DNA methylome analysis reveals novel insights into active hypomethylated regulatory mechanisms of temperature-dependent flower opening in .

Short-term ambient low temperature (ALT) stimulation is necessary for to facilitate continued flower opening after floral bud development reaches maturity. DNA methylation, a vital epigenetic modification, regulates various biological processes in response to temperature fluctuations. However, its role in temperature-driven flower opening remains elusive. In this study, we identified the pivotal timeframe during which promptly detected temperature cues. Using whole-genome bisulfite sequencing, we explored global DNA hypomethylation during this phase, with the most significant changes occurring in CHH sequence contexts. Auxin transport inhibitor (TIBA) application revealed that ALT-induced endogenous auxin accumulation promoted peduncle elongation. In our mRNA-seq analysis, we discovered that the differentially expressed genes (DEGs) with hypo-differentially methylated regions (hypo-DMRs) were mainly enriched in auxin and temperature response, RNA processing, and carbohydrate and lipid metabolism. Transcripts of three DNA demethylase genes (, , ) showed upregulation. Furthermore, all DNA methylase genes, except , also displayed increased expression, specifically with two of them, and , being associated with hypo-DMRs. Promoter assays showed that , with promoters containing low-temperature- and auxin-responsive elements, were activated by ALT and exogenous IAA at low concentrations but inhibited at high concentrations. Overexpression of reduced endogenous auxin levels but enhanced the expression of genes related to auxin response and spliceosome in . Furthermore, promoted sucrose synthesis in petunia corollas. Our data characterized the rapid response of active DNA hypomethylation to ALT and suggested a possible epiregulation of temperature-dependent flower opening in . This study revealed the pivotal role of DNA hypomethylation in during the ALT-responsive phase before flower opening, involving dynamic DNA demethylation, auxin signaling modulation, and a potential feedback loop between hypomethylation and methylation.