Resveratrol accumulation and its involvement in stilbene synthetic pathway of Chinese wild grapes during berry development using quantitative proteome analysis

Abstract

Attention has become focused on resveratrol not only because of its role in grapevine fungal resistance but also because of its benefits in human health. This report describes the Chinese wild grapevine Vitis quinquangularis accession Danfeng-2 in relation to the high resveratrol content of its ripe berries. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) tandem mass spectrometry strategy to quantify and identify proteome changes, resulting in the detection of a total of 3,751 proteins produced under natural conditions. Among the proteins quantified, a total of 578 differentially expressed proteins were detected between Danfeng-2 and Cabernet Sauvignon during berry development. Differentially expressed proteins are involved in secondary metabolism, biotic stress, abiotic stress and transport activity and indicate novel biological processes in Chinese wild grapevine. Eleven proteins involved in phenylpropanoid metabolism and stilbene synthesis were differently expressed between Danfeng-2 and Cabernet Sauvignon at the veraison stage of berry development. These findings suggest that Chinese wild V. quinquangularis accession Danfeng-2 is an extremely important genetic resource for grape breeding and especially for increasing the resveratrol content of European grape cultivars for disease resistance and for improved human nutritional benefits.

Figure 1

Workflow and proteins identified in four developmental stages of berries of Vitis quinquangularis accession Danfeng-2 and control grapevine V. vinifera cv. Cabernet Sauvignon with iTRAQ. (A) iTRAQ 8-plex labeling of different developmental stages (green hard, before veraison, veraison and ripe). DAB, days after blooming; (B) Venn diagram showing proteins among the three iTRAQ data sets of Danfeng-2 and Cabernet Sauvignon; Set1, Set2 and Set3 represented three independent biological replicates. (C) GO term enriched broader functional classification of 3751 proteins identified during four developmental stages of berries of Danfeng-2 and Cabernet Sauvignon. Number of each GO term was next to the corresponding pie.

Figure 2

Determination of metabolites in berry skins of Vitis quinquangularis accession Danfeng-2 and V. vinifera cv. Cabernet Sauvignon at four different development stages. GH, green hard, BV, before veraison, V, veraison, and R, ripe. (A) Trans-resveratrol content; (B) Piceid content. (C) Total flavonoid content; (D) Anthocyanin content. The p-value level less than 0.05 was considered significantly different at each development stage between Vitis quinquangularis accession Danfeng-2 and V. vinifera cv. Cabernet Sauvignon and labeled with an asterisk.

Figure 3

Overlaps of different expression proteins over the developmental stages between Vitis quinquangularis accession Danfeng-2 and V. vinifera cv. Cabernet Sauvignon. GH, green hard, BV, before veraison, V, veraison, and R, ripe. (A) Venn diagram illustrating overlaps of 578 differential proteins at all stages between Danfeng-2 and Cabernet Sauvignon. DEPs-differentially expressed proteins; (B) Venn diagram illustrating overlaps of proteins with higher expression profiles in Danfeng-2 at each stage; (C) Venn diagram illustrating overlaps of proteins with lower expression profiles in Danfeng-2 at each stage.

Figure 4

Enriched GO terms of the different expression proteins related with secondary metabolism over the developmental stages of Vitis quinquangularis accession Danfeng-2 and V. vinifera cv. Cabernet Sauvignon.

Figure 5

Differential expressed proteins involved in phenylalanine metabolism pathway revealed specific protein promoting stilbenes accumulation in V. quinquangularis accession Danfeng-2. (A) C4H, Resveratrol metabolism pathway, (B) RSGT, and flavonoid metabolism (C) CHS, (D) CHI, (E) F3′5′H, (F) F3H, (G) DFR, (H) LDOX, and (I) UFGT, were identified. Especially, a resveratrol glucosyltransferase (A5BIH9) was significantly up-regulated at stage Ripe in Danfeng-2. PAL, phenylalanine ammonia-lyase, C4H, trans-cinnamate 4-monooxyygenase, 4CL, 4-coumarate-CoA ligase, STS, stilbene synthase; RSGT, resveratrol glucosyltransferase, OMT, O-methyltransferase, CHS, chalcone synthase, CHI, chalcone isomerase, F3H, flavanone 3-hydroxylase, F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol-4-reductase; FLS, flavonol synthase, LDOX, leucoanthocyanidin dioxygenase, LAR, leucoanthocyanidin reductase, ANR, anthocyanidin reductase, UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase. Heat maps of proteins were constructed with iTRAQ-derived quantitative data. D-2, Danfeng-2, CS, Cabernet Sauvignon. GH, green hard, BV, before veraison, V, veraison, and R, ripe. The proteins that increased and decreased are displayed in yellow and blue, respectively.

Figure 6

Relative expression levels of related transcripts. Data were normalized with the housekeeping gene GAPDH. Genes involved in stilbene synthesis are (A) RSGT; (B) STS; (C) MYB14; (D) MYB15; Genes involved in flavonoid and anthocyanin synthesis are (E) MYB5a; (F) MYB5b; (G) MYBPA1; (H) MYBA1. 2^−ΔΔCt method was used to calculate the relative expression for each gene. Each value represents the means ± SE of three different experiments.

Figure 7

Correlation of protein and mRNA levels of stilbene and flavonoid biosynthesis related members. C4H, trans-cinnamate 4-monooxyygenase, RSGT, resveratrol glucosyltransferase, CHS, chalcone synthase, CHI, chalcone isomerase, F3H, flavanone 3-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol-4-reductase; LDOX, leucoanthocyanidin dioxygenase. GH, green hard, BV, before veraison, V, veraison, and R, ripe. The proteins that increased and decreased are displayed in red and blue, respectively.