Glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants but its involvement in modulating subcellular Fe homoeostasis remains elusive. In this study, we report that the GSH-deficient mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant downregulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4, and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe levels in vacuoles but lower Fe levels in chloroplasts compared to wild type (Columbia ecotype [Col-0]) under Fe limited conditions. Exogenous GSH treatment enhanced chloroplast Fe contents in Col-0 but failed to do so in the nramp3nramp4 double mutants demonstrating that GSH plays a role in modulating subcellular Fe homoeostasis. Pharmacological experiments, mutant analysis, and promoter assays revealed that this regulation involves the transcriptional activation of Fe transporter genes by a GSH-S-nitrosoglutathione (GSNO) module. The Fe responsive bHLH transcription factors (TFs), AtbHLH29, AtbHLH38, and AtbHLH101 were found to interact with the promoters of these genes, which were, in turn, activated via S-nitrosylation (SNO). Taken together, the present study highlights the role of the GSH-GSNO module in regulating subcellular Fe homoeostasis by transcriptional activation of the Fe transporters AtNRAMP3, AtNRAMP4, and AtPIC1 via SNO of bHLH TFs during Fe deficiency.
Glutathione (GSH) is a ubiquitous molecule known to regulate various physiological and developmental phenomena in plants. Recently, its involvement in regulating iron (Fe) deficiency response was established in Arabidopsis. However, the role of GSH in modulating subcellular Fe homeostasis remained elusive. In this study, we dissected the role of GSH in regulating Fe homeostasis in Arabidopsis shoots under Fe limited conditions. The two GSH depleted mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with smaller rosette diameter and higher chlorosis level compared with the Col-0 plants. Interestingly, the expression of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4, chloroplast Fe importer, AtPIC1, along with AtFer1 and AtIRT1 were significantly down-regulated in these mutants. The expression of these genes were up-regulated in response to exogenous GSH treatment while treatment with BSO, a GSH inhibitor, down-regulated their expression. Moreover, the mutants accumulated higher Fe content in the vacuole and lower in the chloroplast compared with Col-0 under Fe limited condition suggesting a role of GSH in modulating subcellular Fe homeostasis. This regulation was, further, found to involve a GSNO-dependent pathway. Promoter analysis revealed that GSH induced the transcription of these genes presumably via S-nitrosylation of different Fe responsive bHLH transcription factors.
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