Combined Abiotic Stress Rewires DNA Methylation to Modulate Regulatory Potential Rather Than Transcription in Arabidopsis.

Abstract

Plants are simultaneously exposed to multiple abiotic stresses, yet how opposing epigenetic signals are integrated remains unclear. Here, we investigated the combined effects of heat stress and phosphate deficiency in Arabidopsis thaliana, two conditions with antagonistic impacts on DNA methylation. Using an integrated multi-omic approach (WGBS, RNA-seq and small RNA-seq) in roots and shoots under field-mimicking conditions, we show that combined stress establishes a distinct, non-additive epigenomic state characterised by widespread CHH hypomethylation and differential engagement of RdDM and CMT2 pathways. Despite extensive methylation remodelling across transposable elements, genes and intergenic regions, we observe a genome-wide decoupling between DNA methylation dynamics and transcriptional output. Notably, differentially methylated regions are enriched in transcription factor binding sites, particularly GATA motifs, linking CHH hypomethylation to increased chromatin accessibility and activation of GATA-associated regulatory networks. In contrast, nuclear mitochondrial DNA insertions represent a notable exception, where CHH hypomethylation is directly coupled to transcriptional activation. Functional analyses reveal distinct contributions of RdDM and CMT2 to basal silencing and stress responsiveness at these loci. Overall, our results support a model that positions DNA methylation as a regulator of regulatory potential rather than a direct determinant of transcription under combined stress.