In China, Prunus mume is a famous flowering tree that has been cultivated for 3000 years. P. mume grows in tropical and subtropical regions, and most varieties lack cold resistance; thus, it is necessary to study the low-temperature response mechanism of P. mume to expand the scope of its cultivation. We used the integrated transcriptomic and metabolomic analysis of a cold-resistant variety of P. mume ‘Meiren’, to identify key genes and metabolites associated with low temperatures during flowering. The ‘Meiren’ cultivar responded in a timely manner to temperature by way of a low-temperature signal transduction pathway. After experiencing low temperatures, the petals fade and wilt, resulting in low ornamental value. At the same time, in the cold response pathway, the activities of related transcription factors up- or downregulate genes and metabolites related to low temperature-induced proteins, osmotic regulators, protective enzyme systems, and biosynthesis and metabolism of sugars and acids. Our findings promote research on the adaptation of P. mume to low temperatures during wintering and early flowering for domestication and breeding.

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.

CCCH is a zinc finger family with a typical CCCH-type motif which performs a variety of roles in plant growth and development and responses to environmental stressors. However, the information about this family has not been reported for Osmanthus fragrans. In this study, a total of 66 CCCH predicted genes were identified from the O. fragrans genome, the majority of which had multiple CCCH motifs. The 66 OfCCCHs were found to be unevenly distributed on 21 chromosomes and were clustered into nine groups based on their phylogenetic analysis. In each group, the gene structure and domain makeup were comparatively conserved. The expression profiles of the OfCCCH genes were examined in various tissues, the flower-opening processes, and under various abiotic stresses using transcriptome sequencing and qRT-PCR (quantitative real-time PCR). The results demonstrated the widespread expression of OfCCCHs in various tissues, the differential expression of 22 OfCCCHs during flower-opening stages, and the identification of 4, 5, and 13 OfCCCHs after ABA, salt, and drought stress treatment, respectively. Furthermore, characterization of the representative OfCCCHs (OfCCCH8, 23, 27, and 36) revealed that they were all localized in the nucleus and that the majority of them had transcriptional activation in the yeast system. Our research offers the first thorough examination of the OfCCCH family and lays the groundwork for future investigations regarding the functions of CCCH genes in O. fragrans.