Abiotic stress in plants

1. Molecular basis of abiotic stress
in plants- Drought and Heat
Submitted by:
Kirti
Ph.D. (MBB)
Advances in Plant Molecular Biology (MBB 601)

2. 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

3. 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

4. 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)

5. Effect of stress in plants

6. 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%.

7. 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

8. 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

9. 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)

10. 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.

11. 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.

12.  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.
Phytohormone
s

13.  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’

14.  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).

15.  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).

16. Molecular control of abiotic
stress
DREB, MAPK,
Phospholipases
Signaling
cascades & TFs
Water & ion
transport
Functional
proteins
HSP, Chaperones,
LEA, Dehydrins, ROS
scavenger, OA etc..
Ion transporters,
Aquaporins,

17. Molecular Response
Perception by
specific receptor
Initiates/ inhibits
cascade response
Information
transmittedGene expression

18. 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.

19.  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).

20. 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.

21.  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.

22.  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 reductase
L-Glutamate L-Δ1-Pyrroline-5-carboxylate L-Proline
NADH + H+ NAD + 2H2O NADH NAD

23. 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 and
Sawhney, 1990)
Specified protein synthesized under water scarcity/Changes in plant metabolics (Proteins):

24. 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.

25.  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).

27. 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.

28.  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).

29. 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

30. 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).

31. 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)

32. 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.

33.  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)

34. Engineering drought tolerance in plants: discovering and
tailoring 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

35.  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.

36. 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).

37.  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)

38. 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.

39. Role of DREBs in regulation of abiotic stress responses in plants
Charu 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).

40.  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).

41. 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.

42. 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).

43. 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.

44. 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)

45. 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

46. 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

47. 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.

48.  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).

49. 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

50.  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

51. 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.

52. 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).

53.  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.

54.  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.

55. Role of plant heat-shock proteins and molecular chaperones in the abiotic
stress response
Wang 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

57.  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.

58. 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.

59. 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

60. 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)

61. 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

62. -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.

63. 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

64. 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.

65. 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

66. 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.

67.  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).

68. 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).

69. Impact of high-temperature stress on rice plant and its traits related to
tolerance
Shah 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.

70.  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.

71.  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:
Growth
stage
Threshold
Temperature (°C)
Symptoms References
Seedling 35 Poor growth of
the seedling
Yoshida (1978),
Akman (2009)
Flowering 35 Floret sterility Satake &
Yoshida (1978)
Grain
formation
34 Yield
reduction
Morita et al.
(2004)
Grain
ripening
29 Reduced
grain filling
Yoshida
(1981)
Anthesis 33.7 Poor anther
dehiscence
and sterility
Jagadish
et al. (2007)

72. 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).

74. 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).

75. 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).

76. 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

77. Thank you…!!!!!