CP-505 Dr Prakash kalita

Professor
Dept. of Crop Physiology
FA/AAU, Jorhat-13

Involvement of phytohormones in drought tolerance in

plant : ( A signaling perspective and cross talk among
various phytohormones)
In plants, the perception of abiotic stress triggers the activation of signal
transduction cascades that interact with the baseline pathways transduced by
phytohormones. The convergence points among hormone signal transduction
cascades are considered cross-talk, and together they form a signaling network.
Through this mechanism, hormones interact by activating either a common second
messenger or a phosphorylation cascade.

Drought stress causes a wide range of morphophysiological and biochemical

modifications that adversely affect the development as well as the plant
productivity . Generally, drought stress severely restricts plant growth and
development, affecting plant height, leaf dry weight, stem dry weight, leaf area
index, node number, canopy, and root development .
Plant growth is highly sensitive to water deficit, largely due to inhibition of cell
elongation.
Water-stressed plants are shorter, and having a smaller leaf area, which reduces the
amount of photosynthetically active radiations absorbed by leaves, the rate of
photosynthesis and finally the yield.
Water deficit induces stomatal closure which leads to limited CO2 uptake by the
leaves and reduces the potential activity of Calvin cycle enzymes, particularly
ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), due to lack of
substrate.
In addition, the photosynthetic rate is also affected by non-stomata processes; for
example, the reductions in rubisco activity, availability of CO 2 in the chloroplast,
and efficiency of PSII photochemistry gradually progress with intensity of drought
stress and duration .
The photosynthetic apparatus can also be damaged by ROS generation when the
stomata are closed, and consequently reduces photosynthesis and growth .
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Nutrients are very important for plant growth and yield, especially macronutrients
and their uptake are limited under drought stress .
In response to drought conditions, plants have evolved morpho-physiological,
phenological and biochemical mechanisms to maintain constant cellular water
potential and/or relative water content .
These processes are controlled by numerous phytohormones which
are the basic mediators to tolerate or avoid the negative effects of
water deficit.
Phytohormones are the key regulators of plant growth and developmental
processes as well as crucial for biotic and abiotic stress response throughout their
life cycle .
Various plant hormones are synthesized in response to drought stress and manage
processes related to drought tolerance mechanisms.
Phytohormones including, ABA, jasmonic acid (JA), ethylene (ET), and salicylic
acid (SA) are involved in osmotic adjustment and other drought-related processes .
These phytohormones perform as chemical messengers in response to various
abiotic stresses. After stress signal perception, phytohormones activate various
plant physiological and developmental processes including stomatal closure, root
growth stimulation, and accumulation of osmolytes to avoid drought
conditions.
ABA is the major stress-responsive hormone produced after perception of
drought signal. ABA is synthesized in the plastids and cytoplasm and derived from
zeaxanthin, a plant pigment. A cluster of NCED (9-cis-epoxycarotenoid
dioxygenase) genes are thought to be involved in ABA synthesis . The first NCED
gene was identified from the maize vp14 mutant and these genes were induced by
drought stress during seed maturation. Osmotic stress promotes the synthesis of
ABA, which activates gene expression and adaptive physiological changes .

Upon stress arrival, signal perception occurs through plasma membrane, ABA
synthesis is initiated, which occurs mostly in the plastids, with the exception of
xanthoxin conversion to ABA, which takes place in the cytoplasm .

Generally, ABA synthesis occurs in the roots. It is then transported via vascular
tissues, and it shows responses in a variety of cells, such as guard cells and others .
As a weak acid, abscisic acid is a charged anion (ABA−) in the cytoplasm (pH 7).
While in more acidic cell wall (pH 5.5), some left uncharged (ABAH). This
presumably enhances the movement of ABA into but not out of cells, while cell-to-
cell ABA transport is enhanced by transporters, i.e., plasma membrane bound

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ATP-binding cassette (ABC) transporters, which hydrolyze ATP to transport it,
and a family of low-affinity nitrate transporters.
ABA perception and signal transduction are mediated by two pathways, which
are ABA-dependent and ABA-independent.
ABA-dependent signaling pathways play a critical role in stress-responsive gene
expression during various stresses, especially osmotic stress. ABA receptors are
important elements for ABA signal transduction. Various receptors have been
identified in different sub-cellular compartments, including the plasma membrane,
nucleus, cytosol, and chloroplast envelope.
Under normal conditions, ABA content is low, and SnRK2 protein kinase activity
is inhibited by PP2C phosphatases, which leads to dephosphorylation.
When plants suffer from drought stress, the cellular ABA level increases, and
ABA then binds to PYR/PYL/RCARs, which in turn bind and inactivate PP2Cs .
So, PYR/PYL/RCARs ABA receptors necessary for ABA responses and various
genes encode these receptors for example, rice contain 11 genes while Arabidopsis
contains 14 genes .
The SnRK2s are auto activated when they dissociate from PP2Cs.
ABA-dependent signaling pathway has several branches controlled by various
transcription factors including MYB, MYC, and NAC. In addition, ABA-
responsive elements (ABREs) are also involved in ABA signalling . Calcium-
dependent protein kinases (CDPKs) also participate in ABA signaling . Thirtyfour
CDPKs in Arabidopsis, 29 in rice, 20 in wheat and 35 in maize have been reported
. Two CDPKs, CPK4 and CPK11 have been reported in Arabidopsis to be involved
in the regulation of ABA signalling .
Activated SnRK2s phosphorylate downstream targets and trigger ABA-induced
physiological and molecular responses including germination, stomata regulation,
root development and photosynthesis . ABA regulates many stress-related genes to
enhance drought tolerance in plants. There are several ABA-hypersensitive and
reduced sensitivity mutants, such as era1-2, fry, abh1, ABI1 and ABI2 .

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Fig : Signalling pathway of ABA during moisture deficit condition .

Regulation of stomata by ABA and other phytohormones :

Stomata regulation plays a pivotal role in gas exchange between
tissues and the atmosphere. It is one of the key mechanisms that allow plants to
produce energy and maintain cellular function. Ninety percent of water losses
(transpiration) from plants occur though stomata openings . Closure of the
stomata is the first step to reduce water loss during drought conditions, when
the rate of transpiration is very high. Stomatal conductance could be a potential
indicator of drought tolerance in plants as there is a negative correlation between
drought tolerance and stomata conductance .
ABA synthesis occurs upon signal perception during stress conditions, especially
drought stress. Guard cells respond to ABA and reduce water loss by stomata
regulation. Stomatal movements are mediated by cell turgor, which is controlled by
the transport of ions across membranes.
ABA triggers closure of stomatal pores via efflux of potassium (K+) and anions
(A−) from guard cells as well as removal of organic osmolytes . Ion channels are
present on the vacuolar and plasma membranes and include inward and outward
transporting K+, A−, and calcium (Ca2+) channels. ABA promotes opening of the
ion channels in the vacuolar and plasma membranes, releasing ions from the cell,
and inactivates a proton-ATPase. Osmosis let the water out of guard cells and the
cell volume shrinks which lead to the closure of stomata .

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 Cytosolic calcium ion [Ca2+]cyt is a second messenger which leads to the
closing of stomata.
ABA stimulates an increase in cytoplasmic calcium by activating calcium
channels in the plasma and internal membranes. The movement of anions out of
the cell increases the pH and depolarizes the membrane, which activates the pH
and voltage-sensitive potassium channels. Guard cell turgor is regulated by a
complex network of interacting second messengers, pH, membrane potential, the
protein phosphorylation, ion channel activity and other factors . In addition, ROS
also play significant role in guard cell signaling; especially, H2O2 plays central role
in ABA induced stomatal closure .

ABA regulates several types of ion channels, causing anion efflux, K+ efflux, and
the inhibition of K+ import . One mechanism of ABA action begins when ABA
binds to receptors belonging to a protein family known as PYR/RCARs, the
pyrabactin (4-bromo-N-[pyridin-2-yl methyl]naphthalene-1-sulfonamide)
resistance (PYR)/regulatory component of ABA receptor (RCAR) .In Arabidopsis,
14 highly conserved PYR/RCARs have been identified. Upon binding to ABA,
PYR/RCARs inhibit the activity of specific protein phosphatise 2Cs (PP2Cs),
which are negative regulators of ABA signaling. The inactivation of PP2C allows
for the phosphorylation and activation of three sucrose nonfermenting 1-related
protein kinase 2s (SnRK2s), which belong to the SnRK2 subfamily, whose
members are associated with the regulation of abiotic stress .While the precise
mechanism of SnRK2 activation is unknown, it has been shown to
autophosphorylate. SnRK2 acts as a positive regulator of several targets on the
plasma membrane, including the anion exporter slow anion channel-associated 1
(SLAC1) and the K+ channel in Arabidopsis thaliana (KAT1) in guard cells
.SnRK2 activates SLAC1 through phosphorylation, while phosphorylation of
KAT1 by SnRK2 inhibits its function, thus decreasing the influx of K+ into the cell
.Increased SLAC1activity causes an efflux of anions, which depolarizes the
membrane and results in the loss of K+ through the depolarization of activated K+
efflux channel called guard cell outward-rectifying K+ (GORK ). The collective
loss of anions and K+ ions from the guard cells causes water to move out of these
cells, which results in the reduction in turgor that triggers stomatal closure in
response to ABA.

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Fig :Model showing the interaction among hormones in causing stomatal closure
during drought situation .

This model illustrates the interactions between phytohormone signal pathways

during ABA-induced stomatal closure as a result of drought stress. Drought
triggers an increase in ABA concentration in guard cells, where it becomes bound
to its receptor complex, in which SnRK2 is activated. The SnRK2 phosphorylates
and inactivates a potassium import channel and activates an anion efflux channel,
which in turn simulates K+ efflux.
SnRK2 also activates enzymes involved in H2O2 production; the resultant H2O2
activates NO that then triggers the influx of Ca2+ from the vacuole into the
cytoplasm. The Ca2+ activates CDPKs, which then stimulate anion efflux and
inhibit K+ influx.
The resulting ion loss causes water efflux, loss of turgor, and stomatal closure. In
addition, Ca2+ influx from the vacuole into the cytoplasm is induced by ABA-
mediated production of IP3.
Stress-induced JA production interacts with ABA-mediated stomatal closure by
stimulating the influx of extracellular Ca2+ and/or by activating H2O2/NO

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signaling. Ethylene acts as a negative regulator of ABA, and thus, of this pathway.
Steps inferred from experiments exploring ABA function using turgid cells are
indicted with dashed lines .

All root traits are potentially important in the drought stress;

however, hydraulic conductance and plant allometry have been of particular
interest to researchers.
More profuse (higher root length density) and deeper root systems in the soil are
often proposed as desirable characteristics for drought adaptation.

ABA also mediates root elongation to reach deep water in the soil during drought
conditions .
Fluridone, an ABA synthesis inhibitor, inhibits primary root elongation at low
water availability.
Sharp et al. (2004) studied that water deficit increase ABA and ROS, while ABA
act as a suppressing agent of growth-inhibitory ROS and ethylene. `
In another case, lateral roots were partially mediated by ABA under drought stress.
Root characteristics, such as root length density, long roots, and small fine roots,
play critical roles in maintaining the plant productivity under drought stress,
specifically when water is available deeper in the soil .
A continually elevated ABA concentration initiates changes in gene expression
that inhibit shoot growth and maintain root growth, thus increasing the root-to-
shoot ratio.
ABA-Dependent Gene Expression During Drought Stress and
root growth :

Longer-term physiological responses to abiotic stress conditions are caused by

changes in gene regulation through ABA-mediated regulation of transcription
factors that bind to ABA-responsive elements (ABREs) on ABA-regulated genes .
Phosphorylation cascades also lead to changes in ABA-regulated transcription
factors. For example, the ABAresponsive transcription factors (ABFs) ABF1 and
ABF4 are activated when they are phosphorylated by CPK4 or CPK11 (which are
ABA-inducible kinases) .
Zhu et al. (2007) also demonstrated that other kinases can phosphorylate ABF1
and ABF4. For example, ABF1 was reported to be a target of some SnRK2
members (Fujita et al. 2009), and CPK32 phosphorylates ABF4 .

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Osmosensing and subsequent signal transduction require phosphorylation of key
intermediates through the activity of mitogen-activated protein kinases (MAPKs)
(Boudsocq and Laurie`re 2005).
Current models indicate that NO is a central signaling molecule that triggers
phosphorylation events through MAPKs as an early response to drought stress .
In drought-stressed wheat seedlings, elevated NO concentrations enhance the
transcription of specific drought-induced genes .

Fig: Model of involvement of phytohormones in root growth and

architecture under drouht condition.

This model illustrates drought-induced gene regulation that controls root growth
and architecture. Elevated ABA concentration activates H2O2, NO, and Ca2+
signaling. As shown, specific CDPKs and MAPKs activate ABA-regulated
transcription factors, which control the expression of ABA-responsive genes.
These genes inhibit the ethylene response via the downregulation of several
ethylene-responsive and ethylene biosynthetic genes; they also reduce cytokinin

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levels by triggering an increase in the expression of enzymes that oxidize
cytokinin. An increase in the expression of miRNAs associated with auxin
signalling downregulates auxin responses. Collectively, these interactions favour
the maintenance of the root primordia of primary roots and existing lateral roots
while inhibiting lateral root elongation and adventitious root formation.
In Arabidopsis, MPK6 and its associated MAPK cascade components are activated
by drought stress . MPK6 targets two ethylene biosynthetic enzymes, 1-
aminocyclopropane-1-carboxylic acid [ACC] synthase 2 (ACS2) and ACS6, and is
also involved in ethylene signaling that leads to the regulation of ethylene-
responsive genes . In addition, both ACS2 and ACS6 contain ABRE on their
promoters.
Under drought conditions, the region near the root tip continues to grow, while
regions away from the tip are inhibited or cease to grow. The tip region of the
primary root is also the area where ABA and ROS accumulate and where a
decrease in ethylene production is noted 3.5 h after drought stress (Yamaguchi and
Sharp 2010).
Therefore, during drought stress, 2 Cross-Talk Between Phytohormone Signaling
Pathways occur . An increase in ABA concentration inhibits ethylene production
and downregulates genes that respond to the presence of ethylene, thus maintaining
root growth .
Cytokinin is considered to be a negative regulator of root growth and branching,
and root-specific degradation of cytokinin may also contribute to the primary root
growth and branching induced by drought stress .
Werner et al. (2010) demonstrated that an increase in cytokinin degradation in the
roots results in an increase in both primary root length and lateral root formation
during drought conditions. An analysis of the Arabidopsis CKX promoter indicates
that the region contains three ABREs , which implies potential ABA-based
regulation of cytokinin degradation in the roots . This activity would stimulate root
growth and lateral root production; thus, increasing the root to shoot is associated
with drought conditions and with increased drought tolerance .
While auxin is considered to be the primary hormone involved in the initiation and
growth of lateral and adventitious roots, ABA may play a role in regulating lateral
root growth under stress conditions.

During drought conditions, Arabidopsis produces specialized, short lateral roots

that remain in a dormant or non-growing condition while the plant is under stress
These roots replace dehydrated lateral roots once drought conditions are relieved.
Therefore, for Arabidopsis, the additional ABA produced in response to drought
stress inhibits the outgrowth of lateral roots from existing meristems .
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Several miRNAs that are associated with auxin signaling are upregulated by
drought or osmotic stress and may play important roles in the regulation of root
architecture. For example, in Arabidopsis, both miR167 and miR168 are involved
in the regulation of auxin response factors and both contain ABREs, indicating
their own regulation by ABA signaling (Liu et al. 2008).
Drought-associated changes in root architecture are directed by cross-talk between
ABA with ethylene, auxin, and cytokinin .
When Arabidopsis is exposed to drought stress, an increase in ABA in the roots
inhibits ethylene production and stimulates cytokinin degradation, which together
result in the maintenance of primary root growth. ABA-controlled regulation of
genes (such as those that encode ARFs and miRNAs) associated with auxin-
regulated root production inhibits the formation of adventitious roots and the
outgrowth of lateral roots from existing primordia.

The end

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