Integrative methylome and transcriptome analysis reveals genotype and sequence context-specific responses to aluminum stress in rice.

INTRODUCTION: Aluminum (Al) toxicity in acidic soils is a major constraint to rice production worldwide. However, the epigenetic mechanisms underlying genotypic differences in Al tolerance remain largely unexplored.

METHODS: We integrated whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) to characterize DNA methylation dynamics and their relationship with gene expression in two contrasting Oryza sativa genotypes, Al-tolerant Azucena and Al-susceptible BGI, exposed to prolonged Al stress (10 days).

RESULTS: Genome-wide analysis revealed pronounced context-specific methylation changes that differed markedly between genotypes. CHG methylation was broadly reduced under stress in both genotypes, with a stronger response in BGI, whereas CHH methylation showed a genome-wide increase in BGI but only modest changes in Azucena, indicating more extensive epigenomic perturbation in the susceptible background. At the local level, differentially methylated regions (DMRs) were predominantly hypomethylated across all cytosine contexts and, particularly within the CHG and CHH contexts, were significantly enriched within transposable elements and upstream regulatory regions relative to genomic background, suggesting that stress-induced relaxation of TE silencing and regulatory reprogramming of promoter regions are conserved features of the Al epigenetic response. Integration of DMR and differential expression data identified 71 and 93 genes with both methylation and transcriptional changes in Azucena and BGI, respectively, with only three genes shared between genotypes, all showing opposite transcriptional responses, underscoring the near-complete genotype specificity of the methylation-expression interface. In Azucena, the Al-tolerant genotype, methylation and expression changes were targeted in genes directly linked to Al exclusion, including organic acid and MATE transporters, whereas BGI showed a broader and less specific epigenomic response.

DISCUSSION: These results suggest that Al stress triggers genotype- and sequence-context-specific epigenomic reprogramming in rice, and that tolerance is associated with a targeted methylation response rather than a diffuse one. This positions DNA methylation as an additional regulatory layer shaping Al tolerance, and pinpoints a short list of candidate genes as priority targets for epigenome-informed breeding strategies in Al-tolerant rice.