Differential methylation analysis of floral buds between two morphs unravels the contributions of key genes to flowering time in heterodichogamous Cyclocarya paliurus

Abstract

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.

Author summay

^‡ These authors contributed equally: Qian Wang and Yanhao Yu

Acknowledgements

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. We sincerely thank Prof. S. Zhou ([e-mail]) from Nanjing Agricultural University and the colleagues from Tea Research Institute, Anhui Academy of Agricultural Sciences, for their assistance in the revision of this manuscript. Special thanks to Dr. Q. Lu ([e-mail]) from the College of Life and Environmental Science, Huangshan University, for his thoughtful review and invaluable suggestions. We are grateful to N. Gong and B. Wu for their help in this study.

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Acknowledgements

X.X.F. and Q.W. conceived and designed this study. Q.W., Y.H.Y., D.M., Y.B.Y., Y.M.H., and X.M. performed the experiments. X.L.S. guided the experiment. Q.W. and Y.H.Y. analyzed the data. Q.W. wrote the original draft. Y.Q.Q. and X.X.F. supervised and reviewed the manuscript. All authors have read and approved the final manuscript.

Data availability

The reference genome of diploid C. paliurus is available in the Genome Warehouse (GWH, CNCB; https://ngdc.cncb.ac.cn/gwh/Assembly/26382/show) [10]. Whole-genome bisulfite and transcriptome sequencing data of female and male flower buds (both morphs) at the inflorescence elongation stage are deposited in the Genome Sequence Archive (GSA) under accessions PRJCA040703 and PRJCA030315, respectively. Transcriptome data of 5-azaC-treated male flowers (both morphs) of diploid C. paliurus are deposited in GSA under PRJCA045638.

Conflicts of interest statement

The authors declare no conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

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