NAC transcription factors (TFs) participate in multiple biological processes, including biotic and abiotic stress responses, signal transduction and development. Cold stress can adversely impact plant growth and development, thereby limiting agricultural productivity. Prunus mume, an excellent horticultural crop, is widely cultivated in Asian countries. Its flower can tolerate freezing-stress in the early spring. To investigate the putative NAC genes responsible for cold-stress, we identified and analyzed 113 high-confidence PmNAC genes and characterized them by bioinformatics tools and expression profiles. These PmNACs were clustered into 14 sub-families and distributed on eight chromosomes and scaffolds, with the highest number located on chromosome 3. Duplicated events resulted in a large gene family; 15 and 8 pairs of PmNACs were the result of tandem and segmental duplicates, respectively. Moreover, three membrane-bound proteins (PmNAC59/66/73) and three miRNA-targeted genes (PmNAC40/41/83) were identified. Most PmNAC genes presented tissue-specific and time-specific expression patterns. Sixteen PmNACs (PmNAC11/19/20/23/41/48/58/74/75/76/78/79/85/86/103/111) exhibited down-regulation during flower bud opening and are, therefore, putative candidates for dormancy and cold-tolerance. Seventeen genes (PmNAC11/12/17/21/29/42/30/48/59/66/73/75/85/86/93/99/111) were highly expressed in stem during winter and are putative candidates for freezing resistance. The cold-stress response pattern of 15 putative PmNACs was observed under 4 °C at different treatment times. The expression of 10 genes (PmNAC11/20/23/40/42/48/57/60/66/86) was upregulated, while 5 genes (PmNAC59/61/82/85/107) were significantly inhibited. The putative candidates, thus identified, have the potential for breeding the cold-tolerant horticultural plants. This study increases our understanding of functions of the NAC gene family in cold tolerance, thereby potentially intensifying the molecular breeding programs of woody plants.

Prunus mume is an important ornamental woody plant that grows in tropical and subtropical regions. Freezing stress can adversely impact plant productivity and limit the expansion of geographical locations. Understanding cold-responsive genes could potentially bring about the development of new ways to enhance plant freezing tolerance. Members of the serine/threonine protein kinase (CIPK) gene family play important roles in abiotic stress. However, the function of CIPK genes in P. mume remains poorly defined. A total of 16 CIPK genes were first identified in P. mume. A systematic phylogenetic analysis was conducted in which 253 CIPK genes from 12 species were divided into three groups. Furthermore, we analysed the chromosomal locations, molecular structures, motifs and domains of CIPK genes in P. mume. All of the CIPK sequences had NAF domains and promoter regions containing cis-acting regulatory elements of the related stress response. Three PmCIPK genes were identified as Pmu-miR172/167-targeted sites. Transcriptome data showed that most PmCIPK genes presented tissue-specific and time-specific expression profiles. Nine genes were highly expressed in flower buds in December and January, and 12 genes were up-regulated in stems in winter. The expression levels of 12 PmCIPK genes were up-regulated during cold stress treatment confirmed by qRT-PCR. Our study improves understanding of the role of the PmCIPK gene family in the low temperature response in woody plants and provides key candidate genes and a theoretical basis for cold resistance molecular-assisted breeding technology in 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.