Spatiotemporal-specific DNA methylation and small RNAs are involved to regulate cell cycle progression and organ development of cucumber (Cucumis sativus L.).
TL;DR
A dual mechanism in which DNA methylation and CsRDR1a-related small RNAs coordinately regulate cucumber organ development is unveiled, highlighting the importance of epigenetic regulation in cucumber.
Abstract
INTRODUCTION Cucumber fruits, with a relatively high rate of fruit expansion, are harvested at an early developmental stage once moderately expanded. Although DNA methylation regulating fruit ripening has been well studied, its function in organ development of cucumber remains unknown.
Objectives
In this study, the regulation and underlying mechanism of DNA methylation in cucumber fruit development were investigated.
Methods
Chemical treatment with DNA methylation inhibitor 5-aza, together with whole genome bisulfite sequencing (WGBS), flow cytometry, small RNA sequencing, and functional validation via virus-induced gene silencing (VIGS), were comprehensively employed.
Results
Treating cucumber fruits with 5-aza at 0 or 4 days after anthesis (DAA) caused stage-dependent inhibition of fruit expansion, with stronger inhibition observed at 0 DAA. WGBS revealed that DNA methylation at CG and CHG contexts remained at high levels before 4 DAA, while mCG levels peaked at 20 DAA and mCHH levels increased throughout. Before 4 DAA, DNA methylation levels at CHG and CG contexts were higher in fruit than fruit neck, revealing spatiotemporal dynamics of DNA methylation during the development of cucumber fruit. These dynamics were functionally enriched in genes related to cell cycle transition, and this finding was further confirmed by impaired endoreduplication observed in 5-aza treated fruits when compared with control. In addition, CsRDR1a, an RNA-dependent RNA polymerase 1 homolog, displayed fruit stage-specific expression patterns, accompanied by significant accumulation of 19---23 nt small RNAs. Cross-tissue validation by silencing CsCMT3 or CsRDR1a showed reduced leaf size, inhibited endoreduplication, and increased expression of cell-cycle genes with decreased DNA methylation levels at their promoter regions, demonstrating that DNA methylation was essential for cell cycle transition during cucumber organ development.
Conclusion
Our results unveil a dual mechanism in which DNA methylation and CsRDR1a-related small RNAs coordinately regulate cucumber organ development, highlighting the importance of epigenetic regulation in cucumber.