Proteins and enzymatic activities in Erbaluce grape berries with different response to the withering process.

During the off-vine natural withering process of Erbaluce (white) grapes to obtain “Erbaluce Caluso” Passito wine, some berries change in color from green-yellow to blue. This phenomenon appears to a different extent in different years and it might be related to several parameters, such as temperature and humidity during withering, grape composition and Botrytis loading. To better understand the mechanism involved in this phenomenon, the metabolic changes corresponding to this color variation were studied. At the end of the withering process berries with different colors were separated using a reflectance spectrophotometer, obtaining three color classes identified as “green” (L*= 40.3, a*= -0.56, b*= 15.20), “gold”(L*= 37.7, a* = 5.01, b*= 14.12) and “blue” (L*= 28.6, a* = 0.89, b*= -0.67). The three groups of berries had a different water content, the blue berries containing about 30% less water than the green ones. Samples were crushed and the juices were analyzed. The yield in juice for blue berries was less than 50% of that of the other two classes, confirming their higher dehydration level. Protein extraction from de-seeded berries was carried out using two different protocols, the first involving a treatment with phenol (to remove polyphenolic substances) and the second based on an extraction with a mild detergent (to recover the proteins to be used for enzyme analyses). No trace of laccase activity was found in any of the samples, although DNA analysis, by quantitative PCR, suggested the presence of Botrytis cinerea infection in the blue grapes. Chitinase activity of the blue berries was only 30% of that of the other two samples, as confirmed also by zymographic analysis on electrophoretic gels. The same was found also for esterase activity, which was decreased (of about 85%) in the blue berries. In contrast, the highest beta-glucosidase activity was found the in the blue berries. The electrophoretic analysis of the protein extracts revealed strong differences between the profile of the blue berries and those of the other two samples. Compared to the green and gold ones, the blue berries showed a complete absence of bands corresponding to chitinases, and the appearance of unusual protein bands. Mass spectrometry (MS) analysis showed that these latter proteins were of grape origin, the main of them corresponding to the PR-protein β-1,3-glucanase, although no significant increase in glucanase activity was measured in the corresponding protein extract.