Molecular basis of abiotic stress in plants- Drought and Heat

Submitted by: Kirti Ph.D. (MBB)

Advances in Plant Molecular Biology (MBB 601)

What is stress?

• Stress is considered to be a significant deviation from optimal conditions of life.

• A condition that adversely affects growth, development, and/or productivity.

• Stresses trigger a wide range of plant responses: – altered gene expression – cellular metabolism – changes in growth rates and crop yields

Types of Stress

Biotic and abiotic stresses can reduce average plant productivity by 65% to 87%, depending on the crop.

Biotic• imposed by other organisms

Abiotic• arising from an excess or deficit in the physical or chemical

component of the environment

Environmental conditions that can cause stress

Environmental conditions

High/low temperatu

res

Drought

Flooding

Excessive soil

salinity

Inadequate minerals in

the soil

Too much or too

little light

Air pollutants

(ozone, sulfur

dioxide)

Effect of stress in plants

Drought – Serious threat to agriculture

Drought (an abiotic stress) is an extended period of months or

years when a region notes a deficiency in its water supply whether surface or underground water.

Drought stress accounts for more production losses than all other factors combined.

Fraction of world arable land subjected to Drought (abiotic stress) – 26%.

Drought escape- proper timing of lifecycle, resulting in the completion of the most sensitive developmental stages while water is abundant. Dehydration avoidance- Avoiding water-deficit stress with a root system capable

of extracting water from deep soil layers.

Dehydration tolerance- Mechanisms such as osmotic adjustment (OA) whereby a plant maintains cell turgor pressure under reduced soil water potential.

Drought tolerance mechanism is genetically controlled and genes or QTL responsible for drought tolerance have been discovered in several crops which opens avenue for molecular breeding for drought tolerance.

How plants cope with

drought stress

Physiological, biochemical and molecular basis of drought stress tolerance in plants, Shao et al. (2008). Comptes Rendus Biologies, 331:215-225 ©2008, Elsevier Limited, UK

Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell (Chaves et al., 2009)

Water stress reduces photosynthesis by decreasing both leaf area (leaf rolling) and photosynthetic rate per unit leaf area.

Photosynthesis is among the primary processes to be affected by drought decreased CO2 availability caused by diffusion limitations through the stomata and the mesophyll (Flexas et al., 2007).

Supply of CO2 to Rubisco is impaired predisposes the photosynthetic apparatus to increased energy dissipation and down-regulation of photosynthesis.

Drought may be of two kinds: short-term and long-term length of a growing season lasts more than one growing season (stomatal regulation)

In the signaling pathway towards stomatal closure there are several secondary messengers, such as Ca2+, H2O2 and NO (Garcia-Mata and

Lamattina, 2009) that contribute to the stomatal closure.

Passive loss of turgor pressure also results in stomatal closure.

In addition to alterations in photosynthesis and cell growth, stress also induce osmotic adjustment which is considered an important mechanism to allow the maintenance of water uptake and cell turgor under stress conditions.

Soluble sugars (namely sucrose, glucose and fructose) that are altered by water deficits and salinity, also act as signalling molecules under stress (Chaves et al., 2004).

A major source for glucose signals is transitory starch breakdown from chloroplasts during the night.

Huber et al. (1984) concluded that water stress has a larger effect on carbon assimilation than on translocation.

Resurrection plants have been widely used as model plants for dehydration studies (Bartels, 2001).

Drought induces synthesis of both chaperons (which enhance stability of other proteins), and proteases and ubiquitin (which cause degradation of proteins that become denatured during water loss).

- play vital roles in drought tolerance of plants.

Under drought, endogenous contents of auxins, gibberellins, cytokines usually decrease while those of abscisic acid and ethylene increase.

There are many signals that induce stomatal closure, among these the best known signal is ABA.

Phytohormones

The phytohormone ABA is the central regulator of abiotic stress particularly drought resistance in plants, and coordinates a complex gene regulatory network enabling plants to cope with decreased water availability (Cutler et al., 2010; Kim et al., 2010).

Drought and salinity trigger the production of ABA in roots which is transported to the shoots causing stomatal closure and eventually restricting cellular growth.

ABA can also be synthesized in leaf cells and translocated around the plant (Wilkinson and Davies, 2002).

ABA reduces water loss from plants during drought stress via a signal transduction network in guard cells (Schroeder et al., 2001).

The ABA synthesis is probably dependent on plasma membrane-localized pressure sensitive receptors or ion channels (Guerrero et al., 1990).

ABA- ‘stress hormone’

ABA induces K+ and Ca2+ fluxes in guard cells causing stomatal closure ( to improve the water use efficiency (WUE)) and regulating water loss.

Drought induced changes in the photosystem (Giardi et al., 1996) reduce the stromatal pH, which lead to release of the chloroplast compartmentalized ABA into the cytosol.

There it could act upon an intracellular receptor initiating a cascade of signal transduction, or could be directly imported into the nucleus triggering gene activation.

ABA biosynthesis gene (NCED3), a cytochrome P450 CYP707A family has been identified as ABA 80-hydroxylases, which play a central role in regulating ABA levels during dehydration stress conditions (Umezawa et al., 2006).

An insertional mutant of CYP707A3 exhibited elevated drought tolerance with a concomitant reduction of transpiration rate (Kushiro et al., 2004).

Drought stress and ABA application modulate the expression of various constitutively expressed genes (Chen et al., 1996), and are also capable of down regulating transcription.

For example, genes whose products are involved in photosynthesis {cab and rbcS) are negatively- (down-) regulated by ABA in water-stressed tomato leaves (Bartholomew et al.,1991).

Elucidation of ABA-responsive promoter regions revealed an ABA-response element ABRE in the promoter region of several ABA-regulated genes (Chandler and Robertson, 1994).

Molecular control of abiotic stress

DREB, MAPK, Phospholipases

Signaling cascades & TFs

Water & ion transportFunctional

proteins

HSP, Chaperones, LEA, Dehydrins, ROS scavenger, OA etc..

Ion transporters, Aquaporins,

Molecular Response

Perception by specific receptor

Initiates/ inhibits cascade response

Information transmittedGene expression

Aquaporins Aquaporins are water channel proteins, belong to a highly conserved

family of major intrinsic membrane proteins (MIP) family (Tyerman et al., 2002).

In plants they are abundant in plasma membrane and in the vacuolar membrane and function in water transport at the plasma membrane in many plant species (Tyerman et al., 2002).

Aquaporins act by facilitating the permeation of water across membranes driven by differences in water potential (Tyerman et al., 2002).

In tobacco plants, three PIP (Plasma membrane intrinsic proteins)-type genes: NtPIP1, NtPIP2 and NtAQP1, have been identified and characterized.

Siefritz et al. (2002) found that on knocking down the expression of the NtAQP1 gene using NtAQP1 antisense constructs, transgenic tobacco plants showed a higher sensitivity to water stress.

cell to cell water transport in roots is strongly downregulated in response to drought stress.

Grapevine, along with a number of other plant species, demonstrates a reduction in root hydraulic conductivity in response to water stress

reduction was associated with a closure of aquaporins,

suggested mechanism to prevent water loss to the soil, which has a lower water potential than the plant.

(North et al., 2004).

Osmoprotectants

The biosynthesis and accumulation of compatible solutes is an important adaptive mechanism

enable protection of cell turgor and maintains cellular water

potential, stabilize membranes and scavenge ROS.

Many crops lack the ability to synthesize the special osmoprotectants that are naturally accumulated by stress tolerant organisms.

Osmoregulation would be the best strategy for abiotic stress tolerance, especially if osmoregulatory genes could be triggered in response to drought, salinity and high temperature.

In stress-tolerant transgenic plants, many genes involved in the synthesis of osmoprotectants—organic compounds such as amino acids (e.g. proline), amines (e.g. glycinebetaine and polyamines) and a variety of sugars and sugar alcohols (e.g. mannitol, trehalose and galactinol).

Glycine betaine (GB), a fully N-methyl-substituted derivative of glycine, accumulates in the chloroplasts of many species in response to abiotic stress and is considered the major osmolyte involved in cell membrane protection.

Choline is converted to betaine aldehyde by the choline mono oxygenase (CMO) under drought, and salinity stresses.

Betaine aldehyde is then catalyzed into glycine betaine by betaine aldehyde dehydrogenase.

Many important crops, such as rice, potato and tomato, do not naturally accumulate glycine betaine

potential candidates for the engineering of betaine biosynthesis to make them perform better under the drought conditions.

Choline dehydrogenase gene (codA) from Arthrobacter globiformis the gene product catalyzes the oxidation of choline to

glycine betaine via betaine aldehyde, was used to transform rice.

Proline represents an important osmolyte, which has protective role during the drought stress.

Δ1-Pyrroline-5-carboxylate synthase Δ1-Pyrroline-5-carboxylate reductaseL-Glutamate L-Δ1-Pyrroline-5-carboxylate L-Proline

NADH + H+ NAD + 2H2O NADH NAD

Plant proteins: Responses to drought

Protein changes references

Dehydrin tobacco (Sawhney, 1990)

Osmotin tobacco, tomato and maize (Ramagopal,1993)

SAPs (stress associated proeins) accumulation

rice varieties (Pareek et al.,1997)

RAB (responsive to ABA)18 protein accumulation

Arabidopsis thaliana (Mantyla et al., 1995)

LEA (late embryogenesis abundant) group1

cotton, barley, carrot (Ramagopal, 1993)

Proline, glycine betain accumulation, putrescine

tobacco (Galston andSawhney, 1990)

Specified protein synthesized under water scarcity/Changes in plant metabolics (Proteins):

LEA (late embryogenesis abundant) proteins

They are highly hydrophilic, highly soluble, globular proteins, originally characterized as accumulating in seeds during maturation and desiccation.

Having biased amino acid composition (rich in Ala, Gly and lacking Cyt and His) and have high number of polar residues.

LEA proteins participate in protecting cellular components from dehydration (Reyes et al., 2005).

Protect structure of cell membrane.

Prevent aggregation of proteins due to water stress.

Protect enzymatic activities (Reyes et al., 2005), prevent misfolding and denaturation of important proteins.

LEA (late embryogenesis abundant) proteins were first characterized in cotton and wheat.

Produced in abundance during seed development, comprising up to 4% of cellular proteins.

Seven different groups of LEA proteins have been defined on the basis of expression pattern and sequence; the major categories are group 1, group 2 and group 3.

Group 3 LEA proteins play a major role in cellular dehydration (replacement water).

Dehydrins Dehydrins are a subgroup (group 2) of Late Embryogenesis Abundant

(LEA) protein.

Accumulate late in embryogenesis and in all vegetative tissue during normal growth conditions and in response to stress leading to cell dehydration.

Structural and Biochemical Studies indicate that dehydrins are intrinsically disordered proteins (IDPs), i.e. in their functional state they are devoid of single and stable tertiary structure (Tompa, 2009).

Dehyrins interact with membranes in the interior of cells and reduce dehyration induced damage.

They increase water-binding capacity by creating a protective environment for other proteins or structure.

Overproduction of a wheat dehydrin (DHN5) in Arabidposis enhanced the tolerance to osmotic stress.

When compared to wild type plants, dehydrin-5 transgenic plants exhibited stronger growth under water deprivation and more rapid recovery (Brini et al., 2007).

Accumulation of

was identified in plant tissues upon exposure to stress.

acts as a signaling molecule in higher plants under stress

Physiological role in drought tolerance osmotic regulation, detoxication of reactive oxygen radicals and intracellular signal transduction (Kinnersley and Turano, 2000).

Drought stress initiates a signal transduction pathway in which increased cytosolic Ca+2 activates Ca+2 /calmodulin-dependent gluatamte decarboxylase activity, leading to gamma-aminobutyric acid synthesis.

generally decreases during drought stress which has been shown to be associated with inhibition of lipid biosynthesis and stimulation of lipolytic processes.

non-protein amino acid gamma-aminobutyric acid

Membrane lipid content

Reactive Oxygen Species (ROS) Consequences of drought stress is the ROS production in the different

cellular compartments, in the chloroplasts, the peroxisomes and the mitochondria.

There are basically four forms of cellular ROS- singlet oxygen (1O2), superoxide radical (O2-) hydrogen peroxide (H2O2)

and the hydroxyl radical (HO·), each with a characteristic half‑life and an oxidizing potential.

ROS can be extremely reactive, especially singlet oxygen and the hydroxyl radical, can oxidize multiple cellular components like proteins, lipids, DNA, RNA.

Photorespiration generate H2O2 (in peroxisome).

The first plant organ to detect a limitation on the water supply is the root system

Roots send signals to the leaves through the xylem sap

abscisic acid (ABA) is to be one of the major root‑to‑shoot stress signals

stress signal reaches the leaves it triggers stomatal closure (plant shifts to a water‑saving strategy)

By adjusting stomatal opening, plants are able to control water loss by reducing the transpiration flux

but they are concomitantly limiting the entrance of carbon dioxide (CO2)

direct and indirect effects on the reduction of net photosynthesis and on the overall production of ROS by plants under drought stress.

(Drought stress and reactive oxygen species, 2008)

The chloroplast is a quite robust cellular compartment towards ROS because of the different scavenging enzymes and metabolites present.

But under drought stress threats towards the chloroplast is the production of the hydroxyl radical in the thylakoids through “iron‑catalysed” reduction of H2O2 by SOD (superoxide dismutase).

Hydroxyl radical has the shortest half‑life but has an extremely strong oxidizing potential reacting with almost every biological molecule.

There is no enzymatic reaction known to eliminate the hydroxyl radical

its accumulation leads to deleterious reactions which damage the thylakoidal membranes and the photosynthetic apparatus.

Photorespiration shifts H2O2 production to peroxisomes (hydroxyl radical formation occurs) rather than chloroplasts.

Plants keep ROS under control by an efficient and versatile scavenging System.

SOD is the front‑line enzyme in ROS attack, it rapidly scavenges superoxide

active oxygen species is rapidly dismutated by a membrane bound superoxide dismutase (SOD)

producing H2O2 (thiol inhibitor) and oxygen

H2 O2 is then locally converted to water by ascorbate peroxidase (APX).

(Drought stress and reactive oxygen species, 2008)

Engineering drought tolerance in plants: discovering andtailoring genes to unlock the future

Umezawa et al. 2006, Plant Biotechnology (Science direct)

Transcriptome analyses have revealed that dozens of transcription factors

(TFs) are involved in the plant response to drought stress (Altman et al., 2005).

Most of these TFs fall into several large TF families, such as AP2(APETALA 2)/ ERF (ethylene-response factor), bZIP, NAC, MYB, MYC, Cys2His2 zinc-finger and WRKY.

The expression of TFs regulates the expression of downstream target genes that are involved in the drought stress response and tolerance.

Transgenic plants expressing a drought-responsive AP2, SHN1-3 or WXP1, induced several wax-related genes and resulted in enhanced cuticular wax accumulation and increased drought tolerance (Zhang et al., 2005).

Transcription and Signaling Factors

Strategies to produce active forms of transcriptional activators for

engineering drought tolerance:

deletion of the region containing a transcriptional inhibitory region Example: deletion of PEST sequence from DREB2A , which is

generally known to play a role in the degradation of the protein.

point mutation Example: mimicking the phosphorylation of a rice bZIP (basic leucine-

zipper protein) transcription factor, TRAB1, from serine to aspartic acid at a phosphorylation site, increased the level of transcriptional activation.

Engineering drought tolerance using signaling factors

Upstream of TFs, various signal transduction systems function in abiotic stress responses, involving protein phosphorylation and/or dephosphorylation, phospholipid metabolism, calcium sensing, protein degradation and so on (Boudsocq et al., 2005).

Several genes encoding signaling factors function in the drought response.

Mitogen-activated protein kinase (MAPK)- important mediators in signal transmission, connecting the perception of external stimuli to cellular responses.

A tobacco mitogen-activated protein kinase (MAPK), NPK1, which was truncated for constitutive activation, activated an oxidative signal cascade and led to cold, heat, salinity and drought tolerance in transgenic plants (Shou et al., 2004).

In the case of farnesyltransferases, suppression of signaling factors can also effectively enhance tolerance to abiotic stress.

In the regulation of stomatal closure by ABA, a β-subunit of farnesyltransferase ERA1 (enhanced response to abscisic acid) functions as a negative regulator of ABA signaling.

Antisense downregulation of the α or β subunits of farnesyltransferase enhances response to ABA and drought tolerance of plants.

The era1-2 mutation deletes only Ftase β-subunit gene present in Arabidopsis and affects a variety of signal transduction and developmental processes (Ziegelhoffer et al., 2000).

(Hypersensitivity of Abscisic Acid–Induced Cytosolic Calcium Increases in the Arabidopsis Farnesyltransferase Mutant era1-2, Allen et al., 2002)

Enhanced ABA sensitivity in era1-2 guard cells leads to reduced rates of water loss from era1-2 plants compared with wild-type plants under drought stress (Pei et al., 1998).

Increases in cytosolic calcium concentration ([Ca2]cyt) in guard cells have been shown to be early events in the signaling cascade that results in ABA-induced stomatal closure in a number of plant species (Allen et al., 2001).

Increase of cytosolic calcium showed that the activation of S-type anion currents downstream of cytosolic calcium and extracellular calcium-induced stomatal closure were unaffected in era1-2

supporting the positioning of era1-2 upstream of cytosolic calcium in the guard cell ABA signaling cascade.

Role of DREBs in regulation of abiotic stress responses in plantsCharu Lata and Manoj Prasad (NIPGR)Journal of Experimental Botany, 2011

Large-scale transcriptome analysis has revealed that gene products can broadly be classified into two groups (Bohnert et al., 2001).

One group constitutes genes that encode proteins to protect the cells from the effects of water stress, eg, osmotin, LEA etc.

A second group of genes activated by abiotic stresses comprises regulatory proteins that further regulate stress signal transduction and modulate gene expression and function in the stress response.

They include various transcription factors (TFs) such as myelocytomatosis oncogene (MYC), myeloblastosis oncogene (MYB), basic leucine zipper (bZIP), dehydration responsive element binding (DREB).

The dehydration responsive element (DRE) with a 9 bp conserved core sequence (5’-TACCGACAT-3’) was first identified in the promoter of the drought-responsive gene rd29A (Yamaguchi- Shinozaki,1993).

DREs have been reported to be involved in various abiotic stress responses through both ABA-dependent and ABA-independent pathways (Dubouzet et al., 2003).

The DREB proteins namely, DREB1 and DREB2, involved in two separate signal transduction pathways under low temperature and dehydration, respectively, are important ethylene responsive factor (ERF) plant TFs that induce a set of abiotic stress-related genes.

The DREB TFs contain a highly conserved AP2/ERF DNA-binding domain across the plant kingdom including Arabidopsis, rice, soybean, chickpea, tomato, tobacco, and millets (Lata et al., 2011).

DREB TFs Species Stress response References

DREB2A Arabidopsis thaliana

Drought, Salt, ABA Liu et al., 1998

CBF4 Arabidopsis thaliana

Drought, ABA Haake et al., 2002

OsDREB1C Oryza sativa Drought, Salt, Cold, ABA, Wound

Dubouzet et al., 2003

OsDREB2A Oryza sativa Drought, Salt, faintly to Cold, ABA

Dubouzet et al., 2003

HvDRF1 Hordeum vulgare Drought, Salt, ABA Xue and Loveridge, 2004

GmDREBa Glycine max Cold, Drought, Salt Li et al., 2005

PeDREB2 Populus euphratica Drought, Salt, Cold Chen et al., 2009

SbDREB2A Salicornia brachiata Drought, Salt, Heat Gupta et al., 2010

Response of different DREB genes to drought stresses.

Heat stress

Heat stress is often defined as the rise in temperature beyond a threshold level for a period of time sufficient to cause irreversible damage to plant growth and development.

Heat stress due to high ambient temperature is a serious threat to crop production worldwide (Hall, 2001).

At very high temperatures, severe cellular injury and even cell death may occur within minutes (Schoffl et al., 1999).

At moderately high temperatures:

direct injuries include protein degradation, aggregation and increased fluidity of membrane lipids.

Indirect injuries include inactivation of enzymes, inhibition of protein synthesis, protein degradation and loss of membrane integrity (Howrath, 2005).

Heat-stress threshold

It is a value of daily mean temperature at which a detectable reduction in growth begins / the temperature at which growth and development of plant cease.

Upper threshold: is the temperature above which growth and development cease.

Lower threshold (base temperature): is the temperature below which plant growth and development stop.

Threshold high temperatures for some crops plants

Crop plants Threshold temperature (0C)

Growth stage References

Cotton 45 Reproductive Rehman et al., (2004)

Rice 34 Grain yield Morita et al., (2004)

Cow Pea 41 Flowering Patel and Hall (1990)

Pearl millet 35 Seedling Seedling

Corn 38 Grain filling Thompson (1986)

Wheat 26 Post -anthesis Stone and Nicolas (1994)

Brassica 29 Flowering Morrison Stewart (2002)

Heat stress is a complex function of: Intensity (temperature in degrees) Duration Rate of increase in temperature.

Morpho-anatomical and phenological responses Morphological symptoms: -Sunburns on leaves branches and stems -Leaf senescence and abscission -Shoot and root growth inhibition -Fruit discoloration and damage and reduced yield

Anatomical changes: -Reduced cell size -Closure of stomata and curtailed water loss -Increased stomatal density -Damaged to mesophyll cells and increased permeability of plasma

membrane -High temperatures reduced photosynthesis by changing the structural

organization of thylakoids (Karim et al., 1997).

Plant response to heat

Water relations Heat stress perturbed the leaf water relations and root hydraulic

conductivity (Morales et al., 2003).

Enhanced transpiration induces water deficiency in plants, causing a decrease in water potential and leading to perturbation of many physiological processes (Tsukaguchi et al., 2003).

Photochemical reactions in thylakoid lamellae and carbon metabolism in the stroma of chloroplast have been suggested as the primary sites of injury at high temperatures (Wise et al., 2004).

Increasing leaf temperatures and photosynthetic photon flux density influence thermotolerance adjustments of PSII.

High temperature alters the energy distribution and changes the activities of carbon metabolism enzymes, particularly the rubisco, thereby altering the rate of RuBP regeneration by the disruption of electron transport and inactivation of the oxygen evolving enzymes of PSII (Salvucci and Crafts-Brandner, 2004).

Physiological responses

Accumulation of compatible osmolytes

A variety of osmolytes such as sugars and sugar alcohols (polyols), proline, tertiary and quaternary ammonium compounds are accumulated.

Glycinebetaine (GB) plays an important role as a compatible solute in plants under various stresses, such as salinity or high temperature.

Proline is also accumulated in large quantities in response to environmental stresses.

Secondary metabolites

High-temperature stress induces production of phenolic compounds such as flavonoids and phenylpropanoids.

Phenylalanine ammonia-lyase (PAL) is considered to be the principal enzyme of the phenylpropanoid pathway.

Increased activity of PAL in response to thermal stress is considered as the main acclimatory response of cells to heat stress.

Thermal stress induces the biosynthesis of phenolics and suppresses their oxidation, which is considered to trigger the acclimation to heat stress (Rivero et al., 2001).

Carotenoids are widely known to protect cellular structures in various plant species irrespective of the stress type (Wahid and Ghazanfar, 2006; Wahid, 2007).

Cell membrane thermostability

Changes in membrane fluidity can be the result of single or combined effects of the degree of saturation and composition of lipids in the membrane.

Lipid saturation level typically increases whereas unsaturated lipids decrease with increasing temperatures.

Palmitic (saturated fatty acid)- enhances heat tolerance.

The integrity and functions of biological membranes are sensitive to high

temperature Heat stress

Alters the tertiary and quaternary structures of membrane proteins

Enhance the permeability of membranes

increased loss of electrolytes

The increased solute leakage, as an indication of decreased cell membrane thermostability (CMT), has long been used as an indirect measure of heat-

stress tolerance in diverse plant species

The increase in levels of saturated fatty acid have been found in different cellular membrane including

more sensitive to heat stress than changes in the plasma membrane

Increase in saturation increases their melting temperature

this retard the membrane fluidity at higher temperature

Alteration of membrane fluidity has been found to affect plant tolerance to heat stress.

Example: Mutants of Soybean and Arabidopsis that are deficient in fatty acid unsaturation maintained stable membrane fluidity and showed improved tolerance to high temperature.

thylakoid membrane Plasma membrane

Molecular responses

Heat stress may induce oxidative stress: Generation and reactions of activated oxygen species (AOS) singlet

oxygen (1O2), super oxide radical (O2

.-), hydrogen peroxide (H2O2) hydroxyl radical (OH-)

AOS cause -- autocatalytic peroxidation of membrane lipids and pigments leading to the loss of membrane semi- permeability and modifying its function.

The scavenging of O2.- by superoxide dismutase (SOD) results in the

production of H2O2, which is removed by APX (ascorbate peroidase) or CAT (catalase).

Protection against oxidative stress is an important component in determining the survival of a plant under heat stress.

Heat-stress tolerance mechanisms in plants

Proposed heat-stress tolerance mechanisms in plants. MAPK, mitogen activated protein kinases; ROS, reactive oxygen species; HAMK, heat shock activated MAPK; HSE, heat shock element; HSPs, heat shock proteins; CDPK, calcium dependent protein kinase; HSK, histidine kinase. Partly adopted from Sung et al. (2003).

The first response to heat stress such as plasma membrane fluidity disruption and osmotic changes

triggers downstream signaling and transcriptional cascade activating a

stress responsive pathway

leading to reestablishment of cellular homeostasis and separation of damaged proteins and membranes.

Inadequate or delayed response in any step may lead to cell death (Vinocur and Altman, 2005; Bohmert et al., 2006).

Initial effects are on the plasma membrane which becomes more fluid under stress triggering calcium influx and cytoskeletal reorganization, resulting in the upregulation of some mitogen activated and calcium dependent kinases.

Signaling of these cascades at nuclear level leads to antioxidant production and compatible solute accumulation.

Membrane fluidity changes also lead to ROS generation in organelles and signaling in cytoplasm.

Thermotolerance acquirement is correlated with the activities of CAT and SOD, higher ascorbic acid control and less oxidative damage (Tyagi, 2004).

Induction of HSP and other proteins such as LEA and dehydrins interact with other stress response mechanism such as production of osmolytes and anti-oxidants.

HSPs are involved in stress signal transduction and gene activation as well as in the regulation of cellular redox state.

Role of plant heat-shock proteins and molecular chaperones in the abiotic stress responseWang et al., 2004

TRENDS in Plant Science

Heat shock proteins belong to a larger group of molecules called chaperons.

Heat-shock proteins (Hsps)/chaperones are responsible for protein folding, assembly, translocation and degradation in many normal cellular processes, stabilize proteins and membranes, and can assist in protein refolding under stress conditions.

They can play a crucial role in protecting plants against stress by

reestablishing normal protein conformation and thus cellular homeostasis.

Heat shock proteins

Low molecular weight heat shock proteins are generally produced only in response to environmental stress, particularly high temperature (Wahid et al., 2007).

In higher plants, HSPs is usually induced under heat shock at any stage of development.

Expression of stress proteins is an important adaption to cope with environmental stresses.

Most of the stress proteins are soluble in water and therefore contribute to stress tolerance presumably via hydration of cellular structures.

Five major families of Hsps/chaperones are conservatively recognized are the- Hsp70 (DnaK) family; the chaperonins (Hsp60); the Hsp90 family; the Hsp100 family; and the small Hsp (sHsp) family.

The diagram shows the role of heat-shock proteins and a chaperonin in protein folding.  As the ribosome moves along the molecule of messenger RNA, a chain of amino acids is built up to form a new protein molecule.  The chain is protected against unwanted interactions with other cytoplasmic molecules by heat-shock proteins and a chaperonin molecule until it has successfully completed its folding.

List of heat shock proteins

Protein class

Size (kDa)

Location

HSP100 100-114 cytoplasm

HSP90 80-94 cytoplasm, ER

HSP70 69-71 Cytoplasm, ER, mitochondria

HSP60 10-60 Chloroplast, mitochondria

smHSP 15-30 Cytoplasm, ER, mitochondria, Chloroplast

Cooperation in the chaperone network exemplified for the role of Hsp70 chaperone machines in the cytoplasm, ER and organelles for protein import. Nascent protein chains emerging from the ribosomes are bound by cytosolic forms of Hsc/Hsp70 (orange). Depending on the nature of the N-terminal signal sequence, proteins are imported post-translationally into the chloroplasts or mitochondria. In both cases a protein import chanel is formed of an translocase outer membrane (TOC/TOM) and inner membrane (TIC/TIM) multiprotein complex. Partially unfolded proteins are delivered to the pore complex and imported in an ATP consuming process involving the organellar Hsc70. (Baniwal et al., 2004)

functions- preventing aggregation, assisting refolding, protein import and translocation, signal transduction, and transcriptional activation.

- Some family members of Hsp70 are constitutively expressed and are often referred to as Hsc70 (70-kDa heat-shock cognate).

functions- folding and assisting refolding. -Chaperonins play a crucial role by assisting a wide range of newly

synthesized and newly translocated proteins to achieve their native forms.

functions- Facilitating maturation of signaling, molecule, genetic buffering.

HSP70 family

HSP60 family

HSP90 family

-plays a key role in signal-transduction networks, cell-cycle control, protein degradation and protein trafficking.

-Hsp90 is one of the major species of molecular chaperones that requires ATP for its functions.

-It is among the most abundant proteins in cells: 1–2% of total cellular protein.

-AlthoughHsp90 chaperones are constitutively expressed in most organisms, their expression increases in response to stress.

-It was suggested in this work that Hsp90 acts as a ‘buffer’ to sustain the functions of those mutated proteins that participate in the signaling pathways of development and morphogenesis.

-Under normal physiological conditions, the expression of genetic variations that are hidden by the Hsp90 ‘buffering’ effect is suppressed or silenced.

functions- preventing aggregation, stabilizing non- native proteins.

-The sHsps are low-molecular-mass Hsps (12–40 kDa).

- In plants, sHsps form a more diverse family than other Hsps/chaperones with respect to sequence similarity, cellular location and functions.

-Among the five conserved families of Hsps (Hsp70, Hsp60, Hsp90, Hsp100 and sHsp), sHsps are the most prevalent in plants.

sHSP family

-The removal of non-functional but potentially harmful polypeptides arising from misfolding, denaturation or aggregation is important for the maintenance of cellular homeostasis.

-have been reported in many plant species, such as Arabidopsis, soybean, tobacco, rice, maize (Zea mays), etc..

functions- disaggregation, unfolding.HSP100 family

Hsp/chaperone families [e.g. small Hsp (sHsp) and Hsp70] stabilize protein conformation, prevent aggregation and thereby maintain the non-native protein in a competent state

for subsequent refolding, which is achieved by other Hsps/chaperones (e.g. Hsp60, Hsp70 and Hsp90).

When denatured or misfolded proteins form aggregates, they can be resolubilized by Hsp100 followed by refolding, or degraded by protease.

Some Hsps/chaperones (e.g. Hsp70, Hsp90) accompany the signal transduction and transcription activation that lead to the synthesis of other members of Hsps/chaperones.

Hsp/chaperone network in abiotic stress response

Different classes of Hsps/chaperones play complementary and sometimes overlapping roles in protecting proteins from stress.

Hsp synthesis is tightly regulated at the transcriptional level by heat shock factors (HSFs).

HSF-1 main regulator of the short-term induction of Hsp.

The basic modular structure of Hsfs includes a highly conserved DNA-binding domain (DBD), oligomerization domain (OD), nuclear localization signal (NLS), and the least conserved C-terminal activation domain (CTAD).

The transcription activating functions of is related to short peptide motifs with in the CTADs.

during stress converted from a monomeric to trimeric form

The heat responsive elements are always present in the promoter region (TATA box proximal 5’ flanking regions )of heat shock genes.

The HSE is the binding target and the recognition site for trans-active Hsfs.

Hsfs

Other heat induced proteins

Besides HSPs, there are a number of other plant proteins, including ubiquitin, cytosolic SOD and Mn-Peroxidase.

Heat stability is a notable feature of LEA proteins, i.e. they do not coagulate upon boiling (Thomashow,1999).

Group 1 LEA proteins from wheat prevent aggregation and protect the citrate synthase from desiccating conditions like heat and drought-stress (Goyal et al., 2005).

Exhibit protective effect in presence of trehalose and acts synergistically to prevent heat-induced protein aggregation.

Another common characteristic of LEA-type proteins is that, in most cases, their related gene expression is transcriptionally regulated and responsive to ABA (Leung and Giraudat, 1998).

Three low-molecular-weight dehydrins have been identified in sugarcane leaves with increased expression in response to heat stress (Wahid and Close, 2007).

Hormonal changes

Abscisic acid (ABA) and ethylene (C2H4), as stress hormones, are involved in the regulation of many physiological properties by acting as signal molecules.

ABA induction is an important component of thermotolerance, suggesting its involvement in biochemical pathways essential for survival under heat-induced desiccation stress (Maestri et al., 2002).

A gaseous hormone, ethylene regulates almost all growth and developmental processes in plants, ranging from seed germination to flowering and fruiting as well as tolerance to environmental stresses.

Salicylic acid (SA) is also involved in heat-stress responses elicited by plants. SA is an important component of signaling pathways in response to systemic acquired resistance (SAR) and the hypersensitive response (HR) (Kawano et al., 1998).

Impact of high-temperature stress on rice plant and its traits related to toleranceShah et al., 2011

Journal of Agricultural Science

The optimum temperature for the normal development of rice ranges from 27 to 32 °C (Yin et al., 1996).

The predicted 2–4 °C increment in temperature by the end of the 21st Century poses a threat to rice production (IPCC 2007), due to both anthropogenic and natural factors (Eitzinger et al., 2010).

Impact of high temperatures at night is more devastating than day-time or mean daily temperatures.

Flowering and booting are the stages most sensitive to high temperature, which may sometimes lead to complete sterility.

Yields of rice have been estimated to be reduced by 41% by the end of the 21st Century (Ceccarelli et al., 2010).

Pollination contributing factors (pollen production, viability and reception) play a dominant role in productivity of the crop.

Generally, male reproductive development in rice is known to be more sensitive to heat stress (Wassmann et al., 2009).

Prasad et al. (2006) reported that high-temperature stress during rice flowering led to decreased pollen production and pollen shed.

The probable reasons were the inhibition of swelling of pollen grains, poor release of pollen grains (Matsui et al., 2005), and thus fewer numbers of pollen grains were available to be intercepted by the stigma.

High temperature affects almost all the growth stages of rice, i.e. from emergence to ripening and harvesting.

Symptoms of heat stress in rice plants:Growthstage

ThresholdTemperature (°C)

Symptoms References

Seedling 35 Poor growth of the seedling

Yoshida (1978),Akman (2009)

Flowering 35 Floret sterility Satake & Yoshida (1978)

Grainformation

34 Yieldreduction

Morita et al.(2004)

Grainripening

29 Reducedgrain filling

Yoshida(1981)

Anthesis 33.7 Poor antherdehiscenceand sterility

Jagadishet al. (2007)

Role of HSPs in inducing thermo-tolerance

The rapid accumulation of HSPs in the sensitive organs can play an important role in the protection of the metabolic apparatus of the cell, thereby acting as a key factor for plants’ adaptation to, and survival under, heat stress (Wahid et al., 2007).

Considering the crucial role of HSP in imparting thermo-tolerance, Katiyar-Agarwal et al. (2003) developed transgenic rice by the introduction of HSP101 from Arabidopsis thaliana cDNA into the indica rice variety Pusa Basmati 1 via Agrobacterium-mediated transformation.

this transgenic rice showed normal growth and development, and also performed better in the recovery after heat stress when compared with the untransformed rice.

The over-expression of rice OsHSF7 gene in A. thaliana has very been reported to increase the thermo-tolerance by increasing the proportion of plants surviving 42 °C for 16 h, from 0·22 to 0·52 (Liu et al., 2009).

Factors other than HSPs contributing to thermo-tolerance

Disruption of some plant growth hormones such as ethylene, salicylic acid, abscisic acid, calcium and hydrogen peroxide through mutation affected the thermo-tolerance capability of the plants (Larkindale et al., 2005).

When applied exogenously, these chemicals can enhance thermotolerance without an accompanying accumulation of HSPs (Larkindale & Knight, 2002).

This is mainly through increased antioxidant capacity and membrane thermal stability which can reduce the extent of damage caused by ROS (Mohammed & Tarpley, 2009).

Plant architecture can play an important role in high temperature stress tolerance.

Developing plant varieties with appropriate architecture will help to cope with the increase in temperature.

For example, if the plant morphology is such that the panicle is surrounded by many leaves, the plant will be able to withstand high-temperature stress due to increased transpirational cooling and by preventing evaporation from the anther due to its shading by the leaves.

Length of anther

It has been suggested that cultivars with large anthers (having large number of pollen grains) are tolerant of high temperature at the flowering stage (Matsui & Omasa, 2002).

Mitigating strategies for the forthcoming warmer climate

The availability of high-density genetic and physical maps, expressed sequence tags (ESTs), genomic sequences and mutant stocks such as T-DNA insertional mutants (Jeon et al., 2000) have established for the study of heat tolerance among cereals.

Progress in rice breeding has rapidly accelerated due to the availability of the full rice genome sequence (IRGSP 2005) and intensive QTLs mapping efforts for a wide range of traits (Ismail et al., 2007).

And the high degree of homology within the Poaceae family facilitate transfer of identified QTLs and candidate genes from rice to other cereals (Maestri et al., 2002).

rice as an excellent model plant

Thank you…!!!!!