The basis quality analysis of wampee fruit in different development stages
The advancement and growth of wampee fruit could be separated into five steps: cell proliferation and differentiation, expansion (PDQ stage), coloring (ZSQ stage), fruit ripening (CSQ stage), and delay harvest (WSQ stage). This study mainly focused on the changes of seedless wampee fruit during the four stages (PDQ to WSQ stage, Fig. 1). The fruit development of seedless wampee required a 120-day cycle from flowering to fruit maturation.
According to the analysis of fruit development during four stages (Table 1), we found that total acid increased rapidly from 0.09 mg/g FW to 2.34 mg/g FW (PDQ to ZSQ stage), and decreased to 1.50 mg/g FW (ZSQ to CSQ stage), and further reduced to 1.22 mg/g FW (CSQ to WSQ stage). While the total sugar content gradually increased from 27.55 mg/g FW to 104.83 mg/g FW (CSQ to WSQ stage), which was different from total acid. These results demonstrated that the total sugar content gradually increased during the four stages, while the total acid content quickly increased in the early stages (PDQ to ZSQ stage) but maintained a relatively high level after ZSQ stage.
Metabolomics analysis
To explore the quality-related metabolism changes of seedless wampee fruit during development, we detected a number of both primary and secondary metabolites through widely targeted metabolome for the samples at the four stages (PDQmeta, ZSQmeta, CSQmeta, and WSQmeta). A total of 623 metabolites were identified(Supplement 1), containing 158 flavonoids, 81 amino acids and amino acids its subcategories, 58 fatty acids, 57 organic acids and its subcategories, 49 nucleotides and its subcategories, 31 hydroxycinnamic acid its subcategories, 21 quinic acid and its subcategories, 20 sugar, 15 plant hormones, 14 catechins and its subcategories, 14 vitamins, 13 phenolamines, 12 coumarin and its subcategories, 11 benzoic acid its subcategories, 7 indole and its subcategories, 6 choline, 6 pyridine and alcohol, 6 tryptamine and its subcategories, 6 alkaloids, 4 hydrochloric acid and its subcategories, and 3 terpenes .
By comparing the metabolome data in different development stages, 222, 156, and 104 down-regulated metabolites could be observed between PDQmeta and ZSQmeta, ZSQmeta and CSQmeta, CSQmeta and WSQmeta, respectively. From the PDQ stage to ZSQ stage, the most variable substances were flavonoids (43), amino acids (41), lipids (25), organic acids and its subcategories (23), nucleotides and its subcategories (21), carbohydrates (6), and vitamins (5). From the ZSQ stage to the CSQ stage, the most variable substances were flavonoids (24), amino acids (8), lipids (20), organic acids and its subcategories (12), nucleotides and its subcategories (13), carbohydrates (2), and vitamins (1). From the CSQ stage to the WSQ stage, the most variable substances were flavonoids (24), amino acids (7), lipids (20), organic acids and its subcategories (12), nucleotides and its subcategories (13), carbohydrates (2), and vitamins (1).
RNA-Seq and differentially expressed genes (DEGs) studies
To explore the mechanism of metabolic changes in different stages of seedless wampee fruit, especially the mechanism of sucrose and citrate changes, four transcriptome databases (PDQseq, ZSQseq, CSQseq, WSQseq) were established by RNA-sEq. The signals that were larger than 240 million could be detected in each transcriptome data library. The results showed that a total of 7191618051nt of RNA fragments were detected in PDQ stage, of which GC content accounted for 44.11%, 7392980402nt of for ZSQ stage(44.35% GC content), 7270067923nt for CSQ stage(44.25% GC content), and 7487257723nt for WSQ stage(44.43% GC content) (Table 4). According to the analysis of annotation database, such as the annotation COG, GO, KEGG, KOG, Pfam Annotation, Swissprot, eggNOG, NR, a total of 82673 unigenes were thoroughly analyzed, and the number of unigenes with a length over 1000 bp was 21781, and the number of unigenes with a length between 300 and 1000 was 34764. All RPKM values of unigene were shown in Supplement 2.
By comparing different transcriptome data library, 2540 down-regulated and 1134 up-regulated differential genes have ascertained among PDQseq and ZSQseq, 837 down-regulated and, 568 up-regulated differential genes were identified between ZSQseq and CSQseq, and 272 up-regulated and 837 down-regulated differential genes were identified between CSQseq and WSQseq (Fig. 2A). The Venn diagram of differential genes between different databases showed the total of 19 up-regulated (Fig. 2B) and 89 down-regulated differential genes (Fig. 2C) in different developmental stages.
Based on the co-expression analysis, the total of 5722 differentially expressed genes have divided into four categories, containing 452, 705, 1837 and 2728 genes in each group respectively. Group A included the up-regulated genes (Fig. 3A), and Group B included the down-regulated genes (Fig. 3B), while group C and D included the unchanged genes (Fig. 3C, 3D). Since the changed genes were involved in many metabolism pathways, we classified these genes into different pathways by KOG database according to the investigation of genes with up and down regulation, of which energy generation and transfer (C), amino acid metabolism and transport (E), secondary metabolites synthesis, metabolism and transport (Q) were the main classifications, and the sum of down-regulated genes in the aforementioned trajectories was significantly higher than the sum of up-regulated genes, indicating that the content of energy storage substances (sucrose and glucose), amino acids and secondary metabolites (flavonoids) changed significantly during fruit development of seedless wampee.
According to the annotated genomic information of the genes that related to sucrose and citrate metabolism (shown in Table 4 and Table 5), we predicted the location information and the ratio of the expression level at each stage (the ratio > 1 means up-regulation; the ratio < 1 means down-regulation). Among all the genes, nine genes were selected from the expression profile to verify by qRT-PCR with three biological replicates for each gene. Linear analysis showed that the correlation between transcriptome data obtained by RNA-seq and gene expression levels acquired through qRT-PCR, indicating that transcriptomic data was authentic, as well as reliable (Fig. 4).
Regulation of sucrose metabolism
Fruit sucrose content mainly depends on sink strength. Sucrose is absorbed and assimilated by various organs, and it depends on the activity of enzymes (e.g., SPS, SUS, IVR [14–17]) in the sucrose metabolism pathway [18]. Here, the transcriptome data (see Table 4) showed that three SPS genes, five SUS genes, four IVR genes and four TPS genes could be detected. As shown in Fig. 5, with the maturation of wampee fruit, the sucrose and glucose content gradually augmented, which was in agreement with the cumulative trend of sucrose, glucose in other fruits, such as peach [3], pomelo [4], and watermelon [19].
From PDQ stage to ZSQ stage, the increase of sucrose content was mainly due to the up-regulation of SUS gene, leading to a larger amount of sucrose synthesis compared to its degradation caused by up-regulation of IVR gene expression. SPS can regulate the sucrose synthesis, and SUS is able to catalyze sucrose in order to produce fructose and UDP-glucose [17]. During this stage, SPS (c31711.graph_c0) was up-regulated by 2.11-folds, SUS (c49920.graph_c0, c60779.graph_c0) was up-regulated by 4.5-folds and 30.9-folds, respectively. While, the expression level of IVR and TPS did not change significantly. Besides, the sucrose content was increased by 2.05-folds, the glucose content was increased by 1.41-folds, while the Trehalose-6P content did not change. These results indicated that the synthesis rate of sucrose was much higher than the degradation rate. Also, the expression level of HXK, a gene involved in glycolysis, and PEPCK [20], a gene related to the gluconeogenesis, were up-regulated, and the content of Fructose-6P was reduced by 55%, suggesting that the respiration of wampee fruit gradually enhanced from the PDQ stage to ZSQ stage, and the precursors and energy generated by the TCA cycle increased rapidly, providing a material basis and energy for fruit expansion.
The increase rate of sucrose content slowed down during the transitionary period from ZSQ stage to CSQ stage, which might be due to the increased degradation of sucrose caused by upregulation of IVR, SUS and TPS genes. During this period, the upregulation of SUS promoted the decomposition of sucrose into fructose and UDP-glucose, while, the upregulation of TPS contributed to further increasing the Trehalose-6P content. The upregulation of IVR caused the decomposition of sucrose into fructose, which could be further transformed into Fructose-6P via down-regulating FRK. The metabolome database showed that the increase of sucrose (only 66%) and glucose (only 29%) gradually slowed down, while trehalose-6-phosphate, fructose-6-phosphate, and glucose-6-phosphate improved by 81%, 55%, and 37%, respectively, suggesting that some sucrose was converted into monosaccharides, which was further converted into other precursors through TCA cycle to provide energy during this period.
During the period from CSQ stage to WSQ stage, sucrose was further reduced which might be due to slower synthesis and further increased consumption rate. During this period, sucrose content, glucose, fructose-6-phosphate, glucose-6-phosphate, trehalose-6-phosphate increased by 36%, 33%, 64%, 29%, 21%, respectively. Transcriptomic data exhibited that the expression level of SPS was unchanged and the SUS gene was up-regulated during this period, suggesting that sucrose synthesis was further reduced compared to the stage before maturation. The upregulation of IVR involved in sucrose degradation indicated the increase of sucrose consumption. The expression of IVR increased rapidly from PDQ stage to ZSQ stage, then became stable. It has been reported that the acidic IVR activity of citrus fruit was activated before maturation, and then gradually disappeared due to higher fruit pH (> 5) during the CSQ stage [21–22]. While the pH of seedless wampee fruit has been maintained between pH 3 and 5 throughout the development (Table 1), indicating acidic IVR activity maintained a high activity throughout the development. Hence, degradation amount of sucrose from the CSQ stage to WSQ stage was the same with the period from ZSQ stage to CSQ stage, while the expression of sucrose synthesis-related genes (SPS, SUS) was lower than the period before the CSQ stage, leading to a reduced synthesis (only 36%).
According to the transcriptome and metabolome analysis, sucrose content was mainly regulated by SUS, IVR, SPS and TPS during the maturation of wampee fruit. Among these genes, synthesis related genes, such as SUS and SPS were mainly responsible for the period from PDQ stage to ZSQ stage, and the degradation-related genes, such as IVR and TPS mainly functioned after ZSQ stage.
Regulation of citrate metabolism
To explore the citrate mechanism in seedless wampee fruit, transcriptome and metabolome analyses were used to analyze fruit citrate metabolism-related genes and pathways during fruit development (Fig. 6). From PDQ stage to ZSQ stage, MDH expression increased significantly, citrate content increased by 35%, while malic acid content decreased by 66%. These findings indicated that citrate synthesis and malic acid decomposition were positively correlated with MDH expression, which was most likely due to the upregulation of MDH that promote the malic acid to be oxidized by NAD-mtMDH, and further being converted into citrate via pyruvate and stored in vacuole. Besides, there was no differential expression for citrate synthase gene, suggesting the synthesis of citrate in wampee fruit might be regulated by MDH but not citrate synthase gene [4, 23].
After the ZSQ stage, the citrate content decreased slowly, indicating only part of citrate was converted into other substances after transporting out of the vacuole. Generally, the conversion of citrate into other substances was mainly through TCA cycle, glyoxylate cycle, GABA shunt, and acetyl-CoA catabolism [24]. The TCA cycle might not determine the citrate content of wampee fruit. After the ZSQ stage, the malic acid content gradually decreased, the content of fumaric acid did not change significantly, and other genes in TCA cycle, such as citrate synthase gene, isocitrate dehydrogenase gene, and cis-aconitate gene, were also not significantly differentially expressed. Also, it has been reported that citrate could be transported out of the mitochondria in the TCA cycle [25], implying the TCA cycle was closely related to the decomposition of fruit citrate but cannot determine the citrate content [26–27]. Here, the transcriptome data showed that the key gene ICL was not differentially expressed in glyoxylate cycle pathway, indicating that the degradation of citrate was not involved in this pathway. GABA shunt and acetyl-CoA pathways are the main decomposition trajectories for citrate during fruit ripening such as banana [28], tomato [29], and orange [30]. Here, we found that after ZSQ stage, the expression of ACO and IDH (not detected), key genes in GABA shunt pathway, did not change significantly (Table 5), and the content of glutamic acid, glutamine, asparagine in the pathway gradually decreased. These findings strongly suggested that the GABA shunt pathway might not be the main pathway for citrate decomposition after ZSQ stage. The acetyl-CoA pathway is mainly involved in the decomposition of citrate during fruit maturation [31]. In this pathway, ACL, a key gene in acetyl-CoA pathway, could generate oxaloacetic acid and acetyl-CoA by catalyzing citrate, and finally generate fatty acids, amino acids, and flavonoids. Here, the expression of ACLβ was negatively correlated with citrate content, suggesting that the acetyl-CoA pathway might be the main pathway for citrate decomposition after the ZSQ stage (Fig. 6).