Being sessile, plants are constantly exposed to changes in temperature and other abiotic stress factors. The temperature stress experienced by plants can be classified into three types: those occurring at (a) temperature below freezing (b) low temperature above freezing and (c) high temperature. The plants must adapt to them in other ways. The biological substances that are deeply related to these stresses, such as heat shock proteins, glycine betaine as a compatible solute, membrane lipids etc.and also detoxifiers of active oxygen species, contribute to temperature stress tolerance in plants. Rapid advances in Molecular Genetic approaches have enabled genes to be cloned, both from prokaryotes and directly from plants themselves, that are thought to provide the key to the mechanism of temperature adaptation (Iba et al., 2002). The accumulation of heat shock proteins under the control of heat stress transcription factors is assumed to play a central role in the heat stress response and in acquired thermotolerance in plants (Kotak et al., 2007). The pattern of protein synthesis during cold acclimation is very dissimilar to the heat shock proteins in many ways. Different low temperature stress proteins, such as Anti-freeze proteins or thermal hysteresis proteins (THPs) and cold shock domain proteins etc. are accumulated in plant cell and are frequently correlated with enhanced cold tolerance ( Guy, 1999). The heat stress-induced dehydrin proteins (DHNs) expression and their relationship with the water relations of sugarcane (Saccharum officinarum L.) leaves were studied to investigate the adaptation to heat stress in plants (Wahid and Close, 2007). In order to get an in vitro evidence of Hsc70 functioning as a molecular chaperone during cold stress, a cold-inducible spinach cytosolic Hsc70 was subcloned into a protein expression vector and the recombinant protein was expressed in bacterial cells. Results suggest that the molecular chaperone Hsc70 may have a functional role in plants during low temperature stress (Zhang and Guy, 2006). To analyze the least and most strongly interacting stress with Hsps and Hsfs, a transcriptional profiling of Arabidopsis Hsps and Hsfs has been done (Swindell et al., 2007). As plants receive complex of stress factors together, therefore in future research, emphasis should be placed on such cases where tolerance is attempted to different stress factors simultaneously by employing sophisticated techniques.

1. Molecular Basis of Temperature Tolerance
in Plants
Indramohan Singh.

2. INTRODUCTION
 The overpowering pressure that affects the normal functions
of individual life or the conditions in which plants are
prevented from fully expressing their genetic potential for
growth, development, and reproduction.
(Levitt, 1980; Ernst, 1993)

3. Stress Overview
Stress
Biotic Stress Abiotic Stress
Temperature Drought Salinity Metal Stress
High Temperature Low Temperature
Chilling Freezing

4. Stress response relationship

5. Complexity of Plant Response to Abiotic Stresses
(Wang et al., 2003)

6. PSII Reaction Centre

7.  Increase in permeability of plasmalemma
Chilling
 Liquid crystalline phase Solid gel state
 High saturated fatty acids in membranes More chilling
sensitivity
 Depolymerisation of cortical microtubules.

8. Impact of Heat Stress on Plant Cell
 Disruption of normal protein synthesis
 Disruption of splicing of mRNA precursors
 Cessation of pre-RNA processing
 Decline in transcription by RNA polymerase I
 Inhibition of chromatin assembly

9. Factors involved in Thermotolerance
(Kotak et al., 2007)

10. Role of Hsps in Abiotic Stress Tolerance

11. Hsfs as Central Regulators of Heat Stress
(Bharti and Nover, 2002)

12. 1. Lea(s) – also expressed in seeds before dehydration
(protective)
2. Antifreeze proteins - (e.g., kin1 - similar to a fish
antifreeze protein), prevent ice formation
3. Other Hydrophilic proteins
4. Proteases
5. Heat shock protein
6. Regulatory proteins (transcription factors, Ca+2 binding
proteins etc.)
Appearance of many of these gene products correlate
with Cold acclimation

13. Role of miRNA in Abiotic Stress

14. Regulatory Network of Gene Expression in Cold Stress

15. Small RNAs for Stress Response

17. Case Study- 1

18.  Hsp70 molecular chaperones :- Wide role in High
Temperature Stress Tolerance
 Also induced at low temperature, but only limited evidences
for cold responsive Hsp70s.
 Hypothesis:- Denaturation of “cold labile” proteins could
occur at low temperature and Hsp70s could bind unfolded or
non-native proteins.
 The experiment aimed to test this hypothesis.

19. Role of Hsp70 Chaperone machine

20. Subcloning of a cold inducible spinach cytosolic Hsc70 into a
protein expression vector PGex2t
Purification of recombinant Hsc70 (GSTHsc70)
Test for substrate binding activity by SBA using CMLA
(α-carboxymethylated
lactalbumin)
Radiolabelling and immunoprecipitation with the anti-
cytosolic Hsc70 Mab at low temperature

21. Results

23.  CMLA and CS are commonly used substrates for chaperons as
they can bind a number of divergent chaperones.
 The successful binding of the spinach GST-Hsc70 fusion
protein to CMLA suggests that CMLA can be used as a model
substrate for molecular chaperone binding studies with plant
Hsc70.
 Thus the results show that low temperature can cause the
denaturation of certain proteins and that spinach Hsc70 can
function as molecular chaperone at low temperature.

24. Case Study- 2

25.  The heat shock response of Arabidopsis thaliana is dependent
upon a complex regulatory network which involves:-
◦ 21 known transcription factors
◦ 4 heat shock protein families.
 The role of Hsps and Hsfs under cold and non-thermal stress
conditions is not well understood.
 Aim :- To reveal the extensive overlap between heat and non-
heat stress response pathways.

26.  The analysis is based on a total of 22,746 genes, representing
approx. 80% of all known Arabidopsis genes.
 The abiotic stress datasets consist of gene expression
measurements performed on Arabidopsis thaliana roots and
shoots under a control and nine environmental stress
conditions viz.
◦ Cold, osmotic stress, salinity, drought, genotoxic stress,
oxidative stress, UV-B light stress, wounding and high
temperature.

27. Expression Profiles Under Wounding and Heat Stress

28.  All stress treatments interact with Hsf and Hsp response
pathways to varying extents, suggesting a cross-talk between
heat and non-heat stress regulatory networks.
 These results have implications regarding the molecular basis
of cross-tolerance in plant species.
 This cross-tolerance raise new questions for future
experimental studies of the Arabidopsis heat shock response
network.

29. Case Study- 3

30.  The dehydrin proteins (DHNs) are a group of Late
Embryogenesis Abundant (LEA) proteins.
 Referred to as LEA group II .
 Typically accumulate in embryogenesis in response to
environmentally imposed dehydrative forces, such as drought,
salinity and freezing. (Close et al., 1997)
 Thought to protect cellular membranes and organelles during
cellular dehydration induced by salinity, water deficit and low
temperature, but no report of the expression in heat stress.

31.  Sugarcane has high optimum temperature for its growth, but
needs to be frequently irrigated ( Qureshi et al., 2002)
 These is a correlation between changes in RH of the air and
high temperature tolerance ability of plants.
 The aim of the study is to determine the short term effect of
heat stress on sugarcane to monitor:-
◦ DHNs expression
◦ Changes in leaf water relations
◦ Possible relation of DHNs expression with leaf osmotic
potential, when heat stress is the only variable .

32. Single noded sets of sugarcane sown in pots
30 days after sprouting
1/2 of the pots transferred to control condition and rest
1/2 to heat stress condition
Samples taken at 4,12,24,36,48,60 and 72 hrs after
submitting plants to heat stress
Physiological properties measured
Heat stable proteins extracted, separated by
SDS-PAGE and immunoblotted

34. Contd.

35. Contd.
 Despite well-defined humidity conditions, initial effect of heat
stress is the hampered water relations of leaves.
 Increased earlier synthesis of compatible solutes and later
expression of DHNs improved the integrity of cellular
membranes and enabled the sugarcane to maintain φp.
 Results further suggest that expression of DHNs is
independent of dehydration stress and have a definitive
protective role like other heat stress proteins.

36. Case Study- 4

37.  Glycinebetaine (GB) is one of the organic compatible solutes
that can accumulate rapidly in many plants under salinity
stress, drought and low temperature (McCue and Hanson, 1990;
Rhodes and Hanson, 1993; Bohnert et al., 1995).
 GB is in particular effective in protecting highly complex
proteins, such as the PSII complex, against heat-induced
inactivation (Mamedov et al., 1993; Allakhverdiev et al., 1996).

38. GB as a protectant for PSII Complex

39.  Aim:- To genetically engineer tobacco (Nicotiana tabacum)
with the ability to synthesis glycinebetaine by introducing the
BADH gene for betaine aldehyde dehydrogenase from spinach
(Spinacia oleracea).
 The genetic engineering enabled the plants to accumulate
glycinebetaine mainly in chloroplasts and resulted in enhanced
tolerance to high temperature stress during growth of young
seedlings.

40. Western Blot Analysis

41. Comparison of GB Level

42. Effect on CO2 Assimilation

43. Effect on Rubisco Activation

44.  The study demonstrates the importance of transformation with
the BADH gene for enhancing tolerance of growth and
photosynthesis to high temperature stress because
photosynthesis is among the plant functions most sensitive to
high temperature damage.

45. Applied Biotechnology