| Rice(Oryza sativa L) is considered as the most important food crop of the world. It has been the main source of human nutrition for more than10thousand years. Several environmental factors may affect rice yield, among which heat stress is the most important. Enormous increase in ambient temperature can cause significant reduction in grain yield in rice. Global warming has been proved to be a serious threat to the crop plants during previous century and an increase in global mean temperatures is expected by the end of current century in various regions of the world. This situation has propelled the attention of crop scientists towards the development of heat-tolerance, especially in case of cereals. In addition to being a major cereal crop, rice is considered as a model monocots plant for molecular studies, as well. Its genome has been fully sequenced, so it is very reliable to study stress responsive genes/proteins for understanding their involvement in stress response mechanism through molecular characterization and transgenic development. The results can be then simulated for other crops with great confidence. We characterized three gene families involved in stress mechanism of rice, viz. heat shock factor binding protein (HSBP), the Bcl-2-associated athanogene (BAG) and autophagy protein6(ATG6). A detailed functional analysis of rice HSBP genes was done in transgenic and mutant lines of these genes to elucidate their involvement in stress response mechanism. Furthermore, six BAG genes and three ATG6genes were identified in rice using different bioinformatic tools and their expression profile was also studied under heat stress. The main results are described as follows:The role of HSBP genes in rice (OsHSBP) is first unveiled in the present study. Two homologs of OsHSBP (described as OsHSBPl and OsHSBP2in proceedings) were identified in preliminary study. Both proteins were predicted to contain potential coiled-coils, as well as highly conserved a-helix region. OsHSBP1and OsHSBP2proteins were found to be localized in cytoplasm, as well as in nucleus. Yeast two-hybrid assay revealed that both the genes showed self-binding ability, probably making trimer and hexamer. Both the genes exhibited similar functions as both were induced by heat stress. Heat shock (HS) of42℃for1h slightly upregulated the expression of OsHSBPl and OsHSBP2. Their expression was significantly increased during the recovery phase and reached its maximum level at2h after recovery; however, it returned to the basal level at4h after recovery. Both genes were ubiquitously expressed in all the tissues. However, maximum expression of both the genes was observed in panicle. Furthermore, knock-down lines of both the genes showed significant seed abortion, hence can be considered to play an important role during seed development.The survival of over-expressed seedlings of both genes was significantly decreased, whereas those of knocked-down and mutant seedlings significantly increased, as compared to their respective wild-types during thermotolerance assay. Expression analysis of heat specific HSPs showed significant increase in the expression of OsHSP16.9, OsHSP17.5and OsHSP70in OsHSBPl knockdown lines, while OsHSP82in OsHSBP2knock-down lines. This result indicated the negative regulation of heat tolerance by OHSBP1and OsHSBP2. Furthermore, antioxidant activity was determined as an indicator of thermotolerance, which revealed a significant increase in case of knock-down lines. Catalase (CAT) and Peroxidase (POD) activity was significantly increased in knock-down lines of both the genes. Similar results were observed when expression of CAT and POD genes was analyzed. This result further confirms the involvement of OHSBP1and OsHSBP2in thermotolerance as negative regulators. Overall, these studies provide an insight into functional basis of HSBP homologs in rice and reveal that both genes are negative regulators of HSR and required for seed development in rice.In the present study, we identified six BAG genes in rice (OsBAG). These genes showed conservation in HSP70binding domain region. Expression analysis of OsBAG genes through microarray data showed that OsBAG genes are up or down regulated under different biotic and abiotic stresses. OsBAG genes were analyzed for their expression under heat stress, as well as in different tissues in rice through real-time PCR. All OsBAG genes were expressed differentially in different tissues. The expression of OsBAGl, OsBAG4and OsBAG6genes was observed at maximum level in culm, while OsBAG2expressed at very low level with maximum expression in panicle. Expression level of OsBAG3was observed at maximum level in node and panicle, while OsBAG5expressed only in root and leaf with highest expression in leaf. Analysis of OsBAG expression under heat stress showed that maximum expression of all the genes was observed at1h of heat stress treatment (42℃), after which the expression kept on decreasing, and it returned to the basal level at24h treatment (42℃). These results suggest that OsBAG genes might play an important role at the onset of heat stress. Further detailed study will explore the exact function of the members of this gene family and help to make understanding their role in HSR.Accumulation of misfolded proteins in abundance resulting from severe stress triggers autophagosome mediated autophagy, and autophagy related protein6(ATG6) plays a key role in autophagosome formation. Our analysis of ATG6/Beclin-1proteins, revealed significant functional divergence while assessing across the species phylogenetic and evolutionary relationship. Presence of important stress related c/s-acting elements in the promoters region of rice ATG6genes depicted their involvement in abiotic stress responses. Real-time PCR analysis of ATG6genes showed that OsATG6a showed maximum expression in panicle; OsATG6b expressed equally in all the tissues; while, OsATG6c showed maximum expression in root, internode and panicle. Furthermore, the expression profiling of rice ATG6genes based on microarray data, as well as by real-time PCR showed differential expression under different stresses suggesting their involvement in abiotic stresses (heat, cold and drought) responses. Analysis of co-expressed genes showed that most of them annotated to DNA repair pathways and proteolysis, etc. Collectively, these results suggest the involvement of OsATG6genes in different stresses, and provide basis for further functional studies to investigate the biological mechanism of action of these genes under abiotic stresses in rice.Conclusively, we studied three gene families involved in stress response mechanism. HSBPs modulate the expression of HSPs by binding to HSFs, while BAG proteins modulate HSPs negatively or positively during stresses. If damage caused by stress is beyond the limits of protein repair mechanism, autophagy related proteins (ATG) are activated, ATG6take part in the formation of autophagosome, where protein degradation takes place. Our studies revealed the involvement of these three gene families in stress response mechanism. OsHSBPs are found as negative regulators of thermotolerance as well as essential for seed development. In silico studies, phylogeny, protein structural modeling and expression analysis of OsBAG and OsATG6gene families revealed that these families might be functional homologs in rice, and play important role in stress response mechanism. Overall, our results will provide an insight and understanding of the complex stress response mechanism in rice. |
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