Epigenetic changes caused by methylcytosine modification participate in gene regulation and transposable element (TE) repression, resulting in phenotypic variation. Although the effects of DNA methylation and TE repression on flower, fruit, seed coat, and leaf pigmentation have been investigated, little is known about the relationship between methylation and flower color chimerism. In this study, we used a comparative methylomic–transcriptomic approach to explore the molecular mechanism responsible for chimeric flowers in Prunus mume “Danban Tiaozhi”. High-performance liquid chromatography-electrospray ionization mass spectrometry revealed that the variation in white (WT) and red (RT) petal tissues in this species is directly due to the accumulation of anthocyanins, i.e., cyanidin 3,5-O-diglucoside, cyanidin 3-O-glucoside, and peonidin 3-O-glucoside. We next mapped the first-ever generated methylomes of P. mume, and found that 11.29–14.83% of the genomic cytosine sites were methylated. We also determined that gene expression was negatively correlated with methylcytosine level in general, and uncovered significant epigenetic variation between WT and RT. Furthermore, we detected differentially methylated regions (DMRs) and DMR-related genes between WT and RT, and concluded that many of these genes, including differentially expressed genes (DEGs) and transcription factor genes, are critical participants in the anthocyanin regulatory pathway. Importantly, some of the associated DEGs harbored TE insertions that were also modified by methylcytosine. The above evidence suggest that flower color chimerism in P. mume is induced by the DNA methylation of critical genes and TEs.

DNA methylation is a common epigenetic modification involved in regulating many biological processes. However, the epigenetic mechanisms involved in the formation of floral scent have rarely been reported within a famous traditional ornamental plant Prunus mume emitting pleasant fragrance in China. By combining whole-genome bisulfite sequencing and RNA-seq, we determined the global change in DNA methylation and expression levels of genes involved in the biosynthesis of floral scent in four different flowering stages of P. mume. During flowering, the methylation status in the “CHH” sequence context (with H representing A, T, or C) in the promoter regions of genes showed the most significant change. Enrichment analysis showed that the differentially methylated genes (DMGs) were widely involved in eight pathways known to be related to floral scent biosynthesis. As the key biosynthesis pathway of the dominant volatile fragrance of P. mume, the phenylpropane biosynthesis pathway contained the most differentially expressed genes (DEGs) and DMGs. We detected 97 DMGs participated in the most biosynthetic steps of the phenylpropane biosynthesis pathway. Furthermore, among the previously identified genes encoding key enzymes in the biosynthesis of the floral scent of P. mume, 47 candidate genes showed an expression pattern matching the release of floral fragrances and 22 of them were differentially methylated during flowering. Some of these DMGs may or have already been proven to play an important role in biosynthesis of the key floral scent components of P. mume, such as PmCFAT1a/1c, PmBEAT36/37, PmPAL2, PmPAAS3, PmBAR8/9/10, and PmCNL1/3/5/6/14/17/20. In conclusion, our results for the first time revealed that DNA methylation is widely involved in the biosynthesis of floral scent and may play critical roles in regulating the floral scent biosynthesis of P. mume. This study provided insights into floral scent metabolism for molecular breeding.

Trehalose and its key synthase (trehalose-6-phosphate synthase, TPS) can improve the drought tolerance of plants. However, little is known about the roles of trehalose and the TPS family in Prunus mume response to drought. In our study, we discovered that the trehalose content in leaf, root, and stem tissues significantly increased in P. mume in response to drought. Therefore, the characteristics and functions of the TPS family are worth investigating in P. mume. We identified nine TPS family members in P. mume, which were divided into two sub-families and characterized by gene structure, promoter elements, protein conserved domains, and protein motifs. We found that the Hydrolase_3 domain and several motifs were highly conserved in Group II instead of Group I. The distinctions between the two groups may result from selective constraints, which we estimated by the d_N/d_S (ω) ratio. The ω values of all the PmTPS family gene pairs were evaluated as less than 1, indicating that purity selection facilitated their divergence. A phylogenetic tree was constructed using 92 TPSs from 10 Rosaceae species, which were further divided into five clusters. Based on evolutionary analyses, the five clusters of TPS family proteins mainly underwent varied purity selection. The expression patterns of PmTPSs under drought suggested that the TPS family played an important role in the drought tolerance of P. mume. Combining the expression patterns of PmTPSs and the trehalose content changes in leaf, stem, and root tissues under normal conditions and drought stress, we found that the PmTPS2 and PmTPS6 mainly function in the trehalose biosynthesis in P. mume. Our findings not only provide valuable information about the functions of trehalose and TPSs in the drought response of P. mume, but they also contribute to the future drought breeding of P. mume.

DNA methylation is one of the best-studied epigenetic modifications involved in many biological processes. However, little is known about the epigenetic mechanism for flower color chimera of Prunus mume (Japanese apricot, mei). Using bisulfate sequencing and RNA sequencing, we analyzed the white (FBW) and red (FBR) petals collected from an individual tree of Japanese apricot cv. ‘Fuban Tiaozhi’ mei to reveal the different changes in methylation patterns associated with gene expression leading to significant difference in anthocyanins accumulation of FBW (0.012 ± 0.005 mg/g) and FBR (0.078 ± 0.013 mg/g). It was found that gene expression levels were positively correlated with DNA methylation levels within gene-bodies of FBW and FBR genomes; however, negative correlations between gene expression and DNA methylation levels were detected within promoter domains. In general, the methylation level within methylome of FBW was higher; and in total, 4,618 differentially methylated regions (DMRs) and 1,212 differentially expressed genes (DEGs) were detected from FBW vs. FBR. We also identified 82 DMR-associated DEGs, and 13 of them, including PmBAHD, PmCYP450, and PmABC, were playing critical roles in phenylalanine metabolism pathway, glycosyltransferase activity, and ABC transporter. The evidence exhibited DNA methylation may regulate gene expression resulting in flower color chimera of Japanese apricot.

Low-temperature accumulation is one of the essential stages in the growth process of woody ornamental plants. In this study, two different low-temperature treatments, 6 °C and 10 °C, were used to analyze the effects of different low-temperature treatments on dormancy release and flowering of the ‘Gulihong’ plant using artificial low temperatures. Based on the experimental results, four typical early-blooming Prunus mume cultivars widely planted in Yangling area of Henan Province, China, including ‘Zaoyudie’, ‘Zaohualve’, ‘Nanjing gongfen’, and ‘Gulihong’, were selected as the experimental materials. The effects of low-temperature accumulation on the flowering characteristics of different cultivars were analyzed using a 6 °C artificial low-temperature treatment. The suitable cultivation temperature for early-blooming cultivars was screened to provide a theoretical basis for further exploration of P. mume bonsai cultivation techniques. The results showed that the flowering rate, flower diameter, flowering quantity, flowering uniformity, and bud development in the 6 °C treatment were significantly better than those in the 10 °C treatment. Furthermore, under 6 °C low-temperature treatment, the flowering rate and quality of different cultivars showed an increasing trend with the accumulation of low temperature, with ‘Gulihong’ exhibiting the highest flowering rate. Therefore, chill accumulation plays a significant role in promoting flowering quality.