DNA hypomethylation induced by drought preconditioning improved physiological responses and promoted tiller growth of perennial ryegrass

Previous studies have shown that perennial ryegrass exhibits superior performance under drought preconditioning (DP) compared to continuous drought stress (DR) or even well-watered conditions (WW), however, the underlying mechanisms remain unclear. This study aims to elucidate if changes in DNA methylation could be the mechanism behind preconditioning, particularly studying its contribution to plant physiological responses and tiller growth. Dynamic alterations in DNA methylation levels in the crowns of perennial ryegrass were detected at 14, 17, 30, 33, and 44 days under WW, DP, and DR by the methylation-sensitive amplification polymorphism (MSAP) method. DNA methylation level was significantly elevated after 14 days of drought stress. Conversely, DP treatment induced DNA hypomethylation, with MSAP% at DP 30 , DP 33 , and DP 44 being lower than DR and comparable to WW. Supplementing S-adenosylmethionine (SAM), a methyl group donor, counteracted the beneficial effects of DP, evidenced by reduced chlorophyll content, aboveground biomass, net photosynthesis rate (Pn), and tiller number in DPS (DP + SAM) compared to DP. In contrast, applying 5-azacytidine (5-azaC), a DNA methylation inhibitor, mimicked the DP effect, increasing leaf relative water content, aboveground biomass, Pn, and tiller number while reducing electrolyte leakage rate. Relative expression levels of DNA methylase and demethylase genes under SAM and 5-azaC treatments supported the dynamic changes of DNA methylation level in those plants. Meanwhile, expression levels of methylation genes were significantly correlated with the physiological parameters. This study provides the first evidence for the dynamic role of DNA methylation regulating perennial ryegrass physiological and tiller growth underlying the drought preconditioning process. • Drought preconditioning induces DNA hypomethylation in perennial ryegrass crowns. • SAM counteracts, while 5-azaC mimics, the benefits of drought preconditioning. • DNA methylation dynamics critically regulate tiller growth under drought preconditioning. • Methylation-related gene expression correlates with key physiological traits.