Transcriptional analysis of late ripening stages of grapevine berry

Abstract

Background: The composition of grapevine berry at harvest is a major determinant of wine quality. Optimal oenological maturity of berries is characterized by a high sugar/acidity ratio, high anthocyanin content in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition and berry tasting. In this context, it is interesting to identify genes that are expressed/repressed specifically at the late stages of ripening and which may be used as indicators of maturity.

Results: Whole bunches and berries sorted by density were collected in vineyard on Chardonnay (white cultivar) grapevines for two consecutive years at three stages of ripening (7-days before harvest (TH-7), harvest (TH), and 10-days after harvest (TH+10)). Microvinification and sensory analysis indicate that the quality of the wines made from the whole bunches collected at TH-7, TH and TH+10 differed, TH providing the highest quality wines.In parallel, gene expression was studied with Qiagen/Operon microarrays using two types of samples, i.e. whole bunches and berries sorted by density. Only 12 genes were consistently up- or down-regulated in whole bunches and density sorted berries for the two years studied in Chardonnay. 52 genes were differentially expressed between the TH-7 and TH samples. In order to determine whether these genes followed a similar pattern of expression during the late stages of berry ripening in a red cultivar, nine genes were selected for RT-PCR analysis with Cabernet Sauvignon grown under two different temperature regimes affecting the precocity of ripening. The expression profiles and their relationship to ripening were confirmed in Cabernet Sauvignon for seven genes, encoding a carotenoid cleavage dioxygenase, a galactinol synthase, a late embryogenesis abundant protein, a dirigent-like protein, a histidine kinase receptor, a valencene synthase and a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase.

Conclusions: This set of up- and down-regulated genes characterize the late stages of berry ripening in the two cultivars studied, and are indirectly linked to wine quality. They might be used directly or indirectly to design immunological, biochemical or molecular tools aimed at the determination of optimal ripening in these cultivars.

Figure 1

Representativity of berry classes separated according to their density at three stages of Chardonnay ripening. In 2005 (A) and 2006 (B), one thousand berries were harvested at each of the three harvest date and were separated into classes according to their density. TH-7, 7-days before theoretical harvest; TH, theoretical harvest; TH+10, 10-days after harvest.

Figure 2

Venn diagram summary of differentially expressed genes identified in Chardonnay at three stages of ripening. Chardonnay whole bunches and densimetrically sorted berries were harvested at the 7-days before harvest (TH-7), theoretical harvest (TH) and 10-days after harvest (TH+10) stages during the 2005 and 2006 years. Comparisons of the expression profiles of TH versus TH-7 and TH+10 versus TH were made for whole bunches and densimetrically sorted berries. The total numbers of genes differentially expressed are indicated in respective circles (P < 0.05, ≥1.75-fold). The combined number of genes simultaneously up- or down-regulated is given in intersections between circles. Twelve genes were differentially expressed at all stages of late ripening; some genes were stage or sample type specific while others were overlapping in two stage or sample comparisons (for gene identity, see Tables 4, 5 and 6 and Additional files 1 and 2(Tables S1and S2)). WBB, whole bunch berries; DSB, densimetrically sorted berries.

Figure 3

Bar diagram of Vitis vinifera carotenoid cleavage dioxygenase 4a (VvCCD4a) transcript abundance: a comparison of qRT-PCR data of Chardonnay whole bunch and densimetrically sorted berries harvested at three ripening stages in 2005 and 2006. The mRNA level was expressed relative to controls (set at 1), reference gene EF1-α. RT-PCR data are reported as means ± SE (error bars) of n = 3 technical replicates. DSB, densimetrically sorted berries; TH-7, 7-days before theoretical harvest; TH, theoretical harvest; TH+10, 10-days after harvest; WBB, whole bunch berries.

Figure 4

Bar diagram of Vitis vinifera (+)-valencene synthase (VvValCS) transcript abundance: a comparison of qRT-PCR data of Chardonnay whole bunch and densimetrically sorted berries harvested at three ripening stages in 2005 and 2006. The mRNA level was expressed relative to controls (set at 1), reference gene EF1-α. RT-PCR data are reported as means ± SE (error bars) of n = 3 technical replicates. DSB, densimetrically sorted berries; TH-7, 7-days before theoretical harvest; TH, theoretical harvest; TH+10, 10-days after harvest; WBB, whole bunch berries.

Figure 5

Progression of Cabernet Sauvignon berry development and ripening. Changes of various parameters i.e. the berry volume (A), percentage of colored berries (B) and total soluble solids (°BRIX) in the berry juice (C) were investigated in controlled rooms. Cabernet Sauvignon rooted cuttings were subjected to either 30°C days and 25°C nights or 20°C days and 15°C nights temperature regimes. Data are reported as means ± SE (error bars).

Figure 6

Transcript abundances of eight potential ripening indicators in Vitis vinifera cv. Cabernet Sauvignon. The expression profiles of VvCCD4a (A), VvPAL2 (B), VvGolS (C), VvLEA1 (D), VvDIR-like (E), VvHKR (F), VvValCS (G) and VvSAMT (H) were investigated from véraison to harvest. Berries were subjected to either 30°C days and 25°C nights or 20°C days and 15°C nights temperature regimes. The mRNA level was expressed relative to the lowest level of expression detected in any sample for each gene, reference genes Ubiquitin, Actin and EF1-α. RT-PCR data are reported as means ± SE (error bars) of n = 3 technical replicates.