High-temperature stress during grain filling significantly reduces rice grain yield and quality. The heat-tolerant cultivar Kumasannochikara (Kuma) has a lower chalky-grain percentage and better grain quality under heat stress than the heat-sensitive Hinohikari (Hino), owing to repression of the α-amylase gene OsAmy3E during grain filling. Here, we propose that the expression of OsAmy3E during grain filling under heat stress is epigenetically regulated, as the promoter region of OsAmy3E in Kuma is highly methylated. In developing Kuma × Hino F1 seed, hypermethylation of the OsAmy3E promoter suppressed its expression. Screening of DNA methylation levels in F2 plants divided plants into non-methylated and methylated groups. Methylated plants of both Hino × Kuma and Kuma × Hino had repressed OsAmy3E gene expression and better grain quality under heat stress than non-methylated plants. These findings suggest that the suppression of α-amylase gene expression through epigenetic regulation could improve rice grain quality under heat stress.
Supplementary Information: The online version contains supplementary material available at 10.1038/s41598-026-40998-2.