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The contribution of glutathione (GSH) in combating environmental stress in plants has long been known. Previous reports have pointed to the involvement of GSH in inducing various heat shock proteins (HSPs), but the molecular mechanism is yet to be explored. Here, we investigate how GSH induces the expression of important HSP genes in Arabidopsis. Expression of HSP genes BiP3, HSP70B, and HSP90.1 was positively regulated by GSH, and a promoter activation assay suggested a role for GSH in their induction. Lower expression of BiP3 and HSP70B in the GSH-fed Atmyb21 mutant and of HSP90.1 in the GSH-fed Atbzip10 mutant, in comparison with GSH-fed Col-0, revealed a role for GSH in activating their promoters through the transcription factors MYB21 and BZIP10. Co-transfection of transcription factor mutant protoplasts with transcription factor constructs and HSP promoters confirmed the results. Comparative proteomics also revealed proteins whose expression was controlled by MYB21 and BZIP10 in response to GSH feeding. A co-immunoprecipitation assay demonstrated a role for GSH in modulating the level of interaction of glutathione-S-transferase with HSP70. Collectively, our results demonstrate a role for GSH in activating the promoters of BiP3 and HSP70B via MYB21 and of HSP90.1 via BZIP10. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) plays a fundamental role in plant defense. Recent reports showed that enhanced GSH content activates mitogen-activated protein kinases (MPKs). However, the molecular mechanism behind this GSH-mediated MPKs expression during environmental challenges is unexplored. Here, we found that under control and combined abiotic stress-treated conditions, GSH feeding activates MPK3 expression in Arabidopsis thaliana by inducing its promoter, as established through the promoter activation assay. Additionally, transgenic A. thaliana overexpressing the LeMPK3 gene (AtMPK3 line) showed increased γ-ECS expression, which was decreased in mpk3, the MPK3-depleted mutant. An in-gel kinase assay exhibited hyperphosphorylation of Myelin Basic Protein (MBP) in the GSH-fed AtMPK3 transgenic line. Under control and combined abiotic stress treated conditions, expression of transcription factor WRKY40 binding to MPK3 promoter was up-regulated under enhanced GSH condition. Interestingly, GSH feeding was rendered ineffective in altering MPK3 expression in the Atwrky40 mutant, emphasizing the involvement of WRKY40 in GSH-MPK3 interplay. This was further confirmed by a wrky40 co-transformation assay. The immunoprecipitation assay followed by ChIP-qPCR showed a significant increase in the binding of WRKY40 to MPK3 promoter, which further established MPK3-WRKY40 association upon GSH feeding. In conclusion, this study demonstrated that GSH modulates MPK3 expression via WRKY40 in response to stress. • Deciphering the role of GSH-MPK3 interplay in plant defense system during environmental stress. • Altered GSH content in plants positively modulates the expression of MPK3 both at transcript and protein level. • This modulation was observed to take place via WRKY40 transcription factor. • Elevated GSH level increased the WRKY40 binding to MPK3 promoter, consequently activating MPK3 during stress. [ABSTRACT FROM AUTHOR]