The document provides information about plant responses and adaptation to cold stress. It discusses chilling stress which occurs at low but non-freezing temperatures and can cause physiological disorders in plants. It describes the symptoms of chilling injury which includes reduced growth, lesions, and cellular changes like alterations to membranes and metabolism. Freezing stress that occurs below 0°C is also summarized, noting how ice formation can damage plant cells and tissues. The document outlines various strategies plants use to survive cold stress, including accumulating antifreeze proteins, osmoprotectants, and cold-regulated proteins. Signal transduction and transcription factor pathways that sense and respond to cold by regulating gene expression are summarized. Methods to artificially prevent chilling and freezing effects on plants

1. Faculty of Basic Science
Department of Biochemistry
SKUAST JAMMU
Presentation
on
Cold stress: Plant Responses and Adaptation
Presented by
Sheikh Mansoor
PhD Biochemistry

2. CHILLING STRESS
Plants may develop physiological disorders when exposed to low
but non-freezing temperatures.
The German plant physiologist Molisch suggested the term
‘chilling injury’ as long ago as 1897 to describe this phenomenon
The international "star of plant physiology" Hans MOLISCH.
He studied at the university of Vienna. In 1894, he
became professor in Prague and in 1909, in Vienna,
he began research into "frost-resistance" at the cellular
level.
As a microbiologist, he was working with purple and luminescent
bacteria, and has been innovative in photosynthesis research and
chemotaxonomy.

3. How do plants perceive cold?
• As a result of exposure to low temperatures, many physiological and biochemical
cell functions have been correlated with visible symptoms (wilting, chlorosis, or
necrosis) (Ruelland and Zachowski 2010).
• Often, these adverse effects are accompanied by changes in cell membrane
structure and lipid composition (Uemura and Steponkus 1999; Matteucci et al.
2011)
• Cellular leakage of electrolytes and amino acids, a diversion of electron flow to
alternate pathways (Seo et al. 2010), alterations in protoplasmic streaming and
redistribution of intracellular calcium ions (Knight et al. 1998)
• They also involve changes in protein content and enzyme activities (Ruelland and
Zachowski 2010) as well as ultrastructural changes in a wide range of cell
components, including plastids, thylakoid membranes and the phosphorylation of
thylakoid proteins, and mitochondria (Zhang et al. 2011).
. However, prolonged exposure to stress causes plant necrosis or death.

4. Symptoms of Chilling
• Cellular changes : Changes in membrane structure and composition, decreased
protoplasmic streaming, electrolyte leakage and plasmolysis.
• Altered metabolism : Increased or reduced respiration, depending on severity of
stress, production of abnormal metabolites due to anaerobic condition.
• Common Symptoms
– Reduced plant growth and death
– Surface lesions on leaves and fruits
– Abnormal curling, lobbing and crinkling of leaves
– Water soaking of tissues
– Cracking, splitting and dieback of stems
– Internal discolouration (vascular browning)
– Increased susceptibility to decay
– Failure to ripen normally
– Loss of vigour (potato lose the ability to sprout if chilled)

5. A model to explain symptoms of chilling injury in chilling-sensitive plants. Membranes are the primary site of
cold-induced injury, leading to a cascade of cellular processes with adverse effects on the plant. When exposure
to low temperature is brief, the effects may be transitory and plants survive. However, the plant will exhibit
necrosis or die if exposure is maintained (Lyons 1973; Raison and Lyons 1986)

7. Affects on plants
• Cellular Membranes
• The first symptom of chilling injury is the phase transition from liquid crytalline
phase to solid gel state
• Increase in permeability of plasmalemma results in leakage of organic and
inorganic substances

8. Affects on plants
Cellular Membranes(contd..)
• Plasmolysis: Plasmolemma- pressed against the
tonoplast and deleted into the vacuole as sac like
intrusions
• Formation of crystalline deposits in root cells,
epidermal, mesophyll and vascular cells of leaves
-leading to tonoplast disruption.
• Tonoplast injury is irreversible
• During hardening at low or above zero temp the
lipid bodies accumulate in cytoplasm in close
association with plasmalemma.
Lipid composition
– Ratio of Unsaturated to saturated fatty acids is
higher in chilling resistant plants
– Increase in activity of fatty acid de-saturases was
found in chilling resistant plants

9. Cytological Changes
• Swelling of plastid membranes and mitochondrial membranes
• Swelling of chloroplast thylakoids
• Decrease in size and no. of starch grains
• Grana disintegration and increase in size and no.of plastoglobules
• Mitochondria with reduced cristae and transparent matrix
• Mitochondria – double the volume
• Extensive dilation and vesiculation of smooth ER cisternae

11. Freezing stress
When plants are exposed to a low temperature below 00
c. Freezing damage occurs
primarily due to the formation of ice crystals, which damage cell structure when the
temperature falls below 00
C.

13. Freezing stress
Freezing injury in plants can be from two sources:
1.Freezing of soil water
The soil water that is available to plants is found in
the porous regions between soil particles. It freezes at
about -2°C, depriving the plant of its source of water.
2. Freezing of the fluids within the plant.
Freezing of water within the plant can cause
disruption of structure and function of cells and tissues.
Ice usually forms first in the cell walls and intercellular
spaces. Damage occurs when ice crystals grow and
puncture into the cytoplasm.

14. Symptoms of Freezing injury
• Desiccation or burning of foliage
• Water-soaked areas that progress to necrotic spots on
leaves, stems or fruit and death of sections of the plant
or the entire plant.
• Close examination of woody plants several days or weeks
after freezing may reveal a dead or weakened root
system or split bark on stems or branches.
• Obvious symptoms on plant foliage may not be present
until after the plant has been stressed by warm
temperatures.
• Wilting and/or desiccation, as caused by direct drought
stress.

15. Formation of ice intracellularly may be due to
•By internal nucleation (certain large polysaccharides /proteins
serve as nucleating agents to form ice)
•By penetration of external ice crystals into the cells
•Intracellular ice formation is very lethal which causes
immediate disruption of cells.
•It spreads from cell to cell through plasmodesmata
•Formed in the cell wall adjacent to the intercellular spaces
•Originates spontaneously from centers of nucleation in the
cytoplasm.

16. Signal
transduction
Fundamental responses of plants
during cold stress exposure. Cold stress
exposure causes various
physiochemical disturbances, leading to
growth inhibition. Cold stress response
is perceived by plants through a signal
transduction that leads to the
activation of transcription factors and
cold-responsive genes. Such
transcription factors and genes control
the damage due to cold stress and help
in providing tolerance to plants.

17. Chilling tolerance
• The acquisition of chilling tolerance is associated with huge changes in metabolite
contents, such as the accumulation of soluble sugars, dehydrins, RNA-chaperones,
and an increase in reactive oxygen species (ROS) detoxification activities.
• These changes in cellular components are due to a transcriptome rearrangement.
They mean that chilling has been perceived and transduced to the nucleus.
• Chilling is not perceived by a single mechanism in plants but at different sensory
levels, that are the very biological processes disturbed by the temperature
downshift. Once perceived, chilling stress is transduced.
• An increase in cytosolic calcium is the major transducing event that will then
regulate the activity of many signalling components, including phospholipases and
protein kinases. This will end in changes in gene expression.
• The best-documented pathway leading to gene induction in response to cold is the
CBF (C-repeat binding factor) pathway.

18. A Transcription Factor Regulates
cold-Induced gene Expression
Involvement of various transcription
factors in the induction of cold-responsive
genes during cold stress. Many cold
stress–induced genes are activated by
transcriptional activators called C-repeat
binding Factors.

19. Accumulation of antifreeze proteins
(AFPs) in cold-acclimated
plants with antifungal activity
(adapted from Yeh et al. 2000;
Huang and Duman 2002; Griffith
and Yaish 2004; Yaish et al. 2006).
By accumulating PR proteins during
cold acclimation, overwintering
plants may acquire a systemic, non-
specific, pre-emptive defence
against pathogens and exhibit
greater disease resistance

20. SURVIVAL STRATEGIES
Anti freeze proteins (AFP)
- Declines rate of ice crystal growth
- Lowers the efficiency of ice nucleation sites
- Lowers temp. at which ice forms
Osmoprotectants
- Osmolytes- quarternary amines, amino acids, sugar alcohols
- Balances the osmotic potential of externally increased osmotic
pressure
ABA
- Plants develop freezing resistance when treated with
exogenous ABA

21. Ca2? and reactive oxygen species (ROS) responses to chilling in sensitive plants. ROS are not simply toxic by-products of
metabolism; they also act as signalling molecules by modulating the expression of various genes, including those encoding
antioxidant enzymes and modulators of H2O2 production. Changes to the plasma membrane can cause actin cytoskeletal
rearrangements that may be followed by the activation of Ca2? channels and increased cytosolic Ca2? concentrations, triggering a
series of reactions such as the expression of cold-regulated genes

22. COR/LEA and Dehydrins
• The accumulation of hydrophilic proteins predicted to form an amphipathic a-helix
is one of the best documented responses of plants to cold treatment (Eriksson et al.
2011).
Most of these proteins have therefore been named COR (cold responsive), LTI (low
temperature induced), RAB (responsive to abscisic acid), KIN (cold induced) or
ERD (early responsive to dehydration). These proteins include the dehydrins, which
belong to group II of the late embryogenesis abundant (LEA) proteins (Bies-Etheve
et al. 2008).
• The accumulation of one particular dehydrin (WCO R410) is correlated with the
capacity to develop freezing tolerance in wheat. However, the overexpression of
single dehydrins does not necessarily lead to enhanced freezing tolerance.
• For instance, the overexpression of RAB18, a cold-induced dehydrin, has no effect
on freezing tolerance in Arabidopsis (Lang and Palva 1992).

24. ICE1 and CBFs. The CBF genes play important roles in cold acclimation and are regulated by multiple
pathways. They activate the transcription of CBFs and repress MYB15. ICE1, a constitutively expressed
gene, is activated by cold stress via sumoylation and phosphorylation.
HOS1 is a RING-type ubiquitin E3 ligase that negatively regulates cold-induced DREB1/CBF expression.
The constitutive HOS9 and HOS10 regulons have a role in the negative regulation of CBF-target genes.
CBFs regulate the expression of COR genes that confer freezing tolerance.
The expression of CBFs is negatively regulated by MYB15 and ZAT12. HOS1 mediates the ubiquitination
and proteosomal degradation of ICE1 and, thus, negatively regulates CBF regulons. Transcription of
CBFs might be cross-regulated.
Transcription factor binding sites are represented at the bottom of the diagram, with the representative
promoters listed below. Yellow arrows indicate post-translational regulation; solid arrows indicate
activation, whereas broken lines show negative regulation; small circles indicate post-transcriptional
modification, such as phosphorylation; question marks indicates unknown ciselements.
ABRE ABA responsive element, CBF C-repeat binding factor (an AP2-type transcription factor);
COR cold-responsive genes, CRT C-repeat elements, DRE dehydration-responsive elements,
HOS1 high expression of osmotically responsive genes1, HOS9 and HOS10, ICE1 inducer of CBF
expression 1, KIN cold-induced genes, LOS2 low expression of osmotically responsive genes 2
(a bifunctional enolase with transcriptional repression activity), LTI low temperature-induced genes,
MYB myeloblastosis, MYBRS MYB recognition sequence, MYCRS MYC recognition sequence, RD
responsive to dehydration) genes, ROS reactive oxygen species, SIZ1 SAP and MiZ1 (a SUMO E3
ligase), P phosphorylation, S SUMO (small ubiquitin-related modifier), U ubiquitin

25. Prevention & Protection
Some methods to avoid chilling and
freezing effects artificially:
SOIL BANKING
oIt consists of placing a mound of soil
around the tree’s trunk to protect the bud
union and trunk from cold.
oOne of the most efficient cold protection
methods for young trees and has been
used with success for many years

26. Wrapping

27. Thank You