Transcriptome and metabolome combined analysis metabolism change of soluble sugars and organic acids in mulberry fruit during development stages

Abstract: 【Objective】Through the systematic study of the metabolism and molecular mechanism of sugar and organic acids, the mechanism of fruit taste formation was well revealed. In this study, we investigated the metabolism of soluble sugar and organic acid and transcriptome expression levels during the development of mulberry (Morus alba) in order to reveal the molecular mechanism of fruit quality formation of mulberry.【Methods】White mulberry fruits were used as experimental materials at greening stage (W1), transforming stage (W2) and ripening stage (W3) . The content and transcriptome of the soluble sugar and organic acid at three stages were determined separately, and the molecular mechanism of regulation of the soluble sugar and organic acid metabolism were analyzed based on the combination of transcriptome and metabolome. By exploring the key differential genes regulating the synthesis andmetabolism of the soluble sugar and organic acid during mulberry ripening, the metabolic network was proposed to elucidate the influence of fruit development on the metabolism of the soluble sugar and organic acid. The UPLC-MS/ MS targeted metabolomics method was used to detect the changes of metabolites at 3 developmental stages, and the Cluster analysis was performed on the obtained different substances. The expression of the differential genes was analyzed by qRT-PCR and the obtained differential genes were further analyzed through KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analysis.【Results】A total of 64 metabolites were detected, including 52 organic acids and 12 soluble sugars. According to the cluster analysis of different substances, the results showed that there were obvious changes in sugar and acid metabolism during the mulberry fruit development. Through the data analysis, it was found that the sucrose, glucose and D-fructose were the main soluble sugars in mulberry fruits, and their contents continued to increase during the development of mulberry fruits, and reached a peak at W3. The malic acid, citric acid and succinic acid were the main organic acids in mulberry fruits. According to the assembly analysis of the transcriptome sequencing data of the mulberry samples at different developmental stages, a total of 58.65 Gb was obtained. The differential gene analysis of gene expression at different developmental stages showed that W3 vs W1 group had the largest number of differential genes, reaching 9098. The Venn map was drawn for the 3 different genes in comparison combinations, among them 762 genes were expressed in common. The W3 vs W1 group contained the largest number of the unique differential genes, with 2836 differential genes. The second group was W2 vs W1 with 499 unique differential genes, and the least group was W3 vs W2 with 195 unique differential genes. The results showed that transcription and translation of a large number of genes were activated at the beginning of fruit development, while transcription and translation of some genes were inhibited at maturity. The KEGG enrichment analysis showed that the differential genes in W2 vs W1 and W3 vs W2 groups were enriched into carbohydrate- related metabolic pathways, which were mainly starch and sucrose metabolism and tricarboxylic acid cycle pathways. In the W2 vs W1 group, 52 upregulated differential genes were enriched in the starch and sucrose metabolism, and 27 upregulated differential genes were enriched in the citric acid cycle. In the W3 vs W2 group, 27 upregulated differential genes were enriched for the starch and sucrose metabolism. Combined with the differential gene identification, correlation analysis and common KEGG pathway analysis of the differential genes and differential metabolites related to soluble sugar and organic acid metabolism were carried out, there were significant differences in the expression of some candidate genes related to the soluble sugar and organic acid metabolism in mulberry. In this study, four differentially expressed SUSY genes were detected, and their expression levels were high in the early stage of fruit development, but significantly decreased with fruit development; three differentially expressed NINV genes were detected, and their expression increased with the development of fruit. Two differentially expressed FRK genes were identified, which were highly expressed at the early stage of fruit development; one differentially expressed HK gene was identified, and its expression gradually increased with the fruit development. In addition, this study also found that the expression of the two MDH genes increased during fruit ripening, and the expression of the MDH was significantly correlated with malic acid content. These results indicated that these genes play a significant role in the regulation of mulberry maturation. The metabolome and transcriptome association analysis showed that the NINV, HK, CS, ACO, MDH and ICDH were the key regulatory genes of saccharic acid accumulation in mulberry. The qRT- PCR analysis showed that the expression of key regulatory genes was up-regulated at different developmental stages, which was consistent with the expression trend in the transcriptome. The TCA cycle was promoted inthe ripening process of mulberry fruits, and then affected the change of the organic acid content, and the change of the organic acid content ultimately affected the taste difference of the fruits.【Conclusion】 The NINV, HK, CS, ACO, MDH and ICDH would play important regulatory roles in the synthesis and metabolism of the soluble sugars and organic acids during mulberry maturation, which initially revealed the biological basis of mulberry taste change. The rich metabolites and differential genes identified will not only provide a lot of information for high-quality genetic improvement of mulberry, but also provide valuable reference for other mulberry crops.