GSNOR-dependent nitric oxide homeostasis shapes methylome persistence after multi-generational exposure to future climate conditions in Arabidopsis thaliana

Environmental stress can modify plant DNA methylation, but the roles of nitric oxide homeostasis in methylome recovery and multigenerational persistence remain unclear. We used whole-genome bisulfite sequencing to compare Arabidopsis thaliana wild type and the GSNOR-deficient mutant (gsnor1-3) exposed to drought, elevated CO2, ozone, heat, or their combination during generation G1 to G3, followed by two generations under control conditions. Methylomes from G1, G3, and G5 were analysed for global divergence, differentially methylated regions, genomic annotation, and methylation trajectories and were integrated with matched RNA-seq and independent datasets. Control methylomes of gsnor1-3 were already distinct from those of the wild type, and the mutant accumulated substantially more DMRs, with a strong hypermethylation bias. In gsnor1-3, 44.1 to 45.7% of G1 CG DMRs retained their direction through G3 and G5, compared with 0.09 to 1.24% in wild type; persistent CHG and CHH DMRs were also markedly more abundant. Persistent regions occurred at genes, promoters, and transposable elements and were associated with broader expression responses in the mutant. Independent datasets supported a subset of these regions, including directional agreement for 211 of 292 published GSNOR-associated DMRs. These results indicate that GSNOR-dependent nitric oxide homeostasis contributes to methylome stability and resetting after repeated environmental exposure.