Metabolomic analysis of peels at different russet levels of Shine Muscat grape

【Objective】Grape berry russet is caused by adverse environmental stimulation, and seriously affects the appearance and commercial value of grape. The study aimed to explore the mechanism of berry russet formation in grape and provide some scientific guidance and suggestions for its effective prevention and control.【Methods】6-year-old self-rooted Shine Muscat grapevines (Vitis labrusca × V. vinifera) cultivated in rain-shelter were used as the materials. Flower and fruit managements were carried out according to the conventional methods. The flower cluster was kept 6 cm in length through cutting during the initial flowering period. 1 to 3 days and two weeks after blooming period, the flower clusters were treated with 25 mg·L-1 gibberellic acid + 2 mg·L-1 rchlorfenuron for fruit preservation and enlargement, respectively. After softening period, the berries were collected and divided into three groups: no russet (NR), mild russet (MR, the area of berry russet was less than 10% of the whole peel and the color of berry russet was light), and severe russet (SR, the area of fruit russet was more than 25% of the whole peel and the color of berry russet was dark.). The contents of related substances, en-zyme activities, and metabolome in the peel at different berry russet levels were studied through the methods of biochemical and widely targeted metabolomics.【Results】With the increase of berry russet level, the contents of chlorophyll, cellulose, hemicellulose, and total pectin in the peel decreased gradually, while the content of lignin, total flavonoids, and total phenol increased gradually. There were significant differences in the contents of cellulose, hemicellulose, total pectin, lignin, total flavonoids, and total phenol among the berries at three russet levels. The lignin content, total flavonoid content, and total phenolic content of severe russet peel were 71.60, 2.89, and 2.04 mg · g-1 , respectively. Furthermore, the activities of peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, cinnamyl-alcohol dehydrogenase, and 4- coumarate: CoAligase in the peel increased gradually, and the catalase activity increased first and then decreased, while the cinnamate-4-hydroxylase activity decreased first and then increased with the increase of berry russet levels. There were significant differences in the activities of peroxidase, polyphenol oxidase, and 4-coumarate: CoAligase among the berries at three russet levels. There were no significant differences in the activities of phenylalanine ammonia-lyase and cinnamate-4- hydroxylase between the berries with no russet and mild russet. A total of 1372 metabolites in 13 classes (Class Ⅰ) or 52 classes (Class Ⅱ) were identified in the peel of Shine Muscat grapes, including 270 flavonoids, 223 terpenoids, 163 phenolic acids, 127 amino acids and derivatives, 115 lipids, and 106 alkaloids and so on. Among them, there were 690 up-regulated metabolites and 682 down-regulated metabolites in the peel with mild russet vs no russet. There were 920 up- regulated metabolites and 452 down-regulated metabolites in the peel with severe russet vs mild russet. There were 835 up-regulated metabolites and 537 down-regulated metabolites in the peel with severe russet vs no russet. 485 differential metabolites were determined by VIP (VIP ＞ 1) and absolute Log2FC (|Log2FC|≥1) in the peel of Shine Muscat grapes with different degrees of berry russet. And 247, 287, and 386 differential metabolites existed between mild russet and no russet, severe russet and mild russet, and severe russet and no russet, respectively. The venn diagram analysis was performed and it was found that there were 110 common differential metabolites in the three groups. Among them, 105 differential metabolites were upregulated in all three groups, accounting for 95.45% of the common differential metabolites, including 37 phenolic acids, 31 flavonoids, 17 stilbenes, five quinones, three terpenoids, three amino acids and derivatives, two lignans and coumarins, one alkaloid, one tannin, one chromone, and four others. Two differential metabolites were down- regulated in all three groups, including one lipid and one nucleotide and derivative. Three differential metabolites (which were flavonoids) were down-regulated in the berries with mild russet vs no russet, and up-regulated in the berries with severe russet vs mild russet and severe russet vs no russet. The above 110 differential metabolites were mainly enriched in 17 metabolic pathways through KEGG pathway annotation and Metware pathway annotation, including biosynthesis of kaempferol aglycones Ⅱ (six differential metabolites), biosynthesis of kaempferol aglycones Ⅰ (four differential metabolites), biosynthesis of secondary metabolites (four differential metabolites), metabolic pathways (three differential metabolites), biosynthesis of quercetin aglycones Ⅰ (two differential metabolites), stilbenoid, diarylheptanoid and gingerol biosynthesis (two differential metabolites), and tyrosine metabolism (two differential metabolites) and so on. In the biosynthesis of kaempferol aglycones Ⅱ, five differential metabolites of 6- hydroxykaempferol- 3,7,6- O- triglycoside, 6- hydroxykaempferol-7,6-O-diglucoside, 6-hydroxykaempferol-3,6-O-diglucoside, 6-hydroxykaempferol-3, 6- O- diglucoside- 7-O- glucuronic acid, 6- methoxykaempferol- 3- O - glucoside presented up- regulated among the three groups, which might play an important role in berry russet formation of Shine Muscat. While the metabolite of kaempferol-4'-O -glucoside presented down-regulated in the berries with mildrusset vs no russet and up-regulated in the berries with severe russet vs mild russet and severe russet vs no russet.【Conclusion】The occurrence of berry russet in Shine Muscat grape had a greater impact on the metabolites of phenolic acids and flavonoids than others. The biosynthesis of kaempferol glycoside Ⅱ might would play an important role in the formation of berry russet.