BBYCT-137

PLANT PHYSIOLOGY
Indira Gandhi AND METABOLISM
National Open University
School of Sciences

Block

4
NITROGEN METABOLISM AND PLANT
GROWTH REGULATORS
UNIT 12
Nitrogen Metabolism 113
UNIT 13
Biological Nitrogen Fixation 128
UNIT 14
Plant Growth Regulators 148
UNIT 15
Plant Response to Light and Temperature 180
UNIT 16
Plant Stress 213
Course Design Committee
Prof. G.C. Srivastava (Retd.) School of Sciences, IGNOU
Former Head,
Prof. M.S. Nathawat, Director, (Ex.)
Department of Physiology,
IARI, Pusa, Prof. Amrita Nigam
New Delhi-110012
Prof. Jaswant Sokhi (Retd.)
Prof. Vijay Paul Prof. Bano Saidullah (Retd.)
Principal Scientist,
Prof. Neera Kapoor
Division of Plant Physiology
IARI, Pusa, Dr. Eklavya Chauhan (Sr. Consultant)
New Delhi-110012

Block Preparation Team

Prof. Amrita Nigam Editor
SOS, IGNOU, New Delhi-110068
Prof. G.C. Srivastava (Retd.)
Dr. Eklavya Chauhan Former Head,
Sr. Consultant, Department of Physiology,
SOS, IGNOU, New Delhi-110068 IARI, Pusa,
New Delhi-110012

Course Coordinator: Prof. Amrita Nigam

Production
Mr. Hemant Kumar
SO(P), MPDD, IGNOU

Acknowledgement
• Dr. Kumkum Chaturvedi for giving useful inputs.

• Sh. Manoj Kumar, Assistant for word processing and CRC preparation.

April, 2021
Indira Gandhi National Open University, 2021
ISBN :
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any other means, without permission in writing from Indira Gandhi National Open University.
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the University’s office at Maidan Garhi, New Delhi-110 068 or IGNOU website
www.ignou.ac.in.
Printed and published on behalf of Indira Gandhi National Open University, New Delhi by the
Registrar, MPDD, IGNOU.
Printed at
 BLOCK 4 : NITROGEN METABOLISM
AND PLANT GROWTH REGULATORS
In the preceding Blocks 1-3 you have read about the plant water relations, occurrence,
uptake and transport of mineral nutrients; energy conservations by plants through the
fascinating process of photosynthesis; transformation of photoassimilates to various parts of
the plant body; biocatalysts, their structure, properties and mechanism of action; finer details
of cellular respiration involing glycolysis, Krebs Cycle, Oxidative phosphorylation, electron
transport chain and the modern aspects of ATP generation.

In the final fourth block you will study in Unit 12 the sources of nitrogen in the atmosphere
and soil along with the details of its absorption involving nitrification, along with emphasis on
the biochemistry of nitrate and ammonium assimilation. You will read more about the
biological nitrogen fixation carried out by the “gifted” species of nitrogen fixers in nature in
Unit 13. This unit describes the nodule-formation and the Rhizobium - root associations
along with a detailed description of the nitrogenase enzyme complex.

Unit 14 is devoted exclusively to a study of the chemical messengers of a plant – the

hormones. A detailed description of the structure and mode of action of hormones follows
their discovery. Synthetic growth regulations along with some recent discoveries in hormonal
signaling has also been discussed in details.

Plant responses to light and temperature is the theme of Unit 15 where you will study the
phenomenon of photoperiodism, its variations and the role of phytochrome in flowering. In
addition, recent information on the flowering hormones has also been discussed. The
process of vernalization has been described along with a discussion on cryptochromes and
the movements in plants.

The last of the units, Unit 16 deals with a detailed description of abiotic and biotic stress
conditions and the response of plants. Various ways in which the plants adapt to stress have
been described. Examples of specific stress conditions have been highlighted so as to
emphasize the changes in plant morphology and metabolism.

Objectives
After studying this block, you will be able to:

describe the steps involved in the assimilatory reduction of nitrates and the activity of
enzyme nitrate reductase and nitrite reductase;

evaluate the structural and functional aspects of the nitrogenase enzyme;

describe the process of biological nitrogen fixation with special emphasis on the
Rhizobium legume symbiosis;

discuss the history of naturally occurring hormones and their usefulness in agriculture
and horticulture;

appreciate the mode of action of phytohormones;

differentiate between the long-day and short-day plants;

describe the role of florigens and hormones in flowering;

distinguish between abiotic and biotic stress; and

appreciate plant response to osmotic, temperature, wounding and pollution stress. 111
112
Unit 12 Nitrogen Metabolism

UNIT 12
NITROGEN
METABOLISM

Structure
12.1 Introduction Glutamate synthase (GOGAT)
route
Objectives
Glutamate Dehydrogenase
12.2 Nitrate Assimilation
Pathway
Sources of Nitrogen
Uptake of Ammonia
Biochemistry of Nitrate
Ammonia Assimilation and its
Assimilation
Regulation
Regulation of Nitrate
Metabolic Interrelation of
Assimilation
Nitrogen, Carbon and Sulphur
Interaction of Nitrogen and
12.4 Summary
Carbon Assimilation
12.5 Terminal Questions
12.3 Ammonium Assimilation
and Its Regulation 12.6 Answers
Biochemistry of Ammonium
Assimilation

12.1 INTRODUCTION
You have studied about the role of nitrogen as a macronutrient in Unit 3.
Nitrogen is considered to be the fourth most abundant nutrient element after
carbon, hydrogen and oxygen in plants. This element occurs in both inorganic
and organic forms. Nitrogen is an essential constituent of amino acids,
proteins, enzymes, hormones, nucleic acids, alkaloids, chlorophyll, vitamins,
glycosides and other important primary and secondary plant constituents.
Needless to say protoplasm, the physical basis of life consists of a major
portion of proteins.

Although molecular nitrogen (N2 or dinitrogen) constitutes 78% by volume of

the atmosphere, this odorless, colorless gas cannot readily diffuse into the
plant and get utilized directly. This is partly due to the exceptionally stable
N ≡ N bond. No such enzyme is present in higher plants that can reduce this
triple covalent bond. Thus, these plants need to be supplied with nitrogen in a
usable or combined form for the biosynthesis of the above-mentioned
substances. 113
Block 4 Nitrogen Metabolism and Plant Growth Regulators

Objectives
After studying this unit, you should be able to:

describe the steps involved in assimilatory reduction of nitrate and the

requirements of the process;

explain the significance of organelle specific or tissue specific

distribution of nitrate reductase and nitrite reductase;

explain the significance of induction, repression and control of activity of

nitrate reductase;

list the requirements of assimilation of ammonia into organic form; and

discuss the metabolic interrelation of nitrogen, carbon and sulfur.

12.2 NITRATE ASSIMILATION

The higher green plants can utilize nitrogen from soil as nitrates (NO3-), nitrites
(NO2-), ammonium salts (NH4+) and organic nitrogenous compounds.

The higher plants depend on prokaryotic organisms in soil for conversion of

dinitrogen into this usable forms. Only certain microbes are ‘gifted’ to convert
the atmospheric nitrogen into utilizable form. You will read about the biological
nitrogen fixation in the next Unit 13.

Atmospheric nitrogen fixation by some microbes (discussed in Unit 13)

accounts for nearly half of nitrate assimilation by green plants, which is
estimated to be about 2 × 104 Mt/year.

Interestingly, nitrogen is often a limiting nutrient for plants, placed after water,
since the plants and soil microorganisms compete with each other for this
element which is limited in the soil pool.

12.2.1 Sources of Nitrogen

Atmosphere and soil are the main sources of nitrogen. The molecular form of
nitrogen (dinitrogen) is prevalent in the atmosphere while both inorganic and
organic forms of nitrogen are present in soil. Inorganic forms of nitrogen in the
soil are, nitrite nitrogen, nitrate nitrogen and ammonical nitrogen. The
organic forms of nitrogen in soil are in the form of amides, amino acids and
urea.

Remains of dead animals and plants, excretions and nitrogenous wastes in

soil are first acted upon by saprophytic microorganismswhich hydrolyze the
proteins into amino acids. The later are then deaminated to release ammonia.
This process is called ammonification. The liberated ammonia immediately
combines with H+ of water to form NH 4 + ion.

Proteins + Water Bacillus

 sp, Actinomyce s sp.
   → n RH. NH2. COOH
hydrolysis amino acids
114
Unit 12 Nitrogen Metabolism
RH. NH2. COOH + Water deaminatio
 n→ R HOH. COOH + NH3
Amino acid Organic acid
+ +
NH3 + H → NH 4
Ammonium ion

Some of these ammonium ions can be absorbed by roots of some plants.

Remaining ammonium ions get converted into nitrate nitrogen by the microbial
activity. The process is called nitrification which occurs in two steps.

i) Nitrite Formation : The nitrite microorganisms like Nitrosomonas,

Nitrosococcus and Aspergillus oxidize ammonical nitrogen into nitrites.
nitrite microbes
NH 4 + + 2O2    → NO 2 − + 2H2O + Energy

The energy is utilized by chemosynthetic bacteria for their carbon

assimilation.

ii) Nitrate Formation : Both biological as well as non-biological oxidation of

nitrite is possible. Biological nitrate formation is carried out by Aspergillus,
Nitrobacter,Nitrocystis and Penicillium.

Energy released in the process is used for carbon assimilation by the

chemosynthetic nitrifying bacteria.
− nitrate microbes
2 NO 2
+ O2    → 2 NO 3 − + Energy

The nitrates are now available to plants for absorption and assimilation.

Most of the plants absorb nitrogen in the form of nitrates. The mechanism of
absorption of nitrate ions is essentially similar to that of other ions. A small
amount of nitrites can also be absorbed. Interestingly, plants complete with the
denitrifiers in soil like Thiobacillus denitrificans which readily reduce nitrate into
dinitrogen, returning it into the atmosphere. Denitrification itself accounts for a
loss of 93-190 million metric tonnes of nitrogen per year! Thus, in order to
maintain a steady state of the supply, demand, turnover, recycling and growth
of plants, there must be an element of a “nitrogen economy” in the biosphere
which can balance the nitrogen recycling. A relationship between inorganic
and organic nitrogen metabolism is illustrated in Fig. 12.1.

Fig. 12.1: Diagram depicting relationships between inorganic and organic

nitrogen metabolism (From Appling et al). 115
Block 4 Nitrogen Metabolism and Plant Growth Regulators
12.2.2 Biochemistry of Nitrate Assimilation
As you have studied in subsection 12.2.1, nitrate is the most readily available
and preferred source of nitrogen for plant growth. Once inside the roots and
after being transported to leaves through the transpiration stream, the nitrates
are converted into ammonia. This assimilatory reduction of NO3-to NH3 occurs
in two steps as shown below :

i) NO3- + 2H+ + 2e- → NO2- + H2O

Nitrate Nitrite

ii) NO2- + 7H+ + 6e- → NH3- + H2O

Nitrite Ammonia

Recent evidence suggests that this conversion may involve many more steps
as indicated below:

NO3- → NO2- → HNO- → NH2OH → NH3

Nitrate Nitrite Nitroxyl Hydroxylamine Ammonia

The first step is catalyzed by nitrate reductase (NR) in the cytosol which
reduces NO 3- to NO -2 at the expense of two electrons. The second step is
catalysed by nitrite reductase which converts NO -2 into NH3 at the expense of
six electrons. Nitrate reductase is a Mo-enzyme like dinitrogenase and nitrite
reductase is a Fe-protein.

The physiological source of reductant for the two reductive processes could be
reduced ferredoxin –Fd(red) or reduced pyridine nucleotides (NADH or NADPH)
depending upon the system. It is important to point out here that NO 3- is also
known to undergo dissimilation process in which it is reduced to N2 gas. Such
a process of nitrate metabolism is called nitrate respiration or denitrification
and occurs exclusively in certain bacterial forms under anaerobic conditions.
The enzymes of denitrification of nitrate are called dissimilatory nitrate
reductases.

Assimilatory Nitrate Reductase and Nitrite Reductase

There are two kinds of nitrate reductases depending upon their specificity to
reductant. Nitrate reductase of cyanobacterial system requires reduced
ferredoxin (Fd-dependent) to catalyse the reaction while nitrate reductase in
plants and fungi requires reduced pyridine nucleotide (NADH or NADPH –
dependent) to carry out the reaction.

In general, most pyridine nucleotide nitrate reductases are capable of using

both NADH and NADPH as source of reductant. The enzyme from
photosynthetic organisms like higher plants and algae show preference for
NADH but those from fungi show preference for NADPH. This suggests
inherent difference between cyanobacterial nitrate reductase and eukaryotic
nitrate reductase in respect to reductant requirement. The two types of
116 reductant dependent nitrate reduction reactions are show below (Fig. 12.2).
Unit 12 Nitrogen Metabolism

Fig. 12.2: Two major types of reductant dependent nitrate reduction reactions in
eukaryotes and cyanobacteria.

The overall reaction of eukaryotic nitrate reductase is summarized as:

NO 3− + NAD (P )H + H + NO −2 + NAD (P ) + + H 2 O

In comparison to the Fd-dependent enzyme which contains only molybdenum

as prosthetic group, the eukaryotic nitrate reductase is made up of two
identical sub-units (dimer), each containing one molecule of FAD heme, and a
Mo atom as prosthetic group complexed with an organic molecule called
Pterin. Functionally, while the Fd-dependent enzyme catalyses only reduction
of nitrate to nitrite, the NAD (P)H-dependent enzyme catalyses two
independent activities, one called diaphorase activity in which NAD(P)H is
oxidized and one electron acceptor like cytochrome C or flavin mono-
nucleotide (FMN) is reduced, and the other called terminal nitrate reductase
activity which is NAD(P)H independent and in which nitrate is reduced at the
expense of reduced flavins (FMNH2) or viologens. In vivo both activities
participate jointly and sequentially in the transfer of electrons from reduced
pyridine nucleotide to nitrate as shown in reaction (Fig. 12.3). Nitrate
reductase in cyanobacteria lacks diaphorase activity and contains only Mo as
prosthetic group.

+
Fig. 12.3: Activities of NAD (P)-dependent nitrate reductase i) diaphorase
activity; ii) terminal nitrate reductase activity. 117
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Reduction of nitrite to ammonia occurs in chloroplasts. The enzyme nitrite
reductase in cyanobacteria or chloroplasts requires reduced ferredoxin for
reduction. Nitrite reductase of fungi requires NADPH to carry out the reductive
function. The two reactions are shown below (Fig. 12.4).

Fig. 12.4: Nirtite reductase activity in the chloroplast and in fungi.

Since NO −2 is highly reactive and does not accumulate in plant tissue, it is
immediately transported from the cytosol to the chloroplast in leaves and
plastids in roots for reduction. Nitrite is reduced to ammonia by the enzyme
nitrite reductase. This involves the transfer of six electrons:

NO 2− + 6Fd red + 8H + NH 4 + + 6Fd ox + 2H 2 O

Ferredoxins involved in this reaction are obtained from the electron transport
system of chloroplasts (see Unit 5).

Nitrate Uptake
Cells accumulate NO 3− against concentration gradient and this accumulation is
a result of the presence of active NO 3− transport system in the cell membrane.
Nitrate uptake and nitrate reduction are independent processes because
organisms genetically deficient in nitrate reductase activity contain normal
transport activity.

12.2.3 Regulation of Nitrate Assimilation

There are two levels of regulation of nitrate assimilation. One is long-term and
another is short-term. The long-term regulation operates at the level of
enzyme synthesis (transcription level) and the short-term regulation operates
at the level of enzyme activity (translation level).

Enzyme Synthesis

Nitrate assimilating systems in general are known to show an increase in

nitrate uptake system and nitrate reductase in the presence of nitrate. In other
words, nitrate assimilatory system is induced by the presence of nitrate.
Similarly, cells or organisms assimilating NH3 as nitrogen source show lack of
nitrate assimilatory system. Such control of nitrate is called repression
control. Thus, nitrate is an inducer while ammonia is repressor of nitrate
assimilatory system.
118
Unit 12 Nitrogen Metabolism
Red light is known to enhance synthesis of nitrate assimilatory system which
is mediated by well-known photomorphogenic pigment called phytochrome
about which you will learn in detail in Unit 15 of this course.

Enzyme-activity Control
Availability of the substrate, NADH and nitrate would be an important
determinant of the rate of nitrate assimilation. In addition, there are number of
substances which are known to cause reversible inactivation of nitrate
reductase under reducing conditions (Fig. 12.5).

Fig. 12.5: Regulation of assimilatory nitrate reductase (NR). Reversible

inactivation of NR by combination with hydroxylamine, cyanide, or
superoxide and reversal of this inactivation by blue light in the
presence of flavin; inactivation by combination with specific binding
proteins or by limited proteolysis. Enzyme synthesis is regulated by
positive effector-nitrate, plastidic factor, light and negative effectors
derived from ammonia.

Cyanide: Plants generate cyanide from cyanogenic glycides and histidines.

Ethylene biosynthesis is also accompanied by small amount of cyanide
production. Nitrate reductase can occur in reduced form following its
interaction with NADH in the absence of nitrate. The reduced form of the
enzyme has the ability to combine with cyanide forming enzyme-CN complex
which is enzymatically inactive. Nitrate, oxygen or blue light oxidise the
enzyme-CN complex releasing cyanide and making cyanide - free enzyme
active.

Hydroxyl amine or superoxide inactivates the enzyme which on exposure to

blue light gets converted to active form. It is observed that plants grown in blue
light are higher in protein contents.

Inhibitor proteins: One kind of inhibitor protein found in higher plants is an

endopeptidase which degrades nitrate reductase thus causing irreversible loss
of the enzyme. The other kind includes binding proteins that specifically bind
to nitrate reductase leading to permanent inactivation of enzyme. Such
inactivator proteins have been isolated from rice seedings and spinach leaves.
These reactions are explained in Fig.12.6. You will see that the transcription of
enzyme is mediated by light. It is a unique enzyme in this respect. On the
other hand, darkness and Mg2+ stimulate a protein kinase that phosphorylates 119
Block 4 Nitrogen Metabolism and Plant Growth Regulators
serine residues which interact with an inhibitor to inactivate nitrite reductase.
This type of regulation is shown to be a much quicker one.

12.2.4 Interaction of Nitrogen and Carbon Assimilation

Application of nitrogenous fertilizers brings about dramatic effects on
thegrowth and performance of the plant. One of the most important
consequences is the effect on utilisation of carbohydrates in plants. It is known
that the level of carbohydrates in the plants goes down with increase in the
level of N2 supplied to them.The form in which the fertilizers are supplied are
NO 3− , and NH3.In the plant NO 3− is reduced to NH3 and assimilation of NH3
requires carbon-skeleton like α-ketoglutaric acid, an intermediate of TCA cycle
for its incorporation into organic form. Naturally, the rate of NH3 assimilation
into organic form would depend on the rate at which TCA cycle supplies the
carbon skeleton. The C-skeleton removed during the assimilation of NH3 must
be replenished through the catabolism of carbohydrates. Consequently, the
carbohydrate content of the plant decreases in proportion to the amount of
organic N2 produced.

The other places of nitrate assimilation in the plant as you have already seen
in photosynthetic tissue and the reductants are reduced ferredoxin and
pyridine nucleotide. Since such reductants are also required for carbon
assimilation, theproduction of carbohydrates in photosynthesis is bound to go
down in proportion tothe increase in rate of reductive assimilation of NO 3− . In
other words; the level of NO 3− utilisation is inversely related to the level of
carbohydrate in the plant.

We will describe how the practical applications of this knowledge is used in

raising crops with desired protein and carbohydrate content. Take the example
of celery eaten as salad; the stalks of this plant are most edible when soft and
this softness is the result of lower availability of carbohydrates. Now, to raise a
celery crop with soft stalks one uses nitrate fertilizers which divert a major part
of carbohydrate in the synthesis of amino acids and proteins. Another example
is sugarcane, a crop of tropical region. It requires a period of ten months from
the time of planting to the period of harvest. Here, the growers apply nitrogen
fertilizers in the beginning and not near the time of harvest.Similar practices
have resulted in production of beet roots highly rich in sugar concentration.
The reason for rise in sugar in the beet root is again due to withholding of N2-
fertilizer near the harvest time, so that the C-skeleton is not utilized for
producing amino acids and proteins.

SAQ 1
a) Match the characteristics listed below of Column I with enzymes given in
Column II.
Column I Column II
i) Nitrate reductase a) Non-heme Fe-protein
ii) Nitrite reductase b) Present in chloroplast
c) Present in cytoplasm
d) Molybdo-flavo protein
e) Diaphorase activity
120
Unit 12 Nitrogen Metabolism

b) Choose the alternate correct word given in parenthesis in the following

statements:

i) Nitrate assimilation in higher plants occurs mainly in (roots/leaves).

ii) Number of electrons used when NO 3− is reduced to NO 2− (1/2).

iii) Number of electrons used when NO 3− is reduced to ammonia (2/6).

iv) In cyanobacteria nitrate reductase is located in the (photosynthetic

membranes/plasma membrane).

v) In higher plants nitrate reductase is in (cytoplasm/chloroplast) and

nitrite reductase is in (cytoplasm/chloroplast).

vi) In C4 plants nitrate assimilation occurs in (bundle

sheath/mesophyll cells) and CO2 assimilation occurs in (bundle
sheath/mesophyll cells).

vii) Nitrate reductase activity is induced in the presence of

(NO 3− / NH +4 ).

12.3 AMMONIUM ASSIMILATION AND ITS

REGULATION
Atmospheric nitrogen (N2) and NO3 are the most common available source of
inorganic nitrogen. Both are enzymatically reduced to ammonia because it is
only ammonia that is incorporated into organic form. The major product of
ammonia assimilation is usually considered to be amino nitrogen. Ammonium
generated during nitrite assimilation (as discussed in Unit 12.2.2) and during
photorespiration (Unit 7) has to be rapidly converted as its accumulation is
toxic to a plant. Molecular nitrogen fixation has been discussed in Unit 13.2.4.

12.3.1 Biochemistry of Ammonium Assimilation

Ammonia resulting from N2- fixation or nitrate reduction or supplied
exogenously is assimilated in plants by the following two primary pathways, i)
Reductive amination/glutamate dehydrogenase (GDH) pathway, and ii)
glutamine synthetase(GS)-glutamate synthase also called (Glutamine
oxoglutarate aminotransferase-GOGAT) pathway.

i) Reductive Amination (High cellular ammonia Pathway):

ீ஽ு
NHଷ + α − Ketoglutarate + NADPH + H ା ሱۛۛۛۛۛሮ Glutamate + NADP ା + Hଶ O

ii) GS-GOGAT (Low cellular ammonia Pathway): (NADH-GOGAT occurs

in diatoms, fungi and in the nucleus of vascular plants). Fd-GOGAT is
prevalent in photosynthetic eukaryotes and cyanobacteria. NADPH-
GOGAT occurs in mitochondria, chloroplasts, archaea and non-
photosynthetic bacteria. 121
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Glutamate + NH3+ ATP Glutamate
  Synthetase

→ Glutamine + ADP + Pi
Glutamate
Glutamine + α − Ketoglutarate + NADPH + H +   →
synthase
(orFd(red ) )

2Glutamate + NADP + + H2 O
(or Fd( ox ) )

As you may note, both the pathways generate the same amino acid,
glutamate as end product of the reaction. The two pathways may operate
simultaneously as in higher plants and eukaryotic algae or alternatively as in
certain heterotrophic enterobacteria.In cyanobacteria glutamine synthetase-
glutamate synthetase (GS) is the main primary pathway of ammonia
assimilation.

The two enzymes involved in ammonia assimilation differ significantly in their

affinity for ammonia which is very high for glutamine synthetase and
significantly low forglutamate dehydrogenase. In other words, glutamate
dehydrogenase - mediated pathway functions under conditions of high cellular
ammonia and glutamine synthetase-mediated pathway under conditions of low
cellular ammonia.

The GS-glutamate synthase pathway is characteristically cyclic in nature in

which glutamate acts as both the acceptor and product of ammonia
assimilation. This pathway of ammonia assimilation is called glutamate
synthase cycle (Fig. 12.6).

12.3.2 Glutamate Synthase (GOGAT) Route

Fig.12.6 : Glutamine Synthase (GS) Route.

Two classes of GS exist in plants:

1. Cytosolic GS expresses itself in vascular bundles of roots and shoots to

produce glutamine for intracellular nitrogen transport.

2. GS present in root plastids or shoot chloroplasts generate amides and

catalyze reassimilation of photorespiratory NH4+ respectively.

When there is an enhanced concentration of glutamine in the plastids, the

enzyme glutamate synthase (also called glutamine: 2-oxoglutarate
aminotransferase, or GOGAT) getsactivated. The amide group of glutamine is
transferred to 2-oxoglutarate, finally yielding two molecules of glutamate
(Fig. 12.6).

There are two types of GOGAT. One accepts electrons from NADH and the
122 other (Fd).
Unit 12 Nitrogen Metabolism
i) Glutamine + 2 − oxoglutarate + NADH + H ା → 2 glutamate + 2NADା

ii) Glutamine + 2 − oxoglutarate + Fd୰ୣୢ → 2 glutamate + Fd୭୶

The NADH type of enzyme (NADH-GOGAT) is

• Located in plastids of non-photosynthetic tissues e.g., roots and vascular

bundles of developing leaves.

• NADH-GOGAT helps in the assimilation of NH4+ absorbed from the

rhizosphere and also assimilates glutamine which has been translocated
from the senescing leaves or roots.

The Fd-dependent type (Fd-GOGAT) is

• Located in the chloroplasts and help in photorespiratory nitrogen

metabolism.

• Helps to incorporate glutamine generated during nitrate assimilation.

12.3.3 Glutamate Dehydrogenase Pathway

An alternate pathway involving Glutamate dehydrogenase(GDH) can also
assimilate ammonium ions in rice and under anaerobic conditions (Fig. 12.7),
particularly when ammonia concentration is high. This NADH-dependent
enzyme is present in mitochondria whereas its NADPH-dependent enzyme is
present in the chloroplasts of photosynthetic parts. This mechanism of
ammonia assimilation is less expensive than the GS-GOGAT Pathway.

Fig. 12.7 : Glutamate Dehydrogenase Route.

Nutrient assimilation consumes a considerable amount of energy. The

reduction of nitrate to nitrite and subsequently to ammonium involves the
transfer of about 10 electrons. This amounts to nearly 25% of the total energy
expenditure of both roots and shoots. Interestingly, nitrogen accounts for only
2% of the total dry weight of the plant, yet requires nearly one fourth of energy
for its assimilation.

As most of these assimilation reactions occur in the chloroplast stroma, and

utilize the reducing agents of the photosynthetic electron transport chain, the
coupling of the two processes viz., nutrient assimilation and photosynthetic
electron transport chain, is called Photoassimilation. The processes involved
in the assimilation of mineral nitrogen and the energy expenditure is depicted
in Fig. 12.8. 123
Block 4 Nitrogen Metabolism and Plant Growth Regulators

Fig. 12.8: A generalized diagram depicting the processes involved in the

assimilation of mineral nitrogen in the leaf (From Taiz & Zeiger).

Since enhanced CO2 levels inhibit nitrate inhibition in both C3 and bundle
sheath of C4 plants, this phenomenon is bound to affect the plant nutrient
reactions significantly by the end of this century, when the atmospheric CO2
levels are expected to rise further.

12.3.4 Uptake of Ammonia

Ammonia (NH3) diffuses freely across biological membranes according to its
concentration gradient. However, ammonium (NH +4 ) ion requires a specific
transport system to cross the biological membrane.

12.3.5 Ammonia Assimilation and its Regulation

Heterotrophic bacteria like Escherichia coli and Klebsiella aerogenes induce
theoperation of GS-glutamate synthase pathway of ammonia assimilation
underconditions of low ammonia and of GDH-pathway under conditions of
high ammonia.Thus the two pathways are mutually exclusive and the level of
cellular ammonia determines which pathway of ammonia assimilation is likely
to operate.Cyanobacteria like systems have only GS-glutamate synthase
pathway functioning under low or high cellular ammonia. Information about the
role of ammonia in metabolic regulation of its two assimilatory pathways in
higher plants is not clearly understood.

12.3.6 Metabolic Interrelation of Nitrogen, Carbon and

Sulphur
Plants grow and develop into characteristic individuals as a result of a series
of well programmed and regulated biochemical and morphological events. The
main elements that enter into the composition of an individual plant are
carbon, hydrogen, oxygen, nitrogen and sulphur. The three elements are
taken by plants in the oxidized form CO2, NO 3− , SO 24 − , and thus need to be
reduced for assimilation. Assimilation of nitrate and sulphate, like carbon
dioxide, requires ATP and reductants. It seems that plants must have evolved
mechanisms to integrate the three assimilatory reductions with light harvesting
photosynthetic reactions. Eukaryotic algae and higher plants have achieved
this integration by localizing most of these assimilatory reactions within the
chloroplast.

Photosynthetically produced ATP and reductant (ferredoxin or NADPH)

together constitute what is called assimilatory power of the plants. Light
reactions of photosynthesis produce ATP and reductants and dark reactions of
photosynthesis are related to the reductive assimilation of carbon dioxide,
nitrate and sulphate.
124
Unit 12 Nitrogen Metabolism
In plants, since carbon, nitrogen and sulphur also occur together in sulphur
containing amino acids there must be regulatory mechanisms controlling
integrated assimilation of carbon dioxide, nitrate and sulphate into organic
forms. The results of scientific studies also support this view. Inhibition of
photosynthetic carbon dioxide assimilation is known to simultaneously inhibit
nitrate assimilation. This is partly because the carbon skeleton for compounds
like α-ketoglutarate is ultimately produced from photosynthetically generated
organic carbon from carbon dioxide. Assimilation of N2 by nodulated legumes
also depends upon the extent and amount of photosynthate like organic
carbon reaching them. There is evidence which suggests that sulphur also
controls nitrogen metabolism. Protein synthesis is known to decline under
sulphate limitation and comes to a halt after sulphate exhaustion. During
sulphur starvation, nitrate or ammonia is assimilated mainly into free amino
acids and not in proteins. Because, in the absence of sulphur, the sulphur
containing amino acids (cysteine and methionine) cannot be synthesized and
the disulphide bridges cannot be formed in proteins. Some of the amino acids
preferentially synthesized under these conditions are arginine, glutamine and
alanine.

The major regulatory mechanisms controlling interdependence of inorganic

carbon, nitrogen and sulphur metabolism are to be studied and understood in
detail as they hold the key to the desired production of major plant products at
commercial scale.

SAQ 2
a) i) Name the two pathways of ammonia assimilation in plants.

ii) Which of the two pathways operates under high concentration of

cellular ammonia?

iii) Which of the two pathways is costly in respect of energy and

material needs?
b) i) Which of the source among NH 4+ , NO 3− and N 2 is preferred by
plants?
ii) What signal in plant causes repression of both NO 3− assimilation
and N2-fixation?

12.4 SUMMARY
• Nitrate assimilation occurs in two enzymatic steps. Nitrate reduction to
nitrite is catalysed by nitrate reductase and nitrite reduction to NH3 is
catalysed by nitrite reductase. NADH is the reductant for nitrate
reductase in plants, NADPH is the reductant in fungi and reduced
ferredoxin in cyanobacteria. Reductant for nitrite reductase in fungi is
NADPH and in plants and cyanobacteria is reduced ferredoxin.

• Nitrate reductase is predominantly localized in cytoplasm while nitrite

reductase is localized exclusively in chloroplast. 125
Block 4 Nitrogen Metabolism and Plant Growth Regulators
• Nitrate assimilation is regulated by inducer and repressor control at
synthetic level and activity control involving reversible inhibition of
enzyme activity by cyanide, hydroxyl amine, superoxide radical and
reactivation by blue light.

• In C4 plants nitrate assimilatory pathway remains localized within

mesophyll cell and CO2 assimilation in bundle sheath cells.

• Plants prefer ammonia as a source of nitrogen over NO 3− or N2 and this

is achieved by repression control of nitrate assimilation and N2 fixation.

• Glutamate dehydrogenase and glutamine synthatase/synthase are the

two primary enzymes of ammonia assimilation.

• Sulphate like NO 3− is also assimilated reductively. Unlike NO 3− , it is

required to undergo ATP dependent activation before entering into
pathway of assimilatory reduction.

• A clear understanding of metabolic interrelationship of N,C and S

nutrition are a must from agricultural point of view.

12.5 TERMINAL QUESTIONS

1. Trace the steps of nitrification.

2. Distinguish between the eukaryotic and bacterial nitrate reductase

enzyme.

3. What are the three basic modes of NO 3− reduction?

4. Where are the nitrate reductase and nitrite reductase enzymes present
in higher plants (C3)?

5. Explain the control of nitrate reductase by various regulatory factors.

6. Explain the two major pathways for ammonium assimilation.

7. How expensive is the nitrogen uptake?

8. How are the sulphates, phosphates and cations assimilated?

12.6 ANSWERS
Self-Assessment Questions
1. a) i) c, d and e;

ii) a and b

b) i) leaves

ii) 2

iii) 6

iv) photosynthetic membranes

126
Unit 12 Nitrogen Metabolism
v) cytoplasm, chloroplast

vi) mesophyll cells, bundle sheath cells

vii) NO 3−

2. a) i) Glutamate dehydrogenase pathway and glutamine

synthetase-glutamate synthetase pathway;

ii) Glutamate dehydrogenase pathway

iii) GS-glutamate synthase.

b) i) NH4+;

ii) high ratio of glutamine to α-ketoglutarate

Terminal Questions
1. Refer to Subsection 12.2.1.

2. Refer to Subsection 12.2.2.

3. Refer to Subsection 12.2.3.

4. Cytoplasm and chloroplast respectively.

5. Refer to Subsection 12.2.4.

6. Refer to Subsection 12.3.1.

7. Refer to Subsection 12.3.3.

8. Refer to Subsection 12.3.7.

127
Block 4 Nitrogen Metabolism and Plant Growth Regulators

UNIT 13
BIOLOGICAL NITROGEN
FIXATION

Structure
13.1 Introduction Nitrogenase Enzyme Complex
Objectives Factors Influencing Function of
Nitrogenase
13.2 Biological Nitrogen Fixation
Measurement of Nitrogenase
Nitrogen Fixers in Nature – the
Activity
‘Gifted’ Species
13.5 Genetic Control of Nitrogen
Conditions for Nitrogen
Fixation
Fixation
13.6 Summary
13.3 Symbiotic Nitrogen Fixation
– Nodule Formation- 13.7 Terminal Questions
leghemoglobin
13.8 Answers
13.4 Biochemistry of Nitrogen
Fixation

13.1 INTRODUCTION
You have learnt in the preceding Unit 12 how nitrogen gets metabolized in the
plant. No doubt, all plants have the capability of converting NH3 to organic
nitrogen compounds –(with C-N) bonds). But can all organisms synthesize
ammonia from inorganic nitrogen – dinitrogen gas N2, which is the most
abundant component of earth’s atmosphere? Although many plants and
−
microorganisms do have the ability to reduce NO3 to NH3, there is no plant
known so far which can reduce N2 to NH3. This ability is confined only to some
prokaryotes, and these nitrogen-fixing microorganisms are termed
diazotrophs. These nitrogen-fixers or the ‘gifted’ prokaryotes, are both free-
living as well as in symbiotic association with plants.

Objectives
bjectives
After studying this unit, you should be able to:

list and classify the nitrogen fixing organisms based on mode of living;

write the conditions necessary for nitrogen fixation and the essential
128 requirements of the process;
Unit 13 Biological Nitrogen Fixation

describe the metabolic modifications occurring during bacteroid
formation in nodules;

explain the basis of specificity of Rhizobium-legume symbiosis; and

list the components of nitrogenase enzyme, describe the reactions

carried out by each component, and explain the significance of H2
evolution during the process.

13.2 BIOLOGICAL NITROGEN-FIXATION

The process by which molecular nitrogen (N2) is reduced to ammonia (NH3) is
called nitrogen-fixation (N2-fixation).

This is the most important section of this unit. Here we first introduce you to
the species capable of N2-fixation and describe the formation of nodules
during symbiotic association. Then we go through the reactions and enzymes
involved in the conversion of molecular nitrogen to NH3. Brief section on the
genetics of N2-fixation will introduce you to the possibility of transferring N2-
fixing capability to non N2-fixers.

13.2.1 Nitrogen Fixers in Nature - The ‘Gifted’ Species

Biological nitrogen fixation remains mainly confined to a few distinct nutritional
types of prokaryotes, some of which are free-living while others live in
symbiotic association with eukaryotic partners. This process contributes
annually about 60 %( nearly 150-190 million metric tons annually) to the
earth's newly fixed nitrogen budget. Tables 13.1 and 13.2 show the ranges
and types of biological nitrogen fixers. They have been variously classified as
free living (Azotobacter, Nostoc, Anabaena (Fig. 13.1a) or symbiotic
(Rhizobium-legume association Fig. 13.1 b).

Free-living nitrogen fixers can further be classified into phototrophic (Nostoc),

chemotrophic (Klebsiella), aerobic (Azotobacter) or anaerobic (Chromatium)
depending upon the state of existence and mode of nutrition. Agronomically
the most important nitrogen fixing systems are Rhizobium-legume association
(Fig 13. 1 b), Frankia (actinorhizal)-woody plant association, Anabaena-Azolla
and Cyanobacteria-rice system. Rhizobium-legume association invariably
occurs in the form of root nodules. This group of nitrogen fixers has been
divided into three categories: i) Rhizobium which includes fast growing
species, ii) Bradyrhizobium which include slow growing strains and iii)
Azorhizobium which is a combination of traits from Rhizobium and
Bradyrhizobium.

Rhizobium is highly specific with respect to its host and that is why the
nomenclature is based on the specific host which it infects. For example, the
Rhizobium infecting clovers and peas are called trifolii and leguminosarum,
respectively. The genus Bradyrhizobium is not so rigid regarding the host
partners. All the strains of Rhizobium and Bradyrhizobium produce nodules on
the host, but Azorhizobium produces nodules on the stem of Sesbania rostrata
and Aeschynomene afraspera. Parasponia is the only non-legume genus
where the bacterial partner is Rhizobium. This observation has raised the 129
Block 4 Nitrogen Metabolism and Plant Growth Regulators
hope of creating nitrogen fixing associations between Rhizobium and
important crops like cereals. Nitrogen fixing nodules on the roots of woody
plants like Alnus, Casuarina, Myricaare formed by symbiotic association with
Frankia, a member of actinomycetes.Anabaena-Azolla association is
experimentally shown to be a strong source of nitrogen fertilizer in wetland rice
agriculture. Free-living heterocyst- possessing cyanobacteria are also known
to contribute significant amount of nitrogen to the wetland rice ecosystem.
Lichens are associations between fungi and N2-fixing cyanobacteria. They are
very important source of N2 in barren harsh habitats. N2-fixing root nodules are
also known to occur in tropical plants like Psychotria with Klebsiella as the N2-
fixing partner. Several nitrogen-fixing bacteria get associated with C4 plants
like grasses Miscanthus and Sugarcane.

(a) (b)
Fig. 13.1: The gifted N2-fixers (photographs). a) Anabaena (arrow-heterocyst);
b) Symbiotic nitrogen fixation by legume-Rhizobium association(Root
nodules on Soybean)(from Taiz et al )

Table 13.1: Some free-living diazotrophs

Archaebacteria Methanococcus voltae

Halobacterium halobium
Eubacteria
Cyanobacteria
Unicellular Gloeothece
Filamentous forms Oscillatoria, Plectonema
Heterocystous forms Nostoc, Anabaena, Calothrix

Other bacteria

Aerobes Azotobacter, Derxia, Beijerinckia,

Azospirillum lipoferum*

Facultative anaerobes Klebsiella pneumoniae,

Enterobacter, Bacillus

Microaerobes Aquaspirillum, Arthrobacter

Anaerobic Clostridium

Photosynthetic Rhodospirillum rubrum,

Rhodopseudomonas, Chromatium

Chemotrophic Thiobacillus ferroxidans

130
Unit 13 Biological Nitrogen Fixation
Cyanobacterial Associations Co symbionts
Gunnera (Angiosperm)

Cyanobacteria Agathis, Cycas, Microzamia

(Gymnosperms)
Azolla (Pteridophyte)

Blasia, Anthoceros, Caricula

(Bryophytes)
Collema, Lichina, Peltigera (Lichens)

Cyanobacterial Animal Association Siphonochalina (Coral reef sponge)

*SomeAzospirillum spp are often found in association with roots of grasses,

and fix nitrogen microaerobically.

Table 13.2: Some Symbiotic Diazotrophs

a) Legumes N2-fixing Symbiont

Bradyrhizobium japonicum Soybean (Glycine max)

(slow growing)
Peanut (Arachis hypogaea)

Sinorhizobium fredii (Fast growing) Soybean

Sinorhizobium meliloti Alfalfa (Medicago sativa)

Sweet clover (Melilotus)

Rhizobium leguminosarum bv. phaseoli Bean (Phaseolus)

Rhizobium tropicii; Rhizobium etli

Rhizobium leguminosarum bv viciae Pea (Pisum sativum), Sweet pea

(Lathyrus)

Photorhizobium (Photosynthetically Aescheomene (aquatic)

active rhizobia form stem nodules.
Associated with adventitious roots)

b) Non-legumes Rhizobia

Bradyrhizobium spp. Parasponia (Ulmaceae)

Rhizobium loti Lotus (Nelumbo sp.) (Nelumbonaceae)

c) Non-legume Shepherdia (Eleagnaceae),Hippophae

(Elaeagnaceae)
(Actinorhizal Association)
Alnus (Betulaceae),Casuarina
Frankia (Actinomycetales)
(Casuarinaceae), Elaeagnus
(Elaeagnaceae), Myrica (Myricaceae),
Ceanothus (Rhamnaceae), Datisca
(Datiscaceae), Coriarea (Coriariaceae).

d) Endophytic bacteria

Acetobacter diazotrophicus Sugarcane

Azoarcus spp. Kallar grass

Herbaspirillum spp. Rice, maize, sorghum, sugar cane

131
Block 4 Nitrogen Metabolism and Plant Growth Regulators
13.2.2 Conditions for Nitrogen Fixation
The following are the essential requirement for N2-fixation.

i) The enzyme catalyzing the conversion of molecular N2 to NH3 is called

nitrogenase. It is produced by a group of genes called the nif genes.
Expression of the nif genes leading to the formation of functional
nitrogenase is thus one of the essential requirements.

ii) The activity of nitrogenase is extremely sensitive to oxygen. The aerobic

N2-fixing organisms, therefore, must have a cellular mechanism to
protect nitrorgenase activity from oxygen inhibition or creating anaerobic
environment (discussed later in the Unit).

iii) The reduction of N2 to NH3 is a high-energy requiring process consuming

16 to 24 ATP molecules per N2 reduced. Therefore, the organism must
have provision for an abundant supply of ATP.

iv) Mo and Fe are integral constituents of nitrogenase enzyme and

therefore, they must be available to the plant to ensure the formation of
this enzyme complex.

v) Nitrogenase enzyme is produced by bacteria and not produced in the

cells that have access to fixed nitrogen source like nitrate or ammonia.
Hence, the process of N2-fixation occurs in situations devoid of such
fixed nitrogen sources.

13.3 SYMBIOTIC NITROGEN FIXATION –

NODULE FORMATION-LEGHAEMOGLOBIN
As you read in earlier subsection 13.2.1, the symbiotic nitrogen fixation
involves specific associations between bacteria and plants (Table 13.2). Most
important of these are the symbiotic association between rhizobia and
legumes. Facultative organisms preferentially fix N2under anaerobic
conditions. Thus, the Rhizobium-legume association contributes significantly
towards the nitrogen economy of soil. A typical legume-Rhizobium association
is known to fix 25-60 kg ha-1 year1 of nitrogen. In contrast, the contribution by
nonsymbiotic nitrogen fixers is just 5 kg ha-1year-1.

Since more than 90% legumes form nodules, the process of nodulation is itself
of great significance in the sequence of events of nitrogen fixation.

There are three main reasons why legumes are important in agriculture.

i) They fix nitrogen in the soil.

ii) Grains and forage legumes are rich in protein content thus nutritionally
indispensable for humans and livestock.

iii) They provide nitrogen in flows to natural habitats such as tropical forest.

The development and functioning of nitrogen fixing nodules is a complex

process which involves a series of multiple interactions between the bacteria
and the legume host. Sequences of events have been studied from the
132
Unit 13 Biological Nitrogen Fixation
morphological, biochemical, and recently from molecular point of view where
there is an exchange of signals in the form of chemical messages between the
two partners.

The sequence of events has been subdivided into three principal stages.

1. Multiplication and Colonization of Rhizobia in the Rhizosphere

Rhizobia live in the soil as free living, saprophytic bacteria. Since the
symbiosis between rhizobia and the legume roots is not obligatory, the latter
may grow independently throughout their life span. These two partners,
however, seek each other by a series of chemical signals only under nitrogen-
limited conditions. The specificity of symbiotic interaction between Rhizobium
and its host occurs at this very first stage and is called recognition. There are
reciprocal signals between the host and symbiont for determination of host
specificity for root nodule development. The rhizobia are attracted to move
towards the legume roots by positive chemotaxis.

Flavonoids like luteolin secreted by the roots that are believed to act as
chemotactic chemicals. In addition, many amino acids, organic acids, and
sugars also contribute towards this chemotaxis, which help the bacteria to
detect beneficial chemicals required for growth and reproduction. The rhizobia
multiply and may soon get attached to the root hairs constituting the bacteria-
rich rhizosphere (Fig.13.2).

Fig.13.2: Binding of rhizobia to the emerging root hair in response to the

chemotactic substances secreted by the root.

After colonizing the rhizosphere, the rhizobia now begin to synthesize special
morphogenic signal molecules or symbiotic signals in response to the
attractants. The attractants activate the transcription of nod genes which
synthesize nod factors/modulation factors in rhizobia.The nod factors are
lipochito-oligosaccharides (derivatives of chitin, a β – (1-4) linked polymer of
N-acetyl-D-glucosamine. Many genes (viz., nod A, nod B and nod C) have
been isolated which code for enzymes synthesizing the basic structure in nod
factors. Different rhizobia have host-specific nod-genes which allow a specific
response from legume.

The nod-factors are recognized by the host cells and induce many important
changes in the growth and metabolism of the host roots before the bacteria
enter the root hair and before nodulation is initiated. The roots become shorter
and thicker. The root hair production increases, more branches appear in the
root hairs. The root hairs start curling at their tip. The Rhizobia also release
mitogenic signals which stimulate localized cell divisions in the root cortex
(Fig. 13.3). 133
Block 4 Nitrogen Metabolism and Plant Growth Regulators

Fig. 13.3: After being chemotactically attracted to the root hair, the rhizobia
stimulate the root hair (nod factor) to curl and send mitogenic signals
for stimulating cell division in the cortex (from Hopkins & Hüner).

These cells divisions form the primary nodule meristem, which defines the
region of future nodule development.

Attachment of rhizobia to the root hair of specific host involves chemical

recognition. There are reciprocal signals between the host and symbiont for
determination of host specificity for root nodule development. It is known that
this recognition is a function of complementary molecules present on the
surface of the two symbiotic partners. There are two types of molecules
comprising this complementary group; one is the complex polysaccharide and
the other lectins. The polysaccharide component is characteristic of both
partners while the lectin molecules are produced only by the host (legume)
concerned. Lectins are small, non-enzymatic proteins which are synthesized
by the host. Lectins have many sites specific for binding to a variety of surface
polysaccharides of the interacting partners. For example, as shown in Fig.13.4
clover root hair produces a lectin called trifollin with two specific sites, one for
binding to polysaccharides at the surface of bacterium Rhizobium trifolii and
other for binding to polysaccharide on the wall of root hair. Thus, lectin
functions as an anchor to fix Rhizobium on the root surface.

Fig. 13.4: Role of lectins in recognition of host and symbionts. The specific
rhizobia attach to the root hair of the correct species because the
bifunctional lectin molecules recognize antigenic sites of both
partners.

2. Invasion of Root hair and Formation of Infection Thread

As a result of Rhizobium nod-factor induced curling of root hairs, the colonies

134 of bacteria themselves get entrapped within the curls. The nod-factors are
Unit 13 Biological Nitrogen Fixation

thought to release enzymes like pectinases, hemicellulases and cellulases,

which degrade the cell wall material of the host cell walls. As a result of
degradation of some regions of the cell wall the bacterial cells gain access to
the underlying plasma membrane of the root hair.

Upon the arrival of rhizobia on the outer surface of the plasma membrane the
root hair stops growing and the plasma membrane starts invaginating. This
internal tubular extension called the Infection thread is made by the fusion of
membrane vesicles derived from Golgi to the end of the tube (Fig.13.5).

Fig. 13.5: Localized degradation of root hair wall results in formation of infection
thread which is contributed by the Golgi activity. The nodule meristem
is also actively dividing (From Taiz & Zeiger).

As the infection thread grows and elongates through the root hair cell, a thin
layer of new wall material on the inner surface of the membrane is
continuously being deposited which prevents the actual entry of the cells. In
the meantime, cells at the primary nodule meristem start rapid cell division
forming a distinct area within cortex. This zone is called a nodule
primordium,a zone where nodule will develop. Position or site of this
primordium is controlled by the action of signaling compounds like the
nucleoside uridine and ethylene. Infection thread continues to elongate
through the base of the root hair cell where its membrane fuses with the host
cell plasma membrane (Fig. 13.6a). During this process, some bacteria get
released into the apoplastic space. This initiates a new infection thread
(Fig. 13.6 b). The infection thread continues its elongation into successive
cells of the cortex while the bacteria also continue proliferating.

(a) (b)
Fig. 13.6: a) Fusion of infection thread membrane and plasma membrane of the
root hair cell; b) Release of Rhizobia into the apoplast. The bacteria
penetrate the middle lamella to the subepidermal cell plasma
membrane to initiate a new infection thread (From Taiz & Zeiger).

Finally, the infection thread reaches the developing nodule and its tip fuses
with the plasma membrane. As a result, the bacterial cells, which were 135
Block 4 Nitrogen Metabolism and Plant Growth Regulators
enclosed by the membrane derived from the lost cell plasma membrane get
released into the cytosol thereby infecting the going nodule (Fig. 13.7). The
branching of the infection thread ensures spread of infection in many cells of
host at the same time by the bacteria.

Fig. 13.7: Extension and arrival of the infection thread at the target cells, where
membrane-enclosed bacterial cells are released (From Taiz &Zeiger).

The bacteria, after their release continue to divide. The surrounding

membrane gradually increases in surface area to accommodate this growth.

3. Formation of Bacteroids

After some time, when the nodule is full of vesicles, the bacteria stop dividing.
In turn, they start enlarging and finally differentiate into specialized nitrogen
fixing endosymbiotic cells called bacteroids. These bacteroids are still
enclosed by the peribacteroid membrane, which is derived from the infected
root nodule cells. Main function of bacteroids is nitrogen-fixation. As the
infection process continues, nodule gradually increases and finally with its
specialized enzymes the nodule meristem establishes contact with the main
root vascular system. This creates a route to export fixed nitrogen from the
nodule to the plant as well as to import photosynthetic carbon into the nodule
(Fig. 13.8).

Fig. 13.8: Schematic diagram of a cross-section through a mature nodule

showing vascular connections of the import and export of substances
136 between the hosts and the microsymbiont (From Hopkins & Hüner).
Unit 13 Biological Nitrogen Fixation
The bacteroids have functional N2-fixing apparatus and cytochrome
component located in new bacteroid membrane that facilitates respiration at
low O2 concentration. During the time a special protein, leghemoglobin, is
synthesized. It is also localized within peribacteroid membrane (Fig.
13.9).Several new proteins of enzymatic and transport nature is also produced
exclusively by the host. Since these host-produced proteins are nodule
specific, they have been given the name nodulins. Nodules are supplied with
root vascular tissues.

Fig. 13.9: An electron micrograph of soybean root nodules. Many bacteroids are
in groups. Each group is surrounded by a peribacteroid membrane.

Mature bacteroids have enzymes for TCA cycle and oxidative phosphorylation.
The bacteroids are active in ATP production and in supply of reductant for
nitrogenase activity. The carbon requirement to produce ATP and reductant is
met ultimately from photosynthetically produced carbohydrates translocated
through phloem. The differentiation of bacterium into bacteroid is accompanied
by inhibition of glutamine synthetase activity, so that the bacteroid cannot
assimilate ammonia. In other words, the bacteroids function as ammonia
producing factories for the host. Consequently, ammonia is released into the
cytoplasm of the host cell and here it is assimilated into glutamine and
asparagine in the case of pea and clover or it is assimilated into ureides as
happens in soyabean. The reaction of conversion of NH3 to glutamine has
been already discussed in Unit 12.

SAQ 1
a) Given below in Column 1 are some N2-fixing species. Match them with
their symbiotic partner in Column 2.

Column 1 Column 2

i) Anabaena a) Certain leguminous plants

ii) Rhizobium b) Anthoceros

iii) Nostoc c) Azolla (fern)

iv) Frankia-actinorhizal d) Sorghum

v) Azospirillum e) Casuarina 137

Block 4 Nitrogen Metabolism and Plant Growth Regulators
b) Match the asymbiotic N2-fixers in Column 1 with the characteristics given
in Column 2.

Column 1 Column 2

i) Nostoc a) Anaerobic

ii) Azotobacter b) Photosynthetic anaerobe

iii) Clostridium c) Heterocyst

iv) Chromatium d) Aerobic

c) Which among the following statements are true? Write T for true and F
for false.

i) Only prokaryotes have the ability to fix nitrogen.

ii) Leguminous plants themselves cannot grow without Rhizobium.

iii) Enzymes necessary for the fixation of nitrogen are present in the
bacteroids.

iv) Enzymes of TCA cycle and electron transport chain are present in
bacteroids.

v) Bacteroid secretes aspargine into cortical cells.

vi) Infection thread consists of an infolded and extended plasma

membrane of the bacteria.

d) Answer in one word

i) An enzyme produced by nif genes that converts N2 to NH3.

ii) The specificity of symbiotic interaction between Rhizobium and its

host occurs at this stage.

iii) The transcription of these genes is activated by attractants.

iv) The molecules that help in recognition of both symbiotic partners

during N2 fixation.

v) A tubular extension made by fusion of membrane vesicles derived

from Glogi body.

vi) They possess enzymes for TCA cycle and oxidative

phosphorylation.

13.4 BIOCHEMISTRY OF NITROGEN FIXATION

Biological nitrogen fixation is a multistep process, and like the Industrial
fixation, converts molecular nitrogen to diamide, hydrazine and finally to
ammonia.

N2 → HN=NH2 → HN- NH2 → 2NH3

Diamide Hydrazine Ammonia
138
Unit 13 Biological Nitrogen Fixation
Interestingly, very high temperature and pressure are needed to reduce the
dinitrogen triple bond with lot of energy input. The reduction of dinitrogen by
biological means is an equally high energy requiring process, utilizing a large
proportion of the photosynthate provided by the host plant.

13.4.1 Nitrogenase Enzyme Complex

Nitrogen fixation is catalyzed by the enzyme dinitrogenase. The gene for
coding this enzyme is present only in some prokaryotic cells. Nitrogenase is a
multimeric protein complex and is composed of two proteins of different sizes.
The larger protein of the dinitrogenase complex is a MoFe protein (a tetramer
consisting of two pairs of identical subunits with Mo-Fe-S clusters. The two
Molybdenum atoms are present in the form of an iron-molybdenum-sulfur
cofactor. The number of clusters can vary depending upon the physiological
status. The total molecular mass of the tetramer ranges from 180-235 kDa and
a half decay time of 10 minutes in air.

The smaller protein, called Fe protein is a dimer which consists of two identical
subunit polypeptides. The molecular mass of each subunit varies from 24-72
kDa in different species. An iron cluster (4 Fe and 4 S2-) is present in each
subunit. The Fe protein is O2- sensitive and gets inactivated irreversibly. It has
a half-decay time of 30-45 seconds. The Fe protein participates in redox
reactions involving conversion of N2 to NH3.

The overall nitrogen reduction reaction catalyzed by dinitrogenase can be

represented in two steps (Fig. 13.10). In the first step ferredoxin serves as
electron donor and reduces the Fe protein. The reduced Fe protein binds and
hydrolyzes ATP. ATP is believed to cause a conformational change in the Fe
protein itself, altering its redox potential to facilitate the transfer of electrons
the two components of dinitrogenase.

Fig. 13.10: The reactions catalyzed by Fe protein and MoFe protein of the
nitrogenase enzyme complex (From Taiz & Zeiger).

The iron moiety serves as a carrier for electrons either in an oxidized ferric
(Fe3+) or a reduced ferrous (Fe2+) state. You have already studied in Unit 6
that Fd acts as a very important electron carrier during the light reaction of
photosynthesis. 139
Block 4 Nitrogen Metabolism and Plant Growth Regulators
The second step is facilitated by these redox reactions where the reduced Fe
protein passes electrons to the MoFe protein, which in turn catalyzes the
reduction of dinitrogen gas and hydrogen.

The overall reaction of dinitrogenase catalyzed reduction of dinitrogen to

ammonia is summarized in the following equation.

8 H+ + 8 e- + N2 + 16 ATP → 2NH3 + 16 ADP + 16 Pi

In terms of energy, biological nitrogen fixation seems to be an energy requiring

process since the process requires 8 electrons to reduce one molecule of
dinitrogen along with 16 ATP. In addition, if we add another 9 ATP molecules
which are required for reduced ferredoxin, the total requirement for 1 mol of N2
reduction is 25 ATP, which is quit costly. In sharp contrast, fixation of one
molecule of CO2 in photosynthesis requires only 3 ATP molecules. Perhaps
this is the reason why the N2 fixation process is restricted to organisms where
there is more efficient ATP production.

A better method for assessing the cost of nitrogen fixation is the measurement
Dinitrogen =N2
of the amount of carbon utilized. This is because the carbohydrates required to
generate reducing potential ATP in the bacteroid come from photosynthesis of
the host plant. Approximately 12 g of carbon are required to fix a gram of
dinitrogen. The relationship of this costly process with respiration and
photosynthesis is depicted in Fig. 13.11.

Fig. 13.11: Summary diagram illustrating the interactions between nitrogen

fixation, respiration, and photosynthesis in bacteroids (From Hopkins
& Hüner).

As is clear from Fig. 13.11 and also mentioned earlier, the high ATP
requirement alone might have been the reason for restricting N2 fixation
process to organisms efficient in ATP production.You can see that hydrogen
production during N2-fixation seems to be a wasteful reaction energy-wise. It
140 also affects nitrogenase activity because molecular H2 is an inhibitor of N2-
Unit 13 Biological Nitrogen Fixation
fixation. Attention, therefore, is being given to eliminate hydrogen generating
reaction of nitrogenase during N2-fixation.

13.4.2 Factors Influencing he Functions of Nitrogenase

Following are the factors that show considerable influence on the activity of
nitrogenase enzyme.

i) Molybdenum (Mo) and Vanadium (Va)

Molybdenum must be available in nature to meet the demand of Mo for the

formation of nitrogenase necessary for N2-fixation. Now, it is known that Mo
inhibits formation of Va containing nitrogenase. As you may know the relative
distribution of Mo and Va in nature is in favour of Va. Therefore, it is quite
likely that some ecological habitats could be deficient in Mo not in Va and N2-
fixers of such habitat are expected to produce Va containing nitrogenase for
N2-fixation Accordingly, there could be two nitrogen cycles functioning in
nature: one mediated by Va system and the other mediated by Mo system.

ii) Molecular Hydrogen

As mentioned in equation for nitrogen fixation, atleast one molecule of

hydrogen is evolved for every molecule of dinitrogen reduced. Thus, 25-30
percent of ATP and electrons supplied to dinitrogenase are wasted in
hydrogen production.

N2-fixing organisms also produce a membrane bound oxygen dependent

enzyme called uptake-hydrogenase under N2-fixing conditions. The
physiological significance of the presence of uptake hydrogenase lies in its
ability to consume hydrogen produced during nitrogenase activity through
aerobic electron transport chain generating ATP in the process. The electrons
are returned to the reductant pool. Such nitrogenase uptake-hydrogenase
relationship in nitrogen fixing cells provides the following advantages:

i) By aerobic respiration it restores a major part of energy in the form of

ATP.

ii) It offers protection of nitrogenase from oxygen.

iii) It prevents inhibitory effect of H2 on nitrogenase activity.

The expression of genes (the hup genes) is responsible for uptake of

hydrogenase have attracted considerable attention. Biotechnological studies
are targeted towards making rhizobia strains with a higher capacity to recycle
hydrogen thereby increasing overall efficiency of the process of nitrogen
fixation in our principal crops.

Also, it has been observed that the nitrogenase enzyme catalyses exclusive
production of hydrogen provided the N2-fixer is in an atmosphere devoid of
nitrogen. Efforts are being made to exploit this special property of the enzyme
of photoproduction of H2 by heterocysts of cyanobacteria on a commercial
scale. This would require construction of special strains of cyanobacteria that
would be genetically deficient in uptake hydrogenase enzyme. It is expected
that these strains would continuously generate hydrogen in photosynthetic and
N2-fixing conditions. 141
Block 4 Nitrogen Metabolism and Plant Growth Regulators
iii) Oxygen
Molecular oxygen is a strong inhibitor of N2-fixation as it blocks both the
synthesis as well as the activity of nitrogenase. As you have seen in Table
13.1, there are N2-fixers which are anaerobes, and many are aerobes. The
problem of oxygen in anaerobes is taken care of by their very anaerobic mode
of nutrition. If facultative, some organisms fix nitrogen only under anaerobic
conditions. The problem is with aerobes like Azotobacter which require oxygen
to fix nitrogen and grow at the expense of nitrogen. So how do the aerobic
nitrogen fixers balance the conflicting demands of O2 for their respiratory
pathway on one hand and the sensitivity of dinitrogenase to oxygen on the
other? Since both Fe protein and MoFe protein are irreversibly inactivated by
molecular oxygen, the aerobic nitrogen fixers have evolved several strategies
to regulate oxygen levels so that both the above-mentioned processes
continue together.

Aerobic nitrogen fixers like some species of cyanobacteria fulfil this

requirement of creating anaerobic or low O2 conditions by isolating the
nitrogen fixing apparatus in specialized cells called Heterocysts. Heterocysts
are specialized cells formed from ordinary vegetative cells. They possess a
thick, multilayered cell wall that restricts the diffusion of oxygen. Although
heterocysts are photosynthetic, their photosystem II activity is lost during
differentiation. Thus, they cannot produce any oxygen. They also lack Rubisco
enzyme and consequently cannot fix CO2. However, they still retain the
capability to synthesize ATP by cyclic photophosphorylation or PSI (Fig.
13.12). Thus, necessary ATP for nitrogen fixation is provided,and at the same
time the O2 concentration is kept low by the formation of a new glycolipid layer.
The heterocyst represents an early model of cellular specialization and
division of labour normally associated with higher plants.

Fig. 13.12: Diagrammatic representation of carbon and nitrogen exchange

between a heterocyst and vegetative cell of a cyanophycean nitrogen
fixer (From Smith et al 1999).
iv) Leghemoglobin

An oxygen binding protein leghemoglobin is synthesized in the legume

nodules (bacteroid injected host cell), the bacteroid and the host plant. While
the bacteroid forms the haem part, the globin portion is synthesized by the
host plant. Leghemoglobin gives a distinct pink color when the cut surface is
exposed to air. Leghemoglobin is similar in structure to the hemoglobin of
142 mammalian blood. Its function is also somewhat similar as it binds oxygen and
Unit 13 Biological Nitrogen Fixation
controls the release of oxygen in the domain of the bacteroid. The oxygen
concentration in the bacteroid in kept at a level which is sufficient to produce
ATP and reducing potential while at the same time preventing excess oxygen
that could inactivate dinitrogenase. To continue aerobic respiration under low
O2 conditions, the bacteroids, use a specialized electron transport chain where
the terminal oxidases have a much higher affinity for oxygen even higher than
that of leghemoglobin.

13.4.3 Measurement of Nitrogenase Activity

There are various methods to find out whether an organism is a N2-fixer or not.
If an organism can grow in normal atmosphere without any provision of a
nitrogen source like nitrate or ammonia, one infers the organism to be a N2-
fixer. It is important to mention here that N2-fixer heterocysts containing
cyanobacteria or Rhizobium-legume system are known to grow without any
supply of fixed nitrogen.

The other more reliable method is the incorporation of 15N isotope into cellular
The word diazotroph
nitrogen containing compound which can be estimated in a mass is derived from the
spectrometer. This method is every costly and is, therefore, not easily words diazo ("di" =
accessible for day-to-day measurement of nitrogenase activity and N2-fixation. two + "azo" =
nitrogen) meaning
The most extensively used method for measuring nitrogenase activity is the "dinitrogen (N2)"
acetylene reduction method. The method consists of incubating the N2-fixing and troph meaning
system with about 10% acetylene for a period of 10 to 30 min during which "pertaining to food or
nitrogenase converts acetylene into ethylene depending on its efficiency. Gas nourishment", i.e.,
samples are then taken out by syringe for the reaction sample and the dinitrogen utilizing.
acetylene reduced is assayed by injecting the gas sample into a Gas The
Chromatograph which separates quantitatively acetylene and ethylene and word azote means
thus provides easy quantitative assay of ethylene. Nitrogenase catalyzed nitrogen in French
reduction of acetylene to ethylene is not accompanied by reduction of proton and was named by
to molecular hydrogen as happens in the reduction of N2 to NH3. French chemist and
biologist Antoine
The reduction of acetylene to ethylene involves two electrons and if one wants Lavoisier.
to equate the amount of ethylene produced with the amount of N2 fixed then Diazotrophs are
the amount of ethylene must be divided by a factor of 4 to find out the optimum bacteria and archaea
level of N2-fixing activity of nitrogenase. that fix atmospheric
nitrogen gas into a
more usable form
13.5 GENETIC REGULATION OF NITROGEN such as ammonia.
FIXATION These
microorganisms
nif Genes arecapable of
growing without
The genetics of nitrogen fixation is one of the most fascinating aspects and external sources of
holds great promise in gene-transfer to the non-nitrogen fixers for higher fixed nitrogen.
productivity. The free-living nitrogen fixer Klebsiella pneumonieae has been
investigated in detail for its nitrogen fixing genes. Dinitrogenase synthesis in
this species is directed by a set of genes known as nif genes. Atleast 17 nif
genes and a set of 7 operons have been described in this diazotroph. There
are a few regulatory and structural genes which are involved in the different
aspects of nitrogenase functioning. Two genes, the nif D and nif K encode far
two different components of MoFe protein, nif H codes for Fe protein and
ferredoxin by nif F. 143
Block 4 Nitrogen Metabolism and Plant Growth Regulators
nod Genes
Nodulation is regulated by nod genes and nif gens. A set of rhizobial nod
genes get switched on prior to the infection of the root. The triggering factor is
the flavonoids contained in the host root exudates. Three nod genes viz nod A,
nod B and nod C are basic nodulation gens present in most rhizobia. Nod
genes are important in determining host specificity.

fix Genes
These genes are switched on during later stages of nodule development. The
fix genes are restricted to symbiotic nitrogen fixers. One fix X gene encodes
for a ferredoxin while the others are involved in the transport of electrons to
dinitrogenase.

The fix genes are required for the maintenance of N2-fixation by the nodule.
They control oxygen protection of nitrogenase as well as ATP and reductant
supply.

nodulins
These are nodule-specific proteins encoded by nod genes located in the host
cell genome. Two categories of nodulins viz., early and late, perform different
functions. Early nodulins are involved with the infection thread plasma
membrane and in the formation of nodule primordia. On the contrary, the late
nodulins function at the time of nodule formation and nitrogen fixation.
Leghemoglobin is a good example of late nodulin.

hup Genes
These are hydrogen uptake genes which make the nodules more energy
efficient as compared to those which lack these genes. They control the
activity of uptake hydrogenase for recovering some of the energy lost to
hydrogen protection.

The nif, the nod, and the fix genes are located on a plasmid in the fast-growing
rhizobia and on the nucleoid (chromosome) in slow growing rhizobia.

Efforts are on to introduce these genes into other rhizobia strains as well as
non-nitrogen fixers to incorporate the nitrogen-fixing capability in cereals and
other important crops.

SAQ 2
a) Match the characteristics given in Column 2 with enzymes given in
Column 1.

Column 1 Column 2

i) Dinitrogenase reductase a) Fe-Mo protein

ii) Dinitrogenase b) requires ATP for activation

c) transfers one electron at a time

d) Transfer electrons to N2

144 e) Fe-protein
Unit 13 Biological Nitrogen Fixation
b) Fill in the blank spaces with appropriate words in the following
statements.

i) In cyanobacteria nitrogenase is present in ………………………

ii) Uptake-hydrogenase utilizes hydrogen by passing it

to…………………….under aerobic condition and
generates…………………….

iii) In an atmosphere devoid of N2 gas nitrogenase enzyme catalyses

the production of …………………….only.

iv) A cyanobacterial strain deficient in uptake of hydrogenase will

produce……………….continuously in N2- free atmosphere.

v) …………………………protects nitrogenase against oxygen and it

also functions as a ………………………of oxygen.

c) Match the genes of N2-fixation given in Column 1 with the functions

given in Column 2.

Column 1 Column 2

i) nif genes a) Maintenance of N2-fixation

ii) nod genes b) Functional nitrogenase

iii) fix genes c) Formation of root nodules

13.6 SUMMARY
• Biological N2-fixation is characteristic feature of prokaryotes and the
process occurs in free-living and symbiotic state with various kinds of
eukaryotic partners.

• Nitrogenase enzyme is made of two functional components

dinitrogenase reductase (Fe-protein) and dinitrogenase (Fe-Mo
protein).Both of these are essential for N2 fixation, acetylene reduction,
proton reduction and HCN reduction.

• Nitrogenase reaction occurs at the expense of 2-3 molecules of ATP

consumed per electron transferred.

• Oxygen is inhibitor of both nitrogenase activity and its synthesis. The N2-
fixers have evolved a strategy to overcome the O2 problem by having
respiratory and conformational protection in Azotobacter, leghemoglobin
in nodules of legumes and heterocysts in Nostoc.

• Nitrate and ammonia are strong inhibitors of N2- fixation. In their

presence N2-fixing system is not functional at all. Uptake of hydrogenase
activity is a mechanism of removing hydrogen from the site of nitrogen
activity which concurrently carries out reactions of nitrogen fixation and
hydrogen production. 145
Block 4 Nitrogen Metabolism and Plant Growth Regulators
• In Rhizobium-legume symbiotic N2-fixing system, there are three classes
of genes controlling N2-fixation in situ. They are nif genes, nod genes
and fix genes.

• Acetylene reduction technique is most common method of measuring

nitrogenase activity.

13.7 TERMINAL QUESTIONS

1. How will you experimentally separate N2-fixers from non-fixers?

2. Why is heterocyst essential for N2-fixation in Nostoc and Anabaena?

3. What is the role of leghemoglobin in nodules?

4. Why is Mo an essential nutrient for N2–fixation and nitrate assimilation?

5. What are Bacteroids? What roles do they play during nitrogen fixation?

6. Name few factors that influence the functioning of Nitrogenase.

13.8 ANSWERS
Self-Assessment Questions
1. a) i) Azolla (fern)

ii) Certain leguminous plants

iii) Anthoceros

iv) Casuarina

v) Sorghum

b) i) Heterocyst

ii) Aerobic

iii) Anaerobic

iv) Photosynthetic anaerobe

c) i) True; ii) False; iii) True;

iv) True; v) False; vi) False

d) i) Nitrogenase

ii) Recognition

iii) nod genes

iv) Lectins and polysaccharides

v) Infection thread

146 vi) Mature bacteroids

Unit 13 Biological Nitrogen Fixation
2. a) i) b, c and e;
ii) a and d
b) i) heterocyst
ii) electron transport chain, ATP
iii) hydrogen
iv) hydrogen
v) Leghemoglobin, reservoir.
c) i) Functional nitrogenase
ii) Formation of root nodules

iii) Maintenance of N2-fixation

Terminal Questions
1. The nitrogen fixers can be separated from non-nitrogen fixers by
performing the following experiment. Take cyanobacteria like
Nostoc and Oscillatoria and grow them separately in mineral growth
medium which lacks NO3- or NH4+. Incubate these two cultures for
growth under photosynthetic conditions. The culture which will keep
on growing without external nitrogen source would be N2-fixer and
the other which will not grow would be non-N2-fixer.

2. For N2-fixation it is necessary to have O2-protection mechanism for

nitrogenase enzyme. In Nostoc and Anabaena this is provided by
heterocyst which is a specialized structure that has lost PSII activity
during differentiation. Hence in heterocyst nitrogenase is protected
because of reduced oxygen tension. It would not be possible for
these algae to fix N2 without heterocyst because the green parts of
algae evolve oxygen which is an inhibitor of N2-fixing enzyme.

3. Oxygen is inhibitor of N2-fixing enzyme nitrogenase. The primary

role of leghemoglobin is to bind oxygen in the vicinity of the
nitrogenase enzyme. It also serves as a reservoir of oxygen and
supplies oxygen to meet the high energy demand during aerobic
respiration.

4. Mo is part of component of enzyme nitrogenase and nitrate

reductase and is involved in the reductive process of N2 fixation.
Like Fe, the Mo ion can also reversibly change from oxidized to
reduced state and help in the transfer of electrons from reductants
like ferredoxin, flavodoxin, and NAD(P)H to N2 or NO3.

5. Refer to Subsection 13.2.3.

6. Refer to Subsection 13.4.2.

ACKNOWLEDGEMENT
Fig. 13.1 : https://o.quizlet.com/snUZABCUmA6jkPUEy8eAkA.jpg
Fig. 13.9 : From Wikipedia

147
Block 4 Nitrogen Metabolism and Plant Growth Regulators

UNIT 14
PLANT GROWTH
REGULATORS

Structure
14.1 Introduction 14.3 New Class of Plant Hormones
Objectives 14.4 Synthetic Growth
Hormones
14.2 Plant Hormones
14.5 Summary
Auxins
14.6 Terminal Questions
Gibberellins
14.7 Answers
Cytokinins

Abscisic Acid

Ethylene

14.1 INTRODUCTION
In this unit and the coming unit15 we will deal with the control of plant growth
and development. The growth and development of plants is largely controlled
by the plant hormones. Plants produce these hormones by themselves to
control the growth of stems, roots, branches, flowers and fruits and their
development. Some hormones, for example auxins, gibberellins, cytokinins
were discovered by scientists long time ago but now many new hormones are
also being discovered from time to time.

Higher plants are complex organisms and their systematic development

requires synchronization between cells. To coordinate various activities, the
cells need to communicate with each other. Intercellular communication within
plants takes place through hormones. Hormones are the signal molecules
that individually or cooperatively direct the development of individual cells or
transfer the information between the cells and thus, coordinate growth and
development. The regulation and coordination of metabolism, growth and
morphogenesis in higher plants depends upon the chemical signals present in
the cells. Thus we can say that plant hormones are a group of naturally
occurring, organic substances which influence physiological processes at low
148 concentration. Plant hormones are also referred as phytohormones.
Unit 14 Plant Growth Regulators

The present unit will discuss different group of plant hormones, how they were
discovered and various roles they play in growth and development of plants.

Objectives
After studying this unit you would be able to:

enlist major hormones in plants;

appreciate the role of auxins, gibberellins, cytokinins, ethylene and

abscisic acid;

describe the new hormones discovered in plants;

discuss the role of synthetic hormones; and

explain the regulation of various growth processes by hormones.

14.2 PLANT HORMONES

Hormones are the chemical messengers that are produced in a cell. They
modulate the cellular processes in another cell by interacting with specific
proteins that function as receptors linked to cellular transduction pathway. The
concept of hormone in plants can be traced back in 1758 from the work of
Duhamel du Moncaeu. He noted swellings on the roots above the girdle
wounds in woody plants. German botanist Julius Sachs postulated presence
of organ forming substances in plants. He stated that root forming substances
are produced in the leaves followed by their migration to stem. These
substances assist in initiation of roots. Later Charles Darwin, through his
experiments on canary grass, also mentioned about existence of such
chemical substances. About half a century later, F.W. Went in 1926
discovered auxin as the growth hormone in plants.

Hormones that are transported to the sites of action in another tissue distant
from the site of their synthesis are known as endocrine hormones. Some
hormones act on the cells adjacent to their source of synthesis. These are
referred as paracrine hormones. Hormones are organic molecules that
influence the physiological processes in plants even at low concentrations.
The development in plants is regulated naturally occurring hormones such as
auxins, gibberellins, cytokinins, ethylene and abscisic acid.

14.2.1 Auxins
The discovery of auxin is a fascinating chapter in plant physiology. The
hormone auxin was discovered first, through some experiments by Charles
Darwin and his son Francis Darwin around 1880 (Fig 14.1). They found that
if coleoptiles of a grass (Phalaris) were illuminated from one side they bent
towards light. However, if the tip region was covered, no bending occurred. 149
Block 4 Nitrogen Metabolism and Plant Growth Regulators
In his book “The Power of Movement in Plants” published in 1880, Darwin
wrote that some matter in the upper part, which is acted upon by light,
transmits its effect to the lower part causing the latter to bend.

Fig 14.1: Darwin’s experiment on canary grass coleoptiles: a) various parts were
covered with metal caps for determination of photosensitive region;
b) Results observed in each case.

Darwin’s work was further explored by Peter Boysen-Jensen and A. Paal.

They performed experiments to understand the movement of the stimulus.
They decapitated coleoptiles of oat seedlings and (a) introduced a tiny gelatin
block between the stump and cut tip and (b) a thin sheet of mica in the other
(Fig. 14.2). The curvature in coleoptiles occurred in experiment ‘a’ but not in
experiment ‘b’. This happened because the stimulus could pass down through
gelatin but not mica. The curvature occurred only when the mica was inserted
half way on the illuminated side.

The studies suggested that stimulus promotes elongation on dark side. A.

Paal observed that in the dark portion, bending occurred in the opposite
direction of the cut tip. This suggested that the stimulus in the tip affects cells
150 present below.
Unit 14 Plant Growth Regulators

Fig. 14.2: a, b) Experiments on auxins by Peter Boysen-Jensen; and c) A. Paal

experiment.

This stimulus was later discovered to be a diffusible substance by a Dutch

scientist F.W. Went in 1926. He collected the substance in large amounts by
placing decapitated coleoptiles tips on agar blocks. He then took tiny squares
of the agar and placed them eccentrically on the cut end of the decapitated
seedlings. Typically bending occurred within an hour after the blocks were
applied (Fig. 14.3).

Fig.14.3: Went’s experiment on auxins.

He concluded that the substance present in agar block diffused in the

coleoptiles causing elongation of cells in that side resulting in curvature. Went
named it auxin (from the Greek word auxein, “to grow”). Later auxin was
chemically characterized as indole-3-acetic acid or IAA by Thimann.

Auxin is the first plant growth hormone that was discovered. It plays a role in
fundamental growth processes such as enlargement of plant cells. It is
synthesized in the meristematic region and other actively growing regions
such as coleoptile apices, root tips, germinating seeds and apical buds of 151
Block 4 Nitrogen Metabolism and Plant Growth Regulators
stem. Growing leaves, developing embryo, and developing inflorescence are
also the sites of auxin synthesis.

Indole -3-acetic acid (IAA) is the most widely distributed naturally occurring
auxin. Indole-3-butyric acid (IBA) is another compound showing auxin like
activity. It has been isolated from maize and several other species. The
chlorinated analogues of IAA (indoleacetic acid) have been found in extracts of
legume seeds. The amount of IAA produced varies in different tissues. The
vegetative tissues generally produce IAA in the range of 1 to 100 µg kg-1 of
fresh weight. The amount is much higher in seeds.

Synthesis of Auxins

In 1930, K.V. Thimann first observed the synthesis of IAA in the mold
Rhizopus suinus supplied with the amino acid tryptophan. The conversion of
tryptophan to IAA was studied under in vivo conditions in more than 20 plant
species. In most plants, synthesis of IAA occurs in three steps beginning with
removal of amino group on the tryptophan side chain. The product is indole-3-
pyruvic acid (IPA). This reaction is catalyzed by enzyme tryptophan amino
transferase. This enzyme removes amino groups from the structural analogs
of tryptophan such as phenylalanine and tyrosine. In the subsequent step,
decarboxylation of IPA occurs to form indole- 3-acetaldehyde (IAAld). The
enzyme that catalyzes this step is indole-3-pyruvate decarboxylase. Finally,
IAAld is oxidized to form IAA by NAD-dependent indole-3-acetaldehyde
oxidase. IAAld gets reduced to form indole-3-ethanol. IAA can reversibly get
converted to IBA by the enzyme indole-3-butyric acid synthase. The synthesis
of IAA via tryptophan independent pathway has been noted in mutants of
maize and Arabidopsis (Fig. 14.4).

Tryptophan (Trp)

Trp transaminase

Indole-3-pyruvic acid (IPA)

IPA decarboxylase

Indole-3-acetaldehyde (IAld)

IAld dehydrogenase

Indole-3-acetic acid (IAA)

Fig 14.4: Auxin biosynthetic pathway in plants.

Studies have shown that Arabidopsis contains nitrilase enzyme that converts
indole-3-acetonitrile to IAA. It has been suggested that indole-3-acetonitrile
can be derived from glucobrassicin, a glucosinolate present in the members
152 of family Brassicaceae.
Unit 14 Plant Growth Regulators
Auxins can be present in free or bound form in plants. The auxin may be
bound to the cell and can be extracted with solvents or by hydrolysis under
alkaline conditions. Bound auxin forms chemical conjugates with sugars to
form glycosyl esters. The conjugates are formed by esterification of a glucose
or inositol molecule to the acid group of the side chain. These conjugates
release active IAA upon solvent extraction or alkaline hydrolysis. The pool of
IAA conjugates has been reported from seeds of maize and is formed in the
endosperm. They act as the active hormone for the embryo. About 60% of the
IAA requirement of the germinating seed is met through these conjugates.

Functions of auxin
Auxins are also involved in other developmental events such as secondary
root initiation, vascular differentiation, and development of axillary buds,
flowers and fruits. They play a crucial role in regulating various physiological
roles in plants. Some of these include:

• Auxin stimulates cell elongation in stem and coleoptiles. It gets

transported basipetally to the tissues below. Steady supply of auxin
promotes cell elongation.

• The treatment of terminal shoots of fruit trees with α (alpha) naphthalene

acetic acid prevents their elongation resulting in formation of dwarf
shoots. This effect of auxin is due to shortening of internodes.

• The treatment of α- naphthyl acetamide treatment helps in the stiffening

of cells and giving them a woody appearance. This prevents lodging.

• High levels of auxin promote the formation of roots in plants. Studies

have established that auxins increase the formation of root initials.
Thimann et al. showed similarity between the root forming substance
and auxins. Formation of both lateral and adventitious roots get
stimulated by auxins. Adventitious roots arise from the differentiated
cells that begin to divide and develop into a root apical meristem. Some
of the compounds that can be applied to cuttings to induce formation of
roots include phenyl acetic acid, indole acetic acid, indole butyric acid.
Hence auxin plays a major role in root initiation.

• Auxin is also involved in the vascular differentiation in shoots. The

process is regulated by auxin concentration. Vascular differentiation is
the regeneration of vessels and phloem sieve tubes around the wounds.
Leaves are the source of auxin and removal of leaves above the wound
limits vascular differentiation. It has been noted that differentiation of
phloem sieve elements is favoured by low concentration of auxin (0.1%)
while the xylem differentiation is favored by high auxin content (1%).
Auxins promote cell elongation in the sub apical region of the stem.

• Auxins help in cell wall expansion by promoting efflux of protons.

Increased activity of plasma membrane H+-ATPase provides H+. Binding
of IAA to auxin binding protein present in the cytoplasm or cell surface
induces the H+-ATPase activity and increases proton pumping
(activation hypothesis). Hydrogen ions play a role in cell wall expansion.
Protons increase acidity of the cell wall. Increased acidity activates the
activities of various hydrolyzing enzymes such as cellulase, pectinase
and hemicellulase. These enzymes break the bonds of cellulose, pectin 153
Block 4 Nitrogen Metabolism and Plant Growth Regulators
and hemicelluloses and cell wall become loose. The proteins called
expansions accelerate cell wall expansion by weakening the hydrogen
bonds between the polysaccharide components of the wall.

• During cell wall expansion, auxin increases the supply of ADP promoting
the synthesis of ATP, hence stimulating respiration.

• The development of floral mersitem depends on auxin transported to it

from subapical tissues. The auxin transport from these tissues regulates
leaf initiation and phyllotaxy (the pattern of leaf emergence from the
shoot apex).

• Auxin delays the onset of leaf abscission (shedding). Addicott and Lynch
(1951) showed that auxin gradient across the abscission zone controls
abscission. Application of auxin to the cut surface of the petiole prevents
the formation of the abscission layer. The premature fruit drop can be
prevented to a great extent by spraying of 2,4D, IAA or NAA.

• Auxin promotes fruit development. Auxin is produced or mobilized from

storage in pollen and the initial stimulus for fruit growth may result from
pollination. The endosperm contributes auxin during initial stage of fruit
growth and developing embryo takes over as the main auxin source at
later stages.

• Auxins generally inhibit flowering but in pineapple a spray of dilute

solutions of 2, 4-D, NAA has been found to promote flowering by
synthesizing ethylene.

• Auxins promote development of fruits without pollination and fertilization.

Auxins promote growth of the ovary wall leading to formation of fruit. The
fruits that develop from parts of the flower without involving the process
of fertilization are referred as parthenocarpic fruits.

• Very high concentrations of auxins such as 2,4D affect the growth

promoting activities of the root cell. The root cells get distorted, sieve
tubes get blocked and the cell divisions are disturbed. The roots dry and
the plant die. Thus application of auxins can help in eradication
(removal) of weeds.

• Auxins promote cell division. In case of a wound, the proliferation of cells

results in the formation of callus. The callus formation is linked with
cambial activity which is stimulated by auxin. Studies have shown that
cambial activity is promoted by auxin

• With the growth of shoot, the apical meristem forms new primordia. A
group of cells in the axil of the primordial become isolated from the
apical meristem which form the axillary bud. In some plants the bud
continues to grow but in many plants, mitosis and cell expansion in the
bud is arrested and hence the axillary bud fails to grow. The removal of
shoot apex stimulates the growth of axillary bud. It is believed that apical
bud exerts a dominant influence and suppresses the cell division and
enlargement in the axillary bud. This phenomenon is referred as apical
bud dominance. K.V. Thimann and F. Skoog tried to find out the
relationship between auxin and apical bud dominance and stated that
154 axillary bud development remains suppressed in the presence of auxin.
Unit 14 Plant Growth Regulators
Bioassays

The effect of a biologically active substance on living materials can be

checked by performing certain experiments. These are known as bioassay.
Bioassays for auxins include

• Avena curvature test

• Avena section test

• Split pea stem curvature test

• Cressroot inhibition test

Avena curvature test

The assay was proposed by F.W. Went. The measurement of auxin activity
can be done by studying its polar transport. The seedling of Avena is
decapitated. An agar block containing the auxin is placed on the decapitated
stump (Fig. 14.5). Auxin from the agar is transported down the coleoptiles and
causes the side to grow faster than the opposite side, resulting in curvature.
Bending depends upon the hormone concentration. The more of the
concentration of hormone more is the curvature.

Avena section test

The studies suggest that auxin stimulates cell elongation. Avena grains are
germinated in dark and 25-30 mm long coleoptiles are harvested. The apex is
removed and a section of 3-5 mm in length is cut from the coleoptile. The
length is measured. The sections are soaked in distilled water for an hour and
distributed in petriplate having the test solution with various concentrations.
The sections are incubated for 12, 24 and 48 h and the sections are
measured.

Split peas stem curvature test

This test depends upon the differential growth response. It was reported by
Went in 1934. A stem section of pea seedling is split longitudinally and floated
on the test solution. A negative curvature is noted because of uptake of water
by cortical cells. The epidermal cells show increase in length because of auxin
while the cortical cells how increase in width. If the concentration of auxin is
increased, a positive curvature will result.

The pea seeds are grown in dark but exposed to red light for some time (3 h).
The stem is decapitated and the portion between node and internode is
selected. The selected portion is soaked in distilled water and the cut stem is
slit longitudinally and placed in a Petri dish a having auxin solution. The
curvature of the stem is noted.

Cress root inhibition test

The roots show more sensitivity to auxins. Low concentrations of auxin

stimulate root growth. The test is used for measuring low concentrations of
auxin. Seeds are sterilized and germinated on a moist paper. After emergence
of roots (about 1 cm long), they are placed in test solution. The length of the
root is measured in control and treated. 155
Block 4 Nitrogen Metabolism and Plant Growth Regulators

Fig.14.5: Bioassay of auxin. The extent of bending as measured by angle α, is a

function of auxin or IAA in the agar block (Source: Leopold 1955).

Transport of auxins
Auxin generally moves from shoot to root. The polar movement i.e., either up
or down has been noted in coleoptiles, stem and roots (Fig.14.6).

Fig. 14.6: Polar transport of auxin in plants.

When the movement of a compound occurs from the apex towards the base,
the movement is called basipetal while the movement towards the
morphological apex is known as acropetal. The movement of auxins is
generally basipetal i.e. it moves away from the root tip. The movement of
auxin is important because auxin plays a role in various growth and
developmental responses in plants. The polar transport of auxin is a carrier
mediated active transport and depends upon oxidative metabolism in
mitochondria. The polar transport is inhibited by respiratory poisons such as
cyanide and 2, 4 -dinitrophenol. Inhibitors block auxin transport by binding to
156 discrete carrier molecules which are involved in polar transport system.
Unit 14 Plant Growth Regulators
BOX.14.1: Auxin transport in a cell

A chemiosmotic model for auxin transport has been proposed by Rubery and
Sheldrake (1974) and Raven (1975). According to this model
1) A pH gradient or proton motive force across the plasma membrane provides
the driving force for uptake of IAA,
2) An IAA influx carrier and efflux carrier is located at the base of auxin
transporting cells.
IAA is a weak acid and depending upon the pH, it exists in either protonated form
(IAAH) or the unprotonated anionic form (IAA-). The cell wall is moderately acidic
with pH of 5.5. At this pH, 20% of the IAA occurs in the protonated (IAAH) form. The
cell wall space contains both protonated and anionic IAA. A small proportion of the
uncharged IAAH molecule diffuses slowly across the plasma membrane from the cell
- +
wall space into the cell. Inside the cell, IAAH dissociate to form IAA and H . The
-
auxin is now trapped inside the cell because the IAA cannot diffuse across the
membranes. The position of the efflux carrier (generally located in the membrane of
the cell) establishes the polarity in auxin transport. The influx carrier identified as
AUX1 plays a role in auxin metabolism and transport. This protein has been
supposed to function as amino acid/proton symport carrier. PIN genes that encode
putative auxin efflux carriers have been identified in plants. PIN proteins direct the
transport of auxin from one cell surface to another. PIN1 protein has been found to
be localized in the basal membranes of xylem parenchyma cells.

Mechanism of action of auxin

Auxins act in cell by getting adsorbed to the hormone binding site. They
induce acidification of membrane by bringing a change in nucleic acids,
proteins or plasticity of wall. The auxin plays a role in cell growth by increasing
rate of cell expansion. According to R. Cleland and D. Rayle (1970) auxin
causes acidification of the cell wall by stimulating cells to excrete protons. The
proton secretion lowers pH and this activates one or more wall loosening
enzymes. This results in cell enlargement. Studies with Avena coleoptiles
have shown that apoplastic pH decreases from 5.7 to 4.7 on application of
auxin.

A. Hager (1970) proposed that auxin stimulates proton excretion by activating

plasma membrane bound H+-ATPase. Auxin induced increase in cell growth
has been explained through acid growth hypothesis proposed by Cleland
and Hager (1971). According to this hypothesis auxin activates ATP proton
pumps located in the plasma membrane. Enhancement in the activity of H+-
ATPase, pumps more protons inside resulting in acidification of the cell wall,
hence lowering the pH. Lowering of pH supports turgor induced cell expansion
and cell wall extensibility. The activation of enzyme occurs after binding of
auxin to specific transmembrane protein receptors such as ABPI (Auxin
binding protein I).

14.2.2 Gibberellins
They are plant hormones that belong to a large family of plant constituents
called terpenoids. They are biosynthetically related to carotenoid pigments,
sterols, and other isoprene derivatives. More than 135 gibberellins have been
identified in plants. GA1 and GA4 are the two most active commonly occurring
gibberellins reported in higher plants. All the gibberellins are diterpenes and 157
Block 4 Nitrogen Metabolism and Plant Growth Regulators
possess a 20-carbon structure. A C20 gibberellin is referred as gibberellic acid
(GA3). The gibberellin biosynthesis occurs mainly in the developing seeds,
fruits, young leaves and apical region of roots. They are also produced in
developing endosperm, cotyledons of legumes or scutellum of cereals.

During late nineteenth century, the Japanese rice farmers found a disease that
reduced the yield of their crops. Plants were infected with the bakane byo
(foolish seedling disease) which exhibited weak, elongate stems and produced
little or no grain. It was found that the disease is somewhat related to the fungi
Gibberella fugikuroi. In 1926, E. Kurosawa reported the appearance of
symptoms of the disease in uninfected rice plants treated with the filtrates from
the cultures of the fungus. The compound was isolated, crystallized and
named as gibberellin. He isolated three gibberellins viz. gibberellin A1,
gibberellin A2 and gibberellin A3. Gibberellin A3 was found identical to
gibberellic acid.

Synthesis of gibberellins

Gibberellins are synthesised through the normal isoprenoid pathway of

terpene biosynthesis. The biosynthesis of gibberellins occurs mainly in the root
apex, elongating shoots, young leaves, seeds and fruits.

Gibberellins are either synthesised in the regions where they act or are
transported there from some other part of the plant. The translocation of
gibberellins is not polar. It is believed that gibberellins synthesized in the root
tip get translocated to the other aerial parts of the plant through xylem.
Gibberellins can move in several different ways in the plant in a manner which
is similar to other organic metabolites such as the carbohydrates, amino acids
etc. They can either be transported by simple diffusion, or through the phloem
or the xylem. Their transport is not polar like that of auxins.

The synthesis of gibberellins involves three steps- First -formation of two

phophorylated derivatives from acetyl Co A, viz., Isopentenyl pyrophosphate
and Dimethylallyl pyrophosphate. Second- One molecule of Isopentyl
pyrophosphate condenses with the molecule of Dimethylallyl pyrophosphate to
form geranylgeranyl pyrophosphate (C20) (GGPP) through geranyl
pyrophosphate (C10) and farnesyl pyrophosphate (C15) (Fig. 14.7). GGPP
gives rise to GA 12-7, which acts as the source of all gibberellins.

A number of growth retardants also known as antigibberellins have been

applied to potted plants as foliar spray or soil supplement. These retardants
reduce stem elongation, resulting in plants which are shorter, compact with
dense foliage. In roots application of antigibberellins such as 2-chloroethyl
trimethylammonium chloride (CCC) stops the synthesis of gibberellins.
AMO-1618 and phosphon-D are the antigibberellins that inhibit enzymes
involved in the synthesis of kaurene. Ancymidol blocks the oxidation of
kaurene to kaurenoic acid. The effect of these compounds can be reversed by
the application of gibberellins such as GA20 or GA3.

The translocation of gibberellins is not polar. It is believed that gibberellins

synthesised in root tip get translocated to the other aerial parts of the plant
158 through xylem.
Unit 14 Plant Growth Regulators

Acetyl Co-A

Mevalonic acid

Mevalonic acid
5-pyrophosphate

Isopentyl Dimethyllyl
pyrophosphate pyrophosphate

Isopentyl Isopentyl
pyrophosphate pyrophosphate

geranyl farnesyl geranylgeranyl

pyrophosphate pyrophosphate pyrophosphate

pyrophosphate (PPi)

ent-Kaurene

Kaurenoic acid GA12-7 aldehyde

Fig. 14.7: Giberellin biosynthesis in plants.

Functions of gibberellins

Various responses in plants have been observed after treatment with

gibberellins. Some of the major physiological roles of gibberellins include:

• Gibberellins promote seed germination. When the concentration of GA is

increased it removes dormancy and promotes germination. During
germination, they induce the synthesis of hydrolytic enzymes such as
amylases and proteases in cereal grains. These enzymes degrade the
stored food reserves accumulated in the endosperm or embryo. The
degradation of carbohydrates and storage proteins provide nourishment
and energy to the growing seedling. Gibberellins are also involved in
mobilization of endosperm reserves during early stages of embryo
growth, leaf expansion, pollen maturation, flowering and fruit
development.

• GA possesses the capacity to stimulate hyper elongation of stem,

especially in dwarf plants and rosette plants. Hence, gibberellins are
involved in the regulation of stem elongation. B.O. Phinney and P.W.
Brian, while working on maize and garden pea found a direct 159
Block 4 Nitrogen Metabolism and Plant Growth Regulators
relationship between the dwarfing or internode length genes and
gibberellins. Application of exogenous gibberellins to the dwarf mutant
restores the normal length in phenotype. Gibberellins are also supposed
to induce elongation in length of internode as noted in maize. You will
also see a demonstration of this effect of gibberellins (Demonstration 1)
in your practical classes.

• The gibberellins increase the size of leaves in number of plants. The

leaves of pea, beans, cucumber, lettuce and cabbage showed increase
in size after spray of gibberellins. The increase in the photosynthetic
area increases the productivity of plants.

• GA regulates the transition from juvenile to adult phase. In many woody

perennials, the juvenile phase (phase in which no flower or fruits are
produced) can be shortened by treatment with GA3.

• GA promotes flowering in long day plants. GA promotes formation of

staminate flowers and inhibitors of GA promote formation of pistillate
flowers.

• GA promotes pollen development and pollen tube growth in plants.

• GA promotes fruit set and fruit growth in plants. GA induced fruit set
may occur in absence of pollination resulting in parthenocarpic fruits. In
grapes the seedless variety produces small fruits which are enlarged by
treatment of GA3.

• Gibberellins play a role in synthesis of RNA and proteins. Exogenous

application of GA causes an increase in level of α amylase and RNA.
Application of GA results in organization of endoplasmic reticulum into
stacks. These are the site of synthesis of α amylase. According to
Butcher et al. (1970) the hormone, acts as the first messenger which
helps in the synthesis of 3, 5 cyclic AMP (the second messenger). This
messenger activates enzyme precursors for producing active enzymes
required for various physiological functions.

Bioassays
The bioassays for the hormones are based on study of plant responses like
reversal of stem dwarfism, synthesis of α amylase in barley endosperm. A
germinating seed is given gibberellin treatment. The length of the epicotyls
and growth of hypocotyls is measured.

In rosette plants, there is a lack of internode elongation resulting in compact

growth with densely spaced leaves. This condition results from mutation in the
step in the GA1 biosynthetic pathway. Hyper-elongation of stems has been
noted in rosette plants after application of gibberellins. Some plants such as
spinach and cabbage do not flower in rosette form. They undergo internode
elongation before flowering. This is known as bolting. The process is
triggered by an environmental signal, change in photoperiod or low
temperature. Zeevaart et.al (1979) found that spinach contains six gibberellins
including GA19, GA20. The gibberellin GA20 causes bolting in rosette plants of
spinach under short day conditions. Spinach contains high levels of inactive
form of GA19 and low levels of active GA20. The level of GA20 increases while
160 GA19 decreases under long day conditions.
Unit 14 Plant Growth Regulators

The gibberellins initiate the mobilization of nutrient reserves stored in the

endosperm in cereal grains. In cereals gibberellins are released by the
hydrated embryos. Cereal grains such as rye, barley, wheat have a protein
rich layer of cells called aleurone which surrounds the starchy endosperm.
During germination of seed, the cells in the aleurone secrete hydrolytic
enzymes such as α amylase and proteases (Fig. 14.8).

Fig. 14.8: Gibberellin induced seed germination.

These enzymes are involved in the hydrolysis of carbohydrate and proteins

stored in the endosperm. The starch and proteins provide nourishment to the
developing embryo. It is suggested that the germinating embryo sends a
signal which is probably a gibberellin to the aleurone cells. The gibberellins
activate or repress the transcription of genes encoding for hydrolytic enzymes.
It has been demonstrated that GA induced secretion of α amylase could be
blocked by inhibitors such as actinomycin D and cycloheximide. These
chemicals inhibit RNA and protein synthesis.

Spray of gibberellins increases the length of floral stem. Larger fruit size is
obtained by the spray of gibberellin at the time of pollination and fruit set.
Gibberellins enhance seed germination and seedling emergence in species
such as grapes, citrus, apples, peach and cherry. The GA induced α amylase
in barley aleurone led to its widespread use of GA in the malting industry. The
growth of stems can be reduced by using growth retardants or antigibberellins.
The blocking specific steps in gibberellin biosynthesis reduce endogenous
levels of gibberellin suppressing internode elongation. These are used mainly
for ornamental plants. This is done to get shorter, compact and darker green
foliage. 161
Block 4 Nitrogen Metabolism and Plant Growth Regulators

SAQ 1
a) State whether the statements are ‘True ‘or ‘False’

i) Hormones influence the physiological processes in plants even at

low concentrations.

ii) Auxin is synthesized mainly in the non-meristematic regions of the

plant.

iii) Auxins can be found free or bound form in plants.

iv) Low levels of auxin can promote the formation of roots in plants.

v) The translocation of gibberellins is polar.

vi) Gibberellins promote flowering in long day plants.

b) Answer in one word:

i) Who discovered auxin as the growth hormone in plants.

ii) The precursor of auxins in plants.

iii) The mode of auxin transport in plants.

iv) The chemical substances when applied to plants as foliar spray or

soil supplement reduce stem elongation.

SAQ 2
Fill in the blanks:

a) The auxin was discovered first through experiments on ……………… of

a grass (Phalaris) illuminated from one side.
b) ……………….. is the most widely distributed naturally occurring auxin.
c) Auxin stimulates cell elongation in ……………. and ………………… .
d) Auxins promote the cell wall expansion by promoting of …………protons
which increase ………………. of the cell wall.
e) Auxins promote development of fruits without …………. and …………….
f) Gibberellins promote ………………. of stem in dwarf plants.

14.2.3 Cytokinins
The first naturally Cytokinins are the derivatives of nitrogenous base, adenine with isoprene
occurring cytokinins related side chain or aromatic side chain. Accordingly, the cytokinins are
were isolated from
called isopernoid cytokinins or aromatic cytokinins. The most common
milky endosperm of
cytokinins are N6-∆ 2-isopentenyl-adenine (iP), trans-zeatin (tZ), dihydrozeatin
maize and
developing plum (DZ). Kinetin is a synthetic derivative not found in plants.
fruit.
The first experimental evidence for cytokinins came through Haberlandt in
162 1913. He found that a chemical controls the cell division in plants. He
Unit 14 Plant Growth Regulators
demonstrated that phloem sap could cause the non-dividing parenchymatous
tissue to revert to an actively diving meristematic tissue. F. Skoog while
studying the nutritional requirement of tissue cultures found that stem tissue
explants containing vascular tissue proliferate on auxin containing medium.
These explants if freed from vascular tissue exhibited cell enlargement. They
showed cell division if extracts of vascular tissue, coconut milk and yeast were
added to the media. Later C.O. Miller was able to identify the active material
as adenine.

Synthesis of Cytokinins
Two mechanisms have been proposed for cytokinin biosynthesis. One is tRNA
induced cytokinin synthesis and the other is synthesis of free cytokinins.
Studies have shown that significant amount of free cytokinin in plants is
derived from the degradation of tRNA. The isopentenyl groups are transferred
from isopentenyl pyrophosphate (IpPP) using an enzyme prenyl transferase.
This enzyme synthesizes tRNA, and is required for the synthesis of isoprenoid
compounds. It recognizes a specific base sequence in the tRNA molecule and
transfers the isopentenyl group to the adenosine adjacent to the 3′ end of the
anticodon. The cis-zeatin formed subsequently gets converted to active trans-
zeatin by the enzyme cis-trans-isomerase.
During the synthesis of free cytokinins, an enzyme isopentenyl pyrophosphate
plays an important role. AMP isopentenyl transferase, also known as cytokinin
synthase catalyzes the conversion of 5-AMP (adenosine-5’-monophosphate)
and isopentenyl pyrophosphate into i6ADE (adenine). The enzyme is a type of
prenyl transferase and removes phosphate to form isopentyl adenosine. The
enzyme converts AMP and dimethylallyl-diphosphate (DMAPP) to the active
cytokinin isopentenyladenosine-5′-monophosphate (iPMP). The products of
the enzyme activity are isopentenyladenosine-5′-triphosphate (iPTP) and
isopentenyladenosine-5′-diphosphate (iPDP), which subsequently get
converted to zeatin (Fig.14.9).

dimethylallyl-diphosphate + ATP/ADP
(DMAPP)

isopentenyl ATP/ADP

isopentenyl AMP zeatin riboside di/ triphosphate

isopentenyl adenosine zeatin riboside monophosphate

isopentenyl adenine (iP) zeatin riboside

trans zeatin

Fig. 14.9: Cytokinin biosynthesis in plants.

163
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Zeatin and Ip are biologically active cytokinins in plants. Cytokinins are
synthesized in both root and shoot meristems during active growth period.
Cytokinins produced in shoot meristem get distributed to long distances
whereas those synthesized by root meristems get transferred to tissues lying
close to the root. In roots, cytokinins are synthesized primarily in the root tip.
They get translocated to the aerial parts of the plant through xylem. Cytokinins
are transported from the root to the shoot via xylem as zeatin riboside. The
presence of cytokinins in the xylem sap proves the movement of hormone is
polar. Immature seeds and developing fruits also contain high levels of
cytokinins but do not show any apparent movement. After reaching the leaves,
some amount of zeatin ribosides either get converted to free base or to
cytokinin glucosides. These glucosides get compartmentalized within cells and
are not available for physiological functions.

Functions of cytokinins

• Cytokinins regulate cell division or cell cycle. The absence of hormones

causes the cells to be arrested in G1 or G2 phase of the cell cycle. The
cells showing arrested division in G2 phase do not possess cyclin –
dependent kinases (CDK). The activity is reduced due to high level of
phosphorylation on the tyrosine residue. If cytokinin supply is restored
the tyrosine gets dephosphorylated, enzyme gets activated and cell
division is resumed. Thus cytokinin plays a role in generation of active
CDK complex that initiates cell division by catalyzing the transition from
the G2 phase of mitosis or M phase in cultured cells.

Cytokinins are also known to possess high level of D-type cyclin (CycD3)
which controls the transition from G1 to S phase. Cytokinins initiate the
cell division at the G1 to S phase transition through the induction of
CycD3.

• Cytokinins have the capacity to initiate cell division in quiescent and non-
dividing cells. Cytokinins promote shoot growth by increasing cell
proliferation in the shoot apical meristem. The hormone initiates cell
proliferation and division in tissues cultured in media having both auxin
and cytokinin.

• Cytokinins induce organ formation in tissue culture conditions. The ratio

of auxin and cytokinins controls root, shoot initiation in callus tissues and
growth of axillary buds. If the ratio of auxin to cytokinin is high, the
cultures initiate root formation. Conversely, low auxin to cytokinin ratio
promotes shoot formation. Equal proportion of the two causes
continuous proliferation of the callus.

• Kuraishi and Okumara (1956) found that cell enlargement in mature

leaves occurs in the presence of kinetin.

• Cytokinins antagonize the effect of auxin in regulating the growth of

axillary buds or apical dominance. Recent studies have demonstrated
that elevated auxin levels show increased apical dominance while the
overproduction of cytokinins reduces apical dominance. The application
of the cytokinins to shoot apex or axillary bud directly releases the bud
164 from inhibition.
Unit 14 Plant Growth Regulators
• Cytokinins are also considered as the inhibitors of senescence.
Exogenous application of cytokinins delays onset of senescence,
maintain protein levels and prevents chlorophyll breakdown in detached
leaves. Richmond and Lang (1975) reported delay of senescence
(disappearance of chlorophyll and protein degradation) in the detached
leaves of Xanthium treated with kinetin. The hormone proved effective in
removing the effect of ageing which was referred to as Richmond-Lang
effect. Cytokinins are supposed to direct nutrient mobilization and help
in retention of nutrients by stimulating the metabolism in the area of
cytokinin application.

• Cytokinins have been found to be very effective in breaking the

dormancy of seeds and other plant organs. They substitute the red light
requirement for breaking the seed dormancy.

Bioassays

Cell division test

These tests are performed in tissue cultures of tobacco pith tissue and
soyabean callus tissue. The cultures were maintained in media for about 2 to 4
weeks and their fresh and dry weights were compared with controls. The
changes in growth tell about the role of cytokinins.

Chlorophyll preservation test

The effect of delay in senescence was studied using Xanthium

pennsylvanicum by Osborne and McCalla (1961). The leaves of the plants
were allowed to senescence by keeping them in dim light. The leaf discs were
kept in dark for 48 h on the filter paper soaked in the test solution. The
chlorophyll was extracted and their levels were measured. A linear relationship
between the amount of chlorophyll retained and the log of the concentration of
cytokinins was noted.

Germination test

In this bioassay, the promotive effect of cytokinin on seed germination was

checked. The lettuce seeds were kept in the test solution in the dark at 25o C
for 8 h. The seeds were then allowed to germinate for 24 to 60 h. The effects
on seed germination and plant growth were recorded.
Table 14.2: Summary of the effect of Auxins, Gibberellins and Cytokinin.

Activity Auxin Gibberellins Kinin

Cell elongation Yes Yes Yes
Cell division Yes Yes Yes
Polar transport Yes No No
Avena curvature test Yes No No
Apical dominance and Yes No No
Dormancy
Root initiation Yes No No
165
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Prevention of abscission Yes - -
Preventive of lodging Yes No No
Selective herbicide Yes No No
Flowering in pineapple and Yes No No
Litchi
Parthenocarpy Yes Yes No
“Bolting” - Yes -
Flowering of long day plants - Yes -
Substituting vernalisation No Yes No
Dwarf Maize test - Yes -
Breaking dormancy No Yes Yes
Seed germination - Yes Yes
Flowering of short day plants No No Yes
Morphogenesis No No Yes
Richmond-Lang effect No No Yes
Tobacco pith culture test No No Yes

SAQ 3
Fill in the blanks:

a) …………………. is a synthetic derivative which is not found in plants.

b) Immature seeds and developing fruits contain high…….. of……….. ..

c) For initiation of root the ratio of auxin to cytokinin should be……….. .

d) Over production of cytokinins will reduce…………………. .

e) Cytokinins are very effective in ………of seed dormancy.

14.2.4 Abscisic Acid

The compound was discovered accidently as an interfering substance in the
experiments of auxin response in Avena coleoptiles. Bennet-Clarke and
Kefford found that the substance inhibited the growth of coleoptiles sections
and called it as inhibitor β. The compound was supposed to be involved in
apical bud dominance and maintaining dormancy. In 1964, P.F. Wareing
proposed the term ‘dormin’ for the endogenous dormancy inducing substance.
The substance that accelerated abscission was isolated from the senescing
leaves and was named abscission II. The compound was later named as
abscisic acid.

Two pathways have been proposed for the synthesis of abscisic acid (ABA). In
most of the plants, ABA is synthesized directly from a 15 carbon terpenoid
precursor, farnesyl phosphate. Indirect pathway suggests its synthesis from
166 cleavage of carotenoid such as β carotene via 40 carbon terpene violaxanthin.
Unit 14 Plant Growth Regulators
Enzyme nine-cis-epoxycarotenoid dioxygenase (NCED) cleaves the 40 carbon
violaxanthin to produce a 15 carbon product xanthoxin. Xanthoxin gets
converted to abscisic acid aldehyde by an enzyme alcohol dehydrogenase
which further gets oxidized to abscisic acid by abscisic aldehyde oxidase. The
synthesis of enzymes NCED and xanthoxin occurs in the chloroplast while the
rest of the reactions occur in the cytosol (Fig. 14.10).

Fig. 14.10: Schematic representation of ABA biosynthesis in plants.

Studies suggested that ABA precursors originate in the chloroplasts but ABA
is formed in the cytosol. NCED expresses itself in the guard cells under
conditions of senescence in leaves and cotyledons, while the enzyme abscisic
acid oxidase gets induced in guard cells under conditions of water stress.
Thus, the site of ABA synthesis is guard cells under stress conditions, while
vascular tissues are the site of ABA synthesis in non-stressed plants. ABA is
highly mobile and rapidly gets transferred to other parts of the plant.

Functions

• ABA regulates maturation of embryo and seed germination. In seeds,

the cytokinin levels are high during early stages of embryo development
(when cell division is fast). During phase of cell enlargement, the IAA
and GA levels increase but at later stages of embryo development
(maturation) the ABA levels increase. During this stage there is
cessation of embryo growth, accumulation of nutrient reserves in the
endosperm and development of tolerance to desiccation.

ABA plays a critical role in maturation of embryo. It prevents precocious

germination or vivipary. This has been established through viviparous
mutants in maize and Arabidopsis. The mutants show low levels of ABA 167
Block 4 Nitrogen Metabolism and Plant Growth Regulators
in seeds and they germinate prematurely on the cob itself before the
seeds enter the dormancy. ABA stimulates the protein accumulation in
the later stages of embryo development and prevents α amylase
biosynthesis in cereal grains. This shows the role of ABA in seed
maturation and prevention of premature or untimely germination.

• ABA accelerates senescence of leaves.

• It inhibits germination of seeds such as those of lettuce.

• It inhibits gibberellin induced growth in various tests. It is suggested to

be an antagonist to gibberellin. It inhibits gibberellin induced α amylase
in barley aleurone.

• It promotes ageing and abscission of leaves.

• It promotes root growth but inhibits shoot growth.

• ABA accumulates in high amounts in leaves during wilting. Increased

concentrations of ABA have been noted in leaves showing stomatal
closure during water stress conditions. ABA is known to act as an
antitranspirant since it possesses the capacity to induce stomatal closure
under water stress conditions. This reduces water loss through
transpiration. Inhibition of electron transport and photophosphorylation in
chloroplast disrupts the proton accumulation in the thylakoid lumen
which lowers the pH of the stroma. The pH of the apoplast surrounding
the mesophyll cell increases at the same time. The pH gradient
stimulates release of ABA from mesophyll cells into the apoplast which
gets carried to guard cells via transpiration stream.

• ABA is also supposed to play a role in lateral or secondary root

development. Application of hormone to the lateral meristem at early
stages of root growth promotes lateral root formation. Studies suggested
that endogenous ABA inhibit or delay flowering in plants like
Arabidopsis.

It has been suggested that ABA inhibits biosynthesis of several growth

hormones or inactivates them. It inhibits RNA and protein synthesis but at the
same time stimulates the activity of hydrolytic enzymes.

14.2.5 Ethylene
It is a hormone which is represented by a gaseous hydrocarbon. It is
synthesized primarily in response to stress and is produced in high amounts
by tissues undergoing senescence or fruit ripening. It is commonly used to
induce ripening in bananas. It occurs in all plant organs-roots, stems, leaves,
bulbs, fruits seeds, though the rate of production varies in each organ.

The effect of ethylene on plants was described by Dimitry Nikolayevich

Neljubow in 1886. He found abnormal growth of dark grown pea seedlings
due to ethylene emanating from illuminating gas. Later studies reported the
role of this hormone in ripening of fruits. H.H. Cousins reported a volatile
substance to be responsible for fruit ripening in plants. He discovered that ripe
168 oranges released a volatile product that accelerates ripening of bananas.
Unit 14 Plant Growth Regulators
Synthesis of ethylene
M. Lieberman and L.W. Mapson in 1964 demonstrated that methionine gets
converted to ethylene non-enzymatically. D. Adams and F. Yang (1979)
showed that S-adenosylmethionine (SAM) is the intermediate compound
formed during synthesis of ethylene from methionine. Under aerobic
conditions, 1-aminocyclopropane-1-carboxylic acid (ACC) gets converted to
ethylene. The enzyme ACC synthase catalyzes the oxidation of ACC to
ethylene (Fig. 14.11).

Methionine
SAM synthetase

spermine/spermidine
S-adenosyl methionine (SAM) (S-AdoMet)
biosynthetic pathway
ACC synthase

1-aminocyclopropane-1-carboxylic acid (ACC)

ACC oxidase

Ethylene

Fig. 14.11: Ethylene biosynthesis in plants.

The cleavage of SAM to give rise to 5 methylthioadenosine (MTA) and ACC is
mediated by enzyme ACC synthase and is the rate limiting step in ethylene
biosynthesis. Ethylene production is promoted by factors such as IAA,
wounding and water stress. These factors induce the synthesis of enzyme
ACC synthase. Control of the ethylene production is supposed to be regulated
primarily through transcription regulation of the ACC synthase gene.

Functions
Ethylene produces many effects on growth of plants.

• It mainly promotes fruit ripening and senescence. Application of ethylene

promotes climacteric effect and induces ripening. The role of ethylene in
fruit development has been studied in tomato, a climacteric fruit. When
the fruit matures, chemical and physiological changes begin which help
in ripening of fruit. It involves a significant increase in the rate of
respiration followed by a decline. This period is referred as climacteric
rise and it signals the onset of degeneration or senescence. During
climacteric phase, the concentration of ethylene increases several fold.
The complex carbohydrates break into simple sugars, cell walls become
soft due to action of hydrolytic enzymes (cellulase and pectinase).
Chlorophyll degradation occurs and volatile compounds for the flavour
and aroma are produced. This has been noted in fruits such as
bananas, lemons, oranges.

• Generally a compound 2-chloroethanephosphonic acid (CEPA) is

sprayed on the plant to induce the effect.

The ethylene production is followed by expression of set of genes that

enhance ripening related activities such as development of fruit colour,
flavour and texture. In mutant called as never ripe, ethylene is non- 169
Block 4 Nitrogen Metabolism and Plant Growth Regulators
responsive because of defective ethylene receptor protein. In this plant
ripening genes are not expressed and fruit never develops red color and
has no flavor and texture as of a ripe tomato.

• The hormone has been shown to stimulate elongation of stems, petioles,

root and floral structures in aquatic and semi-aquatic plants. The
submergence reduces gas dispersion and maintains high ethylene
levels.

• Ethylene stimulates many inhibitory and abnormal growth responses

such as swelling of stem tissues and downward curvature of leaves
called epinasty. Epinasty occurs due to excessive elongation on adaxial
side of petiole. It is a common response of plants to anoxia under
conditions of water logging. The vertical orientation of epinastic leaves
reduces the absorption of sunlight and transpirational water loss.

• Ethylene also plays a role in seed germination, apical dominance, fruit

ripening, cell death and pathogen responses.

• It stimulates the initiation of root and regulates the formation of root

nodules in legumes. It inhibits the formation of bulbs, tubers in many
flowering plants. The hormone induces several genes related to the
ethylene response factor (ERF).

• Auxin induced ethylene formation Inhibition of stem elongation and

swelling in the cells. Ethylene inhibits transport of auxin.

• One of the effects of the ethylene is called ‘triple response’. It is noted in

etiolated (grown in dark) dicot seedlings. The response is characterized
by inhibition of hypocotyl and root cell elongation. The response may be
produced due to ethylene mutation. In presence of endogenous
ethylene, the triple response is not seen.

SAQ 4
a) Cytokinins play a role in cell division. Explain.

b) State whether the following statements are True and False:

i) P.F. Wareing proposed the term ‘dormin’.

ii) Abscisic acid delays senescence in leaves.
iii) ABA does not play any role in maturation of embryo.
iv) ABA prevents vivipary in fruits.
v) Ethylene is a gaseous hydrocarbon.

c) Fill in the blanks:

i) ……………… mainly promotes fruit ripening.

ii) Ethylene is responsible for…………….……… in leaves.
iii) During climacteric phase the concentration of ethylene
……………………. several folds.
iv) Ethylene also regulates …..………………..formation in legumes.
v) Ethylene inhibits transport of ……..……………… .
170
Unit 14 Plant Growth Regulators

14.3 NEW CLASS OF PLANT HORMONES

Apart from the five hormones which we have described above, there are
several newly discovered hormones. We have discussed these hormones in
coming section. Some of the important ones are brassinosteroids, salicyclic
acid, strigolactones and jasmonic acid. Many new naturally occurring
substances in this category are still being discovered.

Brassinosteroids
These are steroid hormones. They were discovered in 1970. A group of
agricultural researchers showed that pollen possess a rich source of growth
promoting substances. These are a complex mixture of lipids and have shown
to capacity to stimulate elongation of second internodes in bean. The active
substances were isolated from rape plant i.e. Brassica napus and called as
Brassins. In 1979, the active component was identified as brassinolide (BL).
More than 40 analogs of BL have been isolated from 60 plant species and
from tissues such as pollen, seeds, leaves, stem, roots and flowers. The
hormone is active even at very low concentrations (micromolar). It has been
suggested that responses of brassionosteroids (BR) are controlled by auxins.

Brassinosteroids play a role in various developmental activities in plants.

These include:

• stem growth

• pollen tube elongation

• increase in rate of cell division

• seed germination

• leaf morphogenesis

• apical dominance

• inhibition of root elongation

• vascular differentiation

• acceleration in senescence and cell death.

• increase in extensibility of the cell wall by regulating the genes encoding

enzymes such as expansins and cellulose synthase.

• mediate responses to both abiotic and biotic stresses including salt,

drought, temperature and pathogens.

Salicyclic acid
Salicylic acid (SA or 2-hydroxybenzoic acid) is the phenolic compound
synthesized by plants. It plays direct and indirect roles in regulating many
aspects of plant growth and development such as thermogenesis and disease
resistance.

The first evidence that SA is a plant hormone came from studies of voodoo lily
(Sauromatum guttatum). SA initiates thermogenesis in lily. The thermogenic
response was initiated by calorigen which was later characterized as SA. 171
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Thermogenesis is a process that generates heat by uncoupled electron flow
through alternative oxidase pathway in mitochondria. The levels of SA
increase several fold (~100) prior to such event. Externally applied SA also
induced thermogenesis. The hormone induces thermogenesis primarily by
stimulating the mitochondrial alternative respiratory pathway. This pathway
generates one ATP and releases the energy from electron flow as heat. SA
treatment also induces the expression of alternative oxidase and/or the
alternative respiratory pathway in non-thermogenic plant species.

Functions

• The hormone plays a role in defense signaling. Earlier studies

established that application of SA to tobacco plants induces defense
gene expression and enhances resistance to viral infection. It plays a
role in disease resistance. It prevents local pathogen infection but
mediates Systemic Acquired Resistance (SAR) at the sites far from the
site of infection. Rise in levels of SA were detected prior to the
development of local and/or systemic disease resistance in tobacco and
cucumber.

• SA also acts as a signal for flowering. The SA-deficient mutants of

Arabidopsis showed delay in flowering.

• Exogenous SA has been shown to affect resistance to biotic (pathogen-

associated) stress and tolerance to many abiotic stresses (drought,
chilling, heat, heavy metal, UV radiation, and salinity/osmotic stress).

• The hormone affects many aspects of plant growth and development

including seed germination, vegetative growth, flowering, fruit yield,
senescence, stomatal closure, root initiation/growth, photosynthesis,
respiration, glycolysis, Krebs cycle, and the alternative respiratory
pathway. Some of these processes are induced by SA in a
concentration-dependent manner. They get activated by treatment with a
low dose of SA but inhibited by a high dose.

• SA retards senescence of petals by lowering the ethylene levels.

• SA plays a role in regulating cellular redox status. Low concentrations of

SA induce accumulation of ROS which serve as secondary signals to
activate biological processes. In contrast high concentrations of
exogenous SA result in accumulation of ROS at high levels leading to
oxidative stress and cell death.

Strigolactones

Strigolactones (SLs) are the new group of hormones discovered in plants.

Studies have shown that plants can transport strigolactones internally. Recent
studies suggested that they play a major part in the interaction between plants
and their environment.

Low concentrations of strigolactones prove very effective in altering the

growth and development in plants. These hormones have been found to play a
172 crucial role in branching of plants. They play an important role in interaction
Unit 14 Plant Growth Regulators
between plants and symbiotic fungi. The fungi live in a symbiotic relationship
with plants and this interaction is mutually beneficial. Fungi transports minerals
from the soil to the plant and in return the plant gives sugars to the fungi. SLs
released by roots of host plants serve to attract arbuscular mycorrhiza to the
root surface and stimulate branching of fungal hyphae and facilitate the
phosphate uptake from soil. The hormones assist the seeds of parasitic plants
to germinate. The seedlings of the parasite attach to the root of the plant and
use the plant’s nutrients for their own growth and reproduction. The mutant
plants showed low seed germination in parasitic plant.

Jasmonic acid

The jasmonates (JAs) including jasmonic acid and its derivatives are plant
hormones. They control plant defenses against herbivore attack and pathogen
infection, provide tolerance to abiotic stresses (ozone, ultraviolet radiation,
high temperatures, freezing), regulate various aspects of development,
including root growth, stamen development, flowering, and leaf senescence.

Exogenous application of JAs inhibits various aspects of seedling growth,

including primary root growth, leaf expansion, and hypocotyl elongation. They
also repress leaf expansion by inhibiting the activity of the mitotic cyclin
CycB1,2 and cell division.

Nitric oxide

Nitric oxide (NO) is a reactive gas known to be produced by plants. It has been
synthesized during nitrate assimilation but under conditions of low oxygen
availability, the nitric oxide is produced by reduction of nitrite. It can also be
produced non-enzymatically from nitrite in acidic condition found in plant cell
walls.

The main role of NO in plants include regulation of stomatal closure, lateral

root initiation, flowering, seed germination, responses to abiotic stresses and
plant defense against pathogen attack by stimulating production of H2O2.

NO2- + 2e- + 2H+ → NO + H2O

NO2- + H+ → HNO2 → NO + H2O + 2R (biological reductant)

Karrikins

These are plant growth regulators produced from burning plant material. Some
karrikins might be produced by processes such as chemical or microbial
degradation of vegetation in response to disturbance of the soil or removal of
the plant canopy. Karrikins comprise C, H, O and belong to the class of
butenolide compounds. They contain two ring structures, one of which is a
pyran and the other one is a lactone comprising of five membered ring known
as butenolide.

Karrikins are potent in breaking dormancy of seeds of many species adapted

to environments that regularly experience fire and smoke. They trigger seed
germination and control seedling growth in taxa that would rarely experience
fire. Endogenous karrikins present in plants control of seed germination. 173
Block 4 Nitrogen Metabolism and Plant Growth Regulators

SAQ 5
a) Make a list of new plant hormones that play a role in plant development
and growth.

b) List the developmental activities in which brassinosteroids have a role to

play.

c) Write a short note on Karrikins.

14.4 SYNTHETIC GROWTH HORMONES

Synthetic auxins have commercial importance. Some of the important
synthetic auxins include indole-3-acetic acid (IAA), indole-butric acid (IBA),
beta naphthoxy acetic acid (NOA), 1-naphthaleneacetic acid (NAA), (2,4-
dichlorophenoxy) propionic acid (2,4-D). Examples of some commonly used
synthetic cytokinins are 6-benzylaminopurine (BAP) and 6-furfurylaminopurine
or kinetin.

Synthetic auxins such as 2, 4 -D and and 4-chlorophenoxy acetic acid (4-CPA)

are used as herbicides. Synthetic auxins are highly effective because they are
not metabolized by the plant as quickly as IAA. They are retained in shoots for
long time. They induce excessive cell expansion and subsequent cell death.
Indole butyric acid and naphthalene acetic acid are used in vegetative
propagation (through stem and leaf cuttings) and rooting.

4-CPA is sprayed on tomatoes to increase flowering and fruit set while NAA is
used to induce flowering in pineapple. This happens due to auxin induced
ethylene production.

NAA is also used to prevent fruit drop in pear and apples. Spraying in early
stages of fruiting enhances abscission of young fruits while spraying after fruit
maturation prevents premature fruit drop and keeps fruit attached to the tree
till maturity.

14.4 SUMMARY
• Hormones are the chemical messengers produced in one cell and
modulate the cellular processes in another cell by interacting with
specific proteins that function as receptors. Major hormones that
regulate growth and developmental activities in plants include auxins,
gibberellins, cytokinins, ethylene, and abscisic acid.

• Auxin was the first growth hormone discovered in plants. It plays an

important role in enlargement of plant cells. It is synthesized in the
meristematic region and other actively growing regions. Auxin
synthesized in shoot apex gets transported basipetally to the tissues
below. The hormone stimulates cell elongation in stem, coleoptiles and
promotes vascular differentiation in shoots. High levels of auxin promote
174 the formation of lateral and adventitious roots.
Unit 14 Plant Growth Regulators
• Gibberellins are diterpene compounds synthesized mainly in the
developing seeds, fruits, young leaves and apical region of roots. They
promote seed germination. They initiate the mobilization of nutrient
reserves stored in the endosperm in cereal grains. They possess the
capacity to stimulate elongation of stem.

• Cytokinins are the derivatives of adenine with isoprene related side

chain or aromatic side chain. The regulate cell division. They have the
capacity to initiate cell division in quiescent, non-dividing cells and have
been considered as the inhibitors of senescence. Exogenous application
of cytokinins delays onset of senescence, maintain protein levels and
prevents chlorophyll breakdown in detached leaves.

• Abscisic Acid (ABA) is a growth regulator synthesised from cleavage of

carotenoid such as β carotene to produce 40 carbon terpene
violaxanthin. ABA plays a role in maturation of embryo and seed
germination. It prevents precocious germination. It plays a role in closure
of stomata during water stress and acts as an antitranspirant.

• Ethylene is a hormone which is represented by a gaseous hydrocarbon.

It is synthesized primarily in response to stress and is produced in high
amounts by tissues undergoing senescence and ripening. It is commonly
used to induce ripening in fruits and senescence.

• Some new hormones have been discovered recently in plants. They

include brassinosteroids which play a role in pollen tube elongation, leaf
morphogenesis and apical dominance. They also mediate responses to
both abiotic and biotic stresses. Salicylic acid (SA) is the phenolic
compound synthesized by plants. It plays direct or indirect roles in
regulating many aspects of plant growth and development such as
thermogenesis and disease resistance. Strigolactones have been found
crucial for the branching of plants and play a major role in interaction
between plants and symbiotic fungi.

14.5 TERMINAL QUESTIONS

1. Fill in the blanks.

a) Transport of auxin in plants is ……………….. .

b) The most commonly found form of gibberellin ………………. .

c) The precursor of ethylene in plants is…………………… .

d) The steroid hormone found in plants is …………………. .

e) The precursor of abscisic acid in plants is ………………………. .

f) …………………. hormone plays a role in opening and closing of

stomata.

2. Enlist the major role that auxins play in plants.

3. How do gibberellins promote seed germination?

4. What is epinasty? It is linked with which hormone in plants. 175

Block 4 Nitrogen Metabolism and Plant Growth Regulators
5. Give the full form of abbreviations.

a) IAA

b) IBA

c) tZ

d) iP

e) ACC

f) SA

6. Discuss in brief :

a) Auxin and cell elongation

b) Ethylene and ripening of the fruit

c) Discovery of auxins

d) Discuss functions of Gibberellins.

7. Describe the effect of Auxins, Gibberellins and Kinins in a tabular form.

14.6 ANSWERS
Self-Assessment Questions
1. a) i) True; ii) False; iii) True; iv) True; v) False; vi) True

b) i) F.W. Went

ii) Tryptophan

iii) Basipetal

iv) Antigibberellins

2. a) coleoptiles

b) Indole -3-acetic acid (IAA)

c) stem, coleoptiles

d) efflux, acidity

e) pollination, fertilization

f) elongation

3. a) Kinetin

b) levels, cytokinins

c) high

d) apical dominance

176 e) breaking
Unit 14 Plant Growth Regulators

4. a) Cytokinins regulate cell division or cell cycle. In the absence of

gibberellins, cells division gets arrested in G1 or G2 phase of the
cell cycle. The cells showing arrested division in G2 phase do not
possess cyclin –dependent kinases (CDK) activity. The activity
reduced due to high level of phosphorylation on the tyrosine
residue. In presence of cytokinin, tyrosine gets dephosphorylated,
enzyme gets activated and cell division is resumed. Thus cytokinin
also plays a role in generation of active CDK complex that initiates
cell division by catalyzing the transition from the G2 phase of
mitosis or M phase in cultured cells. Cytokinins are also known to
possess high level of D-type cyclin (CycD3) which controls the
transition from G1 to S phase. Cytokinins initiate the cell division at
the G1 to s transition through the induction of CycD3.

b) i) True; ii) False; iii) False; iv) True; vi) True

c) i) Ethylene

ii) epinasty

iii) increases

iv) nodules

v) auxins

5. a) Few newly discovered hormones that play a role in plant growth

and development are

• brassinosteroids

• salicyclic acid

• strigolactones

• jasmonic acid

• nitric oxide

b) Refer to Section 14.3.

c) Refer to Section 14.3.

Terminal Questions
1. a) polar

b) GA3

c) methionine

d) brassinosteroid

e) β carotene

f) abscisic acid 177

Block 4 Nitrogen Metabolism and Plant Growth Regulators
2. The major roles of auxins in plants are

• stimulation in cell elongation in stem and coleoptiles.

• secondary root initiation, vascular differentiation, development of

axillary buds, flowers and fruits.
• vascular differentiation in shoots. The production of xylem strands
depends upon the amount of IAA moving through the petiole. The
extent of vascular regeneration is directly related to the supply of
auxin. The differentiation of phloem sieve elements is favored by
low concentration of auxin (0.1%) while the xylem differentiation is
favored by high auxin content (1%). In culture conditions, the
callus differentiates into vascular tissue in the presence of auxin.
• formation of lateral and adventitious roots.
• delays onset of leaf abscission (shedding). Application of auxin to
the cut surface of the petiole prevents the formation of the
abscission layer.

• promote fruit development.

3. Gibberellins promote seed germination. The gibberellins are released by

the hydrated embryos. Gibberellins initiate the mobilization of nutrient
reserves stored in the endosperm. During germination the cells in the
aleurone secrete hydrolytic enzymes such as α amylase and proteases.
These enzymes are involved in the hydrolysis of carbohydrate and
proteins (stored food reserves) accumulated in the endosperm or
embryo. The degradation of carbohydrates and storage proteins provide
nourishment and energy to the growing seedling. This happens in cereal
grains such as rye, barley, wheat which have a protein rich layer of cells
called aleurone surrounding the starchy endosperm. The starch and
proteins produced provide nourishment to the developing embryo.

4. Ethylene stimulates growth responses such as swelling of stem tissues

and downward curvature of leaves called epinasty. Epinasty occurs due
to excessive elongation on adaxial side of petiole. This response occurs
in response to anoxia under conditions of water logging. The vertical
orientation of epinastic leaves reduces the absorption of sunlight and
transpirational water loss.

5. a) IAA - Indole -3-acetic acid

b) IBA - Indole-3-butyric acid
c) tZ - trans-zeatin
d) iP - isopentenyladenine
e) ACC -1-aminocyclopropane 1-carboxylic acid
f) SA - Salicylic acid
6 a) Refer to Section 14.2.1.

b) Refer to Section 14.2.5.

c) Refer to Section 14.2.1.

178 d) Refer to Section 14.2.2.

Unit 14 Plant Growth Regulators
7. Auxins Gibberellins Cytokinins
Synthesised in the root Synthesised in root apex, Synthesised in the root
tip elongating shoots, young tip
leaves
Transport is basipetal Transport is both basipetal Transport is acropetal
and acropetal.
Promote branching of It has no role in rooting Inhibit root branching
roots
Has no role in Promote flowering in long Promote flowering in
flowering day plants short day plants
Has no role in seed Plays a role in seed Has no role in seed
germination germination germination
Brings about apical Has no role in apical Has no role in apical
dominance dominance. dominance.
Inhibits growth of Does not affect growth of Promotes growth of
lateral buds lateral buds lateral buds
Promotes for cell Does not play role in cell Mainly responsible for
elongation elongation or division cell division and
differentiation
Play no role in bolting Help in bolting of rosette Does not play any role
plants in bolting
Does not play any role Promotes growth of shoot Does not play any role
in growth of shoot segments in growth of shoot
segments segments
Essential for growth of It has no role in callus It has no role in callus
callus growth. growth.
Does not help in Help in breaking seed and Does not play any role
breaking seed and bud bud dormancy in seed and bud
dormancy dormancy
Involved in the Play no role in vascular Play no role in vascular
vascular differentiation differentiation in shoots differentiation in shoots
in shoots
Delays the onset of Does not play any role in Does not play any role
leaf abscission leaf abscission in leaf abscission
Promotes Does not play any role in Does not play any role
development of fruit development of fruit in development of fruit
Does not play any role Increase the size of leaves Does not play any role
in formation of leaves in plants in formation of leaves

179
Block 4 Nitrogen Metabolism and Plant Growth Regulators

UNIT 15
PLANT RESPONSE TO LIGHT
AND TEMPERATURE

Structure
15.1 Introduction 15.5 Plant Responses to
Temperature - Vernalization
Objectives
Mechanism of Vernalization
15.2 Photoperiodism
15.6 Rhythmic Changes -
Long day, Short day, and Day Biological Clocks
Neutral Plants
Factors affecting Rhythms
Importance of Dark Period
15.7 Senescence
Site of Perception of Light
Stimulus Biochemical changes associated
with senescence
15.3 Phytochrome
Regulation of Senescence
Discovery
15.8 Cryptochromes
Properties of Phytochrome
15.9 Plant Movements
Phytochrome-mediated
Responses 15.10 Summary
Mechanism of Action 15.11 Terminal Questions
15.4 Flowering Hormone 15.12 Answers
Biochemical changes

15.1 INTRODUCTION
The developmental phases of a flowering plant involve seed germination,
vegetative growth, flowering, fruiting, and senescence. These phases are
precisely timed and are under strict internal and external controls. You must
have noticed that a seed remains dormant all winter long buried in the sand,
suddenly germinates, develops into a mature plant and flowers in the spring. A
short rainfall in desert brings life to many plants and the dormant winter buds
become active in the spring. What external or internal factors initiate
germination which involves resumption of all the metabolic activities? How
does a seed sense that all the environmental conditions are favourable for its
180 germination and growth and how do buds know that the winter is over?
Unit 15 Plant Response to Light and Temperature
You also know that most fruits, vegetables, and flowers are seasonal.
Chrysanthemums bloom in winters while Gulmohar and Amaltas bloom in the
summer. Why do some plants bloom in the spring and others wait for summer,
fall or winter? Many flowers open and close on a fixed schedule as if a clock is
present in their cells. Now it would be interesting to find out whether it is
possible to grow Chrysanthemums throughout the year or make Morning
glories bloom at dusk? You can ask many such questions regarding
development of a plant.

In the previous unit you have learnt that hormones coordinate growth and
development in different parts of an organism by triggering cellular reactions in
the target cells. In this unit we will discuss the phases of plant development
that are under the control of specific environmental signals. You will see that
plant cells receive environmental signals which governs various
developmental events. The signals are received by some special molecules
and the genes are turned on at a precise time to regulate specific activity.

Objectives
After studying this unit, you should be able to:

describe seed formation and seed germination and the factors that
control these processes;

list the factors that are responsible for flower induction and discuss the
nature of the receptor that perceives external stimuli;

explain the role of phytochrome in controlling the period of light and

dark cycles in flowering;

discuss the role of phytochrome in regulating plant development;

list the various factors that affect onset of senescence;

describe the principle and uses of tissue culture technology; and

Give example of circadian rhythms regulated by biological clocks in

plants.

15.2 PHOTOPERIODISM
One of the major changes that occur during the life cycle of a plant is the
transition from vegetative stage to the reproductive stage. In this transition the
vegetative meristems change into flowering meristems which form sepals,
petals, carpels, and anthers instead of branches and leaves. It is a major
morphogenetic change. In some plants this change occurs once in lifetime and
after flowering, fruit and seed set, the plant dies. Whereas, in others, the
flowers are produced every year, as in trees. Now the question arises, is it due
to an internal annual rhythm in the plants or a requirement for environmental
conditions? In this section we will discuss what controls flowering and how this
transition occurs.

One of the major factors that affects flowering, as we know today is related to
the effects of the environment and to the duration of light/dark 24 hours cycle
or temperature. However, it took several years for scientists to realize this fact. 181
Block 4 Nitrogen Metabolism and Plant Growth Regulators
We will describe two experimental observations made by Garner and Allard
(1920) which suggested that the length of the day determines the behaviour of
plants with respect to flowering.

The phenomenon of
The seeds of soyabean (Glycine max) were planted at different times in the
photoperiodism was month of May, June, and August. Even though planted at different times, all of
discovered by two them flowered in the month of September. The first planted seeds took 125
American scientists, days to flower whereas the last set flowered in only 58 days. These
Garner and Allard in observations show that at a particular time of the year the day length was
1920, working at the suitable for plants to flower. The second observation was made on a mutant of
U.S. Department of
tobacco called Maryland mammoth, because of the large size of its leaves. In
Agriculture at
fields in winter, however, in glass houses when light duration was provided like
Beltsville, Maryland.
that of summer, it was possible to induce flowering even in winter in these
In temperate regions plants. The term photoperiodism was suggested by Garner and Allard to
the day length is more categorize plants based on their response to relative length of day and night
during summer. as manifested by their flowering.

15.2.1 Long day, Short Day and Day Neutral Plants

Based on their requirement for day length (number of hours of light) for
flowering, plants were grouped under three major categories, ‘Short-
day’(SDP), ‘Long-day’(LDP) and ‘Day-neutral’ plants. (Fig. 15.1)A SDP
requires more than a critical period of darkness. In this case SDP needed
more than 8 hours of darkness or less than 16 hours of light to flower. A LDP
requires less than a critical hour of darkness or more than a certain hour of
light. In this case, LDP required more than 10 hours of light to flower.

Fig. 15.1: A diagrammatic representation to show the difference between a

short-day plant (SDP) and a long-day plant (LDP).
1. Short-Day plants (SDP): These plants only flower or flower more
profusely and rapidly when given less than a certain (critical) number of
hours of light in a day (24 hours cycle). The critical period, let it be
clear, is not 12 hours. It can be for example 10 hours. In that case a
plant given less than 10 hours of light (say 8 hours light + 16 hours of
darkness) will flower. And this critical period is determined
experimentally and varies from plant to plant. Short-day plants are found
in tropical regions where day length varies comparatively little during the
year. If the short-day plants are to flower, the day length must not
182 exceed a critical value. The photoperiodic requirement of some plants is
Unit 15 Plant Response to Light and Temperature
controlled by temperature. Some of them have a qualitative photoperiod
requirement at one temperature, while at another temperature; their
requirement for photoperiod becomes quantitative.
Thus, the short-day plants can be further sub grouped into:

i) Obligate short-day plants: Such plants have an absolute requirement

for long nights and short days. They are also called obligates. Any other
condition given to them inhibits flowering. They are also called
Qualitative short-day plants.e.g., Amaranthus candatus, Chenopodium
album (pigweed) (Fig. 15.2a), Coffea arabica (Coffee), Euphorbia
pulcherimma, Frageria (Strawberry), Glycine max var. Biloxi
(Soyabean)(Fig.15.2b), Ipomoea hederacea (Morning glory), Lemna
perpusilla (Duckweed), Nicotiana tobacum Var. Maryland mammoth
(Tobacco), Xanthium strumarium (Cocklebur).

(a) (b)
Fig.15.2: Obligate short-day plants: a) Chenopodium album; and
b) Glycine max var. Biloxi.
ii) Facultative short day plants: are those whose flowering gets
accelerated by short days. Such plants are also known as Quantitative
short-day plants. e.g., Saccharum officinarum (Sugarcane), Gossypium
hirsutum(Cotton) are some of the examples (Fig. 15.3).

Interestingly, Allium cepa (Onion) and Chrysanthemum morifolium are

quantitative SDPs which flower better by low temperature pretreatment.
There are many of the above-mentioned plants that show a transition
between different photoperiods with varying temperature.

(a) (b)
Fig.15.3: Facultative short-day plants, a) Saccharum officinarum; and
b) Gossypium hirsutum. 183
Block 4 Nitrogen Metabolism and Plant Growth Regulators
iii) Short-long day plants (SLDP): These plants flower only when a
sequence of short days is followed by an exposure to long days (e.g.,
June-July). e.g., Campenula medium, Trifolium repens (Clover)
(Fig. 15.4).

(a) (b)
Fig.15.4: Short-long day plants a) Campenula medium; b) Trifolium repens.

2. Long-Day plants (LDP): The definition of this is exactly opposite to

short-day plant. That is those plants which flower when given more than
a certain (critical) number of hours of light (day length). These plants are
found in temperate region where they flower during summer.

The LDPs are also of three types:

i) Obligate/Qualitative long day plants: which must necessarily have

long days and short nights as their absolute requirement to flower? The
day length must always exceed a certain critical photoperiod. They could
even flower even when there is continuous light. e.g., Hyoscyamus
niger(Henbane), Spinacea oleracea (Spinach) (Fig. 15.5), Lolium
temulentum (Ryegrass), Avena sativa (Oat) and more.

(a) (b)
Fig.15.5: Obligate/Qualitative long day plants: a) Hyoscyamus niger
(Henbane); b) Spinacea oleracea (Spinach).

ii) Facultative/Quantitative Long Day Plants: accelerate their flowering in

long days e.g., Brassica rapa (Turnip), Beta vulgaris (Beet), Lactuca
sativa (Lettuce), Pisum sativum (Garden pea), Triticum aestivum (Spring
wheat), Secale cereale (Spring rye),Petunia hybrid (Fig. 15.6), Hordeum
184 vulgare (Spring barley).
Unit 15 Plant Response to Light and Temperature

(a) (b)
Fig.15.6: Facultative/Quantitative
ve/Quantitative Long Day Plants:
Plants a) Secale cereale;
b) Petunia hybrid.
iii) Long-Short (LSDP) These long day plants will flower when
Short day plants (LSDP):
they are exposed to long days prior to exposure to short days (i.e.,
September-October). For example, Aloe bulbifera (Aloe), Cestrum
nocturnum (Fig. 15.7), and Bryophyllum.

(a) (b)
Fig.15.7: Long-Short day plants:
plants a) Aloe bulbifera; b) Cestrum nocturnum.
3. Day-Neutral
Neutral plants/Photoneutrals /Indeterminate plants: Besides
SDP and LDP, those plants that flower irrespective of the length of light
are called day-neutral
neutral plants. For such plants there is no specific
requirement of light duration or temperature. They can flower in any day
length. e.g., Lycopersicon esculentum (Tomato), Mirabilis jalapa
(4O’Clock plant) (Fig. 15.8), Oryza sativa (Rice), Zea mays (Corn),
Pisum sativum (Garden pea).

(a) (b)
Fig.15.8: Long-Short
Short day plants:
plants a) Mirabilis jalapa; b) Lycopersicon
esculentum.
4. Intermediate day length plants: These plants flower only within a
narrow range of long photoperiod i.e., 12 to 14 hours. Shorter or longer
photoperiods than this will not result in flowering. e.g., Coleus hybrida,
Chenopodium album (Fig. 15.9)
15.9 and Saccharum spontaneum. 185
Block 4 Nitrogen Metabolism and Plant Growth Regulators

(a) (b)
Fig.15.9: Intermediate day length plants: a) Coleus hybrida; b) Chenopodium album.
5. Ambiphotoperiodic plants:These plants are inhibited by intermediate
day lengths. They would flower in short days or long days. Chenopodium
rubrum and Setaria verticillata (Fig. 15.10) belong to this category.

(a) (b)
Fig.15.10: Intermediate day length plants: a) Chenopodium rubrum; b) Setaria
verticillata.
The day length requirements vary from species to species (Fig. 15.11). Even
within a single species, different varieties of long-day plant may show a range
of critical day length. It is reported that rice plants can detect changes in day
length of only 15 minutes. Therefore, it seems that plants must have an
extremely accurate time-measuring mechanism within the tissues of the plant.
Flower induction in some plants can be stimulated by day length only if the
plant has been previously chilled, for example daffodil and henbane. While
some plants, for example, primrose flower solely in response to pre-chilling,
otherwise they are day neutral (see Section 15.5).

186 Fig. 15.11: Day length and percentage of flowering in Chrysanthemum and in spinach.
Unit 15 Plant Response to Light and Temperature
15.2.3 Importance of Dark Period
For quite some time the role of light period (photoperiod) was emphasized in
flowering. However, based on certain experiments it was realized that it is the
dark period that is more important than the light period for inducing a plant to
flower. In fact, as early as 1912, from his experiments on flowering, Julien
Tournois concluded that flowering occurred “not so much by shortening of
day as by lengthening of nights”. Much later Hammer and Bonner (1936)
proved through their experiment that dark period is more important than the
light period for inducing flowering in plants. They took a short-day plant,
Xanthium required 16 hours of darkness and 8 hours of light to flower. If the
dark period was reduced, plants did not flower. Interestingly, if the dark period
of 16 hours was interrupted by light, again there was no flowering. However, if
the light period was interrupted by dark period, or if plants were kept less than
8 hours in light there was no effect on flowering. It was clear that altering the
light period had no effect but if the dark period were less than 16 hours, the
plants would not flower. A look at Fig. 15.12 will further clarify this experiment
and confirm that dark period is more important for inducing plants to flower.
The role of dark period is to bring about some changes which trigger the
development from the vegetative to the flowering state. The role of light period
is to realize this change and help in bringing about maximum flowering.

Fig. 15.12: Experiments to show that dark period is important for flowering. If
dark period is interrupted by diffuse light (3) or even a flash of light
(4) during the long dark period SDP, no flowering.

For short day plants (SDPs), it is the uninterrupted long period of darkness
which is more important than the length of the day. Such plants may thus also
be called long-night plants. On the other hand, the long day plants require
either a relatively small period of darkness or no darkness at all, as is clear
from Fig. 15.13 that continuous light exposure to them may result in the best
flowering. Thus, such plants can also be called short-night plants. 187
Block 4 Nitrogen Metabolism and Plant Growth Regulators

Fig. 15.13: Long day plants are in fact short night plants.

15.2.3 Site of Perception of Light Stimulus

Photomorphogenesis
is light-mediated
It has been recognized since long that the photoperiodic stimulus is first
development, where
perceived by leaves of a plant. Experiments on Chrysanthemum plants by the
plant growth patterns
respond to the light
Russian physiologist Chailackhyan demonstrated that even 1/8 of a mature
spectrum. Light induces leaf, when exposed to light, was sufficient to induce flowering. Later work by
various changes in Borthwick and Hendricks in the 1950s at the USDA established that the
gene-expression seed quality of light exposure was also important in the induction of flowering Both
germination, seedling SDPs and LDPs were sensitive to red light in the region of 580 nm - 680 nm in
development, and the causing night break. On the other hand, wavelengths in the far-red region
switch from the (730 nm) had the ability to reverse the red light induced inhibition.
vegetative to the
flowering stage. SAQ 1
Phytochromes, cryptoc
hromes and a) Fill in the blank spaces in the following statements.
phototropins are
photochromic sensory i) The major morphogenetic change in flowering is the change of
receptors that operate ……………… meristems into………………………….meristems.
in the different
portions of the ii) Photoperiodic control of flowering depends on the length of
electromagnetic uninterrupted……………………………period given.
spectrum to show the
iii) ……………………………….. plant is likely to flower at the same
morphogenetic
time of the year whether grown in the house, field, or green house.
responses.
Plants follow a different iv) A short-day plant requires more than a critical period
developmental program of……………………………
in dark with altered
b) State whether the following statements are true (T) or false (F)?
growth patterns. These
changes are studied i) The day neutral plants can flower irrespective of day length but
under require a specific temperature to do so.
skotomorphogenesis.
A seedling may switch ii) Ambiphotoperiodic plants are inhibited by intermediate day
over to either of the lengths.
modes depending on
the availability of light. iii) Even a single young leaf when exposed to light is enough to
induce flowering.
iv) An SDP will flower if the light given to it is less than the critical
period.
v) Coffea arabica is an example of an obligate short-day plant.
188
Unit 15 Plant Response to Light and Temperature

15.3 PHYTOCHROME
You know that plants capture light energy during photosynthesis, now you are
familiarized with another important and interesting role of light in
developmental phenomena. Just as for photosynthesis light is absorbed by
pigments chlorophylls and carotenoids, similarly, to bring about developmental
response light must be absorbed by some pigments. One of the pigments that
detects the quality of light in the range of 600-800 nm region is phytochrome.
It may also be mentioned that some developmental changes do exist even in
total darkness. These processes would be independent of active form of
phytochrome. Such changes are called skotomorphogenetic as against
photo morphogenetic changes that occur in light. There also exists an
unidentified blue light absorbing pigment which brings about phototropism and
nastic movements.

15.3.1 Discovery
We have earlier described the experiment that proved the importance of the
dark period in flowering. In that experiment if dark period was interrupted with
light, the flowering was not induced in a short-day plant. In further experiments
instead of white light, dark period was interrupted by lights of various
wavelengths like red, blue, yellow, and far-red light. These experiments
showed that red light alone was sufficient to inhibit flowering. However, the
effect of red light was not always inhibitory. For different developmental
response, its effect was different. For example, and experiment done to check
effect of red light on lettuce seed germination, it was found to have stimulatory
effect. Another interesting observation made by Borthwick, Hendricks and
Parker (1952) was that any response to red light was nullified by far-red light.
The results for percentage germination of lettuce seed under different
irradiation conditions are shown in Table 15.1.

Table 15.1: The germination (%) of moist lettuce seed irradiated with Red
and FR (far-red) (After Borthwick et al. 1952)

Sequence of light exposure / Final Irradiation Germination

Irradiation (%)
Control (Dark) 8.5
R (660 nm) 1 min. R 98
R (1 min) + FR (730 nm) 4 min. FR 54
R + FR + R R 100
R + FR + R + FR FR 43
R + FR + R + FR + R R 99
R + FR + R + FR + R + FR FR 54
R + FR + R + FR + R + FR + R R 98

From several such experiments, it was concluded that some developmental

response brought about by red light could be reversed if followed by far-red
light and vice versa. Based on these physiological experiments, it was
proposed that both red and far-red lights must be absorbed by the same
pigment. This pigment was discovered by Hendricks and Borthwick at
USDA-ARS during a period from 1940 to early1960s. 189
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Phytochrome, thought to be present on the plasma membrane, exists in two
forms: a red-light absorbing form (Pr), which after absorbing red light gets
converted to far-red light absorbing form (Pfr). When Pfr form absorbs far-red
light, it gets converted back to Pr form as shown in Fig. 15.14. So, the two
forms are interconvertible.

Fig. 15.14: Schematic representation of photoconversion of phytochrome.

Phytochrome is synthesized in dark. On receiving light in red range
(R) it is converted from Pr (red light absorbing form) to Pfr (far-red
light absorbing form). The Pfr form is biologically active and induces
a response. It also undergoes decay (Pfr) i.e., it degrades with the
passage of time. When plants are left in dark for long, Pfr form in
some cases can also return to Pr state. It also can be brought back to
Pr state by irradiation with far-red light.

15.3.2 Properties of Phytochrome

Phytochrome is a chromoprotein: this means it is composed of a protein and a
The experiments on
chromophore. The chromophore is attached to the protein and it responsible
reversible effect of
for its color and light absorption. It is possible to isolate phytochrome in pure
red and far-red light to
form by following the procedures of protein purification. It absorbs red light
study developmental
responses were done (maximum absorption occurs at 660 nm wavelength) and on conversion, far-
by Borthwick and red light (maximum absorption occurs in far-red light region at 730 nm). A
Hendricks at the typical absorption spectrum of phytochrome is shown in Fig. 15.15. It is a
same place where soluble protein and is present in the cytoplasm. It is believed that Pfr form may
Garner and Allard had be associated with the membranes. The intact phytochrome protein has a
discovered the molecular weight of 124,000 Daltons. The total amino acid sequence of the
phenomenon of protein is also known and, using recombinant DNA technology, the gene
photoperiodism. coding for phytochrome has been isolated.

Fig. 15.15: Absorption spectrum of phytochrome in vitro. Phytochrome was

purified and its absorption spectrum was measured by a
spectrophotometer. When Pr was irradiated with red light, the
absorption spectrum (dotted line) was obtained. This shows the
conversion of Pr to Pfr. Note that not all Pr can be converted to Pfr
190 form and one sees two peaks.
Unit 15 Plant Response to Light and Temperature
The chromatophore is a tetrapyrrole molecule (Fig. 15.16) like chlorophyll, but Proteins cannot
unlike chlorophyll it is an open tetrapyrrole and contains no metal ion. It is absorb visible light.
covalently attached to the protein. It has been shown that it is Pfr form of They absorb only UV-
phytochrome that is biologically active. Changes in the structure occur during light. So
conversion of Pr to Pfr which probably make the Pfr form active. chromatophore part is
essential for the
absorption of light in
the visible range.

Fig. 15.16: The structure of chromatophore and its attachment to protein.

Phytochrome chromophore is an open tetrapyrrole. It is attached to
the protein at a specific site and undergoes proton shift on conversion
from Pr to Pfr.
The chromophore of phytochrome in higher plants is tetrapyrrole
phytochromobilin which is covalently linked to the apoprotein and helps the
phytochrome to absorb R/FR light. Phytochrome has several structural
domains (viz., PAS domain, GAF domain and PHY domain) which are
responsible for its synthesis, assembly, and functioning. Phytochrome has
now been shown to be a light regulated protein kinase which is capable of
autophosphorylation. The inactive Pr form is dimerized in the cytosol and at
the time of conversion to Pfr undergoes a conformational change in the
domains and moves from the cytosol into the nucleus. Inside the nucleus the
phytochrome mediates change in gene transcription, serving as a light
activated switch for altered gene activity.

There are two types of phytochromes viz., Type I and Type II. Their
concentration varies. However, type I phytochrome degrades in light as the
dark grown seedlings have nearly 9 times more Type I than Type II.
Phytochromes are encoded by a multigene family whose number varies from
three (Rice) to five (Arabidopsis).

Box 15.1: A method to estimate phytochrome

Since there is no chemical test, the only method available to test and estimate
phytochrome is the spectrophotometric method. Firstly, the difference in the
absorbance between 660 and 730 nm is measured after red light irradiation (∆OD
red, No, 1). Next, the sample is irradiated with far-red light and again the difference in 191
Block 4 Nitrogen Metabolism and Plant Growth Regulators
∆OD is measured (∆OD far-red, No. 2). The content of total phytochrome is
measured by subtracting the value obtained after red light irradiation from far-red
light irradiated value.
Phytochrome total = ∆ (∆OD) = [∆OD far-red - ∆OD red]
1) ∆ (∆OD) = [OD660 - OD730] after red irradiation
2) ∆ (∆OD) far-red = [OD660 - OD730] after far-red irradiation.
Nowadays special spectrophotometers are also available which can measure the
absorption difference between two wavelengths. These are called dual wavelength
spectrophotometers.

15.3.3 Phytochrome-Mediated Responses

Phytochrome responses are those which are controlled reversibly by red and
far-red light.

i) Fast responses: those which occur within a time span of seconds to

minutes and
ii) Slow responses: those that take hour to days to manifest themselves.

Some of the fast responses are discussed below:

i) It was found that when mung bean root tips were kept in a specific
solution (containing ATP, IAA, ascorbic acid, MnCl2, KCl) the root tips
adhered to a glass beaker when irradiated with red light. This effect was
reversed by far-red light within 30 seconds. It is suggested that the quick
response is probably due to change in electric charges on the root tips in
response to red light, so the root tips adhere on to the negatively
charged glass surface. Such a response can be classified as a fast
response and is called Tanada effect (after Takuma Tanada).

ii) Similarly, it was shown that if alga, Mougeotia was irradiated, its single
chloroplast turned in response to red light, but far-red light alone had no
effect (Fig. 15.17). The red-light effect was reversed by far-red light. This
movement was also noticed within minutes of irradiation. In fact, in these
experiments, the light was irradiated on the cytoplasm or membranes
barrier and not onto the chloroplast directly.

Fig. 15.17: An experiment with Mougeotia to demonstrate a fast response.

Filaments of Mougeotia are made up of long cells each of which
contains a single flat plate like chloroplast which can turn inside the
cell in response to light. The light induced chloroplast movement was
shown to be red/far-red light reversible response as shown above. The
192 movement starts with a lag phase of only a few minutes.
Unit 15 Plant Response to Light and Temperature
It is believed now that apart from processes involving wavelengths exceeding
550 nm and photosynthesis, all are phytochrome mediated.

The phytochrome mediated plant response can be categorized in various

ways.

a) Based on time of response — the response are either Developmental

(Slow) or Rapid.

i) Developmental responses are slow as they also involve other

physiological processes like growth and differentiation. As a result,
they take a longer time to manifest. e.g., Flowering, seed
germination, anthocyanin synthesis, chlorophyll synthesis,
etiolation, de-etiolation, hypocotyl hook opening, unfolding of
leaves in seedlings and an array of developmental responses in
lower plants like spore germination in ferns, rhizoid development
from gemmae in liverworts and bud differentiation in protonema of
mosses.

ii) The rapid responses occur within seconds of light irradiation. This
may not involve growth but changes in the permeability to water,
changes in biometric potential between different regions of roots
as well as change in turgidity of motor cells in the pulvinus of
leaflets and orientation of the chloroplasts.

b) Phytochrome responses can also be categorized/classified based on the

amount of light required. The amount of light (number of photons falling
on a unit surface area) required to induce a response is called fluence.

Fluence is measured in terms of micromoles or quanta per square meter (µmol-2).

The light that is incident on the object refers to irradiance and not the intensity, as
generally believed. Irradiance is also called the fluence rate (measured as
micromoles of quanta per square meter per second — µmol m-2s-1).

Since total fluence = fluence rate x length of time of irradiation, the

phytochrome responses can be divided into three types:

i) Very low fluence responses (VLFRs): Which can be initiated by

fluence as low as 0.0001 µmole m-2and saturate at 0.05 µmol m-2. For
example, Arabidopsis seeds can be induced to germinate by an
exposure of red light of 0.001 to 0.1µmol m-2(which is many times lower
than even the light emitted by a firefly in a single flash). These
responses are non-photoreversible as significant amounts of Pfr are not
made.

ii) Low fluence responses (LFRs): Can be initiated only after the fluence
reaches 1.0µmol m-2 and get saturated to 1000µmol-2. Such response
are photoreversible and obey the law of reciprocity which means that the
product of the fluence rate and the time of irradiation regulated the
magnitude of response. For example, lettuce seed germination,
regulation of leaf movements, promotion of de-etiolation.

iii) High irradiance response (HIRs): Require prolonged or continuous

high irradiance exposure. In this case the response is proportional to the 193
Block 4 Nitrogen Metabolism and Plant Growth Regulators
irradiance until the response saturates. Additional exposure will now
have no effect. Such responses are time-dependent and non-
photoreversible and do not obey the law of reciprocity. Some of the
examples are production of ethylene in Sorghum, enlargement of
cotyledons in mustard, induction of flowering in henbane and synthesis
of anthocyanin in apple skin segments and various dicot seedlings.

iv) In many leguminous plants it was found that leaves closed if they were
transferred from light to darkness. It has been shown that this response
is under phytochrome control.

v) Several other responses, which could be classified as slow responses

like seed germination, hypocotyl elongation, leaf expansion, abscission,
flowering, fruiting have been shown to be under phytochrome control.

Several other responses, which could be classified as slow responses like

seed germination, hypocotyl elongation, leaf expansion, abscission, flowering,
fruiting have been shown to be under phytochrome control.

15.3.4 Mechanism of Action

You have learnt that biochemical and molecular changes are brought about by
phytochrome. However, what is not yet clear is the relationship of these
changes to a specific developmental event. Since phytochrome controls many
processes even in one plant (Table 15.18). This makes the analysis of
molecular action a difficult task.

Fig. 15.18: Hypothesis for differential regulation of gene expression. The

biologically active form Pfr can affect the transcription process by
either inducing the synthesis of new mRNA or by repressing the
synthesis of mRNA already being synthesized in dark. By altering the
level and quality of enzymes and proteins, developmental responses
can be controlled by phytochrome.

Table 15.2: List of multiple effects of light on mustard seedlings grown in

dark

1. Inhibition of hypocotyl lengthening

2. Enlargement of cotyledons
3. Opening of the hypocotylar (“Plumular”) hook
194
Unit 15 Plant Response to Light and Temperature
4. Development of primary leaves
5. Synthesis of anthocyanin
6. Increase of the rate of chlorophyll accumulation (in white light)
7. Changes in the rate of cell respiration
8. Changes in the rate of degradation of storage protein Increase of negative
geotropic reactivity of the hypocotyl
9. Increase of protein synthesis in the cotyledons
10. Decrease of RNA contents in the hypocotyl
11. Increase of RNA contents in the cotyledons
12. Differentiation of stomata in the epidermis of the cotyledons
13. Increase in the rate of carotenoid synthesis
14. Increase in the level of mRNA, increase in nitrate reductase, increase in
RuBP carboxylase

Of the various changes brought about by phytochrome many of them occur

within a few minutes and some other have a time-lag of 1-2 h or more. It has
been suggested that responses which occur rapidly, say within 0-15 minutes,
may be considered as ‘membrane associated events’, and the responses
with a lag period of few hours may be considered as ‘gene expression
events’ (Fig. 15.19). The responses which are measurable as changes in
growth may be considered final ‘developmental responses’. It has been
found in several systems that phytochrome does affect membrane related
phenomena. First, it has been reported in a few cases that phytochrome may
be associated with membranes. For example, in Mougeotia, if the membranes
were irradiated with light, chloroplast movements was detectable. Also,
experiments suggest that phytochrome binds to the membranes, but the result
need to be confirmed. However, it has been clearly established for example in
Albizzia that phytochrome controls transport of K+ ions across the plasma
membrane. Similarly, it has been shown that phytochrome also affects the
movement of calcium ions. The exact mechanism by which phytochrome
affects membrane transport proteins is not yet known.

Fig. 15.19: Photoreversible effect of R and FR light on the induction of nitrate

reductase. R and FR lights were given for only 5 minutes to etiolated
(plants grown in the absence of light) maize leaves. 195
Block 4 Nitrogen Metabolism and Plant Growth Regulators
More work has been done on gene expression events. It has been shown that
phytochrome does affect the level and activity of enzymes. In some cases,
however, the pigment increases the enzymes levels as for example, in phenyl
alanine ammonia lyase, nitrate reductase, RuBP carboxylase, light harvesting
chlorophyll a/b protein, whereas in other the phytochrome decreases the
enzyme level, e.g., NADH protochlorophyllide oxidoreductase. An example of
phytochrome regulation of nitrate reductase enzyme is shown in Fig. 15.19.

Recently, it has been shown that phytochrome affects the level of enzymes by
affecting the process of transcription. An increase in the level of mRNA of
several proteins in response to light irradiation has been shown.

A major question that remains to be answered is how does phytochrome

which is present in the cytoplasm affect gene expression in the nucleus? It is
suggested that probably some signals or second messengers are generated
by phytochrome which affect at the level of transcription as shown. Lately the
role of calcium ions as messenger of light mediated responses has been
emphasized. This is based on the findings that in many instances the light
affect could be mimicked by calcium ions.

The molecular properties of phytochromes undergo a change after absorbing

light. As a result, the phytochrome protein interacts with other cellular
component to bring about changes. The signal transduction pathways lead to
response in term of ion fluxes causing rapid turgor changes a slower low term
alteration in gene expression.

Fig. 15.20: A suggested model explaining the transport of floral stimulus and the
induction of flowering genes (after Taiz et al).

It has now been demonstrated that phytochrome A enters the nucleus through
the mediation of Far-red light whereas red light triggers the import of
196 phytochrome B. Some phytochrome interacting factors (PIFs) present in the
Unit 15 Plant Response to Light and Temperature
nucleus bind to the promotor region of the phytochrome-repressive genes.
This causes repression of transcription. Entry of Pfr into the nucleus triggers
degradation of PIF by a 26S-proteosome system. Gene transcription is now
activated, and mRNA is formed. Increase in the concentration of Pr in the
nucleus helps in reassociation of PIF with the promotion region thereby
causing gene repression Fig. 15.20). There are primary response genes
which encode the transcription factors get regulated by a shift from dark to
light. On the other hand, the secondary response genes require new protein
synthesis and are late acting. Thus, a transition from dark modulated
development (skotomorphogenetic) to a light modulated (photomorphogenetic)
on requires a reprogramming of gene expression. In addition, various
phytochrome Interacting factors (PIFs) have been identified. These
proteinaceous transcription factors modify the phytochrome action by acting as
negative regulators of phytochrome response.

15.4 FLOWERING HORMONE

It was in 1880, that Julius Sachs suggested that there is a chemical basis for The concept of
flowering. Later experiment indicated that some hormone was produced which florigen-a flowering
controlled flowering. The hormone was named florigen by Mikhail hormone was given
Chailakhyan. Before we find out what the florigen is, let us first see the by a Russian Botanist
experiments which suggested that a chemical is involved in flowering and that named Chailakhyan
in 1930s.
it is produced in leaves.
The light dark
conditions which
induce plants to
flower are called
inductive
conditions.

Fig. 15.21: Experiments to demonstrate that photoperiodic stimulus is perceived

by the leaf. In a) Kalanchoe blossfeldiana plants begin to flower even
if one single leaf is maintained under short-day conditions. In
experiment b) a leaf from LDP, Nicotiana sylvestris was grafted on
SDP, N. tabacum. The plant started flowering even under long-day
conditions (i). However, if a leaf from N. tabacum was grafted on N.
tabacum itself in a control experiment ii, flowering was not induced.

In an experiment only the leaves of Kalanchoe, a short-day plant, were given

the inductive conditions and not the whole plant. Even then the plant flowered
(Fig. 15.21). More such experiments were done where treatment of light-dark
cycle was given only to the leaves and the plants were induced to flower. 197
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Some scientists performed experiment with a twig, a single leaf of which had
been given the inductive condition. It was grafted onto a plant which was not
given any inductive condition. Interestingly, it was found that the grafted plant
also started flowering (Fig. 15.21). In some cases, grafts were taken from
plants which were induced by short-days, for example from Xanthium
canadense and were put on plants which required long-day conditions e.g.,
Rudbeckia bicolour. Even in such graftings the recipient plants, in the above
case Rudbeckia, started flowering.

If you have understood the above experiments perfectly well, you will come to
the following conclusion.

i) the leaves perceive the light stimulus, and

ii) some chemical moves out of the leaves (as must be occurring in grafts)
and goes to the meristems and changes them from vegetative to
flowering. What is this chemical?

However, some observations are contrary to this view. It has been shown that
florigen can move readily between tissues, but it cannot pass between two
tissues if separated by a strip of agar while a hormone can travel easily
through agar. Further, experiments have suggested that the flowering stimulus
moves through the phloem. This suggests that some chemicals, other than
hormones may also be involved in flower induction in vivo. We can hope that
soon it would be possible to isolate and identify florigen by analyzing and
comparing the content of phloem sap before and after flower induction.

15.4.1 Biochemical Changes

Many workers have tried to follow the biochemical changes that precede
flowering and result in meristems which give rise to flowers instead of
vegetative structures. In Pharbitis, which is a short-day plant and requires only
one dark period for flowering it was found that soon after the dark period, the
flowering stimulus begins to move out of the leaves. In this experiment, the
plant was given inductive conditions and after specific time intervals,
biochemical changes were given inductive conditions and after specific time
intervals, biochemical changes were measured in meristem. An increase in
metabolic activity around 40thhour at the floral apex was manifested by an
increase in the level of RNA, proteins, and ribosomes. Electron microscopic
observations also revealed extensive formation of endoplasmic reticulum.
These activities were followed by an increase in DNA synthesis and mitotic
activity. At about 88th hour after floral induction, the rate of cell division was
noticed particularly in the central zone and peripheral zone of apical
meristems.

Such experiments have also been done in other plants. However, it has not
been possible yet to identify which of the RNA or proteins are responsible for
the onset of flowering. With the application of newer techniques, it has been
possible to suggest that there some specific flowering genes which get
switched on after receiving specific light-dark cycle. Although we do not know
the products of all these genes, some of them have been shown to code for
198 proteins which regulate transcription.
Unit 15 Plant Response to Light and Temperature

SAQ 2
a) In the following sentences, fill in the space with appropriate words:

i) Pr absorbs……………… nm wavelength of light and Pfr absorbs

at…………………… .

ii) The effect of red light could be completely reversed by

…………….. light.

iii) Phytochrome is a …………….. protein. It is present in the

…………………. of cell.

iv) The biochemical signal to the nucleus by phytochrome for making

new mRNA is suggested to be ………………….. .

v) Biological active form of phytochrome ………………….. can affect

…………………. process by induction or repression.

b) List the biochemical changes that precede flowering.

15.5 PLANT RESPONSE TO TEMPERATURE-

VERNALIZATION
Some plants flower only after passing a winter season. For example, winter
Vernalization is a
wheat is sown in the autumn for harvest in the following summer. It needs Latin term, it means
exposure to cold. If winter is mild, plants do not flower, and the crop fails. It “to make springlike”.
has been shown that winter wheat and many other plants require a period of
chilling 0°-2°C for about a week for flower formation. The cold treatment given
to geminating seeds for flower induction is called vernalization. The technique
of vernalization was developed in Russia where winter crop required chilling
for successful cultivation. The seeds are soaked for small period to initiate
germination and then they are buried in the snow. Later, they are planted in
the spring when severely cold conditions are over. Thus, chilling is also a
stimulus for flower induction.

Which part of the plant requires chilling stimulus for floral induction?
Experiments show that only the shoot apex receives the stimulus (needs
vernalization) which then is passed on to the other parts of the plants. Similar
conditions have been made with the help of grafting experiments (in
Henbane). When the shoot apex that has received the stimulus is pinched off,
the lateral shoot flowers. And if the apices of lateral shoots are also pinched
off, the side shoots develop and flowers. Moreover, when extracts of a
vernalized plant are applied to a long-day plant growing under short-days, the
recipient plant flowers. Like light induction, the stimulus can also pass through
a graft to a non-vernalized plant. These results show that flowering stimulus is
transmitted from shoot apex to other tissues. The chilling stimulus was named
vernalin by Melchers (1939). The nature of this compound has yet not been
identified, although it is believed that it could be a gibberellin like substance as
the latter is capable of inducing flowering in some biennials without any cold
pretreatment. 199
Block 4 Nitrogen Metabolism and Plant Growth Regulators
The term vernalization was coined by Russian scientist Lysenko in the 1920s.
A detailed study on the Petkus rye (Secale cereale) was made by Gregory and
Purvis in the 1930s where the cold pretreatment could mimic the flowering
behavior of a spring crop. If a vernalized seed on plant is exposed to high
temperature, it loses its earlier effects, and the effect of low temperature is lost
(Fig.15.22). This is called devernalization. Treatment once again with low
temperature can result in vernalization.

15.5.1 Mechanism of Vernalization

There is growing conception of some hormonal involvement in vernalization.
The responsible substance, Vernalin is believed to be produced in some
biennials in response to low temperatures. According to some workers two
chemicals viz., gibberellins and anthesins - are responsible for flowering.
Anthesins are already abundant in LDPs. So cold treatment to LDPs induces
vernalin which now combines with anthesins to produce gibberellins which
induces flowering (see scheme in Fig. 15.22).

Fig. 15.22: A schematic depiction of low temperature induced flowering.

Vernalization is also Recent investigations on Arabidopsis (LDP and low temperature requiring
known to shorten the type) have revealed changes in the pattern of gene expression in the meristem
flowering time. Hence in response to cold treatment. Such changes in DNA which can pass to the
more than one crop next generation of cells by mitosis or meiosis are called Epigenetic changes.
can be obtained per
A gene flowering locus C (FLC) has been identified which acts as a repressor
year. Crops like
cotton and maize of flowering. Vernalization switches off this gene for the remainder of the
which require long plant’s life cycle. Thus, flowering occurs in response to long day conditions.
stimulus can now be Three genes (VRN1, VRN2 and VRN3) are thought to determine the
grown in temperate vernalization stimulus requirement in cereals.
areas which have
short summers. Vernalization has been practiced extensively in Russia and other countries
with severe winters so that winter crops can be grown in spring season to
200 protect them from freezing injury.
Unit 15 Plant Response to Light and Temperature

SAQ 3
Fill in the blanks with appropriate words/terms.

a) …………………. is the site of perception of cold treatment.

b) A cold temperature treated grain could be ……………… by exposing to

high temperatures.

c) The existence of a transmissible stimulus for vernalization called

…………………. is strongly suggested.

d) In many plants …………………….. can stimulate vernalization.

e) Both ………..….. followed by …………….. are required for flowering.

15.6 RHYTHMIC CHANGES/ BIOLOGICAL CLOCK

Lastly, we are going to learn about a phenomenon which is still not very well
understood by scientists. Like animals, which show rhythmicity in their
behavior, it has been found that some developmental events in plants also
occur with regular periodicity in time. These processes are rhythmic and if
these occur in the absence of external disturbing factor, these are called
endogenous rhythms. This would be clear if you have ever observed sleep
movements of leaves in plants like Phaseolus, Mimosa, Albizzia and
Samanea. In these plants during the daytime the leaves are open and during
the night they droop down. In fact, these movements can be recorded. This
was done by tying a thread to the leaf that shows periodicity. The thread is
rolled over a pulley and then attached to a stylus which is touching a rotating
drum as shown in Fig. 15.23.

Fig.15.23: Methods for recording leaf movements. The leaf of the plant is tied with a thread
to a lever that touches the marker running on a clock driven drum a) Since the
drum is moving the trace of leaf movement can be recorded over a period of day
and night. A time lapse photography camera is fitted which takes pictures of the
plant after short time intervals; b) From the developed film as shown in above
right side, the leaf position with respect to time can be recorded. 201
Block 4 Nitrogen Metabolism and Plant Growth Regulators
How do we know that these movements are independent of external factors?
An experiment was done where after the rhythm was induced by light
exposure, the plants were kept in total darkness for days. It was found that the
leaves would open and close as they would have done under normal
conditions. These oscillations were about 23 h apart. Such rhythms with
oscillations of 20-23 h are called circadian rhythms and the cellular
machinery which helps the plant remember and keep these rhythms in time is
called the ‘Biological clock’. Interestingly, when plants were put on satellites,
the rhythms persisted.

There are some good examples of plants where circadian rhythms have been
noticed and studied. These are sporulation in Oedogonium cardiacum; spore
discharge in Pilobolus sphaeroporus; leaf movement in Phaseolus multiflorus
and CO2 fixation in darkness in Bryophyllum fedtschenkoi.

Very pretty floral clocks have been assembled in tourist places in various parts
of the world. One in Canada near the famous Niagara Falls tells the time from
6.00 a.m. to 6.00 p.m., beautiful flowers open at sharp 6.00 a.m., 7.00 a.m.,
8.00 am., and so on, a glorious sight worth seeing. Fig. 15.24 depicts a floral
clock found in a garden in Europe.

202 Fig. 15.24: The flower clock.

Unit 15 Plant Response to Light and Temperature
15.6.1 Factors Affecting Rhythms
Two factors, more important than others which regulate or affect the time
period and the intensity of a rhythm are temperature and light. It has been
found that once the rhythm is set, many circadian processes are independent
of temperature over a wide range. The effect of light in many cases has been
shown to be regulated by phytochrome, the pigment-protein complex about
which you have studied in section 15.3. The amount of Pfr form changes
during the day, i.e., from sunrise to sunset. At sunset, when red light is less, its
level would decrease which would further go down in the following darkness.
In the morning when red light is more, the Pfr level would rise again. This daily
change could be one of the factors to set the biological clock.

One of the founders of biological clock research in plants, Erwin Bunning of

Botanical Institute in Tübingen, Germany, worked on leaves of different
varieties of soyabean and found some correlation between their photoperiodic
behaviour and sleep movements. He further found that leaf movement was
less pronounced. These observations do suggest that light does influence
rhythmic phenomenon in plants.

At present it is still not clear what is the physical basis of the biological clock.
Certainly, it is not any specialized timing organ. Some processes get affected
at the nuclear, protein and membrane level. However, the exact nature is not
known with certainty. Probably some chemical inside each cell works as a
clock. Although recent work seems to suggest the involvement of membrane
bound glycoproteins which intercept environmental signals and set the clock.

SAQ 4
Match the contents of column I with those given in column II.

Column I Column II

i) Biological clock setting a) Spore discharge in Pilobolus

ii) Sleep movements b) Absence of external factor

iii) Niagara Falls c) Level of Pfr

iv) Circadian rhythm d) Floral clocks

v) Endogenous rhythms e) Phaseolus

15.7 SENESCENCE
Plants follow a common pattern: growth, flowering, senescence and death.
The entire period from the beginning to death is called the longevity or age or
life span. Life span varies from species to species. Some plants like annuals,
complete their life cycle within a few months whereas others may live for a few
centuries. For example, the life of Juniperus scophularium is around 3,000
years. The giant Sequoia also has a long life-span. 203
Block 4 Nitrogen Metabolism and Plant Growth Regulators
The period just before death is called the senescent period. Senescence is
age-dependent degradation and degeneration of cells, organs or the entire
organism, leading to death. During this period deterioration occurs because
there is a consistent decrease in viability and increase in vulnerability. This
phase can be prolonged but cannot be reversed.

Senescence may occur either very quickly or may be a slow process.

Sometimes the individual organs senesce while the whole plant may remain
healthy. Hence, senescence in plants occurs at various levels, both at the
organ and organism level. The senescence at the organ level is evident by the
change in leaf color and the subsequent death of autumn leaves. Cell death in
leaf starts from mesophyll cells and proceeds to other cell types. The most
notable change seen during senescence is the change in the cellular
structures of leaves which occur due to disintegration of intracellular
organelles particularly, the chloroplast.

The process of senescence shows variation in various types of plants (Fig.

15.25). In annuals, the whole plant dies; in biennials, the plant dies only after
two years, whereas in perennials, year after year the leaves and fruits are
shed but the main plant survives. Broadly, senescence has been categorized
into following types.

Fig. 15.25: Diagrammatic representation of different senescence patterns noted

in plants. The dying parts are shaded brown.

Overall senescence (whole plant senescence): Only the parts above the
ground level i.e., the aerial parts die whereas underground parts survive, for
example- potato, rice, wheat, gram.
Deciduous senescence (simultaneous or synchronous senescence):
Only the leaves senesce as in many trees such as elm and maple. The
senescence of leaves, or abscission occurs when at the base of a leaf a layer
of cells is laid which is called abscission layer. An abscission zone or
separation zone formed at the base of the petiole is composed of a top layer
that has cells with weak walls. The weak walls of the cells in the top layer
204 break which allows the leaf to be shed (Fig. 15.26).
Unit 15 Plant Response to Light and Temperature

Fig. 15.26: L.S of leaf base showing a) abscission zone; b) formation of separation layer.

The bottom layer expands in the autumn. The cells of abscission layer show
high activity of cellulase enzyme which degrades cellulose of cell wall. Another
enzyme that increases during abscission is polygalacturonase (which
hydrolyses pectin, a major component of the middle lamella region of the wall).
So, the cells get separated and the leaves fall. Below this layer, the plant
makes a protective layer which has many lignified cells. When a leaf falls, this
layer protects the tissue from desiccation.

Progressive senescence: Here also the leaves are shed but it is gradual
senescence of leaves up the stem, for example- palm trees.

Sequential senescence: It is found in many perennial plants. The tips of the

main shoot and branches continue to produce new buds and leaves. The older
leaves and branches senescence and die, for example-Eucalyptus, Pinus.

15.7.1 Biochemical Changes Associated with

Senescence
During senescence many physiological and biochemical changes take place in
plants. One of the important changes noted in plants is the decrease in
chlorophyll content. The degradation of chlorophyll results in yellowing of
leaves. The overallstructure of chloroplast shows disorganization as the
thylakoids gets disturbed. As a result CO2 fixation capacity of chloroplast
declines. Chloroplast degeneration is accompanied by loss of proteins such as
ribulose biphosphate carboxylase (Rubisco) and chlorophylla/bbinding protein
(CAB) from the chloroplast. The organelles such as mitochondria and nucleus
remain intact. It has been suggested that pheophytinase (PPH), an enzyme
plays a role in chlorophyll degradation. Enzymes such as phospholipase
D,phosphatidic acid phosphatase, lytic acyl hydrolase and lipoxygenase get
involved in hydrolysis and metabolism of the membrane lipid in senescing
leaves. The lipid peroxidation and membrane leakiness also increases during
the process due to increase in generation of reactive oxygen species (ROS). 205
Block 4 Nitrogen Metabolism and Plant Growth Regulators
A dramatic metabolic transition from anabolism (carbon assimilation) to
catabolism has been noted in leaves. This transition to nutrient remobilization
has been suggested as major reason for degradation of cellular structures.
Increased catabolic activity is responsible for converting the cellular materials
accumulated during the growth phase of leaf into nutrients that are supplied to
developing seeds or to other growing organs.The nutrients produced in the
leaf either show degradation or get redistributed to developing seeds during
senescence. The rate of respiration also decreases with time and supply of
ATP is reduced.The hydrolysis of macromolecules such as proteins, lipids,
nucleic acids and pigments occurs as number of degradative enzymes like
proteases, ribonucleases, P-glucan hydrolases(for loosening of cell wall),
chlorophyllase (for degradation of chlorophyll) are produced. These enzymes
increase the rate of catabolism and finally over a period of time, the plant
succumbs and dies. In annual plants, hydrolyzed molecules are relocated to
developing seeds and fruits. In trees, the nutrients that are degraded
eventually get stored in stems or roots and are utilized later for the
development of new leaves or flowers. The nutrients relocated from senescing
autumn leaves help in blooming of spring flowers.

Though leaf senescence is a deleterious process for the leaf organ but
nevertheless critically contributes to the fitness of whole plants by ensuring
optimal production of offspring and better survival of plants in their given
temporal and spatial niches. Leaf senescence is therefore, an evolutionarily
selected and genetically controlled developmental process that constitutes an
important phase in the plant life cycle.

15.7.2 Regulation of Senescence

Senescence is a process which is a part of a developmental sequence of
events and is a controlled process. It is controlled by both external and internal
factors. Of the external factors, plant hormones, length of the day, temperature
and nutrient supply play an important role. Of the internal factors, size and age
of the plant, degree of flowering and time of ripeness of fruits determine the
onset of senescence.

In one experiment where the normal time of senescence was 120 days, when
mature fruits were removed from plants, the time of senescence was delayed.
Senescence occurred after 140 days. And when young fruits and-flowers were
removed from plants as soon as they were formed, the senescence was
delayed to 160 to 180days. This means that the senescence is initiated as
soon as the process of reproduction is set in. This is probably because a plant
needs a lot of nutrition for the growth of flowers and fruits. So to increase the
nutrient supply to fruits, the stored material from leaves and other parts is
translocated to the growing fruits. The demand is so high that the fresh supply
of nutrients and photosynthates cannot be replenished.

It is a simple case of more demand than supply. Naturally, the system

collapses.

However, in the process the plant tries to ensure that the fruits mature and
seeds are set so that it can continue with its progeny.

Internal and external factors get interconnected to form a complex network of

206 regulatory pathways for senescence. Hormone signaling pathways mediate or
Unit 15 Plant Response to Light and Temperature
influence development responses in plants. Hormones play key role in the
progression of senescence. Hormones induce activation of hydrolytic enzyme
synthesis genes and trigger leaf senescence. The hormonal pathways
regulate all the stages of leaf senescence including the initiation phase,
progression, and the terminal phases. Cytokinins have been known to delay
senescence. Cytokinins act as senescence-retarding hormones because the
level of endogenous cytokinins falls during leaf senescence. Molecular
analysis revealed that genes for enzymes viz. cytokinin synthase and
adenosine phosphate isopentenyl-transferase (IPT) involved in cytokinins
synthesis get downregulated and a gene for the enzyme, cytokinin oxidase
(cytokinin degradation) gets up-regulated in senescing leaves. The gaseous
hormone, ethylene is known to play a role in hastening leaf and flower
senescence. Ethylene is supposed to act as an important positive regulator of
leaf senescence. In many plant species, including Arabidopsis where the level
of ethylene increases during leaf senescence. Exogenous application of ABA
has also known to promote leaf abscission and senescence. The ABA level
increases in senescing leaves and exogenously applied ABA induces
expression of several senescence associated genes (SAGs), which is
consistent with the effect on leaf senescence. High salicylic acid (SA) level in
senescing leaves appears to be involved in up-regulation of several SAGs
during leaf senescence.

Leaf senescence can be induced by the interaction of photoperiod and

temperature. Studies have shown that photoperiod reduction increased leaf
senescence by 61%, while low temperatures increased the process by about
39%. Photoperiod reduction triggers leaf senescence in several woody
species. Thus, the photoperiod may be the abiotic factor which activates
senescence. Temperature has been considered as the secondary factor that
accelerates the deterioration process. Low temperatures activate leaf
senescence in most deciduous species. The deciduous plants respond to
these conditions by increasing electrolyte leakage, chlorophyll degradation,
formation of reactive oxygen species and reducing enzyme activities.

Nitrogen deficiency accelerates leaf senescence. During this period, protein

and amino acid degradation is high and this contributes to allocation of
nutrients to developing seeds. The genes involved in the processes and
transport of biosynthetic aromatic amino acids are regulated in early stages of
leaf senescence. High nitrogen levels may delay leaf senescence in non-
perennial species, in addition to maintaining high photosynthetic rates during
reproduction and production. Wheat plants with higher leaf nitrogen content
showed delayed senescence.

Water deficit also accelerates leaf senescence. Under water deficit, plants
regulate dehydration by closing stomata in response to the production of the
chemical messenger Abscisic Acid (ABA). Increased ABA levels increase
ethylene production and consequently, the synthesis of the enzymes that act
on the cell wall and middle lamella increases. Ethylene induces genes that
encode specific hydrolytic enzymes of polysaccharides and cell wall proteins
in the abscission zone. Thus, ABA accelerates leaf senescence, indirectly
inducing abscission.

Besides environmental and endogenous factors, the biotic factors also play a
role in inducing senescence. For example, attack of mites, insects or even
parasitic fungi, hastens the process of senescence. Also, without realising 207
Block 4 Nitrogen Metabolism and Plant Growth Regulators
when you walk in a garden and pluck leaves or break branches and twigs, you
are also contributing to initiating senescence in those plants (induced by
injury). Fig. 15.27 gives a general view of the factors that affect senescence of
various plants.

Fig. 15.27: The effect of various physical and chemical factors on senescence.

SAQ 5
a) Mention the major changes noted in leaves during senescence.

b) List various factors that induce senescence in plants.

c) Name some enzymes that get activated during abscission

15.8 CRYPTOCHROMES
These are blue-coloured photoreceptors present in almost all groups of plants.
Earliest ideas regarding biological responses to the blue light were presented
by Charles Darwin, who demonstrated that when light was passed through a
solution of potassium dichromate, it failed to induce any heliotropic
movements in plants. Plants sense and respond to blue light (400-500 nm) by
production of anthocyanins and carotenoids. Interestingly, there exists a lot of
common ground between the absorption spectrum of Flavin and many
responses of blue light. For several years the nature of this photoreceptor
remained elusive and debatable—a reason why it was called Cryptochrome.
Cryptochrome is also known to regulate other aspects of growth and
development like hypocotyl elongation, opening and closing of stomata,
anthocyanin production, setting of flowering time and clocks. Cryptochromes
function in coordination with phytochromes to bring about many responses.

Cryptochromes were first characterized in Arabidopsis and have helped us to

explain the role of this photoreceptor in flowering by affecting the biological
208 clock.
Unit 15 Plant Response to Light and Temperature
A gene HY4 has been shown to encode for this blue photoreceptor. CRY1
(which works under high blue light fluence rates). Another cryptochrome, viz.
CRY2 has been shown to be important under low blue light fluence rates.

The cryptochrome is believed to be a chromoprotein which is composed of a

light harvesting chromophore- pterin or deazaflavin along with a catalytic part
containing FMA and FAD, and an imino- terminal photolyase region. In
bacteria, these flavoprotein photoreceptors repair the pyrimidine dimers
produced by UV light, i.e., they act as DNA photolyases.

15.9 PLANT MOVEMENTS

Most of the plants, with the exception of Volvocales do not show any bodily
movements, which is common in animals. Such movements of locomotion are
however, absent in higher plants. Such plants respond to external environment
by different type of movements like bending, twisting, elongation of certain
parts, swelling or shrinkage and quite a few more.

Before studying the different movements in plants, let us first get familiar with
some important terms used in plant movements.

• Any factor, external or internal which causes a reaction in a plant is

called a stimulus due to its properties of irritability and sensitivity. The
reaction in a plant due to this stimulus is the response which is
expressed in various ways.

• Only certain specialized regions in a plant are sensitive to this stimulus.

These are called centres/sites of perception.

• The manifestation of response occurs at the site of response. The two

sites viz. site of perception and site of response may be same or
different.

• The minimum time period for which a stimulus needs to be given so as

to produce a response is called presentation time. Presentation time is
inversely proportional to the intensity of stimulus.

• The time take by the plant between the given stimulus and the
appearance of response is called reaction time.

Plant Movements are classified into two types (Fig. 15.28):

1. Physical - expressed by dead parts. These are hygroscopic

movements or mechanical movements/chasic movements. Such
movements are reversible. These are named accordingly to the nature of
the stimulus and response.

Hydrochasic = Swelling of wood.

Xerochasic = Dehiscence of sporangia, elaters of bryophytes,

peristome teeth in moss capsules, pseudo-elaters in Equisetum spores.

2. Vital - Expressed by living cells in response to internal or external

stimuli. Movements taking place without any external stimulus are called 209
Block 4 Nitrogen Metabolism and Plant Growth Regulators
autonomicor spontaneous movements. Movements in response to
external stimuli are termed Paratonic or induced. The paratonic
movements are broadly classified as movements of locomotion and
movements ofcurvature. Whereas the movements of curvature are
generally found in the rooted terrestrial plants, locomotory movements
are shown by some aquatic plants.

Fig. 15.28: Summary of Plant Movements

15.10 SUMMARY
• The development of the plant starts with the formation of seeds. A seed
has stored food material and mRNA which it uses during the process of
germination. By utilizing proteins that are already existing and newly
synthesized on stored mRNA or whose transcription is induced, the
stored food reserves like lipids, starch and seed storage proteins are
degraded to yield compounds that can be utilized by the growing embryo
for its growth and development.

• After a certain span of vegetative growth, plants undergo a reproductive

development. During this process, flowers are produced. The flowering
process is regulated by the duration of light/dark cycles. The dark period
seems to be more critical to the flowering process. It is the leaf which
perceives the photoperiodic stimulus. After perception the signal is sent
to the vegetative meristems which then are converted into flowering
210 meristems.
Unit 15 Plant Response to Light and Temperature
• The photomorphogenetic events in plants are regulated by a red light
and far-red light absorbing pigment called phytochrome, which exists in
two forms/states. The far-red light absorbing state—Pfr is biologically
active and induces a large number of fast and slow responses in plants.
The fast responses could be mediated by bringing changes in
membrane properties. The slow responses may involve formation of new
proteins which would control the transition from one developmental state
to another.

• Each plant undergoes senescence after the completion of their life cycle.
Senescence may be manifested in the form of seasonal leaf-fall or death
of certain parts or the entire plant itself.

• Senescence involves a series of genetically controlled biochemical

changes which also determine the various patterns of programmed
death.

• The phenomenon of flowering itself is under the control of endogenous

rythms and follows a strict biological clock- like precise pattern. Of
course, these developmental processes are influenced by external
factors.

• Many developmental responses are studied using in vitro culture

technique. This technique allows the culture of any type of tissue to
regenerate a complete plant. The tissue culture technique has many
exciting possibilities and commercial application. The process of
senescence shows variation in its pattern. This process also involves a
series of biochemical changes which are genetically controlled.

15.11 TERMINAL QUESTIONS

1. What are the three major categories of plants bases on their day length
requirement?

2. Give one example each of: i) Ambiphotoperiodic plant, ii) obligate short-
day plant

3. Discuss the importance of dark period in flowering.

4. How are the two forms of phytochrome interconvertible?

5. Comment on the chemical nature of phytochrome.

6. Give examples of some phytochrome-mediated responses

7. What are the recent ideas regarding phytochrome action leading to

flowering.

8. Discuss the concepts of florigens.

9. What is Vernalization? How is it useful in agriculture?

10. What are biological clocks? Give some examples. 211

Block 4 Nitrogen Metabolism and Plant Growth Regulators
15.12 ANSWERS
Self-Assessment Questions
1. a) i) Vegetative, floral ii) dark; iii) Day-neutral; iv) darkness

b) i) False; ii) True; iii) False; iv) True; v) True

2. a) i) 660 nm, 730 nm

ii) Far-red

iii) Soluble protein, cytoplasm

iv) Calcium

v) Pfr, transcription

b) Refer to Subsection 15.4.1.

3. a) Shoot apex; b) devernalization; c) Vernalin; d) GA

e) Low temperatures, suitable photoperiod

4. i) Level of Pfr

ii) Phaseolus

iii) Floral clocks

iv) Spore discharge in Pilobolus

v) Absence of external factor

5. a) Refer to Section 15.7.

b) Refer to Section 15.7.

c) Refer to Section 15.7.

Terminal Questions
1. Long day, Short day and Day neutral plants;

2. i) Setaria verticellata, ii) Chenopodium rubrum

3. Refer to Subsection 15.2.2.

4. Refer to Subsection 15.3.1

5. Refer to Subsection 15.3.2.

6. Refer to Subsection 15.3.3.

7. Refer to Subsection 15.3.4.

8. Refer to Section 15.4.

9. Refer to Subsection 15.5.1.

10. Refer to Subsection 15.6.

212
Unit 16 Plant Stress

UNIT 16
PLANT STRESS

Structure
16.1 Introduction 16.5 Plant Responses to
Specific Stress Conditions
Objectives
Water and Osmotic Stress
16.2 What is stress?
Salinity
16.3 Nature of Stress
Pollutant Stress
Physical stress
Temperature Stress
Chemical stress
Stress by Infection and
Biological stress
Wounding
16.4 Ways to Adapt to Stress
16.6 Future Prospects
Changes at Cellular and
16.7 Summary
Molecular Level
16.8 Terminal Questions
Biochemical Alterations
16.9 Answers
Changes in Plant Morphology
and Behaviour

Alternate Metabolic Pathways

16.1 INTRODUCTION
The previous units have provided you information about the various essential
physiological processes in plants. These include photosynthesis, respiration,
transpiration, translocation of solutes and assimilation of nutrients such as
carbon, nitrogen, sulphur. Plants get exposed to environmental variations.
These changes bring alterations in the growth and development of plants by
affecting various biochemical and physiological processes. Exposure to abiotic
and biotic stresses such as alteration in temperature, water availability, soil
pH, salinity, alkalinity, heavy metals, pathogen infestation, infection and injury
brings a change in the metabolic status of the plants. Exposure of plants to
environmental conditions or stress triggers various metabolic responses in
plants. The present unit introduces you to the concept of abiotic and biotic
stress in plants and provides detailed information about plant response to
stress. 213
Block 4 Nitrogen Metabolism and Plant Growth Regulators

Objectives
After studying this unit you would be able to:

define stress and differentiate between abiotic and biotic stress to

which plants gets exposed;

describe changes in morphological, physiological and metabolic

behaviour of plants in response to stress;

explain plant response to water and osmotic stress, temperature

stress;

discuss plant response to pollution; and

explain about plant response to infection and wounding.

16.2 What is stress?

Any change in the surrounding environment that affects the functioning of
plants and alters the homeostasis is defined as biological stress. A sudden
transition in the optimal environmental conditions which disturbs the
homeostasis in plants and induces adverse effects on their physiology is
referred as plant stress. Plant species get exposed to fluctuations in
temperature (chilling, freezing and high temperatures), water availability,
changes in the soil conditions such as salinity, heavy metals etc. Plants have
an optimum requirement for various environmental variables and show
susceptibility to any alteration. These changes impose stress in plants which
include delay or alteration in growth and development, effects on productivity
and in extreme cases, death.

Let us recall what happens within the natural communities occupying the same
habitat. The relative location of two plants may place them under differing
conditions with respect to a given environmental factor such as light. The top
cover of a rainforest, for example, consists of relatively tall trees and receives
maximal irradiance while the floor dwellers manage with sunflecks.

214 Fig. 16.1: Graphic representation of yield gap.

Unit 16 Plant Stress
What will happen if we artificially shadow the outer cover plants and illuminate
the forest floor? Being a deviation from the natural situation, this is likely to
have adverse effects on growth of plants. Fig. 16.1 shows the yield gap due to
biological and environmental constraints. Thus, any deviations from optimal
environmental conditions usually with adverse effects are considered as
stress. This in turn affects the yield of useful agricultural products

We must, however, realise that in no habitat, all conditions - temperature, light,

availability of water, nutrient supply and soil characteristics can be controlled
and fixed to optimal level for any species. But since the species must survive,
it must adapt itself to the deviations in the environmental condition(s). This
could be achieved either by breeding crop varieties tolerant to stress or by
offering conditions that help the plants to withstand the stress. For example, if
there is a deficit of some nutrient one could try to supplement the same. Plants
capable of adapting themselves to changes in environmental conditions
perform the best under stressful conditions.

16.3 NATURE OF STRESS

As mentioned in the preceding section, stress can be considered as any
deviation in environmental condition from optimal for the overall performance'
of the plant species under reference. If you recall the environmental factors,
you can tell the various kinds of stress that plants may be subjected to. A
broad outline on the nature of possible environmental stress is illustrated in
Fig. 16.2.

Fig.16.2: Nature of Environmental Stress Conditions.

16.3.1 Physical Stress

Physical stress originates from environmental conditions such as:

i) Temperature : We are familiar with the plants and other organisms that
live at temperatures close to the temperature range in which we are 215
Block 4 Nitrogen Metabolism and Plant Growth Regulators
adapted to live i.e., 15° to 45° C. However, we know that there is life
below 0° C in the arctic and above 90° C in the sulphur springs. In the
subtropical zones, plants face stress -when they get exposed to freezing
temperatures during the winters while in the deserts of the tropics the
native plants withstand over 55° C during summer. High temperature can
be inhibitory for photosynthesis (Fig. 16.3).

Fig. 16.3: Effect of light intensity and temperature on the rate of photosynthesis.

ii) Osmotic strength of the fluids in immediate surrounding: The

availability of soluble mineral salts varies widely from habitat to habitat.
In fact, a big chunk of land in our country has been classified as
'wasteland' because of high salinity of the soil cover or the water
leachates. Common crop cultivars cannot be grown in such
environments.

iii) Photosynthetically active radiation: This is usually in direct proportion

with the incident solar radiation. You know that tropics are probably the
best illuminated part of the globe. Also, variation in day length is minimal
in this zone. No wonder, light-dependent life has attained maximal
density in the tropics. Within a dense population of plants, different
organisms may have different degrees of exposure. However, if a plant
is exposed to increasing light intensity it shows a corresponding increase
in the rate of photosynthesis upto a certain point, beyond which further
increase in light causes either no change in the rate or causes an
inhibition (Fig. 16.3). Thus, each plant species shows a characteristic
photosaturation and/or photoinhibition of photosynthesis. In other words,
each species has its own optimum with respect to light intensity. Plants
get stressed if they get exposed to intensity above or below the
optimum.

16.3.2 Chemical Stress

Survival of cells is dependent on carrying out of a set of chemical reactions
(metabolic reactions) in a particular order. This results in a net gain in mass as
216 well as energy in a growing cell. Even for dormant cells as in dormant buds
Unit 16 Plant Stress
and seeds, where there may not be any net gain in mass, a certain amount of
energy must be spent to maintain them in a viable state. Various catabolic and
anabolic pathways operate according to the availability of chemical
constituents present in the cytoplasm. This may in turn be influenced by the
composition of the extracellular environment with respect to the following:

i) Nature of the solutes: The acidic or basic reaction of soil and water of a
particular habitat reflects its geochemical history beginning with its
formation and subsequent interactions with other constituents of the
earth up to its current chemical activities. Mineral deposits in their oxide
form ('Bhashma') are usually basic in their reaction. The reaction of
chloride, sulphate and nitrate is acidic or neutral depending upon the
nature of the conjugate ions. You can visualise the reaction of a salt
through its acidic and basic radicals. On a natural course, one would
expect the neutralisation reactions and consequently change in the
character of the habitat towards neutral. However, there are soils which
are very high and others very low in pH and certain plant species survive
in such soils.

ii) Mineral composition: The living systems make use of several mineral
ions that they might have encountered at the very origin or during
evolution, particularly for transformation of matter involving proteins and
nucleic acids. These elements continue to remain essential requirements
for life. You have already learnt in Unit 12 that nitrogen, phosphorus,
calcium, potassium and magnesium are familiar major requirements for
plant growth besides carbon, hydrogen and oxygen. Apart from these,
many other micronutrients such as manganese, iron, zinc, cobalt and
molybdenum are required for healthy plant growth in much smaller
quantities. Availability of these elements in the environment in quantities
smaller than required cause’s deficiency symptoms while a surplus may
cause toxicity leading to stunted growth, necrosis, abnormal
development of vegetative and reproductive parts.

iii) Atmospheric imbalances: At least two components of the environment,

oxygen and carbon dioxide that plants require are predominantly in the
gaseous form. As you have learnt, atmospheric nitrogen can be used by
nitrogen-fixing bacteria, some prokaryotic algae and plant-bacterial
symbiont. The biological cycling of these gases keeps their overall
availability buffered within a reasonably stable range. Yet, intensive
industrial activities that involve emission of one of these gases or high
levels of the oxides of carbon, nitrogen and sulphur have visible effects
on the performance of most plant species.

16.3.3 Biological Stress

Since in nature, the various organisms do not live in complete isolation from
others, stress to a plant species might also be caused by what other
organisms in the community require and consume. The following situations
exemplify biological stress.

i) Population density: You are aware of what might follow an uncontrolled

growth of human population. There will be competition for common 217
Block 4 Nitrogen Metabolism and Plant Growth Regulators
consumables and for space but this is not merely a human problem. Too
thick population densities of plants can resist the competition for
common nutrients, water and photosynthetically active radiation (PAR).
The effect can be a self inhibition or inhibition of other plant species
growing in the same environment. That is one of the reasons why weeds
are removed from croplands.

ii) Parasites: Many insects and microorganisms can feed upon tissues and
saps of living plants. Hence plants must be protected against such
parasites either by expressing evasive devices such as inhibitors and
toxins against the enzymes of parasites or by developing preventive
morphological and biochemical alterations that keep the parasite away
from aggregating near the plant.

iii) Symbiotic interaction: Presence of symbiotic microorganism can result

in differential growth stimulation of those plants which can recognise the
beneficial symbionts and utilise their presence in the environment. This
interaction is a mutual one. For example in Rhizobium-legume
symbiosis, the rhizobium causes a drain on the carbon fixed by the plant
(refer Unit 13) while the plant gains access to the nitrogen fixed by the
bacteria.

SAQ 1
What is the nature of stress faced by the following species if the change
mentioned below is brought about in their environment?

a) A plant species growing in Manali is brought to Jaipur during summer.

b) A sun-loving plant gets shaded by other plants.

c) A wheat crop gets flooded due to heavy rains for several days.

d) Trees growing near the roadway are subjected to heavy traffic.

e) Plants acclimatised to grow near fresh water are grown in coastal areas
of Goa.

16.4 WAYS TO ADAPT TO STRESS

Plant stress responses are dynamic and involve various regulatory pathways.
These include physiological, morphological adaptations and adjustment of
metabolism. Alterations in the physiology or morphology help the plants to
survive the changing environmental conditions (Fig 16.4). Plants respond to
stress by undergoing change in metabolic functions, morphological features
and physiological features. These responses are referred as stress
responses. In salt sensitive plants (glycophytes), a number of physiological
responses get stimulated. These response help plants to cope salinity stress.
This includes SOS pathway (known as salt overlay signaling) that leads to
enhanced efflux of sodium (Na) ions and reduction in toxicity induced by
218 salinity.
Unit 16 Plant Stress

Environmental stress

Abiotic Biotic

Response

Resistance Susceptibility Avoidance

Survival
Survival Death

Fig. 16.4: Effect of stress on plants.

Stress responses require no genetic modification, hence are referred to as

phenotypic plasticity. In phenotypic plasticity, the plants respond to changes
in environmental conditions via changes in morphology and physiology. The
changes are reversible. The ability of the plant to survive and establish in a
given environment is related to balance between the genetic adaptation and
phenotypic plasticity

Abiotic stress produces primary and secondary alterations in plants. Stress

primarily causes reduction in hydrostatic pressure (turgor pressure) which
leads to cellular dehydration. The change in cell hydration potential affects the
physical and biochemical properties of cell. The secondary effects of stress
include reduction in metabolic activities, production of reactive oxygen species
(oxidative stress) and disruption of cellular integrity, ultimately death. Stress
factors such as increase in temperature and exposure to ionizing radiation, UV
light and environmental pollutants leads to generation of reactive oxygen
species (ROS) which causes oxidative stress. The most common ROS formed
include superoxide radical (O2.-), singlet oxygen (1O2), hydrogen peroxide
(H2O2) and hydroxyl radicals (OH.).

Oxidative stress is caused when the ROS are produced and accumulated in
high amounts in a cell or tissues than those that can tackled by the biological
system. Oxidative stress leads to inhibition of major metabolic functions in
plants. These include inhibition of photosynthesis and respiration. This results
from down regulation or disruption of electron transport chains (ETC) in
chloroplasts and mitochondria. Abiotic stresses such as water deficit, salinity
and freezing disrupt cellular structures and impair physiological processes in
plants. The damage to cellular structures leads to reduction in growth, fertility
and cause premature senescence. The cell membranes become disorganized
as the proteins get deactivated or denatured. 219
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Box16.1: Tolerant plants

Two major strategies of combating stress have been recognized in plants. These are
avoidance and tolerance strategies. Stress avoidance includes a various protective
mechanisms that delay or prevent the negative effect of stress on a plant. Stress
tolerance is the potential of a plant to acclimate to a stressful condition. Many plants
have the capacity to tolerate stress. These are referred to as stress
resistant/tolerant plants (Fig. 16.5). These plants exhibit the capacity to adjust or
acclimatize to stress conditions. The plants ability to resist or tolerate stress depends
on their genetic capacity to adjust/overcome stress and establish a new homeostatic
state over a period of time. Some plants escape the stress. These include
ephemerals or short lived plants. They germinate fast, grow and flower quickly. They
complete their life cycle during a period of adequate moisture and form dormant
seeds before the onset of dry conditions. Many arctic plants complete their life cycle
during the short arctic summer and survive during winter in the form of seeds.
Ephemerals never get exposed to stress because they survive the stress by
avoidance. Plants of alfa alfa (Medicago sativa) survive dry habitats by sending their
roots deep into the soil near the water table. In this way the plant ensures survival
under conditions of drought. Other plants develop fleshy leaves that store water, thick
cuticles and pubescence (leaf hairs) to reduce transpiration such as Bryophyllum,
Opuntia etc.

Fig 16.5: Medicago sativa and Opuntia.

16.4.1 Changes at Cellular and Molecular Level

Responses to environmental stresses occur at all levels of organization. The
changes can be at cellular or genetic level.

Cellular level - At this level, plants tackle stress by bringing modifications in

the structure and architecture of the cell wall and membrane, cell cycle and
cell division. Plants bring alteration in the metabolic events. These include
production of compatible solutes such as proline, raffinose, glycine betaine.
These solutes are the organic compounds that are osmotically active in the
cell and play a role in stabilization of proteins, membranes and organelles.
These compounds also contribute to maintenance of cell osmotic status by
removing ROS and re-establishment of redox balance.

Molecular level - At this level, gene expression gets modified due to stress.
Stress-inducible genes i.e. genes that are involved in protection of cell from
stress via synthesis of osmoprotectants, detoxifying enzymes, transporters
and those which encode regulatory proteins such as transcription factors,
220 protein kinases, and phosphatases get induced.
Unit 16 Plant Stress

SAQ 2
Define the following terms:

a) Stress

b) Tolerant plants

c) Ephemerals

d) Phenotypic plasticity

16.4.2 Biochemical Alterations

The plants exposed to stress depict alterations in their metabolic processes.
Drought (water deficit), salinity and low temperature stress leads to change in
osmotic/water potential resulting in loss of turgor in plant cells. The plants
adjust to the condition of loss of turgor by accumulating solutes. The osmotic
adjustment refers to the capacity of the plant cell to accumulate solutes which
help in maintaining the osmotic potential during stress conditions.

High levels of sodium (Na) or chloride (Cl) ions exert detrimental effects in
plants. Accumulation of ions (Na and Cl) in vacuoles (vacuolar
compartmentalization) facilitates osmotic adjustment in halophytes growing in
saline conditions.

Compatible solutes (osmolytes) help in tackling stress. These include a group

of chemically diverse uncharged, polar, and soluble organic compounds and
do not interfere with the cellular metabolism even at high concentrations. They
mainly include proline, glycine betaine, sugar, and polyols. They act as source
of carbon and nitrogen to the cell under normal conditions. Some plants show
accumulation of amino acids on exposure to abiotic stress. This results from
increase in amino acid synthesis or stress-induced protein breakdown.

Carbohydrates such as starch and fructans show accumulation under stress

conditions. They act as storage substances and are used as energy source
during stress. Fructans have high water solubility and possess resistance to
crystallization at freezing temperatures. They stabilize membranes and
indirectly contribute to osmotic adjustment by the release of hexose sugars.
The roles played by these carbohydrates in stress mitigation are
osmoprotection, carbon storage and scavenging of reactive oxygen species.
Sugars function as osmolytes and help in maintaining turgor of cell and protect
membranes and proteins from damage. Salt and drought stress generally
leads to a depletion of starch content but accumulation of soluble sugars in
leaves.

16.4.3 Changes in Plant Morphology and Behaviour

The plants adapt to extreme environmental conditions through modification in
their life cycles. Desert plants have short life cycles and complete their life
cycle when the water is available. The deciduous trees of the temperate zone
shed their leaves before winter so that leaf tissue does not get damaged by
low temperature. Besides these the growth habits of plants also contribute to 221
Block 4 Nitrogen Metabolism and Plant Growth Regulators
tolerance in plants. Studies have shown that plants which flower for long
period of time are more tolerant to changing environmental conditions than
those flowering for short periods.

The leaves of some plants develop certain characters that help them to
escape extreme environmental changes. These include changes in leaf area,
leaf orientation, thickness of leaf, presence of trichomes and cuticle. Large leaf
area provides conditions for production of more photosynthates under stress
conditions. Large surface area proves advantageous for leaf cooling as more
water can be lost via evaporation, though excessive loss of water results in
dehydration.

Plants growing under stress conditions generally reduce their leaf area by
restricting cell division and expansion, altering leaf shapes and initiating
senescence or abscission of leaves. The cell division and expansion of leaf
gets restricted under conditions of water scarcity and salinity. Several
signaling mechanisms slow down or stop cell cycle thereby limiting growth.
Water deficit reduces turgor which affects cell expansion and reduces leaf
expansion. The smaller leaf area reduces transpiration resulting in effective
conservation of water. The thin film of air at the surface of the leaf (boundary
layer) permits the transfer of heat from the leaf to the air. It helps the leaves to
maintain surface temperature close to that of air so that transpiration is
reduced and overheating can be prevented.

Alteration in leaf shape is another means by which plants overcome stress.

Under conditions of water, heat, salinity stress, leaves become narrower or
develop deeper lobes. This leads to reduced leaf area and prevents loss of
water caused due to excessive heating. In some plants, leaf abscission occurs
under conditions of water deficit and reduced leaf area prevents loss of water
effectively. The plants adapted to drought show leaf abscission. Abscission
results from the enhanced synthesis and responsiveness of the plant hormone
ethylene. Some plants particularly crops such as corn, sorghum are able to
maintain green leaf area during grain maturation stage under stress
conditions. The retention of photosynthetically active leaves under stress is
known as stay green. Some varieties maintain green stem and leaves even
during drought conditions. The delay in senescence has been noted in these
varieties.

The change in orientation also allows greater light absorption by leaves. Under
conditions of high temperature and/or soil water deficits, plants can alter their
leaves to avoid excessive heating. Leaves of some plants orient themselves
away from sunlight to protect themselves from overheating. These leaves are
called paraheliotropic. Some leaves gain energy by orienting themselves
perpendicular to the sunlight is called as diaheliotropic. Wilting and leaf rolling
also alters the absorption of light. Wilting changes the angle of the leaf while
leaf rolling minimizes area exposed to sun.

The presence of trichomes on the surface of leaves help in keeping the leaf
surface cool. The presence of densely packed hairs on the leaf surface
reflects light. These hairs give leaf a silvery appearance. The cuticles (a layer
of waxes and hydrocarbons present on the cell wall of epidermis) present on
the leaf surface also reflect light thereby reducing heat. This layer also restricts
diffusion of water, gases and entry of pathogens. Plants exposed to water
222 stress develop thick cuticle to prevent water loss through transpiration.
Unit 16 Plant Stress
Water stress affects the development of roots and shoots. It has been
speculated that the shoot tends to grow until water uptake by the roots
becomes limiting for further growth. In contrast the roots tend to grow until the
demand for photosynthate from the shoot exceeds the supply. The functional
balanced gets disturbed during water stress. When water supply becomes
limiting, leaf expansion is reduced and a greater proportion of the plant
assimilates can be allocated to the root system where they can support root
growth. As the water deficit progresses the upper layers of the soil dry but the
roots proliferate into deeper, moist soil. This change in root architecture is
considered as defensive strategy against drought. Enhanced root growth into
the deeper soil requires allocation of photosynthates to the growing root tips.

SAQ 3
a) Match the statements from the column A with the correct options from
column B.

Column A Column B

i) salt sensitive plants a) water deficit or salinity

ii) dehydration b) osmolyte

iii) results in loss of cell turgor c) trichomes

iv) stabilizes proteins and d) ephemerals

membranes

v) present on the surface of leaves e) glycophytes

vi) short lived plants f) Reduction in water content

b) Differentiate between:

i) abiotic and biotic stress

ii) paraheliotropic and diaheliotropic leaves

16.4.4 Alternate Metabolic Pathways

The metabolic pathways change in response to stress conditions. Water deficit
decreases photosynthesis and consumption of assimilates in the expanding
leaves. The decreased water potential inhibits movement of assimilates and
less amount of photosynthates get exported from leaves. The ability of plants
to continue translocating assimilates under drought conditions is considered
as a key factor for plant resistance.

Some plants such as C4 and CAM plants change their mode of

photosynthesis. In CAM plants (succulents such as cacti), stomata open at
night and close during the day. This prevents loss of water through
transpiration and increases water use efficiency. The change in CAM mode of
photosynthesis has been noted in plants exposed to water deficit or salinity.
This adaptation allows plant to acclimate to arid environments. 223
Block 4 Nitrogen Metabolism and Plant Growth Regulators
During flooding, oxygen levels are low (hypoxia) and roots, stem of the plants
develop interconnected gas filled channels with in cells (aerenchyma) that
provide resistance pathway for the movement of oxygen and other gases. The
gases enter stomata or lenticels in woody stem and roots. They move by
molecular diffusion or by convection driven by small pressure gradients. In
many plants growing in wet habitats, the root cells develop into aerenchyma
cells. When the oxygen supply is insufficient for respiration, roots begin to
ferment pyruvate to lactate through the action of lactate dehydrogenase. The
production of lactate lowers the intracellular pH inhibiting the lactate
dehydrogenase and activating pyruvate decarboxylase. The change in the
enzyme activity leads to ethanol production. Hence during fermentation, 2
moles of ATP are formed per mole of hexose sugar catabolized in comparison
to 36 moles of ATP produced from each hexose catabolized in aerobic
respiration. Thus injury to the root metabolism by oxygen deficiency leads to
lack of ATP which affects essential metabolic processes.

16.5 PLANT REPONSES TO STRESS

CONDITIONS
16.5.1 Water and Osmotic Stress
The condition that arises due to scarcity or excess of water is known as water
stress. Water scarcity results in drought while excess of water leads to
flooding. Water makes a large proportion of the cell volume and is used for
expansion and metabolic processes. Flooding stress leads to anaerobic
conditions i.e. oxygen stress. The decreased supply of oxygen limits
respiration, nutrient uptake and other root functions. During drought
conditions, the plant cells lose water; they shrink because of collapse of cell
wall. The damage to root hairs affects the absorption capacity. The outer
layers of root cortex get covered with suberin, a water impermeable lipid that
increases resistance to water flow in the root. The resistance to water flow
also results from breakage of water columns within xylem under tension.

Plant growth becomes limited under conditions of water deficit. This is

because water deficit leads to cell dehydration. Water deficit causes reduction
in cell turgor and dehydration which results in reduction in cell size and/or
volume. The cell wall extensibility decreases due to increase in cell wall pH. In
long term, water deficit leads to reduction in vegetative growth. Shoot growth
and leaf production gets severely affected. There is reduction in leaf
expansion and growth which is an adaptation in plants to reduce rate of
transpiration. The loss in turgor of cells affects the cell enlargement. Reduction
in low water potential enhances root growth gets thereby changing the root
shoot ratio. Improvement in root shoot ratio improves water supply as the roots
can extract more water by exploring larger volumes of soil.

Water deficit leads to closure of stomata. This has been considered as a

strategy adapted by plants to conserve water. The closure of stomata leads to
low internal CO2 concentration in plants and this limits the photosynthetic
capacity of the plant. Water stress affects the photosynthesis in two ways.
First by blocking supply of carbon dioxide to the chloroplasts due to closure of
224 stomata and second by changing the structural integrity of the photosynthetic
Unit 16 Plant Stress
machinery due to low water potential. The change in the photosynthetic
machinery affects electron transport and photophosphorylation.

The metabolic processes such as accumulation of solutes get triggered by

stress. The solutes which take part in osmotic adjustment include inorganic
ions (such as K+), sugars and amino acids. The solutes maintain hydration of
the protoplasm by decreasing osmotic potential under stress. They prevent
loss of membrane integrity and maintain protein stability. The amino acid
proline, sorbitol (sugar alcohol) and glycine betaine are the other solutes that
help in the recovery of turgor under conditions of water stress.

Anatomical and physiological adaptive mechanisms limit transpiration in

conditions of water deficit. The presence of thick cuticle, trichomes, sunken
stomata limit the loss of water through transpiration.

16.5.2 Salinity
Excessive accumulation of salt in the soil is referred as salinity. High
concentrations of Na+, Cl-, Ca2+, Mg2+ and SO42- are present in saline soils.
High concentration of salts replaces the essential ions present in the soil
resulting in toxicity. Salinity results in accumulation of Na and Cl ions in the
cytosol. The soils having high sodium (Na) content are called sodic soils. They
degrade soil structure by decreasing porosity and water permeability. High
concentrations of salts cause denaturation of proteins and membrane
destabilization by reducing hydration of molecules. The nutrient acquisition
gets disturbed from accumulation of toxic ions in soil.

Some plants growing in saline soils are not or less adapted. They are called
as glycophytes. These plants get exposed to stress referred as salinity
stress. In contrast, some plants are well adapted to saline conditions. These
are called as tolerant plants. For example-halophytes. These plants develop
various physiological and biochemical mechanisms to survive in highly saline
soils. The mechanisms involved in salt tolerance mainly include ion
homeostasis, compartmentalization, biosynthesis of
osmoprotectants/compatible solutes, activation of antioxidant systems
(enzymes and compounds), synthesis of polyamines, generation of nitric oxide
(NO) and hormone modulation.

Halophytes possess specialized salt glands on the surface of leaves that

excrete salt. Some plants accumulate toxic ions in the older leavers which
senesce and abscise to allow younger, more photosynthetically productive
leaves to survive.

Compatible solutes function as a protector or stabilizer of enzymes or

membrane structures that are sensitive to dehydration. Proline provides
tolerance to stress and serves as a nitrogen reserve. It functions as a
quencher of superoxide radical and hence exhibits antioxidant potential. It
provides tolerance to salt stress by increasing the activity of enzymes involved
in antioxidant defense system. Straight-chain polyols such as mannitol,
sorbitol and cyclic polyols such as myo-inositol or its methylated derivatives
increase in response to conditions of salinity. Trehalose accumulation caused
due to salinity stress protects plants against several physical and chemical
damages. Polyamines play an important role inducing tolerance in plants. 225
Block 4 Nitrogen Metabolism and Plant Growth Regulators
Although, abscisic acid (ABA) is a phytohormone which inhibits growth but its
application ameliorates the effect of stress conditions in plants. This hormone
gets upregulated in conditions of water deficit. Increased production of ABA
has been observed in shoots and roots of plants exposed to salinity stress and
water deficit. ABA acts as a vital cellular signal that modulates the expression
of many salt and water deficit-responsive genes. The genes related to
accumulation of K+, Ca2+ and compatible solutes (such as proline and sugars)
get upregulated by ABA. Hormones such as salicylic acid (SA) and
brassinosteroids (BR) are also known to participate in plant responses to
abiotic stresses.

A large number of genes and transcription factors are upregulated in response

to salinity in different plant species. These are called salt-responsive genes.
These genes are mainly involved in ion transport or homeostasis (e.g., SOS
genes, AtNHX1, and H+-ATPase), senescence [senescence associated gene
(SAG)], molecular chaperones (such as heat shock proteins), and
dehydration-related transcription factors (DREB). Upregulation of
metallothionein and water channel proteins has also been noted in plants
exposed to stress.

16.5.3 Pollutant Stress

Plants have adapted various ways to tackle stress caused by exposure of
various gaseous pollutants and toxic trace elements present in the
environment.

Exclusion - The process/means by which the plants maintain level of

contaminants below a toxic threshold. It mainly involves removing the element
out of the cell/body.

Tolerance - The process/means by which plants develop various biochemical

adaptations which allow the plant to survive stress conditions. These mainly
include compartmentalization and chelation.

Plants get exposed to high levels of heavy metals present in the soil. Some
plants show high accumulation of heavy metals such as copper (Cu), nickel
(Ni), zinc (Zn), chromium (Cr), lead (Pb), cadmium (Cd), cobalt (Co), iron (Fe)
in their tissues. These are called hyperaccumulators. High concentration of
heavy metals induces oxidative stress in plants. Antioxidant defensive
mechanisms play an important role in tackling oxidative stress caused by
heavy metal accumulation.

The plants exposed to air pollutants show alterations in the physiological and
biochemical features in plants. These features help in determining
susceptibility or tolerance to stress caused by pollutants. Changes in stomatal
behavior, carbon and nitrogen assimilation have been noted in plants exposed
to air pollutant stress. Air pollution stress leads to stomatal closure which
reduces availability of carbon dioxide to leaves and inhibits carbon fixation. As
a result the net phostosynthetic rate decreases thereby limiting the plant
226 productivity. Absorption of air pollutants such as sulphur dioxide (SO2), oxides
Unit 16 Plant Stress

of nitrogen (NOX) and carbon dioxide (CO2), suspended particulate matter

(SPM) by leaves causes reduction in the concentration of photosynthetic
pigments viz. chlorophyll and carotenoids. This affects the plant productivity.
The plants respond to pollutant stress by closing their stomata. This depicts
the avoidance mechanism adapted by plants. In contrast, synthesis of
antioxidant compounds reflects strategy adapted by tolerant plants.

SAQ 4
Answer in one word:

a) The plant hormone that accumulates in the water stressed leaves.

b) Soils having high concentrations of Na+, Cl-, Ca2+, Mg2+ and SO42-.

c) Solutes that protect or stabilize enzymes or membrane structures during

stress.

d) The process by which plant maintains the level of elements below a toxic
threshold.

e) Some plants show high accumulation of heavy metals in their tissues.

f) Some plants possess specialized salt glands on the surface of leaves

that excrete salt.

g) The condition in which roots and stem of the plants develop

interconnected gas filled channels that provide low resistance pathway
for the movement of oxygen and other gases.

16.5.4 Temperature Stress

Plants exhibit sensitivity to variations in temperature. Each plant requires an
optimum temperature for growth and development. Exposure to extremely
high and low temperatures adversely affects growth in plants.

Low temperature stress

An exposure to moderate chilling temperature may kill or cause injury to

plants. Maize, tomato, soybean, cotton are susceptible to injury when exposed
to temperatures between 10 to 15o C. The seedlings show wilting and
chlorosis in conditions of chilling stress. The low temperature causes
reversible changes in the physical state of the membranes. This occurs due to
change in the proportion of saturated and unsaturated fatty acids present in
the membrane lipids. The temperature at which the transition from liquid to
solid state occurs is called as transition temperature. At temperature above
the transition temperature, the membrane remains fluid but becomes solid or
gel like at temperatures below the transition temperature. With the drop in
temperature, the membranes show a transition from a flexible liquid crystalline
structure to a solid gel state. The rate of transition varies with the species and
depends upon the lipid composition of the membrane. Sensitive plants have 227
Block 4 Nitrogen Metabolism and Plant Growth Regulators
high proportion of saturated fatty acids while chilling resistant species possess
low proportion of saturated fatty acids but more of unsaturated fatty acids. The
membranes with this composition tend to solidify into a semicrystalline state at
temperature above 0º C. For functioning and stability, membrane needs to be
maintained in a liquid gel like state but under stress they are changed to solid
state which affects the membrane structure and activity. Freezing leads to
intra and intercellular crystal formation. Intercellular crystal formation damages
membranes and organelles. Extracellular ice crystal formation cause cellular
dehydration. This is because ice formation lowers the water potential in the
apoplast. It forms a gradient from high water potential gradient in the symplast
to low water potential gradient in the apoplast. Water moves from the symplast
to apoplast resulting in cellular dehydration which affects the membranes. The
formation of a stable ice crystal involves participation of several hundred water
molecules. This is called ice nucleation.

The damage to the plant cell membranes i.e. structure and function affects the
metabolism. Enzymes and enzymatic reactions are sensitive to temperature.
The rate of reaction doubles for each 10oC rise in temperature until an
optimum is reached beyond which the rate of reaction declines. The decline in
enzymatic activity results from unfolding of protein and is referred as thermal
denaturation. The Q10 increases linearly with short term rise in temperature.
Q10 measures change in rate of respiration. An increase in ambient
temperature causes change in the rate of respiration. .

The herbaceous species grown at low temperature exhibit a short, compact

growth habit, thicker leaves due to an increase in leaf mesophyll cell size
and/or an increase in the number of palisade layers.

Deciduous trees, conifers and shrubs such as birch, willow, survive cold
stress because they are able to adapt to low temperature conditions. The
adaptation in woody tissues begins in autumn when the growth and
photosynthesis ceases and plant enters dormancy. Hence the plants enter the
dormant phase prior to the onset of frost to prevent damage caused due to
freezing. During frost the respiratory activities sufficiently provide the energy
required for the numerous metabolic changes to attain maximum cold
acclimated stage. During this period the conversion of starch to sugars and
level of organic phosphate increases. The glycoproteins accumulate and the
protoplasm becomes more resistant to dehydration.

During adaptation to cold conditions, the temperate woody trees withdraw

water from the xylem vessels thereby preventing the stem from splitting in
response to the expansion of water during freezing. The physical properties of
lipids get altered at low temperatures resulting in increased membrane rigidity.

High temperature stress

High temperature (HT) stress is injurious to growth and development of plants.

High temperature stress causes loss of water content from the cell as a result
cell size gets reduced and ultimately the plant growth gets affected. Reduction
228 in seed germination potential, seedling growth, reduced radicle and plumule
Unit 16 Plant Stress
growth has been noted as the major impact of heat stress in plants (Fig.16.6).
Reduction in relative growth rate (RGR) due to decrease in net assimilation
rate (NAR) has been noted in conditions of HT stress. The negative effect of
heat on leaf includes reduction in water potential, leaf area and pre-mature
leaf senescence.

Exposure to high temperature alters the total phenological duration of the plant
life. Increases in temperatures affect the grain filling periods. A short period of
heat stress can cause significant decrease in production of floral buds. The
increase in sterility is caused due to impairment of meiosis in both male and
female organs, negative effects on pollen germination, reduced pollen tube
growth, reduced ovule viability, abnormality in stigmatic and style positions,
disturbance in fertilization process, hindrance in growth of endosperm and
proembryo. High temperature treatment reduces anther dehiscence and pollen
fertility. The reduced fertilization results from decrease in number of pollens on
the stigma.

Oxidative Alteration in
stress phenology
Water loss

High temperature

Reduction in
Reduction in Yield
metabolic events
growth reduction

Fig. 16.6: Major effects of high temperature stress on plants.

The plants that can adjust to high temperature conditions are called
thermotolerant plants. Plants exposed to cold temperature exhibit a lower
optimum temperature for photosynthesis while those growing at high
temperatures exhibit high temperature optima for photosynthesis. The tissues
of the plants are not able to survive temperatures above 45oC but some of the
tolerant species show survival at these temperatures because of their ability to
show of evaporative cooling. Leaf temperatures can raise to 4 to 5oC above
ambient air temperature in bright sunlight when soil water deficit causes
stomatal closure to high relative humidity reduces gradient driving evaporative
cooling.

Plants exhibit various mechanisms to ensure survival under high temperature

conditions. These include phenological changes, morphological adaptations
and short-term avoidance or acclimation (adaptation) mechanisms such as
changing leaf orientation, transpirational cooling, or alteration of membrane
lipid compositions. Closure of stomata and reduced water loss, increased 229
Block 4 Nitrogen Metabolism and Plant Growth Regulators
stomatal and trichome densities and larger xylem vessels are common heat
induced features in plant.

Early maturation noted in many crop plants has been considered as a

mechanism to escape heat stress. Plants growing in a hot climate avoid heat
stress by reducing the absorption of solar radiation. This ability is supported by
the presence of small hairs (tomentose) that form a thick coat on the surface
of the leaf as well as cuticles, protective waxy covering. In such plants, leaf
blades often turn away from light and orient themselves parallel to sun rays
(paraheliotropism). Solar radiation may also be reduced by rolling leaf blades.

Plants with small leaves are also more likely to avoid heat stress. They
evacuate heat more quickly due to smaller resistance of the air boundary layer
in comparison with large leaves. In well-hydrated plants, intensive transpiration
prevents leaves from heat stress, and leaf temperature may be 6°C or even
10-15° C lower than ambient temperature. High temperature can affect the
degree of leaf rolling in many plants

When plantlets or tissues of plants are shifted to 42° C and above, the
synthesis of normal proteins rapidly declines, instead synthesis of new
proteins is induced. These proteins are known as heat-shock proteins (HSP).
These proteins are self-regulatory i.e. their synthesis is switched off after 6 to
8 h of exposure to elevated temperature while synthesis of the normal proteins
resumes. The molecular weight of heat-shock proteins ranges from 15 to 102
kDa. HSPs are known to be induced also by heavy metals and arsenites. The
HSPs occur in representatives of all the major groups of organisms. A pre-
treatment at elevated temperature (at 45°C for 2 h) eliminates the heat-shock
response. It is believed that heat shock protein 2 protects essential enzymes
and nucleic acids from denaturation.

Induction of synthesis of heat-shock proteins has also been observed under

field conditions. In dry fields during summer when the leaf temperature
reaches or exceeds the ambient temperature (>40° C), synthesis of HSPs is
induced as under experimental conditions. The synthesis of HSP shows a
transcriptional as well as translational control.

How do HSPs help in heat-shock avoidance? They probably help important

cellular proteins to acquire conformations that would be safe and functional
under high temperature and the protein will remain in soluble state in the
cytoplasm.

Molecular approaches are also being followed to improve plant tolerance.

Plants tolerate HT stresses by modulating multiple genes involved in various
pathways. Heat stress up-regulates several heat inducible genes referred as
“heat shock genes” (HSGs) which encode HSPs. These proteins protect
intracellular proteins from being denaturation and preserve their stability and
function through protein folding. HSPs act as chaperones. High temperature
also induces synthesis of low molecular mass proteins known as heat shock
proteins (HSPs). The synthesis of these proteins in cells improves thermal
tolerance. HSPs are also induced by water deficit, ABA treatment, wounding,
230 low temperature and salinity. Three distinct classes of these proteins have
Unit 16 Plant Stress
been recognized on the basis of their molecular mass in plants. These include
HSP90, HSP70 and heterogenous group of proteins in the range of 17 to 28
kDa.

16.5.5 Stress by Infection and Wounding

Plants respond to various pathogens through an intricate and dynamic

defense system. The mechanism of defense has been classified as innate and
systemic plant response. An innate defense is exhibited by plant in two ways,
viz., specific (cultivar/pathogen race specific) and non-specific (non-host or
general resistance). A large array of proteins and other organic molecules are
produced prior to infection or during pathogen attack. Constitutive defenses
include morphological and structural barriers (cell walls, epidermis layer,
trichomes, thorns, etc.), chemical compounds (metabolites, phenolics, nitrogen
compounds, saponins, terpenoids, steroids and glucosinolates), proteins and
enzymes. These compounds confer tolerance or resistance to biotic stresses
by not only protecting the plant from invasion, but also giving the plant strength
and rigidity. The production of toxic chemicals, pathogen-degrading enzymes
(chitinases and glucanases) is used by plants. These compounds may be
present in their biologically active forms or stored as inactive precursors that
are converted to their active forms by host enzymes in response to pathogen
attack or tissue damage. Plant defense strategies involving these compounds
can fall in the category of either innate or systemic acquired resistance (SAR).
Innate immunity is of greater efficiency and is the most common form of plant
resistance to microbes.

Imbalances in biotic factors reduce cell proliferation, photosynthesis,

membrane integrity, protein stability and induce production of ROS, oxidative
damage and death. The plant exposed to insects or pathogens responds with
changes in the composition and properties of the cell walls and synthesis of
secondary metabolites that limit the infection.

Secondary metabolites associated with the hypersensitive reaction constitute

signal transduction pathways that prepare cells and tissues to resist secondary
infection. Because of this effect the plant reacts to infection by slowly
developing a general immune capacity. The phenomenon is called as
systemic acquired resistance (SAR). One important component of this
signaling pathway is salicyclic acid (SA). It is a naturally occurring secondary
metabolite with analgesic properties.

Methyl jasmonate is the principle constituent of the essential oil of Jasminium

and high concentrations of Jasmonic acid (JA) has been isolated from fungal
culture filtrates. Both JA and methyl ester of jasmonate mediate insect
resistance. Besides the role in insect and pathogen resistance, jasmonic acid
is supposed to be involved in many physiological processes such as seed and
pollen germination, protein storage, root development and coiling of tendrils.

Plants show specific responses to abiotic or biotic stress. These responses

mainly include production/synthesis of specific hormones, proteins and
accumulation of solutes that helps in maintenance of the physiological and
metabolic processes (Fig. 16.7). 231
Block 4 Nitrogen Metabolism and Plant Growth Regulators

Fig.16.7: Plants responses in various kinds of stress.

SAQ 5
Complete the statements given below:

a) The low temperature causes reversible changes in the ……………… .

b) The chilling injury can result in many ……………………. .

c) The measure of Q10 depicts change in ……………………….. .

d) The negative effect of heat on leaf mainly include ……………………… .

e) High temperature has a greater influence on the photosynthetic capacity
of …………………….. .

f) The plants that can adjust to high temperature conditions are called
……………………….. .

g) Synthesis of low molecular mass proteins in cells that improve thermal

tolerance are …………………….. .

h) …………….. acts as important component of the signaling pathway in

systemic acquired resistance.

i) Hormone that mediates insect resistance in plants is ………………….. .

232
Unit 16 Plant Stress

16.6 FUTURE PROSPECTS

Plants have evolved a variety of mechanisms to withstand stress conditions. In
many cases we can precisely define the way plants enable themselves to
survive and perform well under stress conditions. Based on such knowledge, it
should be possible to construct and breed plants tolerant to many
environmental extremes found in our country and elsewhere on earth while
trying to cultivate plants. There are so many salt affected areas in our country.
Salt tolerant varieties can be planted in large areas affected by salinity.

One of the ways to manipulate stress tolerant varieties is through genetic

engineering technique. With this technique it is possible to isolate a gene and
introduce it in a desired organism. This results in the expression of transferred
gene in the new organism which then starts behaving like the organism from
which this gene was originally isolated. The gene products related to a
particular phenomenon also get synthesised in the organism. The synthesis of
one or more proteins can be achieved easily through genetic engineering
technique. Thus, for instance, if genes for betaine synthesis can be transferred
to a plant sensitive to osmotic stress, it might confer tolerance against such
stress or an elicitor like phytoalexin can be produced by plants by artificial
gene transfer, it would be possible to have disease resistant plant.

These programmes can be directed towards incorporating the following traits

among common crop plants.

1) Non-photoinhibition of photosynthesis particularly in the tropics would

mean several fold increase in biomass produced over the same period of
time as water and carbon dioxide supply are non-limiting.

2) Resistance to high temperature beyond mesophilic ranges can allow

agriculture in several areas that are left uncultivated because of
prohibitive temperatures. To a large extent any strategy to achieve this
would depend on the water status of the environment.

3) Cold-hardiness in cultivars can help curb the losses often incurred

because of extremely low temperatures reached during winters in some
parts of the world.

4) Drought resistance in crop plants will be particularly helpful to our

primarily rain-fed agriculture.

5) Salt tolerance in plants will bring a lot more of territory under green
cover.

6) Disease resistance has always been a trait sought after in plants

adopted for cultivation. This can improve the present yield by 20 to 50%
(depending on the plant in question and the climatic zone).

7) Pest tolerance in the crop plants can be achieved by producing

proteinase inhibitors or by producing bacterial gene coding for pesticidal
protein. This limits their nutritional value but in cases like cotton where
our prime interest is fibre instead of food, this can still be a way to
achieve upto 50% improvement in yield. 233
Block 4 Nitrogen Metabolism and Plant Growth Regulators
16.7 SUMMARY
• Transition in the optimal environmental conditions which disturbs the
homeostasis and induces adverse effects on their physiology in plants is
referred as stress. Stress is of two types: abiotic and biotic. Plant
species get exposed to various stresses such as fluctuations in
temperature (chilling, freezing, high temperatures), water scarcity,
salinity, infections and pathogen infestations etc.
• Plants respond to stress in various ways. These mainly include
physiological, morphological and metabolic adaptations. These changes
help the plants to survive the changing environmental conditions. These
responses require no genetic modification and hence are referred as
phenotypic plasticity.
• Plants combat stress by two major ways i.e. avoidance and tolerance.
Stress avoidance includes a variety of protective mechanisms that delay
or prevent the negative effect of stress on plant. Stress tolerance is the
potential of a plant to acclimate to a stressful condition.
• Abiotic stress produces primary and secondary effects in plants. Primary
effects include reduction in water potential, and cellular dehydration.
These effects alter the physical and biochemical properties of cell. The
secondary effects include reduced metabolic activity, production of
reactive oxygen species (oxidative stress) and disruption of cellular
integrity.
• Plants respond to these stresses at various levels of organization. The
changes can be at cellular or genetic level. At cellular level, plants tackle
stress by alteration in the metabolic events such as production of
compatible solutes.
• Studies on plant responses to stress provide useful information. In
future, it would be possible to take measures by genetic engineering and
other means to prevent losses in yields of crops, fruits, vegetables and
other useful products due to stressful environmental conditions. It would
also be possible to bring into use chunks of land that at present cannot
be used for cultivation.

16.8 TERMINAL QUESTIONS

1. What are the two different strategies adapted by plants to overcome
stress?

2. What is oxidative stress and how does it affects plants?

3. What compatible solutes are synthesized by plants under conditions of

abiotic stress?

4. What are the main components of the antioxidant defense system in

plants?

5. Explain the reason for closure of stomata under conditions of water

stress.

6. What are heat shock proteins? What role do they play in plants exposed
234 to stress?
Unit 16 Plant Stress

16.9 ANSWERS
Self-Assessment Questions
1. a) physical stress i.e., heat/temperature stress
b) physical stress i.e., photosynthetically active radiation
c) physical stress i.e., flooding
d) chemical stress i.e., Pollution
e) physical stress

2. a) Stress-Any change in the surrounding environment that affects the

functioning of plants and disturbs homeostasis in plants is referred
as stress.

b) Tolerant plants – The plants that have the capacity to tolerate

stress by adjusting or acclimatizing to stress conditions are
referred as resistant/tolerant plants. The plants tolerance capacity
depends on their genetic capacity to adjust/overcome stress and
establish a new homeostatic state over a period of time.

c) Ephemerals- Some plants escape the stress. These are called

ephemerals or short lived plants. They germinate fast, grow and
flower quickly. They complete their life cycle during a period of
adequate moisture and form dormant seeds before the onset of
dry conditions. They survive the stress by avoidance. Many arctic
plants complete their life cycle during the short arctic summer and
survive during winter in the form of seeds.

d) phenotypic plasticity- Stress response in which plants respond to

changes in environmental conditions via changes in morphology
and physiology without any genetic modification. The changes are
reversible.

3. a) i) glycophytes
ii) water deficit or salinity
iii) reduction in water content
iv) osmolytes
v) trichomes
vi) ephemerals

b) i) Stress imposed on plants due to change in physical or

chemical environment is called as abiotic stress while the
stress caused by living organisms, specially viruses,
bacteria, fungi, nematodes, insects, arachnids and weeds is
called as biotic stress.

ii) Leaves of some plants orient themselves away from sunlight

to protect themselves from overheating. These leaves are
called paraheliotropic. Some leaves gain energy by orienting
themselves perpendicular to the sunlight is called as
diaheliotropic. 235
Block 4 Nitrogen Metabolism and Plant Growth Regulators
4. a) Abscisic acid (ABA)

b) Saline soils

c) Compatible

d) Exclusion

e) Hyper accumulators

f) Halophytes

h) Hypoxia

5. a) physical state of the membranes

b) metabolic dysfunctions in plants

c) rate of respiration

d) reduction in water potential, leaf area and pre-mature leaf

senescence.

e) C3 plants

f) thermotolerant plants

g) heat shock proteins (HSPs)

h) Salicyclic acid (SA)

i) Jasmonic acid (JA)

Terminal Questions
1. Two major strategies of combating stress recognized in plants are
avoidance and tolerance. Stress avoidance includes a variety of
protective mechanisms that delay, avoid or prevent the negative effect of
stress on plant. The plants which are able to tolerate stress are called as
stress resistant/tolerant plants. The plants ability to resist or tolerate
stress depends on their genetic capacity to adjust/overcome stress and
establish a new homeostatic state over time.

2. Oxidative stress occurs due to generation of reactive oxygen species

(ROS). During oxidative stress, the ROS are produced and accumulated
in high amounts in a cell or tissue than those that can tackled by the
biological system. The most common ROS include superoxide radical
(O2.-), singlet oxygen (1O2), hydrogen peroxide (H2O2) and hydroxyl
radicals (OH.). ROS possess strongly oxidizing capacity and potentially
affect metabolic activities such as photosynthesis, damage cellular
structures leading to reduction in growth, reduction in fertility and
premature senescence.

3. The plants respond to abiotic stress such as drought, salinity by

undergoing metabolic alterations. One of the strategies adapted by
plants to overcome stress is synthesis of compatible solutes. Compatible
236 solutes are the organic compounds which help in curtailing stress. They
Unit 16 Plant Stress
mainly include amino acids such as proline and sugars. Proline acts as a
ROS scavenger, molecular chaperone and helps in stabilization of
protein structure thereby protecting cells from damage caused due to
stress. The non-protein amino acid, γ-aminobutyric acid (GABA) helps in
maintaining carbon–nitrogen balance and ROS scavenging.
Carbohydrates such as starch and fructans act as storage substances
and are used as energy source during stress conditions. They stabilize
membranes and indirectly contribute to osmotic adjustment upon
freezing and dehydration by the release of hexose sugars. Sugars
function as osmolytes to maintain cell turgor and protect membranes and
proteins from damage caused due to stress. Trehalose (non-reducing
disaccharide) functions as an osmolyte and stabilizes proteins and
membranes in desiccation-tolerant plants. The level of trehalose
increases under stress. Polyols play a role in stabilizing macromolecules
and scavenging hydroxyl radicals. Accumulation of polyols such as
mannitol and sorbitol has been noted in several plants species exposed
to stress.

Glycine betaine (GB) is a quaternary ammonium compound produced in

plants exposed to abiotic stress such as cold, drought, and salinity. The
compound protects photosystem II, stabilizes membranes, and mitigates
oxidative damage. Various stresses such as drought, salinity and cold
induce synthesis of polyamines (PA). High PA levels have been
positively correlated with stress tolerance. PAs have been implicated in
protecting membranes and alleviating oxidative stress.

4. Antioxidant system includes antioxidant enzymes and non-enzymatic

compounds. These play critical role in detoxifying ROS. The activity of
antioxidant enzymes, such as superoxide dismutase (SOD), catalase
(CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and
glutathione reductase (GR) and accumulation of nonenzymatic
antioxidant compounds increases under stress conditions. Antioxidant
compounds such as ascorbate, glutathione also help in mitigation of
stress. They react with superoxide radical, hydroxyl radical, and
hydrogen peroxide thereby functioning as a free radical scavenger.

5. Abscisic acid (ABA) accumulates in the water stressed leaves and

mediates the loss of solute from guard cells. The plant regulates ABA
metabolism by modulating ABA synthesis in response to dehydration.
ABA accumulation in the chloroplast lowers the pH. The pH increases in
the region surrounding the mesophyll cells stimulates release of ABA
from the mesophyll to the apoplast. This supports efflux of K ions from
the guard cells. The guard cells lose turgor resulting in closure of
stomata.

6. Heat stress leads to up-regulation of genes referred as “heat shock

genes” (HSGs). These genes encode proteins called heat shock proteins
(HSPs). These proteins protect denaturation of intracellular proteins from
being and preserve their stability, function through protein folding. HSPs
act as chaperones. The synthesis of these proteins improves thermal
tolerance in cells.
237
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Volume 2 Plant Physiology and Metabolism

FURTHER READING
• Appling, D.R., Anthony-Cahill, S.J. and Mathews, C.K .2015.
Biochemistry, Concepts and Connections. Pearson Education
Limited. New Delhi.

• Buchanan, B.B., Gruissem, W., and Jones, R.L. (eds), 2000.

Biochemistry & Molecular Biology of Plants 2nd ed.. Wiley, Blackwell,
U.K.

• Hopkins, W.G. and Hüner, N.P.A. 2008. Introduction to Plant

Physiology.4th ed. Wiley, New York.

• Mohr, Hans and Schopfer, Peter (eds). 1995. Plant Physiology,

Springer-Verlag, Berlin.

• Nelson, D.L. and Cox, M.M. 2017. Lehninger Principles of

Biochemistry.7th ed. W.H. Freeman, U.S.A.

• Ochs. R.S. 2014. Biochemistry. Jones & Barlett Learning, USA.

• Smith, R.J., Lea, P.J. and Galton, J.R. 1999. Nitrogen Fixation. In Plant
Biochemistry and Molecular Biology (P.J. Lea and R.C. Leegood eds.)
2nd Ed. John Wiley and Sons. Chichester, New York.

• Taiz, L., Zeiger, E., Møller, I.M. and Murphy, A. 2015. Plant Physiology
and Development, 6th ed. Sinauer Associates, Inc. Publishers
Sunderland, Massachusets U.S.A.

• Salisbury, F.B. and Ross, C.W. 1969. Plant Physiology, Wadsworth

Publishing Company.

• Verma, V. 2016. Plant Physiology. 2nd Ed. Athena Academic, U.K.

• Woo H.R., Kim H.J., Nam H.G., Lim P.O. 2013. Plant leaf senescence
and death – regulation by multiple layers of control and implications for
aging in general. J. Cell Sci.126: 4823-4833.

• Woo H.R., Masclaux-Daubresse C., Lim P.O. 2018Plant senescence:

how plants know when and how to die. J. Exp. Bot. Volume 69: 715–
718.

• Matos F.S., Borges L.P., Müller C. (2020) Ecophysiology of Leaf

Senescence. Journal of Agronomy & Agricultural Science 3, 22.

• Plaxton. W.C. 1996. The organization and regulation of plant glycolysis.

Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 185-214.

239
Volume 2 Plant Physiology and Metabolism
GLOSSARY
Antioxidant : A substance that reduces damage caused by
free radicals.

Actinorhizal : Pertaining to several woody plant species,

such as alder trees, in which symbiosis occurs
with soil bacteria of the nitrogen fixing genus
Frankia.

Apoplast : Space in between the cells creating a pathway

through which materials may diffuse freely.

Arbuscles : Branched structures of mycorrhizal fungi that

form within penetrated cells; the sites of
nutrient transfer between the fungus and the
host plant.

Arbuscular Mycorrhizal : Symbiotic fungi that form hyphae growing in a

fungi loose arrangement, both within the root itself
and extending outward from the root into the
surrounding soil. The hyphae form unique
structures, such as the arbuscules that
enhance the exchange of nutrients between
the fungus and its host.

Bacteroids : Nitrogen-fixing organelles that develop from

endosymbiotic bacteria upon a signal from the
host plant.

Cessation : The ending of a process.

Chelates : Substances such as EDTA that from a

complex with divalent cations eliminating their
biological activity.

Chelator : A carbon compound that can form a non-

covalent complex with certain cations
facilitating their uptake (e.g., malic acid, citric
acid).

Coleoptile : A sheath protecting a young shoot tip in a

grass or cereal.

Denaturation : Breaking of weak linkages or bonds within a

molecule.

Eccentrically : Deviation from conventional or usual pattern or

style.

Endogenous : Anything that originates internally.

Heterogenous : Consisting of different, distinguishable parts or

240
238 elements.
Volume 2 Plant Physiology and Metabolism
Homeostatic : A stage by which an organism tends to
maintain stability or adjusting to conditions
required for its survival.

Hydroactive : Activated by water

Indeterminate : Not exactly known or defined.

Leghemoglobin : An oxygen-binding heme protein found in the

cytoplasm of infected nodule cells that
facilitates the diffusion of oxygen to the
respiring symbiotic bacteria.

Mesophilic : growing or thriving best in an intermediate

environment (moderate temperature)

Nitrate reductase : Enzyme that reduces nitrate NO3- to nitrite

NO2-. Catalyzes the first step by which nitrate
absorbed by roots is assimilated into organic
form.

Nitrite reductase : The enzyme that reduces nitrite NO2- to

ammonium (NO4+.).

Nitrogenase enzyme : The two-component protein complex that

complex conducts biological nitrogen fixation in which
ammonia is produced from molecular nitrogen.

Nod factors : Lipochitin oligosaccharide signal molecules

active in gene expression during nitrogen
fixing in which ammonia is produced from
molecular nitrogen.

Nodules : Specialized organs of a plant host containing

symbiotic nitrogen-fixing prokaryotes.

Nodulation genes (nod) : Rhizobial genes, the products of which

participate in nodule formation.

Nodulin (Nod) genes : Plant genes specific to nodule formation.

Photomorphogenesis : Light mediated development of plant.

Phytoalexin : substances produced by plants that inhibit the

growth of pathogen (such as a fungus)

Rhizobia : Collective term for the genera of soil bacteria

that form symbiotic (mutualistic) relationships
with members of the plant family
Leguminosae.

Skotomorphogenesis : The development of a seedling in the dark.

Stimulus : Thing that triggers a specific functional

241
reaction in an organ or tissue.
Volume 2 Plant Physiology and Metabolism
Susceptibility : The state of being influenced or harmed by
something.

Symplast : Inner side of the plasma membrane in which

water and low-molecular-weight solutes can
freely diffuse.

Synchronization : Two activities/events happening at a same

time.

242