## 4 Discussions  
The metabolites and transcriptome profiling of red-peel and yellow-peel pitayas are used to elucidate a betalian biosynthesis pathway [3; 32; 39]. Metabolites, including phenol, flavonoid, starch, sugar and acid, are investigated in fruit development of red-peel pitayas [34]. The transcriptome analyses of red-peel pitayas showed that the key enzymes (invertase and sucrose synthase) and gene (_HypVAl1_) 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  
Figure 9: The proposed TCA cycle pathways in yellow pitaya and transcript abundance of their genes in ‘WCHL’ and ‘YCHL’ pitayas. Gene abbreviations are shown in Table S9. Letters a-f represent malic, citric, citromalic, oxalic acid, fumaric, and succinic acids, respectively. The correlation analyses of organic 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. 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 yellow-peel 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 _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.