VvLAR1 and VvLAR2 Are Bifunctional Enzymes for Proanthocyanidin Biosynthesis in Grapevine.

Affiliation

BioDiscovery Institute and Department of Biological Sciences, University of North Texas, Denton, Texas 76203 [Email]

Abstract

Proanthocyanidins (PAs) in grapevine (Vitis vinifera) are found mainly in berries, and their content and degree of polymerization are important for the mouth feel of red wine. However, the mechanism of PA polymerization in grapevine remains unclear. Previous studies in the model legume Medicago truncatula showed that 4β-(S-cysteinyl)-epicatechin (Cys-EC) is an epicatechin-type extension unit for nonenzymatic PA polymerization, and that leucoanthocyanidin reductase (LAR) converts Cys-EC into epicatechin starter unit to control PA extension. Grapevine possesses two LAR genes, but their functions are not clear. Here, we show that both Cys-EC and 4β-(S-cysteinyl)-catechin (Cys-C) are present in grapevine. Recombinant VvLAR1 and VvLAR2 convert Cys-C and Cys-EC into (+)-catechin and (-)-epicatechin, respectively, in vitro. The kinetic parameters of VvLARs are similar, with both enzymes being more efficient with Cys-C than with Cys-EC, the 2,3-cis conformation of which results in steric hindrance in the active site. Both VvLARs also produce (+)-catechin from leucocyanidin, and an inactive VvLAR2 allele reported previously is the result of a single amino acid mutation in the N terminus critical for all NADPH-dependent activities of the enzyme. VvLAR1 or VvLAR2 complement the M. truncatula lar:ldox double mutant that also lacks the leucoanthocyanidin dioxygenase (LDOX) required for epicatechin starter unit formation, resulting in increased soluble PA levels, decreased insoluble PA levels, and reduced levels of Cys-C and Cys-EC when compared to the double mutant, and the appearance of catechin, epicatechin, and PA dimers characteristic of the ldox single mutant in young pods. These data advance our knowledge of PA building blocks and LAR function and provide targets for grapevine breeding to alter PA composition.