Metabolic Profiling of Sugars and Organic Acids, and Expression Analyses of Metabolism-Associated Genes in Two Yellow-Peel Pitaya Species

Sugar and organic acids are important factors determining pitaya fruit quality. However, changes in sugars and acids, and expressions of metabolism-associated genes during fruit maturation of yellow-peel pitayas are not well-documented. In this study, metabolic and expression analyses in pulps of different fruit developmental stages of ‘Wucihuanglong’ (‘WCHL’, Hylocereus undatus) and ‘Youcihuanglong’ pitaya (‘YCHL’, Hylocereus megalanthus) were used to explore the sugar and organic acid metabolic process. Total phenols and flavonoids were mainly accumulated at S1 in pitaya pulps. Ascorbic acid contents of ‘WCHL’ pitaya were higher than that of ‘YCHL’ pitaya during fruit maturation. Starch was mainly accumulated at early fruit development stages while soluble sugars were rich in late stages. Sucrose, fructose, and glucose were the main sugar components of ‘YCHL’ pitaya while glucose was dominant in ‘WCHL’ pitaya. Malic and citric acids were the main organic acids in ‘WCHL’ and ‘YCHL’ pitayas, respectively. Based on the transcriptome analyses, 118 genes involved in pitaya sugar and organic acid metabolism were obtained. Results from the correlation analyses between the expression profiling of candidate genes and the contents of sugar and organic acid showed that 51 genes had a significant correlation relationship and probably perform key role in pitaya sugar and organic acid metabolism processes. The finding of the present study provides new information for quality regulation of pitayas.


Introduction
Pitaya (also known as dragon fruit) belonging to Hylocereus genus within the Cactaceae family (Caryophyllales order) is native to Mexico, and Central and South America. Nowadays, pitayas are widely commercially cultivated in the tropical and subtropical regions. Based on the color of peel and pulp, pitaya is mainly classified into three species, i.e., Hylocereus undatus (H. undatus, red or yellow peel with scales and white pulp), H. monacanthus or H. polyrhizus (red peel with scales and red pulp) and H. megalanthus or Selenicereus megalanthus (yellow peel without scales and with white pulp) [1]. Pitaya is popular with consumers due to its abundant betalains, exotic appearance, fresh and sweet taste, and high nutrients [2][3][4]. Moreover, yellow-peel pitayas have economic potential in the market due to its conspicuous appearance, shocking yellow color and long shelf life. Pitaya cultivars from a different genetic background have different biochemical and Elucidation of key metabolites and candidate genes responsible for fruit quality is beneficial for breeding new pitaya cultivars and improving their nutritional values. To date, candidate genes related to betalain biosynthesis were isolated from transcriptome data of red-peel and yellow-peel pitayas [31,32]. Besides, several candidate genes involved in sugar biosynthesis were also obtained through RNA-Seq [16]. However, compared with red-peel pitayas, genes related to sugars and organic acids in fruit development and maturation of yellow-peel pitaya are rarely studied. In this study, nine fruit development stages of 'WCHL' and 'YCHL' pitaya pulps were collected to investigate the changes in the total phenol, total flavonoid, ascorbate, starch, soluble sugar, major sugar, and organic acid components. Subsequently, the transcription abundance of candidate genes related to ascorbate synthesis and regeneration, starch synthesis and degradation, major sugar synthesis and TCA cycle were analyzed in the three fruit developmental stages of 'WCHL' and 'YCHL' pitaya pulps.
Elucidation of key metabolites and candidate genes responsible for fruit quality is beneficial for breeding new pitaya cultivars and improving their nutritional values. To date, candidate genes related to betalain biosynthesis were isolated from transcriptome data of red-peel and yellow-peel pitayas [31,32]. Besides, several candidate genes involved in sugar biosynthesis were also obtained through RNA-Seq [16]. However, compared with red-peel pitayas, genes related to sugars and organic acids in fruit development and maturation of yellow-peel pitaya are rarely studied. In this study, nine fruit development stages of 'WCHL' and 'YCHL' pitaya pulps were collected to investigate the changes in the total phenol, total flavonoid, ascorbate, starch, soluble sugar, major sugar, and organic acid components. Subsequently, the transcription abundance of candidate genes related to ascorbate synthesis and regeneration, starch synthesis and degradation, major sugar synthesis and TCA cycle were analyzed in the three fruit developmental stages of 'WCHL' and 'YCHL' pitaya pulps.

Measurements of Total Flavonoids
The total flavonoids were measured by aluminum chloride [33]. A total of 0.5 g samples were extracted with 10 mL 80% aqueous methanol (v/v) solution. After centrifugation at 5000 rpm for 10 min, 100 µL supernatants, 1.9 mL 90% aqueous ethanol (v/v) and 300 µL 5% NaNO 2 were stilled for 6 min. After that, 300 µL 10% Al(NO 3 ) 3 and 2 mL 4% NaOH were added to each mixture with an interval of 6 min, and measured by spectrophotometer at 510 nm after standing 10 min. All determinations were performed in three biological repetitions.

Measurements of Starch and Soluble Sugar
Samples of 0.5 g were extracted with 8 mL 80% aqueous methanol (v/v) solution and put in a water bath at 80 • C for 30 min. The supernatants were collected after centrifugation at 5000 rpm for 10 min. A total of 10 mg activated carbon (AC) was added to the supernatants and then incubated in a water bath at 80 • C for 30 min. After filtrating the AC, the supernatants were diluted to 25 mL with distilled water. The supernatants and residues were collected for measuring soluble sugar and starch, respectively.
A total of 2 mL distilled water was added to the residues (dried in 80 • C) and kept in boiling water for 10 min. After cooling, 2 mL 9.2 mol·L −1 perchloric acid and 6 mL distilled water were added with an interval of 10 min. The supernatants were collected by centrifuging at 5000 rpm for 10 min. The resides were subjected to a similar second extraction, and then the supernatants were collected for analyses of the absorbance values at 620 nm by a spectrophotometer.
A total of 1 mL supernatants with 5 mL anthrone (1 µg·mL −1 ) were kept in boiling water for 10 min, and then the absorbance values were measured at 620 nm by a spectrophotometer.

Measurements of Sugars and Organic Acids
A gas chromatography-mass spectrometry (GC-MS) was used to assay sugars and organic acids according to Lisec et al. [34] with minor modifications. Samples of 50 mg were extracted with 1.4 mL 70% aqueous methanol (v/v) (20 • C). Then, 60 µL ribitol (0.2 mg·mL −1 , internal quantitative standard) was added to each mixture and followed by shaking in a Thermomixer Compact (Eppendorf, Germany) at 70 • C with the speed of 950 rpm for 10 min. The supernatants were collected by centrifugation at 11,000× g for 10 min. A total of 750 µL chloroform and 1.5 mL distilled water were added to the supernatants and then centrifuged at 2200× g for 15 min. A total of 100 µL of the upper phase (polar phase) was dried in a 1.5 mL tube by a vacuum concentrator (Eppendorf Concentrator plus, Germany) at room temperature. The dried samples were oximated with 20 µL of methoxyamination reagent in 37 • C for 2 h in a Thermomixer Compact (950 rpm) and the derivatization reaction without samples was used as the control. After oximation, silylation occurred by adding 35 µL of MSTFA to each mixture and incubated in a Thermomixer Compact (950 rpm) at 37 • C for 30 min. A total of 40 µL of each derivatization product were transferred into glass vials for GC-MS analyses using an Agilent 7890A GC system equipped with an Agilent 7693 autosampler and Agilent 5975Cinert MSD with Triple Axis Detector (Agilent, Atlanta, GA, USA). The operating parameters were performed according to Hua et al. [3].

RNA Extraction and Sequencing
Three key fruit development stages (S2, S5, and S7) of 'WCHL' and 'YCHL' pitaya pulps were used for RNA-Seq with three biological repetitions (PRJNA797242). The total RNA was extracted using the RNA Prep Pure Plant Kit (TIANGEN, Beijing, China) according to the manufacturer's instructions. The integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). A total of 1 µg total RNA of each sample was used as input material for the RNA sample preparations. The first-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). The second-strand cDNA was synthesized using DNA Polymerase I and RNase H. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA) to select cDNA fragments of preferentially 370~420 bp in length. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. The library quality was assessed using the Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, CA, USA) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.

Transcriptome Data Analyses
Raw data (raw reads) of FASTQ format were firstly processed through in-house perl scripts. Clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. Q20, Q30, and GC content of the clean data were calculated and listed in Table S1. The datasets were functionally annotated by pitaya genome using Hisat2 (version 2.0.5) [35]. The quantification of each gene expression level (fragments per kilobase millions (FPKM)) was counted using fea-tureCounts (version 1.5.0-p3). The differential expression analyses of two libraries were performed using the DESeq2 package (version 3.10) of R software (version 4.1.2). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using KofamKOALA (https://www.genome.jp/tools/kofamkoala/, accessed on 5 February 2022). The transcript abundance of candidate genes was drawn by Tbtools software [36]. The accuracy of the RNA-Seq data was verified by RT-qPCR with specific primers (Table S2) according to the method of Xie et al. [32].

Statistical Analysis
Comparisons of the groups were performed by a one-way analysis of variance (Oneway ANOVA) with a Duncan test (p < 0.01). The correlation coefficients were calculated by the SPSS 25 software (SPSS Inc., Chicago, IL, USA) through Pearson's correlation and a two-tailed t test (p < 0.05 and p < 0.01).

Changes in Total Phenol, Total Flavonoid and Ascorbic Acid Contents during Fruit Development
Phenol, flavonoid, and ascorbic acid belong to the bioactive compounds which play important roles in plant antioxidant activity [37]. The change in total phenol, total flavonoid, and ascorbic acid contents were analyzed during fruit development of 'WCHL' and 'YCHL' pitayas. Total phenol and flavonoid contents reached their maximum at stage 1 during fruit development of 'WCHL' and 'YCHL' pitayas ( Figure 2A,B). Higher contents of total phenols and flavonoids were detected in 'WCHL' pitaya than that of 'YCHL' pitaya at ripening stages. Meanwhile, higher contents of ascorbic acid were detected in 'WCHL' pitaya than that of 'YCHL pitaya ( Figure 2C). These data suggested that 'WCHL' pitaya accumulates more bioactive compounds than that of 'YCHL' pitaya.

Changes in Sugar Contents during Fruit Development
Sugar contents were analyzed in pulps during fruit development of 'WCHL' and 'YCHL' pitayas. Starch showed an increasing pattern from stage 1 to stage 3, and declined from stage 3 to stage 5 and kept low levels thereafter ( Figure 3A), suggesting that starch was synthesized from stage 1 to stage 3 and degraded after stage 3. Soluble sugar showed an upward trend and higher contents were detected in pulps of 'WCHL' pitaya than that of 'YCHL' pitayas during fruit development ( Figure 3B). These results suggested that starch was mainly accumulated before stage 4 while soluble sugar was highly accumulated after stage 5, indicating that starch synthesis and degradation resulted in the accumulation of soluble sugars during fruit development of the two pitaya cultivars.
The sugar components including glucose, sucrose, fructose, galactose, inositol, and sorbitol were assayed during fruit development of the two yellow-peel pitayas ( Figure  3C-H). Glucose, fructose, sorbitol, and galactose kept increasing tendency while sucrose and inositol began to highly accumulated after stage 5 during fruit maturation. Sucrose, fructose, and glucose were the dominant soluble sugars that highly accumulated at the later stages (from stage 5 to stage 9) in 'YCHL' pitayas ( Figure 3J), while the main soluble sugar was glucose in 'WCHL' pitayas ( Figure 3I). These results suggested that the sweetness of 'YCHL' pitaya is mainly determined by sucrose, fructose, and glucose compared with only glucose for 'WCHL' pitaya.

Changes in Sugar Contents during Fruit Development
Sugar contents were analyzed in pulps during fruit development of 'WCHL' and 'YCHL' pitayas. Starch showed an increasing pattern from stage 1 to stage 3, and declined from stage 3 to stage 5 and kept low levels thereafter ( Figure 3A), suggesting that starch was synthesized from stage 1 to stage 3 and degraded after stage 3. Soluble sugar showed an upward trend and higher contents were detected in pulps of 'WCHL' pitaya than that of 'YCHL' pitayas during fruit development ( Figure 3B). These results suggested that starch was mainly accumulated before stage 4 while soluble sugar was highly accumulated after stage 5, indicating that starch synthesis and degradation resulted in the accumulation of soluble sugars during fruit development of the two pitaya cultivars.
The sugar components including glucose, sucrose, fructose, galactose, inositol, and sorbitol were assayed during fruit development of the two yellow-peel pitayas ( Figure 3C-H). Glucose, fructose, sorbitol, and galactose kept increasing tendency while sucrose and inositol began to highly accumulated after stage 5 during fruit maturation. Sucrose, fructose, and glucose were the dominant soluble sugars that highly accumulated at the later stages (from stage 5 to stage 9) in 'YCHL' pitayas ( Figure 3J), while the main soluble sugar was glucose in 'WCHL' pitayas ( Figure 3I). These results suggested that the sweetness of 'YCHL' pitaya is mainly determined by sucrose, fructose, and glucose compared with only glucose for 'WCHL' pitaya.

Changes in Organic Acids during Pitaya Fruit Development
The changes in six organic acids, i.e., oxalic, malic, fumaric, succinic, citric, and citramalic acids during fruit development of 'WCHL' and 'YCHL' pitayas were presented in Figure 4. Malic and citric acid contents were increasing until stage 4 and then gradually decreased during pitaya fruit development of 'WCHL' and 'YCHL' pitayas ( Figure 4A,B). However, citramalic acids were mainly accumulated in the unripe stages and rapidly degraded in the ripe stages of 'WCHL' pitaya ( Figure 4C). Higher contents of malic, citramalic and fumaric acid were detected in pulps of 'WCHL' pitaya than that of 'YCHL' pitaya while citric acid was higher accumulated in 'YCHL' pitaya than that of 'WCHL' pitaya ( Figure 4A,C,E). Oxalic and succinic acids kept at lowly levels without significant difference between two pitayas ( Figure 4D,F). Thus malic and citric acids were respectively the dominant organic acids in 'WCHL' and 'YCHL' pitayas, and contribute to sourness of pitaya pulps ( Figure 4G,H).

Candidate Genes Involved in Ascorbic Acid Metabolism
Based on the ascorbic acid biosynthesis and regeneration pathway, candidate genes encoding its key enzymes were investigated in 'WCHL' and 'YCHL' pitayas. The L-galactose, L-gulose, myo-inositol and D-galacturonate pathways were proposed to be involved in ascorbate biosynthesis in plants [5,38]. In the two yellow-peel pitayas, all enzymes associated with the L-galactose pathway were found and 61 candidate genes were obtained from the pathway (Table S3). Among these candidate genes, 27 genes were highly expressed in fruit development of yellow-peel pitayas, especially APX2 and MDAR3 that showed a downward trend in 'YCHL' pitayas and highly expressed at stage 5 in 'WCHL' pitaya ( Figure 6; Table S3). According to the correlation analyses between the ascorbic acid contents and expression patterns of these candidate gene, PMM1, GMP3, GMP4, APX1, APX9, and MDAR1 were down-regulated during fruit development, and showed negative correlation relationship with ascorbic acid contents ( Figure 6; Table S4). PMI1, PMI3 and MDAR2 were up-regulated during fruit development and showed significant positive correlation relationship with ascorbic acid contents. These results indicated that PMI1 and PMI3 are the potential key genes involved in ascorbic acid synthesis, while APXs (APX1, APX2, and APX9), and MDARs (MDAR1, MDAR2, and MDAR3) are responsible for ascorbic acid regeneration in 'WCHL' and 'YCHL' pitayas.

Candidate Genes Involved in Ascorbic Acid Metabolism
Based on the ascorbic acid biosynthesis and regeneration pathway, candidate genes encoding its key enzymes were investigated in 'WCHL' and 'YCHL' pitayas. The Lgalactose, L-gulose, myo-inositol and D-galacturonate pathways were proposed to be involved in ascorbate biosynthesis in plants [5,38]. In the two yellow-peel pitayas, all enzymes associated with the L-galactose pathway were found and 61 candidate genes were obtained from the pathway (Table S3). Among these candidate genes, 27 genes were highly expressed in fruit development of yellow-peel pitayas, especially APX2 and MDAR3 that showed a downward trend in 'YCHL' pitayas and highly expressed at stage 5 in 'WCHL' pitaya ( Figure 6; Table S3). According to the correlation analyses between the ascorbic acid contents and expression patterns of these candidate gene, PMM1, GMP3, GMP4, APX1, APX9, and MDAR1 were down-regulated during fruit development, and showed negative correlation relationship with ascorbic acid contents ( Figure 6; Table S4). PMI1, PMI3 and MDAR2 were up-regulated during fruit development and showed significant positive correlation relationship with ascorbic acid contents. These results indicated that PMI1 and PMI3 are the potential key genes involved in ascorbic acid synthesis, while APXs (APX1, APX2, and APX9), and MDARs (MDAR1, MDAR2, and MDAR3) are responsible for ascorbic acid regeneration in 'WCHL' and 'YCHL' pitayas.  Table S3. The correlation analyses of ascorbic acid contents and gene expressions are labeled as * (p < 0.05, two-tailed) or ** (p < 0.01, two-tailed), red indicates positive relationship while blue is negative.

Candidate Genes Involved in Starch Metabolism
The starch synthesis and degradation pathway has been proposed in plants [5]. A total of 90 candidate genes involved in starch synthesis and degradation were obtained in pitayas (Table S5). Among them, 32 candidate genes were highly expressed in pulps, especially five starch degradation related genes (AMY6, BAM10, BAM12, PHS2, and PHS3) which highly expressed at stage 5 during fruit development (Figure 7). A total of 13 candidate genes showed significantly negative correlation, including PGM2, StSy6, GWD2, GWD4, PWD1, BAM10, DPE1, AGL8, AMY6, AMY7, PHS2, PHS3, and PHS4 (Table S6). Additionally, the expression of AGPS2 and AGPS5 exhibited a downward trend and showed positive correlation with starch contents during fruit development. These data demonstrated that AGPSs (AGPS2 and AGPS5) are key genes related to the starch synthesis while AMY6, BAM10, PHS2, and PHS3 are important genes involved in starch degradation in pitaya (Table S6).  Table S3. The correlation analyses of ascorbic acid contents and gene expressions are labeled as * (p < 0.05, two-tailed) or ** (p < 0.01, two-tailed), red indicates positive relationship while blue is negative.

Candidate Genes Involved in Starch Metabolism
The starch synthesis and degradation pathway has been proposed in plants [5]. A total of 90 candidate genes involved in starch synthesis and degradation were obtained in pitayas (Table S5). Among them, 32 candidate genes were highly expressed in pulps, especially five starch degradation related genes (AMY6, BAM10, BAM12, PHS2, and PHS3) which highly expressed at stage 5 during fruit development (Figure 7). A total of 13 candidate genes showed significantly negative correlation, including PGM2, StSy6, GWD2, GWD4, PWD1, BAM10, DPE1, AGL8, AMY6, AMY7, PHS2, PHS3, and PHS4 (Table S6). Additionally, the expression of AGPS2 and AGPS5 exhibited a downward trend and showed positive correlation with starch contents during fruit development. These data demonstrated that AGPSs (AGPS2 and AGPS5) are key genes related to the starch synthesis while AMY6, BAM10, PHS2, and PHS3 are important genes involved in starch degradation in pitaya (Table S6).  Table S5. The correlation analyses of starch content and gene expressions labeled as * (p < 0.05, two-tailed) or ** (p < 0.01, two-tailed), red indicates positive relationship wh blue is negative.

Candidate Genes Involved in Sugar Metabolism
Totally, 73 candidate genes associated with sugar metabolism pathway were o tained in the transcriptome data, of which 18 candidate genes (three SPSs, four Ivrs, th SuSys, four HXKs, three FRKs, and one PGI) were highly expressed and probably coor nately regulate the sugar metabolism in the two yellow-peel pitayas (Table S7; Figure Among them, higher expression levels of SuSy1, SuSy5, SuSy11, FRK5, FRK9, and FRK were detected, of which SuSy5, SuSy11, FRK5, and FRK9 showed a downward trend wh SuSy1 and FRK10 were up-regulated during fruit development. The correlation analy suggested that up-regulated genes, i.e., SPS1 and FRK9 showed positive correlation w sucrose and inositol contents while Ivr4 and HXK1 demonstrated positive correlation w glucose, fructose, sorbitol, and galactose accumulation (Figure 8; Table S8). SuSy5, SuSy  Ivr10, FRK5, FRK10, HXK5, HXK9, and HXK12 had negative correlation with pitaya m sugars. These results suggested that SuSys (SuSy1, SuSy5 and SuSy11) and FRKs (FRK FRK9 and FRK10) are key genes to catalyze the pitaya sugar metabolism.  Table S5. The correlation analyses of starch content and gene expressions are labeled as * (p < 0.05, two-tailed) or ** (p < 0.01, two-tailed), red indicates positive relationship while blue is negative.

Candidate Genes Involved in Sugar Metabolism
Totally, 73 candidate genes associated with sugar metabolism pathway were obtained in the transcriptome data, of which 18 candidate genes (three SPSs, four Ivrs, three SuSys, four HXKs, three FRKs, and one PGI) were highly expressed and probably coordinately regulate the sugar metabolism in the two yellow-peel pitayas (Table S7; Figure 8). Among them, higher expression levels of SuSy1, SuSy5, SuSy11, FRK5, FRK9, and FRK10 were detected, of which SuSy5, SuSy11, FRK5, and FRK9 showed a downward trend while SuSy1 and FRK10 were up-regulated during fruit development. The correlation analyses suggested that up-regulated genes, i.e., SPS1 and FRK9 showed positive correlation with sucrose and inositol contents while Ivr4 and HXK1 demonstrated positive correlation with glucose, fructose, sorbitol, and galactose accumulation ( Figure 8; Table S8). SuSy5, SuSy11, Ivr10, FRK5, FRK10, HXK5, HXK9, and HXK12 had negative correlation with pitaya main sugars. These results suggested that SuSys (SuSy1, SuSy5 and SuSy11) and FRKs (FRK5, FRK9 and FRK10) are key genes to catalyze the pitaya sugar metabolism.

Discussions
The metabolites and transcriptome profiling of red-peel and yellow-peel pitayas are used to elucidate a betalain biosynthesis pathway [3,32,39]. Metabolites, including phenol, flavonoid, starch, sugar and acid, are investigated in fruit development of redpeel pitayas [3,4]. The transcriptome analyses of red-peel pitayas showed that the key enzymes (invertase and sucrose synthase) and gene (HpVAI1) are involved in the sugar metabolism [16]. Those results provide fundamental information for developing pitaya quality and molecular breeding. However, little information is available about sugar and organic acid metabolism in yellow-peel pitayas. In this study, sugars and organic acids, and expression of metabolism-associated genes during fruit maturation of yellow-peel pitayas were analyzed. DEGs related to sugar and acid metabolism from RNA-Seq were verified by RT-qPCR (Pearson's correlation coefficient R 2 = 0.8078; Figure S1).
Phenolics and flavonoids are the antioxidant compounds which can decrease the incidence of oxidative stress and associated with diseases in human health [33]. Phenolics and flavonoids are highly produced in unripe fruits [22] and mature fruits [40]. Besides, ascorbic acid is one of the most abundant antioxidants with various accumulation levels in different species [27,41]. In this study, higher contents of phenols and flavonoids were detected in pulps at the initial fruit development stages compared with low contents in mature stages (Figure 2A,B and Figure 10). A higher content of ascorbic acid was detected in 'WCHL' pitaya than that of 'YCHL' pitaya ( Figure 2C). The L-galactose pathway represents the major route to L-ascorbic acid biosynthesis in higher plants. APX and MDHR are the key components involved in the ascorbate-glutathione cycle which operates in plant chloroplasts for H 2 O 2 detoxifications [29,42]. In our study, 27 candidate genes involved in ascorbic acids synthesis and regeneration were obtained ( Figure 6). APX2 and MDAR3 showed higher expression levels than the other genes during fruit development of yellowpeel pitayas (Table S4). Compared with the expression profiling and ascorbic acid contents in yellow-peel pitayas, MDAR2 were positively regulated the ascorbic acid contents while APX1, APX9, and MDAR1 showed negative correlation. These results indicate that APX and MDHR may play major roles in controlling the level of L-ascorbic acid in yellow-peel pitaya fruits and their gene family members were also identified and characterized in Arabidopsis thaliana [42,43]. and flavonoids are highly produced in unripe fruits [22] and mature fruits [40]. Besides, ascorbic acid is one of the most abundant antioxidants with various accumulation levels in different species [27,41]. In this study, higher contents of phenols and flavonoids were detected in pulps at the initial fruit development stages compared with low contents in mature stages (Figures 2A,B and 10). A higher content of ascorbic acid was detected in 'WCHL' pitaya than that of 'YCHL' pitaya ( Figure 2C). The L-galactose pathway represents the major route to L-ascorbic acid biosynthesis in higher plants. APX and MDHR are the key components involved in the ascorbate-glutathione cycle which operates in plant chloroplasts for H2O2 detoxifications [29,42]. In our study, 27 candidate genes involved in ascorbic acids synthesis and regeneration were obtained ( Figure 6). APX2 and MDAR3 showed higher expression levels than the other genes during fruit development of yellowpeel pitayas (Table S4). Compared with the expression profiling and ascorbic acid contents in yellow-peel pitayas, MDAR2 were positively regulated the ascorbic acid contents while APX1, APX9, and MDAR1 showed negative correlation. These results indicate that APX and MDHR may play major roles in controlling the level of L-ascorbic acid in yellow-peel pitaya fruits and their gene family members were also identified and characterized in Arabidopsis thaliana [42,43].  Carbohydrate was first synthesized as starch and then transferred into soluble sugars during fruit development of 'WCHL' and 'YCHL' pitayas ( Figure 3A,B and Figure 10). The starch-to-sugar conversion is not only responsible for fruit sweetness but it also provides energy to coloration [3,44]. APS is a key enzyme of starch synthesis in seeds, tubers, and fruits [45][46][47]. In this work, 32 candidate genes (four PGMs, four AGPSs, four StSy, four GWDs, two PWDs, two AGLs, AMYs, two BAMs, four DPEs, and four PHSs) involved in starch synthesis and degradation were obtained (Figure 7). Expression patterns of most genes involved in starch degradation had a negative correlation with the changes in starch contents (Table S6). AGPS2 and AGPS5 kept a downward trend and shared a positive correlation with starch contents during fruit development of yellow-peel pitayas suggesting that AGPS is also a key enzyme for pitaya starch synthesis. AMY and BAM play a prominent role in starch breakdown and participate in the regulation of plant growth, development, and stress responses [48]. PHS participates in phosphorolytic degradation of starch [49]. In the present study, higher expression levels of AMY6, BAM10, PHS2, and PHS3 related to starch degradation pathways were detected than other genes and negative correlation with starch contents (Table S5), indicating that these genes probably play important roles in transferring starch into sugar in pitayas.
Malic, citric, and citramalate acids were the three organic acids mainly accumulated at S2 to S5 in pitayas ( Figure 10). Due to the degradation of citramalate acid in maturation stages, malic acid (15.06 mg/g FW) and citric acid (5.76 mg/g FW) were the most abundant organic acids in 'WCHL' and 'YCHL' pitayas, respectively. CMS has been reported as a key enzyme responsible for citramalate synthesis in Escherichia coli and apple [50,51]. In yellow-peel pitayas, the expression patterns of CMS1 and CMS3 had positive correlation with citromalic acid contents, indicating they probably responsible for citromalic acid biosynthesis of pitaya fruits. PEPC catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate which is the substrate for forming citrate [14,52]. In this study, the accumulation of citric acids was significant positive correlation with the expression of PEPC3 and PEPC4 during pitaya fruit development. Furthermore, CS catalyzes the reaction of oxaloacetate and acetyl-CoA convert into citrate and coenzyme A, which is the first step in the TCA cycle [52]. However, no CSs show correlated relationship with citrate in the two yellow-peel pitayas. This suggests that carbon fluxed into TCA cycle mainly via PEPC3 and PEPC4 rather than CSs, probably because rich acetyl-CoA fluxed into the irreversible step catalyzed by CMSs for citramalate acid biosynthesis. MDH catalyzes the conversion between oxaloacetate and malate and FUM catalyzes the reversible interconversion between malate and fumarate [53,54]. A higher content of malic acid in 'WCHL' pitaya were detected than that of 'YCHL' pitaya. Malic acid content had a negative correlation with FUM1 and MDH1. Compared with 'YCHL' pitaya, MDH1 was strongly down-regulated in 'WCHL' pitaya responsible for more malic acid in 'WCHL' pitaya than 'YCHL' pitaya. Further work such as enzyme activity analyses and genetic transformation are necessary to elucidate their roles in modulating the fruit quality of pitayas.

Conclusions
In this study, higher total phenol and flavonoid contents were detected in the stage 1 of yellow-peel pitaya pulps during fruit development. A higher content of ascorbic acid was detected in 'WCHL' pitaya than 'YCHL' pitaya. Starch was mainly accumulated at early stages and transferred to soluble sugars at S5. Six sugars components (glucose, sucrose, fructose, galactose, inositol, and sorbitol) and six organic acids (oxalic, malic, fumaric, glyceric, succinic, citric and citromalic acid) were detected in pitayas. Glucose and malic acid were the key factors contributed to the taste and flavor quality of 'WCHL' pitaya. Sucrose, fructose, and glucose were the main sugars, and citric acid was the dominant acid responsible for fruit quality of 'YCHL' pitaya. Based on the expression profilings, 27 candidate genes probably involved in ascorbate biosynthesis and regeneration were achieved, of which PMIs was vital for ascorbic acid synthesis, and APXs and MDHRs played important roles in ascorbate accumulation. A total of 32 candidate genes probably involved in starch synthesis and degradation pathway were isolated, of which AGPSs were key genes for starch synthesis and AMY, BAM and PHSs were key regulators controlling starch degradation. SPSs Ivrs, SuSys, HXKs, FRKs and PGI coordinately regulated sucrose, fructose, and glucose synthesis, of which SuSys and FRKs were expressed more than the other genes. Organic acids were lowly accumulated in ripening stages associated with downward expression patterns of PGK, Enos, PEPCs, PKs, and PDHs which limited the carbon flux into TCA cycle. Twenty candidate genes involved in organic acid production and degradation (TCA cycle) were obtained in pitayas. The findings of this study provide basic information for improving fruit quality of yellow-peel pitaya.
Supplementary Materials: The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/plants11050694/s1, Figure S1: The RT-qPCR analyses of eleven candidate genes (A-K) in sugar and acid metabolic pathway and the coefficient analyses between FPKM values and RT-qPCR of those genes (L); Table S1: RNA-Seq data and corresponding quality control information; Table S2: Specific primers used in RT-qPCR assay; Table S3: The transcript abundance of genes related to ascorbate pathways in 'WCHL' and 'YCHL' pitayas pulps; Table S4: Correlation analyses between the ascorbic acid contents and expression patterns of candidate genes in 'WCHL' and 'YCHL' pitayas; Table S5: The transcript abundance of genes related to starch synthesis and degradation pathways in 'WCHL' and 'YCHL' pitayas; Table S6: Correlation analyses between the starch contents or total soluble sugar and expression patterns of candidate genes in 'WCHL' and 'YCHL' pitayas; Table S7: The transcript abundance of genes related to major sugar biosynthesis pathways in 'WCHL' and 'YCHL' pitayas; Table S8: Correlation analyses between the main sugar contents and expression patterns of candidate gene in 'WCHL' and 'YCHL' pitayas; Table S9: The transcript abundance of genes related to TCA cycle pathways in 'WCHL' and 'YCHL' pitayas; Table S10: Correlation analyses between the main organic acid contents and expression patterns of candidate genes in 'WCHL' and 'YCHL' pitayas.