DNA methylome analysis reveals novel insights into active hypomethylated regulatory mechanisms of temperature-dependent flower opening in Osmanthus fragrans

Abstract

Short-term ambient low temperature (ALT) stimulation is necessary for Osmanthus fragrans to facilitate continued flower opening after floral bud development reaches maturity. DNA methylation, a vital epigenetic modification, regulates various biological processes in response to temperature fluctuations. However, its role in temperature-driven flower opening remains elusive. In this study, we identified the pivotal timeframe during which O. fragrans promptly detected temperature cues. Using whole-genome bisulfite sequencing, we explored global DNA hypomethylation during this phase, with the most significant changes occurring in CHH sequence contexts. Auxin transport inhibitor (TIBA) application revealed that ALT-induced endogenous auxin accumulation promoted peduncle elongation. In our mRNA-seq analysis, we discovered that the differentially expressed genes (DEGs) with hypo-differentially methylated regions (hypo-DMRs) were mainly enriched in auxin and temperature response, RNA processing, and carbohydrate and lipid metabolism. Transcripts of three DNA demethylase genes ( OfROS1a, OfDML3, OfDME ) showed upregulation. Furthermore, all DNA methylase genes, except OfCMT2b, also displayed increased expression, specifically with two of them, OfCMT3a and OfCMT1, being associated with hypo-DMRs. Promoter assays showed that OfROS1a, with promoters containing low-temperature- and auxin-responsive elements, were activated by ALT and exogenous IAA at low concentrations but inhibited at high concentrations. Overexpression of OfROS1 reduced endogenous auxin levels but enhanced the expression of genes related to auxin response and spliceosome in petunia. Furthermore, OfROS1 promoted sucrose synthesis in petunia corollas. Our data characterized the rapid response of active DNA hypomethylation to ALT and suggested a possible epiregulation of temperature-dependent flower opening in O. fragrans. This study revealed the pivotal role of DNA hypomethylation in O. fragrans during the ALT-responsive phase before flower opening, involving dynamic DNA demethylation, auxin signaling modulation, and a potential feedback loop between hypomethylation and methylation.

Acknowledgements

This work was supported by National Natural Science Foundation of China (Grant Nos. 32072615 and 32302591), the major scientific and technological project of Zhejiang Province (2021C02071), and the Talent Startup Program of Zhejiang A&F University (2021LFR009).

Author contributions

S.Z. planned and designed the research. H.Z. provided financial support and article suggestions. H.Z., W.L., D.W., Y.M., B.D., Y.W., Z.X., Q.F., and J.D. conducted the experiments, carried out the fieldwork, and analyzed the data. S.Z. wrote the manuscript.

Data availability statement

Reference genomes of O. fragrans are available as public genome data at the National Center of Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/genome/?term=txid93977[orgn]) [44]. Whole-genome bisulfite sequencing and transcriptome sequencing data for floral buds subjected to diverse treatment durations at 19°C in comparison with those treated at 23°C have been deposited in the public NCBI BioProject database under accession numbers PRJNA1014796 and PRJNA1014987. Transcriptome sequencing data for corollas of OfROS1a overexpression and controls have been deposited in the public NCBI BioProject database under accession number PRJNA1014991. Reference genomes of P. hybrida were obtained from the public genome website (https://solgenomics.net/ftp/genomes/Petunia_axillaris/assembly/Petunia_axillaris_v1.6.2_genome.fasta).

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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