3.3.5. SPS

The changes in SPS activity are shown in Figure 2e. The dynamic changes in enzyme activity in the high-Ca treatment group and CK group showed a single-peak curve; the peak value of the activity in the high-Ca treatment group occurred at 64 d after anthesis, and the peak value in the CK group was at 50 d after anthesis. There was no significant difference in SPS activity between the high-Ca treatment group and the CK group at 64 d after anthesis, but the activity was significantly lower in the high-Ca treatment group than in the CK group at 64-50 d after anthesis. High-Ca treatment inhibited SPS enzyme activity, resulting in the inhibition of sucrose accumulation.

#### 3.3.6. Synthesis Direction and Net Activity of the Sucrose Invertase System

The characteristics of net activity change in the synthesis direction of the sucrose invertase system are shown in Figure 2f. The change trend of the net activity in the high-Ca treatment group was "up, down, and up", while that in the CK showed the opposite trend. At 71 d after anthesis, the net activity of the sucrose invertase system in the high-Ca treatment group was significantly higher than that in the CK treatment group. At this time, a large amount of sucrose in the CK group was transformed into glucose and fructose, while less sucrose in the high-Ca treatment group was converted. Overall, the sucrose in the CK group decomposed into fructose and glucose, while the high-Ca treatment inhibited the conversion of sucrose to reducing sugars, and the accumulation of reducing sugars was inhibited relative to that in the CK group.

#### *3.4. Transcriptome Analysis of Fruit Pulp*

#### 3.4.1. Assembly and Analysis of Transcriptome Sequencing Data

The sequencing yield of the lychee pulp transcriptome is shown in Table 1. The GC content of each sample is greater than 44.86%, the percentage of Q30 bases is greater than 91.43%, and the error rate is only 0.02%, which indicates that the quality of the bases is high and that the sequencing results are ideal, which ensures the accuracy and reliability of subsequent analysis.

Figure 3a shows that the number of transcripts and unigenes with a length of more than 200 to 2000 bp exceeds 1000, and the number of transcripts and unigenes with a length ranging from 200 to 300, 300 to 400, and 400 to 500 bp exceeds 10,000. Table 2 shows that a total of 306,396 transcripts with an N50 of 1913 bp, an N90 of 456 bp, and an average length of 1119 bp were assembled. A total of 115,413 unigenes were obtained. The average length of these sequences was 894 bp, the N50 was 1581 bp, and the N90 was 335. Taken together, these findings indicate that the sequencing results are ideal and that the analysis results are reliable.

**Table 1.** Statistics of transcriptome sequencing data from the pulp of Feizixiao lychee fruit in the high-Ca treatment and CK treatment groups.


Notes: 35 d-CK, 64 d-CK, and 71 d-CK represent transcripts in the control Feizixiao lychee fruit pulp at 35, 64, and 71 d after anthesis, respectively; 35d-CK, 64d-Ca, and 71d-Ca represent transcripts of Ca-treated Feizixiao lychee fruit pulp at 35, 64, and 71 d after anthesis, respectively.

**Figure 3.** *Cont*.

**Figure 3.** Assembly and annotation of the transcriptome from the pulp of Feizixiao lychee: (**a**) sequence length distribution; (**b**) statistical chart of species with gene homology; (**c**) statistical chart of KOG function classification.


**Table 2.** The statistics of assembly results.

3.4.2. Functional Annotation and Analysis of Unigenes

The unigenes were subjected to functional enrichment analysis, and the results are shown in Table 3. By comparing the unigene sequences with sequences in the KEGG, NR, GO, and other databases, we found that 55,352 unigenes were annotated in at least one database, accounting for 47.96% of the total. Among these unigenes, the number of unigene annotations obtained from the TrEMBL database was the largest at 51,397, accounting for 44.53%; this was followed by the annotations obtained from the NR and GO databases. In addition, 36,242 unigenes were annotated in the SwissProt database, and 41,174 and 34,652 unigenes were annotated in the KEGG pathway and Pfam databases, respectively.

**Table 3.** Unigene annotation results.


The results of our evolutionary analysis are shown in Figure 3b. The number of homologous genes is ranked from large to small. The top ten species with homologous genes were *Citrus sinensis*, *Citrus clementina*, *Citrus unshiu*, *Vitis vinifera*, *Theobroma cacao*, *Quercus suber*, *Populus trichocarpa*, *Hevea brasiliensis*, *Durio zibethinus*, and *Manihot regia*. With 603 homologous genes, Dimocarpus longan, which is closest to lychee, ranks 19th. Based on an in-depth study of citrus fruit growth and development, this study provides a reference method and results.

As shown in Figure 3c, the obtained unigenes were compared with sequences in the EuKaryotic Orthologous Groups (KOG) database to analyze their possible functions. The results showed that these gene sequences were related to 25 biological processes.

Among these biological processes (in terms of general function prediction only), the most abundant terms involved posttranslational modification, protein turnover, chaperones, signal transduction mechanisms, translation, ribosomal structure, and biogenesis. Carbon transport and metabolism; energy production and conversion; and secondary metabolite biosynthesis, transport, and catabolism were the next most abundant terms.

#### 3.4.3. Functional Annotation and Enrichment Analysis of Differentially Expressed Genes

Additionally, we examined the positive and negative regulatory processes related to biological processes and the regulation of enzyme activity related to molecular function. As shown in Figure 4a, there were differences in gene expression during the growth and development of the pulp of the fruit in the high-Ca treatment group and the CK group. With respect to sugar metabolism in the pulp, there were different degrees of gene upregulation and downregulation in the CK group compared with the high-Ca treatment group. In total, 48 genes were upregulated in the high-Ca treatment group compared with the CK group at 50 d after anthesis, and 83 genes were downregulated; at 64 d after anthesis, 92 genes were upregulated, and 19 genes were downregulated. Moreover, at 71 d after anthesis, 49 genes were upregulated, and 38 genes were downregulated, which indicated that high-Ca treatment significantly altered the expression of genes during fructose metabolism.

Figure 4b shows that at 64 d after anthesis, in the biological process category, the differentially expressed genes in the high-Ca treatment and CK groups were involved mostly in metabolic processes, cellular processes, and cellular component organization or biogenesis. In the broad cellular components category, the genes in the high-Ca treatment and CK groups were involved mostly in extracellular regions, cells, and cell parts. In the broad molecular function category, the differentially expressed genes in the high-Ca treatment and CK groups were mostly involved in catalytic activity.

Figure 4c shows that there are many subclasses of genes differentially expressed at 71 d after anthesis compared with those at 64 d after anthesis, but the number is similar to that at 64 d after anthesis. At 71 d after anthesis, there were also a greater number of genes involved in response to stimuli and a smaller number involved in cell components, tissues, or biogenesis. In the cellular component category, the number of genes annotated as extracellular regions was relatively small. In the molecular function category, binding was also notably annotated.

**Figure 4.** *Cont*.

**Figure 4.** Statistics and functional classification of differentially expressed genes: (**a**) statistical chart of gene difference between treatment and control; (**b**) histogram of GO classification of DEGs in CK and treatment at 64 days after flowering; (**c**) histogram of GO classification of DEGs in CK and treatment at 71 days after flowering.

Pathway enrichment analysis was carried out on the genes differentially expressed between the CK group and the high-Ca treatment group. The pathways that were significantly enriched are shown in Figure 5a,b. The fructose content of the high-Ca treatment and CK groups reached a maximum at 64 d after anthesis, and the fructose content of the high-Ca treatment group was significantly lower than that of the CK group at that time. The pathways most significantly enriched were pentose and glucuronic acid interconversion, flavonoid biosynthesis, and others. At 71 d after anthesis, the sugar withdrawal phenomenon was detected in both the high-Ca treatment group and the CK group, and the enriched pathways causing this difference were mainly involved in plant hormone signal transduction and ABC transporters. This indicates that the inhibitory effect of high-Ca treatment on sugar accumulation is mainly reflected in the effects on the above pathways.

**Figure 5.** Enrichment statistics of metabolic pathways involving differentially expressed genes: (**a**) KEGG pathway enrichment of DEGs in CK and treatment at 64 days after flowering; (**b**) KEGG pathway enrichment of DEGs in CK and treatment at 71days after flowering.

Hierarchical cluster analysis was performed on the expression (FPKM values) after analysis and standardization of the differentially expressed genes, and a cluster heatmap of the groups of genes differentially expressed between the high-Ca treatment group and the CK group at 64 and 71 d after anthesis was constructed, as shown in Figure 6a,b. The results corresponding to the two time points and the CK are grouped into one category. The differentially expressed genes could be divided into two categories: those with high expression and those with low expression.

**Figure 6.** Hierarchical clustering heatmap of differentially expressed genes: (**a**) hierarchical clustering heatmap of differential genes between 509−CK and 509−T1; (**b**) hierarchical clustering heatmap of differential genes between 516−CK and 516−T1. Note: The abscissa represents the sample name and hierarchical clustering results, and the ordinate represents the differentially expressed gene and hierarchical clustering results shown in Figure 6. Red indicates high expression, and green indicates low expression; 509-CK and 509-T1 correspond to the control and high-Ca treatment groups, respectively, at 64 d after anthesis; 516−CK and 516−T1 correspond to the control and high-Ca treatment groups, respectively, at 71 d after anthesis.

Figure 7a shows the metabolic pathway of starch and sucrose metabolism, which mainly controls carbohydrate metabolism. In sucrose metabolism, there are three pathways labeled 3.2.1.21 involving β-D-glucoside glucose hydrolase, which involves an enzyme that promotes the conversion of other sugars to glucose. Pathway number 5.3.1.9 involves α-D-glucose-6-phosphate aldose ketone isomerase, which is involved in the catalysis of α-D-glucose 6-phosphate and the β-reversible conversion between D-fructose and furanose 6 phosphate. The pathway number of the SUS-coding gene is 2.4.1.13; this pathway involves sucrose synthase, which is an enzyme that catalyzes the reversible synthesis of sucrose. Interestingly, the expression level of SUS was downregulated. In starch metabolism, the gene expression in pathway number 3.2.1.28 (involving α-trehalose glucose hydrolase) was downregulated; this enzyme is involved in the catalysis of α,α-trehalose. The hydrolysis of the o-bond of glucoside releases the initial equimolar amount of α- and β-D-glucose, which promotes the synthesis of D-glucose. Since the enzymes encoded by these genes directly affect the conversion and accumulation of sucrose, glucose, and fructose, it is suggested that the difference in the expression of these genes may be related to the inhibition of sugar accumulation detected in the high-Ca treatment group. The enzyme encoded by the sus gene is SS, which indicates that high-Ca treatment leads to downregulation of the SS enzyme-encoding gene. In the analysis of differentially expressed genes involved in glucose metabolism in this paper, no significant change was found in the expression of SPS genes, indicating that there may be other physiological and biochemical mechanisms involved in the regulation of SPS activity in the high-Ca treatment group.

Figure 7b shows the biosynthetic pathway of flavonoids, which compose a main category of plant secondary metabolites. Flavonoids are synthesized from phenylpropanoid derivatives by condensation with malonyl-CoA. The genes in pathway numbers 2.3.1.74 (involving 4-β-D-glucan glucose hydrolase) and 2.3.1.170 (involving malonyl-CoA) were downregulated. Both belong to the chalcone synthase (CHS) superfamily. CHS is the first key structural enzyme in flavonoid synthesis and is responsible for catalyzing the formation of chalcone, which, as a precursor of many flavonoids, participates in the formation of downstream secondary metabolites. The downregulation of these genes inhibits the formation of chalcone and may ultimately inhibit the accumulation of flavonoids.

Figure 7c shows a cluster heatmap of the expression levels of the invertase-related genes CIN and VIN. According to the expression level, it can be concluded that the high-Ca treatment affected the expression of the gene-encoding invertase. Moreover, the expression level of the VIN gene in the CK group significantly decreased during sugar withdrawal, while that in the high-Ca treatment group did not significantly decrease. The expression of the CIN gene was significantly increased, but there was no significant change in the high-Ca treatment group. Since the CIN gene encodes NI and the VIN gene encodes AI, high-Ca treatment suppressed the activity of invertase by downregulating the expression of the invertase-encoding gene, thereby inhibiting the accumulation of glucose and fructose.

**Figure 7.** *Cont*.

**Figure 7.** *Cont*.

#### *3.5. qRT–PCR Validation Analysis*

qRT–PCR analysis was performed on eight unigenes. As shown in Figure 8, the linear relationship between the RNA sequencing (RNA-seq) data and qRT–PCR data was significantly positive, indicating that the results of the transcriptome analysis were accurate and reliable.
