Heat stress responses mediated by N6-methyladenine DNA methylation in maize

N6-methyladenine (6mA) plays an important role in eukaryotic development and stress responses, yet its function under heat stress (HS) in crops remains unclear. Here, we show that 6mA dynamics in two maize inbred lines, B73 and Mo17, correlate with their responses to HS. Genome-wide 6mA profiling reveals enrichment in promoters, intergenic regions, and transposable elements (TEs), with inverse correlation with gene/TE expression. Upon HS, heat-tolerant plants show elevated 6mA levels, with differential 6mA patterning on key HS-related genes underlying thermotolerance variation between B73 and Mo17. We identified ZmALKBH1 as a 6mA demethylase, and its mutation enhances HS tolerance. A deep learning model based on 6mA methylomes of B73 and Mo17 accurately predicts and experimentally validates 6mA distribution and HS response in additional W22 and B104 lines. These findings uncover the role of 6mA in transcriptional regulation of crop stress adaptation and offer potential targets for improving thermotolerance in maize.