DNA methylation plays an important role against adverse environment by reshaping transcriptional profile in plants. To better understand the molecular mechanisms of watermelon response to osmotic stress, the suspension cultured watermelon cells were treated with 100mM mannitol, and then a methylated cytosines map was generated by whole genome bisulfite sequencing (WGBS). Combined with transcriptome sequencing, the effects of osmotic stress on differentially methylated expressed genes (DMEGs) were assessed. It was found that genes related to plant hormone synthesis, signal transduction, osmoregulatory substance-related and reactive oxygen species scavenging-related enzyme could rapidly respond to osmotic stress. The overall methylation level of watermelon decreased after osmotic stress treatment, and demethylation occurred in CG, CHG, and CHH contexts. Moreover, differentially methylated expressed genes (DMEGs) were significantly enriched in RNA transport, starch and sucrose metabolism, plant hormone signal transduction and biosynthesis of secondary metabolites, especially in biosynthesis of osmolytes synthase genes. Interestingly, demethylation of a key enzyme gene Cla014489 in biosynthesis of inositol upregulated its expression and promoted accumulation of inositol, which could alleviate the inhibition of cell growth caused by osmotic stress. Meanwhile, a recombinant plasmid pET28a-Cla014489 was constructed and transferred into Escherichia coli BL21 for prokaryotic expression and the expression of ClMIPS protein could improve the tolerance of E. coli to osmotic stress. The effect of methylation level on the expression properties of inositol and its related genes was further confirmed by application of DNA methylation inhibitor 5-azacytidine. These results provide a preliminary insight into the altered methylation levels of watermelon cells in response to osmotic stress and suggest a new mechanism that how watermelon cells adapt to osmotic stress.
Melatonin, a multifunctional signaling molecule, has been shown to play a significant role in response to abiotic stress. Several species have been reported to unveil melatonin’s effect on osmotic stress; however, the signal transduction mechanism of phytohormone-mediated melatonin biosynthesis in plant species remains unclear. In this study, although plants can biosynthesize melatonin, the exogenous application of melatonin to watermelon cells can improve cell growth in response to osmotic stress by regulating the antioxidant machinery of cells. Regarding the melatonin synthesis pathway, ClOMT (ClASMT and ClCOMT) is a multi-gene family, and ClSNAT has two members. Both ClOMTs and ClSNATs harbor the cis-elements in their promoter regions responding to various hormones. Among abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), ABA treatment observably upregulated the expression of ClOMTs and ClSNATs, and the accumulation of melatonin with ABA treatment reached a level comparable to that with osmotic stress by mannitol treatment. Furthermore, when hormone biosynthesis inhibitors were added to cells before osmotic stress, the expression of ClOMTs and ClSNATs, as well as melatonin accumulation, were significantly suppressed with the ABA biosynthesis inhibitor. This study demonstrated the crucial role of melatonin biosynthesis in response to osmotic stress via plant hormone signal transduction. It showed that ABA signaling plays a dominant role in melatonin synthesis under osmotic stress.
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