Proteome changes in the skin of the grape cultivar Barbera among different stages of ripening

Abstract

Background: Grape ripening represents the third phase of the double sigmoidal curve of berry development and is characterized by deep changes in the organoleptic characteristics. In this process, the skin plays a central role in the synthesis of many compounds of interest (e.g. anthocyanins and aroma volatiles) and represents a fundamental protective barrier against damage by physical injuries and pathogen attacks. In order to improve the knowledge on the role of this tissue during ripening, changes in the protein expression in the skin of the red cultivar Barbera at five different stages from véraison to full maturation were studied by performing a comparative 2-DE analysis.

Results: The proteomic analysis revealed that 80 spots were differentially expressed throughout berry ripening. Applying a two-way hierarchical clustering analysis to these variations, a clear difference between the first two samplings (up to 14 days after véraison) and the following three (from 28 to 49 days after véraison) emerged, thus suggesting that the most relevant changes in protein expression occurred in the first weeks of ripening. By means of LC-ESI-MS/MS analysis, 69 proteins were characterized. Many of these variations were related to proteins involved in responses to stress (38%), glycolysis and gluconeogenesis (13%), C-compounds and carbohydrate metabolism (13%) and amino acid metabolism (10%).

Conclusion: These results give new insights to the skin proteome evolution during ripening, thus underlining some interesting traits of this tissue. In this view, we observed the ripening-related induction of many enzymes involved in primary metabolism, including those of the last five steps of the glycolytic pathway, which had been described as down-regulated in previous studies performed on whole fruit. Moreover, these data emphasize the relevance of this tissue as a physical barrier exerting an important part in berry protection. In fact, the level of many proteins involved in (a)biotic stress responses remarkably changed through the five stages taken into consideration, thus suggesting that their expression may be developmentally regulated.

Figure 1

2-DE maps of five stages through the ripening of Barbera. 2-DE maps of five different ripening stages from véraison until full ripeness of cultivar Barbera berry skins. The véraison stage (0 DAV) was considered as the moment when 50% of the berries started to change colour. Proteins (200 μg) were separated by IEF at pH 3–10, followed by 12.5% SDS PAGE and visualized by cCBB-staining.

Figure 2

Clustering analysis of the spots that resulted to change their relative volumes during ripening. Two-way hierarchical clustering analysis of the 80 spots that showed at least a two-fold change in the relative spot volumes (ANOVA, p < 0.01) in the five different ripening stages of grape berry skins of cultivar Barbera. The véraison stage (0 DAV) was considered as the moment when 50% of the berries started to change colour. The clustering analysis was performed with PermutMatrix graphical interface after Z-score normalization of the averages of relative spot values (n = 6). Pearson's distance and Ward's algorithm were used for the analysis. Each coloured cell represents the average of the relative spot value, according to the colour scale at the bottom of the figure.

Figure 3

Protein profiles of identified proteins. Identified proteins are indicated in a 2-DE gel representative of the fifth ripening stage with spot name abbreviation corresponding to those in Table 1, Figure 6 and 7. Spots showing an increased or a decreased expression during ripening are indicated in red and in green, respectively.

Figure 4

Functional categories distribution of the identified proteins. Functional distribution of the identified proteins (Table 1) according to the annotation in the MIPS FunCat.

Figure 5

Changes in the expression of proteins involved in stress response. Changes in the relative spot volumes of the proteins (Table 1) involved in stress responses during five different ripening stages from véraison until full ripening of cultivar Barbera grape berry skins. The véraison stage (0 DAV) was considered as the moment when 50% of the berries started to change colour. Proteins were grouped according to their functions. Values are the mean ± SE of six 2-DE gels derived from two independent biological samples analyzed in triplicate.

Figure 6

Changes in the expression of proteins involved in C- and N-metabolism or with other functions. Changes in the relative spot volumes of the identified proteins belonging to the indicated functional categories (Table 1), during five different ripening stages of cv. Barbera grape berry skins from véraison until full ripening. The véraison stage (0 DAV) was considered as the moment when 50% of the berries started to change colour. Proteins were grouped according to their functions. Values are the mean ± SE of six 2-DE gels derived from two independent biological samples analyzed in triplicate.

Figure 7

Schematic overview of the enzymes involved in sugar and organic acid metabolisms and their connection with some intermediary activities that changed in expression in grape berry skins during five different ripe stages from véraison until full ripening. The expression was evaluated by measuring relative spot volumes in the 2-DE analysis. Green or red arrows indicate whether the abundance of the identified proteins decreased or increased during ripening, respectively. IRV1, cell wall invertase, GIN1, vacuolar invertase; Susy, sucrose synthase; UGP, UDP-glucose-pyrophosphorylase; PGluM, phosphogluco-mutase; PGI, phosphogluco-isomerase; PFK, phosphofructokinase; ALD, aldolase; TPI, triosephosphate-isomerase; G3PDH, glyceraldehyde-3-phosphate-dehydrogenase; PGK, phosphoglycerate-kinase; PGlyM, phosphoglycerate-mutase; ENO, enolase; PK, pyruvate kinase; PDC, pyruvate decarboxylase; NADP-ME, NADP-dependent malic enzyme; ADH, alcohol dehydrogenase; PDH, Pyruvate dehydrogenase.

Figure 8

Biochemical changes occurring during the ripening of Barbera berries. Changes in the physiological parameters were measured during five different ripening stages of cultivar Barbera grape berries from véraison until full ripening. The véraison stage (58 days after blooming) was considered as the moment when 50% of the berries started to change colour. A, total soluble solids; B, titratable acidity; C, berry juice pH; D, total anthocyanin contents. The data are the means ± SE of three experiments run in triplicate (n = 9).