Bernard Donèche

Pectin methylesterase and polygalacturonase in the developing grape skin.

Ripening of grape (Vitis vinifera L.) berry immediately precedes harvesting and the evolution of the skin tissue is important as it contains the key compounds for wine quality. Grape softening is thought to result from extensive cell wall modifications that occur during ripening. These modifications result from the activity of different cell wall-modifying enzymes. Two of the most significant pectin-degrading enzymes are pectin methylesterase (EC 3.1.1.11) and polygalacturonase (EC 3.2.1.15). In this work, the activities of both enzymes were monitored in skin tissue throughout berry development. Pectin methylesterase activity was present before the onset of veraison and increased during skin maturation. No polygalacturonase activity could be detected. The accumulation of mRNA encoding a pectin methylesterase and two polygalacturonase isoforms was examined using RT-PCR. Transcripts for pectin methylesterase were present in all stages analyzed with a maximal accumulation at the end of color change. Accumulation of VvPG1 transcript was closely correlated with berry softening, and expression of this gene was markedly increased during the color change. VvPG2 mRNA accumulation began before veraison and was low during skin ripening. A phylogenic analysis showed that this gene is classified in a different group than VvPG1. These findings suggest that both genes are associated with different mechanisms during skin development. VvPG1, in particular, is most likely to play a role in skin softening and VvPG2 in triggering the ripening process.

Purification and characterization of two isozymes of polygalacturonase from Botrytis cinerea. Effect of calcium ions on polygalacturonase activity

The phytopathogenic fungus Botrytis cinerea produces a set of polygalacturonases (PGs) which are involved in the enzymatic degradation of pectin during plant tissue infection. Two polygalacturonases secreted by B. cinerea in seven-day-old liquid culture were purified to apparent homogeneity by chromatography. PG I was an exopolygalacturonase of molecular weight 65 kDa and pI 8.0 and PG II was an endopolygalacturonase of 52 kDa and pI 7.8. Enzymatic activity of PG I and PG II was partially inhibited by 1 mM CaCl2, probably by calcium chelation of polygalacturonic acid, the substrate of the enzyme.

Impact of foliar symptoms of "Esca proper" on proteins related to defense and oxidative stress of grape skins during ripening.

Esca is one of the major diseases affecting vineyards with direct impact on product yield; nevertheless, scientific studies concerning its impact on grape quality are scarce. As an attempt to better understand the mechanisms behind “Esca proper” development in grapes, this work focused on the identification of proteins whose expression is altered by the disease. 2‐DEs were performed on protein extracts from grape skins at different stages of maturity for two consecutive vintages. Grapes were collected in 2009 and in 2010 from plants that did not present signs of infection by Esca proper since the 2004 vintage and from plants that presented cast leaf symptoms at least once since 2004. For the first time, 13 proteins were shown to be influenced by Esca proper during the ripening process. Extensive bioinformatics analysis allowed the grouping of proteins involved in (i) stress tolerance and defense response, (ii) oxidative phosphorylation, (iii) oxidation–reduction processes in mitochondria, and (iv) oxidation–reduction processes in chloroplasts. Of these 13 proteins, cysteine synthase is the only one implicated in a metabolic pathway of oenological interest. This study shows how foliar symptoms of Esca proper may impact stress‐related pathways in grapes, which are characterized by modifications in the chain of oxidative phosphorylation and redox scavenging.