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 population's diet, but increasingly frequent  droughts threaten its productivity and food security. While massive transcriptional reprogramming under drought in wheat seedlings is well characterised, DNA methylation's 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. Overall design: Fourteen landraces where used to asses the wheat DNA methylation response to drought. The experiment was performed by growing the plant until GS13 (Zadok's growth scale) and applying the drought stress during 10 days (withhold of water) and three days of recovery (normal watering) after that. DNA from 7 landraces (4 replicates per landrace) was pooled per sample per condition in equimolar concentrations. BD contains pooled DNA from tissue collected before drought treatment; AD is from samples after drought. DNA samples were sequenced using whole-genome bisulphite sequencing, aligned using Bismark, and analysed using DSS-Single.