DNA methylation is a conserved epigenetic modification that regulates gene expression and developmental transitions in plants. Although its roles in photoperiodic and hormonal flowering pathways are increasingly recognized, its involvement in heterodichogamy—a mating system characterized by temporal separation of male and female flower function—remains largely unexplored. In Cyclocarya paliurus, two morphs (protogynous and protandrous) differ in flowering sequence, yet low seed set suggests complex regulatory constraints. Previous studies implicate gibberellins and candidate genes such as TPPD in floral differentiation. However, the epigenetic mechanisms coordinating sexual asynchrony are unclear. Based on these challenges, in-depth investigation into DNA methylation dynamics is required to understand flowering regulation in heterodichogamous systems.
Researchers from Nanjing Forestry University and collaborating institutions report (DOI: 10.1093/hr/uhaf296) in Horticulture Research (2026) that DNA methylation dynamically regulates flowering time in diploid Cyclocarya paliurus. By constructing single-base resolution methylomes of male and female floral buds from two morphs, the team discovered elevated CHH methylation in early-flowering samples. Integrative analyses linked methylation shifts to transcriptional changes in flowering pathway genes. Functional validation through heterologous overexpression and field application of the methylation inhibitor 5-azacytidine confirmed that methylation directly influences flowering timing.
Genome-wide methylation profiling revealed that early-flowering floral buds exhibited significantly higher CHH methylation levels than late-flowering counterparts. Notably, differentially methylated regions were enriched in promoter regions, suggesting regulatory effects on gene expression. Transcriptome integration identified methylation-differentially expressed genes (MDEGs) within three major flowering pathways: photoperiod (e.g., CpHd16), gibberellin metabolism (e.g., CpGA2ox family), and trehalose-6-phosphate (Tre6P) signaling (e.g., CpTPPD). Correlation analyses showed that promoter CHH methylation negatively regulated certain flowering repressors while positively correlating with others, indicating complex epigenetic modulation. Expression of the methyltransferase CpDRM-D2 was elevated in early-flowering samples, whereas demethylase CpDME-D1 expression was reduced, suggesting coordinated control of CHH hypermethylation. Functional assays strengthened these findings. Overexpression of CpHd16, CpTPPD, and CpFTIP3 in Arabidopsis delayed flowering and altered FT and SOC1 expression. Moreover, field treatment of C. paliurus with 5-azacytidine reduced promoter methylation in CpTPPD, increased methylation within CpFTIP3, and delayed flowering in both morphs. These results demonstrate that methylation-dependent gene regulation modulates floral timing.
“This study provides the first epigenomic evidence linking CHH methylation dynamics to heterodichogamous flowering,” the corresponding authors note. “Our findings suggest that methyltransferase and demethylase balance fine-tunes flowering timing by modulating key regulatory genes. The coordinated action of CpDRM-D2 and CpDME-D1 appears central to establishing sex-specific methylation states. Importantly, methylation changes are not merely correlative—they actively influence gene expression and flowering behavior, as validated by both transgenic and field experiments.”
Understanding epigenetic regulation of flowering has significant implications for tree breeding and reproductive management. In C. paliurus, low seed fullness limits propagation efficiency, and manipulating methylation states could improve flowering synchrony and seed production. The identification of methylation-sensitive genes such as CpTPPD and CpFTIP3 offers potential molecular targets for breeding strategies. Beyond this species, the findings expand knowledge of how epigenetic mechanisms coordinate complex mating systems in perennial plants. By revealing that CHH methylation dynamics contribute to temporal sexual separation, this work opens new avenues for integrating epigenetic editing into crop and forest tree improvement programs.
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References
DOI
10.1093/hr/uhaf296
Original Source URL
https://doi.org/10.1093/hr/uhaf296
Funding information
This research was funded by the National Natural Science Foundation of China (project number: 32271859), the Postgraduate Research and Practice Innovation Program of Jiangsu Province (No. KYCX22_1109), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data in writing the manuscript.
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.