Exogenous glutathione can alleviate chromium toxicity in kenaf by activating antioxidant system and regulating DNA methylation

Glutathione (GSH) participates in plant response to heavy metals (HMs) stress, however, the epigenetic regulating mechanisms of GSH in HMs detoxification remains unclear. In this study, to reveal the potential epigenetic regulating mechanisms, kenaf seedlings were treated with/without GSH under chromium (Cr) stress. A comprehensive physiological, genome-wide DNA methylation and gene functional analysis were performed. Results showed that external GSH obviously recovered Cr-induced growth inhibition, significantly decreased H2O2, O2.- and MDA accumulation, increased the activities of antioxidant enzymes (SOD, CAT, GR and APX) in kenaf exposed to Cr. In addition, the expression level of the main DNA methyltransferase (MET1, CMT3 and DRM1) and demethylase (ROS1, DEM, DML2, DML3 and DDM1) genes were investigated by qRT-PCR. The result indicated that Cr stress decreased DNA methyltransferase genes expression while increased demethylase genes expression; however, apply exogenous GSH led to the recovery trend. These indicating exogenous GSH alleviation Cr stress on kenaf seedlings by increasing DNA methylation level. At the same time, the MethylRAD-seq genome-wide DNA methylation analysis showed the DNA methylation level was significantly increased after GSH treatment compared with Cr treatment alone. The differentially methylated genes (DMGs) were uniquely enriched in DNA repair, flavin adenine dinucleotide binding and oxidoreductase activity. Furthermore, a ROS homeostasis-associated DMG, HcTrx, was selected for further functional analysis. Results showed that the knock-down of HcTrx kenaf seedlings displayed yellow-green phenotype and impaired antioxidant enzyme activity; in contrast, the overexpression lines of HcTrx improved chlorophyll levels and enhanced Cr tolerance in Arabidopsis. Taken together, our results illustrate the novel role of GSH-mediated Cr detoxification in kenaf by modulating the DNA methylation, and thus further affect the activation of antioxidant defense systems. The present characterized Cr tolerant gene resource could be further used for kenaf Cr tolerant breeding via genetic improvement.