DNA methylation mediates transcriptional stability and transposon-driven trans-regulation under drought in wheat

Bread wheat (Triticum aestivum) is a major component of half the global populations diet, but increasingly frequent droughts threaten its productivity and food security. While massive transcriptional reprogramming under drought in wheat seedlings is well characterised, DNA methylations contribution remains poorly understood. Using paired whole-genome bisulphite sequencing (WGBS) and RNA-seq before and after drought stress in wheat landraces, we probed the nuanced role of DNA methylation in the drought response, uncovering antagonistic trends between cytosine contexts and novel mechanisms, with the ROS1a family potentially playing a key demethylation role under drought. Examination of gene methylation profiles revealed that gene body methylation was strongly positively correlated with gene expression but negatively with stress responsiveness, simultaneously identifying that gene body differentially methylated regions (DMRs) targeted stress-associated genes. Many DMR-associated genes maintained consistent transcription under stress, suggesting a stabilising role for DNA methylation. Most DMRs localised to intergenic regions and transposable elements (TEs), with the ancient LTR retrotransposon RLX_famc9 emerging as a critical target of differential methylation under drought. We propose a model in which the RLX_famc9 family, enriched in differential methylation and exhibiting substantial sequence similarity to drought-responsive genes, is involved in the trans-regulation of stress-associated genes under control conditions through the generation of regulatory siRNA precursors, a mechanism suppressed by drought-inducible hypermethylation. Our findings suggest an intricate regulatory role of DNA methylation under drought, with genic DNA methylation promoting high, stable expression, ROS1a glycosylases coordinating targeted demethylation, and methylation-controlled TEs modulating the expression of downstream genes in trans.