Yuan Li

Potato cultivars are inherently sensitive to high temperatures, and dissecting the mechanisms underlying heat response and tolerance has long been a central focus in potato research. However, the molecular mechanisms governing the short-term heat stress response in potato, as well as the regulatory role of DNA methylation in heat adaptation, remain largely unclear. In this study, we identified breeding line D187 as heat-tolerant and cultivar Q9 as heat-sensitive through microtuber induction under heat stress. We further confirmed that heat-sensitive cultivar Q9 exhibited distinct physiological responses in leaves following 6 h of heat treatment. Comparative transcriptome analysis of leaves exposed to 6 h of heat stress revealed distinct molecular response patterns between D187 and Q9. D187 specifically upregulated genes enriched in heat and other stress response pathways to enhance heat adaptation, whereas Q9 relied on pathways related to RNA modification and splicing, presumably adapting to high temperatures via post-transcriptional regulation. Notably, genes involved in the RdDM pathway were differentially upregulated in both genotypes, and heat stress correspondingly enhanced CHH methylation levels in the vicinity of functional genes in the heat-sensitive cultivar Q9. Treatment with 5-Azacytidine, a DNA methylation inhibitor, exacerbated the inhibition of in vitro tuber formation under high temperatures, indicating that maintaining and enhancing DNA methylation is essential for heat adaptation in potato. Furthermore, overexpression of StAGO4a/b in Nicotiana benthamiana modestly improved heat tolerance, suggesting that StAGO4s act as positive regulators of heat tolerance in potato. Collectively, our results suggest that heat-induced CHH methylation near functional genes via the RdDM pathway contributes positively to heat stress response and tolerance in Q9, providing new insights for identifying heat tolerance regulators from a DNA methylation perspective.