## 4 Discussion  
### Effect of SA on the Flavonoid and Carotenoid Accumulation in Citrus Juice Sacs In Vitro  
Flavonoids and carotenoids are two important secondary metabolites that accumulate in plants. To date, the role of SA in flavonoid accumulation has been investigated in different plant species such as tea and cucumber [15; 26]. SA and flavonoids are both phenylpropanol derivatives, and recent studies have suggested the SA treatment affected the flavonoid biosynthesis in plants [27]. In tea, it was reported that exogenous methyl salicate treatment increased the expression of flavonoid biosynthesis-related genes, such as _CHS_ and _CHI_, and as a result the total content of flavonoid was enhanced by the methyl salicate treatment [15]. In cucumber, 0.5 mM salicylic acid treatment was found to be effective to enhance the contents of total phenolic compound and total flavonoid [26]. In the present study, the results showed that SA treatment induced the accumulation of eriocitrin, narirutin, and poncirin in the citrus juice sacs in vitro (Figure 1). The gene expression analysis showed that the expression of _CitCHS2_, _CitUFIGT2_, and _CitF3H_ was markedly increased by the SA treatment (Figure 3). As previously reported, _CitCHS2_, which was highly expressed in the young fruit, was an important gene regulating the biosynthesis of flavonoid in citrus fruit [28]. In the present study, the high expression of _CitCHS2_ could lead to enhance the contents of eriocitrin, narirutin, hesperidin, and poncirin in the SA-treated juice sacs. In addition, CitF3H, which catalyzes the hydroxylation of \(\beta\)-rings at the 3'-position, is a rate limiting enzyme for flavonoid biosynthesis in plant [29]. In this study, the high expression of _CitF3H_ in the SA treatment could contribute to increasing the content of eriocitrin, which has a hydroxy group on the \(\beta\)-rings at the 3'-position, in the citrus juice sacs.  
Different from flavonoid, the roles of SA in carotenoid accumulation were controversial in plants. In wheat and moong seedlings, total carotenoid content, size of xanthophyll pool, and de-epoxidation rate were induced along with an increase in SA concentration [30]. In plum, in contrast, the preharvest treatment of salicate significantly reduced the total carotenoid content [16]. In the present study, the results showed that the total carotenoid content was not significantly affected by the SA treatment, but the content of \(\beta\)-cryptoxanthin, which was the major carotenoid accumulated in the juice sacs, was clearly enhanced by the SA treatment. The gene expression results suggested that the higher expression of carotenoid biosynthetic genes, _CitPSY_, _CitPDS_, and _CitHYb_ and lower expression of carotenoid catabolic gene _CitNCCD3_ could lead to enhance \(\beta\)-cryptoxanthin content in the juice sacs treated by SA.  
Summarizing, we found that SA treatment was effective to enhance the contents of eriocitrin, narirutin, and poncirin and \(\beta\)-cryptoxanthin in the citrus juice sacs of Satsuma mandarin. Overall, our findings were consistent with a previous study, in which the preharvest treatment of salicate caused an increase of carotenoids and flavonoids contents in navel oranges [19]. Flavonoids and carotenoids are important antioxidant compounds in citrus fruits with beneficial effects to human health [31]. Thus, the results presented in this study suggested that SA treatment could be an effective method to improve the quality and nutritional values of citrus fruit.