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 at different extents in different years and might be related to several parameters, such as temperature and humidity during withering, grape composition and Botrytis cinerea loading. To better understand the mechanism involved in color variation, the metabolic changes corresponding to this event 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 different water contents, the blue berries containing about 30% less water than the green ones. Samples were crushed and the juices were analyzed. The juice yield 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 enzymatic analyses). No trace of laccase activity was found in any of the samples, although DNA analysis, by quantitative PCR, suggested the presence of B. 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 lower (of about 85%) in the blue berries, which, in contrast, showed the highest beta-glucosidase activity. The electrophoretic analysis of the protein extracts revealed strong differences among the 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 analysis showed that these latter proteins were of grape origin, the main of them corresponding to β-1,3-glucanase, although no significant increase in glucanase activity was measured in the corresponding protein extract.