1 .

Plant hormones and Growth regulators

1 1 .Auxin
1.1.2 Introductio
In 1881, Charles Darwin and his son Francis performed experiments on coleoptiles, the sheaths
enclosing young leaves in germinating grass seedlings. The experiment exposed the coleoptile to
light from a unidirectional source, and observed that they bend towards the lightHohm, T;
Preuten, T; Fankhauser, C (2013). By covering various parts of the coleoptiles with a light-
impermeable opaque cap, the Darwins discovered that light is detected by the coleoptile tip, but
that bending occurs in the hypocotyl. However the seedlings showed no signs of development
towards light if the tip was covered with an opaque cap, or if the tip was removed. The Darwins
concluded that the tip of the coleoptile was responsible for sensing light, and proposed that a
messenger is transmitted in a downward direction from the tip of the coleoptile, causing it to
bend(Whippo, CW; Hangarter, RP (2006).

Auxins are compounds that positively influence cell enlargement, bud formation and root
initiation. They also promote the production of other hormones and in conjunction with
cytokinins, they control the growth of stems, roots, and fruits, and convert stems into flowers.
Auxins were the first class of growth regulators discovered Vanneste, S; Friml, J (2012). They
affect cell elongation by altering cell wall plasticity. They stimulate cambium, a subtype of
meristem cells, to divide and in stems cause secondary xylem to differentiate. Auxins act to
inhibit the growth of buds lower down the stems (apical dominance), and also to promote lateral
and adventitious root development and growth. Leaf abscission is initiated by the growing point
of a plant ceasing to produce auxins. Auxins in seeds regulate specific protein
synthesisDharmasiri N, Dharmasiri S, Estelle M (May 2005).as they develop within the flower
after pollination, causing the flower to develop a fruit to contain the developing seeds. Auxins
are toxic to plants in large concentrations; they are most toxic to dicots and less so to monocots.
Because of this property, synthetic auxin herbicides including 2,4-D(2,4-dichlorophenoxyacetic)
and 2,4,5-T have been developed and used for weed control. Auxins, especially 1-
Naphthaleneacetic acid (NAA) and Indole-3-butyric acid (IBA), are also commonly applied to
stimulate root growth when taking cuttings of plants. The most common auxin found in plants is

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indole-3-acetic acid or IAA. The correlation of auxins and cytokinins in the plants is a constant
(A/C = const.

Main article: Auxin

The auxin indole-3-acetic acid

1.1.2. Biosynthesis of Auxin

IAA is chemically similar to the amino acid tryptophan which is generally accepted to be the
molecule from which IAA is derived. Three mechanisms have been suggested to explain this
conversion: Tryptophan is converted to indolepyruvic acid through a transamination reaction.
Indolepyruvic acid is then converted to indoleacetaldehyde by a decarboxylation reaction. The
final step involves oxidation of indoleacetaldehyde resulting in indoleacetic acid. Tryptophan
undergoes decarboxylation resulting in tryptamine. Tryptamine is then oxidized and deaminated
to produce indoleacetaldehyde. This molecule is further oxidized to produce indoleacetic acid.
As recently as 1991, this 3rd mechanism has evolved. IAA can be produced via a tryptophan-
independent mechanism. This mechanism is poorly understood, but has been proven using trp(-)
mutants. Other experiments have shown that, in some plants, this mechanism is actually the
preferred mechanism of IAA biosynthesis.

The enzymes responsible for the biosynthesis of IAA are most active in young tissues such as
shoot apical meristems and growing leaves and fruits. The same tissues are the locations where
the highest concentrations of IAA are found. One way plants can control the amount of IAA
present in tissues at a particular time is by controlling the biosynthesis of the hormone. Another
control mechanism involves the production of conjugates which are, in simple terms, molecules

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which resemble the hormone but are inactive. The formation of conjugates may be a mechanism
of storing and transporting the active hormone. Conjugates can be formed from IAA via
hydrolase enzymes. Conjugates can be rapidly activated by environmental stimuli signaling a
quick hormonal response. Degradation of auxin is the final method of controlling auxin levels.
This process also has two proposed mechanisms outlined below: The oxidation of IAA by
oxygen resulting in the loss of the carboxyl group and 3-methyleneoxindole as the major
breakdown product. IAA oxidase is the enzyme which catalyzes this activity. Conjugates of IAA
and synthetic auxins such as 2,4-D can not be destroyed by this activity. C-2 of the heterocyclic
ring may be oxidized resulting in oxindole-3-acetic acid. C-3 may be oxidized in addition to C-2
resulting in dioxindole-3-acetic acid. The mechanisms by which biosynthesis and degradation of
auxin molecules occur are important to future agricultural applications. Information regarding
auxin metabolism will most likely lead to genetic and chemical manipulation of endogenous
hormone levels resulting in desirable growth and differentiation of important crop species.
Ultimately, the possibility exists to regulate plant growth without the use of hazardous herbicides
and fertilizers (Davies, 1995; Salisbury and Ross, 1992).

1.1.3 . Transport
The integrity of the complicated structure of plants depends, to a great extent, on regulations that
coordinate the various parts of the whole plant. Because production centers and action sites are
often located at different places in the plant body, auxin transport takes place. Ever since Went
first demonstrated the basipolar movement of auxin and its quantitative description by van der
Weij [13],physiologists have been interested in studying various parameters of its transport as a
way to understand the general phenomena of polarity in plant development. With the availability
of high-specificactivity 14C-labeled auxin coupled with liquid scintillation spectrometry (90%
counting efficiency), itbecame possible to perform more complex experiments and to obtain
more reliable data from a singlesegment than was feasible previously . In dicots and monocots
alike, auxin moves predominantly in the basipolar direction . However, in the young vegetative
Coleus blumei internode, theauxin applied moves with a 3:1 ratio in the basipetal to acropetal
direction, but this changed to1.3:1.0 when the plants flowered . Using paper chromatography,
evidence was obtained, for the first time, that the auxin collected apically was the auxin applied
at the basal end . In excised root segments the polar transport was in the acropetal direction, but

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it appears that in the apical segments of intact roots (with caps), auxin moves basipetally .
However, in both organs, polarity was maintained regardless of the tissue orientation.
1.1.3. MODE OF ACTION OF AUXIN
Plants - like other higher organisms - have to possess intraorganismal communication system(s)
working over relatively long distances. As no nervous system is present, the main signalling
systems are hormone-dependent (Libbenga and Mennes, 1995). Auxins are a component of such
systems. Auxins and cytokinins impact at several levels in many different processes of plant
development. At the level of isolated plant cells (grown in cell suspension culture) one can
distinguish two main processes apparently controlled by auxins in collaboration with cytokinins,
i.e.,cell cycle and cell division on one hand, and cell elongation on the other. The auxin:cytokinin
ratio represents an important signal in the formation of cell phenotype and also in the onset and
maintenance of the process of cell division (Stickens et al., 1996; See chapter 10). The ability of
auxins (together with cytokinins) to manage key events in plant morphogenesis was documented,
among others, by Skoog and Miller’s (1957) discovery of the regulation of organogenesis in
vitro by means of the auxin:cytokinin ratio in culture media . It has been further supported by
recent investigations on the relationships between auxin and cytokinin levels and the
morphogenetic response of various plants (e.g., Li et al.,1994; Leyser et al., 1996; Centeno et al.,
1996). However, despite many reports on the physiological action of both individual
phytohormones, the molecular mechanisms of their effect(s) on cell expansion, cell division,
differentiation,organogenesis,andthemechanisms of their interactions have not yet been
elucidated.Nevertheless,themainstepsinauxin-(aswellas otherhormone-)signaling can be
generallydescribedas:1.Thesignal transductioncascade,and 2. the final physiological response 3.
Initial perception of the hormone signal

physiological response. Receptor-like auxin-binding proteins have been identified and

characterised by various techniques (traditional ligand-binding studies, photoaffinity labelling
and genetic approaches)as recently reviewed by Napier et al., (2002), Zazimalova and Napier
(2003),Hagenetal.,(2004).There are some candidates for true auxin receptors, especially
ZmABP1,i.e.ZeamaysAuxin-BindingProtein the major auxin-binding protein from maize
membranes.This protein exists in the form of a dimer of 22-kDa subunits. It has been purified by
several methods and its primary structure was deduced from cDNA clones. Additionally, several

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other gene sencoding this auxin-binding protein (ABP1) have been sequenced from other plants
(Arabidops is thaliana and other dicots including tobacco (reviewed in Napier et al., 2002). All
homologues share acommon primary amino acid sequence containing an N-terminal signal
peptide for transitin to the endoplasmic reticulum,one glycosylation site and the C-terminal
KDEL (LysAsp-Glu-Leu)sequence for retention in the lumen of endoplasmic reticulum. The
crystal structure of ABP1and its interaction with auxin was described by Woo et al., (2002).
Another membrane-associatedABP,showing specificity very similar to that of the maize ABP1,
was detected in tobacco cells cultured invitro(Vreughdenhiletal.,1981andreference therein,
Zazimalova et al., 1995). Never theless,Jones’s(1994)statement:“There isnot enough information
to label any single ABP as the auxin receptor...” is still valid and the auxin-binding protein
story seems to be“curiouser and curiouser”(Timpte,2001).

1.1.4. Auxin Signal Transduction

There are several perception/transduction mechanisms known in animal and plant cells
(Libbenga and Mennes, 1995; Walden and Lubenow,1996). There is some indication that
transduction of the auxin signal might be mediated by mechanisms based on a plasma
membrane-located receptor, a heterotrimeric G protein and phospholipase A2- or C catalysed
hydrolysis of specific membrane lipids(recent reviews by Millner, 2001; Fujisawa et al.,183
2001; Scherer, 2002). However, convincing evidenceis still missing.Recent findings suggested
the involvement of targeted protein degradation in auxin signalling. This
mechanism is based on the regulation of the ubiquitin-conjugating pathway by auxin
(Estelle,1999). Ubiquitin is a small and highly-conserved protein which facilitates protein
degradation. It seems to be a rather unexpected way to explain the mode of auxin action. On the
other hand, if auxin controls the ubiquitin-mediated degradation of those proteins, which are
unique for particular phases of the developmental program me this may be well
in agreement with a multifunctional regulatory role of auxin in plant development (Buchanan et
al., 2000;Leyser, 2001; Kepinski and Leyser, 2002; Hellman and Estelle, 2002; Dharmasiri and
Estelle,2004

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1.1 .5 .Metabolism
The hormone IAA has been studied for more than six decades, yet it remains unclear how it is
synthesized or degraded in plants. Because of the structural similarities, the amino acid
tryptophan is commonly considered to be a precursor to IAA. To date, biosynthetic pathways
from L-tryptophan by way of tryptamine indole-3-pyruvate and indole-3-acetaldehyde , indole-
3-aldoxineandindole-3-acetonitrile and indole-3-acetamide have been proposed. Nevertheless,
some workers have reported that D-tryptophan may also be an effective precursor for IAA
biosynthesis . However, in Lemna gibba, D-tryptophan was not converted to IAA and also the
rate of conversion from L-tryptophan was far in plants.Wright et al. using the tryptophan
auxotroph maize mutant orange pericarp, have questioned the idea that tryptophan is a precursor
of auxin and suggest a nontryptophan pathway as a primary route of IAA biosynthesis. It is
therefore possible that plants and crops use more than one route for invivo IAA biosynthesis.It is
reasonable to assume that plants have mechanisms to regulate the levels of auxin to maintain
balanced growth. This is done by controlling the rate of synthesisas well as by degradation or by
forming conjugates (bound). The enzyme IAA oxidase with its several isoenzymes, which
usually have the characteristics of peroxidases, is known to catalyze the reaction.Two pathway
so degradation are known in many plants. The first involves oxidation by O2, leading to loss of
the carboxyl group as CO2 and usually 3-methyleneoxyindole as a principal product. In the
second pathway the carboxyl group of IAA remains intact, but carbon at the second position of
the heterocyclic ring is oxidizedtooxindole-3-aceticacid.In some species, however, carbons 2 and
3 are oxidized to form dioxindole-3-acetic acid . Lee and Starratt have shown
thatsoybean(Glycinemax)callusandhypocotylstissueswerecapableofoxidizing[14C]IAA via the
carboxylative pathway to indole-3-methanol glucoside as a major product. However, deteils of
these degradative pathways are still unclear. Synthetic auxins and IAA conjugates are also not
destroyed by these enzyme In auxin conjugates the carboxyl group is covalently combined with
other molecules in the cell to form derivatives that do not allow easy extraction. Many IAA
conjugates are known, including the indole- 3-acetylaspartic acid (IAAsp), indole-3-
acetylglutamic acid (IAAGlu), and the esters IAA-myo-inositol (IAIns) and indole-3-
acetylglucose (IAGlu). These conjugates, along with the freeIAA,havealsobeen found in IAA-
overproducing transgenic and wild-type tobacco (Nicotiana tabacum)plants.

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 1.1.6. Conclusion
As the whole Auxins are compounds that positively influence cell enlargement, bud formation
and root initiation. They also promote the production of other hormones and in conjunction with
cytokinins, they control the growth of stems, roots, and fruits, and convert stems into flowers.
Auxins were the first class of growth regulators discovered. changes within the membrane
structure. This in turn might lead to changes in permeability, causing an increased ion flux across
the membrane. If a receptor molecule is located on the plasma membrane, a change in
conformation of the membrane might bring about a release of the receptor. It has been proposed
that this receptor moves into the nucleus of the target cell and once there, increases the activity of
RNA polymerase. This, in turn, leads to the synthesis of messenger-RNA which codes for
proteins and this brings about the net result of auxin increased growth.

In this particular section the effects of cyclic-AMP on auxin controlled growth have not been
covered. When cyclic-AMP was first found to stimulate plant growth, considerable excitement
was generated (Solomon & Mascarenhas, 1971). However, that excitement was short-lived, and
it now appears that cyclic-AMP has little or no effect on plant cells (Ownby et al., 1975).

A number of enzymes have been shown to be affected by auxin. These include the hydrolases,
particularly cellulase, and other enzymes associated with cell wall degradation as well as cell
wall synthesis. Whilst these enzymes are affected by the auxins, it is very likely that this result is
a secondary effect of the auxin and is not related to the primary mechanism of action. In essence
then, it appears that auxins control a multitude of physiological and biochemical responses in
plants. It is possible that the primary site of action is localized within the plasma membrane or
some surface binding site of the cell. A change at this particular level, either in structure or
function, could bring about the release of a factor which moves into the nucleus. As a secondary
amplification, the hormone thus modulates the activity of RNA polymerase. This in turn leads to
the synthesis.

1.2 Abscisic Acid

1 .2.1 Introduction
Abscisic acid (ABA) is a plant hormone. ABA functions in many plant developmental
processes, including bud dormancy, and can be involved in stress responses. It is degraded by the

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enzyme (+)-abscisic acid 8'-hydroxylase into phaseic acid.ABA was originally believed to be
involved in abscission. This is now known to be the case only in a small number of plants. ABA-
mediated signaling also plays an important part in plant responses to environmental stress and
plant pathogens Zhu, Jian-Kang (2002). The plant genes for ABA biosynthesis and sequence of
the pathway have been elucidated. ABA is also produced by some plant pathogenic fungi via a
biosynthetic route different from ABA biosynthesis in plants. Siewers, V.; Smedsgaard, J.;
Tudzynski, P. (2004)

Abscisic acid owes its names to its role in the abscission of plant leaves. In preparation for
winter, ABA is produced in terminal buds.This slows plant growth and directs leaf primordia to
develop scales to protect the dormant buds during the cold season. ABA also inhibits the division
of cells in the vascular cambium, adjusting to cold conditions in the winter by suspending
primary and secondary growth. Abscisic acid is also produced in the roots in response to
decreased soil water potential (which is associated with dry soil) and other situations in which
the plant may be under stress. ABA then translocates to the leaves, where it rapidly alters the
osmotic potential of stomatal guard cells, causing them to shrink and stomata to close. The ABA-
induced stomatal closure reduces transpiration (evaporation of water out of the stomata), thus
preventing further water loss from the leaves in times of low water availability. A close linear
correlation was found between the ABA content of the leaves and their conductance (stomatal
resistance) on a leaf area basis.

Seed germination is inhibited by ABA in antagonism with gibberellin. ABA also prevents loss of
seed dormancy. Several ABA-mutant Arabidopsis thaliana plants have been identified and are
available from the Nottingham Arabidopsis Stock Centre - both those deficient in ABA
production and those with altered sensitivity to its action. Plants that are hypersensitive or
insensitive to ABA show phenotypes in seed dormancy, germination, stomatal regulation, and
some mutants show stunted growth and brown/yellow leaves. These mutants reflect the
importance of ABA in seed germination and early embryo development. Pyrabactin (a pyridyl
containing ABA activator) is a naphthalene sulfonamide hypocotyl cell expansion inhibitor,
which is an agonist of the seed ABA signaling pathway. Park, Sang-Youl. et.al 2009. It is the
first agonist of the ABA pathway that is not structurally related to ABA.

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Structure of ABA(Wikipedia)

1.2.2 . Biosynthesis ABA

The direct cytosolic ABA biosynthesis pathway described for fungal pathogens (Botryotinia
fuckeliana, Botrytis cinerea) starts with farnesyldiphosphate, which is also an intermediate of
the cis neo/violaxanthin biosynthesis pathway . The near ubiquitous presence of the isoprenoid
pathway renders farnesyldiphosphate the core precursor of ABA biosynthesis in plants and fungi
and possibly in other ABA-producing organisms (bacteria and algae , protozoa , sponges and
human .

The degradation of ABA is catalyzed by members of the ancient cytochrome P450 superfamily
(AtCYP707A; reviewed in . Their role in hormone degradation and in particular the degradation
of ABA emerged probably in a later phase of the land transition since the algae Chlamydomonas
reinhardtii and the moss Physcomitrella do not encode orthologs of AtCYP707A .

1 2. 3 . transport
External ly applied ABA- Distributed all direction cell to cell transported is low.ABA synthesis
root cap transport ed centeral vascular tissue and transported mostly in its free form.
Transported in conjugated form as ABA –β-D- glucosly ester redistribution of ABA- ph
griediant. At low protonated or undisociated form of (ABAH).and at high ph dissociated
(ABA).Mutant have bee isolated that can affectes in the conversation of ABA aldeyhed into
ABA. Flacca and sitiens in solonum iycopersicum nar 2a in hordeum valgure Aab3 and aao3
in Arabidopsis

1.2.4. Mode of action ABA

Generally, the physiological processes are related to senescence or abscission and growth
retardation or inhibition. ABA appears to act as an abscission accelerating hormone in many
fruits and leaves. Furthermore, it also tends to induce dormancy in some woody plants. ABA has

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been shown to move from the leaves to the apical bud to bring about a dormant condition. In
potato, the levels of inhibitors, including ABA, decrease during the quiescent period prior to
renewed growth. ABA in extremely low concentration, moreover, prolongs the dormancy of
excised potato buds.

Recently, ABA has been shown to be involved in the responses of many plants to stress
conditions. As the ABA concentration increases in the leaves the stomata close. In this way, it
appears that ABA is directly involved in the opening and closing of the stomata, thus regulating
the rate of transpiration. Through this mechanism, ABA appears to protect the plant through
conditions of water stress or drought.At present the mechanism of action of ABA is not clearly
understood. However, the available evidence indicates that ABA affects transcription as shown
by reduced activity of chromatin-associated RNA polymerases. In other cases the mechanism of
action of ABA appears to involve regulation of the translation of long-lived messenger-RNA,
whilst its effects on stomata probably involve the regulation of membrane permeability.

1.2.5. Signal transduction ABA

signal transduction consists of so-called core signaling components, including Pyrabactin
resistance (pry)/regulatory component of ABA receptor (RCAR) ABA receptorsWildermuth
MC, Dewdney J. et.al 2001. group aprotein phosphatase 2Cs (PP2Cs) Morris K, Mackerness SA-
H.et al 200 and members of the SNF1-related protein protein KINASE 2 (SnRK2) group of
kinases. PYR/RCAR–PP2C complex formation leads to inhibition of PP2C activity thereby
allowing activation of SnRK2s which target ion channels, NADPH oxidases and
ABF/AREB/ABI5 type basic/region leucine zipper (bZIP) transcription factors. The complexity
of ABA signaling in Arabidopsis is reflected by the number of PYR/RCARs 6–9 PP2Cs and 3
SnRK2s, which function in ABA signaling Phosphatidic acid, synthesized by phospholipase
DThorpe MR, Ferrieri AP. et al 2007 binds and inhibits protein phosphatase 1 (PP1) and PP2A
phosphatases, which function in ABA and light signaling and interactions among these stimuli.
Additional enzymes involved in phospholipid metabolism also play a role in ABA signaling

Changes in cellular Ca2+ levels activate Ca2+-DEPENDENT PROTEIN KINASES (CDPKs)] and
CALCINEURIN B-LIKE PROTEIN (CBL) INTERACTING PROTEIN KINASES (CIPKs), the
latter mediated through interaction with CBLs (reviewed in . CIPKs (SnRK3s) appear to be
involved in plant ion homeostasis and abiotic stress tolerance by regulating H+, Na+, Ca2+ and
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NO3− transporters and K+ channels and interacting with transcription factors . In addition, CIPKs
physically interact with PP2Cs but it still needs to be elucidated if CIPKs and PP2Cs can
(de)phosphorylate each other or if both act together as a signaling module regulating target
proteins such as K+ channels (reviewed in. CDPKs function in ABA-induced stomatal closing,
and anion-channel and Ca2+-channel activation and fulfill a dual function, as they directly
phosphorylate PP2Cs and targets of SnRK2s, e.g. SLAC1, NADPH oxidases and ABFs .The
functional consequence of PP2C phosphorylation by CDPKs still needs to be clarified. Ca2+ in
addition may function in parallel to ABA-induced transcriptional activation through calmodulin-
binding transcription activators (CAMTAs) that can bind to ABA-regulated cis-acting elements
(ABREs) (Orti´z-Castro R, Contreras-Cornejo HA. el al 2009).

1.2.6. Metabolism ABA

The typical sesquiterpene nature of ABA indicates that its endogenous synthesis is through
mevalonic acid (MVA) as a precursor. Two pathways for its biosynthesis have been suggested.
First is via farnesyl pyrophosphate, from which GAs are also derived. Through this pathway,
MVA is converted to mevalonate 5-phosphate mevalonate-5-pyrophosphate 3-isopentenyl
pyrophosphate (IPP). This compound is converted either directly to geranyl pyrophosphate or
through 3,3-dimethylallyl pyrophosphate (DMAPP) to geranyl pyrophosphate farnesyl
pyrophosphate and finally, to ABA. However, use of radioactive MVA has yielded low amounts
of ABA in only a few systems . The second pathway is known to occur through the degradation
of certain (40-carbon) carotenoids. Although this pathway is indirect, it seems to produce major
amounts of ABA via ABA-aldehyde in perhaps all plants . Zeevaart et al. [128], using various
tissues incubated in an atmosphere containing 18O2, have demonstrated that xanthophylls rather
than farnesyl pyrophosphate are the precursors of ABA. In the xanthophyll cycle, 9-cis-
neoxanthin is converted to xanthoxin → ABA-aldehyde, which is finally oxidized to ABA.
Abscisic acid is catabolized to more polar compounds by conjugation, oxidation, hydroxylation,
or isomerization. However, it seems that each plant species has its own system
toregulateitsfreeABAlevel.This regulation is further dependent on the kind of organ tested as
well as the physiological state.This regulation of ABA may operate through conjugation with
sugar(s) to form glucoside or glycosylesteroran acylated form. It can also be inactivated by

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oxidation to more polar free acids such as phaseic and dihydrophaseic acids. Both of these
metabolites possess low or no growth-regulating activities and are derived via 6-hydroxymethyl

1.2.7 . Conclusion
Generally absisic acid is plant hormone which has been different functions in many plant
developmental processes, including bud dormancy, and can be involved in stress responses. It is
degraded by the enzyme (+)-abscisic acid 8'-hydroxylase into phaseic acid. The direct cytosolic
ABA biosynthesis pathway described for fungal pathogens (Botryotinia fuckeliana, Botrytis
cinerea) starts with farnesyldiphosphate, which is also an intermediate of the cis
neo/violaxanthin biosynthesis pathway. signal transduction consists of so-called core signaling
components, including Pyrabactin resistance (pry)/regulatory component of ABA receptor
(RCAR) ABA receptors group aprotein phosphatase 2Cs (PP2Cs) and members of the SNF1-
related protein protein KINASE 2 (SnRK2) group of kinases

1.3.Cytokinin
1 3 .1. Introduction
Cytokinins (CK) are a class of plant growth substances (phytohormones) that promote cell
division, or cytokinesis, in plant roots and shoots. They are involved primarily in cell growth and
differentiation, but also affect apical dominance, axillary bud growth, and leaf senescence. Folke
Skoog discovered their effects using coconut milk in the 1940s at the University of Wisconsin–
Madison (Kieber JJ (March 2002).

There are two types of cytokinins: adenine-type cytokinins represented by kinetin, zeatin, and 6-
benzylaminopurine, and phenylurea-type cytokinins like diphenylurea and thidiazuron (TDZ)
Quesenberry, K.; Gallo, M. (2012). Most adenine-type cytokinins are synthesized in roots.
Cambium and other actively dividing tissues also synthesize cytokinins. No phenylurea
cytokinins have been found in plants. Cytokinins participate in local and long-distance
signalling, with the same transport mechanism as purines and nucleosides Sakakibara H (2006).
Typically, cytokinins are transported in the xylem.Cytokinins act in concert with auxin, another
plant growth hormone. The two are complementary, having generally opposite effects.

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the shoots, eventually signaling lateral bud growth. Simple experiments support this theory.
When the apical bud is removed, the axillary buds are uninhibited, lateral growth increases, and
plants become bushier. Applying auxin to the cut stem again inhibits lateral dominance.

While cytokinin action in vascular plants is described as pleiotropic, this class of plant hormones
specifically induces the transition from apical growth to growth via a three-faced apical cell in
moss protonema. This bud induction can be pinpointed to differentiation of a specific single cell,
and thus is a very specific effect of cytokinin Decker EL, Frank W. et al. ( 2006).

Cytokinins have been shown to slow aging of plant organs by preventing protein breakdown,
activating protein synthesis, and assembling nutrients from nearby tissues. A study that regulated
leaf senescence in tobacco leaves found that wild-type leaves yellowed while transgenic leaves
remained mostly green. It was hypothesized that cytokinin may affect enzymes that regulate
protein synthesis and degradation.

Cytokinin signaling in plants is mediated by a two-component phosphorelay. This pathway is

initiated by cytokinin binding to a histidine kinase receptor in the endoplasmic reticulum
membrane. This results in the autophosphorylation of the receptor, with the phosphate then being
transferred to a phosphotransfer protein. The phosphotransfer proteins can then phosphorylate
the type-B response regulators (RR) which are a family of transcriptions factors. The
phosphorylated, and thus activated, type-B RRs regulate the transcription of numerous genes,
including the type-A RRs. The type-A RRs negatively regulate the pathway Hutchison, Claire E.
et al. ( 2002).

1.3. 2. Biosynthesis
Adenosine phosphate-isopentenyltransferase (IPT) catalyses the first reaction in the biosynthesis
of isoprene cytokinins. It may use ATP, ADP, or AMP as substrates and may use dimethylallyl
pyrophosphate (DMAPP) or hydroxymethylbutenyl pyrophosphate (HMBPP) as prenyl donors.
This reaction is the rate-limiting step in cytokinin biosynthesis. DMADP and HMBDP used in
cytokinin biosynthesis are produced by the methylerythritol phosphate pathway (MEP).
Cytokinins can also be produced by recycled tRNAs in plants and bacteria. tRNAs with
anticodons that start with a uridine and carrying an already-prenylated adenosine adjacent to the

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anticodon release on degradation the adenosine as a cytokinin. The prenylation of these
adenines is carried out by tRNA-isopentenyltransferase (Hwang I, Sakakibara H .2006)

1.3.3.Transport
It is paradoxical that plant parts that are meristematic or that otherwise have growth potential
(youngleaves, buds and internodes, and developing fruits and seeds) are known to be the primary
center of production as well as the main sink for the endogenous cytokinins or its metabolites.
The detection of cytokinin, from the xylem exudate and phloem sap of a large number of plants
and crops, clearly indicates that nonliving as well as living tissues are involved in translocation.
The polarity of cytokinin movement is acropetal in the xylem, whereas it moves bidirectionally
in the phloem.Thus,in phloem, cytokinins move not only from organ to organ in the aerial
portion but also from shoot to root and vice versa . Thus, the velocity of its movement is the
same as that of other assimilates. However, under in vitro conditions (donor-tissue-receiver
system), 6benzylaminopurine(BAP)is very poorly translocated .

1.3.4.Mode of action
The ratio of auxin to cytokinin plays an important role in the effect of cytokinin on plant growth.
Cytokinin alone has no effect on parenchyma cells. When cultured with auxin but no cytokinin,
they grow large but do not divide. When cytokinin is added, the cells expand and differentiate.
When cytokinin and auxin are present in equal levels, the parenchyma cells form an
undifferentiated callus. More cytokinin induces growth of shoot buds, while more auxin induces
root formation.

Cytokinins are involved in many plant processes, including cell division and shoot and root
morphogenesis. They are known to regulate axillary bud growth and apical dominance. The
"direct inhibition hypothesis" posits that these effects result from the cytokinin to auxin ratio.
This theory states that auxin from apical buds travels down shoots to inhibit axiliary bud growth.
This promotes shoot growth, and restricts lateral branching. Cytokinin moves from the roots into

1.3.5.Signal Transduction of Cytokinin

Cytokinin induces rRNA transcription: However, the signal transduction pathways responsible
for pol I regulation are poorly understood. We tested the effects of exogenously applied plant
hormones on promoter-dependent rRNA transcription inArabidopsis thaliana Gibberellic acid,

14 | P a g e
abscisic acid, auxin, and ethylene had no detectable effect on rRNA transcription, but kinetin (a
cytokinin) stimulated rRNA transcription within 1 h of treatment. Increased steady-state levels of
accurately initiated rRNA transcripts, detected by S1 nuclease protection, were paralleled by
increased levels of nascent rRNA transcripts in isolated nuclei. Therefore, the primary effect of
cytokinin appears to be at the level of transcription initiation rather than rRNA stability. Pol I
accounts for ∼34% of total nuclear transcription in untreated plants and 60 % following
cytokinin treatment.

1.3 .6.Metabolism
Miura and Miller have suggested that all plant cells are capable of synthesizing cytokinins
provided that the mechanisms to do so are “switched on.” However, this does not mean that
cytokinins are biosynthesized in the entire plant. Evidence suggests that actively dividing regions
of plants are the sites of cytokinin biosynthesis. Because the root system possesses the most
actively dividing regions, these regions are considered to be the major sites of cytokinin
production. Compared with other aspects of cytokinin physiology, little is known about their
biosynthesis, which is comparatively quite complicated. The circumstances of its discovery and
its effect on cell division and protein synthesis have somehow closely associated free cytokinins
with RNA and DNA. The production in plants can be accounted for either by the turnover of
cytokinin-containing transfer RNA (tRNA), by dennovo biosynthesis, or by both mechanisms. It
has also been reported to be present in ribosomalRNA(rRNA. The major cytokinin-active base
in tRNA, [9R]iP, is formed by the condensation of adenine with an appropriate donor of the N6
substituent during posttranscriptional processing. The 2-isopentenyl pyrophosphate (IPP) is the
immediate precursor (donor) of the 2-isopentenyl side chain of N6-(2-isopentenyl)adenosine in
tRNA. A cell-free enzyme system, isopentenyl AMP synthase, has been isolated fromcultured
autotrophic tobacco tissue which forms cytokinin from adenosine monophosphate (AMP) and
IPP as substrate. The 2-isopentenyl pyrophosphate is a product of mevalonic acid (MVA) (an
important precursor of carotenoids, abscisic acid, gibberellins, sterols, and other isoprenoid
compounds) via3-IPP.Besides the formation of free cytokinins from tRNA, there is strong
evidence that they are also formed by de novo biosynthesis. Beutelmann supplied labeled
adenine to moss callus cells and obtained labeled cytokinin that cochromatographed with 2iP, but
no labeled cytokinin was detectable from tRNA. Similar results were obtained from the

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cytokinin-autotroph tobacco callus tissues, Vinca rosea crown gall tissues, and synchronously
dividing to baccocallus cells .The amount of cytokinin present in the tissue is regulated by
conversion to a diversity of metabolites by the following reactions: (1) trans-hydroxylation of the
terminal methyl group on thesidechain, (2)side-chain reduction, (3) isoprenoid side-chain
cleavage, (4) O-glucosylation, (5)N-glucosylation, (6) ring substitution by alanine moiety, and
(7) base-ribonucleoside-ribonucleotide interconversion. These types of reactions have been
observed in a number of plant species as well as in crown gall tissues. These reactions have also
been obtained from tissues exogenously supplied with the hormone. Three enzymes, cytokinin
oxidase [molecular weight (MW) 88,000], cytokinin 7-glucosyltransferase (MW 46,500), and (9-
cytokinin)alanine synthase (MW 64,500), have been purified and characterized . The free base,
nucleotide, and nucleoside forms of cytokinins appear to be easily interconverted in plant tissues.
Incorporation of labeled cytokinin bases into ribosides (ribonucleoside) and ribotides (riboside 5-
phosphates) have been observed in a number of plant species. Five enzyme systems, purified
from wheat germ,may be responsible for this interconversion. These are (1) adenosine
phosphorylase, (2) adenosine kinase, (3)adenine phosphoribosyltransferase, (5-ribonucleotide
phosphohydrolase) 5-nucleotidase.

1.3.7. Conclusion
Cytokinins have been shown to increase the activities of a number of enzymes. These increases
in activity do not appear to be a direct effect of the cytokinin but rather to be an indirect effect of
some kind. Cytokinins increase the amount of DNA produced in a number of tissues.
Furthermore, cytokinins increase the rate of RNA synthesis in ageing tissue. However, a number
of studies have pointed out that the cytokinin effect on nucleic acid synthesis most likely is not
due to a direct effect on the derepression of the DNA within the genome.

Over the past few years there has been a great deal of excitement generated relating to the fact
that the native cytokinin is found in some transfer-RNA isoaccepting species. For a few years
data was presented which showed that cytokinins, particularly the synthetic cytokinin, 6-
benzyladenine, was incorported directly into transfer-RNA. That excitement, however, appears
to be shortlived since other studies have shown that the incorporation of the labelled synthetic
cytokinins is not into tRNA species but into heavier forms of RNA. Therefore, it appears quite
clear that cytokinins are likely not incorporated into tRNA directly. A model is presented .

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which suggests that the free cytokinins act as a protective agent within the target cells of plants
to protect the transfer-RNA molecules which contain the native cytokinin. It is suggested that
there are specific nucleases which degrade only certain species of transfer RNA. The model
implies that free cytokinin inhibits the nucleases through product inhibition, or possibly through
some other mechanism which prevents the enzyme from attacking the tRNAs which contain the
cytokinin. At present there is only a small amount of evidence to substantiate this model.

1.4 .Ethylene
1.4.1 introduction
Despite its simple two-carbon structure, the olefin ethylene is a potent modulator of plant growth
and development Ecker, 1995. The plant hormone ethylene is involved in many aspects of the
plant life cycle, including seed germination, root hair development, root nodulation, flower
senescence, abscission, and fruit ripening reviewed in Johnson and Ecker, 1998. The production
of ethylene is tightly regulated by internal signals during development and in response to
environmental stimuli from biotic (e.g., pathogen attack) and abiotic stresses, such as wounding,
hypoxia, ozone, chilling, or freezing. To understand the roles of ethylene in plant functions, it is
important to know how this gaseous hormone is synthesized, how its production is regulated, and
how the signal is transduced. Morphological changes in dark-grown (etiolated) seedlings treated
with ethylene or its metabolic precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), have
been termed the triple response. The exaggerated curvature of the apical hook, radial swelling of
the hypocotyl, and shortening of the hypocotyl and root are the unmistakable hallmarks of this
ethylene response. Over the past decade, the triple response phenotype has been used to screen
for mutants that are defective in ethylene responses (Bleecker et al., 1988; Guzman and Ecker,
1990. Etiolated Arabidopsis seedlings with minor or no phenotypic response upon ethylene
application are termed ethylene-insensitive (ein) or ethylene-resistant (etr) mutants. Mutants
have also been identified that display a constitutive triple response in the absence of ethylene
Kieber et al., 1993; Roman and Ecker, 1995. This class can be divided into subgroups based on
whether or not the constitutive triple response can be suppressed by inhibitors of ethylene
perception and biosynthesis, such as silver thiosulfate and aminoethoxyvinyl glycine (AVG).
Mutants that are unaffected by these inhibitors are termed constitutive triple-response (ctr)
mutants, whereas mutants whose phenotype reverts to normal morphology are termed ethylene-

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overproducer (eto) mutants, which are defective in the regulation of hormone biosynthesis. The
genetic hierarchy among ethylene biosynthesis and signaling pathway components in
Arabidopsis has been established by epistasis analysis using these mutants (Solano and Ecker,
1998; Stepanova and Ecker, 2000).

The intent of this review is not to cover all aspects of ethylene biology but to focus on recent
findings. In particular, we examine interaction of ethylene and two other plant growth regulators,
jasmonic acid (JA) and salicyclic acid (SA), and their roles in mediating responses to biotic and
abiotic stresses. We begin by summarizing what is currently known about the mechanism and
regulation of ethylene biosynthesis and by providing an update of our current understanding of
the ethylene signaling pathway.

1.4 2.Biosynthesi etheyle

The biochemistry of ethylene biosynthesis has been a subject of intensive study in plant hormone
physiology (reviewed in Kende, 1993). Major breakthroughs in the ethylene synthesis pathway
were the establishment of S-adenosylmethionine (S-AdoMet) and ACC as the precursors of
ethylene reviewed in Yang and Hoffman, 1984). On the basis of this knowledge, the enzymes
that catalyze these reactions were characterized and purified using biochemistry approaches. The
first successes in molecular cloning of the ACC synthase (ACS) (Sato and Theologis, 1989) and
ACC oxidase (ACO) (Hamilton et al., 1991; Spanu et al., 1991) genes led to the demonstration
that these enzymes belong to a multigene family and are regulated by a complex network of
developmental and environmental signals responding to both internal and external stimuli
(reviewed in Johnson and Ecker, 1998).

ACC Synthase: A Multigene Family in Plants Early attempts to purify plant ACC synthases
were hampered by their low abundance and labile nature (reviewed in Kende, 1993). ACC
synthase is encoded by a multigene family whose structure resembles the subgroup-I family of
pyridoxal 5′-phosphate (PLP)–dependent aminotransferases (Mehta et al., 1993). PLP is an
essential co-factor for ACS activity that is pre-bound in the active site of unliganded enzymes.
The crystal structure of ACS from apple has been determined and reveals that the enzyme forms
a homodimer (Capitani et al., 1999). Not only are 11 invariant residues conserved between

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aminotransferases and ACC synthases, but also the tertiary arrangement of the conserved
residues near the active site can be superimposed on that of aminotransferase enzymes. Most of
the conserved residues among the isoforms of the ACS family are located on the dimer surface
and are clustered near the active site of the enzyme. The substrate specificity of these enzymes
may arise from the relative distances between conserved residues in the active site, which is
supported by the different spatial position of Y85 of apple ACC synthase and Y70 of
aminotransferase in the active site. The ACS dimer is aligned by a local twofold axis, with the
most variable carboxylic region protruding away from the center. There are two distinct domains
of each monomer, a large and a small domain that are defined by the tertiary structure. The large
domain spans the central region of the enzyme and contains the strictly conserved secondary
structures found among families of PLP-dependent enzymes. The small domain, which shows
greater variability in structure between ACC synthases and aminotransferases, consists of the
most amino and carboxyl regions of the protein. The active site with a bound PLP cofactor is
predicted to lie between the cleft formed by the two domains. Interestingly, some of the residues
in the active site that interact with PLP (Y85 in apple ACC synthase; Y70 and R292 in
aminotransferase) are provided from the neighboring subunit, supporting the hypothesis that
active ACC synthase functions as a dimer (Tarun and Theologis, 1998). Random and site-
directed mutations introduced in LeACS2 have been used to study the relationship between ACS
structure and function (Tarun et al., 1998). Mutations introduced at these conserved residues
render the enzyme inactive (White et al., 1994; Tarun et al., 1998). Coexpression of two mutated
ACC synthases with compensatory mutations partially rescues the activity in a bacterial system
(Tarun et al., 1998).

Regulation of ACC Synthase: Gene Expression Because of their central role in ethylene
biosynthesis, the regulation of ACC synthases has been an intensively studied. Since the cloning
of ACS from zucchini (Cucurbita) (Sato and Theologis, 1989), many ACS genes have been
identified and cloned from different plant species, including tomato, winter squash, apple,
carnation, mung bean, and Arabidopsis (reviewed in Johnson and Ecker, 1998; Ge et al., 2000).
An emerging paradigm is that different isoforms of ACC synthase are differentially regulated
(Oetiker et al., 1997; Peck and Kende, 1998; Barry et al., 2000). Although studies of ACS genes
from other species, particularly those of the tomato ACS family, have been informative, the ACS
genes identified from Arabidopsis can be used to exemplify this point. In Arabidopsis, seven

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ACS genes have been characterized (Liang et al., 1992; Van der Straeten et al., 1992; Arteca and
Arteca, 1999; Samach et al., 2000). ACS2 is induced by cycloheximide, wounding, and 2 h of
ethylene treatment. The ethylene-induced expression gradually decreases with prolonged
ethylene exposure, suggesting negative feedback regulation of ACS2 (Van der Straeten et al.,
1992; Liang et al., 1996). ACS4 is induced in seedlings by cycloheximide, indoleacetic acid, and
wounding (Liang et al., 1992; Abel et al., 1995). ACS5 is induced by lithium chloride and a low
concentration of cytokinin only in etiolated seedlings (Liang et al., 1996; Vogel et al., 1998b).
ACS6 can be induced specifically by cyanide treatment, exposure to ozone in light-grown leaves,
and mechanical strain by touching; it can also be induced by cycloheximide, indoleacetic acid,
and ethylene (Vahala et al., 1998; Arteca and Arteca, 1999; Overmyer et al., 2000; Smith and
Arteca, 2000). ACS10 was identified as one of the early targets of CONSTANS, which promotes
flowering of Arabidopsis in response to light (Samach et al., 2000). Because cycloheximide
treatment induces most of the ACS isoforms, the implication is that ACS transcripts are short-
lived and negatively regulated by some unknown labile repressor(s) (Liang et al., 1992). An
alternate explanation is that cycloheximide treatment results in retention of mRNA on the
ribosomes; therefore, the steady state of ACS mRNA is relatively increased.

In Arabidopsis, ACS1 and ACS3 do not show ACS activity in either bacterial or yeast expression
systems (Liang et al., 1995). ACS1 is missing a highly conserved tripeptide, TNP (Thr-Asn-Pro),
which is located near the active site and may be essential for ACS activity (Liang et al., 1995).
Deletion of this tripeptide from ACS2 inactivates it. On the other hand, ACS3 is believed to be a
pseudogene resulting from a partial duplication of ACS1. It is intriguing to speculate about the
role of ACS1 in planta, given that it is expressed and induced by several signals that activate
other ACS genes. It is possible that ACS1 may function as a regulator of ACS activity through
dimerization with other ACS enzymes.

1.4.3. Transport
Being a gas, ethylene moves by diffusion from its site of synthesis. A crucial intermediate in its
production, 1-aminocyclopropane-1-carboxylic acid (ACC) can, however, be transported and
may account for ethylene effects at a distance from the causal stimulus

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The effects of ethylene. include: The so called triple response, when, prior to soil emergence,
dark grown seedlings display a decrease in tem elongation, a thickening of the stem and a
transition to lateral growth as might occur during the encounter of a stone in the soil.
Maintenance of the apical hook in seedlings. Stimulation of numerous defense responses in
response to injury or disease. Release from dormancy. Shoot and root growth and
differentiation. Adventitious root formation. Leaf and fruit abscission. Flower induction in
some plants .Induction of femaleness in dioecious flowers. Flower opening, Flower and leaf
senescence. and Fruit ripening

1.4.4.Mode of action of Ethylene

At physiological concentrations, ethylene inhibits stem and root extension growth, but there are
instances where it increases the growth rate in Callitriche platycarpa stem, in Helianthus petiole,
and in rice stems and roots (M Zeroni, MA Hall,1980). The myriad of plant responses and
functions, including seed germination, cell division, epicotyl curvature, seedling growth,
flowering, fruit ripening, response to stress, and senescence, are known to be influenced by
ethylene. The diversity of the processes in a wide variety of plants makes it difficult to assign the
hormone a definitive role.

Its production is regulated by a number of developmental and environmental factors. Ethylene

production is induced at germination, ripening of fruits, and senescence (abscission) by auxins
and by wounding and other chemical stress. It is also produced auto catalytically in the
climactaric fruits. Many of the effects of IAA, such as apical dominance and stomatal movement,
are attributed to IAA-induced ethylene production ( CP Romano,et al,1993, LK Levitt, DB Stein,
B Rubinstein,1987) . However, evidence with transgene-mediated auxin/or ethylene deficiencies
and mutants insensitive to either of the hormones has ruled out the notion that auxin-induced
ethylene was involved in the inhibitory influence that the apical bud exerts on the growth of
lateral buds (CP Romano,et al,1993).

1.4.5.ETHYLENE SIGNALING tranduction

After its synthesis, ethylene is perceived and its signal transduced through transduction
machinery to trigger specific biological responses. On the basis of the highly reproducible triple

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response in dark-grown Arabidopsis seedlings, a number of mutants impaired in their response to
ethylene have been identified. Cloning and characterization of the genes disrupted in these
mutants are leading to a complete picture of the ethylene signal transduction pathway .

Ethylene is perceived by a family of five membrane-localized receptors that are homologous to

bacterial two-component histidine kinases involved in sensing environmental changes. The
system typically consists of two proteins: a histidine kinase as the sensor that autophosphorylates
an internal histidine residue in response to environmental signals, and a response regulator that
activates the downstream components upon receiving a phosphate from the histidine residue of
the sensor on its aspartate residue (Wurgler-Murphy and Saito, 1997; Pirrung, 1999). Five
ethylene receptors exist in Arabiodpsis: ETR1, ETR2, ERS1, ERS2, and EIN4 (Chang et al.,
1993; Hua et al., 1995; Hua and Meyerowitz, 1998; Sakai et al., 1998). Among these receptors,
only ETR1, ETR2, and EIN4 contain a receiver domain that shows similarity to bacterial
response regulators at the C-terminal part of the protein. Since homodimerization of ETR1 and
ERS1 has been observed in plants (Schaller et al., 1995; Hall et al., 2000), receptors that do not
have receiver domain, ERS1 and ERS2, have been postulated to use the receiver domains of
other proteins by forming heterodimers with them (Hua et al., 1998). On the basis of the
structural similarities of the sensor domain, regardless of the presence of the receiver domain, the
receptor family can be further divided into two subfamilies. The ETR1-like subfamily, consisting
of ETR1 and ERS1, features three membrane-spanning regions at the N-terminal region, where
ethylene binding occurs (Schaller and Bleecker, 1995; Hall et al., 2000), and a well-conserved
histidine kinase domain at the C-terminal part of the protein. The ETR2-like subfamily, which
includes ETR2, EIN4, and ERS2, is predicted to have four hydrophobic extensions at the N
terminus and a degenerate histidine kinase domain that lacks one or more elements considered
necessary for catalytic activity, implying that these receptors may function differently. The fact
that members of a family of photoreceptors, the phytochromes, have a histidine kinase domain
related to two-component systems but exhibit serine/threonine kinase activity (Fankhauser et al.,
1999) supports the notion that the ETR2 class of receptors may function not as histidine kinases
but possibly as serine/threonine kinases.

Genetic and biochemical analyses of the ethylene receptors have lent insight into the mechanism
of regulation in planta. etr1, etr2, and ein4 were initially identified as dominant ethylene-

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insensitive plants (Bleecker et al., 1988; Roman et al., 1995; Hua et al., 1998; Sakai et al., 1998).
Similar missense mutations introduced into the N-terminal transmembrane domain of ERS1 and
ERS2 cause the same ethylene insensitive phenotype, suggesting their role in ethylene perception
(Hua et al., 1995). Isolation of the loss-of-function alleles of ETR1, ETR2, EIN4, and ERS2 by
screening for intragenic suppressors of the dominant receptor mutants provides genetic evidence
of how the ethylene receptors actually work (Hua and Meyerowitz, 1998). The absence of
phenotypes in single-receptor mutants suggests that in spite of the structural differences, there is
functional redundancy (or compensation of function) among the receptors. The constitutive triple
response observed in a quadruple-receptor mutant indicates that the receptors negatively regulate
this ethylene response. Consistent with these elegant genetic studies is the observation that the
dominant ethylene-insensitive mutant etr1 binds less ethylene (Schaller and Bleecker, 1995;
Rodriguez et al., 1999). The synthesis of the results from both genetic and biochemical studies
leads one to conclude that ethylene receptors are inactivated by ethylene binding. Interestingly,
members of both ETR1 and ETR2 subfamilies have also been identified in other plant species. In
tomato, the Never Ripe (NR) gene encodes a receptor similar to the ETR1 class with no receiver
domain, whereas LeETR4 is an ETR2 class member with a receiver domain. Reduction in the
expression level of LeETR4 leads to enhanced ethylene responses in tomato plants, and
overexpression of NR can compensate for the loss of LeETR4 and eliminates the ethylene
sensitivity. These results reveal that mechanisms of ethylene perception are likely conserved
among flowering plants (Tieman et al., 2000).

Further characterization of ethylene binding to ETR1 has revealed that it occurs in a hydrophobic
pocket located at the N terminus of the receptors and requires a transition metal, copper, as a
cofactor (Schaller and Bleecker, 1995; Rodriguez et al., 1999). Addition of copper ions was
required for the recovery of ETR1 ethylene binding activity in yeast extracts. Subsequently, it
was shown that copper copurifies in stoichiometric amounts with the ethylene binding domain
extracted from membranes of yeast overexpressing the ETR1 binding domain. Both ethylene
binding activity and copurification of copper were eliminated when the etr1-1 mutation, a
conversion of Cys-65 to a Tyr, was introduced into the protein. Further evidence for a role of
copper in ethylene signaling comes from the characterization of the Arabidopsis RESPONSIVE-
TO-ANTAGONIST (RAN1) gene (Hirayama et al., 1999). Two weak mutant alleles, ran1-1 and
ran1-2, were identified in a screen for mutants that displayed an ethylene-like triple response in

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response to treatment with the potent ethylene antagonist transcyclooctene. More importantly,
the mutant allele ran1-3/ctr2 or co-suppression of the RAN1 gene led to a constitutive ethylene
response phenotype. This is consistent with a loss-of-receptor function (Hirayama et al., 1999;
Woeste and Kieber, 2000). This phenotype can be partially rescued by exogenous copper
application. Cloning and subsequent functional analysis of RAN1 revealed that it encodes a
copper transporter that shares similarity with copper-transporting P-type ATPases such as the
yeast Ccc2p and human Menkes/Wilson disease proteins (Hirayama et al., 1999). Taken
together, these findings indicate that RAN1 is involved in delivery of copper to the ethylene
receptor and that this copper-delivery pathway is required to create functional ethylene receptors
in plants.

1.4.6. Metabolism
The task of unraveling the biosynthesis of ethylene was not an easy one. Feeding experiments
using various radioactive materials with ethylene-producing plant tissues were unsuccessful in
identification of the pathway. However, based on model nonenzymatic system, Lieberman and
Mapson proposed methionine as the precursor. Subsequently, Lieberman et al.[96]demonstrated
the in vivo conversion of [14C]methionine to [14C]ethylene in apple (Malus domestica) tissues.
Studies with [14C]methionine have shown that the C-1 atom is converted to CO2, C-2 to formic
acid, and C-3 and C-4 to ethylene and the sulfur atom is retained in the tissue. Because the
ethylene production system is extremely labile and is completely lost by tissue disruption, the
characterization has been madeat the living tissue level .In these studies, climactaric fruit slices
or plugs and auxin-treated stem segments of etiolated pea and mungbean (Phaseolus aureus)
seedlings have been used extensively .Earlier studies on the metabolism of ethylene, conducted
with improper precautions, had led to the conclusion that the compound was metabolically inert.
However, Beyer [99], employing proper precautionary measures, convincingly demonstrated that
ethylene was metabolized by plants ,and the metabolic products of the dark-grown aseptic pea
seedlings were identified as CO2 and ethylene oxide. In addition to these two gaseous
metabolites, ethylene was metabolized to a number of nonvolatile soluble products,

1.4.7. Conclusion
Even though little is known about the binding site(s) of ethylene, in a few cases such as ripening,
abscission, swelling and senescence there is reason to believe that the combination of ethylene

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with a site results in the regulation of protein or enzyme synthesis which in turn accounts for the
observed responses. In these cases RNA synthesis, presumably messenger-RNA, with the
accompanying support of soluble- and ribosomal-RNA, is an essential or early step suggesting
regulation of gene action. However, in other cases, especially those associated with the
physiology of excised tissue, control is probably not exerted at the level of RNA or protein
synthesis. In the case of the inhibition of elongation, the action is apparently directed toward
blockage of DNA metabolism. The site of action of ethylene in epinasty, root initiation,
intumescence formation and floral initiation is even more poorly understood. The only valid
conclusion appears to be that a number of essential features of plant growth and development are
susceptible to ethylene action. In the final analysis it is concluded that there may be as many
mechanisms of ethylene action as there are modes of ethylene operation.

1.5.Gibberellin
1.5.1.Introduction
Gibberellins (GAs) are plant hormones that regulate growth and influence various
developmental processes, including stem elongation, germination, dormancy, flowering, sex
expression, enzyme induction, and leaf and fruit senescence.

Gibberellin was first recognized in 1926 by a Japanese scientist, Eiichi Kurosawa, studying
bakanae, the "foolish seedling" disease in rice. It was first isolated in 1935 by Teijiro Yabuta and
Sumuki, from fungal strain (Gibberella fujikuroi) provided by Kurosawa. Yabuta named the
isolate as gibberellins.

Interest in gibberellins outside Japan began after World War II. In the United States, the first
research was undertaken by a unit at Camp Detrick in Maryland, via studying seedlings of the
bean Vicia faba. In the United Kingdom, work on isolating new types of gibberellin was
undertaken at Imperial Chemical Industries. Interest in gibberellins spread around the world as
the potential for its use on various commercially important plants became more obvious. For
example, research that started at the University of California, Davis in the mid-1960s led to its
commercial use on Thompson seedless table grapes throughout California by 1962. [clarification
needed. A known gibberellin biosynthesis inhibitor is paclobutrazol (PBZ), which in turn inhibits
growth and induces early fruitset as well as seedset.

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 1.5.2. Biosynthesis Gibberellins
Most bioactive GAs are located in actively growing organs on plantsHedden, G.; S. Thomas
(2012).. Both GA20ox and GA3ox genes (genes coding for GA 20-oxidase and GA 3-oxidase)
and the SLENDER1 gene (a GA signal transduction gene) are found in growing organs on rice,
which suggests bioactive GA synthesis occurs at their site of action in growing organs in plants.
During flower development, the tapetum of anthers is believed to be a primary site of GA
biosynthesis. (Kaneko, M.; H. Itoh;etal 2003).

Differences between biosynthesis in fungi and lower plants Arabidopsis, a plant, and Gibberella
fujikuroi, a fungus, possess different GA pathways and enzymes. P450s in fungi perform
functions analogous to the functions of KAOs in plants. The function of CPS and KS in plants is
performed by a single enzyme, CPS/KS, in fungi. In fungi, the GA biosynthesis genes are found
on one chromosome, but in plants, they are found randomly on multiple chromosomes. Plants
produce low amount of GA3, therefore the GA3 is produced for industrial purposes by
microorganisms. Industrially the gibberellic acid can be produced by submerged fermentation,
but this process presents low yield with high production costs and hence higher sale value,
nevertheless other alternative process to reduce costs of the GA3 production is Solid-State
Fermentation (SSF) that allows the use of agro-industrial residues.

1.5.3.Transport gebberellins
Gibberellins are known to be synthesized in all young, actively growing organs, vegetative or
reproductive, including immature and mature seeds. Understanding their transport within the
plant pertains primarily to work with excised coleoptile,stem,orpetiole segmentsina donor-tissue-
receiversystem. Transport has generally been observed to be nonpolar, but occasionally,
basipolar movement has been reported with a velocity up to 1 mm/hr. However, information
regardingen do genous movement is rather indirect. It has been noted to occur in the phloem by
the same mechanism and in apattern similar to that with which other assimilates move.
Gibberellins have been isolated from phloem sieve tube saps as well as from the xylem stream.
1.5 .4.Mode Of Action of Gibrellins
The action of many GAs is similar to that of IAA, including cell elongation, promotion of
cambial activity, induction of parthenocarpy, and stimulation of nucleic acid and protein
synthesis. The GAs vary greatly in their biological activity, and GA3 and GA7 are considered to

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have the widest range. In ferns, algae, and fungi, GAs have also been shown to influence growth
and development.

The content of GAs varies depending on the types of tissues and their stages of growth. Tissues
other than seeds usually contain very low amounts (e.g., 0.3 _g/kg in young bamboo shoots) (JM
Clarke and RA Richards,1988). Roots are considered to be the richest source of GAs, and most
GAs, as such or in bound form, are supplied to the shoot.

2.6.5.Signal Transduction
Physiological studies and phenotypic characterization of mutants with impaired GA biosynthesis
revealed that GA plays an important role in internode elongation. (Hooley R,1994, Swain SM,
Olszewski NE,1996, Ross JJ, Murfet IC, Reid JB,1997) It stimulates cell division and expansion
in response to light or dark (photo-morphogenesis and skoto-morphogenesis). (Ogawa M, et
al,2003, Alabadí D, et al,2008, Feng S, et al,2008, de Lucas M, et al,2008., Gallego- Bartolomé
J, et al,2011) Despite complexity, the GA biosynthetic pathway has been well characterized.36 It
is very difficult to determine precisely the site of bioactive GA biosynthesis in plants. Very little
is known about level of GA in plants and still much remain to understand the signal transduction
pathways leading to elongation of stems and leaves with response to different environmental
factors. Various studies on gene expression and characterization of GA deficient mutants
revealed GA signaling and bioactive sites in plants.37, 38A model proposed by Sakamoto39
depicted relationship between GA biosynthesis and cell fate determination at the apical region of
tobacco shoot. A KNOTTED1-like homeobox (KNOX) protein, NTH15 is present at the corpus
region of the shoot apical meristem (SAM). An interaction with the cis-acting element results a
negative regulation of the GA 20-oxidase gene. When NTH15 expression is controlled, GA
biosynthesis starts and finally stimulates cell division and determines cell fate. In rice, the
expression of GA related genes is restricted to the basal and peripheral region of the SAM rather
than corpus region.40 In rice corpus region of SAM expressed OSHI and KNOX type home box
genes to determine cell fate. 41 Another report also revealed the expression of GA regulated
genes in growing tissues of Arabidopsis.42 GA promotes cell elongation through releasing
DELLA mediated inhibition of BZR1 transcription factor.43

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1.6.7.Gibberellin metabolism

One or two genes encode the enzymes responsible for the first steps of GA biosynthesis in
Arabidopsis and rice. The null alleles of the genes encoding CPS, KS, and KO result in GA-
deficient Arabidopsis dwarves. Multigene families encode the 2ODDs that catalyze the
formation of GA12 to bioactive GA4.

AtGA3ox1 and AtGA3ox2, two of the four genes that encode GA3ox in Arabidopsis, affect
vegetative development. Environmental stimuli regulate AtGA3ox1 and AtGA3ox2 activity
during seed germination. In Arabidopsis, GA20ox overexpression leads to an increase in GA
concentration. (Huang, S.; A. S. Raman.et.al.1998 )

1.6.8.Conclusion
The gibberellins comprise a complex and large group of related compounds which control cell
elongation and enzyme secretion. The mechanism by which the hormone turns on the synthesis
of enzymes has been studied in fairly close detail. Initially, it was felt that the hormone regulated
the synthesis of RNA at the level of transcription. Through the careful and dedicated work of
Varner and his associates it appears reasonably clear that the hormone does not control the
secretion of enzymes by controlling the synthesis of messenger-RNA. However, it is clear
primarily from the work on amylase, that inhibitors of nucleic acid synthesis and protein
synthesis disrupt the production of the enzyme. The recent experiments of Paleg and his
associates bring a new dimension to the question of gibberellin action by their demonstration that
GA3 affects the membrane permeability of fluidity. Thus, a new range of possible hormonal
mechanisms can now be envisaged and explored.

1.7. Jasmonates
1.7 .1.Introduction
In 1962, jasmonic acid methyl ester (JAME was isolated for the first time from the essential oil
of Jasminum grandiflorum (Demole et al., 1962). The first physiological effects of JAME,
however, and its free acid (JA) were only observed two decades later. In 1980, Ueda's group in
Osaka, Japan, described a senescence-promoting effect of JA, and Sembdner's group in Halle,
Germany, isolated and characterized these compounds as growth inhibitors (Ueda and Kato,
1980; Dathe et al., 1981). Inhibition of root growth became the most prominent assay for

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screening of mutants affected in respect of JA signalling. The biosynthesis of JA was elucidated
by Vick and Zimmermann (1984). The functional analysis of the mode of action was initiated by
the observation in Parthier's group in Halle that application of JA or its methyl ester to barley
leaves leads to an altered protein pattern. The synthesis of abundantly accumulating proteins, so-
called jasmonate- induced proteins (JIPs) and degradation of housekeeping proteins such as
Rubisco were described for the first time in 1987 (Weidhase et al., 1987a, b). In 1990, another
dramatic alteration of gene expression by JAME was observed in tomato plants (Farmer and
Ryan, 1990). Here, a proteinase inhibitor (PIN2) was found to accumulate upon wounding (such
as that caused by herbivore attack) or by treatment with airborne JAME. Subsequently, JA-
induced alteration of gene expression and metabolite patterns became the focus of studies
examining accumulation of vegetative storage proteins in Staswick's group (Staswick et al.,
1992) and alkaloids in Zenk's group (Gundlach et al., 1992). In the latter case, an endogenous
rise of JA was demonstrated upon elicitation of cell suspension cultures, thus indicating JA as a
putative signal between the external stimulus and the response by cells of alkaloid formation.
Other breakthroughs were the first cloning of a JA biosynthetic enzyme, allene oxide synthase
(AOS) in Brash's lab (Song et al., 1993) and isolation of the first JA-insensitive mutant in
Turner's lab (Feys et al., 1994). This mutant was screened by use of coronatine, a bacterial toxin
with structural and functional similarity to JA (coronatine-insensitive1, coi1). Four years later,
Turner's lab were able to clone the corresponding gene. COI1 codes for an F-box protein, which
is part of the ubiquitin-mediated proteasome pathway (Xie et al., 1998). In the same year, the
corresponding F-box protein in auxin signaling, TIR1 was cloned. Consequently, much effort
was spent in trying to understand hormone signalling in molecular terms and this led to the
identification of TIR1 as one of the auxin receptors (Dharmasiri et al., 2005; Kepinski and
Leyser, 2005). The JA receptor, however, is still unknown. Other important steps in
understanding the action of jasmonates were the identification of mutants affected in JA-
biosynthesis (cf. Turner et al., 2002), the identification of the first transcription factors regulating
JA-responsive gene expression by Memelink's group (van der Fits and Memelink, 2001), the
proof that fatty acid β-oxidation acts in JA biosynthesis (Li et al., 2005), and the cloning of JA
amino acid conjugate synthase (JAR1) by Staswick's lab (Staswick et al., 2002; Staswick and
Tiryaki, 2004). Subsequent to the latter, interest was shifted to JA metabolism and the action of
JA metabolites

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1.7.2. JA BIOSYNTHESIS

The AOS branch in the LOX pathway: JA biosynthesis

Conversion of 13-HPOT, the first step in JA biosynthesis, is carried out by 13-AOS, leading to
an unstable allene oxide (Vick and Zimmermann, 1987). AOSs belong to the family of CYP74A
enzymes, which are independent from molecular oxygen and NADPH, exhibit low affinity to CO
and use the hydroperoxide group as a source for reducing equivalents and oxygen (Song et al.,
1993; Feussner and Wasternack, 2002; Howe and Schilmiller, 2002). Based on structural
information on AOS, an effective and selective inhibitor, JM-8686, has been designed (Oh et al.,
2006). First cloned from flax seeds, more than 20 AOS sequences have been included in a
phylogenetic tree analysis in which 9-AOSs and 13-AOSs were grouped separately (Stumpe and
Feussner, 2006). With the exceptions of the AOS of guayule (Pan et al., 1995) and barley
(Maucher et al., 2000), all of the 13-AOSs carry a plastid transit peptide. Plastid localization has
been shown by immunolocalization and import studies, even for the barley AOSs that lack the
transit peptides (Maucher et al., 2000; Froehlich et al., 2001). This compartmentation
corresponds to the plastid location of 13-LOX, whereas 9-LOX activity was found in the cytosol
(Feussner and Wasternack, 2002) . The picture is, however, more complicated. The recently
cloned potato 9-AOS is associated with amyloplasts and leucoplasts (Stumpe et al., 2006), and
the recombinant AOS of A. thaliana exhibits dual 9-/13-specificity regulated by monomer-
micelle association (Hughes et al., 2006). This raises the possibility that in vivo an AOS can be a
versatile enzyme in respect to ‘substrate-specific’ membrane association and oligomeric state
(Hughes et al., 2006). The subsequent enzyme of the AOS branch, the allene oxide cyclase
(AOC) is also plastid-located (Ziegler et al., 2000). Using the highly unstable allene oxide, the
AOC establishes the enantiomeric structure at the cyclopentenone ring that occurs in JA. Non-
enzymatic side-reactions in the absence of AOC are the formation of racemic OPDA and the
cleavage to α- and γ-ketol (Fig. 1). Due to the instability of the allene oxide and the common
location of AOS and AOC, both enzymes were thought to be in close contact. But so far there is
no proof on an AOS–AOC protein interaction (Zerbe et al., 2007). The AOC product cis(+)-12-
oxophytodienoic acid (OPDA) is the final product of the plastid-located part of JA biosynthesis.
The subsequent step, reduction of the cyclopentenone ring, is catalysed by a peroxisomal OPDA
reductase (OPR; Strassner et al., 2002). In Arabidopsis and tomato there are more than three

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enzymes, but only OPR3 exhibits specificity for cis(+)-OPDA (Schaller et al., 2000; Strassner et
al., 2002).

Initial experiments with labelled OPDA revealed β-oxidative side-chain shortening as essential
steps in JA biosynthesis (Miersch and Wasternack, 2000). More recently, for Arabidopsis and
tomato genetic evidence has been described showing that enzymes of fatty acid β-oxidation, such
as acyl-CoA oxidase of tomato (ACX1), a multifunctional protein (MFP) and a L-3-ketoacyl
CoA thiolase all have a function in JA biosynthesis (Fig. 1) (Cruz Castillo et al., 2005; Li et al.,
2005; Wasternack et al., 2006). Participation of ACX1 in JA biosynthesis was clearly shown by
isolation of an acx1 mutant of tomato exhibiting JA deficiency but accumulating OPDA upon
wounding (Li et al., 2005). In Arabidopsis only the double mutant acx1/5 shows less JA
formation upon wounding, indicating redundancy among the five ACX genes (Schilmiller et al.,
2007). Further proof for role of fatty acid β-oxidation enzymes in JA biosynthesis has come from
analysis of the aim1 mutant, affecting one of the MFPs, and the pex6 mutant, affecting
peroxisome biogenesis (C. Delker et al., unpubl. res.; Leibniz Institute of Plant Biochemistry,
Halle/Saale, Germany). β-oxidative steps take place only with the corresponding CoA ester. The
formation of the OPDA–CoA ester in the chloroplast has been suggested, but not proven
experimentally. There are, however, several indications for synthesis of OPDA–CoA ester in
peroxisomes by 4-coumarate:CoA ligase-like (4-Cl-like) enzymes encoded by a small gene
family in A. thaliana (Schneider et al., 2005). Interestingly, this type of enzyme is also able to
activate downstream intermediates in JA biosynthesis such as OPC-8 (Schneider et al., 2005;
Koo et al., 2006) or OPC-6 (E. Kombrink, pers. comm.; Max Planck Institute for Plant Breeding
Research, Cologne, Germany). These data suggest different import reactions (free OPDA versus
OPDA–CoA) and activation of OPDA (or its products) where it carrys a different carboxylic acid
side-chain to the corresponding CoA ester. It is, however, still unclear whether OPR3 can
convert the OPDA–CoA ester. The existence of gene families for 4-CL-like enzymes and for
genes encoding enzymes in β-oxidation, such as acyl CoA oxidase, suggest distinct and/or
overlapping roles in β-oxidation of fatty acids and in the final steps of biosynthesis of JA.
Similar β-oxidation steps were found for the final steps in auxin biosynthesis. Consequently,
mutant analysis requires double or triple mutants in order to dissect the role in JA-dependent,
auxin-dependent or β-oxidation (development)-dependent processes. These aspects have been
recently reviewed (Baker et al., 2006).

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Figure 1 . Annals of Botany, Volume 100 , Pages 681–697,

1.7.3. Mode Of Action

Jasmonate modulates the expression of numerous genes and influences specific aspects of plant
growth, development, and responses to abiotic and biotics tresses Many of these responses were
identified by application of jasmonate to plants, sometimes at non physiological levels.
Interactions between JA and other plant growth regulators make assignment of physiological
roles for JA even more complicated. In the section below, proposed actions of JA in plants are
related to the level of JA in plant tissues, the activity of JA responsive genes, and insights
provided by JA insensitive and JA deficient plants. JA and MeJA inhibit the germination of non
dormant seeds and stimulate the germination of dormant seeds. Application of JA to leaves

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decreases expression of nuclear and chloroplast genes involved in photosynthesis (22, 122). JA
treatments also cause a loss of chlorophyll from leaves or cell cultures (122). Jasmonate might be
expected to play a role in formation of flowers, fruit, andseed because of the relatively high
levels of this compound in developing plantreproductive tissues

1.7.4. METABOLITES OF JA
Since the first isolation of JAME as a constituent of the oil of Jasminum grandiflorum (Demole
et al., 1962), numerous jasmonate compounds have been detected in diverse plant phyla (Meyer
et al., 1984). Jasmonates occur in algae, mosses, fungi, gymnosperms and angiosperms. The
capacity to form or to convert various jasmonates is remarkably high in fungi; for example, more
than 20 jasmonates were detected in culture filtrate of Fusarium oxysporum (Miersch et al.,
1999a), whilst Aspergillus niger grown on liquid medium could form more than 25 jasmonate
compounds upon application of JA, 9,10-dihydro-JA and their methyl esters (Miersch et al.,
1999c). Some fungi, such as Botryodiplodia theobromae, are able to accumulate up to 500 µg
mL−1 of culture medium of (+)-7-iso-JA, the initial product in JA biosynthesis (Miersch et al.,
1989). Whereas in fungi the biological function of various jasmonates is unknown, application of
jasmonates and their structural analogs to higher plants led to the first insights into their
structural requirements for biological activity. Based on different assays, such as alkaloid
formation (Blechert et al., 1995), tendril coiling (Blechert et al., 1999), tuber formation (Koda,
1992), or gene expression (Miersch et al., 1999b), the following structural requirements have
been found (Fig. 3). (−)-JA and its derivatives are more active than (+)-derivatives.An intact
pentenyl side-chain is most active, whereas hydroxylation at C-11 or C-12 and reduction
between C-11 and C-12 reduce biological activity.Chain elongation of the carboxylic acid side-
chain retains activity only with an even number of C-atoms.Activity is retained or increased by
methylation or conjugation to amino acids.A cyclopentanone ring carrying a keto group at C-6 is
essential.Structure–activity relationships among jasmonates. The various structural analogs and
enantiomers were tested with respect to JA-induced gene expression in barley (data from
Miersch et al., 1999b). ↓, decreased expression; ↑, increased expression; –, inactive; +, active in
gene expression More recently, highly active JA compounds such as coronalon (Schüler et al.,
2004) and other 6-substituted 4-oxo-indanoyl-isoleucine conjugates have been synthesized
(Mithöfer et al., 2004). The construction of these compounds was deduced by modelling based
on the structure of the bacterial toxin coronatine. The extremely high biological activity of

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coronatine has been used as a tool and has led to isolation of the first JA-insensitive mutant of A.
thaliana, coi1 (Feys et al., 1994). Recent data have revealed distinct responses for JA and
coronatine (Uppalapati et al., 2

Metabolic fate of JA :Homeostasis between various metabolites is a common mechanism in

plants to sustain the level of active hormones such as auxins, cytokinines, gibberellins and
jasmonates. In the case of JA, seven different metabolic routes have been suggested by
identification of the corresponding products, but only a few enzymes have been cloned so far
(Fig. 4). Formation of amino acid conjugates by a JA conjugate synthase (JAR1) (Staswick and
Tiryaki, 2004) upon adenylation at the carboxylic acid side-chain of JA by the AMP-transferase
activity of JAR1. Methylation of JA by a JA-specific methyl transferase (Seo et al., 2001).
Hydroxylation at C-11 or C-12 of the pentenyl side-chain and subsequent O-glucosylation
(Sembdner and Parthier, 1993; Swiatek et al., 2004) or sulfation (Gidda et al.,
2003).Decarboxylation of JA to cis-jasmone (Koch et al., 1997).Formation of cucurbic acids by
reduction of the keto group of the cyclopentanone ring (Sembdner and Parthier, 1993).Formation
of jasmonoyl-1-β-glucose, jasmonoyl-1-β-gentiobiose and hydroxyjasmonoyl-1-β-glucose
(Swiatek et al., 2004).Conjugation of the ethylene precursor ACC to JA (Staswick and Tiryaki,
2004).

Figure 2.Metabolic fate of jasmonic acid. The carboxylic acid side-chain can be conjugated to
the ethylene precursor 1-amino cyclopropane-1-carboxylic acid (ACC), methylated by JA methyl

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transferase (JMT), decarboxylated to cis-jasmone, conjugated to amino acids such as Ile by JA .
(Annals of Botany, Volume 100)

1.7.5. Signal transduction pathway

One of the best-studied signal-transduction pathways of jasmonates is that of wound-response,
which was initially studied mainly in tomato. Briefly, a sequential action of the 18-aa peptide
systemin, cleaved from prosystemin upon local wounding, activates JA biosynthetic enzymes
such as AOC and leads to local rise in JA (Fig. 5). An amplification in JA formation has been
suggested due to JA-dependent PROSYSTEMIN expression, a systemin-dependent AOC
expression, and their common location in vascular bundles (Stenzel et al., 2003a; Narváez-
Vásquez and Ryan, 2004). It is possible that JA acts as a systemic signal, leading also to
systemic expression of genes encoding proteinase inhibitors (PINs) and other foliar compounds
with negative effects on herbivore performance. Consequently the plant becomes immunized
against a subsequent herbivore attack. Grafting experiments with JA-deficient and JA-signalling
mutants in G. Howe's laboratory have shown that JA signalling – but not JA biosynthesis – is
necessary in the systemic leaf. The numerous data on the wound response pathway have been
reviewed repeatedly, including discussions on cross-talk to other signals, further elements of the
wound-response pathway such as MAP kinases, and additional herbivore-induced compounds
such as volatiles and their consequences for diverse plant–insect interactions, together with
overlap to plant pathogen–interactions (Farmer et al., 2003, Howe, 2004; Schilmiller and Howe,
2005; Wasternack, 2006). Here, I want to mention only few important new developments.

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Figure 3. Local and systemic wound response in tomato and cell-type-specific

Local and systemic wound response in tomato and cell-type-specific occurrence of AOC, AOS
and LOX (after Hause et al., 2003; modified from Wasternack et al., 2006).For a long time, an
active systemin was found only in tomato (Solanum lycopersicon), but not in tobacco. Even in
the closely related Solanum nigrum the systemin-homolog of Solanum lycopersicon is not active
in defence responses (Schmidt and Baldwin, 2006). In tobacco, hydroxyproline-rich homologs of
tomato systemin have been found that do not induce PIN expression in tomato (Pearce et al.,
2001; Pearce and Ryan, 2003). Recently, peptides active in wound-signalling have been
identified outside the Solanaceaen species. In A. thaliana a 23-aa peptide (AtPEP1) processed
from a 92-aa precursor is part of the innate immune response (Huffaker et al., 2006). PROPEP1
expression is induced by wounding, JA and ethylene leading to H2O2 formation and PDF1·2
expression. Arabidopsis thaliana plants over-expressing PROPEP1 show altered root

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development and enhanced resistance to the root pathogen Pythium irregulare. PROPEP1
belongs to a small gene family, and AtPEP1 exhibits striking similarities in structure and
function to tomato systemin (Huffaker et al., 2006). The receptor of AtPEP1 has been isolated
and characterized as a functional LRR receptor, which is active even if transformed in tobacco
(Yamaguchi et al., 2006). The link between defence against pathogens and wound signalling
provided by AtPEP1 suggests that receptor-mediated defence-signalling peptides generated upon
wounding may amplify defence against pathogens in A. thaliana (De Vos et al., 2006).
Resistance against pathogens can also be increased by modified emission of green-leaf volatiles
initially released upon herbivory (Shiojiri et al., 2006).

Arabidopsis :Analyses of transcript accumulation upon wounding or pathogenic attack have

greatly improved our understanding of plant responses to biotic and abiotic stress (Pieterse et al.,
2006). Mutants in JA biosynthesis and JA signalling have become an important tool in dissecting
signalling pathways and in determining signalling properties of JA and related compounds
(Turner et al., 2002; Lorenzo and Solano, 2005; Delker et al., 2006; Wasternack, 2006). Initial
expression analyses with the JA-deficient but OPDA-accumulating opr3 mutant revealed OPDA-
specific gene expression (Stintzi et al., 2001). Following the first large-scale transcription
analysis, genes could be grouped into COI1-dependent and COI1-independent types (Reymond
et al., 2000, 2004). Recent transcription analyses have shown distinct and overlapping groups of
genes being expressed dependently and independently with respect to COI1, OPDA and JA
(Devoto et al., 2005; Taki et al., 2005). Similar differences and overlaps in action have been
found at the level of transcription factors acting in JA-/OPDA-dependent processes (Fig. 6).
Among transcription factors acting downstream of JA in stress responses are the ethylene
response factor 1 (ERF1), the bHLHzip-type transcription factor ATMYC2 (Lorenzo et al.,
2004), WRKY70 and the newly found family of ORAs (identified by J. Memelink's group).
ORAs are the Arabidospis homologs of ORCAs initially identified in Catharanthus roseus cell
suspension cultures (Memelink et al., 2001). Among them, ORA47 is a COI1-dependent positive
regulator of JA biosynthesis, whereas ORA59, ERF1, ORA37, MYC2 and WRKY70 act
positively or negatively on different groups of defence genes (Fig. 6). The antagonistic action of
MYC2 and ERF1 may cause the independence between wound signalling and pathogen-defence
signaling (Lorenzo et al., 2004; Lorenzo and Solano, 2005)

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1.7.6. Conclusion
In the last few years interesting new data on JA biosynthesis and action have appeared. Crystal
structures for several JA biosynthetic enzymes now allow molecular explanations for substrate
specificity. Fatty acid β-oxidation enzymes and 4-Cl-like CoA ligases have been identified to
function in JA biosynthesis. New esterified oxylipin derivatives such as the arabidopsides
suggest new functions in defence reactions. Occurrence and specific functions of JA metabolites
have been detected. Transcription factors acting in JA signalling have shed new light on cross-
talk in signalling pathways that are active following biotic and abiotic stresses, as well as in
developmental processes. Finally, it is possible that a putative role of jasmonates in cancer
formation may be substantiated.

Future work will address questions on specific actions of JA metabolites, on JA and oxylipin
perception, on MAP kinases active in JA signalling, on the diversity of action of JA in plant–
plant and plant–microbe interactions; and, finally, cell- and organ-specific functions of
jasmonates in plant development will be analysed.

1.8.Salicylic Acid
1.8.1. Introduction
Plants are the source of many substances used in treating human disease and discomfort. One of
the earliest known plant-derived therapeutic compounds originated from the bark of willow
trees , which intraditional medicine was chewed to provide relief from pain and inflammation, a
practice that can be traced to over two thousand years ago. This ancient remedy was described in
writings by the Greek physician Hippocrates (Fifth Century B.C.) and the physician and botanist
Pedanius Dioscorides (First Century A.D.), who described the ingestion of willow leaves and
bark as a means for relieving pain . In the 1820s, the predominant active ingredient in this
natural product was identified as salicin, and the presence of this compound was discovered in
several other plant species including meadowsweet (Spirea spp.)andmyrtle(Myricaspp.).In the
following decade, salicin from natural sources was shown to consist of both a sugar, and an
aromatic component initially called spirsaure and later salicylic acid (SA1), named for the source
genera Spirea and Salix, respectively. In 1852, the first de novo synthesis of SA was described
and the chemical structure of SA deduced as 2-hydroxybenzoic acid . In addition to SA, a
number of different SA-related compounds are found in plants, such as salicin, the glycosylated

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derivative of SA, and the fragrant oil of wintergreen, a methylated form of SA obtained from
plants such as teaberry Gaultheria spp.. Though salicin from natural sources was used as an
analgesic and antipyretic through the 19th century, ingestion of plant materials that contained
this compound frequently produced stomach irritation that limited the usefulness of salicin as a
pain and fever-reducing drug. The synthesis of a less irritating acetyl-SA derivative led to the
popularization of this form of SA as a drug and to early growth in the pharmaceutical industry,
exemplified by the patenting in 1899 of Aspirin by the Bayer Company in Germany . While
humans have exploited the therapeutic powers of willow for centuries, plants have for millions
of years employed SA as an endogenous signaling molecule active in defense and other
processes. In addition to its role in controlling defense against pathogens, SA signaling in plants
can also influence the expression of defenses against insect pests, as well as modifying
physiology and reproductive development in some plants . This chapter will discuss these roles
of SA in plants, the synthesis and storage of SA, and the signal transduction pathways that are
responsive to SA signals.

1.8.2.Biosynthesis Salicylic

Much of the organic carbon on our planet is a product of the shikimic acid pathway, as this is the
source of aromatic amino acids that are precursors of lignin, a stable and abundant constituent of
wood. Shikimic acid lies at the top of a linear pathway that leads to chorismic acid, which is a
common intermediate for several branched pathways that yield tyrosine, tryptophan,
phenylalanine, and many other aromatic compounds formed in plants. Of these, two pathways
have been implicated to play a role in SA synthesis in plants.
Biochemical labeling studies of TMV-infected tobacco plants showed that SA in these plants is
produced from the shikimate pathway’s phenylalanine branch through the phenylpropanoid
pathway. Another pathway from chorismate leads to production of isochorismic acid by action of
isochorismate synthesis (ICS). In microorganisms, this pathway has been shown to play a role in
SA synthesis.

The shikimic acid pathway produces chorismic acid, which is at the top of several pathways that
yield aromatic compounds, including the aromatic essential amino acids. One branch from

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chorismate produces phenylalanine, which by action of PAL is converted to trans cinnamic acid,
which can form two compounds implicated in SA synthesis, ortho coumeric acid and benzoic
acid. The latter compound is hydroxylated by BA2H, to yield SA. Another pathway from
chorismate yields isochorismic acid through action of ICS, and the product further modified
by IPL to form SA. The ICS gene has been shown in genetic studies to be required for most
SA production in parasitized Arabidopsis plants. Abbreviations: BA2H, benzoic acid 2
hydroxylase; ICS, isochorismate synthase; IPL, isochorismate pyruvate lyase; PAL,
phenylalanine ammonia lyase; SA, salicylic acid. Most arrows imply action of more than one
enzymatic conversion.

1.8.3.SALICYLIC ACID SIGNAL TRADUCTION

Genetic Dissection of SA-Mediated Defenses Because of the important role played by SA in
activating defenses against pathogens, the identity of signaling pathway components that respond
to SA signals have been sought using genetic and biochemical methods. Several groups looked
for Arabidopsis mutants defective in the irresponsetothe synthetic SA analog INA, in mutant
screens that focused on the failure of IN A to induce resistance to P. parasitica or activate
expression of reporter genes driven by PR gene promoters . These efforts led to discovery of
over a dozen independent mutants called nim1 (non-inducible immunity 1), npr1 (non-
expresserofPRgenes1)orsai1(SAinsensitive1),which fail to respond to INA, SA or BTH in the
induction of PR gene expression and resistance. Complementation analysis showed all mutants
to involve the same gene now called NIM1 or NPR1. In later screens for enhanced disease
susceptibility mutants, several other mutant alleles at this locus were isolated . The NIM1/NPR1
gene was position ally cloned and found to encode a protein containing ankyrin repeat and
BTB/POZ domains , motifs that in many proteins have been shown to mediate interactions with
other proteins. The NIM1/NPR1 protein show possible homology to Iκ-B , a protein that binds to
and inhibits activity of the transcription factor Nf-κB in vertebrates. The Nf-κB pathwa is
homologous to the Dorsal pathway in Drosophila, pathways that in both animals regulate
expression of the innate immune system that is activated by pathogen exposure and controls
expression of broad-spectrum disease resistance, analogous to SAR in plants. In plants, a number
of genes involved in pathogen defense have been found to be homologous to components of the
animal Nf-κB and Dorsal pathways, suggesting that the pathways that regulate innate immunity

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in animals and plants may be conserved, and thus of ancient origin . Using the NIM1/NPR1
protein as a probe in yeast two-hybrid screens, several TGA proteins, members of the bZIP
family of transcription factors, were identified that bind to NIM1/NPR1, implicating these
factors in SA mediated changes in gene expression . Elucidating the specific roles played by
these factors in defense is complicated by their redundancy (53) and ability to impinge upon
defense pathways that function independent of SA orNIM1/NPR1activity.Anothertwo-
hybridscreen revealed several related novel proteins that may act to bridge between NIM1/NPR1
and TGA factors, suggesting that a complex of proteins may be involved in mediating SA signal
transduction . Additional studies of NIM1/NPR1 action have examined the role of post-
translational modifications on the function of this protein in activating defense. Intriguing results
from Dong and coworkers have shown that specific cysteine residues in NIM1/NPR1 are
important for its function, and can mediate formation of disulfide bonds and multimerization of
the protein. The authors of this study speculate that inducers of SAR act by Salicylic Acid.
To fully understand SA responsive signaling pathways, it is also essential to define the molecules
that act as receptors for SA, and trigger downstream responses in response to elevated SA levels
in plants. The mutant screens descry bed above did not reveal obvious SA receptors, but
biochemical approaches to identify SA-binding activities have recently provided clues to the
identity of the elusive SA receptor.
Salicylic acid binding protein:The Search for an SA Receptor The features expected for an SA
receptor are that it binds specifically to SA,that its binding affinity to SA be relevant to
concentrations of SA that occur in planta, and that its function be required for SA-mediated
responses. Also,because biologically active synthetic analogs of SA are known, such as INA and
BTH, an SA receptor may also bind to and be activated by these compounds, but not by other
chemically similar but inactive compounds.To identify potential SA receptors, K lessig and
colleagues sought proteins that bind in vitro to radiolabeled SA. Several SA-binding proteins
(SABPs) that vary in properties and structure were found in tobacco; the twobest characterized
will be discussed here. SABP1 has a coefficient of SAbinding (Kd) of approximately 14 µM (4),
and upon cloning was found to be a catalase enzyme whose activity is reduced by SA binding
but not bybiologically inactive analogs of SA . This discovery led to speculation that SA-
inactivation of the SABP1 catalase leads to accumulation of H2O2, which then acts as a second
messenger for SAR activation . This hypothesis was examined further and excluded based on

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therelatively low binding affinity of SABP1 to SA relative to SA amounts found in systemic
tissues of infected plants, the lack of a measurable increase in H2O2 levels after induction of
SAR with SA, and the demonstration that H2O2 induction of SAR genes is associated with and
requires SA accumulation (19). A role for SABP1 in defense seems more likely to involve local
responses, such as hypersensitive cell death, as tissues surrounding an HR can accumulate
sufficient SA to inhibit SABP1 activity . The group also characterized another SA-binding
protein called SAPB2 , which is distinguished from SABP1 by its lower abundance and 150- fold
higher affinity for SA (Kd=90 nm).The SABP2 protein was recently cloned and shown to be a
lipase with an activity stimulated by SA . The importance of SABP2 in defense is indicated by
the phenotype of tobacco plants in which the SABP2 gene was silenced. SABP2 silenced plants.
1.8.4. Transportation
Transport of Labeled SA shows that SA Moves in Phloem .Although the experiments described
above are strong evidence that SA is not the systemic signal in SAR, in vivo 18O2 labeling
experiments show that SA made in an infected leaf can move systemically in tobacco, consistent
with SA playing a long distance signaling role. These studies examined SA levels in
uninoculated leaves having a strong vascular connection with the infected leaf, and showed that
in these leaves, a substantial amount (approx. twothirds) of the nascent SA originates from
infected leaves .Thus, while labeling experiments show that SA movement occurs in plants
undergoing pathogen attack, grafting studies indicatethat SA accumulation in rootstock leaves is
not required for export of the systemic SAR-inducing signal. It is possible that SA may be
transported in parallel with another systemic signal, and that SA itself is not sufficient to activate
SAR in uninfected leaves. A definitive test of whether SA is the primary long distance inducer of
SAR may require grafting experiments with plants unable to manufacture SA, or identification of
an authentic long distance signal in SAR through mutant or biochemical analyses. SA-deficient
mutants have been found in Arabidopsis, but thus far few workers have
tackled grafting experiments in this small rosette species.

1.8.5. Mode Of Action

Jasmonate modulates the expression of numerous genes and influences specific aspects of plant
growth, development, and responses to abiotic and biotics tresses Many of these responses were
identified by application of jasmonate to plants, sometimes at non physiological levels.
Interactions between JA and other plant growth regulators make assignment of physiological
roles for JA even more complicated. In the section below, proposed actions of JA in plants are
related to the level of JA in plant tissues, the activity of JA responsive genes, and insights
provided by JA insensitive and JA deficient plants. JA and MeJA inhibit the germination of non

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dormant seeds and stimulate the germination of dormant seeds. Application of JA to leaves
decreases expression of nuclear and chloroplast genes involved in photosynthesis . JA treatments
also cause a loss of chlorophyll from leaves or cell cultures. Jasmonate might be expected to play
a role in formation of flowers, fruit, and seed because of the relatively high levels of this
compound in developing plant reproductive tissues.

1.8.6..Metabolism Salicylic
It is recognized that salicylic acid potentially generates a wide array of metabolic responses in
plants and also affects photosynthetic parameters and plant water relations. These responses
could be either pathogen induced mal-physiological responses; restricting plant growth and
survival based upon pathogen type, could be root mal-nutritional responses Krouk G, Ruffel S. et
al 2011 or due to plant incompetency to catch up well compatible level of plant hormone
network to push abiotic stress(es) as indicative with several studies showing favorable responses
of stage, and dose specific responses of different plant hormones; or could be combinations of
either of these cases. A range of favorable physiological responses have been generated by
external application to incompatible (sensitive) plants or while these were promoted in
compatible (resistant) cultivars. These responses include shift in active biosynthesis of pigments
and their subsequent accumulation, photosynthesis related parameters Kumar P, Lakshmi NJ,
Mani VP. 2000 and the activity of associated enzymes and energy economy at the cost of ROS
production (discussed later in detail Plant physiology encompasses the functional domain of
metabolic manifestation of biochemical processes within the plant which in turn get regulated at
molecular level. Despite being visibly demarked as compartmentalized cells, tissue types and
different systems etc., a remarkable coherence is obvious within such subsystems.

During cellular metabolic processes, toxic level of reactive oxygen species (ROS) such as
superoxide anion (O2-), hydroxyl ions (OH·), and hydrogen peroxides (H2O2) are generated in
aerobic conditions and can cause damage to these cells, a phenomenon known as oxidative
stress. When plants are exposed to various environmental stresses they produce range of ROS in
large quantities sufficient to disrupt cellular and metabolic functions of the plant. To prevent
oxidative injury by the toxic, reactive oxygen species, the activity of antioxidant enzymes such
as SOD, APX, GR, POX, CAT and low molecular weight compounds like AsA and GSH is
increased normally. For the survival of the plants appropriate functioning of the antioxidant
system is important i.e., to maintain a balance between ROS and production and scavenging

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system. Gill SS, Tuteja N. 2010. Stress tolerance is therefore related with improved antioxidant
system in plants. Previous studies imprints salicylic acid enhances plant antioxidant protective
system following logistic growth curve thereby inducing stress tolerance. However, several
studies show that higher endogenous concentration of salicylic acid interplays with ROS pushing
the cell toward death. Higher concentration of salicylic acid appears to deactivate the antioxidant
activity in synergy with ROS. The analysis of peroxysomal antioxidant enzymes viz. SOD, CAT,
GPX (Glutathione peroxidase) and AsAGSH cycle activities in tomato infected with B. cineria
Kuzniak E, Sklodowska M. 2005, clearly indicated that initial infection induced increase in SOD,
CAT and GPX indicating antioxidant defense activation. It was followed by a progressive
inhibition concomitant with disease symptom development.

1.8.7. Conclusion

Salicylic acidic Much of the organic carbon on our planet is a product of the shikimic acid
pathway, as this is the source of aromatic amino acids that are precursors of lignin, a stable and
abundant constituent of wood. Shikimic acid lies at the top of a linear pathway that leads to
chorismic acid, which is a common intermediate for several branched pathways that yield
tyrosine, tryptophan, phenylalanine, and many other aromatic compounds formed in plants. Of
these, two pathways have been implicated to play a role in SA synthesis in plants. Despite the
fact that several SA-binding proteins (SABPs) have been identified, the identification and
characterization of the SA receptor is probably the most anticipated discovery. Although NPR1 is
not a receptor itself, it is the only known gene that, when mutated, generates plants insensitive to
SA (Canet et al., 2010,b) and causes a clear phenotype on plant defence response and some
effects on development. However, not all SA-induced genes depend on a functional NPR1, as
demonstrated in microarray analysis in wild-type and npr1 genotypes. In Arabidopsis it is clear
that NPR1 subcellular localization is regulated through a redox-sensitive mechanism mediated
by conserved cysteine residues that form intermolecular disulphide bonds that upon SA
accumulation are reduced and the monomers translocated into the nucleus (Mou et al., 2003).
Once in the nucleus, the NPR1 monomer functions as a co-activator of gene transcription, and
the nuclear levels of this protein are kept in check by proteasome-mediated degradation (Spoel et
al., 2009). However, this might not be a universal mechanism in all plant species as it has
recently been shown that tobacco NPR1 lacks the conserved cysteine residues, and differs in

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subcellular localization and transactivation potential from AtNPR1, as well in its sensitivity to
SA (Maier et al., 2011). Thus future research should emphasize the functional genomics of
NPR1 paralogues in various species, as well as the mechanism through which SA modulates
redox potential in the plant cell.

The role of SA in plant growth and development is still a controversial field in plant biology;
however, various phenotypes are associated with deregulated SA levels and new discoveries and
mutant characterization should shed more light on this topic. SA's complex role is not limited
only to its canonical signal transducer, NPR1, but also involves its role in modulating the plant
cell red ox statu.

1.9. Brassinosteriods

1.9.1. Introduction

Brassinosteroids (BRs) are a class of polyhydroxysteroids that have been recognized as a sixth
class of plant hormones. These were first explored nearly 40 years ago, when Mitchell et al.
reported promotion in stem elongation and cell division by the treatment of organic extracts of
rapeseed (Brassica napus) pollen. Brassinolide was the first isolated brassinosteroid in 1979,
when pollen from Brassica napus was shown to promote stem elongation and cell divisions, and
the biologically active molecule was isolated. The yield of brassinosteroids from 230 kg of
Brassica napus pollen was only 10 mg. Since their discovery, over 70 BR compounds have been
isolated from plants.

1.9.2.Biosynthesis
The BR is biosynthesised from campesterol. The biosynthetic pathway was elucidated by
Japanese researchers and later shown to be correct through the analysis of BR biosynthesis
mutants in Arabidopsis thaliana, tomatoes, and peas. The sites for BR synthesis in plants have
not been experimentally demonstrated. One well-supported hypothesis is that all tissues produce
BRs, since BR biosynthetic and signal transduction genes are expressed in a wide range of plant
organs, and short distance activity of the hormones also supports this. Experiments have shown
that long distance transport is possible and that flow is in an acropetal direction, but it is not

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known if this movement is biologically relevant. Brassinosteroids are recognized at the cell
membrane, although they are membrane-soluble.

1.9.3 . Mode of action Brassisterniod

A mode of action (MoA) describes a functional or anatomical change, at the cellular level,
resulting from the exposure of a living organism to a substance. In comparison, a mechanism of
action (MOA) describes such changes at the molecular level.

A mode of action is important in classifying chemicals as it represents an intermediate level of

complexity in between molecular mechanisms and physiological outcomes, especially when the
exact molecular target has not yet been elucidated or is subject to debate. A mechanism of action
of a chemical could be "binding to DNA" while its broader mode of action would be
"transcriptional regulation". However, there is no clear consensus and the term mode of action is
also often used, especially in the study of pesticides, to describe molecular mechanisms such as
action on specific nuclear receptors or enzymes.

1.9.4 .Brassinosteroids Signaling tranduction

In the recent past, with the use of various biochemical, genetic and proteomic approaches, a great
advancement has been made in our understanding of the BR signaling pathway (Zhu et al.,
2013b; Fàbregas and Caño-Delgado, 2014; Belkhadir and Jaillais, 2015). At the plasma
membrane BR are perceived by the extracellular domain of the plasma membrane localized
leucine rich repeat receptor like kinase (LRR-RLK) BRI1 and its two closely related homologs,
BRI1-LIKE 1 (BRL1) and BRI1 LIKE 3 (BRL3), in the nanomolar range (Cano-Delgado et al.,
2004). With the recent elucidation of atomic structures of the BRI1 and BRL1 in complex with
BL has facilitated in structural understanding of BR perception at the cell surface (Hothorn et al.,
2011; She et al., 2011, 2013). BRI1 and BRL1 have similar basic skeleton with 25 and 24 units
of LRRs, respectively with a great degree of similarity in overall shape and curvature of the
horseshoe-like structure of extracellular domain which is the seat for binding of BR. However,
minor structural differences in the BR binding pocket of BRL1 and BRI1 reveals that BR have
less affinity to BRI1 as compared to BRL1 as BR binds more strongly to BRL1 as compared to
BRI1 (She et al., 2011, 2013). BRL2, which is considered as another BRL1 homolog and
responsible for vascular development has also been studied for its ability to bind to BR (Ceserani
et al., 2009). It is interesting to note that despite having high sequence identity of BRL2 with
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BRL1 and possessing conserved Arg588 and Gly690 residues responsible for higher affinity of
BRL1 for BL, BRL2 lacks the ability to bind to BR. This could be due to the presence of heavily
negatively charged residues located at the inner side of the binding pocket in BRL2 that result in
the change in its hydrophobicity to BRs (She et al., 2013).

receptor BRI1-associated receptor kinase1 (BAK1), also known as somatic embryogenesis

receptor-like kinase 3 (SERK3) (Li et al., 2002; Nam and Li, 2002). Binding of BR to the 70-
amino acids island domain of BRI1 triggers a change in the receptor either in the form of
conformational change in the preformed homodimer or receptor dimerisation (Wang et al.,
2005a). It results in the autophosphorylation of BRI1 kinase domain activation loop which
induces a partial kinase activity of BRI1 leading to the transphosphorylation at Tyr211 residue of
BKI1 and resulting in its dissociation from the membrane and thus allowing BRI1 to interact
with BAK1 (Wang et al., 2005b; Wang and Chory, 2006). Thus, BR induces the conformational
changes in its receptor that are necessary for BRI1-BAK1 interaction (Santiago et al., 2013; Sun
et al., 2013). BKI1 also promotes BRI1 signaling by binding to 14–3–3 protein and repressing
their negative functions in BR signaling (Wang et al., 2011). BRI1 then interacts with BAK1 to
transphosphorylate each other at multiple residues (Wang et al., 2008). Forster resonance energy
transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) approaches have shown
that a significant amount of BRI1-BAK1 hetero-oligomers are present at the cell surface even in
the absence of BR (Bucherl et al., 2013). It is hypothesized that preassembled BRI1-BAK1 after
conjugation with ligand might undergo discrete rearrangements of their respective intracellular
kinase domains. This hypothesis derives its significance as similar signaling paradigms is
observed in receptor tyrosine kinases in animals (RTKs) which undergo ligand-independent
receptor dimerization, followed by dimer reorganization upon ligand binding (Lemmon and
Schlessinger, 2010; Bucherl et al., 2013). Activated BRI1 initiates a cascade of phosphorylation
events of its downstream plasma membrane bound receptor-like cytoplasmic kinases (RLCKs),
BR signaling kinases (BSKs) and constitutive differential growth 1 (CDG1) to transmit the
signal from membrane receptors to cytoplasmic regulators of BR signaling (Tang et al., 2008;
Kim et al., 2011; Sreeramulu et al., 2013). Mass-spectrometric analysis has shown that BRI1
phosphorylates CGD1 and BSK1 at Ser-234 and Ser-230, respectively and subsequently
phosphorylate and activate BRI1-suppressor 1 (BSU1). It has been found that BSU1 can be
activated by BRI1 either through BSK1 or CDG1. Furthermore, BSK1 induced activation

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requires BRI1 kinase activity, whereas CDG1 induced activation does not require BRI1 but is
enhanced by BRI1. Thus, either of one set of interacting partners, BSU1-CDG1 or BSUI-BSK1
is the minimum set of components required for transducing the signal from the receptor kinase
BRI1 to the GSK3-like kinase BIN2 (Kim et al., 2011). BSU1 then dephosphorylates GSK3 like
kinase BR insensitive 2 (BIN2) (Kim and Wang, 2010) to inhibit its function and relieves the
inhibitory effect on two master transcription factors of BR signaling, BZR1 and BZR2 also
known as BRI1-EMS suppressor1 (BES1) (Wang et al., 2002; Yin et al., 2002, 2005; He et al.,
2005). BZR1 and BES1 are then dephosphorylated by protein phosphatase 2A (PP2A) and
released from 14–3–3 proteins (Tang et al., 2011), resulting in their nuclear localization to bind
to the promoter of their target genes to regulate their gene expression (Sun et al., 2010; Yu et al.,
2011; Wang et al., 2012b). Other BRI1 substrates like Arabidopsis TGF-β receptor-interacting
protein-1 (TRIP-1) and transthyretin-like protein (TTL) have also been identified with TTL
being putatively linked to inhibition of BRI1 signaling as it binds with higher affinity to kinase-
active BRI1 while TRIP-1 being an essential subunit of eIF3 protein translation initiation
complex, its phosphorylation by BRI1 is thought to modulate its activity and influence protein
translation (Ehsan et al., 2005). Though both BRI1 and BAK1 are classified as serine/threonine
kinases, the studies done in past decade has shown that BRI1 has structural features reminiscent
of both serine/threonine and tyrosine kinases like insulin receptor, thus providing insights into
the evolution of dual-specificity kinases in plants (Wang et al., 2005b; Oh et al., 2009; Macho et
al., 2015). Infact, BRI1 possess significant tyrosine kinase activity and it can undergo
autophosphorylation on tyrosine residues within the kinase and juxtamembrane domains and can
lead to transphosphorylation of Tyr211 in BKI1 as well as tyrosines in other proteins (Oh et al.,
2009; Jaillais et al., 2011; Wu et al., 2012). Tyr-831 and Tyr-956 are identified as
autophosphorylation sites in vitro and in vivo with Tyr-956 in kinase subdomain V being
essential for activity, while Tyr-831 in the juxtamembrane domain is not essential for kinase
activity but plays an important role in BR signaling in vivo (Wang et al., 2005b; Oh et al., 2009;
Macho et al., 2015).

1.9.5. Metabolism
Brassinosteroid catabolism/metabolism involving various process like acylation, sulphonation,
glycosylation etc. play a crucial role in maintaining the optimum levels of bioactive BR in the

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cell. Novel genes belonging to the family of BAHD acyltransferases, brassinosteroid
inactivator1 (BIA1) and abnormal shoot-1 (abs-1) (Roh et al., 2012; Wang et al., 2012a) have
been identified to be involved in BR acylation to inactivate BR. Another BAHD acyltransferases
pizza (PIZ), have a redundant role with BIA1 in BR inactivation (Schneider et al., 2012).
Similarly, bri1-5 enhanced 1 (BEN1) and Brassica napus sulfotransferase 3 (BNST3) possessing
differential specificities to castesterone and BL inactivate active BR by various mechanism
involving reduction and sulfonation (Marsolais et al., 2007; Yuan et al., 2007). A set of
glycotransferases enzymes UGT73C6 and its close homolog UGT73C5, catalyze the 23 O-
glycosylation of CS and BL as part of the inactivation process (Poppenberger et al., 2005).
Evidence reveal that these conjugations may serve as temporary storage forms of pool of inactive
BR and believed to serve additional functions such as irreversible inactivation, transport,
compartmentalization, and protection against cellular removal (Bajguz, 2007; Husar et al., 2011;
Piotrowska and Bajguz, 2011). Recent studies have also shown that BR biosynthesis can be
regulated by external stimuli like salt and temperature stress (Maharjan and Choe, 2011; Sharma
et al., 2013).

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