DNA methylation-triggered ABA regulation drives contrasting osmotic stress responses in soybean cultivars

Soybean, a globally important food crop, is highly susceptible to abiotic stresses, which have intensified due to climate change. DNA methylation plays a central role in regulating adaptive responses to these stresses. This study explored the relationship between DNA methylation patterns and the regulation of abscisic acid (ABA) pathways in two contrasting soybean cultivars: EM48 (drought-tolerant) and BR16 (drought-sensitive). Seedlings were exposed to osmotic stress through mannitol, DNA methylation inhibitor 5-azacytidine (5-azaC), and the combination of both. Treatment with 100 μM 5-azaC led to significant growth inhibition, particularly a reduction in the number of lateral roots, whereas 25 μM had no effect on biomass in either cultivar but also impacted the number of lateral roots. Under treatments with 5-azaC and 5-azaC + mannitol, EM48 showed reductions in dry mass of the aerial part and lateral roots, while BR16 exhibited greater sensitivity in all measured parameters. Global DNA methylation profiling revealed cultivar-specific patterns, BR16 showed dynamic methylation changes throughout the stress and 5-azaC treatments, whereas EM48 maintained stable levels. EM48 also accumulated more carotenoids and ABA under stress, in contrast to BR16, which showed lower ABA levels and stable carotenoid content. Gene expression analysis indicated that EM48 activated key ABA pathway genes in response to 5-azaC and combined treatments, while BR16 showed gene-specific, limited responses. Promoter methylation analysis revealed CHH context predominance, especially in the ABA3 gene, and greater 5-azaC-induced demethylation in BR16, while EM48 maintained more stable methylation. This study advances the understanding of epigenetic regulation in soybean stress tolerance, offering insights for biotechnological strategies aimed at improving crop resilience.