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Molecular Basis of Temperature Tolerance
               in Plants




             Indramohan Singh.
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)
Stress Overview
                        Stress



           Biotic Stress          Abiotic Stress




              Temperature    Drought     Salinity   Metal Stress




     High Temperature            Low Temperature



                             Chilling          Freezing
Stress response relationship
Complexity of Plant Response to Abiotic Stresses




                                          (Wang et al., 2003)
PSII Reaction Centre
   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.
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
Factors involved in Thermotolerance




                                      (Kotak et al., 2007)
Role of Hsps in Abiotic Stress Tolerance
Hsfs as Central Regulators of Heat Stress




                                       (Bharti and Nover, 2002)
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
Role of miRNA in Abiotic Stress
Regulatory Network of Gene Expression in Cold Stress
Small RNAs for Stress Response
Temperature tolerance
Case Study- 1
   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.
Role of Hsp70 Chaperone machine
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
Results
Temperature tolerance
   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.
Case Study- 2
   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.
   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.
Expression Profiles Under Wounding and Heat Stress
   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.
Case Study- 3
   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.
   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 .
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
Temperature tolerance
Contd.
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.
Case Study- 4
   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).
GB as a protectant for PSII Complex
   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.
Western Blot Analysis
Comparison of GB Level
Effect on CO2 Assimilation
Effect on Rubisco Activation
   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.
Applied Biotechnology
Temperature tolerance
Temperature tolerance
Temperature tolerance

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Temperature tolerance

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