Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves

Abstract

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.

Figure 1.

Scheme of the flavonoid pathway leading to synthesis of anthocyanins and PA polymers. Leucocyanidin is a precursor to synthesis of anthocyanins such as cyanidin-3-glucoside but also contributes the extension subunits of the PA polymer. LAR catalyzes reduction of leucocyanidin to catechin and ANR (BANYULS) catalyzes reduction of cyanidin to epicatechin, both of which can form the terminal unit of the PA polymer. FLS, Flavonol synthase.

Figure 2.

Phylogenetic tree showing LAR and IFR proteins as well as distantly related ANR and DFR proteins of the RED superfamily. The ClustalW multiple sequence alignment (shown in Supplemental Fig. 1) was formed using the default parameters of the MEGA package (Kumar et al., 2004). The tree was constructed from the ClustalW alignment using the unweighted pair group using arithmetic averages method by the MEGA program. The scale bar represents 0.2 substitutions per site. The tree includes all related proteins from the Arabidopsis genome (designated according to their Arabidopsis Genome Initiative number) to give an indication of how the proteins from other species fit into a complete genome. Also included are enzymes for which a catalytic function has been shown such as IFR, pinoresinol-lariciresinol reductase (PLR), and phenylcoumaran benzylic ether reductase (PCBER) in the IFR branch; Arabidopsis and Medicago ANR; and Arabidopsis and maize (Zea mays) DFR. The IFR branch includes six grape IFR-like (IFRL) proteins found in the grape EST collections (IFRL_Vitvi1-6, BN000706-11). The ANR/DFR branch shows only a small fraction of the proteins in this branch of the RED superfamily. The Arabidopsis proteins most closely related to ANR (At4g27250) and DFR (At2g45400) are also present to show that grape ANR and DFR are more closely related to their Arabidopsis orthologs than to the next most closely related Arabidopsis protein. The proteins are labeled according to their proposed catalytic activity followed by the species they come from, e.g. LAR_ Desun is LAR from D. uncinatum (Q84V83). Other species represented in the tree and database accession numbers are Cicer arietinum (IFR, Q00016), Forsythia x intermedia (PLR, AAC49608; PCBER, AAF64174), G. arboretum (ANR CAD91910; LAR1, BN000695; LAR2, BN000699), G. raimondii (LAR1, BN000700; LAR2, BN000701), H. vulgare (LAR, BN000696), M. truncatula (ANR, AAN77735; LAR, BN000703), O. sativa (LAR, BN000704), P. coccineus (ANR, CAD91909; LAR, BN000698), P. taeda (LAR, BN000697), V. shuttleworthii (LAR1, BN000702), grapevine (ANR, CAD91911; DFR, P51110; LAR1, AJ865336; LAR2, AJ865334), and Z. mays (DFR, CAA28734).

Figure 3.

Functional characterization of VvANR by its ectopic expression in tobacco. Flowers of untransformed tobacco (Samsun) and four transgenic lines were analyzed for expression of VvANR by northern analysis (A), flower color (B), anthocyanins by HPLC (C), PAs by DMACA (D), and flavonols by HPLC (E).

Figure 4.

Enzyme activity of recombinant grapevine LAR (VvLAR1). HPLC chromatography of LAR reactions of E. coli transformed with a control pET plasmid (A) or the pET LAR1_Vitvi plasmid (B). The chromatograms show the elution profiles of the ³H-labeled reaction mixture obtained and processed as described in “Material and Methods.” The retention times for the substrate (leucocyanidin) and the product (catechin) are indicated by arrows.

Figure 5.

Accumulation of PAs and gene expression of VvLDOX, VvANR, VvLAR1, and VvLAR2 in grapes during the early stages of berry development. Flowering (indicated by down arrow) occurred 8 weeks before véraison, which is the onset of ripening in grapes. A, Flavan-3-ol accumulation and composition. B, Accumulation and composition of PA extension subunits. C, Gene expression of VvLDOX, VvANR, VvLAR1, and VvLAR2. Expression was determined by real-time PCR and is shown relative to expression of VvUbiquitin1 in each sample. All data is presented as mean of three replicates.

Figure 6.

Accumulation of PAs and gene expression of VvLDOX, VvANR, VvLAR1, and VvLAR2 in grape skin during berry development. No skin sample was obtained for grapes 6 weeks before the onset of ripening. Time point −6 is included only for better comparison with Figure 5. A, Flavan-3-ol accumulation and composition. B, Accumulation and composition of PA extension subunits. C, Gene expression of VvLDOX, VvANR, VvLAR1, and VvLAR2. Expression was determined by real-time PCR as described for Figure 5.

Figure 7.

Accumulation of PAs and gene expression of VvLDOX, VvANR, VvLAR1, and VvLAR2 in grape seeds during berry development. A, Flavan-3-ol accumulation and composition. B, Accumulation and composition of PA extension subunits. C, Gene expression of VvLDOX, VvANR, VvLAR1, and VvLAR2. Expression was determined by real-time PCR as described for Figure 5.

Figure 8.

Composition of PAs in grape leaves during development. A, Flavan-3-ol content as mg/g. B, Flavan-3-ol content as mg per leaf. C, Extension subunits of PAs shown as mg/g. D, Extension subunits of PAs shown as mg per leaf. The stages of leaf development are defined in “Materials and Methods.” All data is presented as mean of three replicates.

Figure 9.

Accumulation of PAs and gene expression of VvLDOX, VvANR, VvLAR1, and VvLAR2 in grape leaves during development. A, PAs measured as mg/g. B, PAs measured as mg per leaf. C, Gene expression of VvLDOX, VvANR, VvLAR1, and VvLAR2. Expression was determined by real-time PCR as described for Figure 5.