Cyclocarya paliurus is a medicinal plant traditionally used in China to treat hypertension and diabetes. It exhibits heterodichogamy, a dimorphic mating system with protogynous and protandrous morphs, which are based on the maturation sequence of female and male flowers within the same plant. DNA methylation, a crucial epigenetic modification in regulating plant flowering, is poorly characterized in heterodichogamous species. Here, whole-genome bisulfite sequencing and transcriptome analyses were performed on female and male flower buds from two morphs during inflorescence elongation in diploid C. paliurus. Single-base methylation maps revealed higher DNA methylation levels in early-flowering samples, particularly in CHH contexts, which may be dynamically regulated by the interplay between CpDRM-D2 and CpDME-D1. Candidate genes involved in the photoperiod, gibberellin, and trehalose-6-phosphate signaling pathways were identified based on their transcriptional and methylation dynamics across floral buds. Heterologous overexpression of CpHd16, CpTPPD, and CpFTIP3 in Arabidopsis delayed flowering. Furthermore, field application of the DNA methylation inhibitor 5-azacytidine to diploid C. paliurus delayed the flowering of both male and female flowers and altered methylation levels within the CpTPPD promoter and CpFTIP3 gene body. These epigenetic changes, accompanied by downregulated CpTPPD and upregulated CpFTIP3 expression, suggest that these genes may mediate C. paliurus flowering via methylation-dependent regulation. This study provides novel insights into the molecular regulatory mechanisms of heterodichogamous flowering and lays a theoretical foundation for epigenetic research in C. paliurus.

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