MOLECULAR BIOLOGY OF ABIOTIC STRESSES LIKE DROUGHT, SALINITY, HEAT AND LOW TEMPERATURE AND THEIR EFFECT ON THE PLANT GROWTH

1. ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY
S.V. AGRICULTURAL COLLEGE, TIRUPATI.
Course No : MBB – 601
Course Title: Advances in Plant Molecular Biology
Topic : Molecular Biology of Abiotic stresses
Submitted to
Dr.V.L.N. Reddy,
Associate Professorand Head,
Department of MolecularBiologyand Biotechnology
Submitted by
A. Govardhani,
TAD/2020-028
Department of MolecularBiologyand Biotechnology

2. Introduction:
Necessity to study the molecular biology of the plants:
As human populations grow, agricultural systems must feed more people while competing with urban
development for premium arable land. This increasing demand, coupled with shrinking resources, has fueled
research into elucidating mechanisms by which plants respond to stress and manipulating these mechanisms to
enhance plant productivity in suboptimal environments.
Molecular Biology of salinity stress tolerance in plants
Contents under salt salinity stress
• Introduction
• Adverse Effect of Salinity Stress
• Generic Pathway for Plant Response to Stress
• Ion Pumps, Calcium, and SOS Pathways in Relation to Salinity Stress
• Abscisic Acid and Transcription Factors in Salinity Stress Tolerance
• Mitogen-Activated Protein Kinases and Salinity Stress
• Glycine Betaine and Proline in Salinity Stress
• Reactive Oxygen Species in Salinity Stress
• DEAD-Box Helicases in Salinity Stress Tolerance
Introduction to stress:
Plants are frequently exposed to environmental stress, that adversely affect
growth, development, or productivity. Of the environmental stress salinity,
drought, heat, low temperature, flooding and oxidative stresses are major stresses.
When plant exposed to stresses, many factors determine how plants respond to
environmental stress: the genotype and developmental conditions of the plant, the
duration and severity of the stress, the number of times the plant is subjected to
stress, and any additive or synergistic effects of multiple stressors. Plants respond
to stress through a variety of mechanisms. Failure to compensate for severe stress
can result in plant death
Salinity stress:
 Salinity is a major stress limiting the increase in the demand for food crops. More than 20% of cultivated
land worldwide (∼ about 45 hectares) is affected by salt stress and the amount is increasing day by day.
 Furthermore, there is a deterioration of about 2 million ha (1%) of world agricultural lands because of
salinity each year
 Salinity stress is also considered as hyperosmotic / hyperionic stress
During the initial phases of salinity stress, water absorption capacity of root systems decreases and water
loss from leaves is accelerated due to osmotic stress of high salt accumulation in soil and plants, and therefore
salinity stress is also considered as hyperosmotic stress.
Salinity stress is also considered as a hyperionic stress. One of the most detrimental effects of salinity stress is
the accumulation of Na+
and Cl-
ions in tissues of plants exposed to soils with high NaCl concentrations. Entry of
both Na+
and Cl-
into the cells causes severe ion imbalance and excess uptake might cause significant
physiological disorder(s). High Na+
concentration inhibits uptake of K+
ions which is an essential element for
growth and development that results into lower productivity and may even lead to death.
• Causes of salinity stress:poor water management practices,high evaporation and previous exposure to
seawater.

3. Based On Adaptive Mechanism to salinity stress:
Many crop species are very sensitive to soil salinity and are known as glycophytes, whereas salt-tolerant plants
are known as halophytes. In general, glycophytes cannot grow at 100 mM NaCl, whereas halophytes 420
Narendra Tutejacan grow at salinities over 250 mM NaCl. Salinity-sensitive plants restrict the uptake of salt and
strive to maintain an osmotic equilibrium by the synthesis of compatible solutes such as prolines, glycine betaine
(GB), and sugars. Salinity-tolerant plants have the capacity to sequester and accumulate salt into the cell
vacuoles, thus preventing the buildup of salt in the cytosol and maintaining a high cytosolic Kþ/Naþ ratio in their
cells.
Adverse Effect ofSalinity Stress:
• High salinity interferes with plant growth and development and can also lead to physiological drought
conditions and ion toxicity
• Decreasesthe water absorption capacity of root systems
• water loss from leaves is accelerated due to osmotic stress
• Cell division and expansion, membrane disorganization, and osmotic imbalance
• reduction in photosynthesis
• Production of reactive oxygen species
• High Na+
concentration inhibits uptake of K+
ions
• Alterations in K+
ions can disturb the osmotic balance, the function of stomata, and the function of some
enzymes.
Generic Pathway for Plant Response to Stress:
A generic stress signal transduction pathway for the plant stress
response is depicted in Fig. The stress signal is first perceived at the
membrane level by the receptors (G-protein-coupled receptors, ion
channel, receptorlike kinase, or histidine kinase), which results in the
generation of many secondary signal molecules, such as Ca2þ, inositol
phosphates, ROS, and abscisic acid ABA. The stress signal then
transduces inside the nucleus to induce many stress responsive genes, the
products of which ultimately lead to plant adaptation to stress tolerance.
The stress responsive genes could be either early or delayed induced
genes. Early genes are induced within minutes of stress perception, often
express transiently, and their products (e.g., various transcription factors)
can activate the expression of delayed genes (e.g., RD [responsive to
dehydration], KIN [cold induced], COR [cold responsive]). Overall,
gene products are either involved directly in cellular protection against
the stress (e.g., late embryogenesis abundant proteins, antifreeze
proteins, antioxidant, chaperons, and detoxification enzymes) or
involved indirectly in protection (e.g., transcription factor, enzyme of PI
metabolism).

4. Ion Pumps, Calcium, and SOS Pathways in Relation to Salinity:
High salinity stress causes an imbalance in sodium ions (Na+
) homeostasis, which is maintained by the
coordinated action of various pumps, ions, Ca2+
sensors, and its downstream interacting partners, which
ultimately results in the efflux of excess Na+
ions. The roles of various ion pumps/ channels are depicted in Fig.
2. Certain channels show more selectivity to K+
over Na+
. These include the K inward-rectifying channel, which
mediates the influx of K+
upon plasma membrane hyperpolarization and selectively accumulates K+
over Na+
ions. The histidine kinase transporter (HKT) is a low-affinity Na+
ion transporter, which blocks the entry of Na+
ions into the cytosol. The nonspecific cation channel (NSCC) is a voltage-independent channel, which acts as a
gate for the entry of Na+
into plant cells. Moreover, there is the K+
outward-rectifying channel, which opens
during the depolarization of the plasma membrane and mediates the efflux of K+
and the influx of Na+
ions,
leading to Na+
accumulation in the cytosol. The vacuolar Na+
/H+
exchanger (NHX) helps push excess Na+
ions
into vacuoles. Na+
extrusion from plant cells is powered by the electrochemical gradient generated by H+
-
ATPases, which permit the NHX to couple the passive movement of H+
inside along the electrochemical
gradient and extrusion of Na+
out of the cytosol. Another pump, the H+
/Ca2+
antiporter (CAX1), helps in Ca2+
homeostasis
Regulation of ion homeostasis by various ion pumps. The salinity stress signal is perceived by a receptor
or salt sensor present at the plasma membrane of the cell.This signal is responsible for activating various ion
pumps present at plasma and vacuolar membranes.This signal also activates the SOS pathway, the components
of which help in regulating some of these pumps. The various pumps/channels are the K+
inward-rectifying
channel (KIRC), histidine kinase transporter (HKT), nonspecific cation channels (NSCC), K+
outward-rectifying
channel (KORC),Na+
/H+
antiporters (SOS1), vacuolar Na+
/H+
exchanger (NHX), and H+
/Ca+
antiporter (CAX1).
Na+
extrusion from plant cells is powered by the electrochemical gradient generated by H+
-ATPases, which
permits the Na+
/H+
antiporters to couple the passive movement of H+
inside along the electrochemical gradient
and extrusion of Na+
out of the cytosol. The stress signal sensed by SOS3 activates SOS2, which activates SOS1.
Regulation of ion (e.g., Na+
, K+
, and Ca2+
) homeostasis by SOS and related pathways in relation to
salinity stress tolerance. High salinity (Na+
) stress initiates a calcium signal that activates the SOS pathway.The
signal first activates phospholipase C (PLC), which hydrolyses phosphatidylinositol bisphosphate (PIP2) to
generate inositol trisphosphate (IP3), and diacylglycerol (DAG) resulting in an increased level of Ca2+
ions. This
change in cytosolic Ca2+
ions is sensed by a calcium sensor such as SOS3, which interacts with the SOS2 protein
kinase.This SOS3^SOS2 protein kinase complex phosphorylates SOS1, a Naþ/Hþ antiporter, resulting in an
efflux of excess Naþ ions. The SOS3-SOS2 complex interacts with and influences other salt-mediated pathways,
resulting in ionic homeostasis.This complex inhibits HKT1 activity (a low-affinity Na+
transporter), thus
restricting Na+
entry into the cytosol. SOS2 also interacts and activates the vacuolar Na+
/H+
exchanger (NHX),
resulting in the sequestration of excess Na+
ions, further contributing to Na+
ion homeostasis. Calnexin and
calmodulin (CaM) or other calcium-binding proteins can also interact and activate the NHX or other

5. transporters.The H+
/Ca2+
antiporter (CAX1) has been identified as an additional target for SOS2 activity
reinstating cytosolic Ca2+
homeostasis
SOS pathway:
The SOS signalling pathway consists of three major proteins, SOS1, SOS2, and SOS3. SOS1, which encodes a
plasma membrane Na+
/H+
antiporter, is essential in regulating Na+
efflux at cellular level. It also facilitates long
distance transport of Na+
from root to shoot. Overexpression of this protein confers salt tolerance in plants. SOS2
gene, which encodes a serine/threonine kinase, is activated by salt stress elicited Ca+
signals. This protein
consists of a well-developed N-terminal catalytic domain and a C-terminal regulatory domain. The third type of
protein involved in the SOS stress signalling pathway is the SOS3 protein which is a myristoylated Ca+
binding
protein and contains a myristoylation site at its N-terminus. This site plays an essential role in conferring salt
tolerance. C-terminal regulatory domain of SOS2 protein contains a FISL motif (also known as NAF domain),
which is about 21 amino acid long sequence, and serves as a site of interaction for Ca2+
binding SOS3 protein.
This interaction between SOS2 and SOS3 protein results in the activation of the kinase. The activated kinase
then phosphorylates SOS1 protein thereby increasing its transport activity. SOS1 protein is characterised by a
long cytosolic C-terminal tail, about 700 amino acids long, comprising a putative nucleotide binding motif and
an autoinhibitory domain. This autoinhibitory domain is the target site for SOS2 phosphorylation. Besides
conferring salt tolerance it also regulates pH homeostasis, membrane vesicle trafficking, and vacuole functions.
Thus with the increase in the concentration of Na+
there is a sharp increase in the intracellular Ca2+
level which
in turn facilitates its binding with SOS3 protein. Ca2+
modulates intracellular Na+
homeostasis along with SOS
proteins. The SOS3 protein then interacts and activates SOS2 protein by releasing its self-inhibition. The SOS3-
SOS2 complex is then loaded onto plasma membrane where it phosphorylates SOS1. The phosphorylated SOS1
results in the increased Na+ efflux, reducing Na+
toxicity.

6. Abscisic Acid and Transcription Factors in Salinity Stress Tolerance:
ABA biosynthesis pathway and its regulation by osmotic stress. ABA is synthesized from β-carotene via the
oxidative cleavage of neoxanthin and conversion of xanthoxin to ABA via ABA-aldehyde. Stresses, including
salinity stress, stimulate ABA biosynthesis and accumulation by activating genes involved in the ABA
biosynthetic pathway, which itself could be mediated by a calcium-dependent phosphorylation cascade. ABA
can also upregulate the expression of ABA biosynthetic genes via calcium signaling pathways. ZEP, zeaxanthin
epoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase; AAO, ABA-aldehyde oxidase; MCSU, molybdenum
cofactor sulfurase.
Transcriptional factors in response to salinity stress:
Transcriptional regulatory network of cis-acting elements and ABAdependent transcription factors involved in
salinity stress gene expression. Osmotic stress signaling generated via salinity stress seems to be mediated by
transcription factors such as DREB2A/DREB2B, AREB1, and MYC/MYB transcription activators, which
interact with DRE/CRT, ABRE, and MYCRS/MYBRS elements in the promotion of stress genes, respectively.

7. AtMYC2 and AtMYB2 act cooperatively to activate the expression of ABA-inducible genes such as
RD22.Transcription factor-binding sites are represented as rectangles at the bottom of the figure, with the
representative promoters.
Mitogen-Activated Protein Kinases and Salinity Stress:
Mitogen-activated protein kinases are a specific class of plant serine/ threonine protein kinases that play a central
role in the transduction of various extracellular and intracellular signals, including stress signals. These generally
function as a cascade where MAPK is phosphorylated and activated by MAPK kinase (MAPKK), which itself is
activated by MAPKK kinase (MAPKKK). All three of these kinases are interlinked together and are also called
extracellular receptor kinases.
Glycine Betaine and Proline in Salinity Stress:
Plants initiate some defensive machinery in order to cope with stress; one of them is associated with changes in
metabolites. Glycine betaine (N, N,N-trimethylglycine betaine) and proline are two major osmoprotectant
osmolytes, which are synthesized by many plants (but not all) in response to stress, including salinity stress, and
thereby help in maintaining the osmotic status of the cell to ameliorate the abiotic stress effect
Glycine betaine:
Glycine betaine (N,N,N‐ trimethylglycine, GB) is synthesized under various types of environmental stress and
functions as a major osmoprotectant. It stabilizes the quaternary structures of the PSII protein‐ pigment under
high salinity and maintains the ordered state of membranes at extreme temperatures. GB is synthesized mainly
from choline via betaine aldehyde (BA), with the first and second steps in the pathway catalyzed by choline
monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively.

8. Functions of Glycine betaine:
Glycine betaine is an amphoteric quaternary ammonium compound ubiquitously found in microorganisms,
higher plants and animals, and is electrically neutral over a wide range of pH. It is highly soluble in water but
also contains nonpolar moiety constituting 3-methyl groups. Because of its unique structural features it interacts
both with hydrophobic and hydrophilic domains of the macromolecules, such as enzymes and protein
complexes. Glycine betaine is a nontoxic cellular osmolyte that raises the osmolarity of the cell during stress
period; thus it plays an important function in stress mitigation. Glycine betaine also protects the cell by osmotic
adjustment, stabilizes proteins, and protects the photosynthetic apparatus from stress damages and reduction of
ROS
Proline:
Proline is synthesized by glutamic acid by the actions of two enzymes,
pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate
reductase (P5CR).
During seedling development and in stress conditions, proline accumulation
occurs through Ornithine pathway.
Uses: The amino acid proline accumulates (normally in cytosol) under stress
and is correlated with osmotic adjustment to improve plant salinity tolerance.
Proline also plays roles in scavenging free radicals, stabilizing subcellular
structures, and buffering cellular redox potential under stresses.
Reactive Oxygen Species in Salinity Stress:
Reactive oxygen intermediates (ROI) typically result from the excitation of O2 to form singlet oxygen (O2 1) or
the transfer of one, two, or three electrons to O2 to form superoxide radical (O 2
), hydrogen peroxide (H2O2), or a hydroxyl radical (OH ), respectively. The
enhanced production of ROIs during stresses can pose a threat to plants because they are unable to detoxify
effectively by the ROI scavenging machinery. The unquenched ROIs react spontaneously with organic
molecules and cause membrane lipid peroxidation, protein oxidation, enzyme inhibition, and DNA and RNA
damage.
MPO-myeloperoxidase SOD-superoxide dismutase

9. DEAD-Box Helicasesin Salinity Stress Tolerance:
Possible mechanism of stress tolerance by a helicase. Eukaryotic initiation factor 4A (eIF4A) is a prototypic
member of the DEAD-box RNA helicase family. Abiotic stresses enhance formation of the inhibitory secondary
structure at the 5’ UTR of mRNA. This protein is responsible for removal of the secondary structure of the
mRNA. eIF4A, along with eIF4B, binds to 5’ UTR and unwinds the inhibitory secondary structure in an ATP-
dependent manner.This facilitates the binding of ribosome. After this ribosome scans for the start codon (AUG)
and protein synthesis begins normally, which was inhibited due to negative impact of the stress.
Refernces:
Tuteja, N. 2007. Mechanisms of High Salinity Tolerance in Plants. Osmosensing and Osmosignaling.419-438.
doi:10.1016/s0076-6879(07)28024-3
Gupta, B., Huang, B. 2014. Mechanism of Salinity Tolerance in Plants: Physiological, Biochemical, and
Molecular Characterization. International Journal of Genomics. 1–18. doi:10.1155/2014/701596
Buchanna B, Gruissen W and Jones R. 2015. Biochemistry and Molecular Biology of plants. American Society
of Plant Physiologists, USA.
MOLECULAR BIOLOGY OF DROUGHT STRESS TOLERANCE IN PLANTS
Contents:
• Drought
• Effect of drought stress
Water relations
Cell membrane damage
Photosynthesis
Stomata
ABA
• Adaptive responses to drought tolerance
Osmotic adjustment
Phytohormones
Antioxidant activity
Gene expression
• Drought tolerance mechanisms
• Cross- tolerances between stresses

10. Drought:
Drought is termed as meteorological term that involves complex physical and chemical process. It is the
Interaction of reduced rainfall, ground water table, water deficit with increased temperature.
Effects ofDrought stress:
Water deficit conditions stimulate several plant responses, such as morphological, physiological,
biochemical and molecular alterations, which ultimately result in disturbing plant functioning . As depicted in
Figure, drought events limit plant performances in different developmental stages. Limited water availability can
indeed reduce the germination rate and the development of young plants. During the progression of plant growth,
drought basically influences the plant water relations, which in turn cause severe perturbation to the whole plant
metabolism (at physiological, biochemical and molecular levels), depending to the stress severity and duration.
Water deficit conditions alter several activities of plant, but one of the main effects is the decline of
photosynthetic activity and finally the plant yield. During drought stress conditions, oxidative stress, directly or
indirectly generated in plants, is one of the main drivers of plant responses and results in damage to cell
membrane, altering membrane integrity, physiological and biochemical alterations which lead to acute metabolic
disorders and eventually alter the plant productivity.
Effect On The Water Relations:
• Water deficit occurs due to drought
• It can also occur in environments in which water is not limited
• Drought stress reduces the soil water potential, leaf water potential, relative water content (RWC) and
osmotic potential.
• Water potential
The Ψw of a plant equals the sum of various component potentials. Solute potential, Ψs , is dictated by
the number of particles dissolved in water. Water potential decreases as solute concentration increases. Pressure
potential, Ψp , reflects physical forces exerted on water by its environment. When water is subjected to negative
pressure (tension), Ψp is less than 0 MPa (megapascals) and Ψw is diminished. (Note that water potential is
typically defined in units of pressure rather than energy.) In contrast, water potential is increased by positive
pressure (turgor, Ψp > 0 MPa). Gravitational potential, Ψg , can have a substantial effect when water is
transported over vertical distances greater than 5-10 meters, but this term can be omitted when describing
transport between cells or within small plants. A fourth factor, matric potential, Ψm, accounts for how solid

11. surfaces (e.g., cell walls and colloids) interact with water and depress Ψw. Because Ψm values are small and
difficult to measure, however, its impact on plant water potential is usually ignored. For conditions under which
Ψg and Ψm are insignificant, the water potential equation is frequently simplified as follows:
Relative water content (RWC):
RWC explains about the water content in the samples. It explains about cellular volume.
Osmotic Potential:
Osmotic adjustment occurs when the concentrations of solutes within a plant
cell increase to maintain positive turgor pressure within the cell. As the cell
actively accumulates solutes, Ψs drops, promoting the flow of water into the
cell. In cells that fail to adjust osmotically, solutes are concentrated passively,
but turgor is lost.
Cell Membrane Damage:
• Membranes are fundamental for sustaining the biological processes of living organisms.
• Act as a selective barrier
• Responsible for triggering signal transduction as a first response to stress conditions
• A decrease in membrane lipid content is correlated to an inhibition of lipid biosynthesis
• In chloroplast, balance between digalactosyldiacyloglycerol (DGDG) and
monogalactosyldiacyloglycerol (MGDG), together with sulfoquinovosyldiacylglycerol (SQDG) and
phosphatidylglycerol (PG) is required to maintain membrane stability
Effect Of Drought Stress On Photosynthesis:

12. Major consequence of water deficit in plants is the decrease or suppression of photosynthesis. Reduced
leaf area, increased stomata closure and consequent reduced leaf cooling by evapotranspiration increases osmotic
stress leading to damages to the photosynthetic apparatus are among the major constraints for photosynthesis.
Among these, the decrease in photosynthetic process in plants under drought is mainly attributable to the decline
in CO2 conductance via stomata and mesophyll limitations. Decrease in photosynthetic activity due to drought
may also be due to reduced ability of stomatal movement. Declined activity of photosynthesis is triggered by the
loss of CO2 uptake, whose drop has been shown to affect Rubisco activity and decrease the function of nitrate
reductase and sucrose phosphate synthase and the ability for ribulose bisphosphate (RuBP) production.
Supportively, CO2 enrichment eliminated many early responses of maize metabolites and transcripts attributable
to drought stress. Water deficit also resulted in decreased leaf area per shoot, and, thus, modification in canopy
architecture, and this feature can alter gas exchange, water relations, vegetative growth and sink development
(e.g., fruits or grains).
Decline In Photosynthetic Pigments:
Water stress inhibits chlorophyll synthesis at four consecutive stages: 1) formation of 5-aminolevuliniuc acid
(ALA); 2) ALA conversion into porphobilinogen and primary tetrapyrrol, which is transformed into
protochlorophyllide; 3) lightdependent conversion of protochlorophyllide into chlorophyllide; and 4) synthesis of
chlorophylls a and b along with their inclusion into developing pigmentprotein complexes of the photosynthetic
apparatus
Stomatal Regulation:
Stomata regulation was carried out by the concentrations of solutes
in the guard cells. If the concentrations of the solutes Ca2+
in the
guard cells increases. Ca2+
inhibits the OST2 functioning, K+
channels. Allows the ions to enters into the vacuole and allows the
water influx leading to turgid of the guard cells (opening of the
stomata). In the process of stomata closing, when the Ca2+
enters
into the guard cells, The outflux of the Ca2+
occurs through the S-
type, R-type anion channels, K+
out channels. From the vacuole the
K+
ions also flows out through the channels leading to the increase
in the water potential. So, that water moves out leading to closing
of the stomata.

13. ABA Regulation In Response To Drought :
ABA is primarily synthesized de novo in response to drought and high salinity, and the genes involved in ABA
biosynthesis and catabolism have been identified mainly through genetic and genomic analyses. Xanthoxin, a C15
precursor of ABA, is produced by direct cleavage of C40 carotenoids by 9‐ cis‐ epoxycarotenoid dioxygenase
(NCED) in plastids. This step is critical for ABA stress responses. NCED is encoded by a multigene family, and
the stress‐ inducible NCED3 gene plays a key role in ABA biosynthesis under stress conditions. In Arabidopsis,
overexpression of NCED3 increases endogenous ABA levels and improves drought stress tolerance, whereas
disruption results in defective ABA accumulation under drought stress and impairs drought stress tolerance. At
least two regulatory pathways exist for ABA catabolism: the oxidative pathway and the sugar conjugation
pathway. The oxidative pathway is catalyzed by ABA C‐ 8′ hydroxylase to produce phaseic acid. This enzyme
belongs to a class of cytochrome P450 monooxygenases, the CYP707As, of which there are four members in
Arabidopsis. Among the four CYP707As, CYP707A3 is a major enzyme for ABA catabolism during the
osmotic stress response. The CYP707A3 gene is induced by rehydration after
exposure to conditions of dehydration, and CYP707A3 knockout mutants show
increased endogenous ABA and dehydration tolerance. In the sugar conjugation
pathway, ABA is inactivated in sugar‐ conjugated forms, such as ABA glucosyl
ester, and stored in vacuoles or apoplastic pools. Under conditions of dehydration,
ABA is released from the glucosyl ester form by β‐ glucosidase. Regulation of
the genes involved in ABA synthesis and catabolism by transgenic technology can
improve drought tolerance. ABA transport is also thought to be important for
plant responses to abiotic stress. NCED3 is mainly expressed in vascular tissues,
and endogenous ABA is mainly synthesized in vascular tissue of leaves. ATP
binding cassette (ABC) transporters function as ABA transporters in both the
export and import of ABA. Regulation of ABA transport is important for both
inter‐ and intracellular signalling in plants.
Uses Of ABA In Response To Drought:
• ABA is useful in plant drought tolerance by triggering diverse signalling mechanisms
• stimulate stomatal movement
• Maintain root architecture
• Regulate photosynthesis
• ABA-induced genes encoding drought-related proteins such as dehydrins, ROS-detoxifying enzymes,
regulatory proteins and phospholipid signalling enzymes can improve drought stress tolerance.
• Improved amount of ABA induced a signalling pathway in guard cells which results in outflow of guard
cells K+ and reduced turgor pressure, ultimately causing stomata closure
Adaptive Responses To Drought Tolerance:
• Osmotic adjustment
• Phytohormones
• Antioxidant activity
• Gene expression
Osmotic Adjustment:
• Osmotic adjustment has been considered as one of the vital processes in plant adaptation to drought.

14. • compatible solutes such as amino acids (proline, aspartic acid, and glutamic acid), glycine betaine,
sugars (sucrose), cyclitols (mannitol and pinitol) plays an important role in maintaining the osmotic
adjustment.
Proline:
proline acts as an important signaling moiety against drought stress to stimulate mitochondria functioning and
alter cell proliferation, stimulating particular drought stress recovery genes. Proline accumulation helps to
maintain membrane integrity by diminishing lipids peroxidation by defending cell redox potential and declining
ROS level.
• Mannitol and Sorbitol are the most frequent polyols found in plants. Accumulation of these two polyols
under drought may account for upto 80 % of the total solutes involved in the osmotic adjustment.
Trehalose under drought stress aids to stabilize macromolecules such as lipids, protein and other
biological moieties to enhance photosynthetic functioning, thereby conferring drought tolerance.
Phytohormones:
• Increased level of Cytokinin amount in xylem sap induced stomata opening by diminishing its sensitivity
to ABA.
• Jasmonic acid synthesis- Root growth, Decreased level of ROS, Promoting stomatal closure
• Auxin- Regulates root development, Functioning of ABA related genes, ROS metabolism
• Ethylene affects stomata closing by suppressing drought mediated ROS formation
Antioxidant Activity:
Scavenging reactive oxygen species by enzymatic and non-enzymatic antioxidant systems. AA - ascorbate, APX
- ascorbate peroxidase, CAT - catalase, DHA - dehydroascorbate, DHAR - dehydroascorbate reductase, GPX -
glutathione peroxidase, GSSG - glutathione disulfide (oxidised), LOO - lipid peroxide radical, LOOH - lipid
hydroperoxide, MDHA - monodehydroascorbate, MDHAR - monodehydroascorbate reductase, SOD -
superoxide dismutase.
ABA Pathway:
ABA pathway occurs through ABA dependent and ABA independent pathways. In ABA dependent
pathway, MYB/MYC and bZIP, ABRE transcriptional factors gets activated further activating RD22, RD29B
genes. These genes increases the antioxidant and osmoprotectant of the cells leading to ROS scavenging and
osmotic adjustment. In ABA independent pathway, transcriptional factors like DREB 2A, 2B gets activated,
leading to activation of the gene RD29A. The activation of the gene increases the dehydrin and LEA protein,
increasing membrane stability and protein folding.

15. Mechanisms of drought stress tolerance in plants :
Plant drought tolerance encompasses alterations at morphological, biochemical and molecular levels. Exhibition
of single or multiple tolerance factors governs the plant capability to survive under adverse drought conditions.
Reference:
Mahmood., Khalid., Abdullah., Ahmed., Shah., Ghafoor., Du. 2019. Insights into Drought Stress Signaling in
Plants and the Molecular Genetic Basis of Cotton Drought Tolerance. Cells. 9(1): 105.
doi:10.3390/cells9010105
Kaur, G., Asthir, B. 2017. Molecular responses to drought stress in plants. Biologia Plantarum. 61: 201-209.
https://doi.org/10.1007/s10535-016-0700-9.
Kapoor, D., Bhardwaj, S., Landi, M., Sharma, A., Ramakrishnan, M and Sharma, M. 2020. The Impact of
Drought in Plant Metabolism: How to Exploit Tolerance Mechanisms to Increase Crop Production. Applied
Sciences.
Kim, T.-H., Böhmer, M., Hu, H., Nishimura, N., Schroeder, J. I. 2010. Guard Cell Signal Transduction Network:
Advances in Understanding Abscisic Acid, CO2 , and Ca2+
Signaling. Annual Review of Plant Biology, 61(1):
561-591. doi:10.1146/annurev-arplant-042809-112226
HEAT SHOCK RESPONSES IN HIGHER PLANTS
Introduction:
Heat stress may occur under numerous temporal and developmental conditions, with results ranging
from retarded growth to damaged organs and plant death. In the field, leaves may experience heat stress when
transpiration is insufficient (i.e., when water is limiting and temperature is high) or when stomata are partially or
fully closed and irradiance is high; in germinating seedlings, when the soil is warmed by the sun; in organs with

16. reduced capacity for transpiration (e.g., fruit); and overall, from high ambient temperatures. The duration and
severity of stress, susceptibility of different cell types, and stage of development all influence the ability of a
particular genotype to survive heat stress.
The signature response to acute heat stress is a rapid and transient reprogramming of gene expression, including
a decrease in the synthesis of normal proteins and accelerated transcription and translation of heat shock proteins
(HSPs). This response is observed when plants are exposed to temperatures 5°C or more above their optimal
growing conditions.
Different types of HSPs and their characteristics:
Model of HSP gene expression :
The HSF of Arabidopsis can only bind DNA as trimers, and heat stress is required for trimerization. The
oligomerization and DNA‐ binding domains of HSF are conserved among different organisms. Trimerization
depends on the presence of a leucine zipper configuration of hydrophobic heptad repeats located adjacent to the
DNA binding domain. The mechanism that controls trimerization is poorly understood, but recent studies have
indicated that trimerization, DNA binding, and transcriptional activity are repressed in the absence of heat stress.
In the unstressed cell, HSF is maintained as a monomer and cannot bind DNA. Upon heat shock, the HSF is
assembled into a trimer capable of binding a specific DNA sequence. This model is mainly based on the research
of bacterial HSFs.
Reference:
Buchanna B, Gruissen W and Jones R. 2015. Biochemistry and Molecular Biology of plants. American Society
of Plant Physiologists, USA.

17. MOLECULAR BIOLOGY OF LOW TEMPERATURE STRESS IN PLANTS
Low temperature can be categorized into chilling and freezing stresses. Freezing and chilling stress impose both
direct and indirect effects on plant health. Direct effects include solidification of membrane lipids and reductions
in enzymatic reaction rates, and these occur over a relatively short time. Indirect (or secondary) injury
symptoms, on the other hand, appear gradually over time and include solute leakage from cells, respiration and
photosynthesis imbalance, ATP depletion, accumulation of toxic substances, and wilting by water loss.
Plants differ in their tolerance to chilling (0-15ºC) and freezing (< 0ºC) temperatures. Plants from temperate
regions are chilling tolerant, although most are not very tolerant to freezing but can increase their freezing
tolerance by being exposed to chilling, non freezing temperatures, a process known as cold acclimation, which is
associated with biochemical and physiological changes.
Effects of chilling stress :
• Causes membrane destabilization and metabolic dysfunction
• Lipids with high melting temperatures begin to solidify
• Membranes become leaky/dysfunctional
• Intracellular water and solutes are lost
• Membrane‐ associated reactions such as carrier‐ mediated transport, enzyme‐ mediated processes and
receptor function are inactivated
• Its effect on photosynthesis
• Production of ROS
• D1 protein, a major component of the PSII reaction center, is affected at low temperature
• Chilling injury occurs due to the lack of or impaired cellular recovery functions
Effects of freezing stress :
• Causes membrane destabilization and damage due to osmotic and mechanical stress
• Chemical potential of ice is lower than that of unfrozen water
Exposure of plants to freezing temperatures causes a cellular water deficit as water travels down its potential
gradient, crossing the plasma membrane into the cell wall and intercellular spaces. When the rate of freezing is
sufficiently slow to prevent formation of ice crystals in the cytoplasm, the cell dehydrates and freezing occurs in
the apoplast.

18. Diagrammatic representation of the transition of membrane structure from Lamellar to Hexagonal II
phase at freezing temperatures and vice versa at warm temperatures.
• Osmotic dehydration increases solute concentrations in the cytoplasm and other intracellular
compartments.
• This can inactivate membrane‐ associated enzyme and transporter activities.
• Direct interaction of solutes with the membrane results in dissociation of membrane proteins due to
changes in electrostatic and hydrophobic interactions
• Cell wall–plasma membrane interactions are altered due to freezing
• Freezing leads to acidification of the cytoplasm, probably as a result of disturbance of H+
‐ transport
systems associated with the vacuolar membrane (tonoplast).
The Plasma membrane‐ associated protein that affects freezing tolerance is synaptotagmin1 (SYT1).
Synaptotagmins are a family of membrane‐ trafficking proteins that function as calcium sensors in plasma
membrane vesicle fusion processes mediated by the SNARE protein complex. The level of Arabidopsis SYT1
increases rapidly in the plasma membrane in parallel with the development of freezing tolerance during cold
acclimation.
Model for Ca2+
‐ and SYT1‐ induced membrane resealing occurring during freeze/thawing. (A) The plasma
membrane is mechanically punctured by ice crystals, and (B) Ca2+
moves from the extracellular space into the
cytoplasm through the damaged sites. (C) Endomembranes may then fuse at the site of the damaged plasma
membrane via Ca2+
binding SYT1. (D) The damaged site is resealed.
Acclimation to Low Temperature :
• Membrane fluidity are considered to play a role in sensing a temperature drop outside the cell
• Freezing tolerance develops in a process known as cold acclimation
• After cold acclimation, an increase in proportion of plasma membrane phospholipids and decrease in the
proportion of glucocerebrosides
• glucocerebrosides and sterol lipids are enriched in plasma membrane microdomains.

19. LT-induced adaptive and protective changes in plants leading to cold acclimation:
• Phospholipases influence freezing tolerance probably through alterations in the plasma membrane lipid
composition
• In Arabidopsis, antisense suppression of phospholipase Dα1 (PLDα1), the most abundant plant
phospholipase, increases freezing tolerance.
• Arabidopsis mutant eskimo1, which is constitutively freeze‐ tolerant, overproduces proline at warm
temperatures
Cold signal perception and responsive pathway in Arabidopsis:
At the onset of cold stress, Ca2+
channels and the plasma membrane protein CaM-regulated receptor-like kinase
(CRLK)1/2 are activated, followed by rapid activation of Ca2+
signals and mitogen-activated protein kinase
(MAPK) cascade pathways. Ca2+
signals are decoded by a series of Ca2+
binding proteins, leading to downstream
signal transduction. ICE1, which is a key regulator in the ICE1-CBF-COR pathway, is regulated by multiple
post-translational modifications. OST1 phosphorylates ICE1 to activate its transcriptional activity. ICE1 can be
ubiquitinated by HOS1, leading to its degradation. However, the sumoylation of ICE1 by SIZ1 and

20. phosphorylation by OST1 can inhibit the degradation of ICE1. BTF3 and BTF3L can also be phosphorylated by
OST1 and their interactions with C-repeat binding factors (CBFs) are enhanced to stabilize CBFs under cold
stress. During cold acclimatization, ICE1 can be phosphorylated by MPK3/6 in the MAPK cascade, leading to
the degradation of ICE1 by an unknown E3 ligase. Meanwhile, 14-3-3 proteins, which are phosphorylated by
COLD-RESPONSIVE PROTEIN KINASE 1 (CRPK1), shuttle from the cytosol to the nucleus to promote the
degradation of CBFs and thus, regulate the duration of the cold defense response. Expression of CBF genes is
also regulated by other positive and negative regulators. Phytochrome B (phyB) participates in temperature
perception through its temperature-dependent reversion from the active PFR state to the inactive PR state.
Cold tolerance in plants:
The mechanism of cold tolerance in plants. A short day in early autumn represents the first initiation of cold
stress. AFPs and PIP2-7 slow ice crystal formation to maintain cell membranes and reduce membrane injury.
However, low temperature (LT) still causes some changes in membrane structure, sugar concentration, and
production in cry proteins. LT initiates the increase of ABA, EL, (Ca2+)cyt, and ROS accumulation but
decreases chloroplast number. LT induces the expression of some genes, such as NIA, MAPK, TPS11, SMT1,2,
ICE1 and antioxidant enzyme coding genes. Antioxidant enzyme coding genes reduce the EL and increase the
activity of the antioxidant enzymes in cold stress plants. Meanwhile, NIA genes and NR initiate NO as a result of
LT. ICE1, CAMTAs, NIA, and hormones induce the expression of CBFs, which bind to CRT/DRE cis-elements
to enhance cold tolerance. ICE1-CBFs induce expression of cold-responsive genes such as KIN1, RD29A,
COR47A and LEA during the stress.
ICA-CBF-COR pathways in plants tolerance to cold stress:
ICA-CBF-COR pathways in plants tolerance to cold stress. The expression of CBFs is mainly mediated by
DELLA signaling and induced by ICE1. DELLAs contribute to the cold induction of CBF genes through
interaction with JaZs signaling. CBFs activate the expression of COR genes via binding to cis-elements in the
promoter regions of COR genes and results in the enhancement of cold tolerance in the plants .

21. LEA Proteins:
References:
Guo, X., Liu, D., Chong, K. 2018. Cold signaling in plants: Insights into mechanisms and regulation. Journal of
Integrative Plant Biology. doi:10.1111/jipb.12706
Ritonga, F. N., Chen, S. 2020. Physiological and Molecular Mechanism Involved in Cold Stress Tolerance in
Plants. Plants. 9(5): 560. doi:10.3390/plants9050560
MOLECULAR BIOLOGY AND GENOMICS OF FLOODING AND RESPONSE AND
ADAPTION BY PLANT TO FLOODING STRESS
Flooding Introduction :
When flooding occurs, soil gases are replaced with water, thereby reducing entry of oxygen into the
soil and making it difficult for roots and other organs to carry out respiration. oxygen deficit associated
with flooding can also prevent plants from obtaining adequate water from the soil due to gating of root
cell aquaporins. Plant or cellular oxygen status can be defined as normoxic, hypoxic, or anoxic
conditions.

22. Plant species are categorized into wet land, flood tolerant and flood susceptible by sensitivity to
flooding
Wet land Flood tolerant Flood sensitive
possess anatomical,
morphological, and
physiological features that
permit survival in
waterlogged soils and
partial submergence
plants can endure flooding and
anoxia only temporarily. Like
wetland species, these plants
generate ATP through anaerobic
metabolism during short‐term
flooding
Flood‐sensitive plants exhibit an
injury response to anoxia
a thickened root
hypodermis to reduce O2
loss to the anaerobic soil
root elongation is inhibited cytoplasmic acidification,
diminished protein synthesis,
degradation of mitochondria,
inhibited cell division and
elongation, disrupted ion transport,
and cell death within root meristems
some plants develop
specific structures:
aerenchyma, lenticels,
pneumatophores
Aerenchyma development No aerenchyma development
Eg- Echinochloa crusgalli,
Oryza sativa
Solanum tuberosum (potato)
Zea mays (corn)
Hordeum vulgare
Glycine max
Pisum sativum
Genomics of Flooding:
• Global gene profiling provides comprehensive understanding of the networks of genes, proteins,
and small molecules that underlie plant stress and defense responses
• The strategies currently followed for identifying differentially expressed transcripts include
differential display (DD), representational difference analysis (RDA), serial analysis of gene
expression (SAGE), global transcript analysis by microarray and through construction of
subtractive hybridization libraries
• For Arabidopsis, Klok et al. (2002) used oligonucleotide array containing 3500 cDNA clones
and identified 210 genes differentially regulated by hypoxia
Adaptive strategies:
To facilitate transport of O2 from aerial structures to submerged roots and thereby maintain
aerobic metabolism and growth, some plants develop specific structures: aerenchyma, adventitious
roots from the hypocotyl or stem, lenticels, periderm that allow gas exchange, shallow roots, and
pneumatophores. Other adaptive strategies include elongation of stems or leaf petioles towards the
water surface and thinning of leaves to improve underwater photosynthesis.

23. • The escape strategy includes ATP production through active consumption of available carbohydrates
coupled to glycolysis, fermentation, and a partially functioning citric acid cycle.
• Flooding stimulates an increase in glycolytic flux known as the Pasteur effect, where sucrose or glucose
from the phloem is directed toward glycolysis in flooded organs
• Starch is slowly hydrolyzed by amylases in rhizomes of flood‐ tolerant Acorus calamus
Metabolic acclimation under O2 deprivation. Plants have multiple routes of sucrose catabolism, ATP
production, and NAD(P)+
regeneration. These include ethanol and lactate production as well as a modified
noncyclic citric acid flux mode that is both an alanine and 2‐ oxoglutarate shunt and a γ‐ aminobutyric acid
(GABA) shunt. Blue arrows indicate reactions that are promoted during anaerobic stress, and gray dashed lines
indicate reactions that are inhibited during the stress. Metabolites indicated in brown boxes are major or minor
end products of metabolism under hypoxia. Metabolites indicated in orange boxes decrease under hypoxia.
ADH, alcohol dehydrogenase; GAD, glutamic acid decarboxylase; GDH, glutamate dehydrogenase; INV,
invertase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; PDC, pyruvate decarboxylase; SCS,
succinyl CoA synthetase; SUS, sucrose synthase.
• Plants generally increase cellular levels of PDC and ADH in response to flooding
• In Maize, Arabidopsis ADH is translated efficiently in anoxic/hypoxic cells
• Submergence of plants- increase in ethylene, GA and decrease in ABA
Formation of aerenchyma :
Flooding or submergence stimulates production and limits outward diffusion of the gaseous hormone
ethylene, a key trigger for adaptive responses to submergence and low oxygen levels. The levels of 1‐
aminocyclopropane‐ 1‐ carboxylic acid (ACC) synthase and ACC oxidase, enzymes of the ethylene
biosynthesis pathway, increase in response to hypoxia in maize root tips. In hypoxic roots, ethylene promotes the

24. formation of aerenchyma in the central portion of the root cortex; anoxic roots develop fewer aerenchyma
because O2 is essential for ethylene synthesis.
Elongation Growth:
Ethylene and flooding responses in rice. The elongation of stem and leaf cells is positively regulated by
GA. Under normal growth conditions, ABA inhibits GA activity. When plants are submerged, cellular ethylene
levels rise due to biosynthesis and entrapment by the surrounding water. This promotes the breakdown of ABA,
increasing cellular responsiveness to GA, which then stimulates cell elongation. In deepwater rice (Oryza sativa
L. var. Indica), this is accompanied by increased accumulation of bioactive GA. (A) The escape strategy of
deepwater rice involves rapid stem elongation to maintain leaf tissue above the water level, in response to a
slow, progressive flood. This growth is mediated by GA biosynthesis and action. Quantitative trait locus (QTL)
mapping identified two closely linked ethylene responsive factor (AP2/ERF) family transcription factors,
SNORKEL1 and 2 (SK1 and SK2), on chromosome 12 that drive the deepwater escape response. Ethylene
accumulation in underwater stems promotes transcription of SK1 and SK2, triggering internode elongation.
These genes are present in some wild rice species, but are absent or nonfunctional in non‐ deepwater
domesticated rice. (B) In the quiescence strategy of submergence‐ tolerant varieties, shoot elongation is
suppressed to conserve carbohydrates and increase survival under short‐ term complete inundation conditions.
GA signalling and cell elongation are inhibited by the ethylene‐ induced action of SUBMERGENCE‐ 1A
(SUB1A‐ 1 allele) on the growth‐ inhibition genes SLENDER RICE‐ 1 (SLR1) and SLR LIKE‐ 1 (SLRL1).
Submergence‐ induced SUB1A is present in a cluster of two or three ERF transcription factor on rice
chromosome 9 in submergence‐ tolerant rice accessions.
• In the wetland species Rumex palustrus, ethylene entrapment within the leaf petiole inhibits the ABA
biosynthetic enzyme 9‐ cis‐ epoxycarotenoid dioxygenase (NCED) and promotes ABA catabolism to
phaseic acid

25. • Additional plant proteins associated with signal transduction processes that control survival of low‐
oxygen conditions include the monomeric RHO of plant (ROP) G‐ proteins, CIPK, and SnRK1A
Reference:
• Buchanna B, Gruissen W and Jones R. 2015. Biochemistry and Molecular Biology of plants. American
Society of Plant Physiologists, USA.
OXIDATIVE STRESS
Oxidative stress results from conditions that promote formation of ROS, which can damage or kill cells.
Environmental factors that cause oxidative stress include air pollution (increased amounts of ozone or sulfur
dioxide), oxidant‐ forming herbicides such as Paraquat (methyl viologen, 1,1′‐ dimethyl‐ 4,4′‐ bipyridinium),
heavy metals, drought, heat and cold stress, wounding, the transition to anoxia and reoxygenation, UV light, and
intense light conditions that stimulate photoinhibition. Oxidative stress also occurs in response to senescence and
pathogen infection
Environmental factors that increase the concentrations of reactive oxygen species (ROS) in plant cell
ROS signal transduction pathway:
ROS can be detected by at least three mechanisms: ROS receptors, redox‐ sensitive transcription factors,
and phosphatases. Detection by ROS receptors generates Ca2+
signals and activates phospholipase C/D
(PLC/PLD) to generate phosphatidic acid (PA). PA and Ca2+
are thought to activate the protein kinase OXI1,
which then activates a mitogen‐ activated‐ protein kinase (MAPK) cascade (MAPK3/6) to induce or activate
different transcription factors that regulate the ROS‐ scavenging and ROS‐ producing pathways. Activation or
inhibition of redox‐ sensitive transcription factors by ROS might also affect the expression of OXI1 or other
kinases, and the induction of ROS‐ specific transcription factors. Inhibition of phosphatases by ROS might
result in the activation of kinases, such as OXI1 or MAPK3/6. Two loops are involved in the ROS signal
transduction pathway: a localized or general defense response (a negative feedback loop; solid green line) can be
activated to suppress ROS, and a localized amplification loop (positive feedback loop; red dashed line) can be
activated to enhance ROS signals via NADPH oxidases. Salicylic acid (SA) and nitric oxide (NO) might be
involved in this amplification loop as enhancers. HSF, heat shock factor; PDK, phosphoinositide‐ dependent
kinase; TF, transcription factor.

26. • Reference: Buchanna B, Gruissen W and Jones R. 2015. Biochemistry and Molecular Biology of plants.
American Society of Plant Physiologists, USA.
MOLECULAR RESPONSE TO DEHYDRATION STRESS IN PLANTS
• Dehydration stress can be defined as water deficit conditions induced by either drought, salt stress,cold
or high temperature stress that has adverse impact on plants growth.
• Resurrection plants, are able to withstand prolonged periods of dehydration and to recover within hours
to a few days once water is available
• Reference: Buchanna B, Gruissen W and Jones R. 2015. Biochemistry and Molecular Biology of
plants. American Society of Plant Physiologists, USA.