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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]

Abstract: The role of glutathione (GSH) in plant defense is an established fact. However, the association of GSH with other established signaling molecules within the defense signaling network remains to be evaluated. Previously we have shown that GSH is involved in defense signaling network likely through NPR1-dependent salicylic acid (SA)-mediated pathway. In this study, to gain further insight, we developed chloroplast-targeted gamma-glutamylcysteine synthetase (γ-ECS) overexpressed transgenic Nicotiana tabacum (NtGp line) and constructed a forward subtracted cDNA (suppression subtractive hybridization (SSH)) library using NtGp line as a tester. Interestingly, in addition to SA-related transcripts like pathogenesis-related protein 1a (PR1a) and SAR8.2 m/2l, 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase), a key enzyme of ethylene (ET) biosynthesis, was identified in the SSH library. Besides, transcription factors like WRKY transcription factor 3 (WRKY3), WRKY1 and ethylene responsive factor 4 (ERF4), associated with SA and ET respectively, were also identified thus suggesting an interplay of GSH with ET and SA. Furthermore, proteomic profiling of NtGp line, performed by employing two-dimensional gel electrophoresis (2-DE), corroborated with the transcriptomic profile and several defense-related proteins like serine/threonine protein kinase, and heat shock 70 protein (HSP70) were identified with increased accumulation. Fascinatingly, induction of 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) was also noted thus demonstrating the active involvement of GSH with ET. Protein gel blot analysis confirmed the enhanced accumulation of ACC oxidase in NtGp line. Together, our data revealed that GSH is involved in the synergistic multiple steps crosstalk through ET as well as SA to combat environmental stress. [Copyright &y& Elsevier]