CsROS1b regulates autophagy through inducing hypomethylation of <i>CsATG6</i> to enhance salt tolerance in cucumber

Autophagy, an evolutionarily conserved mechanism for cellular homeostasis, is essential for plant adaptation to abiotic stresses. DNA methylation is well-established as an epigenetic regulator of development and stress responses; however, its direct modulation of autophagy under salt stress remains unexplored. In this study, we found that a salt-sensitive cucumber (Cucumis sativus L.) cultivar exhibited higher accumulation of ubiquitinated proteins correlating with a lower level of autophagic activity compared to a salt-tolerant cucumber cultivar under salt stress. Whole-genome bisulfite sequencing analysis revealed that salt stress induced CG hypomethylation in the coding sequence (CDS) region of Autophagy-related gene 6 (CsATG6) in cucumber, coinciding with its upregulation and autophagy activation. The CDS hypomethylation of CsATG6 was induced by DNA methylation inhibitor 5-azacytidine (5-Aza), resulting in its transcriptional upregulation, which was accompanied by increased autophagic activity and decreased accumulation of ubiquitinated proteins. Silencing of CsATG6 decreased autophagic activity and salt tolerance, whereas its overexpression enhanced both. Loss of function of CsATG6 compromised 5-Aza-induced autophagic activity and salt tolerance. Furthermore, salt stress and 5-Aza co-induced Repressor of silencing 1b (CsROS1b) expression, which actively demethylated CsATG6 at CG sites and increased CsATG6 transcript abundance, enhancing autophagosome formation and reducing ubiquitinated protein accumulation. Our study reveals a mechanism by which CsROS1b-mediated CG hypomethylation of CsATG6 activates autophagy to enhance salt stress tolerance in cucumber.