Variations in the physical and chemical characteristics of melon fruit during ripening
For comparisons of fruits of the two assessed Hami melon varieties, we examined the physiochemical characteristics of fruit collected at different stages of development (Fig. 1). The color of the fruit peel of the two varieties was found to be similar at 5 and 10 days after pollination (DAP). Thereafter, however, the peel of Yaolong fruit turned a darker green compared with that of Guimi from 15 to 20 DAP. With continued growth, Guimi melons developed as medium-sized oval fruits with a yellow skin. In both varieties, the juicy flesh underwent a change in color from white to yellow during ripening.
Trends in the changes in physical and chemical characteristics of the fruits, including weight, size, and soluble sugar contents, are shown in Fig. 2. Similar trends in the two varieties were observed with respect to the weight and size of fruit during repining. Interestingly, fruit weight increased rapidly from 35 to 40 DAP, whereas there were only slight increases in size. At 40 DAP, the soluble sugar contents of Guimi fruit had reached a value 12, which was higher than that obtained for Yaolong, although differences in the brix values of the two varieties were found to be non-significant.
Global analysis of the RNA-Seq data
To determine gene expression patterns, we performed RNA-seq analysis using the C. melo reference genome. After filtering out rRNAs and low-quality reads, a total of 111 million reads were mapped to the reference genome (Table S1). For these clean reads, we obtained average mapped reads per sample greater that 90%. The one exception in this the regard was the low (74.44%) alignment ratio obtained for Yaolong at 40 DAP. In total, we detected 21,172 expressed genes among the melon fruit samples.
To determine differences in the expression of genes between two close sampling times, we identified the DEGs in Guimi and Yaolong based on the threshold criteria of a log2 fold change ≥ 1 and FDR ≤ 0.05 (Table S2). For this purpose, we defined the earliest time point of paired groups as the control sample for subsequent measurements. The number of DEGs in Guimi was found to be markedly higher than that in Yaolong at 20 and 30 DAP, while DEGs being identified in Guimi less than Yaolong (223 vs 95) at 40 DAP (Fig. 3A). These finding would thus appear to indicate that during fruit development the constituents of Guimi fruit undergo more pronounced changes than those of Yaolong fruit. Over time, however, there was a gradual reduction in the number of DEGs in both melon varieties during the fruit development, indicting a corresponding reduction in the speed of fruit growth and that the fruit was fully mature at 40 DAP. For both Guimi and Yaolong, we also detected perturbations in gene expression at the same time points. The number of genes differentially expressed between Guimi and Yaolong initially increased, reaching a peak at 20 DAP, after which the number declined to 458 at 40 DAP (Fig. 3B).
For the purpose of functional annotation, identified DEGs in the two melon varieties were assigned to GO terms and KEGG pathways. We accordingly found that for Yaolong melon, the majority of enriched GO terms between 10 and 20 DAP were classified in the category biological process, including ‘histone lysine methylation’, ‘peptidyl-lysine methylation’, and ‘DNA alkylation’, whereas in the case of Guimi melon, the most significantly enriched GO terms were ‘polysaccharide metabolism’, ‘cell wall biogenesis’, and ‘external encapsulating structure organization’ (Fig. S1 and S2). For the second comparison (20 DAP vs. 30 DAP), the DEGs in Guimi melon were found to be significant enriched in the processes of ‘response to acid chemical’, ‘plant-type cell wall biogenesis’, and ‘response to chemical’, whereas for Yaolong, the three most enriched biological processes were ‘hemicellulose metabolism’, xylan metabolism’, and ‘cell wall polysaccharide metabolism’. Comparing between 30 and 40 DAP samples revealed that the DEGs of Guimi melon were notably enriched with respect to ‘amine metabolism’, ‘cellular amine metabolism’, and ‘jasmonic acid metabolism’, whereas those of Yaolong melon were found to be enriched in the cellular component categories ‘external encapsulating structure’, ‘cell periphery’, and ‘vacuole’. With regards to KEGG pathway annotation, we found that in the 10 to 20 DAP comparison, DEGs in Yaolong were enriched with respect to ‘DNA replication’, ‘ABC transporters’, and ‘flavone and flavonol biosynthesis’, whereas ‘metabolic pathways’, ‘biosynthesis of secondary metabolites’, and ‘phenylpropanoid biosynthesis’ were found to be enriched with Guimi DEGs (Fig. S3 and S4). Interestingly, for the 20 to 30 DAP comparison, we observed that for both melon varieties, the three most significantly DEG-enriched KEGG pathways were ‘biosynthesis of secondary metabolites’, ‘metabolic pathways’ and ‘phenylpropanoid biosynthesis’. For the third comparison between 30 and 40 DAP, Guimi DEGs were found to be enriched in pathways such as ‘valine, leucine, and isoleucine biosynthesis’, ‘biosynthesis of secondary metabolites’ and ‘cyanoamino acid metabolism’, whereas pathways enriched with Yaolong DEGs included ‘biosynthesis of secondary metabolites’ and ‘metabolic pathways’.
Comparison of trends in temporal gene expression during melon fruit ripening
To gain further insights into the change trends in gene expression during fruit development, we clustered a total of 4,731 DEGs from Guimi melon and 3,198 DEGs from Yaolong into 38 profiles using the STEM algorithm. Among these, 2,897 Guimi DEGs were significantly clustered into the following six profiles: two upregulated profiles (Profiles 17 and 12), three downregulated profiles (Profiles 0, 7, and 2), and one biphasic expression pattern profile (Profile 18) (Fig. 4A). Similarly, 2,217 Yaolong DEGs were classified into the following six profiles based on P values ≤ 0.05: two upregulated patterns, one biphasic expression pattern, and three downregulated patterns (Fig. 4B).
To systematically investigate the biological functions of candidate genes, we extracted DEGs from up- and downregulated cluster groups for further GO term and KEGG pathway analyses. GO analysis revealed that Yaolong DEGs assigned to profiles 17 and 19 were separately categorized into 18 biological processes and two molecular processes, whereas Guimi DEGs in profile 12 were enriched with respect to 110 major functions in the Biological process, Cellular component, and Molecular function categories (Fig. S5). However, we detected no significant enrichment of the Guimi DEGs clustered in profile 17. The GO terms with most representation for downregulated cluster groups are shown in Fig. S6. Among biological functions, ‘gibberellin metabolic process’ (GO:0009685), ‘cytoskeleton’ (GO:0005856), and ‘cell wall organization or biogenesis’ (GO:0071554) were the most significantly enriched functions in Guimi profiles 7, 2, and 0, respectively. For profile 0, 2, and 7 of Yaolong DEGs, ‘cell wall’ (GO:0005618), ‘histone lysine methylation’ (GO:0034968), and ‘phenylpropanoid biosynthetic process’ (GO:0009699), respectively, were most enriched biological functions.
On the basis of KEGG analysis, we identified nine KEGG pathways enriched with downregulated Guimi DEGs including ‘metabolic pathways’ (ko01100), ‘biosynthesis of secondary metabolites’ (ko01110), ‘phenylpropanoid biosynthesis’ (ko00940), and ‘biosynthesis of various secondary metabolites - part 2’ (ko00998) (Figure S7). In contrast, only two pathways were enriched with upregulated Guimi DEGs, namely, ‘carbon fixation in photosynthetic organisms’ (ko00710) and ‘galactose metabolism’ (ko00052). In total, we identified 12 pathways with significant enrichment of upregulated Guimi DEGs (Fig. S8). These results thus indicate that most of the DEGs regulated during fruit development appear to be associated with the functioning of metabolic pathways.
Analysis of cell wall biogenesis during melon fruit ripening
Fifty-one DEGs in Guimi, clustered in Profile 0 (n = 28) and Profile 2 (n = 23), were found to be significantly associated with cell wall biogenesis, showing downregulated gene expression patterns, whereas in Yaolong, 48 DEGs showing downregulated trends, clustered into Profiles 0, 2, and 7, showed similar associations. Fig. 5 shows the differences in the expression trends of these DEGs between Guimi and Yaolong. We observed 33 common DEGs in cell wall biogenesis-related profiles of Guimi and Yaolong, a majority of which showed similar patterns of expression regulation in the two melon varieties at different stages of fruit development and ripening (Fig. 5A). For example, gradual reductions in the levels of GUX3 expression were observed during fruit development in both germplasms (Fig. 5B and 5C). A similar reduction in expression was detected for ODO1, which peaked during the early stages of fruit development, after which there was a slight reduction, which became more pronounced prior to maturity. Although melon type-specific DEGs involved in cell wall biogenesis showed an overall downregulated pattern, differences in the changes in the direction of gene expression were still different between consecutive stages of development. For example, in Guimi melon, UAM1 was downregulated from stage 1 to 2, then constantly expressed at a stable level from stage 2 to 3, prior to undergoing a decline in the mature stages. These findings thus indicate differences in the expression patterns of melon cultivar-specific DEGs associated with cell wall synthesis during fruit development.
Analysis of sugar metabolism during melon fruit ripening
Some of the genes involved in sugar metabolism were found to be differentially expressed during melon fruit development, including those associated with ‘galactose metabolism’, ‘starch and sucrose metabolism’, ‘fructose and mannose metabolism’, and ‘amino sugar and nucleotide sugar metabolism’ (Fig. 6A). However, in the case of Yaolong, only Profile 0 DEGs were found to be significantly associated with the starch and sucrose metabolism pathway. Contrastingly, in Guimi, ‘amino sugar and nucleotide sugar metabolism’ was identified as being enriched with Profile 2 DEGs. For a majority of the DEGs associated with the ‘amino sugar and nucleotide sugar metabolism’ pathway, including MUR4, UGD1, and UPTG2, the lowest levels of expression were detected in ripe fruit (Fig. 6B). Difference in gene expression were also detected during the different stages of fruit development. For example, there was a marked reduction in the expression of GAUT6 during in the middle phase of fruit development, followed by a slight increase in pre-mature fruit, before declining to the lowest levels in mature fruit. Although the change trend in UGD1 expression in young fruit was similar to that observed for GAUT6, it differed in that it was expressed at a constantly stable level in the pre-mature fruit. In Yaolong, a total of 11 Profile 0 DEGs were found to be significantly associated with the starch and sucrose metabolism pathway. In contrast to trends in the expression of sugar metabolism-related genes in Guimi, nine of these DEGs were characterized by gradual downregulated expression trends during fruit development and ripening (Fig. 6C). For example, the lowest level of At4g02290 expression in Yaolong was detected at stage T3. Conversely, however, whereas INV1 was characterized by a downregulated expression trend in young fruit, high levels of expression were detected during the expansion stage, prior to a subsequent decline in expression, reaching the lowest values in ripe fruit.