Research Projects

This project mainly aims to identify specific CBL interacting protein kinases, operative under heat stress and how they can possibly drive thermotolerance responses by regulating the HS “effectors”, specifically the master HS regulator, transcription factor, HsfA1d in Arabidopsis as a model plant system. It is speculated that HsfA1d regulates transctivation of heat shock proteins (Hsps) and it is most possibly regulated by a yet unknown phosphoregulatory mechanism. This project focuses on identifying and functionally characterizing the calcium signaling modules and how they are involved in mediating HS responses via phosphoregulation of HsfA1d. We hypothesize that CIPKs, especially AtCIPK21 may be involved in the phosphorylation of key residues of HsfA1d and may possibly be involved in the disssociation of Hsp70 from the TDR (temperature dependent repression) region to relieve HsfA1d repression under HS conditions. We intend to phenotypically screen different CBLs/CIPKs mutant lines (available in our laboratory) for the basal thermotolerance stress regime and analyze for sensitivity/tolerance followed by validating interactions between putative kinases and HsfA1d using yeast two-hybrid system, BiFC, GST pull-down assays and Co-immunoprecipitation.We will identify the phosphosites on HsfA1d phosphorylated by CIPKs via LC/MS and generate phosphomutants for those particular sites using site-directed mutagenesis which will be characterized for their thermotolerance, Hsp101 transctivation, protein stability and subcellular localization. Prior work in the our lab has revealed that mutants of CIPK21, were found to be sensitive to heat stress when compared to Col-0. Yeast two-hybrid screening has revealed that AtCIPK21 interact with HsfA1d. AtCIPK3 was also found to interact with AtHsfA1d, which is also indicated to be a negative regulator of HS responses via preliminary phenotypic screening done in our lab. This project is a novel attempt to identify specific Calcineurin B-like interacting protein kinases (CIPKs) that may be playing a role in HS signal transduction. Also, it will be interesting to check whether any HS elements, particularly Class A Hsfs, are putative targets of these kinases. To date, no studies have clearly established a link between any calcium signaling component and HS-specific genes.Consequently, there is no clarity on how these transcription factors, especially HsfA1d, a master HS response co-ordinator in Arabidopsis are being regulated via phosphorylation. As we expect to gain insights into this very aspect from this project, it will provide a detailed understanding of the operation of this module in the model plant system Arabidopsis. These modules can then be extrapolated and validated to identify orthologous members in rice/wheat and other crop plants, which can be utilized to generate gene-edited rice lines for improved thermotolerance and yield. Identification of precise residues that, when phosphorylated, enhance HsfA1d activity in Arabidopsis, will help develop a strong base for identification of similar modules operative in crop plants like rice and wheat, enabling precise gene editing to develop gene-edited varieties tolerant to heat stress under field conditions.