Secondary Metabolism and Defense Responses Are Differently Regulated in Two Grapevine Cultivars during Ripening

Abstract

Vitis vinifera 'Nebbiolo' is one of the most important wine grape cultivars used to produce prestigious high-quality wines known throughout the world, such as Barolo and Barbaresco. 'Nebbiolo' is a distinctive genotype characterized by medium/high vigor, long vegetative and ripening cycles, and limited berry skin color rich in 3'-hydroxylated anthocyanins. To investigate the molecular basis of these characteristics, 'Nebbiolo' berries collected at three different stages of ripening (berry pea size, véraison, and harvest) were compared with V. vinifera 'Barbera' berries, which are rich in 3',5'-hydroxylated anthocyanins, using transcriptomic and analytical approaches. In two consecutive seasons, the two genotypes confirmed their characteristic anthocyanin profiles associated with a different modulation of their transcriptomes during ripening. Secondary metabolism and response to stress were the functional categories that most differentially changed between 'Nebbiolo' and 'Barbera'. The profile rich in 3'-hydroxylated anthocyanins of 'Nebbiolo' was likely linked to a transcriptional downregulation of key genes of anthocyanin biosynthesis. In addition, at berry pea size, the defense metabolism was more active in 'Nebbiolo' than 'Barbera' in absence of biotic attacks. Accordingly, several pathogenesis-related proteins, WRKY transcription factors, and stilbene synthase genes were overexpressed in 'Nebbiolo', suggesting an interesting specific regulation of defense pathways in this genotype that deserves to be further explored.

Keywords: Vitis vinifera; anthocyanin profile; defense metabolism; flavonoid 3′,5′-hydroxylases; high-throughput sequencing; stilbene synthases.

Figure 1

Transcriptome analysis of ‘Nebbiolo’ CVT71 (NE) and ‘Barbera’ CVT115 (BA) berries collected in 2013 at three developmental stages: pea size (E-L31), véraison (E-L35), and harvest at 24° BRIX (E-L38). The number of differentially expressed genes (DEGs), up (UP) or down (DOWN) regulated, is shown for each RNA-seq comparison near the bar charts. Significantly enriched Gene Ontology (GO) biological functional categories were identified for each group of DEGs belonging to downregulated genes at E-L31, upregulated genes at E-L35 and E-L38 in ‘Barbera’ using Cytoscape with the BINGO plug-in according to enrichment p-value (p ≤ 0.05).

Figure 2

Transcriptional reprogramming of secondary metabolism genes in ‘Nebbiolo’ and ‘Barbera’ during ripening. ‘Nebbiolo’ CVT71 (NE) and ‘Barbera’ CVT115 (BA) berries were collected in 2013 and 2014 at three developmental stages: pea size (E-L31), véraison (E-L35), and harvest at 24° BRIX (E-L38). (a) Clusters of differentially expressed genes (DEGs) showing upregulated (Cluster 1) and downregulated (Cluster 2) genes in ‘Barbera’. (b) Results of candidate gene expression analysis performed by RT-qPCR assay. Cluster 1: anthocyanin O-methyltransferase (VvAOMT, VIT_01s0010g03510), UDP-glucose:flavonoid 3-O-glucosyltransferase (VvUFGT, VIT_16s0039g02230), flavonoid 3′,5′-hydroxylase (VvF3′5′H, VIT_06s0009g02810), anthocyanin acyltransferase (Vv3AT, VIT_03s0017g00870) and stilbene synthase VvSTS48 (VIT_16s0100g01200). Cluster 2: stilbene synthase VvSTS16 (VIT_16s0100g00840). Ubiquitin and Actin genes were used as endogenous controls for the normalization of transcript levels. Three independent biological replicates with three technical replicates each were used for analysis. Statistically significant differences between ‘Nebbiolo’ and ‘Barbera’ in each developmental stage were attested by analysis of variance for p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

Figure 3

Transcriptional reprogramming of defense responses in ‘Nebbiolo’ and ‘Barbera’ during ripening. ‘Nebbiolo’ CVT71 (NE) and ‘Barbera’ CVT115 (BA) berries were collected in 2013 and 2014 at three developmental stages: pea size (E-L31), véraison (E-L35), and harvest at 24° BRIX (E-L38). (a) Clusters of differentially expressed genes (DEGs) showing upregulated (Cluster 1) and downregulated (Cluster 2) genes in ‘Barbera’. (b) Results of candidate gene expression analysis performed by RT-qPCR assay. Cluster 1: peroxidase (VvPOX, VIT_18s0072g00160) and Dehydrin (VvDH, VIT_04s0023g02480). Cluster 2: β1-3 glucanases (Vvβgluc, VIT_08s0007g06040) and thaumatin (VvTHAU, VIT_02s0025g04310). VvWRKY18 (VIT_04s0008g05760) and sugar transporter VvSTP13 (VIT_05s0020g03140). Ubiquitin and Actin genes were used as endogenous controls for the normalization of transcript levels. Three independent biological replicates with three technical replicates each were used for analysis. Statistically significant differences between ‘Nebbiolo’ and ‘Barbera’ in each developmental stage were attested by analysis of variance for p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).