Long-day induced flowering requires DNA hypermethylation in orchardgrass.

Flowering, a pivotal plant lifecycle event, is intricately regulated by environmental and endogenous signals via genetic and epigenetic mechanisms. Photoperiod is a crucial environmental cue that induces flowering by activating integrators through genetic and epigenetic pathways. However, the specific role of DNA methylation, a conserved epigenetic marker, in photoperiodic flowering remains unclear. This study integrated methylome, transcriptome, and gene expression analyses in orchardgrass (Dactylis glomerata) to elucidate the molecular mechanisms underlying long-day (LD) flowering. We found that LD treatment led to CHH hypermethylation, which was associated with the increased expression of RNA-dependent DNA methylation pathway components. LD-induced CHH hypermethylation in promoters correlated with upregulated photoperiod pathway genes and down-regulated miRNAs. The suppression of DNA methylation under LD conditions delays flowering, highlighting the critical role of hypermethylation. Additionally, the novel-miR1736-3p was identified as a negative regulator of DgFT. These findings elucidate the promotion of flowering through LD-induced CHH hypermethylation and provide insights into using epigenetic techniques to control plant flowering time.