**4. Discussion and Analysis**

Soluble sugars are important quality indicators of fruits [20]. In lychee, the main sugars are glucose, fructose, and sucrose [21]. Different kinds of lychees have different types of sugars that accumulate as a result of sugar metabolism, and Feizixiao lychees accumulate reducing sugars [15]. In addition, the sugar withdrawal phenomenon can occur [13], which needs further attention. The results of the present study showed that the soluble sugars in lychee pulp were mainly glucose and fructose, and the sucrose content was significantly lower than that of the two monosaccharides, the results of which were consistent with our previous results. The results also showed that the high-Ca treatment inhibited the accumulation of soluble sugars, which was consistent with data from pineapple treated with high Ca [8].

AI, NI, SS-I, SS-II, and SPS [22–25] play an important role in sugar metabolism. The results showed that the high-Ca treatment obviously inhibited the activities of AI and NI during the sugar accumulation period, inhibited the conversion of sucrose to glucose and fructose, and reduced the accumulation of reducing sugars in the fruit pulp. The high-Ca treatment increased SS-I activity and decreased SS-II activity in the early stage of sugar accumulation, and it also decreased SS-I activity and enhanced SS-II activity after sugar withdrawal; that is, at the early stage of sugar accumulation, SS mainly catalyzed the decomposition of sucrose into fructose, and at the time of sugar withdrawal, SS mainly catalyzed the synthesis of sucrose from fructose. At the same time, SPS activity was inhibited, resulting in a decrease in overall sugar accumulation in the pulp. The Feizixiao lychee fruit pulp industry is mainly controlled by the use of these key enzymes. This result is the same as those in previous reports that sugar metabolism of Feizixiao lychee fruit is mainly regulated by AI and SS [26].

Transcriptome analysis showed that high-Ca treatment did not cause changes in the expression of SPS-encoding genes, indicating that the inhibition of the activities of these two enzymes may occur posttranscriptionally, during translation and posttranslation; nonetheless, the underlying molecular mechanism needs further study.

Among the differentially expressed genes encoding α- and β-D-glucose-6-phosphate aldose ketone isomerase [27], the upregulation of D-glucoside glucohydrolase gene expression [28] suggests that the content of reducing sugars in the fruit pulp under high-Ca treatment remains relatively stable. α-Trehalose and glucose hydrolase can catalyze the

decomposition of trehalose to produce glucose, and the downregulation of related genes directly causes a reduction in glucose synthesis. SUS-encoding genes control SS enzyme synthesis, which catalyzes the NDP-α-D-glucose + D-fructose to ribonucleoside 5 - diphosphate + H<sup>+</sup> + sucrose (reversible) conversion reaction [29]. The expression of the SUS gene was downregulated in the high-Ca treatment group, which was consistent with the change trend of SS-I and SS-II activities in the high-Ca treatment group, the findings of which were opposite those of the CK group. In the forward direction, SS-II led to a decrease in the synthesis of sucrose in the pulp, and in the reverse direction, SS-I led to a decrease in the amount of sucrose converted to fructose; thus, the accumulation of soluble sugars in the pulp was inhibited.

High-Ca treatment upregulated VIN gene expression and downregulated CIN gene expression. Combined with the measurements of the sugar components, the sugar withdrawal phenomenon occurred after the sugar content peaked. At that time, the VIN expression in the CK group was significantly decreased, while the VIN expression in the high-Ca treatment group was not significantly decreased. VIN gene expression positively regulated AI activity [30], which was consistent with the AI activity trend. Moreover, AI promoted sucrose decomposition to provide a carbon source for growth; that is, by inhibiting the downregulation of VIN expression, high-Ca treatment may cause AI activity to remain at a high level, thus reducing sucrose accumulation. The expression of the CIN gene in the CK group significantly increased when sugar withdrawal occurred; the CIN gene positively regulated NI activity, but there was no change in CIN activity in the high-Ca treatment group. Among them, the upregulation of the CIN gene promoted the accumulation of reducing sugars [25], while the high-Ca treatment in this experiment inhibited the upregulation of NI expression, thus inhibiting the accumulation of reducing sugars. The results of this paper also confirm that invertase plays an important role in fruit sugar metabolism [31].

By analyzing the transcriptome results at the critical stage of fructose accumulation, we found that the genes of the CHS family involved in the flavonoid biosynthesis pathway were downregulated [32]. CHS is mainly responsible for catalyzing the conversion of coumaroyl-CoA to synthesize naringenin chalcone, which, as a precursor, participates in the formation of flavonoids. According to the results of a test analysis, the downregulation of related genes caused a reduction in chalcone synthesis, which indirectly inhibited the accumulation of flavonoids. High-Ca treatment inhibited the accumulation of glucose, fructose, sucrose, and soluble sugars. It can thus be inferred that the content of flavonoids was reduced [32], and the pulp was damaged by reactive oxygen species, thus inhibiting the accumulation of sugars. However, this needs to be further confirmed.

#### **5. Conclusions**

The physiological mechanism that occurs in response to excessive Ca fertilizer being sprayed onto leaves to inhibit sugar accumulation is as follows: First, Ca causes the expression of trehalase-encoding genes and SUS genes to be downregulated and inhibits the activities of trehalase, SS-I, and SS-II, thus inhibiting the accumulation of glucose, fructose, and sucrose. It does not cause the expression of SPS-related genes to change but does inhibit the accumulation of this sugar by inhibiting SPS activity. There may be other mechanisms involved in inhibiting the activity of this kind of enzyme, which needs further study (glucose metabolism). High-Ca fertilizer also causes decreased expression of the VIN gene, thus inhibiting AI activity and inhibiting glucose and fructose accumulation. In turn, these phenomena inhibit the upregulation of CIN gene expression in the fruit during the fruit growth and expansion period, thereby inhibiting the increase in NI activity in the pulp and causing a decrease in glucose and fructose accumulation in the treated pulp relative to that in the fruit in the CK group. Inhibition of CIN expression possibly causes a downregulation of CHS family gene expression, causing a decrease in chalcone accumulation, which may lead to damage caused by active oxygen production in the pulp, thus inhibiting the accumulation of fructose. However, these phenomena need further confirmation.

**Author Contributions:** Writing—original draft, X.S.; Data curation, W.W.; Data curation, W.M.; Data curation, C.Y.; Data curation, K.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** National Natural Science Foundation of China (NSFC) (No. 31960570).

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
