7.seminar mawuli k. azameti

Heat wheat

1.   AKSHAY S. SAKHARE SUDHIR KUMAR DIVISION OF PLANT PHYSIOLOGY ICAR - INDIAN AGRICULTURAL RESEARCH INSTITUTE ROLE OF HEAT RESPONSIVE GENES FOR STRESS TOLERANCE IN WHEAT

2. INTRODUCTION Its production is essential for global food supply Wheat is one of the most widely grown crops in the world

3. Wheat is a cool season crop Optimal daytime growing temperature during reproductive development is 15 °C For every degree Celsius above this optimum a reduction in yield of 3%–4% has been observed Global temperature is reported to be increasing at a rate of 0.18°C every decade 

4. Impact of heat stress on wheat

5. .. A: Avoidance T: Tolerance Different adaptation mechanisms to high temperature

6. Basal thermotolerance Intrinsic tolerance to high temperatures induced damage Acquired thermotolerance Resources mobilization and gene expression changes to cope with heat stress related injury Thermotolerance

7. Stress response: Up-tick in the synthesis and the accumulation of heat shock proteins (HSPs) HSP synthesis and accumulation during high temperature stress has frequently been linked to the expression of thermotolerance A characteristic of all sHSPs is the presence of N-terminal domains and C-terminal domains The N terminal domain participates in substrate binding and binds denatured proteins while the C terminal domain is involved in the homo- oligomerization and the formation of high temperature stress granules

8. Classification and Functions of HSPs

9. Farooq et al., 2011 Signalling cascades for thermotolerance

10. A subset of protective proteins response to heat stress has been identified in wheat For example; Small heat shock protein (sHSP26) involved in seed maturation and germination, and imparted tolerance to heat stress Over expressing TaHsfA6f in wheat showed improved thermotolerance TaWRKY1 and TaWRKY33 transgenic wheat plants exhibited enhanced tolerance to heat stress More remain to be identified and characterized to effectively elucidate the mechanism of thermotolerance

11. CASE STUDY

12. The family of gibberellic acid-stimulated transcript (GAST) have been found in various plant species and are common among eukaryotes All code for small protein with signal peptide at the N terminus and a conserved C-terminal region of about 60 amino acids containing 12 cystein residues in conserved positions Most GAST homologues have been found to be GA-responsive genes The first GAST gene was isolated from tomato ( Lycopersicon esculentum ) Several studies have suggested that these proteins have diverse roles in plant development and response to environmental stress

13. Isolation and Characterization of TaGASR1 A probe “ Ta.11162 ” was previously identified The probe shared high degrees of homology to GAST family proteins The associated 297bp ORF of this gene encoded a 98-kDa protein with 51.52% identity to OsGASR1 Probe named as TaGASR1

14. GAST proteins belong to two clades TaGASR1 , OsGASR1 and SNAKIN-1 were clustered into one group Phylogenetic relationship between well characterized GAST family proteins TaGASR1 might carry out a similar function with the other GAST family genes

15. Cloning and Cis -element Analysis of the 5’-flanking Region of TaGASR1 A 1343-bp genomic fragment upstream of the ATG was obtained The sequence was then submitted to PlantCARE The sequence contained one heat shock element (HSE), and several other abiotic stress and hormone responsive elements

16. Abiotic stress and hormone responsive elements include One TC-rich repeat involved in defense and stress responsiveness Two motifs (CGTCA-motif and TGACG-motif) involved in MeJA -responsiveness One TCA element involved in SA One gibberellin responsive element (GARE)

17. Expression Patterns of TaGASR1 under Heat Stress The expression levels of TaGASR1 immediately increased after heat treatment

18. The transcript abundance of TaGASR1 was also strongly induced by other stress factors TaGASR1 gene might be involved in a series of abiotic stress response and hormone signal transduction pathways

19. A vector for TaGASR1 -GFP fusion proteins were expressed under the regulation of the cauliflower mosaic virus ( CaMV ) 35S promoter Transiently expressed in onion epidermal cells Subcellular Localization of TaGASR1

20. The transformed cells showed fluorescence of the green fluorescent protein (GFP) in the edge of the cell area, including the cell wall, apoplasm , cell membrane and cytosol The control GFP protein was uniformly localized in the nucleus and cytoplasm

21. Precise position of TaGASR1 TaGASR1 proteins were localized to the cell membrane or cytosol The fluorescence occurred mainly in the cell membrane and slightly in cytosol

22. Ectopic Expression of TaGASR1 Enhanced Thermotolerance in Arabidopsis The gene was over expressed in two Arabidopsis transgenic lines with high expression levels of TaGASR1 The transgenic lines showed a WT phenotype during all phases of the growth cycle

23. After heat stress, the TaGASR1 overexpressing plants exhibited more resistant to heat, compared with WT Approximately 80% of transgenic plants survived after 5 d of recovery, while less than 60% of WT plants survived

24. The leaves’ electrical conductivity for each transgenic line before (T1) and after heat stress (T2) was measured and the CMT was calculated according to the equation: CMT= T1/T2 Cell Membrane Thermostability (CMT) The transgenic lines showed higher level of CMT than WT, which means the cell membrane was more stable in overexpressed plants

25. Using 3,3’- diaminobenzidine (DAB) staining and Nitrotetrazolium blue chloride (NBT) as the chromogenic substrate DAB is oxidized by H 2 O 2 in the presence of peroxidases and produces reddish brown precipitate NBT reacts with O 2 − to form a dark blue insoluble formazan compound 3-week-old Arabidopsis leaves were used for the staining before or after heat treatment for 8 hrs under dark conditions Ectopic Expression of TaGASR1 in Arabidopsis Reduced Accumulation of ROS after Heat Stress

26. The staining in the TaGASR1 -overexpressing leaves was weaker than that in WT after heat stress The results showed that TaGASR1 overexpressing plants have a strong ability to resist ROS accumulation induced by heat stress and therefore can survive by the low level of ROS accumulation after heat stress.

27. Overexpression of TaGASR1 in wheat Enhanced Tolerance to Heat Stress and Oxidative Stress Three transgenic lines (#1, #2 and #3) with higher expression levels were chosen

28. No visible phenotypic differences between transgenic lines and the control YM16 seedlings under normal conditions

29. Grain weight of the main spike under normal and heat stress conditions Transgenic lines and YM16 showed the same level in grain weight of the main spike Transgenic plants displayed higher levels of grain weight of the main spike These results suggested that overexpressing TaGASR1 enhanced tolerance to heat stress in wheat

30. Treated 10- d-old wheat by mM H 2 O 2 for 12 hrs and measured the primary root length Overexpression of TaGASR1 in wheat reduce the damage caused by H 2 O 2 There was no obvious difference between transgenic lines and control YM16 under normal conditions

31. After treatment, the primary root was significantly longer in transgenic lines than that of YM16 These results suggested the possible ability of TaGASR1 in defending the oxidative stress

32. Future Studies... Additional studies must be conducted to illustrate whether the TaGASR1 gene affects the accumulation of heat shock proteins or other proteins Further research must also be carried out to determine the mechanism of ROS removal from the cell by the TaGASR1 gene

33. Heat tolerance is quantitatively inherited and is strongly affected by environmental factors Further, heat tolerance is sensitive to growth stage and tolerance at one growth stage may not be correlated with tolerance at other growth stages In addition, there is no unfailing selection criterion for thermotolerance that may be used over the years and for the generations Hence, direct selection for heat tolerance becomes extremely difficult by traditional breeding because of large genotype × environment interaction, lack of effective tolerance genes in different genetic backgrounds, different expression of tolerance CONCLUSION

34. Increasing knowledge about molecular mechanisms of heat tolerance is likely to pave the way for engineering wheat with satisfactory economic yield under heat stress Although several genes have been successfully engineered in wheat to enhance tolerance to heat stress their function in different genetic backgrounds and under different heat stress conditions needs to be investigated