VvLAR1 and VvLAR2 Are Bifunctional Enzymes for Proanthocyanidin Biosynthesis in Grapevine

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 4b-(S-cysteinyl)-epicatechin (Cys-EC) is an epicatechintype 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 4b-(S-cysteinyl)-catechin (Cys-C) are present in grapevine. Recombinant VvLAR1 and VvLAR2 convert Cys-C and Cys-EC into (1)-catechin and (2)-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 (1)-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.

Integrating metabolomics and targeted gene expression to uncover potential biomarkers of fungal/oomycetes-associated disease susceptibility in grapevine

“…Albeit all these genes showed expression differences between susceptible and resistant/partial resistant Vitis, LAR2 (catechin biosynthesis pathway) seems to present a higher discriminative potential. In fact, recent functional genomic studies in grapevine LAR enzymes confirmed that LAR2 is involved in the conversion of leucocyanidin into ( +)-catechin and (−)-epicatechin 54 . Moreover, catechin is a naturally occurring flavonol with high antioxidant properties.…”

Coexpression of PalbHLH1 and PalMYB90 Genes From Populus alba Enhances Pathogen Resistance in Poplar by Increasing the Flavonoid Content

“…Pathogen infection might have induced differential expression of only 17 genes, including PalbHLH1, two late embryogenesis abundant (LEA) genes and the gene encoding glucosyl transferase 8 ( Figures S4D-F). Furthermore, with reference to the latest research (Yu et al, 2019), we focus on the expression levels of genes coding for PalF3'H, PalDFR, PalANS, and PalANR, which catalyze the initial, middle, and final steps of anthocyanin and PA biosynthesis, respectively, were significantly upregulated in MYB90/bHLH1-OE poplar after infection by D. gregaria but not after infection by B. cinerea ( Figure 5).…”

Azolla ferns testify: seed plants and ferns share a common ancestor for leucoanthocyanidin reductase enzymes

“…By contrast, dihydroflavonol 4-reductase (DFR) may have evolved in the plant lineage (Campanella et al, 2014). Enzymes shared between PA and anthocyanidin biosynthesis, including DFR and Recent advances have revealed a dual role of leucoanthocyanidin reductase (LAR) from an array of seed plants beyond the synthesis of (+)-catechin from leucocyanidin: the enzymes also accept 4b-(S-cysteinyl)-epicatechin (Cys-EP) which they cleave to Cys and (À)-epicatechin, the polymer starter subunit (Yu et al, 2019). Cys-EC has been detected in several plant extracts and both genetic and biochemical evidence supports a role for LAR in determining DP (Liu et al, 2016(Liu et al, , 2018.…”