1. IntroductionThe plum (Prunus salicina L.) is an economically important stone fruit tree in China. The fruit can be eaten fresh or processed into many value-added products, such as juice, fermented wine, and preserves [1]. When the fruit matures, the pulp color can be green, yellow–green, yellow, red, or purple based on the difference cultivar of the plum. The fruit color is mainly determined by the level of anthocyanin accumulation [2]. Anthocyanins have been extensively studied, not only because they result in the production of red, purple, and black pigments, but also in the contexts of their diverse roles in UV protection and pathogen defense, as well as their nutritional value in the human diet [3].Anthocyanins are formed by the combination of anthocyanidin and one or more carbohydrates through glycosides bonds which then stably exist in plant cells, which make the fruit appear bright colors [4]. Anthocyanins are synthesized through the flavonoid pathway which has been well-studied in many plants. Many structural genes of the flavonoid pathway have been isolated and identified as the critical gene for anthocyanin biosynthesis in plants, such as chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid 3′-hydroxylase (F3′H), anthocyanidin synthase (ANS), and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) [4,5,6,7]. In addition, transcription factors play important roles in modulating anthocyanin biosynthetic pathway activity and color changes, including MYB proteins, basic helix–loop–helix (bHLH) proteins, and WD40 proteins [8]. These regulators form an MYB–bHLH–WD40 (MBW) complex that binds to promoters and regulates the expression of structural genes of the anthocyanin biosynthetic pathway [9,10]. The role of MBW in anthocyanin biosynthesis has been elucidated in fruit trees such as apple [11], pears [12,13], grape [14], strawberry [15], Chinese bayberry [16], kiwifruit [17], litchi [18], blueberry [19], sweet cherry [20] peach [21,22], plum [1], and fig [23]. In addition, other regulatory factors also affect anthocyanin biosynthesis via interaction with the MBW complex or by modulating the transcription of structural genes directly [1]. A SQUAMOSA promoter-binding protein-like (SPL) transcription factor (PpSPL1) has been shown to inhibit the expression of anthocyanin biosynthetic genes and negatively regulate anthocyanin accumulation through the destabilization of MBW [24]. MADS-box and NAC transcription factors also are reported to be involved in the regulation of anthocyanin accumulation [25,26].In recent years, RNA sequencing (RNA-Seq) transcriptome analysis has been extensively used for the identification of functional genes in fruit trees, including plum. The genome of Chinese plum ‘Sanyueli’ and the chloroplast genome sequence of ‘Wushan plum’ (P. salicina) have been published [27,28]. The transcriptome analysis of ‘Siyueli’-pollinated and ‘Yinhongli’-pollinated fruits revealed 2762 and 1018 differentially expressed genes (DEGs) involved in the response to different pollen sources [29]. Recently reported are the fruit skin transcriptome assembly of ‘Angeleno’ and ‘Lamoon’ Japanese plum cultivars with different skin color [30], and the transcriptomic changes during fruit-ripening in the red-fleshed plum cultivar ‘Furongli’ (P. salicina) [1]. During the ripening and development of plum fruits, chlorophyll degradation, the content of organic acids decreases, while anthocyanins accumulate rapidly in the colored varieties, and this phenomenon is especially obvious in the late stage of plum fruit-ripening [31]. In this study, as a first step towards understanding gene expression during fruit-ripening in plum, the three mature stages of ‘Huaxiu’ plum fruits with pulp colors of yellow, orange, and red were collected for transcriptome sequencing. This study presents the results of a comprehensive analysis of transcriptome data from fruits at three representative developmental stages. The results will help us to understand the gene expression difference and explore the molecular mechanisms of the biosynthetic pathways in secondary metabolites in plum. 2. Materials and Methods 2.1. Plant Materials

Fruits of ‘Huaxiu’ plum (P. salicina) were collected from 6-year-old field-grown trees in an orchard in Donghai County, Jiangsu Province, China. In July 2007, a natural-bud mutation for early maturing fruit of ‘Qiuji’ was found by a farmer and named as ‘Huaxiu’. According to our observations, ‘Huaxiu’ characteristics are mid-early ripening (fruits mature in late-July in this area, about 108 days after flowering), where the skin is dark purple and the pulp orange–red. These mature fruit samples were divided into three different stages based on pulp color and termed the ‘Yellow stage’ (YS), ‘Orange stage’ (OS), ‘Red stage’ (RS), respectively.

During the fruit ripening periods, three biological replicates were collected per sample, each with 20 fruits randomly collected from two trees in order to decrease background variation. Then, three pulps were peeled and sliced into appropriate pieces after measuring the weight and diameter, and then immediately frozen in liquid nitrogen for the determination of chlorophyll, carotenoid, anthocyanins, and flavonoids. Two biological replicated samples were used at each stage for transcriptome sequencing due to the background and limited yield of fruits.

2.2. Total Anthocyanin, Flavonoid, and Chlorophyll MeasurementsTotal anthocyanin levels were determined according to the HPLC system [32]. Approximately 1.5 g of the sample was ground to a fine powder in liquid nitrogen and extracted with a 1% HCl-methanol solution at 4 °C for 24 h to obtain the extract by filtration. Then, 20 µL of the samples were injected into a C18 column (Agilent Zorbax Eclipse SB-C18, 4.6 mm × 100 mm, 1.8 µm, Santa Clara, CA, USA). The binary solvent system was 5% formic acid in water as mobile phase A and methanol as mobile phase B. The gradient elution was 20% B at 0–30 min, 40% B at 30–40 min, 100% B at 40 min. The flow rate was kept at 10 mL/min, and the column temperature was maintained at 40 °C. The chlorophyll and carotenoids levels were measured according to the absorbance at 663 nm, 645 nm, and 470 nm (A663, A645, and A470) [33]. The flavonoid levels were measured as previously described [34]. Each sample comprised three biological replicates and the analysis represented the results of three independent experiments. 2.3. RNA Extraction, cDNA Library Construction, and Sequencing

Total RNA from approximately 100 mg of frozen fruits of YS, OS, and RS was extracted using the TRIzol 1 Reagent (Invitrogen, Waltham, MA, USA) according to manufacturer’s protocol. Genomic DNA was eliminated by using RNase-free DNase I (TaKaRa, Dalian, China) and then RNA integrity and purity was confirmed on 1% agarose gels and NanoDrop™ 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). All RNA extracts showed a 260/280 nm ratio from 1.9 to 2.2. Approximately 25 μg total RNA of no less than 600 ng/μL concentration was used for the cDNA library construction. The steps of the cDNA library construction and the subsequent sequencing using an Illumina HiSeq™ 4000 (San Diego, CA, USA) were performed by staff at Gene Denovo Biotechnology Co. (Guangzhou, China).

2.4. De Novo Assembly and Functional AnnotationFirst, the raw data were filtered to remove reads with unknown sequencing ‘N’ and low-quality reads with more than 50% bases with a quality value ≤5. Then, mixing the clean reads from the three samples, the de novo assembly was performed using the assembly software Trinity (Trinityrnaseq_r2013_08_14 version) to obtain transcript sequences, and the longest transcript in each gene was taken as a single gene cluster (unigene) [35]. To annotate unigenes sequences of ‘Huaxiu’ plums, Blastx search (E-value −5) was used to search against the NCBI nonredundant protein database [36] (NR, http://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins, last accessed on 23 August 2022), the SwissProt protein database (http://www.expasy.ch/sprot, last accessed on 24 August 2022), the Kyoto Encyclopedia of Genes and Genomes database [37] (KEGG, http://www.genome.jp/kegg, last accessed on 24 August 2022), and the Clusters of Orthologous Groups of proteins database [38] (COG, http://www.ncbi.nlm.nih.gov/COG, last accessed on 24 August 2022). These annotation databases were used for comparisons with homologous genes. GO function classification was performed using Blast2GO [39] (http://www.blast2go.com/b2ghome, last accessed on 14 September 2022) with E ≤ 10−5, the distribution of the GO functional classifications of the unigenes was plotted using WEGO software [40], which allowed categorization into three different GO terms, including molecular function, cellular component, and biological process. The KEGG pathway annotation was performed using the Blast software against the KEGG database [41]. 2.5. Expression AnalysisAfter assembly, unigene expression levels were quantified using fragments per kilo base of transcript per million mapped reads (FPKM). FPKM values were calculated using RSEM (RNA-Seq by Expectation Maximization) [42]. Differentially expressed genes (DEGs) analysis of two samples was performed using the DEG Seq R package [43]. The differentially expressed unigenes between two samples were screened using a false discovery rate (FDR), which is used to determine p-value thresholds in multiple testing [44]. The significance of the DEGs were determined based on a threshold of FDR p-value 45] (https://github.com/tanghaibao/GOatools, last accessed on 16 September 2022).Twelve DEGs involved in flavonoid biosynthesis were selected for validation by real-time quantitative RT-PCR (qRT-PCR). Total RNA was extracted and qRT-PCR was performed with a method modified as previously described. The primer sequences used for qRT-PCR are listed in Table S1. The relative gene expression level was calculated according to the 2−ΔΔCt method. To visualize the relative expression levels data, YS was normalized as “1”. Three biological and three technical replicates were performed in these experiments. 2.6. Statistical Analysis

Statistical analysis of variance (ANOVA) with Duncan’s new multiple range test was carried out to compare cultivar mean values using SPSS Version 16.0 (Chicago, IL, USA). The significance level was set to p < 0.01. Graph Pad Prism version 6.0 (Graph Pad Software, San Diego, CA, USA) and Photoshop CS5 (Microsoft, Redmond, WA, USA and Adobe, San Jose, CA, USA) were used for graph plotting.

4. DiscussionThe color of the pulp cannot only be attractive in appearance, but also has higher nutritional value [1]. Therefore, it is very important to elucidate the genetic mechanism of pulp color regulation. It is generally believed the color of the pulp is determined by the comprehensive performance of chlorophyll, carotenoids, anthocyanins, and other pigment substances. Anthocyanin biosynthesis is fairly complex and is associated with flavonoids [46]. In our study, it was found that, during the coloring process of the pulp of ‘Huaxiu’ plums, the chlorophyll was gradually degraded and the synthesis of anthocyanin increased (Figure 1). When the comprehensive performance of anthocyanins and carotenoids made the pulp orange, then anthocyanins were synthesized in large amounts, the color of carotenoids was masked by anthocyanins, and the pulp was red. The main objective of this study was to identify the genes involved in anthocyanin biosynthesis in plums.In this study, we have performed transcriptome sequencing of the stages of three fruit pulp colors of ‘Huaxiu’ plums with the use of advanced high-throughput Illumina RNA-Seq technology. The results not only enrich the gene information of P. salicina, but also can help explore the molecular genetics and biochemical characteristics of Prunus salicina Lindl. and its related species with the generated transcriptome data. In total, 57,119 unigenes were assembled, with a mean length of 953 bp (Table 1), which is comparable to 944 bp for sweet cherry (P. avium L.) [47], and with the longer to the previously reported other species such as 872 bp for cultivar ‘Furongli’ (P. salicina L.) [1] and 531 bp for Chinese bayberry (Myrica rubra) [48]. Approximately 61.6% of the unigenes were annotated to public databases (NR, Swiss-Prot, GO, COG, and KEGG), which means that more than one-third of the unigenes have no apparent homologs, with similar results seen in other no model plant species [49]. The unannotated unigenes could be plum-specific genes with novel functions, which may be related with some unique biosynthesis processes and pathways in the results. Furthermore, the annotated unigenes of P. salicina L. indicated the highest homology to those of Prunus mume (55.0%), followed by Malus domestica (4.8%) and Prunus persica (3.4%) (Figure 2B and Table S4), which may indicate the evolutionary relationship among these species. In spite of a large number of unigene sequences that indicated no matches, many of the unigenes were still assigned to a wide range of GO and KEGG classifications. The KEGG function annotation analysis showed that 7595 unigenes were involved in 128 biosynthesis process. The largest number of unigenes was associated with the ribosome, carbon metabolism, and biosynthesis of amino acids. However, the smallest number of unigenes was associated with anthocyanin biosynthesis, which have only one matching unigene. All of these data contribute to the study of the metabolic and biosynthesis mechanisms in P. salicina.The RNA-Seq analysis revealed that the numbers of DEGs differed at the coloration stages, and we identified 1095, 3414, and 4947 DEGs between yellow and orange, orange and red, and yellow and red stages, respectively (Figure 5). More DEGs were detected at the yellow and red stage than at the yellow and orange stage, suggesting greater changes in the pulp color during the final ripening stage. Anthocyanin, the most important metabolite in flavonoid production, is an essential nutritional component in P. salicina fruits and their products. In our study, the flavonoid pathways were significantly enriched in the KEGG pathway. We identified many DEGs between different stages of the pulp color involved in anthocyanin and flavonoid biosynthesis, which mainly were structural genes, including CHS, CHI, DFR, F3H, F3′H, and LDOX, and were significantly upregulated during the pulp color of yellow vs. red color stages (Table 2). These observations agree well with those qRT-PCR results mentioned above. This is in accordance with findings for other fruits. To date, most of the structural genes in the anthocyanin biosynthetic pathway are upregulated during the fruit development of red/green skin color mutations of pear [50]. Similarly, coordinated expression changes of ANS, DFR1, F3H, F3′H, and UFGT have also been demonstrated in differently colored plum, sweet cherry, Chinese bayberries, and other plants [47,51].CHS is considered to be a key enzyme in the anthocyanin biosynthesis of Rosaceous plants, which have diverse functions such as defense against pathogens and pigment biosynthesis. CHS proteins have been found responsible for the red coloration in crabapple cultivars [52], and the CHS protein of Japanese morning glory was also found to enhance both flavonoid production and flower pigmentation [53]. In the present study, we found that two CHSs (Unigene0001957 and Unigene0043265) were significantly upregulated at each stage, with the highest expression in the red stage. However, they do not correlate exactly with the increased concentration of anthocyanin content and total flavonoids during ‘Huaxiu’ plum coloration (Figure 1). This was probably due to the complicated composition of flavonoids. DFR and LDOX/ANS are late anthocyanin biosynthetic genes. F3H was one of three main enzymes in the primary phases of the flavonoid pathway. Transcript levels of F3H were greater in RS than YS, and the upregulation of F3H genes in RS indicates that it contributed to the accumulation of anthocyanin, which led to the red pigmentation in the plums. The expression of F3H also proved that it could make apples red [40]. In this study, these two structural genes (Unigene0012798 and Unigene0001105) showed the highest expression levels in the red stage, with the highest anthocyanin concentration (Figure 1, Table 2), which is consistent with findings for apple skin [54]. Thus, we believe that these genes may be play an important role in regulating anthocyanin biosynthesis in ‘Huaxiu’ plum fruit pulp.The red hue of plant organs is caused due to anthocyanins, and the accumulation of these pigments is also regulated by transcription factors (TFs). In Rosaceae species, MYBs play a critical role as key transcription factors for all of the anthocyanin biosynthetic pathway genes or for the regulation of single key genes in fruit and flower color formation, particularly MYB10 genes, which are responsible for part of the natural variation in anthocyanin colors [10]. The bHLH proteins and NAC proteins have also been reported to be involved in anthocyanin synthesis [1]. The exact roles of these candidate transcription factor should be investigated in further studies.