anion channels in guard cells. growth, and gene expression.

The prolonged opening of these slow anion channels permits Heterotrimeric G‐proteins may mediate the effects of ABA on
large quantities of Cl– and malate2– ions to escape from the stomatal movements. For example, in Vicia faba most studies
cell, moving down their electrochemical gradients. (The have shown that G‐protein activators, such as GTPαS, can
inside of the cell is negatively charged, thus pushing Cl – and inhibit the activity of the inward K+ channels. Consistent
malate2– out of the cell, and the outside has lower Cl– and with the inhibitor results, ABA failed to inhibit inward K+
malate2– concentrations than the interior.) The outward flow channels or light‐induced stomatal opening in an Arabidopsis
of negatively charged Cl– and malate2– ions generated in this mutant with a defective Gα subunit. However, ABA still
way strongly depolarizes the membrane, triggering the promoted stomatal closure in this mutant, indicating that
voltage‐gated K+ efflux channels to open. inhibition of opening and promotion of closing take two
distinct paths to the same end point—that is, closed stomata.
In support of this model, inhibitors that block slow anion
channels, such as 5‐nitro‐2,3‐phenylpropylaminobenzoic Other potential second messengers mediating the ABA
acid (NPPB), also block ABA‐induced stomatal closing. response, such as phosphatidic acid and myo‐inositol‐
Inhibitors of the rapid (R‐type) anion channels, such as 4,4′‐ hexaphosphate (IP6) have been identified, but the
diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), have no relationship of these compounds to IP3 and Ca2+ signaling is
effect on ABA‐induced stomatal closing . not yet known. All of these experiments indicate that

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stomatal guard cells respond to multiple signals, possibly dephosphorylating specific serine or threonine residues, but
involving multiple receptors and overlapping signal none of their substrates have been definitively identified.
transduction pathways. Because the abi1­1 and abi2­1 mutations result in decreased
response to ABA, it was initially assumed that the wild‐type
Protein Kinases and Phosphatases Participate in ABA genes promote the ABA response. However, the original
Action mutations turned out to be dominant rather than recessive,
and recent studies have shown that they act as “dominant
Nearly all biological signaling systems involve protein negatives”; that is, one defective copy of the gene is sufficient
phosphorylation and dephosphorylation reactions at some to disrupt the ABA response by poisoning the activity of the
step in the pathway. Thus we can expect that signal functional gene products from the remaining wild‐type
transduction in guard cells, with their multiple sensory allele.
inputs, involves protein kinases and phosphatases.
Artificially raising the ATP concentration inside guard cells Subsequently, recessive mutants of ABI1 were obtained that
by allowing the cytoplasm to equilibrate with the solution exhibited a simple loss of ABI1 activity. These recessive
inside a patch pipette strongly activates the slow anion mutants of ABI1 actually showed increased ABA sensitivity.
channels. Furthermore, overproducing the wild‐type gene products or
their homologs (closely related proteins) by reintroducing
This activation of the slow anion channels by ATP is the gene into plants, under control of a highly expressed
abolished by the inclusion of protein kinase inhibitors in the promoter, confers reduced ABA sensitivity. Thus the wild‐
patch pipette solution. Protein kinase inhibitors also block type function of these protein phosphatases is to inhibit the
ABA‐induced stomatal closing. In contrast, lowering the ABA response.
concentration of ATP in the cytosol inactivates the slow
anion channels. Additional experiments confirm that this ABA Signaling Also Involves Ca2+­Independent Pathways
inactivation is due to the presence of protein phosphatases,
which remove phosphate groups that are covalently Although an ABA‐induced increase in cytosolic calcium
attached to proteins. In view of these results, it appears that concentration is a key feature of the current model for ABA‐
protein phosphorylation and dephosphorylation play induced guard cell closure, ABA is able to induce stomatal
important roles in the ABA signal transduction pathway in closure even in guard cells that show no increase in cytosolic
guard cells. calcium. In other words, ABA seems to be able to act via one
or more calcium‐independent pathways.
There is now direct evidence for an ABA‐activated protein
kinase (AAPK) in Vicia faba guard cells. AAPK activity In addition to calcium, ABA can utilize cytosolic pH as a
appears to be required for ABA activation of S‐type anion signaling intermediate. As previously discussed, a rise in
currents and stomatal closing. This enzyme is an cytosolic pH can lead to the activation of outward K+
autophosphorylating protein kinase that either forms part of channels, and one effect of the abi1 mutation is to render
a Ca2+‐independent signal transduction pathway for ABA, or these K+ channels insensitive to pH.
acts farther downstream of calcium‐induced signaling
events. (The presence of both Ca2+‐dependent and Ca2+‐ Such redundancy in the signal transduction pathways
independent pathways for ABA action will be discussed explains how guard cells are able to integrate a wide range
shortly.) In addition, two Ca2+‐dependent protein kinases, of hormonal and environmental stimuli that affect stomatal
as well as MAP kinases, have been implicated in the ABA aperture, and such redundancy is probably not unique to
regulation of stomatal aperture. guard cells.

The analysis of ABA‐insensitive mutants has begun to help in A simplified general model for ABA action in stomatal guard
the identification of genes coding for components of the cells is shown in Figure. For clarity, only the cell surface
signal transduction pathway. The Arabidopsis abi1­1 and receptors are shown. (see next page)
abi2­1 mutations result in insensitivity to ABA in both seeds
and adult plants. These abi mutants display phenotypes ABA Regulation of Gene Expression Is Mediated by
consistent with a defect in ABA signaling, including reduced Transcription Factors
seed dormancy, a tendency to wilt (due to improper
regulation of stomatal aperture), and decreased expression Downstream of the early ABA signal transduction processes
of various ABA‐inducible genes. already discussed, ABA causes changes in gene expression.
ABA has been shown to regulate the expression of numerous
The defects in stomatal response include the ABA genes during seed maturation and under certain stress
insensitivity of S‐type anion channels—both inward and conditions, such as heat shock, adaptation to low
outward K+ channels—and actin reorganization. Although temperatures, and salt tolerance. The ABA and stress‐
non‐responsive to ABA, the mutant stomata will close when induced genes are presumed to contribute to adaptive
exposed to high external concentrations of Ca2+, suggesting aspects of induced tolerance.
that they are defective in their ability to initiate Ca2+
signaling. Consistent with this finding, ABA does not induce They include genes encoding proteases, chaperonins,
Ca2+ oscillations in these mutants. proteins similar to LEA proteins, enzymes of sugar or other
compatible solute3 metabolism, ion and water channel
ABI Protein Phosphatases Are Negative Regulators of proteins, enzymes that detoxify active oxygen species, and
the ABA Response regulatory proteins such as transcription factors and protein
kinases.
The Arabidopsis ABI1 and ABI2 genes have been cloned and
identified as encoding two closely related serine/threonine In a few cases, stimulation of transcription by ABA has been
protein phosphatases. This finding suggests that ABI1 and demonstrated directly. Gene activation by ABA is mediated
ABI2 regulate the activity of target proteins by by transcription factors. Four main classes of regulatory

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sequences conferring ABA inducibility have been identified, factors act in complexes made up of varying combinations of
and proteins that bind to these sequences have been regulators, whose composition is determined by the
characterized. Under stress conditions, induction of gene combination of available regulators and binding sites. To
expression may be ABA dependent or ABAindependent, and date, the protein ABI3/VP1 has been shown to interact
additional transcription factors have been identified that physically with a variety of proteins, including ABI5 and its
specifically mediate responses to cold, drought, or salt . rice homolog (TRAB1). ABI5 also forms homodimers and
heterodimers with other bZIP family members. There is
A few DNA elements have been identified that are involved additional evidence for indirect interactions that may be
in transcriptional repression by ABA. The best characterized mediated by 14‐3‐3 proteins, a class of acidic proteins that
of these are the gibberellin response elements (GAREs) that dimerize and facilitate protein–protein interactions in a
mediate the gibberellin‐inducible, ABA repressible variety of signaling, transport, and enzymatic functions (see
expression of the barley α‐amylase gene . been characterized. Under stress conditions, induction of
gene expression may be ABA dependent or ABAindependent,
Four transcription factors involved in ABA gene activation in and additional transcription factors have been identified
maturing seeds have been identified by genetic means; that specifically mediate responses to cold, drought, or salt.
mutations in the genes encoding any of these proteins
reduce seed ABA responsiveness. The maize VP1 A few DNA elements have been identified that are involved
(VIVIPAROUS­1) and Arabidopsis ABI3 (ABA­INSENSITIVE3) in transcriptional repression by ABA. The bestcharacterized
genes encode highly similar proteins, and the ABI4 and ABI5 of these are the gibberellin response elements (GAREs) that
genes encode members of two other transcription factor mediate the gibberellin‐inducible, ABA repressible
families. VP1/ABI3, and ABI4 are members of gene families expression of the barley α‐amylase gene.
found only in plants. In contrast, ABI5 is a member of the
basic leucine zipper (bZIP) family, whose members are Four transcription factors involved in ABA gene activation in
present in all eukaryotes. maturing seeds have been identified by genetic means;
mutations in the genes encoding any of these proteins
Additional members of the ABI5 subfamily have been reduce seed ABA responsiveness. The maize
identified by nongenetic means and are also correlated with VP1(VIVIPAROUS­1) and Arabidopsis ABI3 (ABA­
ABA‐, embryonic‐, drought‐, or salt stress–induced gene INSENSITIVE3) genes encode highly similar proteins, and the
expression. Characterization of vp1, abi4, and abi5 mutants ABI4 and ABI5 genes encode members of two other
has shown that each of these genes can either activate or transcription factor families. VP1/ABI3, and ABI4 are
repress transcription, depending on the target gene. Because members of gene families found only in plants. In contrast,
the promoter of any given gene contains binding sites for a ABI5 is a member of the basic leucine zipper (bZIP) family,
variety of regulators, it is likely that these transcription whose members are present in all eukaryotes.

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Additional members of the ABI5 subfamily have been defects in ABAand ethylene responses, mutations in this
identified by nongenetic means and are also correlated with gene result in defects in the responses to auxin, jasmonic
ABA‐, embryonic‐, drought‐, or salt stress–induced gene acid, and stress. This gene encodes a membrane‐bound
expression. Characterization of vp1, abi4, and abi5 mutants protein that appears to represent a point of “cross‐talk”—
has shown that each of these genes can either activate or i.e., a common signaling intermediate— mediating the
repress transcription, depending on the target gene. Because responses to many different signals.
the promoter of any given gene contains binding sites for a
variety of regulators, it is likely that these transcription IP3 catabolism. Other screens have identified ABA signaling
factors act in complexes made up of varying combinations of mutants on the basis of incorrect expression of reporter
regulators, whose composition is determined by the genes controlled by ABA‐responsive promoters. Although
combination of available regulators and binding sites. the defects in some of these mutants are limited to gene
expression, others affect plant growth responses. One such
To date, the protein ABI3/VP1 has been shown to interact mutant, termed fiery (fry) to reflect the intensity of light
physically with a variety of proteins, including ABI5 and its emission by its ABA/stress‐responsive luciferase reporter, is
rice homolog (TRAB1). ABI5 also forms homodimers and also hypersensitive to ABAand stress inhibition of
heterodimers with other bZIP family members. There is germination and growth. The FIERY gene encodes an
additional evidence for indirect interactions that may be enzyme required for IP3 catabolism. The mutant phenotype
mediated by 14‐3‐3 proteins, a class of acidic proteins that demonstrates that the ability to attenuate, as well as induce,
dimerize and facilitate protein–protein interactions in a stress signaling is important for successful induction of
variety of signaling, transport, and enzymatic functions. stress tolerance.
These studies demonstrate the capacity for specific binding
among a variety of transcription factors predicted to interact Similar to the signaling mechanisms documented for other
as components of regulatory complexes involved in ABA‐ plant hormones, ABA signaling involves the coordinated
induced gene expression. action of positive and negative regulators affecting
processes as diverse as transcription, RNA processing,
Other Negative Regulators of the ABA Response Have protein phosphorylation or farnesylation, and metabolism of
Been Identified secondary messengers. As the signaling components are
identified, and often are found to function in responses to
As described already, negative regulators of the ABA multiple signals, the next challenge is to determine how they
response (protein phosphatases) have been identified by can lead to ABA‐specific responses.
isolation of dominant negative mutants such as abi1 and
abi2 that result in ABA‐insensitive phenotypes (analogous to Other potential plant hormones
the dominant negative effects of the ethylene receptor
mutant etr) As it is so difficult to write a comprehensive definition of a
plant hormone it is not surprising that many other plant
Other negative regulators have been identified through compounds might fall within this group. In recent years
isolation of mutants exhibiting enhanced responses to ABA. many signaling molecules have been discovered which may
Mutants showing increased sensitivity to ABA during act as hormones. For some molecules the evidence is good,
germination include era (enhanced response to ABA) and with production, transport, a receptor and responses having
abh (ABA hypersensitive). The era and abh mutants both been demonstrated. For other substances the evidence is
confer ABA hypersensitivity in both stomatal closing and less conclusive, although rapid progress is being made.
germination, making these mutants resistant to wilting and
mildly drought tolerant.

Farnesyl transferase. The ERA1 gene was cloned, and its

protein product was identified as a subunit of the enzyme
farnesyl transferase. Farnesyl transferases catalyze
attachment of the isoprenoid intermediate farnesyl
diphosphate to proteins that contain a specific signal
sequence of amino acids. Many proteins that have been
shown to participate in signal transduction are farnesylated.
Farnesylated proteins are anchored to the membrane via
hydrophobic interactions between the farnesyl group and
the membrane lipids. The identification of ERA1 as part of
farnesyl transferase suggests that a protein that normally
suppresses the ABAresponse requires farnesylation and is
possibly anchored to the membrane.

mRNAprocessing. ABH1 encodes an mRNA 5′cap–binding

protein that may be involved in mRNA processing of
negative regulators of ABA signaling. (Recall that eukaryotic
messenger RNAs have a “cap” consisting of methylated
guanosine at the 5′end.) Comparison of transcript
accumulation in wild‐type and abh1 plants showed a small
number of misexpressed genes in the mutant, including
some encoding possible signaling molecules.

Ethylene insensitivity. ERA3 was found to be allelic to a

previously identified ethylene signaling locus,
ETHYLENEINSENSITIVE 2 (EIN2). In addition to displaying

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Peptides Salicylic acid/jasmonic acid

In animals numerous peptide hormones have been These two compounds have been considered together as
discovered, and it is surprising that so little is known about although they are structurally quite different they appear to
equivalent molecules in plants. However, there is good interact closely to coordinate plant defence responses.
evidence that an 18‐amino‐acid peptide, systemin (Fig. 4), Jasmonic acid (JA) is a 12 –carbon fatty acid derivative of
acts as a hormone in wounded leaves of tomato linolenic acid whereas salicylic acid (SA) is a simple aromatic
(Lycopersicon esculentum). This peptide is released from ring with a hydroxyl and carboxylic acid sidegroup (Fig. 4).
wounded cells and is transported through the phloem to
unwounded leaves where it induces proteinase inhibitor SA is involved in the expression of many pathogenesis‐
(pin) gene expression. The proteinase inhibitors impair related (PR) proteins which have antifungal and
digestion in herbivores and hence have an antifeedant role. antibacterial activities in response to pathogen attack. JA has
Systemin is active at very low (femtomolar) concentrations been implicated in wound responses such as those caused by
and a receptor has been isolated. Peptides closely related to herbivores. It has been proposed that many SA‐induced
systemin have been discovered in other members of the genes are repressed by JA and vice versa, and in this way a
Solanaceae. more ‘directed’ response is achieved without the diversion
of resources into unnecessary protein production. Ethylene
Another peptide, sulfokin, has been implicated in the control also plays a role in this coordinated gene expression. Some
of cell division in asparagus (Asparagus officinalis) and rice pathogens may exploit this interaction – the bacterium
cell cultures. This peptide is even smaller than systemin, Pseudomonas syringae produces corotanine, an analogue of
being only 4 –5 amino acids long, two of which are sulphated JA. This compound may repress the expression of the PR
(Fig. 4). Other biologically active peptides have been proteins, allowing infection to proceed. The complexity of
described. signaling mechanisms in plant defence is beginning to be
unravelled using a combination of mutants and gene
However, in many cases, these peptides may act at the cell expression profiling, allowing interactions between these
surface, performing an important role in signalling pathways to be elucidated.
positional information. Whilst this is a fascinating area of
plant biology, these substances cannot be considered plant Both SA and JA can be methylated in vivo and are volatile in
growth hormones without evidence of transport within the this form. In the laboratory it has been demonstrated that
plant. Nevertheless, there are undoubtedly many other gene expression in neighbouring plants can be induced by
peptide hormones in plants awaiting discovery. these volatile compounds, although this remains to be
convincingly demonstrated in field conditions. Plants
RNA contain many volatile compounds, a number of which have
the potential to act as signalling molecules. Interestingly, this
Recently, small RNA molecules have been identified in the signalling may extend to organisms other than plants.
phloem which may act as transportable signals regulating Compounds released from herbivore‐damaged plants have
gene expression. To date, these have been shown to be the potential to attract predators and parasites of the
important in plant defence responses to viruses and in the herbivores.
silencing of synthetic genes introduced by genetic
modification, both of which may be considered ‘foreign’ Brassinosteroids
genes. This phenomenon is called post‐transcriptional gene
silencing (PTGS) and involves small double‐stranded RNA More than 40 different brassinosteroid molecules have been
molecules, known as short interfering RNAs (siRNAs), which found within plants (Fig. 4). These are all thought to be
are approximately 25 nucleotides long, and which mediate synthesized from the precursor campesterol.
the destruction of specific target mRNAs. In addition, a Brassinosteroids have been implicated in many roles
number of groups have identified a separate class of including stem elongation, pollen tube growth, leaf bending
microRNAs (miRNAs) which may regulate normal plant and unrolling, stimulation of ethylene production, tracheary
development. These are single‐stranded RNAs of 21–22 element differentiation and cell elongation. Plants with
nucleotides which are complementary to the genes encoding mutations in brassinosteroid metabolism are dwarf and
many regulatory proteins. Recently, the importance of exhibit unusual development in darkness. The transport of
miRNAs in leaf development has been demonstrated. As these molecules has not been well documented, but clearly
many hundreds of these miRNAs have been identified it they have a profound effect on plant development.
seems possible that they may represent a more general
means of regulating gene expression, and hence these small Other plant growth hormones will undoubtedly be found
RNAs may be considered to be plant growth regulators. and, in time, the boundaries between the traditional
hormones and the plethora of signalling molecules which
exist within, and pass between, the cells of plants will
become more blurred.

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E. Sensory photobiology: Structure, function and mechanisms of action of phytochromes, cryptochromes and phototropins;
photoperiodism and biological clocks. Stomatal Opening

Photomorphogenesis

1 Introduction chloroplast development and stimulating the synthesis of

secondary products such as anthocyanin
Light is critically important to plants. The majority of pigments. These processes can be initiated at light
them are photosynthetic and light provides the energy energies far below that necessary for photosynthesis, in
source required for growth. However, light is equally many cases by brief exposure only, and can occur in
important for the normal development of plants as an plants where the photosynthetic apparatus has not yet
information medium. In the environment light is a very developed.
complex and dynamic signal. It varies in quantity, quality
(colour) and direction over timescales ranging from
seconds to months (Fig. 1). These different variables can
indicate the passing of the seasons, the availability of new
habitats for growth or the presence of neighbouring
vegetation which may compete for resources. Therefore it
is not surprising that many aspects of plant growth and
development are strongly influenced by light. The plant,
too, is a complicated and ever-changing system, and the
response of a plant to a given set of environmental
conditions will depend upon its developmental state.
Plants pass through a juvenile state where their response
to environmental signals differs from that of mature
plants. Likewise, signals which stimulate a mature plant
to flower may cause the seed of the same species to
germinate – radically different developmental pathways.
Similarly, plant responses are species-specific. Whilst a
fast-growing weed such as Chenopodium album will
respond to shaded conditions (i.e. low light) by elongating
rapidly, rainforest tree seedlings can persist under a
vegetation canopy for many years and commence rapid
growth only when a gap opens in the forest canopy.

The complex nature of light as an environmental signal

and the complex ways in which plants respond to it are
reflected in the multitude of photoreceptors which have
been identified. We are now beginning to understand how
some of these photoreceptors work and interact with each
other to coordinate plant development. Much of this
research has, necessarily, been performed under highly
artificial conditions allowing the roles of single (or at
most, a few) photoreceptors to be studied. However, the
information derived from these experiments is
increasingly being applied to plants growing in a natural
or agricultural environment.

2 The switch from etiolated to de-etiolated growth

Light is perceived by a series of photoreceptors, the best
A very simple, yet informative, experiment is to grow two studied of which are the phytochromes. However, plants
sets of the same species of a plant in light and in darkness. contain several distinct families of photoreceptors in
The two sets of plants may then be scarcely recognizable addition to phytochrome – these include the blue/UV-A
as belonging to the same species. Whilst the light-grown photoreceptors (cryptochromes), poorly characterized UV-
individuals will be green, sturdy, with expanded leaves B photoreceptors and the phototropins (involved in
and ofmoderate height, the specimens grown in the dark phototropic responses).
will be etiolated – unpigmented, very tall, thin and weak
with rudimentary or folded leaves. A plant which has 3 Phytochrome and photomorphogenesis
been grown in darkness and then exposed to light will
switch from etiolated to de-etiolated growth – the control 3.1 The discovery of phytochrome
of development by light being termed
photomorphogenesis (photo = light, morpho = form, Our understanding of plant photoreceptors has been
genesis = origin). Light regulates many aspects of plant revolutionized in recent years by the use of molecular
development, inhibiting internode elongation, promoting genetic techniques coupled with careful biochemical and
leaf expansion (dicotyledons) or leaf unrolling physiological measurements. By isolating and
(monocotyledons), promoting chlorophyll synthesis and characterizing the genes encoding the different

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photoreceptors, and by producing mutant and transgenic

plants with lesions in photoperception, it has been
possible to build a framework describing some of the
mechanisms by which plants respond to light. However,
this current revolution is based upon a long, and
distinguished, history of studying plant responses to light.

The existence of phytochrome was first deduced from

studies of certain varieties of lettuce (Lactuca sativa) seeds
which require light for germination. These seeds are
induced to germinate by brief (a few minutes) exposure to
red light of low irradiance, whilst a similar exposure to
far-red light is inhibitory to germination. Careful
measurements made at many different wavelengths
allowed the action spectra of these two responses to be
established (Fig. 2 ).

Subsequently phytochrome was isolated from plant

tissues. It is alow-abundance, blue-green chromoprotein
and, in solution, shows photoreversibility between the PR
and PFR forms with absorption maxima at the same peaks
as required for the physiological response (Fig. 4).

The stimulation of germination was found to have a

maximum in the red at 660 nm whilst the inhibition of
germination by far-red light had a maximum at 730 nm. A
key breakthrough came with the discovery that At its heart is a chromophore composed of a linear
alternating exposure to red and far-red light can be given tetrapyrrole molecule. With specially designed equipment
formany cycles and whichever wavelength is given last it is possible to detect the change in the phytochrome
determines the response. These observations led absorption spectrum on illumination in an intact etiolated
Hendricks and Borthwick, working in Beltsville, Maryland seedling, although similar changes are masked in mature
in the 1950s, to postulate the existence of a photoreceptor tissues which contain chlorophyll with its strong
named phytochrome, existing in two absorption peak in the red.
photointerconvertible forms. One form, termed PR,
absorbs red light and is converted to the other form PFR. 3.2 Low-fluence responses
The spectral properties of PFR are such that it absorbs
light with a peak in the far-red and under far-red The control of the germination of lettuce seeds by
illumination it is converted to PR again (Fig. 3). PFR also phytochrome was termed a low-fluence response (LFR)
reverts slowly to PR when plants are placed in complete because it is governed by exposures to low fluences of red,
darkness. Many models of phytochrome action have or far-red, light. Many aspects of plant development
proposed that PFR is the active form, whilst PR is inactive. exhibit low-fluence phytochrome responses including
However, as will become apparent later in this chapter, seed germination, seedling development, internode
such a simple interpretation may not always be entirely elongation and flowering in short day plants. Other key
accurate. features of the LFR were determined using such
experimental systems. As already described, low-fluence
phytochrome responses exhibit photoreversibility.
However, far-red light can reverse the effect of red light
only if given within a certain period of time known as the

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escape time (i.e. escape from phytochrome control). the proportions of PR and PFR at photoequilibrium are
Likewise there is a delay – the lag time – following approximately as indicated in Table 1.
exposure to light before the response is observed. This
might be a matter of a few minutes or hours, as in the case
of the etiolation/de-etiolation responses, or may be days
or even weeks in the case of floral induction or seed
germination. Also it is the total number of photons that
the sample receives, rather than the rate at which they are
received, which determines whether a LFR occurs. A brief
exposure to relatively intense red light has the same effect
as exposure to dimmer red light for a longer period of
time. This characteristic is called reciprocity, and low- The proportion of the total phytochrome which is present
fluence phytochrome responses are often reported in in the PFR form is termed f – the Greek letter Φ ‘phi’ is
mmol photons m–2 , i.e. without a time component. often used in biology as a shorthand for ‘proportion’.
Although these proportions may be constant, individual
However, as the number of studies examining plant phytochrome molecules will be continually switching
responses to phytochrome expanded it became apparent between the two forms. This switch is not instantaneous,
that not all phytochrome responses had the characteristics and a significant proportion of the phytochrome
of a low-fluence response. Two other types of response, molecules may exist in an intermediate state. There is
the very-low-fluence response and the high-irradiance increasing evidence that such intermediates may play an
response, were also described. important role in phytochrome responses.

3 .3 Very-low-fluence responses 3 .5 The high-irradiance responses

The very-low-fluence responses (VLFR) are observed only Another class of phytochrome-mediated responses, the
in dark-grown seedlings or imbibed seeds. They are high irradiance responses (HIR), are maximized by
triggered by very low fluences (hence the name!), brief exposure to continuous illumination, although repeated
exposures to light, and exhibit reciprocity. pulses of light can trigger such responses if given
frequently enough. H.Mohr, working with mustard
However, they differ from the LFR in that either red or seedlings (Sinapis alba), found that cotyledon expansion
far-red light triggers the same response. For example, was elicited by brief red illumination and that this was
dormant Arabidopsis seeds can be triggered to germinate prevented by brief exposure to far-red light (a classic low-
by red light but this is prevented by far-red light – a fluence response). However, if exposure to far-red light
classic LFR. However, after 48 hours imbibition these was continued for 2–3 hours the cotyledons proceeded to
same seeds exhibit a VLFR. They will germinate when expand even more than under red light. This is a far-red
exposed to either red or far-red light at fluences 100-1000 high irradiance response (FR-HIR).
times lower than that required for the LFR. So how can we
understand very-low-fluence responses in terms of If dark-grown seedlings of most dicotyledonous plants are
phytochrome acting as a photointerconvertible switch? exposed to continuous far-red light there is a strong
Figure 4 shows the absorption spectra of PR and PFR. inhibition of seedling elongation. Typically the maximal
Although the different forms of phytochrome have inhibition results at far-red wavelengths (FR-HIR, Fig. 5)
absorption maxima in thered and far-red, the peaks are but this is species-dependent, with some plants exhibiting
quite broad and overlap. So although PR is preferentially maxima in the red (R-HIR). As seedlings green these
converted to PFR by red light, a small proportion will also maxima often shift, indicating the operation of more than
be converted by far-red light. In dark-grown plants one inhibitory mechanism. Current theories favour cycling
phytochrome is synthesized as PR but even brief exposure between PR and PFR as central to the operation of the
to dim far-red light will convert a small amount of PR to HIR, but the mechanisms underlying this remain unclear.
PFR. Even though some of this will quickly be re- The peaks in the UV/blue of the action spectrum shown
converted to the PR form, enough PFR is present to trigger in Fig. 5 are thought to result from the action of specific
a very-low-fluence phytochrome response. In this case UV-blue light photoreceptors which interact with
phytochrome is acting as a detector of the amount of light phytochrome to coordinate plant development.
falling on the sample without discriminating between
different colours of light. Such responses are important in
the very earliest responses of plants to light.

3.4 The photostationary state

The concept of phytochrome switching between different

forms applies to more than just the low- and very-low-
fluence responses.

Under any illumination conditions phytochrome will exist

in both forms, although the precise proportion depends
upon the precise spectral properties of the light. An
equilibrium is established called the photostationary state.
With monochromatic illumination of 660 nm and 730 nm,

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developmental state. Consider the example of a small-

seeded plant such as Arabidopsis. Seeds of Arabidopsis have
limited nutrient reserves, which will support growth for
only a few days. Once these reserves are exhausted the
seedling must photosynthesize if it is to continue to grow.
The low-fluence response of these seeds stimulates
germination if at, or near, the soil surface unless the
sunlight has been filtered through a vegetation canopy
and is thus enriched in far-red light. If the seed does
germinate, etiolation must be considered a ‘last chance’
response – the hypocotyls elongates rapidly until the
cotyledons are above the soil. The HIR is then triggered
even if the seedling is shaded. De-etiolation, resulting
from exposure to far-red light, is thus a shade-tolerance
response.

Some plants, e.g. dog’s mercury (Mercurialis perennis), can

tolerate shade throughout their lives and do not respond
greatly to alterations in the ratio of red : far-red light.
However, mature plants of many species exhibit a shade-
avoidance response if exposed to elevated far-red light.
Typically, the shade-avoidance response results in an
3.6 What is the significance of far-red light? increase in apical dominance, an increase in stem growth
as a result of internode elongation, increased petiole
Low ratios of red : far-red light are a signal of vegetation elongation and accelerated flowering. The morphological
shade Full sunlight contains approximately equal changes may enable the plant to grow out of the shade of
amounts of all visible wavelengths of light, including red other plants. These responses can be triggered by a
and far-red components (Fig. 1). However, plant leaves decrease in the ratio of red : far-red light falling from
are rich in chlorophyll, which strongly absorbs blue and above, but can also be triggered by far-red reflected from
red light, green light to a lesser extent, but is relatively surrounding vegetation. A single, brief exposure to far-red
inefficient at absorbing far-red light. Fig. 6 shows the light can also trigger the shade-avoidance response if
spectral composition of sunlight after it has passed given at the end of the photoperiod (the ‘end-of-day far-
through one or two leaves. Absorption of light by the red effect’, EOD-FR). Mature Arabidopsis plants exhibit a
canopy reduces the total irradiance markedly and has clear shade-avoidance response with increased petiole
greatly reduced the amount of red light relative to far-red extension and accelerated flowering, although, as this
light. This shift in red : far-red ratio from values close to 1 plant grows as a rosette, the internodes do not elongate.
in full sunlight to values nearer 0.1 is a strong signal Clearly, the shade-tolerance response of seedlings and
indicating vegetation shade. This signal is not restricted to shade-avoidance response of mature plants are
light falling directly from above. Light reflected from antagonistic; hence a switch between the two must occur
neighbouring vegetation will also be enriched in the far- as the plant develops. The role of phytochrome in these
red and has the potential to act as a signal of future responses is becoming increasingly clear, and these
competition for resources. responses can be manipulated to control plant
development in natural environments.

3.7 Phytochrome is encoded by a multigene family

This plethora of phytochrome responses is difficult to

interpret if phytochrome is a single entity. However, it has
become apparent that plants contain multiple
phytochromes. Work with etiolated oat seedlings (Avena
sativa) indicated the presence of light-labile (‘Type I’) and
light-stable (‘Type II’) forms. Then Sharrock and Quail
(1989) showed that Arabidopsis thaliana contains five
phytochrome genes, which they named PHYA, PHYB,
PHYC, PHYD and PHYE, and showed that whilst
etiolated seedlings of Arabidopsis contained phyA, B and
C, upon illumination there was a rapid decline in phyA.
This indicated that phyAwas light-labile, corresponding to
‘Type I’ phytochrome described in oat, whilst phyB and C
were light stable (‘Type II’). PhyD and E were also
demonstrated to fall into the latter class in subsequent
studies.
Shade tolerance vs. shade avoidance
Although the phyA content of seedlings declines rapidly
The response of plants to the red : far-red ratio is both upon illumination, mature plants exhibit responses
species-specific and highly dependent upon attributable to phyA action; therefore a small amount

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must still remain in light-grown plants. The expression of

phyA is regulated by light by both transcriptional and Traditional biochemical approaches were used firstly to
post-transcriptional processes. The transcription of the isolate the phytochrome protein. This strategy succeeded
PHYA gene decreases markedly (but is not abolished) because the unique spectral properties of phytochrome
upon illumination. At the post-transcriptional level phyA enabled it to be identified in subcellular fractions even
protein is readily degraded in the light after its though it is a very low abundance protein. This approach
photoconversion to the phyAFR form. Most plants contain failed with the UV-A/blue light photoreceptor as many
multiple phytochromes, which appear to have arisen by other proteins within the cell, which have no role in
gene duplication. For example tomato (Lycopersicon photoperception, absorb blue light owing to bound
esculentum) contains five (or perhaps six) phytochrome cofactors. However, the identification of a mutant (hy4 )
genes. The nomenclature has become somewhat confused, which had a lesion in blue-light responses allowed the
but comparisons of phytochrome amino acid sequences gene encoding a UV-A/blue photoreceptor to be
have indicated that four major subfamilies, each with one identified. This mutant was renamed cry1 as
or more members, can be identified in herbaceous dicots. cryptochrome (crypto = hidden) is an alternative name for
These subfamilies have been named PHYA, PHYB, PHYC this class of photoreceptors.
and PHYE, based on comparisons with the genes from
Arabidopsis. PHYD is closely related to PHYB and is placed The Arabidopsis CRY1 gene has been sequenced and
in the same subfamily. In some species, e.g. black encodes a protein with similarity to a class of microbial
cottonwood (Populus trichocarpa), not all of the subfamilies DNA repair enzymes called DNA photolyases. Whilst
have been detected. Monocots also contain multiple biochemical measurements of the plant protein showed no
phytochromes, although an extensive survey of grasses by evidence of DNA repair activity, these enzymes are
Mathews and Sharrock (1996) found evidence of only the known to bind a flavin cofactor (flavin adenine
PHYA, B and C subfamilies. dinucleotide) which absorbs blue light, consistent with its
proposed role as a blue-light receptor. It has also been
Unravelling the roles of these different phytochromes is a suggested that cry1 binds a second chromophore – a
challenging task but has been greatly facilitated by the use pterin (5,10 methenyltetrahydrofolate). The identification
of mutant plants which have lesions in their phytochrome of CRY1 in Arabidopsis quickly led to the identification of a
genes. Likewise the use of transgenic plants where the second, related gene called CRY2 , indicating that the
content of an individual phytochrome has been increased cryptochromes are encoded by a small, multigene family.
has been highly informative. Likewise, two related cryptochrome genes have recently
been identified in tomato.
Screening for Arabidopsis plants which exhibited elongated
hypocotyls when grown under white light revealed a Plants contain other, unrelated, blue-light receptors in
number of different mutants, the hy mutants. The mutants addition to the cryptochromes. Blue-light phototropic
hy1, hy contain phytochrome apoprotein but do not curvatures, chloroplast and stomatal movements are
contain the chromophore – these are mutations affecting stimulated, in part, by phototropin(s) and a separate, as
tetrapyrrole synthesis. Therefore these mutants are often yet unidentified, photoreceptor most probably mediates
considered to lack all functional phytochrome, although responses to UV-B.
some residual activity may remain. The hy3 mutant lacks
PHYB and the hy5 mutant has a lesion in the 5 Genes controlling etiolated growth
phytochrome signal transduction chain rather than in a
phytochrome gene itself. Although many of these initial Clearly, the switch from etiolated to de-etiolated growth
mutations exhibited lesions in aspects of phytochrome requires many aspects of plant development to change.
responses, because the screen was performed under white The emphasis in this chapter so far has been on the
light other photomorphogenic mutants were also development of plants in the light (photomorphogenesis)
identified. The power of this approach was amply but it is equally important to remember that seedlings are
demonstrated when it was found that the hy4 mutant following a developmental pathway when growing in
contained a lesion in the blue-light receptor which had darkness. This is sometimes referred to as
previously proved impossible to identify. More directed skotomorphogenesis (skoto = dark). Just as plants with
screens using continuous far-red illumination identified mutations in light perception have proved invaluable in
plants with lesions in the PHYA gene, and other understanding photomorphogenesis, valuable insights
molecular genetic approaches have been used to identify into skotomorphogenesis have been obtained by isolating
Arabidopsis plants with lesions in PHYC, D and E. Such plants which exhibit some, or all, of the characteristics of
mutants allow the functions of individual phytochromes de-etiolated plants even though they have not been
to be assessed, and many plants with lesions in the exposed to light. A number of laboratories have isolated
phytochrome signal transduction chain have also been such mutants, calling them det (de-etiolated) and cop
isolated. (constitutively photomorphogenic). A third class of
mutants, fus ( fusca), also develop in a de-etiolated
Our understanding of the phytochrome gene family is manner when grown in darkness but were originally
currently most advanced in Arabidopsis but mutations isolated on the basis of increased anthocyanin
have also been found in other species which are often accumulation when grown in the light. Many of these
more useful in ecophysiological studies including pea mutants (involving lesions in at least 11 different genes)
(Pisum spp.), tomato, cucumber (Cucumis sativus), tobacco show an almost complete photomorphogenic
(Nicotiana tabacum), Brassica napus and Sorghum bicolor. development in total darkness. They have short
hypocotyls, the cotyl edons open and true leaves form,
4 UV-A/blue light photoreceptors (cryptochrome) chloroplasts develop and many light-regulated genes are

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expressed. It is quite remarkable that these plants can not photoreversible. The action spectrum closely
complete their entire life cycle in darkness, from resembles the absorption spectrum of phyAR, which is
germination to flowering and seed production, if consistent with the view that absorption of light by
provided with a suitable nutrient source (e.g. glucose). phyAR leads to the formation of phyAFR, which triggers
They do not green under these conditions, however, as the the response. In contrast, the LFR requires much more
biosynthesis of chlorophyll includes a light-dependent light (10–1000 mmol m–2 ), is stimulated by red light (550–
step. The det, cop and fus genes are obviously 690nm), and reversed by far-red light (700–800 nm).
fundamental in maintaining the seedling in an etiolated
state. Other mutations result in only partial
photomorphogenesis in darkness.

Some of the genes which have been mutated in these

plants have been identified and sequenced, providing
valuable insights into this aspect of plant development.
The det2 mutant contains a lesion in the gene encoding a
cytochrome P450. This class of enzymes plays a central
role in many aspects of secondary metabolism and plants
contain many different cytochrome P 450s (at least 30 in
Arabidopsis).

This particular cytochrome P450 is required for

brassinosteroid biosynthesis and the phenotype of this
mutant reverts to that of the wild type if supplied with
appropriate brassinosteroid precursors. Det2 mutants
grown in the light are severely dwarfed, indicating that
these compounds are important in both dark- and light-
regulated developmental pathways. A number of other
dwarf plants have since been found to have lesions in
other parts of the brassinosteroid biosynthetic pathway.
Interestingly, wild-type plants treated with cytokinins also
develop in a de-etiolated manner when grown in
darkness, although the significance of this is not yet clear.

Other det/cop/fus mutations are thought to be

components of a signal transduction chain involved in
both skoto- and photomorphogenesis.

6 Unravelling photomorphogenesis

Given that plants contain so many light-responsive In this example the different responses can be attributed
pathways, the ability to inactivate individual components quite readily to the action of either phyA or phyB, and
of specific pathways selectively provides a powerful tool there appears to be little interaction with either each other
with which to study these aspects of plant biology. This or cryptochrome. However, in many cases, interactions
approach can be readily extended to examine multiple within and between the families of photoreceptors is the
pathways by crossing different mutants together and norm. The responses of seedlings to light are complex, and
selecting offspring which have lesions in two, three or our understanding of the regulation of de-etiolation is far
even more photoreceptors. This is amply demonstrated in from complete.
the examples described in the following section.

6.1 Phytochrome A and B regulate seed germination in 6.2 Cryptochrome and phytochrome interact to regulate
Arabidopsis de-etiolation

As described earlier, the germination of recently imbibed When Arabidopsis seedlings are grown under continuous
seeds of Arabidopsis is controlled by a low-fluence far-red light, a high-irradiance response causes them to
phytochrome response (LFR) whilst imbibition for 4 0 de-etiolate. However, phyA-deficient plants are etiolated
hours results in a shift to control by a very-low-fluence in all respects under these conditions, clearly identifying
response (VLFR). In phy B-deficient mutants the initial phyA as an essential component of the HIR signal
photoreversible LFR is abolished. This tells us not only transduction pathway.
that phyB is responsible for the LFR but also that the other
phytochromes (phyA, phyC–E) cannot substitute for it. For seedlings grown under continuous red light the
With phyA deficient mutants the situation is reversed. The situation is more complex. Someaspects of red-light-
VLFR is lost, showing that this response requires phyA. induced de-etiolation, such as the inhibition of hypocotyl
Since wenowhave seeds which show just one or other elongation, can be attributed to phyB. However, other
response we can measure the properties of the lowand responses, such as the opening of the apical hook, can be
very-low-fluence responses independently (Fig. 7). The mediated by phyA or phyB. Experiments under a variety
VLFR is triggered by 1–1000nmolm–2 (at 660nm) and is of illumination conditions have shown that apical hook

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opening (and other de-etiolation responses) are controlled example, the action of one photoreceptor may be greatly
bymultiple redundant pathways (Table 2 ). enhanced by the activity of another, beyond that expected
from simple additive effects.
In an attempt to unravel the roles of different
photoreceptors in the control of de-etiolation responses 6.3 Phytochrome responses throughout the life cyle
Neff & Chory (1998) examined the development of
Arabidopsis seedlings with lesions in phyA, phyB and cry1. Wild-type seedlings of Arabidopsis exhibit shade tolerance
The single mutants were also crossed together so that (i.e. they initially de-etiolate when exposed to far-red
additional plants with double and triple mutations could light) whilst mature plants exhibit shade avoidance (the
be studied. Whereas the studies of seed germination leaf petioles elongate and flowering is accelerated under
measured an ‘all-or-nothing’ response (i.e. a seed far-red light). Therefore the response of the plant clearly
germinated or it did not), de-etiolation is much more depends upon its developmental state. The photoreceptor
complex, consisting ofmultiple responses, each mutants have helped us to understand how these different
ofwhichmay be quantified precisely. responses are controlled at different stages of the plants’
life cycle. When a seedling first develops it contains
relatively high amounts of phyA and phyB. Therefore the
seedling will de-etiolate under red light (a LFRmediated
by phyB) and far-red light (a HIR mediated by phyA) and
is thus shade tolerant. However, continued illumination
leads to a decline in phyA content. The seedling will
remain de-etiolated under red light (due to the continued
action of phyB) but will now etiolate under far-red light;
i.e. it now exhibits shade avoidance.

A phyB-deficient plant will exhibit a constitutive shade-

Wild-type plants which have been grown in white light avoidance response both as a seedling and a mature plant.
for five days have short hypocotyls, an open apical hook Under white light the phyB mutant has elongated petioles
and open, expanded cotyledons which have accumulated but if exposed to a 10-minute pulse of far-red light at the
chlorophyll and anthocyanin. In contrast, dark-grown end of the day, a more extreme shade-avoidance response
plants have a much longer hypocotyl, a welldeveloped is triggered, indicating that other photoreceptors are still
apical hook and folded, unexpanded cotyledons without responsive. If phyD is also mutated, the petioles become
chlorophyll and anthocyanin (Table 3 ). further elongated under white light, whilst mutations in
phyE cause the rosette habit to be lost. The role of these
The triple mutant lacks cry1, phyA and phyB. When minor phytochromes is normally masked by phyB.
grown in white light some de-etiolation responses are Although mature plants of many species do not exhibit
entirely abolished. For example, the apical hook, shade tolerance, it can be re-established using genetic
cotyledon expansion and anthocyanin content are the engineering techniques. Although the phyA content of a
same as those of a wild-type plant grown in darkness, mature plant is normally low, an artificial gene can be
which suggests that these three photoreceptors control the made where a strong, unregulated promoter is fused to
development of these features. However, the cotyledons the coding region of the PHYA gene. When this over-
have opened slightly and a small amount of chlorophyll is expression construct is introduced into plants they will
still produced, indicating that other photoreceptors have a nowcontain much more phyA than normal. Vierstra and
minor role in de-etiolation (other studies have implicated coworkers used this approach to produce shade-tolerant
phyD). The hypocotyl is even longer than that of an tobacco plants. Wild-type tobacco plants normally show a
etiolated wild-type plant, probably because limited strong shade-avoidance response when they are
photosynthesis can occur, providing additional energy for illuminated with far-red light from the side, or are grown
growth. This approach allows the potential role of in dense canopies. The internodes elongate and the
individual photoreceptors to be investigated, although specific leaf area (the area of leaf per unit dry weight)
care must be taken in interpreting the results. When one increases; i.e. the leaves become thinner. These responses
photoreceptor has been inactivated by mutagenesis, were abolished in transgenic plants which contained five
another may take over its function. However, just because times asmuch phyA as the wild type. However, increasing
a photoreceptor can substitute for another is not the phyA content by this much resulted in dwarf plants,
necessarily proof that it does fulfil the same role in a presumably because they became hypersensitive to far-red
normal plant. Other studies have shown that the light. Smith and coworkers found that transgenic tobacco
interaction between photoreceptors may be complex. For plants which contained lower amounts of oat phyA
developed normally under white light, but showed a
reduced shade avoidance response. This ability to
manipulate plant responses subtly to shade has obvious
agricultural applications as it allows plants to be grown
closer together.

7 Phytochrome signal transduction

So far our discussion of photomorphogenesis has focused

on signal perception (i.e. the photoreceptors) and the
responses observed. Clearly, a complex set of signal

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transduction pathways must link the two. Although our which exhibited impaired responses to red and far-red
understanding of these pathways is far from complete, we light. PIF3 is, therefore, likely to be an important
can trace certain components of them. Our understanding component of the phytochrome signal transduction chain.
of phytochrome signal transduction is considerably more Mutagenic studies have identified many plants with
advanced than that of cryptochrome, so this will be lesions in phytochrome signal transduction chains,
discussed further. indicating that many proteins play a role in signal
transduction. In some cases mutations affect responses to
just phytochrome A or B, whereas other mutants affect
responses to both phytochromes. In addition,
microinjection and pharmacological studies have
implicated a number of small molecules and other
proteins in early signalling processes including Ca2+, cyclic
AMP and calmodulin (a protein which binds Ca2 +). Again,
the role of these compounds is not yet understood but, by
analogy with other systems, they are likely to have a role
in very rapid responses occurring immediately following
phytochrome activation.

7.2 Phytochrome A and B move into the nucleus upon

illumination

Many of the changes in plant development which occur

during photomorphogenesis result from changes in gene
expression. Many hundreds, if not thousands, of genes are
light-regulated, which is not surprising given that light
triggers the development of the photosynthetic apparatus
and the expression of many enzymes of metabolism. The
majority of these light-regulated genes are located in the
nucleus, although a proportion of the photosynthetic
apparatus is encoded in the chloroplast genome. The
regulation of chloroplast gene expression is quite different
from that of nuclear-encoded genes.

Early studies proposed that a proportion of phytochrome

was associated with membranes, but this is now thought
either to be an artefact resulting from the preparation of
cell extracts or to represent a pool of physiologically
irrelevant phytochrome in the process of being degraded.
Current theories favour a model where phytochrome A
and B are initially located in the cytoplasm and then move
into the nucleus upon activation. Studies of this
movement are possible because phytochrome is still
physiologically active even if fused to another protein.
Genetically modified plants have been produced where
7.1 How does phytochrome detect light? the coding region of PHYA or PHYB has been fused to a
reporter gene. One such reporter gene encodes green
The first step of photoperception by phytochrome is the fluorescent protein (GFP) which fluoresces green when
absorption of a photon by the chromophore, but how does illuminated with blue light, allowing its position to be
this initiate subsequent responses? The chromophore is a determined by microscopy. When plants are grown in
linear tetrapyrrole molecule, covalently attached to the darkness, phyA::GFP fusions (or phyB::GFP fusions) are
phytochrome apoprotein, and it undergoes a reversible found in the cytoplasm. When treated with light which
conformational change when it absorbs light. In PR, each will trigger a phytochrome response, the fusion proteins
bond linking the pyrrole groups is in the cis configuration move into the nucleus. The movement of phyB::GFP is
(Fig. 8). Upon conversion to PFR, one of these bonds shifts rapid (it occurs within 15 minutes), red : far-red reversible
to the trans configuration, i.e. part of the chromophore has and requires the presence of the chromophore. The
moved. We do not know precisely how this movement phyA::GFP fusion protein enters the nucleus under
initiates subsequent events, but it alters the absorption illumination conditions which would trigger VLFR and
spectrum, and presumably other properties, of the HIR. Having entered the nucleus the phytochromes
protein. We do know that both phyA and phyB interact accumulate in small patches or ‘speckles’. In contrast, phy
with other proteins, and that both possess a kinase activity C, D and E are constitutively localized in the nucleus,
– they can phosphorylate themselves although speckle formation results fromillumination with
(autophosphorylation) and other proteins. A number of red light and can be reversed by far-red light.
proteins have been identified which interact with
phytochrome, including PIF3 (phytochrome interacting At least two pathways have been identified by which
factor 3). The gene encoding PIF3 was also found, in an phytochrome can stimulate nuclear gene expression, and
independent set of experiments, to be mutated in plants others undoubtedly exist. In both cases, phytochrome

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enters the nucleus and interacts with other proteins, and as interacting with phytochrome, PIF3 will also bind to
these then stimulate gene expression by binding to short another class of LRE. Although PIF3 will bind to the LRE
stretches of DNA (light-responsive-elements – LREs) in both the light and the dark, it forms
found in the promoters of light-responsive genes. In this
way the expression of many different genes can be a complex with phytochrome only in the PFR form. This
stimulated by the action of a few signal transduction complex of PIF3 and PFR is thought to regulate the
chains. recruitment of other protein factors required for
transcription.
The first pathway involves a number of proteins whose
role was first identified in mutagenic studies (Fig. 9). These models go some way to illustrate how phytochrome
These include COP1, other members of the cop/det/fus can trigger gene expression, but undoubtedly much
family, and HY5. In the dark, a large multiprotein remains to be discovered. Many other proteins can
complex (referred to as a signalosome) is found in the interact with these signal transduction chains and some
nucleus which includes the COP1 protein. This interacts light-regulated genes are regulated by other pathways.
with HY5 and prevents HY5 from binding to the LREs in However, the principles of these models are likely to be
the promoters of lightregulated genes. In the absence of repeated in other signal transduction chains.
HY5, transcription does not occur. When phytochrome is
activated, it enters the nucleus and breaks down the 7.3 Photoreceptors’ interactions with plant growth
association between HY5, COP1 and the signalosome. hormones
HY5 binds to the LREs and the transcription of light-
regulated genes is stimulated, triggering de-etiolation. Although the emphasis of this chapter has been on the
COP1 leaves the nucleus and enters the cytoplasm, perception of light by photoreceptors and the ways in
although the rest of the signalosome remains. which these signals affect gene expression, and
subsequently plant development, photomorphogenesis
represents a profound change in plant development. It is
not surprising therefore that plant growth hormones play
a central role in mediating many of these responses. We
have already seen how cytokinin and brassinosteroids
influence skotomorphogenesis. Recently, a protein
induced by cytokinin treatment (ARR4 ) has been shown
to bind to PhyB and stabilize the FR form and over-
expression of ARR4 results in plants hypersensitive to red
light. This demonstrates a direct link between a
photoreceptor and a plant growth hormone.

Another example is provided by the interaction between

the shade-avoidance response and ethylene. Ethylene
production in sorghum (Sorghum bicolor) is under
circadian control, the peak amplitude of which is strongly
increased by shade conditions. A phyB mutant of
sorghum that exhibits a constitutive shade-avoidance
response also produces much more ethylene than normal,
even when grown under full sunlight. In tobacco, Pierik et
al. (2003 ) have demonstrated that many features of the
shade-avoidance response can be stimulated by exposing
plants to ethylene, and that ethylene-insensitive tobacco
plants show a delayed shade-avoidance response when
grown at high densities or when exposed to EOD-FR light.
The molecular basis of this interaction is yet to be
understood.

As auxin and gibberellic acid are important in stimulating

plant growth, it is to be expected that there will be many
interactions between these plant growth hormones and
light.

As our understanding of the signal transduction chains of

individual photoreceptors improves, the interactions
This model elegantly explains the phenotypes of the between them will become clearer. The challenge will still
various mutants. Plants which lack phytochrome or HY5 remain of integrating this detailed molecular information
will be etiolated in the light as the transcription of the into a broader ecophysiological context.
light-regulated genes is not stimulated. On the other hand,
the absence of COP1, or other components of the
signalosome, causes the plants to be constitutively
deetiolated as HY5 is always able to stimulate
transcription. The second pathway involves PIF3 . As well

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8. Ecological functions: Circadian rhythms

Various metabolic processes in plants, such as oxygen

evolution and respiration, cycle alternately through high-
activity and low-activity phases with a regular periodicity
of about 24 hours. These rhythmic changes are referred to
as circadian rhythms (from the Latin circa diem, meaning
“approximately a day”). The period of a rhythm is the
time that elapses between successive peaks or troughs in
the cycle, and because the rhythm persists in the absence
of external controlling factors, it is considered to be
endogenous.

The endogenous nature of circadian rhythms suggests that

they are governed by an internal pacemaker, called an
oscillator. The endogenous oscillator is coupled to a
variety of physiological processes. An important feature of
the oscillator is that it is unaffected by temperature, which
enables the clock to function normally under a wide
variety of seasonal and climatic conditions. The clock is
said to exhibit temperature compensation.

Light is a strong modulator of rhythms in both plants and

animals. Although circadian rhythms that persist under The physiological mechanism of leaf movement is well
controlled laboratory conditions usually have periods one understood. It results from turgor changes in cells located
or more hours longer or shorter than 24 hours, in nature on opposite sides of the pulvinus, called ventral motor
their periods tend to be uniformly closer to 24 hours cells and dorsal motor cells (Figure 11). These changes in
because of the synchronizing effects of light at daybreak, turgor pressure depend on K+ and Cl– fluxes across the
referred to as entrainment. Both red and blue light are plasma membranes of the dorsal and ventral motor cells.
effective in entrainment. The red-light effect is Leaflets open when the dorsal motor cells accumulate K+
photoreversible by far-red light, indicative of and Cl–, causing them to swell, while the ventral motor
phytochrome; the blue-light effect is mediated by blue- cells release K+ and Cl–, causing them to shrink. Reversal
light photoreceptor(s). of this process results in leaflet closure. Leaflet closure is
therefore an example of a rapid response to phytochrome
8.1 Phytochrome Regulates the Sleep Movements of involving ion fluxes across membranes.
Leaves

The sleep movements of leaves, referred to as nyctinasty,

are a well-described example of a plant circadian rhythm
that is regulated by light. In nyctinasty, leaves and/or
leaflets extend horizontally (open) to face the light during
the day and fold together vertically (close) at night (Figure
17.12). Nyctinastic leaf movements are exhibited by many
legumes, such as Mimosa, Albizia, and Samanea, as well as
members of the oxalis family. The change in leaf or leaflet
angle is caused by rhythmic turgor changes in the cells of
the pulvinus (plural pulvini), a specialized structure at the
base of the petiole.

Once initiated, the rhythm of opening and closing persists

even in constant darkness, both in whole plants and in
isolated leaflets (Figure 10). The phase of the rhythm,
however, can be shifted by various exogenous signals,
including red or blue light. Light also directly affects Gene expression and circadian rhythms.
movement: Blue light stimulates closed leaflets to open,
and red light followed by darkness causes open leaflets to Phytochrome can also interact with circadian rhythms at
close. The leaflets begin to close within 5 minutes after the level of gene expression. The expression of genes in
being transferred to darkness, and closure is complete in the LHCB family, encoding the lightharvesting chlorophyll
30 minutes. Because the effect of red light can be canceled a/b–binding proteins of photosystem II, is regulated at the
by far-red light, phytochrome regulates leaflet closure. transcriptional level by both circadian rhythms and
phytochrome. In leaves of pea and wheat, the level of
LHCB mRNAhas been found to oscillate during daily
light–dark cycles, rising in the morning and falling in the
evening. Since the rhythm persists even in continuous
darkness, it appears to be a circadian rhythm. But

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phytochrome can perturb this cyclical pattern of In the following sections we will discuss two central
expression. aspects of the stomatal response to light, the
osmoregulatory mechanisms that drive stomatal
When wheat plants are transferred from a cycle of 12 movements, and the role of a blue light–activated H+-
hours light and 12 hours dark to continuous darkness, the ATPase in ion uptake by guard cells. Light is the
rhythm persists for a while, but it slowly damps out (i.e., dominant environmental signal controlling stomatal
reduces in amplitude until no peaks or troughs are movements in leaves of well-watered plants growing in
discernible). If, however, the plants are given a pulse of natural environments. Stomata open as light levels
red light before they are transferred to continuous reaching the leaf surface increase, and close as they
darkness, no damping occurs (i.e., the levels of LHCB decrease. In greenhouse-grown leaves of broad bean (Vicia
mRNA continue to oscillate as they do during the light– faba), stomatal movements closely track incident solar
dark cycles). In contrast, a far-red flash at the end of the radiation at the leaf surface.
day prevents the expression of LHCB in continuous
darkness, and the effect of far red is reversed by red light. Early studies of the stomatal response to light showed that
Note that it is not the oscillator that damps out under DCMU (dichlorophenyldimethylurea), an inhibitor of
constant conditions, but the coupling of the oscillator to photosynthetic electron transport (see Figure 7.31), causes
the physiological event being monitored. Red light a partial inhibition of light-stimulated stomatal opening.
restores the coupling between the oscillator and the These results indicated that photosynthesis in the guard
physiological process. cell chloroplast plays a role in light-dependent stomatal
opening, but the observation that the inhibition was only
8.2 Circadian Clock Genes of Arabidopsis Have Been partial pointed to a nonphotosynthetic component of the
Identified stomatal response to light. Detailed studies of the light
response of stomata have shown that light activates two
The isolation of clock mutants has been an important tool distinct responses of guard cells: photosynthesis in the
for the identification of clock genes in other organisms. guard cell chloroplast, and a specific blue-light response.
Isolating clock mutants in plants requires a convenient
assay that allows monitoring of the circadian rhythms of The specific stomatal response to blue light cannot be
many thousands of individual plants to detect the rare resolved properly under blue-light illumination because
abnormal phenotype. blue light simultaneously stimulates both the specific blu-
light response and guard cell photosynthesis (for the
To allow screening for clock mutants in Arabidopsis, the photosynthetic response to blue light, see the action
promoter region of the LHCB gene was fused to the gene spectrum for photosynthesis in Figure 7.8). Aclear-cut
that encodes luciferase, an enzyme that emits light in the separation of the responses of the two light responses can
presence of its substrate, luciferin. This reporter gene be obtained in dual-beam experiments. High fluence rates
construct was then used to transform Arabidopsis with the of red light are used to saturate the photosynthetic
Ti plasmid of Agrobacterium as a vector. Investigators were response, and low photon fluxes of blue light are added
then able to monitor the temporal and spatial regulation after the response to the saturating red light has been
of bioluminescence in individual seedlings in real time completed. The addition of blue light causes substantial
using a video camera. further stomatal opening that cannot be explained as a
further stimulation of guard cell photosynthesis because
A total of 21 independent toc (timing of CAB [LHCB] photosynthesis is saturated by the background red light.
expression) mutants have been isolated, including both
short-period and long-period lines. The toc1 mutant in An action spectrum for the stomatal response to blue light
particular has been implicated in the core oscillator under background red illumination shows the three finger
mechanism. pattern discussed earlier. This action spectrum, typical of
blue-light responses and distinctly different from the
9. Blue Light Stimulates Stomatal Opening action spectrum for photosynthesis, further indicates that,
in addition to photosynthesis, guard cells respond
We now turn our attention to the stomatal response to specifically to blue light.
blue light. Stomata have a major regulatory role in gas
exchange in leaves, and they can often affect yields of When guard cells are treated with cellulolytic enzymes
agricultural crops. Several characteristics of blue light– that digest the cell walls, guard cell protoplasts are released.
dependent stomatal movements make guard cells a Guard cell protoplasts swell when illuminated with blue
valuable experimental system for the study of bluelight light, indicating that blue light is sensed within the guard
responses: cells proper. The swelling of guard cell protoplasts also
• The stomatal response to blue light is rapid and illustrates how intact guard cells function. The light-
reversible, and it is localized in a single cell type, the stimulated uptake of ions and the accumulation of organic
guard cell. solutes decrease the cell’s osmotic potential (increase the
• The stomatal response to blue light regulates stomatal osmotic pressure). Water flows in as a result, leading to an
movements throughout the life of the plant. This is unlike increase in turgor that in guard cells with intact walls is
phototropism or hypocotyl elongation, which are mechanically transduced into an increase in stomatal
functionally important at early stages of development. apertures. In the absence of a cell wall, the blue light–
• The signal transduction cascade that links the mediated increase in osmotic pressure causes the guard
perception of blue light with the opening of stomata is cell protoplast to swell.
understood in considerable detail.

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9.1 Blue Light Activates a Proton Pump at the Guard Cell mV have been measured. In addition, proton pumping
Plasma Membrane generates a pH gradient of about 0.5 to 1 pH unit.

When guard cell protoplasts from broad bean (Vicia faba) The electrical component of the proton gradient provides
are irradiated with blue light under background red-light a driving force for the passive uptake of potassium ions
illumination, the pH of the suspension medium becomes via voltage-regulated potassium channels. Chloride is
more acidic (Figure 18.13). This blue light–induced thought to be taken up through anion channels. Thus, blue
acidification is blocked by inhibitors that dissipate pH light–dependent stimulation of proton pumping plays a
gradients, such as CCCP, and by inhibitors of the proton- key role in guard cell osmoregulation during light-
pumping H+-ATPase, such as vanadate This indicates that dependent stomatal movements
the acidification results from the activation by blue light of a
proton-pumping ATPase in the guard cell plasma membrane Guard cell chloroplasts contain large starch grains, and
that extrudes protons into the protoplast suspension their starch content decreases during stomatal opening
medium and lowers its pH. In the intact leaf, this blue- and increases during closing. Starch, an insoluble, high-
light stimulation of proton pumping lowers the pH of the molecular-weight polymer of glucose, does not contribute
apoplastic space surrounding the guard cells. The plasma to the cell’s osmotic potential, but the hydrolysis of starch
membrane ATPase from guard cells has been isolated and into soluble sugars causes a decrease in the osmotic
extensively characterized. potential (or increase in osmotic pressure) of guard cells.
In the reverse process, starch synthesis decreases the sugar
9.2 Blue Light Regulates Osmotic Relations of Guard concentration, resulting in an increase of the cell’s osmotic
Cells potential, which the starch–sugar hypothesis predicted to
be associated with stomatal closing. With the discovery of
Blue light modulates guard cell osmoregulation via its the major role of potassium and its counterion in guard
activation of proton pumping (described earlier) and via cell osmoregulation, the sugar– starch hypothesis was no
the stimulation of the synthesis of organic solutes. Before longer considered important. Recent studies, however,
discussing these blue-light responses, let us briefly described in the next section, have characterized a major
describe the major osmotically active solutes in guard osmoregulatory phase of guard cells in which sucrose is
cells. The botanist Hugo von Mohl proposed in 1856 that the dominant osmotically active solute.
turgor changes in guard cells provide the mechanical force
for changes in stomatal apertures. The plant physiologist Sucrose Is an Osmotically Active Solute in Guard Cells
F. E. Lloyd hypothesized in 1908 that guard cell turgor is
regulated by osmotic changes resulting from starch–sugar Studies of daily courses of stomatal movements in intact
interconversions, a concept that led to a starch–sugar leaves have shown that the potassium content in guard
hypothesis of stomatal movements. The discovery of the cells increases in parallel with early-morning opening, but
changes in potassium concentrations in guard cells in the it decreases in the early afternoon under conditions in
1960s led to the modern theory of guard cell which apertures continue to increase. The sucrose content
osmoregulation by potassium and its counterions. of guard cells increases slowly in the morning, but upon
potassium efflux, sucrose becomes the dominant
Potassium concentration in guard cells increases osmotically active solute, and stomatal closing at the end
severalfold when stomata open, from 100 mMin the closed of the day parallels a decrease in the sucrose content of
state to 400 to 800 mMin the open state, depending on the guard cells (Figure 13).
plant species and the experimental conditions. These large
concentration changes in the positively charged potassium These osmoregulatory features indicate that stomatal
ions are electrically balanced by the anions Cl– and opening is associated primarily with K+ uptake, and
malate2– (Figure 12A). In species of the genus Allium, such closing is associated with a decrease in sucrose content.
as onion (Allium cepa), K+ ions are balanced solely by Cl–. The need for distinct potassium- and sucrose dominated
In most species, however, potassium fluxes are balanced osmoregulatory phases is unclear, but it might underlie
by varying amounts of Cl– and the organic anion malate2– . regulatory aspects of stomatal function. Potassium might
be the solute of choice for the consistent daily opening that
The Cl– ion is taken up into the guard cells during occurs at sunrise. The sucrose phase might be associated
stomatal opening and extruded during stomatal closing. with the coordination of stomatal movements in the
Malate, on the other hand, is synthesized in the guard cell epidermis with rates of photosynthesis in the mesophyll.
cytosol, in a metabolic pathway that uses carbon skeletons Where do osmotically active solutes originate? Four
generated by starch hydrolysis (see Figure 12B). The distinct metabolic pathways that can supply osmotically
malate content of guard cells decreases during stomatal active solutes to guard cells have been characterized (see
closing, but it remains to be established whether malate is Figure 12):
catabolized in mitochondrial respiration or is extruded
into the apoplast. Potassium and chloride are taken up 1. The uptake of K+ and Cl– coupled to the biosynthesis of
into guard cells via secondary transport mechanisms malate2–
driven by the gradient of electrochemical potential for H+, 2. The production of sucrose from starch hydrolysis
∆mH+, generated by the proton pump discussed earlier in 3. The production of sucrose by photosynthetic carbon
the chapter. Proton extrusion makes the electric-potential fixation in the guard cell chloroplast
difference across the guard cell plasma membrane more 4. The uptake of apoplastic sucrose generated by
negative; light-dependent hyperpolarizations as high as 50 mesophyll photosynthesis

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Depending on environmental conditions, one or several osmoregulatory pathways can be selectively activated
pathways may be activated. For instance, red light– under different experimental conditions. Current studies
stimulated stomatal opening in detached epidermis are beginning to unravel the mysteries of guard cell
depends solely on sucrose generated by guard cell osmoregulation in the intact leaf.
photosynthesis, with no detectable K+ uptake. The other

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F. Solute transport and photoassimilate translocation: Uptake, transport and translocation of water, ions, solutes and
macromolecules from soil, through cells, across membranes, through xylem and phloem; transpiration; mechanisms
of loading and unloading of photoassimilates.

1. Water relations free energy. If we consider a plant cell and its

environment, we can therefore say:

When the free energy of cell water is lower than the free energy
1.1 Introduction
of external water, net flux will be into the cell.
Liquid water is absolutely necessary for life as we know it.
When the free energy of cell water is higher than the free energy
Firstly it is the solvent and reaction medium of all living
of external water, net flux will be out of the cell.
cells, which contain some 75–90% water by weight;
secondly it is a reactant in many metabolic processes; and
When the free energy of water is equal inside and outside, there
thirdly, as the hydration water of macromolecules, it will be no net movement in or out, and if water movement
forms part of the structure of protoplasm, existing as results in equalization of the energy levels, net movement will
‘liquid ice’ in a labile but ordered structure. The cease. In both cases, however, a flux (exchange) of water may
physicochemical properties of water (H2 O) are unique; still proceed, equal quantities moving in each direction per unit
heavy water (D2O or DHO), containing deuterium, the time. (This can be demonstrated by applying radiolabelling to
heavy isotope of hydrogen, differs sufficiently to be toxic. the water.)
In multicellular organisms, water provides the transport
medium. Additionally, for plants, water is one of the raw In order to predict the direction of movement of water
materials for photosynthesis and produces the turgor into/out of plants, plant cells or tissues, we therefore need
pressure of water-filled vacuoles which gives mechanical a measure for the free energy of water. This measure is the
rigidity to thin-walled tissues, while some movements of water potential,denoted by the Greek letter C (psi), or Cw.
plant organs occur as a result of turgor pressure changes. Water moves along gradients of water potential, from
Plant cell expansion is driven by turgor pressure and higher to lower water potential. Although Cw is basically
hence growth rates depend on hydration levels. a measure of free energy, for plant physiology it is most
often expressed in pressure units, since hydrostatic
On ‘dry’ land, the highly hydrated body of a terrestrial pressures and tensions (negative pressures) contribute to
plant in many situations tends to lose water to the water potential and play a very important part in the
environment, especially to the atmosphere, in accordance water relations of plants.
with gradients of free energy of water. There are few
habitats where plants do not suffer some water shortage at The pressure unit is the pascal Pa and its multiples, the
least intermittently. The necessity for maintaining an pascal being rather small. Throughout this text, the
adequate internal water content has been a major factor in megapascal MPa (106 Pa) is used. For the derivation of
the evolution of land plants with respect to structure and water potential from basic principles. It should be noted
numerous aspects of physiology. It is not an exaggeration that water potential applies to water in any situation, and
to say that the colonization of land by plants has in any form, liquid, ice, or water vapour. Wherever there
depended upon the evolution of systems for the is water, it has a water potential.
absorption and conservation of water. Another difficulty
for land plants is the transport of water from the 1.3 Water potentials of plant cells and tissues
underground supply tapped by the roots to the aerial
shoots. For the tallest flowering plants this may mean 1.3 .1 Forces determining cellular water potential
moving water some 100 m against gravity.
The water potential of a plant cell is determined by three
This chapter deals with the forces and factors involved in kinds of forces which affect the free energy of the cellular
water uptake and loss in flowering plants, the water.
mechanisms of water movement within the plants, and (1) In plant cells, the cell wall exerts a hydrostatic
the controls exerted on water exchange and water pressure, the turgor pressure (wall pressure) on the
transport by the plant and the environment respectively. protoplast; a cell within a compact tissue may also be
under pressure from surrounding cells. Hydrostatic
1.2 Water movement and energy: the concept of water pressure in excess of atmospheric increases the free energy
potential and raises water potential; thus the pressure potential ψp
is a positive value.
Water moves into plants, in the case of terrestrial plants (2) Plant cells contain low-molecular-weight solutes,
mainly from the soil; and water moves out of plants, mainly vacuolar in a vacuolated cell. These exert osmotic
mainly into the atmosphere. There is also much forces, which decrease the free energy and lower the water
movement of water within plants. Movement implies the potential; the osmotic potential ψπ is therefore a negative
involvement of energy. Metabolism is driven by changes value. (Osmotic pressure is numerically equal to osmotic
in free energy. Water movement, too, is driven by energy potential, but has a positive sign.)
levels. Water will move from a system or area where it is (3) Plant cells contain high-molecular-weight colloids, in
at a higher free energy, to a system where it is at a lower the cytoplasm and the cell wall. Matric forces exerted by
colloids decrease the free energy of water and lower the

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water potential; their effect is represented by the matric

potential Cm. Surface tension forces at air/water
interfaces in cell wall capillary spaces also contribute to
ψm. The overall water potential of a plant cell is the sum of (4)
these three quantities:
If still more water is removed, the effect depends on the
mode of removal (Fig. 1).
(1)

One can think in terms of the wall pressure tending to

squeeze the water out, while osmotic and matric forces
tend to draw the water in. In vacuolated cells of high
water content, the matric potential is thought to make a
relatively minor contribution and, for such cells, the water
potential is often given simply as

(2)

But matric potential is important in controlling water

uptake and retention by tissues of low water content, such
as ‘dry’ and partly imbibed seeds and in the soil. In the
soil, colloid content also can be high.

Osmosis of water is water movement driven by an

osmotic potential gradient through a semipermeable
membrane, i.e. a membrane permeable to water but
impermeable, or very much less permeable, to solutes that
are present. Osmosis drives water movement from a lower
to a higher solute concentration: a high solute
concentration gives a low osmotic potential, hence a low ψ
. All membranes of living cells are semipermeable towards
nearly all the solutes within the cell, or encountered in the
environment, hence osmosis is important in plant water
relations. However, as the preceding discussion shows,
osmosis is not the only process involved in plant water
relations. Hydrostatic pressure (or tension) can overrule
osmotic forces. A cell with a positive ψp is said to be
turgid, though the degree of turgidity varies according to
water content. As a cell takes up water, its ψ rises and its
volume expands as the wall is stretched (Fig. 1). When a
cell is fully water-saturated, i.e. the wall can yield no
more, the cell can take up no more water even from pure
water. Its ψ is zero (= that of pure water, with which it is
in equilibrium), the osmotic potential of the cell contents
being balanced by the pressure potential: If the water is removed by evaporation, drying out in air,
the cell shrivels in size and the wall caves in or folds as the
shrinking protoplast pulls on it. The degree of flexibility of
(3) the wall may determine how much water can be removed.
The tissue becomes wilted. If, however, the water is
This cell is at full turgidity, at maximal volume, maximal removed by immersion of the cell in a solution of low ψπ,
wall pressure and maximal water potential – of 0 MPa! plasmolysis results: the protoplast shrinks away from the
The values of plant cell C usually vary from 0 down, i.e. wall and the external solution fills the space between the
are negative, and negative values are less than zero. plasma membrane and the wall; there is no further
decrease in overall cell size. The relationships between ψ,
On the other hand, as water is progressively lost from a ψπ and ψp are shown graphically in Fig. 2 . Under field
cell, its ψ decreases, both as a result of the reduction of the conditions, wilting is more usual than plasmolysis. Loss of
pressure, as the shrinking cell contents press less strongly water beyond a limit, which varies with the tissue, is fatal.
on the wall, and as a consequence of the solutes being
concentrated into a smaller volume and lowering the ψπ Actual values of cell ψ of plants growing in the field in a
component. When the stage is reached where the temperate climate and with a fairly adequate supply of
protoplast no longer presses against the wall, the cell is water fall mostly in the range of –0.1 to –2.0 MPa, but may
said to be flaccid; now fall well below this in times of water shortage, and in
extreme climates or habitats such as deserts or salt

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marshes; in the latter the apparently generous external 1.3.2 Measurement of water potential and its
water supply is at a low ψ owing to the osmotic effect of components in plant cells and tissues
the salt. Plant ψ values below –10 MPa have been
recorded. The overall ψ of a cell or tissue can be measured by
exposing replicate samples of the tissue to a graded series
of ψ, either by immersing the samples in solutions of
known ψ, or by enclosing them in atmospheres of known
ψ (vapour pressure). Changes in water content of the
samples are detected by weighing the samples before and
after the incubation period; the tissue ψ equals that of the
environment in which it neither gains nor loses water.
Another method for measuring tissue water potential is
the thermocouple psychrometer. The tissue is allowed to
equilibrate with the atmosphere in a small chamber which
houses a thermocouple junction and which is incubated at
constant temperature. The ψ of the chamber atmosphere
will become equal to that of the tissue. A small drop of
pure water is then introduced on to the thermocouple
junction. As this water evaporates, it causes cooling of the
thermocouple, causing a current to flow. The current is
proportional to the rate of cooling; the rate of cooling
depends on the rate of evaporation, which in turn
depends on the ψ of the vapour in the chamber – and this
equals the ψ of the tissue. The instrument is calibrated
with material of known ψ, so that the tissue ψ can be
obtained from the current reading.

The pressure bomb method for obtaining water

potentials, which is more controversial, is discussed later
in connection with xylem transport . It may also be
relevant to measure the individual components of ψ. The
osmotic potential can be obtained by finding the point of
limiting plasmolysis, when the ψp has been just reduced
to 0 and the cell’s ψ equals its ψπ (Equation 4 and Fig. 2).
This is done by immersing the plant material samples in a
series of graded ψ. Since the precise point at which a cell
just becomes flaccid is in practice almost impossible to see,
the point is found at which 50% of the cells can be seen to
be plasmolysed, i.e. have gone beyond the limiting point,
while the remainder still appear turgid. This point is taken
as equivalent to an average limiting plasmolysis value for
all the cells. The method does have an inherent error. By
the time the point of limiting plasmolysis is reached, the
cell volume has shrunk somewhat because of the loss of
water and cellular solutes have become more
concentrated, lowering the value of the ψπ. Fortunately
the volume change is small and the error is estimated at
less than 10 %. An alternative method for obtaining the ψπ
is to express the cell sap and to measure its ψπ by
physicochemical methods applicable to any solution.
Errors can be introduced here through dilution of the sap
with water from the cell walls, and by chemical changes
Quantitative consideration of plant water potentials needs resulting from the mixing of the vacuolar contents with
clear thinking. Because values of ψ of plants and in the the cytoplasm.
natural environment are negative, one must be careful to
keep in mind that e.g. –2 MPa is lower than –1 MPa. The pressure potential is more difficult to measure and is
Sometimes the terms ‘higher’ and ‘lower’ are avoided by often taken as the difference between the overall ψ and the
referring to ‘less negative’ and ‘more negative’ values. ψπ. However, there is an instrument, the pressure probe,
One must also accustom oneself to thinking of zero as a which does measure pressures directly. It consists of a
high value, the highest that the ψ of plant or soil in most very fine hollow glass capillary needle, oil- or water-filled,
cases can attain. (Plant tissues under high pressure, such which is inserted into the cell and at the other end joined
as squirting glands or squirting fruits, which can eject to a pressure sensor. The operation is followed under a
their contents to considerable distances, may have positive microscope, and requires skill. It may be difficult to see
ψ values. But these play no significant part in overall plant precisely where the probe tip is positioned and not all
water relations.) types of cell are amenable to this method.

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1.3 .3 Water permeability of plant membranes and tonoplast towards water are so high that the water
would leak out very fast; the amount of metabolic energy
Gradients of ψ determine the direction of water needed to pump it against the leakage would be
movement between cell and environment, and between unrealistic.
cell and cell. The speed of water movement into or out of
living cells is controlled by membrane permeability. In
plant cells, two membranes need to be considered: the 1.4 Water relations of whole plants and organs
plasma membrane (plasmalemma) which surrounds the
cell; and the tonoplast, the membrane surrounding the The water relations of a whole plant, or even an organ
vacuole, which contains the bulk of the water in most such as a leaf, are much more complex than those of
mature plant cells. The permeability of biological individual cells. The formulae given in the preceding
membranes towards water is generally high. The section, relating water potential to pressure and osmotic
permeability of plant cell plasma membranes towards (and matric) potentials are applicable only at the cellular
water has been estimated to be from several hundred to level. There is no such thing as the ψp of a whole plant;
several thousand times higher than for such small the value will vary between different tissues. The overall
uncharged organic compounds as urea, glycerol or sugars, ψ, too, usually varies between different parts of a plant.
and for charged ions the difference is greater still. Most flowering plants are land plants and, in the
Nevertheless, the resistance of biological membranes terrestrial environment, the ψ of the atmosphere is nearly
towards water is not negligible. The quantitative measure always much lower than that of plant tissues, often by
of the permeability of a membrane towards a chemical is tens of MPa, and hence there is a great tendency for water
the permeability coefficient, the rate of diffusion per unit loss from the plant. The large surface area necessitated by
area, unit time and unit concentration gradient. The the photosynthetic mode of life provides a large surface
permeability coefficients of biological membranes towards for evaporation of water, transpiration. This loss must be
water are so high that they are difficult to measure; the made good from the soil, which is generally at a much
concentration gradients are small and equilibration higher ψ than the atmosphere or the plant. Hence for most
between external and internal water is reached rapidly. of the time there is a flow of water through the plant,
Very variable values have been reported for the along a ψ gradient, as below:
permeability coefficients of plant cell plasma membranes.
Because of the practical problems in measuring the soil Æ rootÆstemÆleafÆair
permeability coefficient for water, the hydraulic
conductivity is often measured instead, the rate of This flow is frequently termed the transpiration stream.
diffusion of water per unit area and unit time per unit Only in times of a water-saturated atmosphere, or in times
hydrostatic pressure gradient. This can, however, be used of extreme drought, may there be equilibrium, more or
only to compare water permeabilities of various systems, less, between plants and the environment, with gradients
not for comparing the permeabilities of water and other within the plant eliminated and water movement nearly at
chemicals. a standstill. Of the water absorbed by the roots, only a
very small fraction is retained by the plant in temperate
The ease with which water, and other small hydrophilic habitats. For maize, an annual, this fraction has been
molecules with masses not above 46 Da, traverse estimated at less than 1% of the water absorbed during its
biological membranes was first attributed to minute pores growing season. During one bright sunny day leaves may
opening up transiently in the bimolecular lipid leaflet of transpire several times their own weight of water; a leaf of
the membrane by the random thermal vibrations of the Senecio jacobaea growing on a sand dune can transpire its
lipid molecules. Subsequently membrane proteins named own weight in water in 45 minutes. The water content of
aquaporins, which make water-specific channels through aerial organs of a plant is generally lower in the daytime,
biological membranes, have been identified in all types of when the rate of transpiration is high, than during the
organisms. Plants contain a greater variety of aquaporins night, when the transpiration rate is much lower (owing to
than organisms from other kingdoms; over 30 aquaporins the lower temperature and closure of stomata) and the
are known from maize, for instance. Aquaporins show deficit is made good. In the roots, which experience much
specificity with respect to organ and membrane, i.e. less temperature change over the diurnal cycle and which
plasma membrane or tonoplast. Evidence is accumulating have no stomata, the water content fluctuates much less.
that the variability of the reported values of the water
permeability coefficients of different plant cells results at 1.4 .1 Absorption of water by roots
least partly from differences in the concentrations (and
may be types) of aquaporins. Development and The root systems of plants are often very extensive. Roots
differentiation are associated with changes in aquaporin of some plants extend much further underground than the
density; e.g. during the cell elongation stage which is shoot rises into the air. The roots of apple tree (Malus
accompanied by rapid water uptake, aquaporins are domestica) may go down to about 10 m, and even in
particularly abundant. A diurnal rhythm in expression of herbaceous plants such depths can be reached, e.g. in
aquaporin genes has been correlated with leaf movements alfalfa (Medicago sativa). The actual area of a root system is
which depend on turgor changes in their motor cells, formidable. It has been reported that a rye (Secale cereale)
mediated by water movements in and out of the cells. It plant had a root surface area of over 600m2 , of which two-
must be emphasized that all water movement into or out thirds was root hair area, and the total length of the root
of plant cells is along the C gradient. There is no active system was over 11000 km, including 10000 km of root
pumping of water against its free energy gradient, at the hairs. The total area of the shoot (including areas of cells
expense of metabolic energy, as occurs with many other bordering leaf air spaces) was only 28 m2 . Most of the
metabolites. The permeabilities of the plasma membrane water absorption takes place near the root tips, where

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there is a thin epidermis with root hairs. Not only do the cells; these end walls are then known as perforation
root hairs provide a large area, but they make intimate plates. Their possession is the distinguishing feature of the
contact with the soil, bending around soil particles and vessel element. The diameters of vessel units range from
penetrating into tiny crevices. below 10 mm to several hundred mm, even 1000 mm in
some lianas. Tracheid diameters overlap with those of the
As the root tissues mature, the epidermis with its hairs is narrower vessel elements. Any one piece of xylem has
replaced by a more impermeable suberized periderm. For conducting elements of varied width; this may be of
efficient water uptake, root growth must continuously functional importance. The lengths of vessels range from
regenerate the absorbing zone behind each growing apex. under 1cm to 10 m or more, and are very variable even
Continuous growth is also necessary to invade new areas within the same plant. In some trees some continuous
of soil, for there is little lateral movement of water in soil vessels run right from the crown to the roots, but most
compared with downward drainage directly after water vessels are shorter than the height of the plant, and even
addition. Water will not move to the roots, so roots must in trees many vessels measure only a few centimetres. The
grow to the water. Positive hydrotropism may direct root possession of vessels making long continuous channels for
growth towards water. Roots can grow rapidly; a rate of water movement is considered to be one of the advanced
10 mm per day is common for grasses; maize (Zea mays) features of flowering plants; the earliest land plants had
roots can extend by as much as 50–60mm per day. The only tracheids, and while the evolution of vessels has
average daily increase in length of the total root
system of the rye plant discussed above (hairs
excluded) was almost 5 km.

Though the root hair zones provide the most

efficient water absorbing surfaces, uptake in older
regions is still appreciable, particularly during
conditions of water shortage and at times when root
growth is slow, such as in winter. Points of
emergence of lateral roots break the suberized
layers and enable the entry of water.

1.4 .2 The route of water movement through the

plant

The xylem as the water-transporting system

The main channel for upward/long-distance

movement of water in the plant is the xylem, the
wood. When the tissues outside the xylem are
peeled off over a short length of woody stem where
the xylem is central, the conduction of water
beyond the stripped region continues unimpeded.
The non-living cells of the xylem are filled with a
watery sap, at least in young wood, and dyes and
Indian ink can be seen to move in the xylem. Toxic occurred in several divisions of land plants they are
solutions have been shown to pass from roots to leaves, lacking in the conifers. Tracheids offer much more
indicating that the route does not involve living cells; resistance than vessels to water movement.
heat-killed stems, too, can conduct water. Chilling does
not stop water movement as long as no freezing occurs. In woody perennials, new layers of xylem known as
All this points to nonliving cells of the xylem as the water- annual rings are produced each year during the growing
conducting cells. When the lumina of these cells are season. The older regions of xylem eventually lose their
blocked with mercury or cocoa butter, water movement is water-conducting capability and become air-filled or
inhibited. blocked by ingrowths (tyloses) from adjacent living
The xylem is a complex tissue. In addition to water- parenchyma cells, or by gums, resins and tannins. The
conducting cells and lignified fibres which all are dead at water then moves only through the young, outer xylem,
maturity, it contains living parenchyma cells and the sapwood, which may comprise only the current year’s
sometimes also living transfer cells. Functional xylem is growth or include a few youngest annual rings. The inner
accordingly not a dead tissue, though it contains a large non-conducting xylem is known as the heartwood.
proportion of dead cells. In flowering plants there are two
kinds of conducting cells, the tracheids and the vessel Living parenchyma cells make up rays in secondary
elements. They have lignified secondary walls with the wood, running radially through the xylem from the pith
secondary thickening laid down in distinctive patterns towards the cortex (Fig. 3) and lying also among the
leaving areas of primary wall as pits; these facilitate the conducting cells. They store organic nutrients, and some
passage of water from cell to cell. Somewhat confusingly, botanists have assigned a role in water transport to them.
the thin primary wall across the pit is often called the pit Transfer cells are not always present in xylem, but may
membrane. The tracheids function as single cells, but occur next to conducting cells especially in leaf veins; they
vessel elements are joined to make elongate vessels by the have highly involuted cell walls, which gives them a large
perforation or partial breakdown of end walls in files of surface area of plasma membrane, and they function in

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the transfer of solutes into and out of the conducting cells. in the material studied. Decreased aquaporin content in
The movement of water into and out of the xylem plasma membranes of tobacco roots (Nicotiana tabacum),
achieved by antisense repression of an aquaporin mRNA
synthesis, has decreased greatly the roots’ hydraulic
conductivity. This indicates that there is movement
through the plasma membranes and certainly supports
the symplast route. But the data do not exclude the
transcellular route. This route has been criticized as being
the path of greatest resistance in view of the large number
of membranes traversed. However, the density of
aquaporins in tonoplasts is very high (up to 40% of total
tonoplast protein), which may give them a much higher
permeability towards water than is shown by the plasma
membranes, and crossing the vacuole may offer less
resistance than originally supposed. The three routes are
not mutually exclusive and it is quite possible that all
three contribute to water movement in proportions
varying according to circumstances. When the rate of
water movement is slow, most of the movement might be
along the low-resistance apoplastic route, the higher
resistance pathways beginning to contribute when
demand increases. But the opposite has also been
suggested, with a strong transpiration pull increasing the
apoplastic flow. In a herbaceous plant, where the vascular
strands of the stem have not been joined to a continuous
In the root, the xylem is the central tissue; to reach it, vascular cylinder by secondary growth, any part of the
water must pass radially through the epidermis, cortex, root system normally supplies those parts of the shoot
endodermis and pericycle (Fig. 3). The precise radial which are directly above it, these being the parts with
movement pathway is still under discussion. There are which it is in direct vascular connection; lateral movement
three possible routes for the water: the apoplastic route, does not occur or is very restricted. But if a part of the root
the symplastic route and the transcellular route (Fig. 4). system is deprived of water, lateral movement is activated
and the overlying aerial parts receive a water supply from
The apoplast is the collective term for all the non-living other root sectors. In trees with a continuous cylinder of
parts of the plant body: cell walls, intercellular spaces and wood, dye injection has shown that the path of water
xylem conducting cells. Apoplastic water movement movement frequently spirals round the stem, following a
would occur in the capillary spaces of the root cell walls helical arrangement of the conducting cells around the
and perhaps in the intercellular spaces; there are reports of trunk. The route of water movement through stem and
these spaces in roots containing fluid. The symplast is the leaf tissue after its exit from the xylem is also problematic.
collective living part of the plant; nearly all the living cells Fluorescent dyes introduced into the xylem to act as
of the plant body are joined by plasmodesmata, tracers for water movement move from the conducting
submicroscopic protoplasmic connections of diameters cells into cell walls or crystallize out in intercellular spaces
around 50 nm. In the symplastic route, water has to cross in the shoot. Such observations have been interpreted as
a plasma membrane to enter the cytoplasm of an outer indicating an apoplastic route for water passing out of the
root cell; it would then move in the cells within the xylem. However, careful analysis of the data points to the
cytoplasm, around the vacuoles, and from cell to cell opposite view: the dye becomes concentrated in the
through the plasmodesmata without the necessity to cross apoplast precisely because the water passes into the living
more membranes, till it exited into a xylem conducting cells near the xylem, leaving behind the dye to which the
cell. The transcellular route envisages movement ‘straight’ plasma membranes are impermeable. It is therefore likely
through the vacuoles, crossing the tonoplasts of each cell; that water in leaves and stems first moves symplastically
cell-to-cell movement could be via plasmodesmata or from the xylem, before it finally passes into cell walls
crossing the plasma membranes. again and evaporates.

The apoplast route has been supported on the grounds

that it is the path of least resistance, with minimal
traversing of membranes. However, the radial walls of
endodermal cells at the level of most active water
absorption develop strips of wall thickening, the
Casparian strips, chemically resembling the water-
impermeable suberin (Fig. 5). In older parts of the root, all
endodermal walls except the outer tangential ones
become heavily thickened. It is therefore frequently
suggested that at least at the endodermis water must pass
through living protoplasts, and that this layer regulates
water movement to the root xylem. There is some
evidence to support this idea, but also data to the
contrary, probably reflecting the extent of wall thickening

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1.4.3 The motive forces for water movement: root hypothesis is the fact that when the ψ of the medium
pressure and transpiration pull around the roots is suddenly lowered, reversing the
gradient, exudation rate falls and may even become
The rate of water movement through plants is very negative, so that externally applied liquid is sucked in at
variable; Table 1gives examples of maximum rates the cut stump. Root pressure can result in guttation, drops
attained in a number of types of flowering plants, and of liquid appearing at leaf tips and edges where the xylem
conifers for comparison. In any individual plant the rate is sap is forced out through pores overlying vein endings.
of course also highly variable, depending on This liquid is much more dilute than bleeding sap at a
environmental conditions. The order of increasing stump, solutes having been absorbed by leaf cells. The
maximal speed of water movement is the order of manifestation of root pressures in temperate climates is
anatomical development towards wider and more most frequent during warm humid weather and the
numerous vessels in the xylem. Conifers, with the lowest pressure shows a diurnal rhythm with maxima at nights,
maximal speeds, lack vessels, having only tracheids, while often dropping to near zero by day. The drops of guttation
lianas may have very wide vessel elements. Water moves fluid are easily mistaken for dew in the morning, but an
through the xylem along a ψ gradient, from roots to attentive examination shows that the drops are arranged
all parts of the shoot, most of the flow terminating in the regularly, corresponding to the positions of the pores. In a
leaves. There must be some motive force, some energy strawberry (Fragaria ananassa) leaf, for instance, there is a
input, which maintains the ψ gradient and overcomes the neat droplet at the tip of every tooth of the leaf edge. In
frictional and gravitational resistances along the way. tropical rainforests, where it is warm and humid all the
There are two possibilities: the xylem sap could be time, guttation fluid drips from shrubs and small trees,
pumped up under pressure, or it could be pulled up mimicking rain.
under tension, negative pressure. Capillary rise (a surface
tension phenomenon) could not account for a rise of more Although the development of root pressures is well
than 1 m in the finest conducting elements; many plants authenticated, in the majority of cases root pressure
are much taller than this, trees reaching 90–100 m. cannot account for water movement. Some species
apparently never develop root pressure. The observed
pressures are too low to raise water to the required level
in tall plants. A pressure of 0.2 MPa can raise water
maximally to 10 m, so root pressures in the usual range
could raise water no higher than 10 m. The maximal
values of 0.6 MPa could raise water to 30 m, still far short
of the height of many trees. The quantity of water that can
be moved by root pressure is small: e.g. wheat seedlings
(Triticum aestivum), which transpired about 3 mL water
per hour, exuded only 0.5 mL per hour by root pressure.
In many instances maximum bleeding rates are only 1–2 %
of the water loss occurring by transpiration. Root pressure
persists only as long as the water-yielding capacity of the
soil is high, but plants can still extract and transport water
after root pressure becomes inactivated through a
Root pressure lowering of the soil ψ. As stated, root pressures in
In certain circumstances, the xylem sap is under positive temperate climates are most frequently developed during
hydrostatic pressure; when the plant is decapitated just warm nights; most of water transport occurs during
above root level, the stump exudes sap, a phenomenon daytime. It is moreover mostly herbaceous species that
often called ‘bleeding’. A manometer fitted over such a develop root pressures. In deciduous trees root pressures
bleeding stump registers a pressure known as the root are demonstrable in the spring before the buds open, but
pressure, usually in the range of 0.1 to 0.2 MPa, once the leaves have expanded and rapid water
exceptionally reaching 0.5 to 0.6 MPa. The development of movement through the plant begins, root pressures can no
this pressure is dependent on the metabolic activity of the longer be detected. During the periods of rapid water
roots. No positive root pressure is found when the roots movement associated with rapid transpiration, the vast
are subjected to treatments inhibiting metabolic activity, majority of evidence in fact indicates that the water is not
such as lack of oxygen, application of respiratory under positive pressure but under tension. In such
inhibitors, low temperature, or starvation. The mechanism circumstances, a cut in the xylem does not result in sap
is thought to be osmotic. The exuded sap has a ψ value exudation, but if the cut is made under water, the water is
below that of the soil because of a higher concentration of sucked in (as seen if a dye is added to the water).
solutes, mainly inorganic ions, but sometimes including Transpiring twigs can pull water against an artificial
organic solutes, too. It is postulated that the living resistance more effectively than a vacuum pump; leafy
parenchyma and transfer cells of the xylem secrete the twigs can raise a column of mercury to heights greater
solutes into the conducting cells using respiratory energy. than can be supported by atmospheric pressure. On the
basis of such evidence it is generally accepted that water
The lowering of ψ causes water to follow the ions into the
movement in plants, particularly in woody species, is the
xylem, building up a pressure – the classical osmotic
result of water being pulled up to replace that lost by
pressure – which pushes up the sap. The xylem ψ does not
transpiration. We therefore need to explain how water can
equilibrate with that of the soil, since the ions are
be pulled up under tension to the topmost leaves of the
continuously swept away with the water movement, and
tallest trees.
the root cells continuously secrete more. In favour of this

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The cohesion–tension theory (transpiration–cohesion A second requirement for this mechanism of water
theory) for the ascent of xylem sap movement is that the water in the xylem must be able to
withstand the necessary tensions without the columns
A theory of the ascent of sap based on the cohesive breaking. Here the situation is more complicated. The
properties of water was advanced independently in 1894 cohesive power of water is great, resulting from hydrogen
by Dixon and Joly, and in 1895 by Askenasy. This theory, bonding between the molecules: theoretical calculations of
as described below, is still supported in its essentials by the tensile strength of pure water have given values as
most plant physiologists, although alternatives have also low as –1400 MPa. Tensions of –20 to –30 MPa are claimed
been postulated. to have been withstood experimentally. But xylem sap is
not pure water: it contains dissolved materials. Taking
The motive force for root pressure is generated at the root into account the presence of these, together with the
end of the plant. The generation of tension takes place at diameters of xylem cells and the adhesive properties of
the leaf end. The living cells of the leaf contain solutes and their walls, the expected tensile strength of xylem sap in
commonly have a ψ well below 0, say down to –2 MPa situ has been put at about only –3 MPa. This, however,
under ‘average’ conditions in a temperate climate. But would still be enough to raise the sap to 150 m, higher
they are still relatively water-saturated compared with the than the tallest trees.
atmosphere for most of the time in most climates. The ψ of
the atmosphere is usually very much lower than that of The experimental measurement of tensions in the xylem is
the leaf cells, say –10 to –50 MPa. To put it another way, not easy and most workers have relied on indirect
the atmospheric vapour pressure is usually very much estimates. One technique is known as the pressure bomb
lower than would be in equilibrium with the leaf cells. method of Scholander, who introduced it in the 1960s.
There are of course occasions when the atmosphere is very Branches or leaves are cut from transpiring plants, where,
humid and water may condense out as mist or dew. But if the cohesion theory is correct, the xylem fluid should be
generally, there being a tremendous drop in ψ between under tension. The leaf/branch is enclosed in a pressure
leaf and atmosphere, amounting to tens of megapascals, chamber with the cut end protruding and nitrogen gas at
there is a great tendency for water to evaporate, or increasing pressures is applied inside the chamber;
transpire from the leaf. The cells in contact with air lose eventually a liquid droplet is seen appearing at the cut
water, mainly into the intercellular air spaces: it should be end. The xylem tension before cutting is taken to equal the
remembered that most of the leaf surface is inside the leaf. pressure at this point, only with a minus sign. The
Moreover the external surfaces are covered by a cuticle argument is that, when the cut is made, the water column
which strongly impedes the passage of water vapour. As in the xylem snaps and the water retreats deep into the
the cells bordering the air spaces lose water, their ψ drops xylem strand; to force it back to its original position, a
and water moves into them by osmosis from the deeper- pressure is needed numerically equal to the original
seated cells with which they are in contact. These in turn tension. With this technique, tensions of – 0.5 to –8.0 MPa
replenish their loss from cells still deeper in the tissue, have been recorded, the highest values occurring in
until water is extracted from the xylem conducting cells, halophytes and desert plants, which must extract their
especially at the veinlet endings. The ‘pull’ on the water is water against a very low ψ in the soil. Twigs of trees have
transmitted right down the xylem and the water loss is been centrifuged, the centrifugal force exerting a tension
made good by further uptake of water from the soil by the on the xylem water until the water columns broke;
roots. There is thus envisaged an uninterrupted column of according to species, tensions from –0.4 to below –3.5
water being pulled under tension from the roots to the MPa were endured.
leaves. The energy for the movement comes from solar
heat, which provides the latent heat of evaporation: Another indirect method for estimating the xylem tension
transpiration is the one plant process which uses solar is to measure the water potentials of living leaf cells in
energy directly without the intervention of contact with the xylem. In wilted plants of tomato
photosynthesis. Lignification gives the xylem conducting (Lycopersicon esculentum), privet (Ligustrum lucidum) and
cells the strength to endure the tensions without cotton (Gossypium barbadense), leaf ψ values were found to
collapsing inwards. This briefly summarizes the reach –4.1, –7.0 and –14.3 MPa respectively. It was then
transpiration–cohesion theory for the movement of water assumed (without direct proof) that the xylem sap was
in a qualitative way. The quantitative aspects are now under a tension of the same magnitude, since the tension
considered. It was stated that the positive pressure needed must be balanced by the ψ of the leaf cells if any water is
to pump water up the xylem by 10m is 0.2 MPa; the to move into the leaves. If numerical values for xylem
negative tension needed to pull it up by 10m is tensions of magnitudes as quoted above are accepted,
numerically equal but opposite in sign, –0.2 MPa. Of this, these support the cohesion theory. There have also been
–0.1 MPa is needed to counteract the force of gravity and a measurements showing gradients of tension or leaf water
further –0.1 MPa is needed to overcome frictional forces potential, with the values becoming progressively more
opposing the flow. If 100m is taken as the height of the negative higher up the plant – as is of course required for
tallest tree, the mechanism for xylem transport under the flow.
tension requires that tensions of at least –2 MPa must be
generated by transpiration. That is perfectly feasible. The There is nevertheless a serious problemwhich has led to
final evaporation of water takes place from the capillary reservations in accepting the transpiration–cohesion
spaces between cell wall fibrils; these spaces may be as theory. Water columns under tension are liable to cavitate
narrow as 5 nm and the tensions generated at the tiny (embolize) on mechanical disturbance, breaking up into
menisci (the curved air–water interfaces) can be calculated droplets of water and water vapour. When water is sealed
to reach –2 9 MPa. There is accordingly no problem with into a glass capillary under tension, the slightest tapping
the generation of tensions.

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or shaking will bring about cavitation. Plants are buffeted transpiration pull, but the magnitude of the tension would
by the wind, sometimes very violently. Any introduction be reduced and hence there would be no high tensions
of an air bubble would similarly break up the column. and less risk of cavitation. Zimmermann claims that xylem
Twigs, even large branches, are broken off by the wind; sap is quite rich in solutes, contrary to the generally
animals bite away pieces; any such damage might be accepted view that it is extremely dilute (except in early
expected to let air spread through large expanses of spring, before transpiration commences). Another idea
xylem, if not the whole plant. Even without external (Canny 1995) is that, whilst transpiration provides the
damage, air seeding, i.e. the sucking in of air through the driving force, there is no building up of large tensions
cell walls, is very likely when the xylem water is under because the living turgid cells of the xylem exert a positive
high tension. Yet the plants go on conducting water. The pressure on the vessels and tracheids, as does the phloem,
actual great stability of the water-conducting system where cells are under a high positive pressure. In defence
under natural conditions seems at variance with the of the cohesion–tension theory The majority of plant
metastable state of water columns under tension. Twigs physiologists nevertheless support the cohesion– tension
which are cut so as to allow air into the vessels will theory. The new postulates of Zimmermann and Canny
resumewater uptake when the severed end is placed in rely on tension measurements from one single type of
water. In the winter xylemsap may freeze; the dissolved instrument, the pressure probe, whereas evidence for
air trapped as bubbles in the ice should remain as bubbles strong tensions has been obtained with several methods,
when the sap thaws – yet sap flow is resumed in the with consistent results. The discrepancy may be due to
spring. On the other hand, cavitation does certainly occur problems with handling the pressure probe. Its insertion
in plants. Each cavitation event in the xylem makes a could cause cracks in the xylem cell walls, letting air in
minute noise that can be amplified by means of suitable and causing cavitation. The probe diameter of 10 mm is
electronic equipment to an audible ‘click’. These clicks can not negligible compared with the diameters of the xylem
then be counted and recorded over time. During rapid vessels probed, 50–90mm; the insertion of the probe could
drying out one can count several hundred clicks per do appreciable damage. With living cells, the plastic cell
minute in a wilting leaf. When the water supply is contents pressing against a pierced wall could seal up
restored, cavitation ceases and water uptake is resumed. cracks, but in the xylem conducting cells there is nothing
Another method for detecting cavitation is to freeze plant to protect against air entry. Claims for a high solute
material very rapidly with liquid nitrogen and then to content could derive from the probe sampling
examine the xylem in the frozen state by cryoscanning preferentially the young xylem (which is outermost),
electron microscopy. Empty (i.e. embolized) and ice-filled where immature cells might still contain solutes derived
vessels are easily distinguished. Cavitation has turned out from the breakdown of cell contents; such cells might also
to be frequent in the field under natural conditions. These be under more positive pressures. Later experiments by
observations are difficult to reconcile with the cohesion other workers have succeeded in measuring stronger
theory and some plant physiologists postulate alternative tensions in the xylem with the probe, down to –1 MPa,
mechanisms. and have shown agreement between pressure-bomb and
pressure-probe data. The osmotic theory implies a high
1.4 .4 Validity of the cohesion–tension theory energy expenditure by the living xylem parenchyma cells
for pumping solutes into the xylem, but the low O2 level
Alternative hypotheses in the xylem of woody axes precludes high aerobic
respiration rates and the limited volume of living cells
All the experimental values for xylem tensions quoted would moreover have to control a large volume of dead
above in support of the cohesion–tension theory have conducting cells (Richter 1997). There would also have to
been obtained by indirect means. It was considered for be a mechanism for recycling the solutes. Mercury is
many years that the direct measurement of tensions in the pulled up by a leafy, transpiring twig inserted into the top
xylem was not practicable. Since 1990, however, a group of a water-filled glass tubing, which has its bottom end in
of workers led by U. Zimmermann has published a reservoir of mercury. The twig sucks the mercury into
measurements which are claimed to be direct readings of the xylem and right into very narrow tapering cell tips,
pressures within intact, water-conducting xylem using a which would require a tension of –2 MPa, and there is of
pressure probe. It is not feasible to insert this delicate glass course no question of osmotic forces acting on the
needle, 10 mm in diameter, into thick wood, but fine veins mercury. Strong positive pressures, postulated to be
such as in leaf midribs were probed and the probe failed exerted by living xylem parenchyma and by the living
to register tensions as strong as obtained by the indirect phloem, would probably have little effect on the
methods, –0.4 MPa being the usual limit. Occasionally conducting cells with their rigid walls.
down to –0.6 MPa was registered, but then the xylem
vessels cavitated within minutes of probe insertion. At present, the bulk of the evidence seems to be in favour
Moreover, many of the probe readings failed to register of the cohesion–tension theory; but then one has to
any tensions at all but recorded weak positive pressures, account for the fact that airlocks can be introduced into
up to 0.1 MPa. the xylem in nature by cavitation owing to water stress,
freezing, mechanical damage – or experimentally, without
On the basis of the pressure probe readings, alternative permanently or even temporarily stopping the overall
mechanisms for xylem transport have been put forward. flow. How is this to be reconciled with the need of
Zimmermann and coworkers suggest that the mass flow continuous water columns for the movement under
of sap in the xylem is aided osmotically (in a manner cohesion?
similar to the building up of root pressures), by the
secretion of solutes into the conducting cells, from the To account for the resumption of sap flow in the spring
living cells of the xylem. There would still occur a after freezing, the suggestion has been made that the air

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bubbles released from thawing ice might be forced into

solution again by root pressures, which are highest in
early spring, although root pressures tend to be low in
trees. Another mechanism by which cavitated vessels
could be refilled at any season is by capillary forces.
Perhaps refilling of cavitated cells is a situation where the
activities of the living cells of the xylem and of the
phloem, emphasized by Canny and Zimmermann, do
come into play in building up pressures and forcing fluid
into the empty vessels. Counts of embolized vessels in
sunflower leaves at different times of day, and at various
stages of drying out, have revealed far fewer empty
vessels than expected from the rate of cavitation. From
this it was deduced that the embolized vessels were
refilled within minutes by pressure from living cells. A
similar conclusion was reached from scoring maize roots
from dawn to dusk for embolized vessels. It is also
possible that many airlocked conduits never resume water
conduction, since in perennials a newring of fresh
sapwood is produced annually; in some species, only the
current year’s growth is active in transport. Redundancy
of xylem does occur. The total amount of xylem in a plant
is generally much in excess over that needed to satisfy the
plant’s water requirements, as shownwhen large amounts
ofwood are removed. The presence of quite large numbers
of cavitated vessels can therefore be tolerated without too
much ill effect.

There is also evidence that airlocks produced by cavitation

do not necessarily spread from their origins over large
volumes of xylem. Vessels are formed by perforation of
end walls between individual vessel element cells, but
very often a perforation plate is crossed by bars of cell
wall material which break up the opening into numerous
fine slits. It is suggested that in such vessels an airlock
may remain confined to the single vessel element in which Moreover some vessel elements are extremely narrow,
it forms, unable to pass the perforation plate due to water there being normally a range of widths of vessel elements
menisci holding firm in the narrow openings because of in the same plant organ. Volume flow through a tube is
surface tension forces. Even if a whole vessel cavitates, its proportional to the fourth power of the radius of the tube.
length may be limited compared with the height of the Hence a two-fold increase in the radius of a vessel from
plant. In trees, some vessels may extend to over 10 m, but say, 5 mm to 10 mm would increase volume flow (for the
most are much shorter; e.g. in a holly (Ilex verticillata) the same tension) 16-fold (24 = 16). Since wide vessels are so
maximum vessel length was 1.3 m, but 99.5% of the much more efficient for a bulk flow of water, why should
vessels were under 5cm long. Similar data can be quoted a vascular bundle contain, in addition to wide vessels,
from other species. Cavitations of limited extent can be narrow vessels and tracheids? The most probable answer
bypassed by lateral flow through the cell walls (Fig. 6), is that this gives the plant the flexibility to react efficiently
mainly through pits. Water movement can continue even to varying environmental water status. When the soil ψ is
in the presence of two overlapping cuts from two sides of high the plant does not require very high tensions in the
a stem, as long as the distance between the two cuts is xylem to extract the water; in such a situation, most of the
great enough to leave some intact vessels within this transpiration stream would pass through the widest
region. An increase in the resistance to flow can be vessels, which offer the least resistance. But when water
detected when such cuts are made, as would be expected: stress sets in and xylem tension increases, it will be the
cell walls offer more resistance to flow than the lumina of widest vessels that are the most vulnerable to cavit ation.
vessel elements. But the cohesive system as a whole is not The narrower vessels and the tracheids then can take over
seriously reduced. the function of water conduction; the same high tensions
which cause vessels to cavitate also overcome the
The necessity to protect the xylem transport system resistance in the narrower conducting cells. In tracheids,
against the effects of cavitation and airlocks may explain any cavitation event is confined to one single cell, and
why tracheids have persisted in the flowering plants tracheids are also relatively narrow, so they are the least
alongside vessels. The vessels, with no barriers to water vulnerable to water stress.
flow between cell and cell, offer much less resistance to
water flow than tracheids, where water must pass from The movement of cohesive water columns in the xylem
tracheid to tracheid through the cell wall, mainly through under transpiration pull may thus be buffered against
pits. serious disruption from cavitation by excess capacity; by
the regular annual replacement of old xylem by new in
perennials; by refilling of air-filled vessels by root

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pressures, by capillarity and by pressure from adjacent to the soil, important considerations are the amount and
living cells; by the bypassing of airlocks in cell walls; and availability of soil water, soil temperature and soil
by the presence in xylem of tracheids and narrow vessels, aeration. Above ground, the relevant factors are
which are less susceptible to cavitation. There is not atmospheric humidity, temperature, wind speed and
enough evidence for accepting alternative theories for the light. The plant factors are the area and water
ascent of sap which deny the existence of high tensions in permeability of the absorbing surface in the roots; the area
the xylem. Nevertheless, data from the pressure probe and water permeability of the evaporating surfaces of the
measurements, and other apparently anomalous shoot; the frequency of stomata and the degree of their
observations, are drawing attention to the possible opening.
functions of the living cells of the xylem, and of the
neighbouring phloem, in water movement. 1.6.1 Soil water and uptake by the roots

1.5 The transport of solutes in the xylem The soil is a complex system. Physically, it consists of
particles with sizes ranging from large stones to
Xylem transport is often considered primarily in terms of submicroscopic colloidal material, and it contains pores of
water movement, but the xylem also has an important varied dimensions. Chemically the particles are of various
function in the transport of solutes. Samples of xylem sap composition, organic and inorganic, and there are many
can be obtained for analysis by collecting the exudate solutes in the soil water. The high colloidal content of
when it is under pressure (the bleeding sap); or it can be most soils (coarse sand is an exception) gives it a
sucked out under vacuum, pressed out or centrifuged out, significant matric potential; solutes such as mineral ions
from pieces of wood. The concentration of dry matter in give it an osmotic potential. The pressure potential is
xylem sap is low, commonly 0.1 to 0.5%, but the total represented by tensions (negative), i.e. the surface-tension
volume of sap moved in the xylem is high, so the amount forces at water menisci in small pores. Electrostatic forces
of solute that is carried in the xylem is significant. The around particles and capillary forces in pores also
mineral ions absorbed by the roots are distributed around decrease free energy and help to retain water in the soil.
the plant in the xylem; this can be shown by tracing the When a soil is saturated with water, all the pores are
pathways of the ions with radioactive labelling, as well as filled, but a well-drained soil does not remain water-
from sap analysis. From two-thirds to three-quarters of the saturated for long. Water drains away quickly under
solids are, however, organic, including amino acids, gravity from the larger spaces, but some is retained in the
amides and carboxylic acids, giving the sap a pH of about smaller pores by the colloidal, surface-tension and
5. The xylem sap composition in any individual plant capillary forces, and as adsorbed surface films around soil
varies with the environmental conditions; during rapid particles. When a soil contains as much water as it can
transpiration, when large amounts of water are passing hold against gravity, it is said to be at field capacity. The
through the xylem, the concentration of solids may fall amount of water present at field capacity depends on the
very low. There are also more regular seasonal variations soil type. Soils with fine particles have many small pores
correlated with plant development. In woody perennials, and much total particle surface area, and can hold more
the mineral content of xylem sap is highest in the spring, water than coarse soils. A clay soil at field capacity may
when active growth is resumed. The carbohydrate content hold 55% water on a dry-weight basis (i.e. 55 g water per
of xylem sap is usually below 0.05% and may be 100 g dry soil), while a coarse sand may hold only 17%.
undetectable. But in woody species in the early spring, Once the water content has fallen to field capacity, there is
before leaf expansion and the onset of transpiration, there almost no movement of liquid water in the soil, though
may be a period of high sugar content in the xylem sap, water evaporates to the atmosphere. TheCof a soil at field
up to 0%. This sugar is derived from reserve starch, stored capacity is very high, just below zero (unless the soil is
in the woody stem over the winter. During this period of highly saline) and uptake by plants can proceed freely. As
sugar mobilization, the xylem sap acquires a positive the water content of a soil falls, its C decreases
pressure. The sugary sap flows out in quantity from cuts progressively. The concentration of solutes rises and the
made into the wood, and the tapping of birch (Betula spp.) Cp falls and the smaller volumes of water between soil
and sugar maple (Acer saccharum) in the spring has been a particles have more curved menisci; this increases the
tradition in northern regions of Europe and America surface tension forces and lowers the C. Also, as the outer
probably for millennia. Maple syrup is commercially layers of water are removed from the surface films, the
produced from sugar maple sap. Plant hormones can also inner layers are held more strongly by electric charges and
move in the xylem; e.g. the sensing of water stress in roots van der Waals forces. At first the lowering of the soil C is
stimulates the synthesis of abscisic acid, which is matched by lowering of the Cin the plant and water
transported in the transpiration stream to leaves, where it absorption continues. Eventually a stage is reached,
induces stomatal closure. Other hormones transported up however, when the soilCfalls so low that the plant can no
in the xylem are important in growth correlations between longer obtain enough water to compensate for
root and shoot. transpirational losses, and it wilts. At first this may be
temporary, the plant wilting by day but recovering at
1.6 Water uptake and loss: control by environmental and night, when the transpiration is low and water uptake
plant factors catches up with water loss. Eventually there comes the
permanent wilting point (PWP), defined as the stage when
The rates of water absorption and water loss, and the plant will not recover from wilting unless more water
consequently of water movement through the plant, are is added to the soil. Numerically the PWP is expressed as
determined by an interaction between plant and the percentage of water left in the soil. The value of the
environmental factors. The environmental factors can be PWP depends on the species as well as on the soil,
classified as soil (edaphic) and atmospheric. With regard

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different species reaching the PWP at different values of temperature may thus considerably reduce the uptake of
soil ψ. water by roots under transpiration pull, leading to
‘physiological drought’ when there is water available in
Water uptake does not totally cease at the PWP but leaf the soil, yet the plants suffer water stress. Root pressures
turgor pressure remains at zero. Once the PWP has been develop only in warm, well-aerated soils of favourable
reached, removals of very small amounts of soil water moisture content.
cause very large decreases in the soil ψ: the relationship
between soil water content and soil C is not a linear one
(Fig. 7). Whether the plant can survive wilting depends on 1.6.2 The atmosphere and transpiration
the species, on the degree of water loss and on the length
of time in the wilted state. The daily course of water absorption by plants closely
follows, with a time lag, the course of transpiration. Thus
the atmospheric factors which determine the rate of
transpiration also largely determine the rate of water
uptake.

The rate of transpiration, the outward diffusion of water

vapour from plants, is subject to the same physical laws as
the inward diffusion of CO2. Transpiration is directly
proportional to the water potential gradient ∆ψ between
the leaf and the air. Or, since atmospheric water status is
often expressed in terms of vapour pressure ∆e, which is
proportional to ψ , we can substitute ∆e, the water vapour
pressure gradient, for ∆ψ. Transpiration, on the other
hand, is inversely proportional to Ra (boundary layer
resistance) and Rs (stomatal resistance). Denoting the rate
of transpiration by T, we have:

There is no term in this equation to correspond with the

mesophyll resistance which is involved in CO2 diffusion,
for water vapour does not pass through cells on its way to
the outside. Transpiration rate increases with increasing
temperature, a rise in temperature resulting in a steeper
concentration gradient of water vapour out of the leaf, i.e.
∆e increases. The air spaces within the leaf are normally at
near saturation vapour pressure, c. 100% RH (relative
humidity, the ratio of actual vapour pressure to saturation
vapour pressure as a percentage). The absolute
concentration of water vapour at a given RH increases
with increasing temperature, i.e. air at 100% RH at 20 0C
will contain more water vapour than air at 100% RH at
Most of the water uptake by a plant takes place when the
100C. A rise in leaf temperature therefore increases the
soil moisture is between field capacity and PWP. As the
vapour pressure in the leaf without a corresponding rise
soil dries out, the forces opposing plant water uptake
in the external air. The gradient is approximately doubled
increase; the rate of water uptake, plant hydration and
for a 100C rise in temperature. Transpiration decreases
plant growth rate can be impaired by water stress even if
with increasing atmospheric humidity, for this decreases
wilting is not reached. This reduction of growth rate can
the ∆e. Wind stimulates transpiration, decreasing Ra as it
aggravate the effect of water shortage, for the slowing
sweeps away the water vapour accumulating in the
down of root growth decreases the rate at which new
boundary layer at the leaf surface. By causing bending of
areas of the soil are tapped by the roots.
the leaf it may cause mass flow of air into and out of the
leaf, thereby enhancing water loss. Light has no direct
Soil aeration affects water uptake. Field capacity is the
effect on water loss, but it does have a profound effect on
ideal state of soil for plants since it has a high C but also
transpiration indirectly: it warms up the tissues,
has air-filled spaces. A fully water-saturated soil has an
increasing transpiration, and it promotes stomatal
even higher C, but it is waterlogged, without air spaces.
opening. The combined effects of light and temperature
An adequate O2 supply is necessary for root growth; a
result in the diurnal changes in the rates of transpiration
lack of O2 and a high concentration of CO2 accumulating
(and hence of water uptake).
from anaerobic respiration of roots and soil
microorganisms) are moreover reported to decrease the
1.6.3 Stomatal control of transpiration
permeability of roots to water. Soil temperature affects
root growth and root permeability, both being decreased
The features of the plant which control the rate of passage
at low temperatures. The viscosity of water on the other
of water through it act largely through controlling the rate
hand increases as the temperature falls; low soil
of transpiration. If comparison is made between plants of

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different species, or different individuals of the same pulled apart by the shrinking of surrounding epidermal
species (which can show variation according to their cells, which lose water more rapidly. In extreme wilting,
growing conditions), the following shoot characters are too, the protective mechanism may break down as the
seen to favour rapid transpiration: a thin cuticle; lack of epidermal cells shrink and again pull the pores open.
hairs; a high stomatal frequency per unit area; a large
surface area and a large ratio of internal to external 1.6.4 Waterproofing the surface: cuticle and wax
surface area. The water uptake capacity of the roots can
also limit the rate; when half the leaves are removed from One of the key features that arose during the evolution of
a plant, the remainder may transpire more rapidly per terrestrial plants is the xylem, which makes possible the
unit area of leaf, being now able to draw on the whole root transport of water to parts of the organism not in contact
system rather than on half of it. with a water supply. Another key feature was the
evolution of waterproofing chemicals which cut down the
However, when an individual plant is considered over a rate of evaporation from plant surfaces. The control of
short term so that developmental changes in, say, the water loss by stomata can be significant only if the rest of
extent of the root system do not come into play, the degree the surface is waterproofed to some extent. This is
of opening of the stomata is often the most important achieved by the presence of the cuticle and wax.
single plant factor directly controlling the rate of
transpiration. By far the greater proportion of the water The whole outer surface of all land plants (even the
lost from the leaf comes from the leaf air spaces via the Bryophyta) is covered by a cuticle. In the flowering plants,
stomata, although the stomatal pore area may be only 1–2 the cuticle covers not just the external surface, but the
% of the total leaf surface. The daily course of walls of cells lining internal air spaces, although here it is
transpiration rate follows closely the course of stomatal extremely thin; there is a very thin cuticle on the root
opening. Where there is a midday closure of stomata, this epidermis, too. A cuticular ridge commonly overarches
is always accompanied by a reduction of transpiration stomata. The cuticle is a continuous skin over an organ,
rate. In plants with a thick cuticle, such as the bay laurel not separate for each individual cell; it can be detached by
(Laurus nobilis) water loss through the epidermal surface chemical treatment in one piece from e.g. a leaf. The
excluding stomata, the ‘cuticular transpiration’, may be as cuticle is made up of several layers which differ in
low as 2 % of the total transpiration. When the cuticle is chemical composition, but the main component in all
thinner, cuticular transpiration can constitute up to about layers is cutin, a complex hydrophobic polymer of mainly
50% of the total; the cuticular transpiration of the ‘average’ hydroxy fatty acids. Some wax (see below) may be present
mesophyte is 10–2 5% of the total. Closing of the stomata in the cuticle, and secondary compounds such as tannins.
will therefore, according to species, reduce transpiration The innermost layer of the cuticle is rich in pectins; when
to some 2 –50% of that occurring with fully open stomata. the pectins are hydrolysed, the cuticle is detached. The
thickness of the cuticle varies from a fraction of a mm to
Transpiration is slowest when stomata are completely over 10 mm, being thick in plants of dry habitats.
closed and increases with increasing stomatal opening. If
the atmospheric conditions favour rapid transpiration, the On top of the cuticle aerial organs have a layer of
increase continues right up to maximum opening, the epicuticular wax. This is attached only loosely and can be
stomatal aperture being the limiting factor. wiped or rubbed off. The wax gives the ‘bloom’ to
glaucous leaves and fruits; some dark plums and so-called
But if the atmospheric conditions do not favour rapid black grapes look almost sky-blue when untouched,
transpiration, maximal transpiration rate may be reached owing to the wax, but much of that gets rubbed off when
when the stomata are only partly open, the external the fruit is handled. The sheen and texture of many floral
conditions becoming limiting. In the pathway of water parts result fromtheir wax layer. The wax is not a pure
movement from soil to atmosphere, the stomata are chemical, but a mixture of long-chain hydrocarbons, long-
situated between the leaf air spaces and the atmosphere: chain fatty acids, long-chain hydroxy acids, esters,
alcohols, aldehydes and terpenoids. Each species
SoilÆRootÆStemÆleaf Æ StomataÆair produces its own mixture; about 50 different chemicals
have been detected in apple wax. Even as for the cuticle,
That is the point where the drop in ψ is the greatest, and the thickness varies from a fraction of a mmupwards. The
therefore the stomata can exert very effective control over wax palm(Klopstockia cerifera) native to the Andes, has
water movement when, in moving air, the boundary layer 5mmof wax on its leaves; carnauba wax, from the leaves
resistance is low. In still air, however, the boundary layer of Copernicia cerifera, is harvested commercially in Brazil.
resistance may become the limiting factor and be more
important than the stomatal resistance. Partial stomatal The thickness and chemical composition of the cuticle and
closure cuts down the rate of transpiration more than the wax determine the extent of transpiration with closed
rate of CO2 diffusion; there being no mesophyll resistance stomata, the ‘cuticular transpiration’. The more
for water vapour movement, the stomatal resistance impermeable these layers, the smaller is the cuticular
assumes proportionally more importance. transpiration (and the more complete is the control
exerted by the stomata). The cuticle and wax also prevent
Stomata are highly sensitive to water stress and some the entry of water. It would not be a healthy situation if
species react by (partial) closure at quite low levels of rain penetrated plants freely, flooded the air spaces,
water deficit, protecting the plant against further water soaked the cell walls and leached out solutes from the
loss. In wilted plants the stomata are shut. The early apoplast. Raindrops largely roll off owing to the
stages of wilting may, however, be accompanied by a hydrophobic nature of the surface. The wettability is
widening of the stomatal aperture, the guard cells being determined mainly by the epicuticular wax, its thickness

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and even more by its structure. To the eye a waxy leaf or transmission of hormonal stimuli in situations such as the
fruit looks smooth. Microscopy, especially scanning sensing of drought by the roots.
electron microscopy, reveals that the wax is present as
plates, rods, granules or tufts. These formations increase The cooling effect of transpiration is also very important
the hydrophobicity; even smooth wax is water-repellent, in keeping down the temperatures of plant organs,
but a surface bristling with small wax projections is almost especially in hot weather. Throughout the day, a leaf
unwettable. Agricultural and horticultural sprays are absorbs radiant energy. The amount of energy stored in
mixed with detergent to lower the surface tension and photosynthate is about 1% of that absorbed, maybe up to 4
enable the fluid to wet plant surfaces. The cuticle and wax % in some C4 species. The rest of the absorbed energy is
also give some protection against the penetration of converted to heat. If the leaf is not to heat up steadily, this
pathogens, and against ultraviolet radiation. The mode of heat must be dissipated, and it has been calculated that
production of these extracellular layers by the plant is transpirational cooling accounts for about half of this heat
something of a puzzle. dissipation. (The remainder is lost through convection and
conduction.) Hence transpiration is generally regarded as
The components are generally assumed to be secreted as a ‘necessary evil’.
liquid precursors by the epidermal cells and to solidify on
the outside, otherwise continuous layers cannot be 1.7 Water conservation: xerophytes and xeromorphic
produced, nor can solid wax rods be transported through characters
a cell wall. The most difficult problem is the wax, which
must move not only through the cell wall, but through the Plants growing in extremely dry habitats (‘xeric habitats’)
cuticle as well. It might be suggested that the wax layer is are termed xerophytes. They exhibit a number of
formed first and then the cuticle under it; but when the structural features which are regarded as potentially
wax is wiped off, leaving the cuticle intact, a new layer of conserving water and are called xeromorphic characters.
wax is formed by young leaves (and in some species by Some of these features may also be found in plants of
mature leaves also). Searches for channels in the habitats which are not particularly deficient in water. It is
epidermal cell walls have not given clear-cut results. common to find xeromorphic characters in evergreen
There have been reports of channel-like structures in cell plants of temperate and cold climates, where the plants
walls seen by light microscopy, but these have not been may suffer water shortage during cold seasons owing to
confirmed by electron microscopy. Another idea is that an inability to absorb water adequately by chilled roots, or
the wax molecules are carried along with water vapour even because of the soil water freezing. The character of
molecules during cuticular transpiration, a process that succulence is typical of plants of saline habitats, where
has been compared to steam distillation. water is abundant, but the plants must absorb it from a
medium of low C. Xeromorphic characters include:
The patterns of the wax can be produced physically, with (1) Deep and/or extensive root systems. These enable the
no control from the living cells. Waxes from various plant to reach water at considerable depths. In the
species dissolved in organic solvents and allowed to Mediterranean region, tree roots grow right into the
crystallize out as the solvent evaporates have been found porous limestone rock. In some species, the ratio of root
to crystallize into the pattern originally exhibited on the weight to shoot weight is very high, as is root length to
epidermis. However, in some cases the pattern has shoot height.
differed from the original. (2) Water storage organs and tissues: succulence. Any
part of the plant may store water – root, stem or leaf. The
1.6.5 Is transpiration really necessary? To put it another storage organs are succulent; they contain large, highly
way, could water be moved to the tops of plants in vacuolate cells which swell up when water is available
sufficient quantities by any other mechanism, root and gradually release water to growing regions when
pressure having been shown to be insufficient? there is no external supply, shrinking in the process.
Continuous columns of water under tension could in fact Examples of stem succulents are cacti, whilst Aloe species
be maintained as water is used up in photosynthesis, and and stone crops (Sedum spp.) have succulent leaves.
in any other chemical reactions where it is a reactant. As (3) Low surface : volume ratio of shoot organs. Leaves
long as something removes water at the leaf end, the usually are the organs with the highest surface : volume
water would be pulled up; it does not have to be removed ratios. Xeromorphic plants tend to have small leaves, or
by evaporation. Osmotic forces in the leaf cells could also succulent leaves, succulence leading to a decrease in the
maintain ψ gradients between the leaf and the root (and ratio and hence a low water loss by transpiration.
soil). But the amount of water moved in such Sometimes the leaves lose photosynthetic activity
circumstances would be no more than is used up in altogether and are replaced by scales or spines – the cacti
growth, in chemical reactions, and in maintaining the are the classic example of the latter. The stem then
hydration levels of the leaf cells. Xylem transport is becomes the photosynthetic organ. The possession of
important also in distributing mineral ions, organic spines discourages animals from using the plants.
nitrogenous compounds and hormones. The critical (4 ) Hidden stomata. The stomata may lie in deep
question is whether the much slower water movement depressions or grooves, which trap a volume of still air
that would be maintained in the absence of transpiration and increase the boundary layer resistance. In dry
would suffice for the distribution of these solutes. conditions, the leaves may roll up and protect the stomata.
Information on this is limited. One investigation with Sometimes the surface cuticle and wax form a dome over
sunflower plants indicated that the suppression of the stoma, with a small aperture, again enclosing a region
transpiration had no adverse effect on mineral ion uptake of still air.
and distribution over 30 days. But one wonders whether
rapid xylem transport might be advantageous for the

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(5) Thick cuticle and wax. These clearly serve to conserve element, the test plants must be placed in an environment
water by preventing evaporation through the outer totally free from that element. In practice this means
epidermal surface. growing the plants in a liquid culture medium of precisely
(6) Hairiness. The hairs trap air and effectively increase known composition. Some elements are required in such
the boundary layer. The hairs also help to keep the minute amounts that they are very difficult to eliminate
temperature down since they are usually colourless or from solutions to levels below those required by plants.
weakly pigmented, resulting in a pale surface which Even distilled water, glass of containers, gaseous pollution
reflects light and decreases heat absorption. of the atmosphere and dust particles may provide enough
(7) Lignified leaves. When the tissues do become of certain elements to support plant life. Specially purified
dehydrated, lignification prevents collapse.Several of water, spectroscopically pure chemicals and a filtered air
these features can be present simultaneously. It may be supply must be used. As techniques have been refined,
noted that the xeromorphic features are incompatible with elements have been added gradually to the list of those
fast growth rates. A high root-to-shoot ratio puts a burden needed. Whilst the macronutrient elements (see below)
on the limited amount of photosynthetic tissues. A low were known by the second half of the nineteenth century,
surface : volume ratio as in cacti (and other succulents) the essentiality of chlorine was not established until 1954 .
results in slow diffusion of CO2 into the photosynthetic Nickel was added to the essential list even later, in 1987.
organs and only the outermost cell layers are well There is the further problem of the minerals already
illuminated, so that photosynthesis is confined to these present in the propagule used to start the culture; the
layers. In succulent leaves and stems, the ratio of supply in this may suffice for a considerable period of
photosynthetic cells to total mass is low. The net growth. More elements may possibly be added to the
assimilation rate is consequently low and growth is slow; current list, which stands at 18–21 (Table 1). The precise
cacti are notorious for their low growth rates. The sinking number depends on the species studied, some elements
or overarching of stomata, and hairy surfaces, increasing being apparently essential for certain species but not for
the boundary layer, slow down the inward diffusion of others; it also depends on how strictly the criteria for
CO2 even as these features protect against excessive essentiality are applied. It may well be that some
outward diffusion of water. That is the price the plants elements, hitherto known to be required by only a few
pay for survival in xeric habitats. plants, may eventually be found to be universally
essential. As shown in Table 1, the essential elements are
2. Mineral nutrition classified as macronutrients and micronutrients. The
macronutrients are required in large amounts relative to
2.1 Introduction the micronutrients; in culture solutions, macronutrients
are supplied at 10–3 to 10–2 mol L–1 , whilst the
Of the naturally occurring 92 elements of the periodic micronutrient concentrations may be as low as 10–7 mol L–
1. Most of the micronutrients become toxic at quite
table, about a quarter are essential to plants. Water and
CO2 provide the plant with the elements C, H and O; the moderate concentrations, say above 10–4 mol L–1 .
remaining necessary elements are obtained by flowering
plants as inorganic mineral ions, mostly from the soil
solution. Water uptake and ion uptake are to some extent
linked, e.g. water uptake mediated by root pressure
depends on ion uptake, and the rate of ion uptake tends to
increase with increasing rate of transpiration. But the
uptake of mineral ions differs greatly from water uptake
in that it proceeds against the free energy gradient of the
ions and is dependent on metabolic energy. The transport
of ions through cellular membranes is mediated by
numerous membrane-bound transport proteins which
enable the plant to exert considerable control and
selectivity over the process. This is vital if the nutritional
needs of the plant are to be satisfied. Heterotrophic
organisms obtain nearly all their essential elements via
plants and the element composition of plants is
accordingly of major interest and importance also for
human nutrition.

2.2 Essential elements

2.2.1 Definition:
macronutrients and micronutrients

An element is classed as essential to a plant if the plant

cannot complete its life cycle without it and no other
element can substitute for it. The effect of the element
must also be direct, i.e. it should not act by promoting the
uptake of another essential element, or by retarding the
In addition to essential elements there are beneficial
absorption of a toxic one. To test for the essentiality of an
elements, as indicated in Table 1, which are not absolutely

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necessary for survival but promote the growth and vigour membranes. Numerous flowering plants contain pungent
of plants. Non-essential elements are also taken up by secondary S-containing compounds appreciated as
plants; any element present in the environment will be flavours; these are very common in the Brassicaceae
absorbed at least in small amounts. For plants grown in (cabbage family) which includes mustard (Sinapis alba).
the soil, large amounts of Al and Na are frequently Onions (Allium cepa), garlic (Allium sativum) and related
present as these are common in soils. Though inessential, species are also flavoured with S-containing chemicals.
such elements are far from being inert. They influence the The presence of such compounds may deter some
ionic balance and osmotic potential of the cells and may herbivores.
affect the uptake of essential ions. Many non-essential
elements are toxic in quite low amounts and their uptake Phosphorus is contained in nucleic acids and also in
is detrimental to the plants and to the animals which feed membrane phospholipids which make up the bimolecular
on them. lipid leaflet of biological membranes. As a component of
the adenosine phosphates (ATP, ADP and AMP) and
2.2.2 The physiological functions of the elements in related nucleotides, the phosphate group is involved in
plants ‘energy metabolism’ and intermediary metabolism
involves many phosphorylated intermediates. In
The roles of the essential elements in plants are at least metabolically active cells there is a continuous turnover of
partly known; the majority of the essential elements are phosphate from organic combination to Pi (inorganic
indeed universal for all living organisms and many of phosphate) and back again.
their functions are the same in plants, animals, fungi and
prokaryotes. In discussing the functions of the elements, Calcium, as in cells of other kingdoms, contributes to
most emphasis is here put on those functions that are membrane stability in plant cells by its association with
characteristic of flowering plants. The macronutrient membrane phospholipids, and it is necessary for the
elements are constituents of cellular macromolecules, maintenance of the normal permeability of the
including all the major building blocks of protoplasm, plasmalemma. In plants it also contributes to cell wall
whereas many micronutrients are enzyme cofactors or structure as calcium pectate; this is a major component of
occur as parts of prosthetic groups of enzymes. Carbon the middle lamella which cements adjacent cell walls
and hydrogen are of course constituents of all organic together. The Ca2+ ion is extremely important in stimulus
molecules and the majority of organic molecules of living perception; one of the first effects in the chain of reactions
cells contain oxygen as well; these three elements are set off by a stimulus, environmental or hormonal, is very
present in the greatest amounts. often a change in the cellular concentration of Ca2+ which
is termed a ‘second messenger’. Ca2+ further acts as
Nitrogen, too, is a constituent of many cellular molecules, activator to some enzymes – amylases, ATPases and
in particular proteins and nucleic acids, the key phospholipases.
macromolecules of life as we know it. There are many
lower molecular weight nitrogenous organic compounds Potassium is something of a mystery element. It is present
vital to cell metabolism – vitamins, cofactors, hormones, in cells as the free K+ ion; it does not enter into organic
the chlorophyll pigments and the phytochrome combination. It is known to be the activator of some
photoreceptors. Flowering plants additionally contain an enzymes, but other elements which act as enzyme
extraordinary variety of nitrogenous secondary activators are required only in micronutrient amounts.
compounds not involved in basic metabolism. These The affinity of proteins for K is, however, low and it may
include alkaloids, among which are compounds used as be that fairly high concentrations are needed to make
drugs, e.g. morphine, nicotine and quinine. Plants also enzymepotassium complexes. It is the chief cation of
contain numerous non-protein amino acids, which are not protoplasm and as such it balances the charges on
incorporated into normal proteins. There has been much cytoplasmic anions, organic cations being few.
dispute about the possible physiological functions of the Chloroplasts have a high content of K+; the movement of
secondary chemicals. Both the alkaloids and the non- H+ from the stroma into the thylakoid lumen during
protein amino acids are toxic and often bitter tasting; one photosynthesis is electrically balanced by a movement of
possible function is protection against herbivores. In K+ into the stroma from the cytosol and a shortage of the
seeds, non-protein amino acids, with a high proportion of element leads to a low rate of photosynthesis. The K+ ion
N by weight, can act as N storage compounds. Some non- is very important in controlling C (the water potential)
photosynthetic pigments contain N, e.g. betacyanin, the and hence the water content of plant cells. Cell expansion
red pigment of beetroot (Beta vulgaris). is associated with the accumulation of K+ in vacuoles,
which induces water uptake into the vacuoles and an
Sulphur performs an important structural role in proteins increase in size. In plant cells which function in
where the disulphide bridges –S–S– stabilize tertiary movements involving turgor changes, K+ ions are
protein structures. Sulphydryl groups, –SH, are found in concerned in turgor control; such cells are stomatal guard
the active sites of many enzymes. There are also –SH- cells and the pulvinar cells (hinge cells) of leaves and
containing coenzymes, e.g. coenzyme A, whilst petioles. In these cells, increases and decreases of turgor
glutathione, again with a –SH group, is an important are achieved by K+ moving in or out, water following
antioxidant. Several iron–sulphur proteins, e.g. according to the resulting C gradient. Since so many of the
ferredoxins, occur in the electron transfer systems of effects of K+ are physical effects on C or electric potential,
chloroplasts and mitochondria; these proteins contain the question is why K+ should be so specifically required
clusters of linked S and Fe atoms at their reactive sites. and why the very similar Na+ can replace it to only a
Membrane sulpholipids are structural molecules which limited extent.
contain a sulphate group, found in chloroplast thylakoid

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Silicon in the form of silica gel, a hydrated oxide of Si, fixation is very important not only for the species in which
gives the cell walls of grasses, including cereals, their it occurs, but in the overall ecological context. Hence it
characteristic rigidity; this is very conspicuous in the seems appropriate to include Co in the essential list.
dried-out straw. Si is not known to take part in any
biochemical reactions within cells and Si-requiring plants Chlorine is required for the O2 -evolving system of
can be nursed to maturity in culture in the absence of the photosynthesis. For this it is needed in only micronutrient
element. Lack of Si does, however, result in some wilting, amounts. However, the element is taken up by cells in
withering and necrosis, and under natural conditions large quantities and the chloride ion Cl– is the chief
such Si-deficient plants would have little chance of inorganic anion in cells, often accompanying K+, e.g.
survival; hence it is reasonable to consider Si as an during K+ fluxes in stomatal guard cells, so that it is
essential element for species which normally have highly beneficial in much larger amounts than required to fulfil
silicified cell walls, e.g. wetland grasses. Many other its essential biochemical role.
species contain smaller amounts of Si in their walls and Si
can be regarded generally as a beneficial element. It can Sodium is required for C4 photosynthesis in some C4
ameliorate toxic effects of Al and Mn and can increase species where it seems to be involved with the conversion
resistance to fungal disease. Many of the micronutrients of pyruvate to PEP. It is present in cells as the free Na+ ion
have been identified as enzyme activators or as parts of and like Cl– is tolerated in relatively high concentrations.
the prosthetic groups of enzymes. Iron and copper are Chemically it is very similar to K and to some extent it can
present in the respiratory and photosynthetic electron interchange with that element; e.g. in Commelina
transfer chain cytochromes. They are also needed for other benghalensis Na can replace K in the control of turgor of
oxidative enzymes: Fe for catalase and peroxidase, Cu for the stomatal guard cells. In succulent halophytes, plants
ascorbic acid oxidase and polyphenol oxidase; Fe is which live in saline habitats, Na+ acts as an
present in iron–sulphur proteins, as mentioned in osmoregulatory ion, with Cl–.
connection with S and Fe is necessary for chlorophyll
synthesis. Boron is the element for which the physiological role has
proved most difficult to elucidate. Much of the B in the
Magnesium and manganese activate many plant is associated with cell walls where it cross-links cell
dehydrogenases and phosphate transfer enzymes and are wall polymers, such as pectins. It also is needed for
also important in photosynthesis, a Mg atom being part of normal membrane function. In B-deficient roots, ion
the chlorophyll molecule whereas Mn is present in the O2 - uptake capacity deteriorates but when such roots are
evolving complex. All the three elements Fe, Cu and Mn supplied with B, recovery is considerable by 20 minutes
are transition metals, able to change valency as they lose and complete within an hour. Such fast action suggests a
or gain an electron; hence their association with primary action at the membrane level, B either affecting
oxidoreduction activities, which involve the transfer of membrane permeability or acting on membrane- bound
electrons between reactants. enzymes. There is also some evidence for B affecting
enzymes of auxin and ascorbate metabolism. The greatest
Zinc is again an activator for many enzymes. Particularly demand for B is during the reproductive phase; in the
important in plants are alcohol dehydrogenase, absence of B, pollen grain formation fails, and pollen tubes
superoxide dismutase (which degrades the highly reactive germinating in the absence of B swell and burst.
and dangerous superoxide radicals formed during certain
oxidative and photosynthetic reactions), and carbonic Regarding the beneficial elements, individual species
anhydrase. The last-named catalyses the reaction differ in their requirements. Sodium benefits many
species, being able to substitute to some extent for K.

Rubidium and strontium also probably owe their

In aquatic plants, where carbonate is the main C source for beneficial effect to an ability to replace some of a plant’s
photosynthesis, this reaction produces CO2 as substrate requirements for K and Ca respectively. Rubidium
for Rubisco. In C4 plants the reverse reaction occurs, enhancesgrowth most markedly in K-deficient media.
resulting in the formation of carbonic acid which Aluminium is more limited in its beneficial effects; it is
dissociates to form bicarbonate, the substrate for PEP beneficial to tea (Camellia sinensis), Fallopia sachalinensis,
carboxylase. and a number of grasses. Selenium is accumulated in large
Nickel is a constituent of the enzyme urease, which amounts by some ‘accumulator species’ e.g. certain
hydrolyses urea; the enzyme is needed for N metabolism species of Astragalus growing in Se-rich soils, also by
in plants. Molybdenum is present in the enzyme nitrate Lupinus albus and Phleum pratense; it affords some
reductase, which is needed to utilize nitrate, the major protection against insect attack and protects against
source of inorganic N for most plants, and it is needed for toxicity from excess Pi. The beneficial effects of silicon
symbiotic N2 fixation. It is also part of the cofactor (Moco) have already been discussed in connection with its
for aldehyde oxidase, an enzyme involved in the synthesis essentiality for some species.
of ABA, and in a few other oxidases. The amount required
is extremely small. Cobalt also is needed in minute Complex interactions take place between mineral
quantities only, and is known to be needed for symbiotic elements and metabolism over and above the primary
N2 fixation, which involves the Cocontaining vitamin B12 . roles of the minerals. Several examples of interactions
Since plants normally associated with symbiotic N2 fixers between mineral supply and growth hormone metabolism
can survive without the symbionts, Co might be argued to have been reported. In sunflower plants, deficiency of N,
be beneficial rather than essential. However, symbiotic N2 P or K in the rooting mediumhas been found to decrease
the flow of cytokinin hormones from the roots to the

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shoots. Macronutrient deficiency can thus act on plant 2.3.2 Ion uptake by the root Adsorption, absorption and
development not only through direct shortage of accumulation
elements, but via the hormone supply. The biosynthesis of
the gaseous hormone ethylene is promoted by Ca2+ ions. The region of most active ion uptake by roots is the same
Ethylene acts antagonistically to the hormone auxin in a as for water uptake, i.e, the young region of the root
number of effects. Thus Ca2+ antagonizes auxin by behind the apical meristem, the root hair region. The
promoting ethylene biosynthesis. Cobalt inhibits the uptake of cations begins with the adsorption of the cations
biosynthesis of ethylene and hence Co salts are used to to the cell walls where polysaccharides carry negative
prolong the life of cut flowers, ethylene being normally charges, which attract H+ ions moving out of cells by the
produced by the flowers and promoting senescence. More action of proton pumps. These H+ are then accessible to be
such interactions have been reported and undoubtedly exchanged for soil solution cations, which thus become
more still remain to be noted. electrostatically bound to the cell walls. The binding sites
lie not just on outer surfaces of walls, but inside the
2 .3 Ion uptake and transport in the plant capillary spaces within the cell walls; a living cell’s wall
2 .3 .1 Ions in the soil may be thought of as a hydrated sponge, with much ‘wet
space’ and much adsorptive surface inside it. There is also
With the exception of C, H and O, which are derived from the possibility of some cation exchange, termed contact
water and CO2 and are incorporated by photosynthesis, exchange, between H+ ions adsorbed on the root surface
plants acquire all other elements as inorganic ions. Even C and the cations adsorbed onto solid colloidal soil particles.
can be obtained as the carbonate or bicarbonate ion. Anions have few adsorption sites in cell walls; they must
Organic nitrogenous compounds can act as the N source, simply diffuse into the water-filled capillary spaces of the
but normally are available to plants in limited amounts if cell walls before passing through the plasmalemma. To
at all. The ions which serve as sources of the essential some extent, they ‘follow’ the cations by electrostatic
elements for flowering plants are listed in Table 1. For attraction.
terrestrial flowering plants the chief source of mineral ions
is the soil. The mineral rock particles of the soil yield ions The adsorption of ions may then be succeeded by
by weathering which gradually brings them into solution; absorption, passage through the plasma membrane. There
ions are also released by the action of microorganisms on is an important difference in meaning between these very
dead organic material. The ion concentration of the soil similar words, adsorption for attraction outside the
solution rises as the water content of the soil falls, but plasma membrane, absorption for actual entry into the
except under very dry conditions the solution is very cell, to the inside of the plasma membrane. When the ions
dilute. It has been shown that a solution corresponding in are taken up to a greater concentration inside the plasma
ionic composition to that of a soil solution will support membrane than outside, this is termed accumulation (Fig.
good growth of crop plants provided it is frequently 1).
renewed or applied as a flowing solution so that it is not
depleted. In a natural soil the ions in the solution are
constantly being replenished. The laws of physicochemical
equilibrium ensure that, as ions derived from soil solids
are removed from the solution, more ions dissolve from
the rock particles. Soil Pi concentration is always low, 1
mg L–1 or less; it has been calculated that the Pi of the soil
solution may need to be renewed 10 or more times per
day to meet the P demands of a growing crop. Nitrate also
needs rapid replenishment; it is absorbed rapidly by
plants, N being needed in larger quantities than most
other elements, and being extremely soluble, nitrate is
easily washed downwards in rainwater. Not all the ions in
the soil are totally free in the soil solution. The colloidal
matter in the soil, both inorganic clay particles and
organic particles, ‘humus’, which help to retain water in
the soil also serve to retain ions by adsorption. The
Uptake of ions is by no means confined to root cells. Roots
colloidal constituents of the soil usually carry a net
take up the ions in the first instance, but these ions are
negative charge; cations, being positively charged, are
distributed around the whole plant in the xylem, where
adsorbed to the negatively charged groups on the clay and
they are in the apoplast, outside living cells, and cells in
on the organic particles. These ions are held at the surface
various parts of the plant have to take up ions from the
of the soil particles by electrostatic attraction only loosely
supply carried up in the xylem sap. All living cells of the
and can be exchanged for other cations; by washing a soil
plant are capable of ion uptake from their environment; in
with a concentrated solution of a salt such as NH4 NO3 the
the natural state, for cells other than the outer root cells,
soil cations can be displaced into the solution in exchange
the environment for ion uptake is the apoplast.
for the ammonium ions (NH4+). For most of the anions,
there is little adsorption because of the lack of positive
Compartmentation of the plant cell and the concept of
charges on the soil colloids. There may be some
free space
adsorption of phosphate ions, especially of the trivalent
PO4 3 – which has a high electric charge density, and
Throughout discussions of ion uptake reference is made to
phosphate ions can also replace hydroxyl and silicate
‘inside’ and ‘outside’. Plant cells are, however, more
anions in clays.

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complicated than a sac surrounded by a membrane. When larger pore spaces between the wall macromolecules reach
plant tissue is thoroughly prewashed and then immersed dimensions up to about 5 nm; the diameters of the ions,
in a solution containing mineral ions, there is first a very including water of hydration, are below 1 nm. The wall
rapid uptake, followed by a much slower rate (Fig. .2 ). does slow down the diffusion of chemicals with masses
When the immersion takes place at low temperature, above some 200–300 Da, but usually transmits molecules
around 40 C, or under anaerobic conditions, only the initial up to several thousand Da. The plasma membrane is
rapid uptake occurs, suggesting that the initial uptake is a generally regarded as the primary barrier to ion
physicochemical process not requiring metabolic energy. movement. There are, however, further barriers within
When the tissue is washed in distilled water after a period plant cells. In mature cells the vacuole forms a
of uptake, some of the ions taken up are quickly washed compartment occupying 80–90% of the protoplast volume
out. These ions are said to occupy the ‘water free space’ and the tonoplast (vacuolar membrane) is an important
which seems to correspond to the cell walls of the tissue. barrier to ion movement, for a large proportion of the free
Some more cations can be removed from the tissue by inorganic ions in a plant cell passes into the vacuole. The
immersion in a solution of cations, e.g. K+ might be organelle membranes constitute further barriers limiting
washed out in exchange for excess NH4 +. The the free diffusion of ions.
exchangeable cations are again present in the cell walls,
but they are associated with the negative charges on the 2 .3 .3 The transport of ions within the plant
cell wall polysaccharides and at the plasma membrane
surface; these ions are described as being in the ‘Donnan The long-distance transport of ions takes place in the
free space’, named after Donnan, an investigator in this xylem concurrently with water transport. Just as for water,
field. A certain fraction of ions is, however, retained it has been questioned whether the ions move through the
firmly; these are the ions which have passed through the outer root tissues by an apoplastic, symplastic or
plasma membrane. Quantitatively, the fraction that is transcellular route. Apoplastic ion movement could be
washed or exchanged out with ease corresponds to the partly by diffusion, partly along with the flow of water
fraction acquired during the rapid uptake phase; the moving into the transpiration stream. The endodermal
firmly held fraction corresponds to what was taken up in Casparian strips are believed to form a barrier to ion
the slow, energy-requiring process. Diffusion and movement, as for water. Indeed for ions there is more
adsorption into the free space or apoplast, being direct evidence for this being so. Solutions of salts of the
physicochemical processes, do not use metabolic energy heavy elements lanthanum (La), lead (Pb) and uranium
and hence proceed even at low temperature or under (U) have been used as tracers for ion movement. These
anaerobic conditions, but passage through the plasma elements can be located by electron microscopy because of
membrane, into the symplast, requires ATP. Only what their high atomic masses (La = 139, Pb = 207, U = 238) and
has passed through the plasma membrane is truly inside can be seen in cell walls of the root cortex and in the
the living cell, although free space ions are inside the plant endodermal cell walls as far as the Casparian strips, but
or tissue as a whole. not beyond; La and U are apparently unable to cross the
plasmalemma and are excluded from the stele; Pb,
however, enters the endodermal cell cytoplasm and also
the stele. The above observations strongly support the
suppositions that (1) ions move in the apoplast; (2 ) the
Casparian strip is an effective barrier to ion movement;
and (3 ) the endodermis can be crossed by ions only if they
can enter the symplast. The weakness of the argument lies
in extrapolating from data obtained with these ions of
high atomic weight to the behaviour of ions of essential
elements, with lower masses. There is, however, indirect
evidence for similar behaviour by the nutrient ions. In
barley (Hordeum vulgare) roots, Pi ions and K+, which
readily enter the symplast, are translocated to shoots even
from the older parts of the roots, where the endodermal
Casparian strips are fully developed. The ion Ca2+, which
penetrates the symplast with difficulty, is translocated to
the shoots mainly from apical regions where the
endodermal cell walls are still permeable.

When the cortex was stripped from a segment of barley

roots (still attached to the shoots) so as to break the
endodermal cells across at the Casparian strips, the
concentrations of 32 Pi and 85Sr2+ in the transpiration
stream equalized with the external medium, which was
not the case when the endodermis was intact. The
endodermis can thus regulate the entry of ions into the
stele. The endodermis also acts as a barrier against the
outward leakage of ions from the stele. Experiments with
45Ca2+ in barley roots have shown that over 60% of the
The cell wall is not a highly selective barrier to movement
of solutes except in so far as, owing to negative charges on radioactivity was exchanged out from the cortex for excess
the wall polysaccharides, it tends to attract cations. The unlabelled Ca2+ions in 10 minutes; but from the pericycle

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(the cell layer just inside the endodermis), only 19% was and the soluble amino acids transported to regions of
lost. In summary, the mineral ions absorbed by roots can growth and storage, so that most of the N is conserved.
travel to the xylem in both the apoplast and the symplast.
In the extreme apices of the roots, the apoplastic pathway The constant supply of minerals in the xylem stream leads
is continuous, but once the Casparian strips have fully to the accumulation of ions in the leaves. It has been
developed (this may occur within a few millimetres of the suggested that the shedding of leaves is equivalent to
tip) the endodermis can be crossed only via the symplastic excretion, ridding the plant of waste minerals (and organic
route. In regions of the root where secondary rootlets waste, too). There occurs also some leaching of minerals
emerge, the endodermis may be interrupted, giving freer by rain. Around trees one can sometimes distinguish a
access to the stele again. Control of what passes into the drip-zone flora, which thrives on the leachings from the
xylem is possible by the selective transfer of ions to the tree.
symplast anywhere between the epidermis and the
endodermis, by the retention of ions by living root cells, 2.3 .4 Ion transport across cellular membranes
and perhaps by a filtering action in the endodermis. The
sap composition is further modified during long-distance Active accumulation: the electrochemical potential
transport by the absorption of ions from the flowing gradient
stream by living xylem parenchyma cells adjacent to the
conducting cells. Ion uptake usually is a process of ion accumulation, plant
cellsacquiring a total ion content greater than that in their
When the rate of water transport from the soil to the environment, and this applies for many individual ions as
xylem is increased, particularly by increased transpiration, well. For some ions, the internal concentration can be very
ion uptake and transport to the shoots are also increased. much higher than the external; several hundred-fold
It may be that the additional ions entering the xylem accumulation is common. Phosphate, which is present in
represent a passive mass flow of ions carried along in the very low concentrations in natural soils, can be
water current through the apoplast. Alternatively, it has accumulated several thousand-fold. This immediately
been suggested that the dilution of the xylem sap by an suggests movement against a free energy gradient, for
increased rate of water uptake into the conducting cells higher concentration of a solute means higher free energy.
stimulates a higher rate of ion secretion into the sap from However, there is another component to the free energy
the symplast of living xylem cells. A still further gradient of ions, namely the electric field. For electrically
possibility is that the tension pull created by a high charged particles, an electric potential gradient is a free
transpiration rate lowers the resistance to ion (and water) energy gradient. A cation, being positively charged, will
movement through membranes and thereby increases the move towards a more electronegative region. An anion,
rate of secretion or of passive leakage from the symplast. being negatively charged, will move towards an
There is evidence that tensions developed in the xylem electropositive region. For ions therefore the free energy
vessels are transmitted across the root diameter. gradient is the combined electrochemical potential
gradient to which both the concentration of the ion
Movement in the xylem is one-way traffic. In leafy plants, (determining its chemical potential) and the electric
most of the solution in the xylem eventually arrives in the potential contribute. This is highly relevant to ion uptake
leaves, though some solutes and water are of course by plant cells, because there does exist an electric charge
absorbed on the way by older root segments, stems and difference across the membranes of cells. As mentioned
petioles. For the ions moving away from the conducting earlier, positively charged protons are pumped to the
cells, there are again the two possible pathways: via outside of plant cells, into the walls; this results in the
diffusion through the apoplast, or by absorption into inside of the plasma membrane being left electronegative
adjacent living cells with subsequent distribution through with respect to the outside. The potential difference is in
the symplast. the range of 100–250 millivolts (mV). Similarly, pumping
of protons from the cytoplasm across the tonoplast into
Some of the elements are retained in the tissues to which the vacuole makes the cytosol side of the tonoplast
they were first transported; these are known as immobile electronegative with respect to the vacuolar side.
elements. One such example is Ca. Crystals of calcium
carbonate or calcium oxalate are often precipitated in The cytoplasm is accordingly more electronegative than
vacuoles of older cells of leaves and stems. Silicon is the apoplast outside it. Therefore, when a cation, say K+, is
deposited as the insoluble silica and is immobile. In many found to be in a higher concentration within a cell than
species B also is not relocated; a common symptom of B outside it, the question arises: has it been moving against
deficiency is the death of meristems: once the external the free energy gradient (as suggested by the
supply fails, the newly formed tissues immediately run concentration gradient) or along the free energy gradient
short, while the older tissues still have the B originally (as suggested by the electric potential gradient)? When the
deposited in them. There are, however, species which can overall electrochemical potential gradient is taken into
translocate and redistribute B in the phloem; in these account, it is found that the accumulation of cations, even
species the phloem sap contains sugar alcohols, with allowing for the electronegativity of the cytoplasm, is very
which B can form complexes. Other ions, the mobile ones, often, though not exclusively, against the electrochemical
can be transported out of old, senescing tissues and potential gradient. For anions, any accumulation into the
relocated to young, growing regions; K+ is a mobile ion. cytoplasm must be against the free energy gradient, for
This transport occurs in the phloem. Some elements can be anions are negatively charged particles moving into a
moved around the plant in organic combination: N can more electronegative area as well as against the
move as amides or amino acids, S as S-containing amino concentration gradient. Movement against the free energy
acids. Proteins in senescing tissues are largely hydrolysed gradient is often termed active and requires an input of

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energy by the cell. Where an ion is accumulated along the The two main characteristics that determine the ease with
free energy gradient, the cell is still not getting something which a particle can diffuse through a biological
for nothing, since maintenance of the potential difference membrane are the lipidsolubility of the particle and its
across membranes requires energy. As knowledge of the molecular size.The more lipid-soluble (lipophilic) and the
mechanisms of ion uptake has developed, the distinction smaller the particle, the easier the penetration. The
between active and passive accumulation has become mineral ions are very hydrophilic, so they do not dissolve
rather blurred. It is perhaps best to regard accumulation in the lipid bilayer. The atomic weights of some of the
generally as an active process, and then to consider, for nutrient ions are quite low. However, the electric charge
individual cases, precisely how the energy input is on the ions attracts hydration shells of water molecules;
achieved, directly or indirectly. e.g. K+ (mass 39 Da) carries 4 molecules of water whereas
the divalent Ca2+ (mass 40 Da) has about 12 associated
When concentrations of ions in the cytoplasm and vacuole water molecules. These hydration shells increase the
are analysed separately, and the electric charge difference effective size of the ion considerably. The permeability of
across the tonoplast is measured, it is found that the biological membranes towards ions is therefore very low.
movement of ions from the cytoplasm to the vacuole is The flux of ions across membranes is enabled by specific
also an active process. In mature vacuolated plant cells transport proteins in the membranes which facilitate the
most of the ions of the cell are in fact transferred to the movement of ions and not only provide the physical
vacuole, which occupies much of the volume inside the means of passage for the ions, but utilize the energy of
cell wall. The vacuolar store is tapped as required. It is ATP to transport the ions against their electrochemical
also found that ions may be actively transported out of potential gradients. The total number of ion transport
cells. This is commonly the situation with Na+. Although proteins in plant membranes is much greater than the
the cytoplasmic concentration of Na+ is generally higher number of nutrient ions, there often being more than one
than the external, it is usually lower than would be in transport protein for the same ion. In Arabidopsis, 16 genes
electrochemical equilibrium with the external medium. have been identified coding for proteins involved with
Indeed the active transport of ions into the vacuole can nitrate uptake, and the same number for phosphate. The
also be regarded as being out of the cytoplasm into a non- specificity of transport proteins varies; some are highly
living space. Some cells are specialized for the outward specific to single ions, but often they can transport several
transport of ions: these include the cells of salt glands of related ions, i.e. ions of similar physicochemical properties
halophytes, which secrete excess Na+ and Cl– ions from such as valency and size. The rubidium ion, for instance,
the plants; and xylem parenchyma and transfer cells, is transported by a number of cellular systems for K+
which secrete ions into the xylem conducting cells. transport, and the radioactive 86Rb is often used in
Directions of active flux of some nutrient ions across the experiments as a substitute for K+, there being no
plasmalemma and the tonoplast of plant cells are shown convenient K radioisotope available.
in Fig. 3 .
The ion transport proteins can be divided into the pumps,
the porters (carriers) and the channels; their main
characteristics are summarized in Table 2 and Fig. 4 . The
term ‘carrier’ was originally used for all membrane
transport proteins, before different types were
distinguished.

The maintenance of physiological concentrations of ions in

plant cells is thus an active process requiring an energy
supply in the form of ATP. It is estimated that up to half of
the energy from root respiration is expended on
membrane transport of ions. In photosynthetic tissues,
ATP from the photochemical reactions can be utilized to
power ion transport.

Mechanisms of membrane transport

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believed to occur between the ion and the channel protein

as the ion moves through: ‘Passage of an ion through a
channel may be likened more to a python swallowing its prey
than to a ball rolling through a drainpipe’. Passage through a
channel has been termed uniport since only one chemical
moves.

A very important feature of ion channels is that they open

only transiently, in response to some stimulus. Any one
channel is estimated to be open over only a few per cent of
(1) Pumps.These are transport proteins which hydrolyse the lifetime of a cell. A permanently open ion channel
ATP and simultaneously transfer an ion across the would be fatal, the cell losing control of its ion
membrane. The energy is derived from the ATP concentration. Movement through a channel is along a
hydrolysis, directly. Pumps are vectorial, i.e. a particular free energy gradient, so active accumulation would
pump can move an ion only in one direction. The become impossible. (Aquaporins are permanently open
hydrolysis of the ATP results in a conformational change and active water accumulation does not occur.) The
in the pump protein, which causes the transmembrane antibiotic gramicidin acts by inserting into the plasma
passage of the ion. Two of the most important ion pumps membrane and forming permanently open channels for
of plant cells are noted in Table 2 . Ca2+ pumps are situated K+ which leak out of the cells. Over 20 ion channels are
in both the plasma membrane and the tonoplast and they known from plants. The Ca2+ pumps keep the cytosolic
pump Ca2+ ions out of the cytosol (Fig. 3 ), keeping the Ca2+ concentration below the apoplastic and vacuolar
cytosolic concentration very low, 1-5 X10–7 mol L–1, much levels. Numerous stimuli cause a transient opening of Ca2+
lower than in the vacuole and the apoplast. Another channels and flooding of Ca2+ ions from the apoplast or
supremely important ion pump is the proton pump, also internal compartments into the cytosol, where a reaction
known as the proton ATPase. One might not tend to think chain is started. The Ca2+ pumps then restore the cytosolic
of H+ as an important metabolite. In fact the proton concentration to its previous level. K+ channels are very
pumps constitute the metabolic machinery driving the important in turgor control of stomatal guard cells. The
porters and, in some instances, controlling the channels. rapid changes in turgor of motor cells also depend on K+
Most of the energy utilized in membrane transport in movements through K+ channels.
plant cells is via the proton pumps. The previously
mentioned movement of protons out of cytoplasm across 2.3.5 Control of ion uptake by plant and environment
the plasmalemma and the tonoplast, building up the interaction
potential differences across these membranes, is achieved
by proton pumps. Their activity of course builds up also a The ion content and the elemental composition of a plant
proton gradient. In combination the electric gradient and reflect an interaction between the plant and its
proton concentration gradient make up an electrochemical environment. Plants show great selectivity: ions are not
potential gradient for protons, favouring the inward taken up in the proportions in which they are present in
movement of the protons. This free energy gradient is the surroundings (Table 3). For example, most flowering
harnessed by the porters. plants show a strong preference for K+ over Na+ and
maintain a higher internal K+ concentration irrespective of
(2) Porters are mostly transport proteins which couple the the external proportions of the two ions. There are
transport of an ion with the inward movement of a proton accumulator species which concentrate some element to a
or protons. The transport can be symport (cotransport), particularly high degree; the selenium accumulators
the proton and the ion moving in the same direction, or mentioned earlier accumulate Se to 200 times higher levels
antiport (countertransport), the two moving in opposite than non-accumulators in the same habitat. When several
directions (Fig. 4). The carrier in the plasma membrane for species are grown with an identical external ion supply,
Cl–, for instance, is a symporter, transporting one Cl– in each shows a different internal content of mineral
with 2H+. The outward pumping of Na+ is achieved by an elements (Table 3). Sometimes a specific preference can be
antiporter, which exchanges one proton going in for one interpreted in terms of function; in a pasture, the grasses
Na+ moved out. There is also a H+/ K+ symporter for the contain much higher levels of Si than other herbs, and this
entry of K+ ions into the cytoplasm, amongst the is correlated with the presence of silica in the grass cell
numerous transport systems existing for this ion. The walls, on which the grasses depend for rigidity. But the
energy input occurs during the activity of the proton physiological significance of, say, maize sap having
pumps which build up the proton gradients. double the K+ content of bean sap in the experiment
illustrated in Table 3 is unknown.
(3) Channels. As the name suggests, channels are formed
by proteins with several subunits enclosing an aqueous The rate of uptake of an ion is dependent on the
pore. An open channel is an open hole and movement physiological requirements of the plant. The rate of nitrate
through a channel is along the free energy gradient; it is uptake by a grass has been found to vary with the diurnal
very much faster than movement mediated by pumps and growth rhythm of the plants, the highest uptake rates
porters (Table 2); presumably because of that, channel coinciding with the maxima of growth rate. There are
proteins are present in low numbers per cell (except for reports of nitrate uptake remaining approximately
aquaporins). The channels nevertheless show specificity; constant over a wide range of external concentrations, and
some are extremely specific, for one single ion; others will of some tendency to maintain more or less constant
permit passage according to size. Some interaction is internal concentrations of several ions – e.g. K+, Cl–,
phosphate and nitrate. There is a negative feedback

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between the tissue content of an ion and the rate of its shoot ratios, increases in number and length of root hairs
uptake: other things being equal, plants with a high and increased development of mycorrhiza.
content of an ion take it up at lower rates than plants with
a low internal concentration. Higher external 2.3 .6 Mycorrhiza
concentrations do usually promote higher ion uptake
rates, and result in higher internal concentrations, but not The word mycorrhiza means ‘fungus-root’. It is the name
in direct proportion to the external. In one experiment, as given to a symbiotic association between a plant root and
the medium Pi concentration was raised from 0.03 to 3 0 a fungus, which in most cases enhances the mineral
mmol L–1 , a thousand-fold increase, the shoot P content nutrient supply of the plant, whilst the fungus benefits
rose only four-fold from 0.23 to 0.96% of dry weight. from a supply of organic C from the plant. Mycorrhizal
Physiological control of ion uptake is thus well associations in some species have been known for many
documented, but the selectivity of plants is far from years; gradually it has become apparent that, far from
absolute and the environment also exerts a very being an odd exception, the formation of mycorrhizal
considerable influence on a plant’s elemental composition. associations occurs in most species of flowering plants in
It was noted above that representatives of different species the field and it is highly beneficial. Fossil evidence
in the same environment differ in their ion contents; it is indicates the presence of mycorrhiza-like associations
equally true that specimens of the same species in already in the primitive terrestrial plants of the Devonian
different ionic environments acquire distinctive elemental era.
compositions. Inessential ions and toxic ions are absorbed.
Not only do these have direct effects, but their uptake may
compete with that of essential elements; e.g. selenate
competes with phosphate for transport proteins, and
arsenate competes with sulphate. Any elements present in
the environment will be found in plant tissues, even the
artificially produced transuranium elements such as
plutonium.

In a mycorrhizal association, part of the fungal mycelium

is free in the soil, part is closely associated with roots. In
ectomycorrhiza there is a thick sheath of fungal mycelium
around the outside of young roots, and from this sheath
hyphae grow into the intercellular spaces of the root
cortex. In endomycorrhiza, all the plant-associated part of
the mycelium is inside the root. The most common type of
endomycorrhiza is the vesicular–arbuscular (VA) type.
The hyphae grow into the cortex cells, penetrating the
walls and branching greatly to form the arbuscules (‘little
trees’, Fig. 5), but they do not penetrate the plasma
membrane and the cells remain alive. The plasma
membrane grows to surround the arbuscules and there is
The uptake capacity of a plant adapts to the current a very large area of surface contact between the plant cell
environmental concentration of that ion. Plants grown at and the fungal arbuscule. Some of the fungal hyphae swell
low concentrations of an ion develop an enhanced into vesicles, hence the name of vesicular–arbuscular. It is
capacity for absorbing that ion compared to plants grown estimated that about 00% of field-grown plants have VA
with an abundant supply. For any one ion there may be mycorrhiza.
several transport systems available, with different
affinities for the ion. At low external concentrations, high- There is also ectendomycorrhiza, with limited cell
affinity systems are activated, which are efficient under penetration by the fungus. Special associations occur in
these conditions. For K+, the high-affinity system some plant groups, e.g. orchids. Exchange of nutrients
predominates at external concentrations below 0.5 mmol takes place over the large area of contact between the
L–1 . The mobilization of high-affinity transport systems fungus and the root cells. The surface area of fungus
involves transcription of genes for the high affinity exposed to the soil is also very large, enabling efficient
transport proteins. These can be numerous; in Arabidopsis, mineral absorption.
6 genes for high-affinity phosphate transporters have been
identified. Other responses also enhance uptake in a Mushrooms (agarics) beneath trees are often the fruiting
nutrientdeficient environment, including increased root: bodies of ectomycorrhizal fungi. Some fungal species are

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associated with particular genera of trees: the brown birch Regions of defoliated stem separated from all leaves by
bolete (Leccinum scabrum) is confined to birch (Betula spp.) such ‘bark rings’ become deficient in carbohydrates, but
whereas others have a broad host range; e.g. the famous the transport of water and minerals continues, showing
red-and-white patterned fly agaric mushroom (Amanita that xylem transport is still functional. This suggests that
muscaria) is associated with birch, oak (Quercus spp.), the transport of carbohydrates occurs in the phloem.
pine (Pinus spp.) and other trees. Admittedly the cortex (if present) and periderm are also
removed in the bark ring; but these tissues do not contain
Most flowering plants can survive without mycorrhiza, cells which are structurally suited for long-distance
and in laboratory culture with abundant nutrients they transport. A variation on debarking is to kill the living
grow vigorously, although even in well-nourished tissues in a length of woody stem by the application of hot
cultures the presence of mycorrhiza may enhance growth. steam. The heat-killed ring stops the translocation of
But in the field, especially if the soil is deficient in mineral organic substances whilst still permitting xylem transport.
nutrients, growth is very much poorer without This type of experiment shows that organic material is
mycorrhiza and the plants are less tolerant of stress. The translocated through living cells, as are found in the
VA association is particularly important in transferring Pi phloem, in contrast to xylem, which functions with dead
to the plant, whereas ectomycorrhiza is known to provide conducting cells. The high concentration of organic
increased access to both N and P. compounds in phloem sap also strongly supports the
postulate of its function in the transport of organic
The occurrence of mycorrhiza demonstrates how closely nutrients.
organisms in an ecosystem interact. Flowering plants are
autotrophic organisms and their basic needs are just light, The most direct evidence for phloem as the channel of
CO2 , water and some 20 mineral ions. But as parts of organic translocation comes from the use of tracers.
natural communities, the majority of flowering plants are Various fluorescent dyes, such as fluorescein and its
provided with an appreciable proportion of their mineral derivatives, can be directly observed under the
requirements by mycorrhizal fungi. microscope to move in the phloem. At first this
observation was regarded with caution, since these are
3. Translocation of organic compounds artificial compounds and might move along paths
different from those of natural metabolites. Final
confirmation has come from radioactive labelling. When
3.1 Introduction
radioactive CO2 is supplied to photosynthesizing leaves,
Flowering plants are described as being autotrophic, ‘self- the radioactivity soon appears in the phloem of the petiole
feeding’, capable of synthesizing all their organic material and the stem, as radioactive sugars. Here there has been
via photosynthesis. But a flowering plant is a complex no introduction of any foreign substance, nor any
organism with cells and organs specialized for diverse interference with the plant’s normal activity, and the data
functions, and only the green photosynthetic cells are prove that the products of photosynthesis move from their
truly autotrophic; they must accordingly supply all the sites of production in the phloem. Radiolabelling has
nonphotosynthetic parts with organic carbon. Over small shown that the phloem is also the pathway of
distances, i.e. between individual cells and within small translocation out of non-photosynthetic storage organs.
groups of cells, chemicals can move by diffusion through Since it has been established that naturally transported
plasmodesmata, or across plasma membranes by diffusion metabolites and numerous fluorescent dyes move along
and by active transport. But organic materials must move the same pathway, the dyes are now frequently used as
for long distances; the growing tips of the roots of a tree tracers for phloem transport.
are many metres away from the nearest photosynthetic
leaves and even in a herbaceous plant diffusion would be 3.2 .2 The structure of phloem
too slow for the distances involved. We have already seen The phloem of flowering plants consists of several types of
how water moves in plants over long distances in a cell. Tracer experiments have shown that, at the cellular
specialized transport tissue, the xylem. The subject of this level, translocation proceeds through the sieve tubes, built
chapter is the long-distance, multidirectional movement of longitudinal files of individual sieve tube elements
or translocation of organic compounds which takes place (sieve tube cells). The sieve tube diameter usually lies
in the phloem. between 10 and 50 mmand the length of individual cells is
150–1000 mm, but in palms diameters of 400 mm and
3.2 Phloem as the channel for organic translocation lengths of 5000 mm have been reported; in minor veins,
however, sieve tubes can be very narrow, below 2 mm.
3.2 .1 Evidence for translocation in the phloem The transverse or oblique end walls between the
In flowering plants, the xylem is regularly associated with individual sieve tube cells, some 0.5–2 mm thick, are
the phloem, the two together making up the vascular pierced by pores giving them a sieve-like appearance and
tissues. In young organs the two tissues are in contact; are known as the sieve plates, hence the cells’ name (Fig.
when secondary growth occurs they become separated by 1). The diameter of the sieve plate pores is extremely
the vascular cambium, the meristem which then adds variable between species and in different parts of a plant.
xylem to one side and phloem to the other. In woody The narrowest are only 0.1 mm wide; 0.5–1.0 mm might be
stems, where the vascular tissues form complete cylinders, considered an ‘average’ value. But in the Cucurbitaceae
it is fairly easy to cut through the outer stem tissues down (marrow family) pore diameters up to 10 mmare found,
to the vascular cambium and to remove the ‘bark’, which and 14 mm has been reported for Ailanthus altissima (tree
includes the phloem, leaving the central xylem intact. of heaven). Associated with the sieve tube elements there
often are companion cells, one or more to each sieve tube
cell, lined up longitudinally beside the conducting cells.

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The sieve tube elements have a very unusual structure. In with the contents of other cells. Very often sap fails to
the mature state they lack nuclei and the only organelles exude at all from cut phloem, although exudation may be
identifiable are small plastids, sparse mitochondria and elicited by mechanical stimulation prior to cutting. The
endoplasmic reticulum, but these organelles do not most reliable method of obtaining sieve tube sap from
occupy much of the cellular volume, lying next to the cell numerous species is to use phloem feeding aphids to tap
walls. Most of the lumen is filled with a kind of sap but the sieve tubes. These insects feed by inserting their stylets
there is no tonoplast and no demarcation of a vacuole. The into sieve tubes from the outside. When the insertion is
cells are still, however, bounded by a functional accomplished, the insect is cut away under anaesthesia.
plasmalemma which also lines the sieve plate pores. The Sap will then continue to drip from the stylets for up to
sap contains P-proteins (P for phloem), sometimes visible several days, at a rate of 1–2 mL per hour. This liquid has
by light microscopy as protein bodies or longitudinal been assumed to represent more-or less unadulterated
strands, though at least some of the reported strands are sieve tube sap. There is evidence that the saliva initially
now thought to be damage artefacts. The companion cells released by the aphid exerts no digestive function on the
on the other hand have a full complement of organelles phloem and the composition of the sap certainly remains
(Fig. 5.1 A, D). They are rich in cytoplasm, with small unchanged over many hours of collection. There still does
vacuoles and very numerous, highly cristate remain the possibility of some continuous of liquid from
mitochondria, which gives them a high potential for surrounding tissues into the tapped sieve tube units.
metabolic energy production. The sieve tube cell and its
adjacent companion cell are derived by longitudinal Sap analyses vary markedly from species to species. The
division of the same mother cell and the pair is often sap is quite viscous, reflecting its high content of organic
referred to as the sieve element–companion cell (SE–CC) solutes; sugars generally make up 90% of the solids and
complex. It is generally believed that the companion cell may be present at concentrations of 2 –25% w/v; the water
with its nucleus exerts control over the enucleate sieve potential is correspondingly low, from –0.6 to –3.4 MPa.
element. The companion cells are joined to sieve tube cells In the majority of species, sucrose is the main sugar, often
by abundant plasmodesmata that are unusually large on present at 0.4 –0.5 mol L–1 , with traces of the
the sieve element side and branched on the companion oligosaccharides raffinose (a trisaccharide), stachyose or
cell side (Fig. 1 C). These plasmodesmata also have a very verbascose (two tetrasaccharides); but in some species one
high molecular exclusion limit, i.e. a high limit for the of these oligosaccharides predominates. In yet other
molecular mass that can pass through, much higher than species, the predominant carbohydrate is a polyol (sugar
in other plant tissues. The materials that are translocated alcohol) such as sorbitol, dulcitol or mannitol. Glucose on
are found in the companion cells, too; when radioactive the other hand is found only in very low concentrations
photosynthate is being translocated, the companion cells and may be undetectable.
also become radioactive. Other cell types foundin the
phloem are phloem parenchyma cells and fibres. Amino acids and amides are regularly present in phloem
sap, amounting to 0.2 –12 % of the transported solutes,
There is some difference in the structure of phloemin with glutamate, glutamine, aspartate and asparagine
various parts of the plant. Photosynthate is loaded into the being the most abundant. In the Cucurbitaceae soluble
phloemin the fine minor veins of leaves, and this loading nitrogenous compounds make up a high percentage of the
(collecting) phloem has sieve tubes narrower than the solids, and in perennials nitrogenous compounds are
companion cells. In the petioles, stems and older parts of abundant at certain times. Phloem which translocates
roots, the transport phloem sieve tubes are wider than the materials out of seed storage tissues can have high levels
companion cells. In the unloading (release) phloem, where of nitrogenous compounds; in Ricinus communis
the solutes leave the transport system, the companion cells seedlings, a total amino acid/amide content of 0.16 mol L–
are very small and may be absent altogether. There are 1 has been found, more than half the sucrose content of

moreover two basic mechanisms of phloem loading, and 0.27 mol L–1 . In species where nitrate reduction takes
phloem structure varies accordingly. place mainly in the leaves, roots are dependent for their N
supply on amino acids and amides translocated down in
In most perennials with secondary growth, the sieve tubes the phloem. Protein is detectable in the sap in variable
and their companion cells die after one growing season amounts and will be discussed separately. Sulphate
and are replaced by the cambium in the next season. In reduction is located in leaves, roots receiving S-containing
perennial monocotyledons, such as the palms, which lack amino acids via the phloem. In some plants, different
secondary growth, sieve tubes persist many years; in regions of the phloem transport different materials; in
palms 50-year-old sieve tubes have been identified. In a cucurbits, phloem within minor leaf veins
few dicotyledons, too, e.g. lime (Tilia sp.) and grapevine transportsmainly carbohydrate, whereas phloem strands
(Vitis vinifera), the phloem conducting cells survive for outside the veins carry mainly amino acids.
several seasons. In such species, the sieve plate pores may
be blocked during the winter by a deposit of dormancy Some mineral elements are found in the phloem sap; K+ is
callose, a polysaccharide of glucose subunits. the predominant ion, reaching concentrations of 0.03 –0.5
mol L–1 . ATP is a regular constituent. In small quantities,
3.2 .3 The composition of phloem sieve tube sap many other compounds have been detected including
organic acids, hormones and secondary products;
Obtaining uncontaminated samples of phloem sieve tube numerous plant viruses, too, spread in the phloem. The
sap in quantities adequate for analysis presents problems. pH is usually alkaline at 7.5–8.6, although in perennials it
Species differ greatly in the ease with which they yield may be faintly acid in the spring. This contrasts with
phloem sap. Sometimes exudate is obtained by cutting xylem sap and vacuolar saps, which are typically acid.
into the phloem, but this poses the risk of contamination

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The carbohydrate concentration of phloem sap derived and together with others as yet unidentified are
from photosynthesizing leaves is strongly dependent on presumably necessary for the maintenance of the life
the rate of photosynthesis, and hence on weather. It also functions of the sieve tube cells. The question then arises
exhibits regular diurnal variation. In the cotton plant how such macromolecules avoid being swept with the
(Gossypium barbadense), the highest concentration in the translocation stream into the sinks: maybe by adsorption
stem is recorded in the latter part of the day. In a number on the P-protein fibrils, or to the peripheral ER
of trees, the highest concentration occurs at night and (endoplasmic reticulum)? But proteins do move in the
close to the leaves, a concentration wave moving down phloem, even through graft unions. Evidence is mounting
the tree. that some sieve tube sap proteins and RNA are
information molecules destined for transport to the sinks,
In perennials there are seasonal patterns in phloem where they are unloaded. These information molecules
exudation and sap composition. In several tree species, include, for instance, signals (believed to be small RNA
abundant exudate is obtained in late summer but no flow molecules) which can suppress the activity of specific
occurs before about mid-June. Marked seasonal changes genes in the sinks. The phloem is emerging as an
are found with respect to amino acid content; this is high important carrier of macromolecular information as well
in spring, drawing on N stored in woody tissues over as hormonal signals.
winter, falls in the summer, and rises to a second peak in
the autumn, when leaf proteins break down prior to 3.3 The rate and direction of translocation
abscission. The N translocated out of leaves at this time is
deposited as organic nitrogenous compounds in the 3.3 .1 The rate of translocation: velocity and mass
stems, where it remains stored during dormancy. transfer
Carbohydrate, too, may be transported to woody stems
for storage. One measure of the rate of translocation is velocity, the
distance moved by the translocated material per unit time,
Proteins and RNA in phloem sap expressed in, say, cm h–1 . This seems a simple value, but it
is difficult to measure. Most estimates to date have been
Phloem sap contains proteins, mostly not more than 0.1 made by introducing a marker into the phloem at a
mg mL–1 , but in the Cucurbitaceae the values reach 10–60 specified point and noting the time of its arrival at another
mg mL–1 . Gel chromatographic methods have revealed a specified point. Fluorescent dyes have been timed, but
large number of sap proteins, with molecular masses from there is a time delay of unknown length between the
9000 to 2 00 000 Da; over 2 00 protein species have been external application of the dye and its entry into the sieve
detected in wheat (Triticum aestivum) phloem sap, and tubes, and the dye, as a foreign substance, might
individual species exhibit specific patterns. RNA conceivably adversely affect the phloem and change the
including mRNA can also be detected. Since the sieve tube velocity of translocation. The timing of the movement of
elements lack a system for transcription or translation, the radioactivity from 14C-labelled photosynthate avoids the
proteins and RNA must be synthesized in the companion introduction of foreign material, but is not
cells and enter through the connecting plasmodesmata. straightforward; among other problems, 14C is a weak b-
Such movement is vividly demonstrated in transgenic emitter and to detect its presence, plant segments have to
plants programmed to synthesize green fluorescent be extracted for assay; it is not feasible to hold a detector
protein (GFP) in leaf companion cells; the fluorescence to the plant. More recently the measurement of nuclear
appears in the sieve tubes. A very interesting finding is magnetic resonance (NMR) has been utilized; NMR
that some proteins in phloem sap have the property of depends on the magnetic properties of atomic nuclei. This
increasing the exclusion limits of plasmodesmata. Proteins technique is used on living plants and causes no damage.
from Cucurbita maxima phloem sap were microinjected The specimen is placed within a detector which gives
into individual cells in Cucurbita cotyledon mesophyll, information about the presence, concentration and
along with fluorescently labelled dextrans, i.e. high movement of selected atoms in known locations in the
molecular mass carbohydrates. These dextrans showed no plant – in this case, the H atoms of the water molecules in
cell-to-cell movement if injected by themselves. But in the the phloem. Actual values of the velocity of translocation,
presence of the phloem sap proteins, the spread of measured in different specimens and using a variety of
fluorescence indicated that dextrans with masses of at methods, range from below 10 to 660 cm h–1 , with woody
least 2 0 000 Da moved through up to 2 0 cells from the plants coming in the lower part of the range, at 20–30 cm
site of injection within 1–2 minutes. The accompanying h–1 . NMR measurements have yielded a value of 200 cm
phloem proteins, with masses of up to 100 000 Da, must h–1 for castor bean (Ricinus communis) seedlings and 90 cm
have moved, too. The exclusion limit for numerous h–1 for the adult plants.
‘ordinary’ plant tissues is 800–1000 Da. Phloem proteins
from Cucurbita and castor bean (Ricinus communis) also A somewhat different measure of the rate of translocation
increased the exclusion limits of plasmodesmata in the is mass transfer, the total mass of translocate moved per
leaf mesophyll of other species, e.g. Nicotiana tabacum. The unit time. If traffic is used as an analogy, the mass transfer
presence of such proteins in the SE–CC complex would be equivalent to counting the total number of cars
presumably maintains the high exclusion limits of the passing an observation point in a given time interval; the
plasmodesmata. Additionally, some proteins in the SE–CC velocity is equivalent to the speed at which the cars pass
complex may act as chaperonins and unfold large proteins the observation point. The units for mass transfer are g h–1
for passage. . If the mass transfer is calculated per unit area of phloem,
it is termed specific mass transfer (SMT) and expressed in
Some of the proteins in phloem sap have been identified g cm–2 h–1 . The (specific) mass transfer is measured by
as enzymes, or as components of the P-protein filaments, increases in dry weight in growing organs. It also is not

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easy to measure accurately, for a correction must be

applied for the amount of translocate respired away in the
organ, and for SMT precise measurements of transporting
cell cross-sectional areas are required.

3.3.2 The direction of translocation

In contrast to movement in the xylem, which is a strictly

one-way traffic, the direction of translocation in the
phloem is variable. Physiologically it is described as
passing ‘from source to sink’, i.e. from organs of synthesis
(or organs of storage) to organs of utilization. In a more-
or-less mature leafy plant, the mature leaves are sources
by virtue of their photosynthetic activity. The main sinks
are the growing regions, which are numerous (Fig. 2 ). The
roots and underground perennating organs such as
tubers, corms and rhizomes require downward
translocation through the shoot axis. In a young
vegetative plant, most of the total translocate goes to
support the rapidly growing root system. The growing
shoot tips, on the other hand, require upward
translocation, as do young leaves before they have
developed their full photosynthetic capacity. In the
reproductive stage, flowers and fruits need upward
translocation, fruits in their most rapid growth stage
receiving nearly all the translocated nutrients. In the
appropriate season, underground storage organs mobilize
their reserves and translocate the soluble products
upwards to growing shoot organs. Mature axial tissues in
stems, petioles and roots of course also need organic
nutrients for their maintenance. They draw on the
translocation flow as it passes through the axis and are
termed axial sinks, to distinguish them from the terminal
sinks in the growing regions. Generally, a sink is supplied
by the nearest source; thus upper leaves tend to feed the
shoot growing points, whilst the roots are fed mainly by
the lower leaves. If, however, leaves close to fruits are
removed, the fruits start to receive translocate from
further away. Tracer experiments have shown that
simultaneous upward and downward translocation can
occur in the same part of an axis. Since a source = organ of The overall direction of phloem translocation is under
production where solutes are passed into the phloem, and metabolic control; the precise pattern of movement is
a sink = organ of utilization where solutes are removed determined by the arrangement of the vascular tissue.
from the phloem, it might be postulated that the source- Leaves generally export photosynthetic products to young
to-sink translocation represents movement along a leaves or floral organs directly above them, and to parts of
concentration gradient. There is typically indeed a the root system directly below them. This corresponds to
concentration gradient of translocated material, the pattern of vascular connections. Lateral transport from
decreasing from source to sink. However, the situation is one vascular strand to another hardly ever occurs in either
more complicated. If a mature leaf is shaded so that it falls root or stem, and usually the removal of leaves from one
below its light compensation point it might be expected to side of a plant results in asymmetric, lop-sided growth as
act as a sink, but in fact shaded mature leaves starve, a consequence of onesided transport. Lateral diversion is
senesce and die. Sugar transport towards a sink is possible in species with a suitably anastomosing vascular
sometimes faster than its utilization in the storage or system, as occurs in e.g. the beetroot (Beta vulgaris).
reproductive organ to which it is moving and temporary
accumulation of sugar occurs in petioles and fruit stalks 3.4 Phloem loading and unloading
without preventing or reversing the flow. It is obvious
that there are metabolic controls for the direction of flow 3.4 .1 Phloem loading
over and above simple concentration gradients. There
must also be control of the amount of mass transfer to Phloem loading is the transfer of material into the phloem
various sinks. at the source; unloading is the removal of this material
from the phloem in the sink. Of the various organs that
can act as sources, the minor veins of leaves, which collect
the photosynthate from the mesophyll cells, have been
studied in most detail. Flowering plant species can be
broadly divided into two groups according to their

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method of phloem loading in the minor veins: the The loading of amino acids has been studied less intensely
apoplastic loaders and the symplastic loaders. than that of sucrose, but a number of amino acid/H+
symporters are found in mature leaves.
In the apoplastic loaders, sucrose is discharged fromthe
mesophyll cells into the apoplast (the cell walls), possibly
via carriers. Subsequent uptake into the SE–CC complexes
is mediated by sucrose-proton symporters in the plasma
membranes (Fig. 3); the loading process is thus driven by
the proton pumps which pump protons into the apoplast
at the expense of ATP hydrolysis. The apoplastic loaders
can be anatomically recognized by having very few
plasmodesmatal connections between the companion cells
and phloem parenchyma or mesophyll cells; their SE–CC
complex is a highly isolated unit, relying on membrane
transport to move sucrose against a concentration
gradient into the phloem. The companion cells of the
apoplastic loaders are typically of transfer cell structure,
with wall infoldings and hence a large surface (i.e. plasma
membrane) area. A number of sucrose transport proteins
and their genes has been identified. For example, in rice
seedlings (Oryza sativa), the sucrose transporter SUT1 is
confined to the companion cells, implying that (most of)
the sucrose is taken up first by the companion cells and
then passes into the sieve tube elements via
plasmodesmata. In the potato (Solanum tuberosum), tomato
(Lycopersicon esculentum) and tobacco (Nicotiana tabacum),
however, SUT1 is distributed throughout the SE–CC
complex, so that the sieve elements can take up sugar
directly. The expression of the sucrose porter genes occurs They are not specific for single amino acids. In symplastic
when leaves mature and begin to export photosynthate. loaders (Fig. 4) the photosynthate moves to the phloem
The repression of sucrose porter gene expression by through plasmodesmata, and there are abundant
transforming potato plants with antisense DNA leads to plasmodesmata between companion cells and phloem
inhibition of sucrose transport, a great accumulation of parenchyma, and between companion cells and mesophyll
starch and lipid in the leaves, and severe reduction of root cells if in contact; the sieve elements must receive their
and tuber growth. supply via plasmodesmata from the companion cells since
they do not have direct plasmodesmatal connections to
other cells. The companion cells lack transfer cell
characteristics. The precise mode of movement of the
solutes through the plasmodesmata is not certain. It has
been noted that symplastic loaders translocate mainly
galactose-containing oligosaccharides rather than sucrose,
predominantly raffinose and stachyose. Chemically, these
are equivalent to sucrose with respectively one and two
galactose residues added. It has therefore been suggested
that, in symplastic loaders, sucrose diffusing into
companion cells is there converted to oligosaccharides,
and these are transferred to the sieve element (Fig. 4). This
would keep up a concentration gradient for sucrose from
mesophyll to the companion cells. This ‘polymer trap’
hypothesis implies that the oligosaccharides cannot back-
flow into the mesophyll through the plasmodesmata,
which in the relevant cell walls must have rather low
exclusion limits, permitting the passage of sucrose
(molecular mass 342 Da) but not of raffinose (504 Da) or
stachyose (666 Da).

In some symplastic loaders sucrose is the main

carbohydrate translocated. There are not many data on
such species, but in two cases at least, willow (Salix
babylonica) and poplar (Populus deltoides) there is evidence
that the mesophyll cytosol has a high sucrose
concentration and there is a diffusion gradient from
mesophyll cytosol to the SE–CC complex. Apoplastic
loading is found in many herbaceous plants and in
families generally regarded as more highly evolved:
Asteraceae (= Compositae), Brassicaceae (= Cruciferae),

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Chenopodiaceae, Fabaceae (= Leguminosae) and the steep concentration gradient. This leakage would in
Solanaceae.Apoplastic loadingis consideredmore efficient. fact deplete the translocation stream, if it were not
It achieves higher loading rates, is relatively insensitive to counteracted by a continuous (partial) retrieval of the
low temperatures and is found in temperate-zone species. sugar by the sucrose-proton symport system. In the
Symplastic loading is characteristic of the more primitive terminal sinks, the retrieval capacity of the phloem is low,
families, including tropical and subtropical families with favouring exit of solutes from the phloem.
many tree species; it is cold-sensitive. Apoplastic loaders,
transporting sucrose, can build up higher sugar After unloading from the transporting cells has taken
concentrations in the phloem sap since sucrose is highly place, the nutrients must still move from cell to cell, for
soluble; concentrations of 0.5 mol L–1 are not rare in distances ranging from a few hundred micrometres (e.g.
phloem sap and values up to 0.9mol L–1 have been apical meristems) to several centimeters (e.g. some fruits).
reported, but raffinose saturates at 0.25 mol L–1 . This post-phloem movement is mainly symplastic. In
immature sinks, the exclusion limit of plasmodesmata is
Osmosis depends on the number ofmolecules per unit very high, up to about 50 000 Da in young leaf tissue (cf.
volume of solution, so that for the same weight, sucrose, about 1000 Da for mature leaf mesophyll) and 27 000 in
with the smaller molecular mass, gives a lower osmotic developing wheat grains. This enables the distribution of
potential and therefore a higher turgor pressure, which is protein and small RNA molecules from the sap through
postulated to drive translocation in the phloem. Table 5.1 the sink; GFP can be seen to move from the sieve tubes
summarizes the features of apoplastic and symplastic into all tissues of immature leaves. There may be bulk
loaders.The two types of loading are not mutually fully flow of solution through the plasmodesmata, especially in
exclusive, certain extent of apoplastic transfer occurring in apical meristematic regions, where there is as yet no
symplastic loaders, and vice versa. functional xylem and the phloem, which differentiates
much closer to the apex, must supply water as well as
nutrients. In root apices, the first sieve tubes differentiate
within 250–750 mm of the tip of the meristem, but the first
protoxylem matures at 400–8500 mm, or even further
away. The possibility of bulk flow in the apoplast, too, has
been suggested. The driving force for solute movement
would be the diffusion gradient, from the high
concentration in the sieve tubes to the sink cells, where the
concentration is kept low by utilization in growth and
metabolism. Bulk flow could be maintained by the
utilization of the water.

3.5 Partitioning of translocate between sinks: integration

at the whole-plant level

In addition to control of the direction of translocation,

there must be control of how much passes to different
sinks at any particular time – i.e. how nutrients are
partitioned. Balanced, integrated growth of a plant would
otherwise be impossible. The photosynthetic potential of a
plant depends on its leaf area; translocation of organic C
to the leaf primordia enables leaf growth and increases the
photosynthetic potential of the plant. But the growth and
subsequent maintenance of the leaves need mineral and
3.4.2 Phloem unloading and post-phloem transport in water supplies from the roots: during leaf growth there
the sinks must be translocation of enough photosynthate to the root
meristems to support a concomitant increase in the mass
Unloading of translocate from the SE–CC complex can (and maintenance cost) of the root system. The organic
also proceed either symplastically or apoplastically, but in nutrients available at any particular moment must be
this respect no taxonomic division of species according to partitioned in appropriate proportion between the shoot
unloading type has become apparent; rather, the type of and root sinks. In fact the growth rates of various parts of
unloading depends on the plant organ, and sometimes a plant can keep in proportion with mathematical
also on physiological conditions. In apical meristems and precision. This implies that partitioning of organic
in fruits, unloading is mainly symplastic but with some nutrients, too, is precisely controlled. There must be
apoplastic contribution. Symplastic movement has been signalling between the sinks and the sources. Partitioning
traced in root apices with fluorescent and radioactive control is a complex process. Farrar (1996) lists the steps
labelling. In unloading phloem the companion cells are involved in C flow to a sink: ‘phloem loading in a source
small and there is some direct contact between sieve tube leaf, phloem transport into the sink, unloading and short
elements and other cells. Plasmodesmata pass from sieve distance transport within the sink and then metabolism
tube cells in the root apex protophloem to meristematic and storage in the sink.’ Just about everything that affects
cells. In mature stems, unloading from the transport the physiology of a plant has the potential to affect
phloem (feeding theaxial tissues) tends to be apoplastic. partitioning, and control of partitioning is far from being
The movement of sugar out of the SE–CC complex into the fully understood. One important signalling molecule is
apoplast is a simple leakage process, diffusion- driven by sucrose itself (Fig. 5). Sucrose acts in growing regions as a

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promoter of the expression of genes involved in growth Hormones are molecules that transmit various signals
and respiration (which provides the energy for growth); in from one part of the plant to another. Translocates can be
the photosynthetic leaves, sucrose represses the attracted to a cut stem stump by applying the hormone
expression of genes involved in photosynthesis, including auxin to it, or diverted away from a growing wheat ear by
the gene coding for the small subunit of Rubisco. Leaf the application of auxin to a point remote from the ear.
growth leads to an increased production of sucrose and its The hormone, however, appears to act by stimulating
translocation to the roots, where the sugar would growth and/or metabolic activity at its point of
stimulate the activity of genes which promote growth application, creating a new sink. Cytokinins, too, can
ofmore root biomass – corresponding to the growth of the induce sink activity. There seems to be no evidence of a
leaves. A fall in the sink demand has been shown to hormonal signal specifically and directly controlling
decrease the rate of loading at the source,although it is not loading or unloading.
clear how the messageis transmitted.Turgor has been
suggested, a change in turgor at the sink being 3.6 The mechanism of phloem translocation
transmitted to the source and suppressing loading
activity. For the movement of any substance in solution, there are
two possibilities. Either it is carried along by mass flow
(bulk flow) of the solution, or else only the solute moves,
e.g. by diffusion, while the solvent remains stationary. The
rates of phloem translocation are of the order of 105 times
faster than diffusion, so this mechanism can be
discounted: there must be either mass flow, or some
special, rapid transport of the solutes. Movement in the
xylem is undisputedly a mass flow of the whole solution,
and the motive force is either the tension pull of
transpiration, or root pressure. For phloem translocation,
too, the current consensus is that mass flow occurs in the
sieve tubes, driven by an osmotic gradient. But the
structure of sieve tube cells is less obviously suited for
mass flow than that of the xylem conducting elements.
The hypothesis of mass flow in the phloem needs to be
examined critically.

3.6.1 The Munch hypothesis of mass flow driven by an

osmotic gradient

One of the earliest theories for the mechanism of phloem

translocation was Munch’s 1930 hypothesis, which
postulated a mass flow along a turgor pressure gradient,
driven by a physiologically maintained gradient of
osmotic potential (Fig. 6). At the source end, sugars (or
other solutes) are loaded into the sieve tubes; this lowers
their water potential and induces an inflow of water. The
turgor pressure in the sieve tubes rises and the solution is
pushed along the phloem by the pressure. At the sink end
the solutes are unloaded and water moves out with them,
according to the water potential gradient, so that the
turgor pressure here is kept low. A lateral movement of
The consequent increase in sugar concentration in the
solutes along the way into surrounding tissues of stem
mesophyll would eventually repress the rate of
and root, which all need to be supplied, would also work
photosynthesis. Some of the proteinsmobile in the
to maintain a solute concentration gradient (and hence
phloemmay also act as signals.
pressure gradient) from source to the final sink. Water
would be absorbed into the phloem from the xylem at the
The axial sinks can act as buffers between the sources and
source and released at the sink, returning into the xylem,
the terminal sinks: when demand by terminal sinks falls,
or the water might be utilized at the sink end, as noted
more photosynthate is unloaded laterally from the
earlier.
transport phloem into the surrounding tissues. The status
of various nutrient elements affects the partitioning of
Evidence in favour of the mass-flow hypothesis
photosynthate. When plant growth is limited by a low
supply of the elements N, P, S or Fe, the ratio of root to
Many observations can be quoted in favour of a mass flow
shoot mass rises, more organic C being delivered to the
in the phloem. When one single sieve tube unit is pierced
roots, whereas limitation of the supply of Mg, Mn or K
by an aphid stylet, the volume of sap exuded in an hour is
leads to a decrease of the root : shoot mass ratio. Water
around 1 mL, equivalent to the volume of some 2500
stress increases the root : shoot ratio, root growth being
individual sieve tube cells; the flow can continue for days
stimulated while shoot growth is reduced.
at this rate. On a macroscopic scale, the phloem sap of
sugar palms and agaves is tapped for sugar production or
fermentation into an alcoholic drink. One tree can exude

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for several months and yield several thousand litres of sap to force the sap through the stylets. Direct measurements
during this period. This is manifestly a mass flow of of the turgor of sieve tubes have been achieved, either by
liquid! Moreover, the sugar concentration remains steady insertion of sensitive pressure probes, or by the external
during the period of exudation in both the aphid stylet application of pressure cuffs (analogous to the apparatus
exudate and in the palm sap, so that the flow of liquid used for measuring blood pressure). Such measurements
cannot be attributed to a leakage of water into punctured are in practice fraught with great difficulty; nevertheless,
sieve tube cells from the nearby xylem. If mass flow is not only have positive pressures been recorded, but some
rejected, all the above-mentioned exudations of sap must workers have succeeded in demonstrating gradients of
be ascribed to an injury reaction unrelated to normal decreasing turgor passing down trees, away from source
translocation in the phloem. NMR techniques have leaves. Gradients of sugar concentration have been
demonstrated water flow in the phloem in intact plants. observed. In soybean (Glycine max(soja)) stalks, for
example, the sugar concentration in the sieve tubes of the
leaflet stalk has been found to be 10.5–12 .5%, when the
sieve tubes of the root contained only 4 .4 –6.3 %.

All this is good evidence for the Mu¨ nch mass-flow

hypothesis. But the hypothesis cannot be accepted until it
has been shown to be feasible quantitatively as well as
qualitatively: until it is shown that mass flow at the
experimentally measured velocities can be driven by the
actually existing pressure gradients, through channels of
the dimensions present in the sieve tubes. It is difficult to
prove this with complete certainty, the main problem
being the interpretation of the fine structure of the sieve
plate area.

The dimensions of transport channels in the sieve tube

cells Mass flow requires continuous open channels, the
wider the better, for the more narrow the diameter, the
greater is the frictional resistance to the flow, and the
greater is the force that is required to drive the liquid
along. The diameter of the sieve tube cells at 10–50 mm
would pose no problems. But at each sieve plate the
solution must pass through the sieve plate pores. The
crucial question for mass flow is: are these pores open
channels of sufficient cross-section to permit transport at
the observed rates?

In mature sieve tubes the pore diameters, as stated, range

from 0.1 to over 10 mm. These are the diameters of the
holes in the wall. If it is assumed that the entire hole is
open to flow, a hydrostatic pressure gradient of 0.06–0.10
MPam–1 has been regarded as adequate to drive mass flow
at the measured rates even for narrow pores. (That means,
for every metre length along the phloem towards the sink,
the pressure in the phloem must decrease by the above
value.) Some workers have put the value higher, up to 0.5
MPa m–1 . Experimentally measured pressure gradients
vary from 0.02 to 0.55 MPam–1 , which lie within the
required range. But if there is any cytoplasmic structure
within the pores, much higher pressure gradients become
necessary, of tens of MPa m–1 . It is clear that turgor-driven
mass flow is possible only if the pores are more or less
fully unobstructed, at least for species with narrow pores.

Sieve plates and their pores are usually seen lined with a
distinctive polysaccharide, callose (a glucose polymer),
The Munch hypothesis requires that sieve tube contents
which may narrow the pore diameters to 0.1 mm even
should be under a positive turgor pressure, and that there
when the aperture in the wall proper is over 1 mm, but it
should be a gradient of turgor pressure (and of
is arguable how much of the callose seen in any
osmotically active solutes), decreasing from source to sink.
preparation was there in the living state, for injury, e.g.
The sieve tubes are certainly under positive turgor
resulting from excision, has been shown to induce callose
pressure; this is demonstrated even by the simple fact that
deposition on sieve plates within seconds. Because of this,
sap can exude from cuts. Aphid stylets contain only a very
it can be practically impossible in many instances to know
narrow channel, offering a considerable resistance to the
the precise diameter of the pores in a functional sieve
flow of the viscous sap; a pressure of 1–3 MPa is required
plate.

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It is even more difficult to decide on the content of a sieve According to the original mass-flow hypothesis, the sieve
plate pore. The pore diameters are often too close to the tubes act as passive conducting channels, with no
limit of resolution of the light microscope for a satisfactory necessity for the expenditure of metabolic energy on the
in vivo examination. Sieve tubes cannot be dissected out way, for the energy input would take place at the source
singly undamaged, and when whole vascular bundles are during loading and possibly also at the sink during
viewed by conventional light microscopy, clarity is lost. unloading. Normally the transport phloem has a high rate
Confocal laser microscopy, however, enables the observer of respiration; sieve tube sap contains ATP at an average
to focus on a narrow plane even in a thick specimen concentration of 0.4 mmol L–1 and the turnover rate of
without interference from surrounding material. This ATP is high, suggesting an expenditure of ATP all along
method has been used to study living phloem in a leaf still the pathway. This has been quoted as an argument against
attached to the plant and actively translocating. As far as the mass-flow hypothesis. It has become clear, however,
could be seen the pores were unobstructed, but that all along the transport route there is leakage of
submicroscopic contents cannot be ruled out. Electron translocate, and whilst some of this goes to nourish the
microscopy overcomes the problems of resolution and axial tissues, a major proportion is retrieved by energy-
interference by surrounding structures, but the results requiring membrane pumps. A high rate of turnover is
remain disputable. The sieve tubes are extremely delicate, claimed for phloem proteins and protein synthesis
being highly hydrated and under high pressure; this requires an energy supply. Perhaps the unusual state of
makes them very susceptible to fixation damage. In the sieve tube cells, lacking nuclei, and traversed by a
particular, as the pressure is released by cutting, the rapid flow of sap, requires a higher than usual rate of
contents surge towards the cut. According to the method maintenance respiration. The high respiratory rate in the
of preparation and the species studied, the appearance of phloem therefore cannot be taken as a contraindication to
the pores varies. At one extreme pores appear quite mass flow driven by an osmotic gradient built up locally
empty, or contain some sparse P-protein filaments; at the in the loading region.
other extreme, the pores are completely packed with plugs
of P-protein and sometimes also with endoplasmic The function of the sieve plates
reticulum.
For mass flow, sieve plates appear to be obstructions and
Proponents of the mass-flow theory can regard the empty their presence has been interpreted as being against the
pores as the natural state and dismiss the densely filled mass-flow hypothesis. They do nevertheless perform an
pores as artefacts, containing material swept in as turgor important function in preventing sap loss from damaged
was released on cutting, and/or material coagulated on phloem: on injury, the deposition of callose narrows the
fixation. Opponents of the mass-flow theory are, however, pore diameters, P-protein surges towards the cut because
equally free to argue that empty-looking pores have had of release of turgor and then piles up against the sieve
their natural contents destroyed by the fixative! Freeze- plates forming slime plugs over the already narrowed
fracture electron microscopy, where the tissue is frozen pores, making them impassable. Plastids burst, releasing
extremely rapidly by being plunged into a liquid nitrogen- starch grains which help to plug the pores, and possibly
cooled bath at about –196 0C, avoids the artefacts caused they also release chemicals which promote P-protein
by chemical fixatives. But in sieve tubes, ice crystal coagulation.
formation has obscured the cell contents and, where
structure could be discerned, it has been very variable. In As noted, from most species there is little exudation from
the same sieve plate, some pores may look empty, others cut phloem in spite of the fact that the sieve tube contents
are traversed by various amounts of filaments, and some are under considerable hydrostatic pressure. The system
have filaments lying across them. Even ‘instantaneous’ can be desensitized by repeated rubbing or even beating
freezing may not be fast enough to show the true structure in the case of robust woody specimens so that cutting no
for sieve tubes pores through which material is moving longer induces blockage, and this effect is utilized when
rapidly. A translocation velocity of 100 cm h–1 is slow on a sugar palms are tapped. The sieve plates may also give
macroscopic scale; a snail can crawl faster. But relative to some mechanical support to the sieve tubes. Different
cellular dimensions, translocation is very fast. A particle functions for the sieve plates have been suggested in
would traverse a 1-mm-thick sieve plate in 0.003 6 seconds connection with alternative views on the mechanism of
(indeed probably less: the measured velocity is an average phloem translocation.
for the whole sieve tubes and the rate should be even
faster in the narrow pores). A fraction of a second would Alternatives to the mass-flow hypothesis
permit structural disturbances to occur in the pores
between the start of freezing and final total solidification. Alternatives to mass flow have from time to time been
proposed. The concept of spreading along interfaces
Current view inclines to accept the pores as more or less visualizes the translocated molecules forming a
unoccluded. When damage has been identified with monomolecular film on surfaces (P-protein fibrils?), like
certainty, it has resulted in formation of P-protein strands oil films at an air–water interface. This film would then
and deposition of P-protein material on sieve plates rather remain intact, with molecules added at the source end and
than loss of discrete structures. This was observed by removed at the sink. The hypothesis does not account for
confocal microscopy when sieve tubes were deliberately the flow of water, which does occur in the phloem. It is
wounded. The blocking of pores on wounding confines also difficult to conceive of surfaces able to form films
leakage of sap to the damaged cell. with all the variety of compounds that can be carried in
the phloem– sugars, amino acids, mineral ions, ATP,
Metabolic activity in the phloem fluorescent dyes, hormones, etc. Some sieve tubes

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moreover contain very little P-protein. Protoplasmic 3.6.2 The translocating system in the plant kingdom and
streaming has been claimed to proceed in the sieve tubes, macroalgae
with transcellular strands running fromcell to cell through
the sieve plate pores, but after the original observations All vascular terrestrial plants possess a phloem of
were published in the 1960s and 1970s, other workers elongated living cells joined by some kind of ‘sieve areas’,
have been unable to confirm them. The apparent i.e. fields of pores on lateral walls, on end walls or on
streaming rates of 3 .5 cm h–1 are also far too slow both. In the Psilotaceae, probably the most primitive
compared with the velocity of translocation which reaches group of living vascular plants, the sieve cells of the
several m h–1 . Submicroscopic tubules passing through phloem are described as just elongated parenchyma cells
the sieve plate pores, and pumping the tubule contents with lateral sieve areas. True sieve tube cells, with
along by contractions (as in peristalsis) have been transverse sieve plates and pores reaching 10-mm
proposed without any solid evidence; the same goes for dimensions, are found only in the flowering plants.
the idea of lashing protein filaments, like miniature cilia, Highly differentiated companion cells are also confined to
anchored to the outsides of the hypothetical tubules and the flowering plants, although the sieve cells of conifers
to sieve tube walls, wafting along a flow in the bulk of the have associated cells known as albuminous cells. It is
cell. The theory of electroosmosis regards the sieve plates reasonable to suppose that the mechanism of organic
as pumping stations for ions: an electric potential translocation is essentially the same throughout the
difference (PD) is built up across the plate, by pumping of kingdom Plantae.
H+ ions from companion cells into the sieve tube
upstream of the plate, and out of the sieve tube into the The brown algae (Phaeophyceae) include species with
companion cells downstream of the plate. This would differentiated bodies measurable in metres, and these
result in the movement of the positively charged K+ ions macroalgae translocate organic compounds through sieve
in the phloemsap across the plate, towards the negatively elements, elongate cells joined by pores. In most of the
charged downstream side and the K+ ion flow would algal species these cells have nuclei and are filled with
carry with it water and other solutes. The attractiveness of cytoplasm containing small vacuoles, small plastids and
the electroosmotic hypothesis is that it ascribes both a many mitochondria; the sieve plate pore diameters range
specific function to the high K+ content of the phloem sap, from 0.03 to 0.1mm. But in the giant kelp (Macrocystis
and a physiological function to the sieve plates. But the pyrifera), which can grow to a length of 50 m, the cells are
idea has no experimental data to support it. The energy enucleate, with cytoplasm confined to a peripheral layer,
expenditure for the pumping would be high; there would and the pores are much larger, 2 .4 –6.0 mm wide; the
have to be localized differentiation of sieve elements and pores are lined with callose and callose deposits block
companion cells with the direction of H+ ion movement themon injury – features strikingly similar to those
between the cells reversed over a micrometre or two near exhibited by flowering-plant sieve tube cells. The
sieve plates, and all other reported cases of H+ pumping compounds translocated are the algal photosynthetic
involve pumping of the ions out of cells. Quantitatively, products – mannitol (and amino acids) in Macrocystis.
too, with sieve tube cells often over 100 mm long, it seems The direction of translocation is ‘source to sink’, from
hardly credible that so much force could be built up by mature photosynthetic regions to meristems, and the algal
sieve plates so far apart. In summary, no viable alternative translocation velocities fall within the range measured in
to turgor-driven mass flow in the phloem has been flowering plants, 10 cm h–1 in Laminaria, 70 cm h–1 in
proposed. But it is conceivable that in species with very Macrocystis. There seems to have been a high degree of
narrow sieve plate pores there is facilitation of mass flow parallel evolution of the translocating systems in
through the pores from some mechanism like terrestrial plants and these macroalgae. Both groups share
electroosmosis. With sieve pore diameters in the flowering the photosynthetic mode of life, and have a common
plants covering two orders of magnitude, from 0.1 mm to fundamental structural feature in the possession of a cell
over 10 mm, the forces required to drive mass flow wall. When walled cells form a multicellular body, the
through the narrowest pores must be very much larger only possibility of direct intercellular communication is
than for the widest ones. via pores in the cell wall. Once the basic structure of cells
joined by communicating pores was established, it is not
surprising that these pores should have evolved in several
evolutionary lines into a means of large scale transport of
nutrients, when transport was necessitated by size
increase accompanied by functional differentiation of the
body of the green terrestrial plant or alga.

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G. Secondary metabolites - Biosynthesis of terpenes, phenols and nitrogenous compounds and their roles.

Plants produce a large, diverse array of organic compounds

that appear to have no direct function in growth and
development. These substances are known as secondary
metabolites, secondary products, or natural products.
Secondary metabolites have no generally recognized, direct
roles in the processes of photosynthesis, respiration, solute
transport, translocation, protein synthesis, nutrient
assimilation, differentiation, or the formation of
carbohydrates, proteins, and lipids.

Secondary metabolites also differ from primary metabolites

(amino acids, nucleotides, sugars, acyl lipids) in having a
restricted distribution in the plant kingdom. That is,
particular secondary metabolites are often found in only one
plant species or related group of species, whereas primary
metabolites are found throughout the plant kingdom.

Secondary Metabolites Defend Plants against

Herbivores and Pathogens

Many secondary metabolites have been suggested to have

important ecological functions in plants:
• They protect plants against being eaten by herbivores
(herbivory) and against being infected by microbial
pathogens.

• They serve as attractants for pollinators and

I. TERPENES
seeddispersing animals and as agents of plant–plant
competition.
The terpenes, or terpenoids, constitute the largest class of
secondary products. The diverse substances of this class are
Plant Defenses Are a Product of Evolution
generally insoluble in water. They are biosynthesized from
acetyl‐CoA or glycolytic intermediates.
• According to evolutionary biologists, plant defenses
must have arisen through heritable mutations, natural
1. Terpenes Are Formed by the Fusion of Five­ Carbon
selection, and evolutionary change. Random mutations
Isoprene Units
in basic metabolic pathways led to the appearance of
new compounds that happened to be toxic or deterrent
All terpenes are derived from the union of five‐carbon
to herbivores and pathogenic microbes.
elements that have the branched carbon skeleton of
isopentane:
• As long as these compounds were not unduly toxic to
the plants themselves and the metabolic cost of
producing them was not excessive, they gave the plants
that possessed them greater reproductive fitness than
The basic structural elements of terpenes are sometimes
undefended plants had. Thus the defended plants left
called isoprene units because terpenes can decompose at
more descendants than undefended plants, and they
high temperatures to give isoprene:
passed their defensive traits on to the next generation.

• Interestingly, the very defense compounds that

increase the reproductive fitness of plants by warding Thus all terpenes are occasionally referred to as isoprenoids.
off fungi, bacteria, and herbivores may also make them
undesirable as food for humans. Many important crop Terpenes are classified by the number of five‐carbon units
plants have been artificially selected for producing they contain, although extensive metabolic modifications
relatively low levels of these compounds, which of can sometimes make it difficult to pick out the original five‐
course can make them more susceptible to insects and carbon residues. Ten‐carbon terpenes, which contain two C5
disease. units, are called monoterpenes; 15‐carbon terpenes (three C5
units) are sesquiterpenes; and 20‐carbon terpenes (four C5
Secondary Metabolites Are Divided into Three Major units) are diterpenes. Larger terpenes include triterpenes (30
Groups carbons), tetraterpenes (40 carbons), and polyterpenoids
([C5]n carbons, where n > 8).
Plant secondary metabolites can be divided into three
chemically distinct groups: terpenes, phenolics, and 2 There Are Two Pathways for Terpene Biosynthesis
nitrogen‐ containing compounds. Figure 1 shows in
simplified form the pathways involved in the biosynthesis of Terpenes are biosynthesized from primary metabolites in at
secondary metabolites and their interconnections with least two different ways. In the well‐studied mevalonic acid
primary metabolism. pathway, three molecules of acetyl‐CoA are joined together
stepwise to form mevalonic acid (Figure 2. This key six‐

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carbon intermediate is then pyrophosphorylated, species show very striking insecticidal activity. Both
decarboxylated, and dehydrated to yield isopentenyl natural and synthetic pyrethroids are popular ingredients
diphosphate (IPP2). in commercial insecticides because of their low
persistence in the environment and their negligible
IPP is the activated five‐carbon building block of terpenes. toxicity to mammals.
Recently, it was discovered that IPP also can be formed from
intermediates of glycolysis or the photosynthetic carbon
reduction cycle via a separate set of reactions called the
methylerythritol phosphate (MEP) pathway that operates
in chloroplasts and other plastids. Although all the details
have not yet been elucidated, glyceraldehyde­3­phosphate
and two carbon atoms derived from pyruvate appear to
combine to generate an intermediate that is eventually
converted to IPP.

3 Isopentenyl Diphosphate and Its Isomer Combine to

Form Larger Terpenes

Isopentenyl diphosphate and its isomer, dimethylallyl

diphosphate (DPP), are the activated five‐carbon building
blocks of terpene biosynthesis that join together to form
larger molecules. First IPP and DPP react to give geranyl
diphosphate (GPP), the 10‐carbon precursor of nearly all
the monoterpenes (see Figure 2). GPP can then link to
another molecule of IPP to give the 15‐carbon compound
farnesyl diphosphate (FPP), the precursor of nearly all the
sesquiterpenes. Addition of yet another molecule of IPP
gives the 20‐carbon compound geranylgeranyl diphosphate
(GGPP), the precursor of the diterpenes. Finally, FPP and
GGPP can dimerize to give the triterpenes (C30) and the
tetraterpenes (C40), respectively.

4 Some Terpenes Have Roles in Growth and

Development

Certain terpenes have a well‐characterized function in plant

growth or development and so can be considered primary
rather than secondary metabolites.

• For example, the gibberellins, an important group of

plant hormones, are diterpenes.
• Sterols are triterpene derivatives that are essential
components of cell membranes, which they stabilize by
interacting with phospholipids.
• The red, orange, and yellow carotenoids are
tetraterpenes that function as accessory pigments in
photosynthesis and protect photosynthetic tissues from • In conifers such as pine and fir, monoterpenes accumulate
photooxidation. in resin ducts found in the needles, twigs, and trunk.These
• The hormone abscisic acid is a C15 terpene produced by compounds are toxic to numerous insects, including bark
degradation of a carotenoid precursor. beetles, which are serious pests of conifer species
• Long‐chain polyterpene alcohols known as dolichols throughout the world.
function as carriers of sugars in cell wall and glycoprotein
synthesis. • Many plants contain mixtures of volatile monoterpenes
• Terpene‐derived side chains, such as the phytol side and sesquiterpenes, called essential oils, that lend a
chain of chlorophyll, help anchor certain molecules in characteristic odor to their foliage. Peppermint, lemon,
membranes. basil, and sage are examples of plants that contain
essential oils. The chief monoterpene constituent of
Thus various terpenes have important primary roles in peppermint oil is menthol; that of lemon oil is limonene.
plants. However, the vast majority of the different terpene
structures produced by plants are secondary metabolites • Essential oils have well‐known insect repellent
that are presumed to be involved in defense. properties. They are frequently found in glandular hairs
that project outward from the epidermis and serve to
5 Terpenes Defend against Herbivores in Many Plants “advertise” the toxicity of the plant, repelling potential
herbivores even before they take a trial bite. In the
Terpenes are toxins and feeding deterrents to many plant glandular hairs, the terpenes are stored in a modified
feeding insects and mammals; thus they appear to play extracellular space in the cell wall. Essential oils can be
important defensive roles in the plant kingdom. extracted from plants by steam distillation and are
important commercially in flavoring foods and making
• For example, the monoterpene esters called pyrethroids perfumes.
that occur in the leaves and flowers of Chrysanthemum

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• Recent research has revealed an interesting twist on the may interfere with sterol uptake from the digestive
role of volatile terpenes in plant protection. In corn, system or disrupt cell membranes after being absorbed
cotton, wild tobacco, and other species, certain into the bloodstream.
monoterpenes and sesquiterpenes are produced and
emitted only after insect feeding has already begun. These II. PHENOLIC COMPOUNDS
substances repel ovipositing herbivores and attract
natural enemies, including predatory and parasitic Plants produce a large variety of secondary products that
insects, that kill plant‐feeding insects and so help contain a phenol group—a hydroxyl functional group on an
minimize further damage. Thus, volatile terpenes are not aromatic ring:
only defenses in their own right, but also provide a way
for plants to call for defensive help from other organisms.
The ability of plants to attract natural enemies of plant‐
feeding insects shows promise as a new, ecologically
sound means of pest control.
These substances are classified as phenolic compounds.
Plant phenolics are a chemically heterogeneous group of
• Among the nonvolatile terpene antiherbivore compounds
nearly 10,000 individual compounds: Some are soluble only
are the limonoids, a group of triterpenes (C30) well
in organic solvents, some are water‐soluble carboxylic acids
known as bitter substances in citrus fruit. Perhaps the
and glycosides, and others are large, insoluble polymers.
most powerful deterrent to insect feeding known is
azadirachtin (Figure 3A), a complex limonoid from the
In keeping with their chemical diversity, phenolics play a
neem tree (Azadirachta indica) of Africa and Asia.
variety of roles in the plant. After giving a brief account of
Azadirachtin is a feeding deterrent to some insects at
phenolic biosynthesis, we will discuss several principal
doses as low as 50 parts per billion, and it exerts a variety
groups of phenolic compounds and what is known about
of toxic effects. It has considerable potential as a
their roles in the plant. Many serve as defense compounds
commercial insect control agent because of its low
against herbivores and pathogens. Others function in
toxicity to mammals, and several preparations containing
mechanical support, in attracting pollinators and fruit
azadirachtin are now being marketed in North America
dispersers, in absorbing harmful ultraviolet radiation, or in
and India.
reducing the growth of nearby competing plants.

1 Phenylalanine Is an Intermediate in the Biosynthesis

of Most Plant Phenolics

Plant phenolics are biosynthesized by several different

routes and thus constitute a heterogeneous group from a
metabolic point of view. Two basic pathways are involved:
the shikimic acid pathway and the malonic acid pathway
(Figure 4). The shikimic acid pathway participates in the
biosynthesis of most plant phenolics. The malonic acid
pathway, although an important source of phenolic
• The phytoecdysones, first isolated from the common secondary products in fungi and bacteria, is of less
fern, Polypodium vulgare, are a group of plant steroids significance in higher plants.
that have the same basic structure as insect molting
hormones (Figure 3B). Ingestion of phytoecdysones by
insects disrupts molting and other developmental
processes, often with lethal consequences.

• Triterpenes that are active against vertebrate

herbivores include cardenolides and saponins.

• Cardenolides are glycosides (compounds containing

an attached sugar or sugars) that taste bitter and are
extremely toxic to higher animals.
The shikimic acid pathway converts simple carbohydrate
• In humans, they have dramatic effects on the heart precursors derived from glycolysis and the pentose
muscle through their influence on Na+/K+­activated phosphate pathway to the aromatic amino acid. One of the
ATPases. In carefully regulated doses, they slow and
pathway intermediates is shikimic acid, which has given its
strengthen the heartbeat. Cardenolides extracted from name to this whole sequence of reactions. The well‐known,
species of foxglove (Digitalis) are prescribed to millions broadspectrum herbicide glyphosate (available
of patients for the treatment of heart disease. commercially as Roundup) kills plants by blocking a step in
this pathway. The shikimic acid pathway is present in plants,
• Saponins are steroid and triterpene glycosides, so fungi, and bacteria but is not found in animals.
named because of their soaplike properties. The
presence of both lipid‐soluble (the steroid or Animals have no way to synthesize the three aromatic
triterpene) and water soluble (the sugar) elements in amino acids—phenylalanine, tyrosine, and tryptophan—
one molecule gives saponins detergent properties, and which are therefore essential nutrients in animal diets.
they form a soapy lather when shaken with water.
The most abundant classes of secondary phenolic
• The toxicity of saponins is thought to be a result of compounds in plants are derived from phenylalanine via the
their ability to form complexes with sterols. Saponins elimination of an ammonia molecule to form cinnamic acid

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(Figure 5). This reaction is catalyzed by phenylalanine Reactions subsequent to that catalyzed by PAL lead to the
ammonia lyase (PAL), perhaps the most studied enzyme in addition of more hydroxyl groups and other substituents.
plant secondary metabolism. PAL is situated at a branch Trans‐cinnamic acid, p‐coumaric acid, and their derivatives
point between primary and secondary metabolism, so the are simple phenolic compounds called phenylpropanoids
reaction that it catalyzes is an important regulatory step in because they contain a benzene ring:
the formation of many phenolic compounds.

and a three‐carbon side chain. Phenylpropanoids are

important building blocks of the more complex phenolic
compounds.

Now that the biosynthetic pathways leading to most

widespread phenolic compounds have been determined,
researchers have turned their attention to studying how
these pathways are regulated. In some cases, specific
enzymes, such as PAL, are important in controlling flux
through the pathway. Several transcription factors have
been shown to regulate phenolic metabolism by binding to
the promoter regions of certain biosynthetic genes and
activating transcription. Some of these factors activate the
transcription of large groups of genes.

2 Some Simple Phenolics Are Activated by Ultraviolet

Light

Simple phenolic compounds are widespread in vascular

plants and appear to function in different capacities. Their
structures include the following:
• Simple phenylpropanoids, such as trans­cinnamic acid, p‐
coumaric acid, and their derivatives, such as caffeic acid,
which have a basic phenylpropanoid carbon skeleton
(Figure 6A).

• Phenylpropanoid lactones (cyclic esters) called coumarins,

also with a phenylpropanoid skeleton (Figure 6B).

• Benzoic acid derivatives, which have a skeleton: which is

formed from phenylpropanoids by cleavage of a two‐carbon
fragment from the side chain (Figure 6 C).

As with many other secondary products, plants can

elaborate on the basic carbon skeleton of simple phenolic
compounds to make more complex products.

• Many simple phenolic compounds have important roles in

plants as defenses against insect herbivores and fungi. Of
special interest is the phototoxicity of certain coumarins
The activity of PAL is increased by environmental called furanocoumarins, which have an attached furan
factors, such as low nutrient levels, light (through its effect ring.
on phytochrome), and fungal infection. The point of control
appears to be the initiation of transcription. Fungal invasion, • These compounds are not toxic until they are activated by
for example, triggers the transcription of messenger RNA light. Sunlight in the ultraviolet A (UV‐A) region (320–400
that codes for PAL, thus increasing the amount of PAL in the nm) causes some furanocoumarins to become activated to
plant, which then stimulates the synthesis of phenolic a high‐energy electron state. Activated furanocoumarins
compounds. can insert themselves into the double helix of DNA and
bind to the pyrimidine bases cytosine and thymine, thus
The regulation of PAL activity in plants is made more blocking transcription and repair and leading eventually
complex by the existence in many species of multiple PAL to cell death.
encoding genes, some of which are expressed only in specific
tissues or only under certain environmental conditions. • Phototoxic furanocoumarins are especially abundant in
members of the Umbelliferae family, including celery,
parsnip, and parsley. In celery, the level of these
compounds can increase about 100‐fold if the plant is

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stressed or diseased. Celery pickers, and even some phenomenon has been hard to obtain. It is easy to show that
grocery shoppers, have been known to develop skin extracts or purified compounds from one plant can inhibit
rashes from handling stressed or diseased celery. Some the growth of other plants in laboratory experiments, but it
insects have adapted to survive on plants that contain has been very difficult to demonstrate that these compounds
furanocoumarins and other phototoxic compounds by are present in the soil in sufficient concentration to inhibit
living in silken webs or rolled‐up leaves, which screen out growth. Furthermore, organic substances in the soil are
the activating wavelengths. often bound to soil particles and may be rapidly degraded by
microbes.

In spite of the lack of supporting evidence, allelopathy is

currently of great interest because of its potential
agricultural applications. Reductions in crop yields caused
by weeds or residues from the previous crop may in some
cases be a result of allelopathy. An exciting future prospect is
the development of crop plants genetically engineered to be
allelopathic to weeds.

4 Lignin Is a Highly Complex Phenolic Macromolecule

After cellulose, the most abundant organic substance in

plants is lignin, a highly branched polymer of
phenylpropanoid groups

that plays both primary and secondary roles. The precise

structure of lignin is not known because it is difficult to
extract lignin from plants, where it is covalently bound to
cellulose and other polysaccharides of the cell wall.

Lignin is generally formed from three different

phenylpropanoid alcohols: coniferyl, coumaryl, and sinapyl,
alcohols which are synthesized from phenylalanine via
various cinnamic acid derivatives.

The phenylpropanoid alcohols are joined into a polymer

through the action of enzymes that generate free‐radical
intermediates.

The proportions of the three monomeric units in lignin vary

among species, plant organs, and even layers of a single cell
wall. In the polymer, there are often multiple C—C and C—
O—C bonds in each phenylpropanoid alcohol unit, resulting
3 The Release of Phenolics into the Soil May Limit the in a complex structure that branches in three dimensions.
Growth of Other Plants
Unlike polymers such as starch, rubber, or cellulose, the
From leaves, roots, and decaying litter, plants release a units of lignin do not appear to be linked in a simple,
variety of primary and secondary metabolites into the repeating way. However, recent research suggests that a
environment. Investigation of the effects of these guiding protein may bind the individual phenylpropanoid
compounds on neighboring plants is the study of units during lignin biosynthesis, giving rise to a scaffold that
allelopathy. then directs the formation of a large, repeating unit .

If a plant can reduce the growth of nearby plants by Lignin is found in the cell walls of various types of
releasing chemicals into the soil, it may increase its access to supporting and conducting tissue, notably the tracheids and
light, water, and nutrients and thus its evolutionary fitness. vessel elements of the xylem. It is deposited chiefly in the
Generally speaking, the term allelopathy has come to be thickened secondary wall but can also occur in the primary
applied to the harmful effects of plants on their neighbors, wall and middle lamella in close contact with the celluloses
although a precise definition also includes beneficial effects. and hemicelluloses already present.

Simple phenylpropanoids and benzoic acid derivatives are The mechanical rigidity of lignin strengthens stems and
frequently cited as having allelopathic activity. Compounds vascular tissue, allowing upward growth and permitting
such as caffeic acid and ferulic acid occur in soil in water and minerals to be conducted through the xylem
appreciable amounts and have been shown in laboratory under negative pressure without collapse of the tissue.
experiments to inhibit the germination and growth of many
plants. Because lignin is such a key component of water transport
tissue, the ability to make lignin must have been one of the
In spite of results such as these, the importance of most important adaptations permitting primitive plants to
allelopathy in natural ecosystems is still controversial. Many colonize dry land.
scientists doubt that allelopathy is a significant factor in
plant–plant interactions because good evidence for this

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Besides providing mechanical support, lignin has significant The colored pigments of plants are of two principal types:
protective functions in plants. Its physical toughness deters carotenoids and flavonoids.
feeding by animals, and its chemical durability makes it
relatively indigestible to herbivores. By bonding to cellulose Carotenoids, as we have already seen, are yellow, orange,
and protein, lignin also reduces the digestibility of these and red terpenoid compounds that also serve as accessory
substances. Lignification blocks the growth of pathogens and pigments in photosynthesis.
is a frequent response to infection or wounding.
Flavonoids are phenolic compounds that include a wide
range of colored substances.
5 There Are Four Major Groups of Flavonoids
The most widespread group of pigmented flavonoids is the
The flavonoids are one of the largest classes of plant anthocyanins, which are responsible for most of the red,
phenolics. The basic carbon skeleton of a flavonoid contains pink, purple, and blue colors observed in plant parts. By
15 carbons arranged in two aromatic rings connected by a coloring flowers and fruits, the anthocyanins are vitally
three‐carbon bridge: important in attracting animals for pollination and seed
dispersal.

Anthocyanins are glycosides that have sugars at position 3

This structure results from two separate biosynthetic (Figure 8B) and sometimes elsewhere. Without their sugars,
pathways: the shikimic acid pathway and the malonic acid anthocyanins are known as anthocyanidins (Figure 8A).
pathway (Figure 7).

Anthocyanin color is influenced by many factors, including

the number of hydroxyl and methoxyl groups in ring B of the
Flavonoids are classified into different groups, primarily on anthocyanidin (see Figure 8A), the presence of aromatic
the basis of the degree of oxidation of the three‐carbon acids esterified to the main skeleton, and the pH of the cell
bridge. We will discuss four of the groups shown in Figure 5: vacuole in which these compounds are stored.
the anthocyanins, the flavones, the flavonols, and the
isoflavones. Anthocyanins may also exist in supramolecular complexes
along with chelated metal ions and flavone copigments. The
The basic flavonoid carbon skeleton may have numerous blue pigment of dayflower (Commelina communis) was
substituents. Hydroxyl groups are usually present at found to consist of a large complex of six anthocyanin
positions 4, 5, and 7, but they may also be found at other molecules, six flavones, and two associated magnesium ions
positions. Sugars are very common as well; in fact, the The most common anthocyanidins and their colors are
majority of flavonoids exist naturally as glycosides. shown in Figure 8 and Table 1.

Whereas both hydroxyl groups and sugars increase the

water solubility of flavonoids, other substituents, such as
methyl ethers or modified isopentyl units, make flavonoids
lipophilic (hydrophobic). Different types of flavonoids
perform very different functions in the plant, including
pigmentation and defense.

5.1 Anthocyanins Are Colored Flavonoids That Attract

Animals

In addition to predator–prey interactions, there are

mutualistic associations among plants and animals. In return Considering the variety of factors affecting anthocyanin
for the reward of ingesting nectar or fruit pulp, animals coloration and the possible presence of carotenoids as well,
perform extremely important services for plants as carriers it is not surprising that so many different shades of flower
of pollen and seeds. Secondary metabolites are involved in and fruit color are found in nature. The evolution of flower
these plant–animal interactions, helping to attract animals to color may have been governed by selection pressures for
flowers and fruit by providing visual and olfactory signals. different sorts of pollinators, which often have different
color preferences.

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5.2. Flavonoids May Protect against Damage by 6 Tannins Deter Feeding by Herbivores
Ultraviolet Light
A second category of plant phenolic polymers with defensive
Two other major groups of flavonoids found in flowers are properties, besides lignins, is the tannins. The term tannin
flavones and flavonols (see Figure 5). was first used to describe compounds that could convert
raw animal hides into leather in the process known as
These flavonoids generally absorb light at shorter tanning. Tannins bind the collagen proteins of animal hides,
wavelengths than anthocyanins do, so they are not visible to increasing their resistance to heat, water, and microbes.
the human eye. There are two categories of tannins: condensed and
hydrolyzable.
However, insects such as bees, which see farther into the
ultraviolet range of the spectrum than humans do, may Condensed tannins are compounds formed by the
respond to flavones and flavonols as attractant cues. polymerization of flavonoid units. They are frequent
constituents of woody plants. Because condensed tannins
Flavonols in a flower often form symmetric patterns of can often be hydrolyzed to anthocyanidins by treatment
stripes, spots, or concentric circles called nectar guides. with strong acids, they are sometimes called pro­
These patterns may be conspicuous to insects and are anthocyanidins.
thought to help indicate the location of pollen and nectar.
Hydrolyzable tannins are heterogeneous polymers
Flavones and flavonols are not restricted to flowers; they are containing phenolic acids, especially gallic acid, and simple
also present in the leaves of all green plants. These two sugars. They are smaller than condensed tannins and may be
classes of flavonoids function to protect cells from excessive hydrolyzed more easily; only dilute acid is needed. Most
UV‐B radiation (280–320 nm) because they accumulate in tannins have molecular masses between 600 and 3000.
the epidermal layers of leaves and stems and absorb light
strongly in the UV‐B region while allowing the visible Tannins are general toxins that significantly reduce the
(photosynthetically active) wavelengths to pass through growth and survivorship of many herbivores when added to
uninterrupted. In addition, exposure of plants to increased their diets. In addition, tannins act as feeding repellents to a
UV‐B light has been demonstrated to increase the synthesis great diversity of animals. Mammals such as cattle, deer, and
of flavones and flavonols. apes characteristically avoid plants or parts of plants with
high tannin contents. Unripe fruits, for instance, frequently
Arabidopsis thaliana mutants that lack the enzyme chalcone have very high tannin levels, which may be concentrated in
synthase produce no flavonoids. Lacking flavonoids, these the outer cell layers.
plants are much more sensitive to UV‐B radiation than wild‐
type individuals are, and they grow very poorly under Interestingly, humans often prefer a certain level of
normal conditions. When shielded from UV light, however, astringency in tannin‐containing foods, such as apples,
they grow normally. A group of simple phenylpropanoid blackberries, tea, and red wine.
esters are also important in UV protection in Arabidopsis.
Recently, polyphenols (tannins) in red wine were shown to
Other functions of flavonoids have recently been discovered. block the formation of endothelin­1, a signaling molecule
For example, flavones and flavonols secreted into the soil that makes blood vessels constrict. This effect of wine
by legume roots mediate the interaction of legumes and tannins may account for the often‐touted health benefits of
nitrogen­fixing symbionts. Recent work suggests that red wine, especially the reduction in the risk of heart disease
flavonoids also play a regulatory role in plant development associated with moderate red wine consumption.
as modulators of polar auxin transport.
Although moderate amounts of specific polyphenolics may
5.3 Isoflavonoids Have Antimicrobial Activity have health benefits for humans, the defensive properties of
most tannins are due to their toxicity, which is generally
The isoflavonoids (isoflavones) are a group of flavonoids in attributed to their ability to bind proteins nonspecifically. It
which the position of one aromatic ring (ring B) is shifted has long been thought that plant tannins complex proteins
(see Figure 5). in the guts of herbivores by forming hydrogen bonds
between their hydroxyl groups and electronegative sites
Isoflavonoids are found mostly in legumes and have several on the protein (Figure 9A).
different biological activities. Some, such as the rotenoids,
have strong insecticidal actions; others have anti‐estrogenic More recent evidence indicates that tannins and other
effects. phenolics can also bind to dietary protein in a covalent
fashion (see Figure 9B). The foliage of many plants contains
For example, sheep grazing on clover rich in isoflavonoids enzymes that oxidize phenolics to their corresponding
often suffer from infertility. The isoflavonoid ring system has quinone forms in the guts of herbivores.
a three‐dimensional structure similar to that of steroids (see
Figure 3B), allowing these substances to bind to estrogen Quinones are highly reactive electrophilic molecules that
receptors. readily react with the nucleophilic —NH2 and —SH groups
of proteins (see Figure 9B). By whatever mechanism
Isoflavonoids may also be responsible for the anticancer protein–tannin binding occurs, this process has a negative
benefits of food prepared from soybeans. In the past few impact on herbivore nutrition. Tannins can inactivate
years, isoflavonoids have become best known for their role herbivore digestive enzymes and create complex aggregates
as phytoalexins, antimicrobial compounds synthesized in of tannins and plant proteins that are difficult to digest.
response to bacterial or fungal infection that help limit the
spread of the invading pathogen.

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1. Alkaloids Have Dramatic Physiological Effects on

Animals

The alkaloids are a large family of more than 15,000

nitrogen‐ containing secondary metabolites found in
approximately 20% of the species of vascular plants. The
nitrogen atom in these substances is usually part of a
heterocyclic ring, a ring that contains both nitrogen and
carbon atoms.

As a group, alkaloids are best known for their striking

pharmacological effects on vertebrate animals. As their
name would suggest, most alkaloids are alkaline. At pH
values commonly found in the cytosol (pH 7.2) or the
vacuole (pH 5 to 6), the nitrogen atom is protonated; hence,
alkaloids are positively charged and are generally water
soluble.

Herbivores that habitually feed on tannin‐rich plant material

appear to possess some interesting adaptations to remove
tannins from their digestive systems. For example, some
mammals, such as rodents and rabbits, produce salivary
proteins with a very high proline content (25–45%) that
have a high affinity for tannins. Secretion of these proteins is
induced by ingestion of food with a high tannin content and
greatly diminishes the toxic effects of tannins. The large
number of proline residues gives these proteins a very
flexible, open conformation and a high degree of
hydrophobicity that facilitates binding to tannins. Alkaloids are usually synthesized from one of a few common
amino acids—in particular, lysine, tyrosine, and tryptophan.
Plant tannins also serve as defenses against microorganisms. However, the carbon skeleton of some alkaloids contains a
For example, the nonliving heartwood of many trees component derived from the terpene pathway. Table 2 lists
contains high concentrations of tannins that help prevent the major alkaloid types and their amino acid precursors.
fungal and bacterial decay.
Several different types, including nicotine and its relatives,
III. NITROGEN­CONTAINING COMPOUNDS are derived from ornithine, an intermediate in arginine
biosynthesis.
A large variety of plant secondary metabolites have nitrogen
in their structure. Included in this category are such well‐ The B vitamin nicotinic acid (niacin) is a precursor of the
known antiherbivore defenses as alkaloids and cyanogenic pyridine (six‐membered) ring of this alkaloid; the
glycosides, which are of considerable interest because of pyrrolidine (fivemembered) ring of nicotine arises from
their toxicity to humans and their medicinal properties. ornithine (Figure 13.18). Nicotinic acid is also a constituent
Most nitrogenous secondary metabolites are biosynthesized of NAD+ and NADP+, which serve as electron carriers in
from common amino acids. metabolism.

In this section we will examine the structure and biological The role of alkaloids in plants has been a subject of
properties of various nitrogen‐containing secondary speculation for at least 100 years. Alkaloids were once
metabolites, including alkaloids, cyanogenic glycosides, thought to be nitrogenous wastes (analogous to urea and
glucosinolates, and nonprotein amino acids. uric acid in animals), nitrogen storage compounds, or
growth regulators, but there is little evidence to support any
In addition, we will discuss the ability of systemin, a protein of these functions.
released from damaged cells, to serve as a wound signal to
the rest of the plant. Most alkaloids are now believed to function as defenses
against predators, especially mammals, because of their
general toxicity and deterrence capability.

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Large numbers of livestock deaths are caused by the Like monoterpenes in conifer resin and many other
ingestion of alkaloid‐containing plants. In the United States, antiherbivore defense compounds, alkaloids increase in
a significant percentage of all grazing livestock animals are response to initial herbivore damage, fortifying the plant
poisoned each year by consumption of large quantities of against subsequent attack.
alkaloid‐containing plants such as lupines (Lupinus),
larkspur (Delphinium), and groundsel (Senecio). For example, Nicotiana attenuata, a wild tobacco that grows
in the deserts of the Great Basin, produces higher levels of
This phenomenon may be due to the fact that domestic nicotine following herbivory. When it is attacked by
animals, unlike wild animals, have not been subjected to nicotine‐ tolerant caterpillars, however, there is no increase
natural selection for the avoidance of toxic plants. Indeed, in nicotine. Instead, volatile terpenes are released that
some livestock actually seem to prefer alkaloid containing attract enemies of the caterpillars. Clearly, wild tobacco and
plants to less harmful forage. other plants must have ways of determining what type of
herbivore is damaging their foliage.
Nearly all alkaloids are also toxic to humans when taken in
sufficient quantity. For example, strychnine, atropine, and Herbivores might signal their presence by the type of
coniine (from poison hemlock) are classic alkaloid damage they inflict or the distinctive chemical compounds
poisoning agents. At lower doses, however, many are useful they release.
pharmacologically. Morphine, codeine, and scopolamine are
just a few of the plant alkaloids currently used in medicine. Recently, the oral secretions of caterpillars feeding on corn
leaves were shown to contain a fatty acid–amino acid
Other alkaloids, including cocaine, nicotine, and caffeine, conjugate that induced the plant to produce defensive
enjoy widespread nonmedical use as stimulants or terpenes when applied to cut leaves.
sedatives.
2. Cyanogenic Glycosides Release the Poison Hydrogen
On a cellular level, the mode of action of alkaloids in animals Cyanide
is quite variable. Many alkaloids interfere with components
of the nervous system, especially the chemical transmitters; Various nitrogenous protective compounds other than
others affect membrane transport, protein synthesis, or alkaloids are found in plants. Two groups of these
miscellaneous enzyme activities. substances— cyanogenic glycosides and glucosinolates—are
not in themselves toxic but are readily broken down to give
One group of alkaloids, the pyrrolizidine alkaloids, illustrates off volatile poisons when the plant is crushed. Cyanogenic
how herbivores can become adapted to tolerate plant glycosides release the well‐known poisonous gas hydrogen
defensive substances and even use them in their own cyanide (HCN).
defense. Within plants, pyrrolizidine alkaloids occur
naturally as nontoxic N‐oxides. In herbivore digestive tracts, The breakdown of cyanogenic glycosides in plants is a two‐
however, they are quickly reduced to uncharged, step enzymatic process. Species that make cyanogenic
hydrophobic tertiary alkaloids, which easily pass through glycosides also make the enzymes necessary to hydrolyze
membranes and are toxic. the sugar and liberate HCN:
1. In the first step the sugar is cleaved by a glycosidase, an
enzyme that separates sugars from other molecules to which
they are linked (Figure).

2. In the second step the resulting hydrolysis product, called

an α‐hydroxynitrile or cyanohydrin, can decompose
Nevertheless, some herbivores, such as cinnabar moth spontaneously at a low rate to liberate HCN. This second
(Tyria jacobeae), have developed the ability to reconvert step can be accelerated by the enzyme hydroxynitrile lyase.
tertiary pyrrolizidine alkaloids to the nontoxic N‐oxide form
immediately after its absorption from the digestive tract. Cyanogenic glycosides are not normally broken down in the
These herbivores may then store the N‐oxides in their intact plant because the glycoside and the degradative
bodies as defenses against their own predators. enzymes are spatially separated, in different cellular
compartments or in different tissues. In sorghum, for
Not all of the alkaloids that appear in plants are produced by example, the cyanogenic glycoside dhurrin is present in the
the plant itself. Many grasses harbor endogenous fungal vacuoles of epidermal cells, while the hydrolytic and lytic
symbionts that grow in the apoplast and synthesize a variety enzymes are found in the mesophyll. Under ordinary
of different types of alkaloids. conditions this compartmentation prevents decomposition
of the glycoside. When the leaf is damaged, however, as
Grasses with fungal symbionts often grow faster and are during herbivore feeding, the cell contents of different
better defended against insect and mammalian herbivores tissues mix and HCN forms.
than those without symbionts.
Cyanogenic glycosides are widely distributed in the plant
Unfortunately, certain grasses with symbionts, such as tall kingdom and are frequently encountered in legumes,
fescue, are important pasture grasses that may become toxic grasses, and species of the rose family. Considerable
to livestock when their alkaloid content is too high. evidence indicates that cyanogenic glycosides have a
protective function in certain plants. HCN is a fast‐acting
toxin that inhibits metalloproteins, such as the iron‐

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containing cytochrome oxidase, a key enzyme of Plant breeders have tried to lower the glucosinolate levels of
mitochondrial respiration. The presence of cyanogenic rapeseed so that the high‐protein seed meal remaining after
glycosides deters feeding by insects and other herbivores, oil extraction can be used as animal food. The first low‐
such as snails and slugs. As with other classes of secondary glucosinolate varieties tested in the field were unable to
metabolites, however, some herbivores have adapted to feed survive because of severe pest problems. However, more
on cyanogenic plants and can tolerate large doses of HCN. recently developed varieties with low glucosinolate levels in
The tubers of cassava (Manihot esculenta), a high‐ seeds but high glucosinolate levels in leaves are able to hold
carbohydrate, staple food in many tropical countries, contain their own against pests and still provide a protein‐rich seed
high levels of cyanogenic glycosides. Traditional processing residue for animal feeding.
methods, such as grating, grinding, soaking, and drying, lead
to the removal or degradation of a large fraction of the 4. Nonprotein Amino Acids Defend against Herbivores
cyanogenic glycosides present in cassava tubers.
Plants and animals incorporate the same 20 amino acids into
However, chronic cyanide poisoning leading to partial their proteins. However, many plants also contain unusual
paralysis of the limbs is still widespread in regions where amino acids, called nonprotein amino acids, that are not
cassava is a major food source because the traditional incorporated into proteins but are present instead in the
detoxification methods employed to remove cyanogenic free form and act as protective substances. Nonprotein
glycosides from cassava are not completely effective. In amino acids are often very similar to common protein amino
addition, many populations that consume cassava have poor acids. Canavanine, for example, is a close analog of arginine,
nutrition, which aggravates the effects of the cyanogenic and azetidine‐2‐carboxylic acid has a structure very much
glycosides. like that of proline (Figure).

Efforts are currently under way to reduce the cyanogenic

glycoside content of cassava through both conventional
breeding and genetic engineering approaches. However, the
complete elimination of cyanogenic glycosides may not be
desirable because these substances are probably responsible
for the fact that cassava can be stored for very long periods
of time without being attacked by pests.

3. Glucosinolates Release Volatile Toxins

A second class of plant glycosides, called the glucosinolates, Nonprotein amino acids exert their toxicity in various ways.
or mustard oil glycosides, break down to release volatile Some block the synthesis or uptake of protein amino acids;
defensive substances. Found principally in the Brassicaceae others, such as canavanine, can be mistakenly incorporated
and related plant families, glucosinolates give off the into proteins. After ingestion, canavanine is recognized by
compounds responsible for the smell and taste of vegetables the herbivore enzyme that normally binds arginine to the
such as cabbage, broccoli, and radishes. arginine transfer RNA molecule, so it becomes incorporated
into proteins in place of arginine. The usual result is a
The release of these mustard‐smelling volatiles from nonfunctional protein because either its tertiary structure or
glucosinolates is catalyzed by a hydrolytic enzyme, called its catalytic site is disrupted. Canavanine is less basic than
a thioglucosidase or myrosinase, that cleaves glucose from arginine and may alter the ability of an enzyme to bind
its bond with the sulfur atom (Figure). substrates or catalyze chemical reactions.

Plants that synthesize nonprotein amino acids are not

susceptible to the toxicity of these compounds. The jack
bean (Canavalia ensiformis), which synthesizes large
amounts of canavanine in its seeds, has protein‐synthesizing
The resulting aglycone, the nonsugar portion of the machinery that can discriminate between canavanine and
molecule, rearranges with loss of the sulfate to give pungent arginine, and it does not incorporate canavanine into its own
and chemically reactive products, including isothiocyanates proteins. Some insects that specialize on plants containing
and nitriles, depending on the conditions of hydrolysis. nonprotein amino acids have similar biochemical
adaptations.
These products function in defense as herbivore toxins and
feeding repellents. Like cyanogenic glycosides, 4.1 Some Plant Proteins Inhibit Herbivore Digestion
glucosinolates are stored in the intact plant separately from
the enzymes that hydrolyze them, and they are brought into Among the diverse components of plant defense arsenals are
contact with these enzymes only when the plant is crushed. proteins that interfere with herbivore digestion. For
example, some legumes synthesize α‐amylase inhibitors that
As with other secondary metabolites, certain animals are block the action of the starch‐digesting enzyme α‐amylase.
adapted to feed on glucosinolate‐containing plants without Other plant species produce lectins, defensive proteins that
ill effects. For adapted herbivores, such as the cabbage bind to carbohydrates or carbohydrate‐containing proteins.
butterfly, glucosinolates often serve as stimulants for
feeding and egg laying, and the isothiocyanates produced After being ingested by an herbivore, lectins bind to the
after glucosinolate hydrolysis act as volatile attractants. epithelial cells lining the digestive tract and interfere with
nutrient absorption .
Most of the recent research on glucosinolates in plant
defense has concentrated on rape, or canola (Brassica The best‐known antidigestive proteins in plants are the
napus), a major oil crop in both North America and Europe. proteinase inhibitors. Found in legumes, tomatoes, and other

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plants, these substances block the action of herbivore In plants, jasmonic acid is synthesized from linolenic acid
proteolytic enzymes. After entering the herbivore’s digestive (18:3), which is released from membrane lipids and then
tract, they hinder protein digestion by binding tightly and converted to jasmonic acid.
specifically to the active site of protein‐hydrolyzing enzymes
such as trypsin and chymotrypsin. Jasmonic acid is known to induce the transcription of a host
of genes involved in plant defense metabolism. The
Insects that feed on plants containing proteinase inhibitors mechanisms for this gene activation are slowly becoming
suffer reduced rates of growth and development that can be clear.
offset by supplemental amino acids in their diet.
For example, recent research on the Madagascar periwinkle
The defensive role of proteinase inhibitors has been (Catharanthus roseus), which makes some valuable
confirmed by experiments with transgenic tobacco. Plants anticancer alkaloids, identified a transcription factor that
that had been transformed to accumulate increased levels of responds to jasmonic acid by activating the expression of
proteinase inhibitors suffered less damage from insect several genes encoding alkaloid biosynthetic genes.
herbivores than did untransformed control plants.
Interestingly, this transcription factor also activates the
4.2 Herbivore Damage Triggers a Complex Signaling genes of certain primary metabolic pathways that provide
Pathway precursors for alkaloid formation, so it appears to be a
master regulator of metabolism in Madagascar periwinkle.
Proteinase inhibitors and certain other defenses are not
continuously present in plants, but are synthesized only Direct demonstration of the role of jasmonic acid in insect
after initial herbivore or pathogen attack. In tomatoes, insect resistance has come from research with mutant lines of
feeding leads to the rapid accumulation of proteinase Arabidopsis that produce only low levels of jasmonic acid.
inhibitors throughout the plant, even in undamaged areas Such mutants are easily killed by insect pests, such as fungus
far from the initial feeding site. gnats, that normally do not damage Arabidopsis. However,
application of exogenous jasmonic acid can restore
The systemic production of proteinase inhibitors in young resistance nearly to the levels of the wild‐type plant.
tomato plants is triggered by a complex sequence of events:

• Wounded tomato leaves synthesize prosystemin, a large

(200 amino acid) precursor protein.

• Prosystemin is proteolytically processed to produce the

short (18 amino acid) polypeptide called systemin, the
first (and so far only) polypeptide hormone discovered in
plants (Pearce et al. 1991) (Figure 9).

• Systemin is released from damaged cells into the

apoplast.

• Systemin is then transported out of the wounded leaf via

the phloem.

• In target cells, systemin is believed to bind to a site on the

plasma membrane and initiate the biosynthesis of
jasmonic acid, a plant growth regulator that has wide‐
ranging effects (Creelman and Mullet 1997).

• Jasmonic acid eventually activates the expression of genes

that encode proteinase inhibitors (see Figure 9). Other
signals, such as ABA (abscisic acid), salicylic acid, and
pectin fragments from damaged plant cell walls also
appear to participate in this wound signaling cascade, but
their specific roles are still unclear.

4.4 Jasmonic Acid Is a Plant Stress Hormone That

Activates Many Defense Responses

Jasmonic acid levels rise steeply in response to damage

caused by a variety of different herbivores and trigger the
formation of many different kinds of plant defenses besides
proteinase inhibitors, including terpenes and alkaloids.

The structure and biosynthesis of jasmonic acid have

intrigued plant biologists because of the parallels to some
eicosanoids that are central to inflammatory responses and
other physiological processes in mammals.

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PLANT DEFENSE AGAINST PATHOGENS Active oxygen species may contribute to cell death as part of
the hypersensitive response or act to kill the pathogen
Even though they lack an immune system, plants are directly.
surprisingly resistant to diseases caused by the fungi,
bacteria, viruses, and nematodes that are ever present in the Many species react to fungal or bacterial invasion by
environment. In this section we will examine the diverse synthesizing lignin or callose. These polymers are thought to
array of mechanisms that plants have evolved to resist serve as barriers, walling off the pathogen from the rest of
infection, including the production of antimicrobial agents the plant and physically blocking its spread.
and a type of programmed cell death called the
hypersensitive response. Finally, we will discuss a special A related response is the modification of cell wall proteins.
type of plant immunity called systemic acquired resistance. Certain proline‐rich proteins of the wall become oxidatively
cross‐linked after pathogen attack in an H2O2‐mediated
1. Some Antimicrobial Compounds Are Synthesized reaction (see Figure 10). This process strengthens the walls
before Pathogen Attack of the cells in the vicinity of the infection site, increasing
their resistance to microbial digestion.
Several classes of secondary metabolites that we have
already discussed have strong antimicrobial activity when Another defensive response to infection is the formation of
tested in vitro; thus they have been proposed to function as hydrolytic enzymes that attack the cell wall of the pathogen.
defenses against pathogens in the intact plant. Among these An assortment of glucanases, chitinases, and other
are saponins, a group of triterpenes thought to disrupt hydrolases are induced by fungal invasion. Chitin, a polymer
fungal membranes by binding to sterols. of N‐acetylglucosamine residues, is a principal component of
fungal cell walls.
Experiments performed in the laboratory of Anne Osbourn
at the John Innes Centre (Norwich, England) utilized genetic These hydrolytic enzymes belong to a group of proteins that
approaches to demonstrate the role of saponins in defense are closely associated with pathogen infection and so are
against pathogens of oat. Mutant oat lines with reduced known as pathogenesis­ related (PR) proteins.
saponin levels had much less resistance to fungal pathogens
than wild‐type oats. Interestingly, one fungal strain that
normally grows on oats was able to detoxify one of the
principal saponins in the plant. However, mutants of this
strain that could no longer detoxify saponins failed to infect
oats, but could grow successfully on wheat that did not
contain any saponins.

2. Infection Induces Additional Antipathogen Defenses

Some defenses are induced by herbivore attack or microbial

infection. Defenses that are produced only after initial
herbivore damage theoretically require a smaller investment
of plant resources than defenses that are always present, but
they must be activated quickly to be effective. Like
proteinase inhibitors, other induced defenses appear to be
triggered by complex signal transduction networks, which
often involve jasmonic acid.

After being infected by a pathogen, plants deploy a broad

spectrum of defenses against invading microbes. A common
defense is the hypersensitive response, in which cells
immediately surrounding the infection site die rapidly,
depriving the pathogen of nutrients and preventing its
spread. After a successful hypersensitive response, a small 3. Phytoalexins.
region of dead tissue is left at the site of the attempted
invasion, but the rest of the plant is unaffected. Perhaps the best‐studied response of plants to bacterial or
fungal invasion is the synthesis of phytoalexins.
The hypersensitive response is often preceded by the Phytoalexins are a chemically diverse group of secondary
production of reactive oxygen species. Cells in the vicinity metabolites with strong antimicrobial activity that
of the infection synthesize a burst of toxic compounds accumulate around the site of infection. Phytoalexin
formed by the reduction of molecular oxygen, including the production appears to be a common mechanism of
superoxide anion (O2–), hydrogen peroxide (H2O2) and the resistance to pathogenic microbes in a wide range of plants.
hydroxyl radical (.OH). However, different plant families employ different types of
secondary products as phytoalexins.
An NADPH‐dependent oxidase located on the plasma
membrane (Figure 10) is thought to produce O2–, which in For example, isoflavonoids are common phytoalexins in the
turn is converted to .OH and H2O2. The hydroxyl radical is the legume family, whereas in plants of the potato family
strongest oxidant of these active oxygen species and can (Solanaceae), such as potato, tobacco, and tomato, various
initiate radical chain reactions with a range of organic sesquiterpenes are produced as phytoalexins.
molecules, leading to lipid peroxidation, enzyme
inactivation, and nucleic acid degradation. Phytoalexins are generally undetectable in the plant before
infection, but they are synthesized very rapidly after
microbial attack because of the activation of new

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biosynthetic pathways. The point of control is usually the

initiation of gene transcription. Thus, plants do not appear to Studies of plant disease have revealed complex patterns of
store any of the enzymatic machinery required for host relationships between plants and pathogen strains.
phytoalexin synthesis. Instead, soon after microbial invasion Plant species are generally susceptible to the attack of
they begin transcribing and translating the appropriate certain pathogen strains, but resistant to others. This
mRNAs and synthesizing the enzymes de novo. specificity is thought to be determined by interaction
between the products of host R genes and pathogen avr
Although phytoalexins accumulate in concentrations that (avirulence) genes believed to encode specific elicitors.
have been shown to be toxic to pathogens in bioassays, the According to current thinking, successful resistance requires
defensive significance of these compounds in the intact plant the elicitor, a product of the pathogen avr gene, to be rapidly
is not fully known. Recent experiments on genetically recognized by a host plant receptor, the product of an R
modified plants and pathogens have provided the first direct gene. Despite their name, avr genes appear to encode factors
proof of phytoalexin function in vivo. that promote infection.

For example, when tobacco was transformed with a gene 5. Exposure to Elicitors Induces a Signal Transduction
catalyzing the biosynthesis of the phenylpropanoid Cascade
phytoalexin resveratrol, it became much more resistant to a
fungal pathogen than non‐transformed control plants were. Within a few minutes after pathogen elicitors have been
In contrast, Arabidopsis mutants deficient in the tryptophan‐ recognized by an R gene, complex signaling pathways are set
derived phytoalexin camalexin were more susceptible than in motion that lead eventually to defense responses (see
the wildtype to a fungal pathogen. In other experiments, Figure 10).
pathogens that had been transformed with genes encoding
phytoalexindegrading enzymes were then able to infect A common early element of these cascades is a transient
plants that were normally resistant to them. change in the ion permeability of the plasma membrane. R
gene activation stimulates an influx of Ca2+ and H+ ions into
4. Some Plants Recognize Specific Substances Released the cell and an efflux of K+ and Cl– ions.
from Pathogens
The influx of Ca2+ activates the oxidative burst that may act
Within a species, individual plants often differ greatly in directly in defense, as well as signaling other defense
their resistance to microbial pathogens. These differences reactions. Other components of pathogen‐stimulated signal
often lie in the speed and intensity of a plant’s reactions. transduction pathways include nitric oxide, mitogen‐
Resistant plants respond more rapidly and more vigorously activated protein (MAP) kinases, calcium‐dependent protein
to pathogens than susceptible plants. Hence it is important kinases, jasmonic acid, and salicylic acid (see the next
to learn how plants sense the presence of pathogens and section).
initiate defense.
6. A Single Encounter with a Pathogen May Increase
In the last few years, researchers have isolated over 20 Resistance to Future Attacks
different plant resistance genes, known as R genes, that
function in defense against fungi, bacteria, and nematodes. When a plant survives the infection of a pathogen at one site,
Most of the R genes are thought to encode protein receptors it often develops increased resistance to subsequent attacks
that recognize and bind specific molecules originating from at sites throughout the plant and enjoys protection against a
pathogens. This binding alerts the plant to the pathogen’s wide range of pathogen species.
presence (see Figure 10). The specific pathogen molecules
recognized are referred to as elicitors, and they include This phenomenon, called systemic acquired resistance
proteins, peptides, sterols, and polysaccharide fragments (SAR), develops over a period of several days following
arising from the pathogen cell wall, outer membrane, or a initial infection (Ryals et al. 1996). Systemic acquired
secretion process. resistance appears to result from increased levels of certain
defense compounds that we have already mentioned,
The R gene products themselves are nearly all proteins with including chitinases and other hydrolytic enzymes.
a leucine‐rich domain that is repeated inexactly several
times in the amino acid sequence. Although the mechanism of SAR induction is still unknown,
one of the endogenous signals is likely to be salicylic acid.
Such domains may be involved in elicitor binding and The level of this benzoic acid derivative,
pathogen recognition. In addition, the R gene product is
equipped to initiate signaling pathways that activate the
various modes of antipathogen defense. Some R genes a compound rises dramatically in the zone of
encode a nucleotide‐binding site that binds ATP or GTP; infection after initial attack, and it is thought to establish
others encode a protein kinase domain. SAR in other parts of the plant, although salicylic acid itself is
not the mobile signal.
R gene products are distributed in more than one place in
the cell. Some appear to be situated on the outside of the In addition to salicylic acid, recent studies suggest that its
plasma membrane, where they could rapidly detect elicitors; methyl ester, methyl salicylate, acts as a volatile SAR
others are cytoplasmic to detect either pathogen molecules inducing signal transmitted to distant parts of the plant and
that are injected into the cell or other metabolic changes even to neighboring plants. Thus, even though plants lack
indicating pathogen infection. R genes constitute one of the immune systems like those present in many animals, they
largest gene families in plants and are often clustered have developed elaborate mechanisms to protect
together in the genome. The structures of R gene clusters themselves from disease‐causing microbes.
may help generate R gene diversity by promoting exchange
between chromosomes.

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H. Stress physiology: Responses of plants to abiotic (water, temperature and salt) stresses; mechanisms of tolerance
to abiotic stress

1. External conditions that adversely affect growth, ƒ the rate of onset

development, or productivity are termed as stress. ƒ if the plant was acclimated to water stress

2. Stresses trigger a wide range of plant responses such as 14. Tolerance to drought and salinity
ƒ altered gene expression ƒ Osmotic adjustment: a biochemical mechanism
ƒ cellular metabolism that helps plants acclimate to dry and saline
ƒ changes in growth rates and crop yields conditions
ƒ Many drought‐tolerant plants can regulate their
3. Stress may be broadly categorized into two groups solute potentials to compensate for transient or
ƒ Biotic ­ imposed by other organisms extended periods of water stress by making
ƒ Abiotic ­ arising from an excess or deficit in the osmotic adjustments, which results in a net
physical or chemical environment increase in the number of solutes particles
present in the plant cell
4. Biotic and abiotic stresses can reduce average plant ƒ Osmotic adjustment occurs when the
productivity by 65% to 87%, depending on the crop. concentrations of solutes within a plant cell
increases to maintain a positive turgor pressure
PLANT RESPONSE TO ABIOTIC STRESS within the cell
ƒ The cell actively accumulates solutes and as a
5. Environmental conditions that can cause stress are result the solute potential (Ψs) drops, promoting
water‐logging, drought, high or low temperatures, excessive the flow of water into the cell
soil salinity, inadequate mineral in the soil, too much or too
little light and phytotoxic compounds such as Ozone 15. Compatible solutes that contribute to osmotic
adjustments: Proline, Glycine betanine,β­alanine
6. Resistance or sensitivity of plants to stress depends on betanine, Dimethyl sulphonioproponate, Proline
the species, the genotype and development age betanine, Choline­O­sufate, mannitol and pinnitol

7. Stress resistance mechanisms 16. Compatible solutes (osmolytes): tend to be neutrally

ƒ Avoidance mechanisms: prevents exposure to charged at physiological pH, either non‐ionic or zwitterionic,
stress and are excluded from hydration shells of macromolecules.
ƒ Tolerance mechanisms: permit the plant to ƒ In contrast, many ions can enter the hydration
withstand stress shells of a protein and promote its denaturation
ƒ Acclimation: alter their physiology in response ƒ Membrane‐associated carriers and transporters
stress are probably involved in differentially
distributing osmolytes within the cell and
8. Regulation of plant stress responses is mediated by throughout the plant.
Abscisic acid (ABA), Jasmonic acid, Ethylene and Calcium
17. Proline: distribution of proline in osmotically stressed
9. Changes in gene expression to stress plants involves a transporter. Two Arabidopsis cDNA
ƒ A stress response is initiated when plants encoding proline transporters were cloned by functional
recognizes stress at the cellular level complementation
ƒ Stress recognition activates signal transduction
pathways that transmit information within the 18. Glycine Betanine: Glycine betaine accumulation in
individual cell and throughout the plant osmotically stressed plants resulted from increased rates of
ƒ Changes in gene expression may modify growth synthesis, whereas, with proline, synthesis and catabolism
and development and even influence reproductive appears to be co‐ordinately regulated in response to water
capabilities stress
ƒ Glycine betaine is synthesised and accumulated
10. Gene expression results in: by many algae and higher plants and is not
ƒ Increase amounts of specific mRNA broken down by plants
ƒ Enhance translation ƒ Genetic evidence indicates that accumulation of
ƒ Stabilize proteins glycine betaine promotes salt tolerance
ƒ Altered protein activity
ƒ A combination of the above 19. Mannitol is the reduced form of the sugar mannose and
is broadly distributed among plants.
11. Stresses involving water deficit: Water related ƒ salt stress inhibits sucrose synthesis and
stresses could affect plants if the environment contains promotes accumulation of mannitol
insufficient water to meet basic needs ƒ mannitol concentrations increase in response to
osmotic stress
12. Environmental conditions that can lead to water deficit ƒ mannitol accumulation appears to be regulated by
are drought, hypersaline conditions, low temperatures and competing pathways and decreasing rates of
transient loss of turgor at midday mannitol consumption and catabolism
ƒ In celery, salt stress inhibits sucrose synthesis but
13. Factors that can affect the response of a plant to does not seem to affect the enzymes that
water deficit synthesise mannitol
ƒ duration of water deficiency

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ƒ Salt stress also down‐regulate NAD+ ‐dependent ƒ Avoidance involves physically excluding the
mannitol dehydrogenase, the enzyme that pollutant by closing the stomata, the principal site
oxidises mannitol in celery at which ozone enters the plant
ƒ Transgenic tobacco and Arabidopsis plants ƒ Tolerance ‐ biochemical responses that induce or
engineered to express the E. coli gene for NAD+‐ activate the antioxidant defence system and
dependent mannitol‐1‐phosphate dehydrogenase, possibly also various repair mechanisms
which converts fructose 6‐phosphate to mannitol‐
1‐phosphate, synthesised mannitol, although at 29. Tolerance to oxidative stress
low concentrations. In transgenic tobacco salt
tolerance was improved, and seeds able to
germinate in the presence of salt

20. D­Pinitol : cyclic sugar alcohol, is a major solute in

members of the Pine Family and Bean Family.
ƒ its concentrations are higher among halophytic
species and those adapted to drought.
ƒ in leaves, pinitol is localised to the chloroplast and
cystol but not in the vacuoles
30. Salicylic acid and ethylene: Ozone exposure results in
21. Osmotin is an abundant alkaline protein discovered in increased amounts of H2O2, which stimulate the production
cultured tobacco cells that had been acclimated to 428 mM of SA. This results in a transient increase in the number of
NaCl. Its molecular size of 26‐kDa transcripts that encode defence­related secondary
ƒ localised in the vacuole metabolites e.g. phytoalexins, cellular barrier molecules e.g.
ƒ classified as a pathogenesis‐related (PR) protein lignins, callose, and extensins, PR proteins e.g. (1→3) β‐
ƒ Transcription of an osmotin gene is induced by at glucanase, chitinase, gluthatione S‐transferase and
least 10 signals: phenylalanine ammonia lyase. Increases ethylene
ƒ ABA, ethylene, auxin, infection by TMV, salinity, production by inducing increases in ACC synthase and ACC
lack of water, cold, UV light, wounding, and fungal oxidase gene transcription
infection
ƒ Many of the stress‐induced genes are regulated by 31. Heat Stress: The typical response to heat stress is a
ABA decrease in the synthesis of normal proteins, accompanied
ƒ ABA plays a role in stomata closure and induction by an accelerated transcription and translation of new
of gene expression proteins known as heat shock proteins (HSPs)

22. Oxidative Stress: Oxidative stress results from 32. Heat shock may arise in leaves when transpiration is
conditions promoting the formation of active oxygen species insufficient or when stomata are partially or fully closed and
that damage or kill cells irradiance is high or may arise in germinating seedlings
when the soil is warmed by the sun and even may arise in
23. Environmental factors that cause oxidative stress are organs with reduced capacity for transpiration e.g. fruits
air pollution (increased amounts of ozone or sulfur dioxide),
oxidant forming herbicides e.g. paraquat dichloride, heavy 33. Resistance or sensitivity of plants to heat stress
metals, drought, heat and cold stress, wounding, UV light depends on Duration, Severity of the stress, susceptibility of
and intense light that stimulate photoinhibition different cell types and Stage of development

24. Reactive oxygen species (ROS): Formed during certain 34. Classes of Heat Shock Proteins (HSPs)
redox reactions and during incomplete reduction of oxygen
or oxidation of water by the mitochondrial or chloroplast
electron transfer chain. Examples: Singlet oxygen, hydrogen
peroxide, superoxide anion, hydroxyl and perhydroxyl
radicals

25. Ozone and oxidative stress: Hydrocarbons and oxides

of nitrogen (NO, NO2) and sulfur (SOx) react with solar UV
radiation to generate ozone (O3). Ozone is a highly reactive PLANT RESPONSE TO BIOTIC STRESS
oxidant
1. Pathogen attack strategies
26. The negative effects of ozone on plants ƒ necrotrophy, in which the plant cells are killed
ƒ decreased rates of photosynthesis ƒ biotrophy, in which the plant cells remain alive
ƒ leaf injury ƒ hemibiotrophy, in which the pathogen initially
ƒ reduced growth of shoots and roots keeps cells alive but kills them at later stages of
ƒ accelerated senescence infection
ƒ reduced crop yield
2. Cause of failure of a pathogen to cause disease
27. Ozone alters ion transport, increases membrane ƒ The plant species attacked is unable to support
permeability, inhibits H+‐pump activity, collapses membrane the life‐strategy of the particular pathogen
potential, increases Ca2+ uptake from the apoplasm and ƒ The plant possesses preformed structural barriers
Oxidative damage to biomolecules or toxic compounds
ƒ Defence mechanisms are activated such that the
28. Resistance to ozone: Utilizes either avoidance or invasion remains localized
tolerance

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ƒ Environmental conditions change and the ƒ Nitric Oxide has the capacity to potentiate
pathogen perish induction of plant cell death by ROS.
ƒ Nitric Oxide is known to bind heme and could
3. Successful pathogen infection and disease occurs: inhibit catalase and ascorbate peroxidase, which
ƒ Only if the environmental conditions are detoxifies H2O2
favourable ƒ In the presence of inhibitors of Nitric oxide
ƒ If the preformed plant disease defenses are production, the HR diminishes, disease symptoms
inadequate become more severe, and bacterial growth is
ƒ If the plant fail to detect the pathogen increased
ƒ If activated defense responses are ineffective ƒ Nitric oxide and Reactive Oxygen Speceis play an
important synergistic role in the rapid activation
4. Preformed defense is present in form of Secondary of a wide repertoire of defence responses after
metabolites pathogen attack
ƒ Plants possess different secondary metabolites
with antimicrobial properties 10. Benzoic acid and salicylic acid: Both SA and BA are
ƒ may be present in their biological active form or derived from the phenyl‐propanoid pathway and have many
may be store as inactive precursors that are roles in plant defence responses. Accumulate to high
converted to their active forms by host enzymes concentrations in the vicinity of incompatible infection sites
in response to pathogen attack or tissue damage
11. Jasmonic acid: Jasmonic acid (JA) is an oxylipin‐like
5. Secondary metabolites: pre‐formed inhibitors are the hormone derived from oxygenated linolenic acid.
saponins and the glucosinolates. ƒ Increases in JA in response to pathogen/insect
ƒ Saponins are glycosylated compounds, classified attack occur both locally and systematically
as either triterpinoids, steroids, or steroidal ƒ Spraying methyl‐JA onto plants increases their
glycoalkaloids. resistance to some (but not all) necrotrophic
ƒ A biologically active triterpinoid saponin found in fungi, but not to biotrophic fungi or bacteria
the roots of oat plants, avenacin A­1, is highly
effective against the root infecting fungus, a major 12. Ethylene: Ethylene is frequently synthesised during
pathogen of cereal roots. both incompatible and compatible interactions
ƒ This pathogen affects wheat and barley, but not ƒ Ethylene is required to mediate both resistance
oat plants due to avencin A1. against necrotrophic fungal pathogens and
against soil borne fungal species that are not
6. Hypersensitive response ordinarily plant pathogens
ƒ 1st line of activated defence, occurs within 24hr ƒ Ethylene and JA are required for activation of
ƒ Recognition of a genetically incompatible proteinase inhibitor (PI) genes and certain PR and
pathogen chitinase genes
ƒ Creates unfavorable conditions for pathogen
growth and reproduction 13. Pathogenesis­related (PR) proteins include fungal cell
ƒ Impair the spread of harmful enzymes and toxins wall‐degrading enzymes, chitinases, glucanases,
ƒ Leads to localised cell and tissue death lipoxygenase, anti‐microbial polypeptides and components
of signal transduction cascades.
7. Reactive oxygen species (ROS): the production of ROS ƒ Salicylic Acid‐mediated signal transduction
such as superoxide and hydrogen peroxide (H2O2) is often cascades regulate the transcriptional activation of
the first response detected, occurring within 5 min on many Pathogenesis Related genes
pathogen infection. The mechanism plants have for ƒ Ethylene and SA have been shown to act
producing superoxide from molecular oxygen probably synergistically, further enhancing the expression
involves a plasma membrane‐associated NADPH oxidase of PR genes

8. Role of ROS in plant defence 14. Plant defensins: Type of defence‐related genes with
ƒ H2O2 may be directly toxic to pathogens demonstrated antimicrobial activity codes small (<7 kDa)
ƒ may contribute to the structural reinforcement of cysteine‐rich peptides that accumulate in the periphery of
plant cell walls, either by cross‐linking various the plant plasma membrane. This is frequently found in dry
hydroxyproline and proline rich glycoprotein to plant seeds. Induction of the defensin PDF1­2 gene transcript
the polysaccharide matrix or by increasing the require ethylene or Me‐jasmonate
rate of lignin polymer formation by way of
peroxidase enzyme activity 15. Pytoalexins: Low‐molecular‐mass, lipophilic
ƒ make the plant cell wall more resistant to antimicrobial compounds that accumulate rapidly at sites of
microbial perpetration and enzymatic incompatible pathogen infection.
degradation ƒ Biosynthesis occurs only after primary metabolic
ƒ H2O2 induces benzoic acid 2 hydrolase (BA 2‐H) precursors are diverted into a novel secondary
enzyme activity, which is required for metabolic pathway e.g. phenylalanine is diverted
biosynthesis of Salicylic Acid. into the synthesis of various flavonoid
ƒ H2O2 is known to induce genes for proteins phytoalexins by the de novo synthesis of
involved in certain cell protection mechanisms phenylalanine ammonia lyase (PAL).
e.g. glutathione S‐transferase
16. Systemic plant defence responses
9. Nitric oxide synthesis (NO): In plants, rapid de novo ƒ Defence responses elaborated in tissues far from
synthesis of nitric oxide accompanies the recognition of the invasion site and even in neighbouring plants
avirulent pathogenic bacteria. ƒ The type of response is determined by the
attacking organism

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CSIR NET LIFESCIENES NOTES‐UNIT 6

ƒ Responses to fungi, bacteria, and viruses are ƒ SA concentrations increase and volatile methyl‐SA
distinct from the response to insect is released in distal plant tissue
ƒ Nematodes appear to induce a mixture of ƒ PR proteins in the non‐invaded parts of the
responses plants are synthesised resulting in reduction in
ƒ Root colonising non‐pathogenic bacteria induce disease symptoms after subsequent infection of
another type of response many pathogenic species

17. Systemic acquired resistance (SAR) : Fungi, bacteria, 18. Induced systemic resistance (ISR):
and viruses activate systemically a specific subset of PR‐type ƒ Non‐pathogenic root‐colonizing rhizobacteria
gene by a mechanism known as systemic acquired cause induced system resistance
resistance (SAR) in which local necrosis formation at the ƒ Rhizobacteria that promote specific plant growth,
initial site of pathogen invasion triggers both a local increase for example P. fluorescens, induce a systemic
in SA accumulation and the formation of a phloem‐mobile resistance response that does not depend on SA
signal or PR protein accumulation
ƒ For SAR to occur, the initial infection must result ƒ Instead, ISR requires both JA and ethylene
in formation of necrotic lesions, either as part of signalling and also the SAR regulatory protein
the HR or as symptom of disease NPRI

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