DNA Methylation Dynamics Reveal Unique Plant Responses and Transcriptional Reprogramming to Combined Heat and Phosphate Deficiency Stress

Abstract Plants adapt to environmental challenges through epigenetic mechanisms that modulate gene expression without altering DNA sequence. Among these, DNA methylation is central to balancing genome stability and transcriptional flexibility. We analyzed methylation dynamics in Arabidopsis thaliana under heat, phosphate deficiency, and their combination—conditions that frequently co-occur in nature—using whole-genome bisulfite sequencing, small RNA-seq and RNA-seq of shoots and roots in a setup closely mimicking field conditions. Stress-specific patterns emerged: heat and combined stress led to CHH hypomethylation in both shoot and root, while phosphate deficiency triggered hypermethylation in shoots but hypomethylation in roots. Importantly, the epigenetic response to combined stress was not a mere additive effect of individual stresses but displayed a distinct methylation signature. While both RdDM and CMT2 pathways contributed to heat-induced changes, CMT2 predominated under phosphate deficiency and combined stress, underscoring mechanistic specificity. Methylation changes concentrated in transposable elements (TEs) and intergenic regions, yet TE methylation shifts showed limited correlation with TE or adjacent gene expression, suggesting methylation does not act as a direct transcriptional switch. Instead, stress-induced methylation may influence chromatin accessibility at regulatory regions, particularly transcription factor binding sites; GATA motifs appeared as especially relevant in our analyses. A striking signature emerged in the nuclear mitochondrial DNA (NUMT) region, where hypomethylation under heat and combined stress correlated with upregulation of oxidative phosphorylation genes, critical for thermotolerance. Our findings highlight DNA methylation as an intricate regulatory layer integrating environmental signals into plant adaptive responses, offering a foundation for strategies to harness epigenetic plasticity for crop resilience under climate change. Competing Interest Statement The authors have declared no competing interest.