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

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.     

An integrated approach to demonstrating the ANR pathway of proanthocyanidin biosynthesis in plants

Proanthocyanidins (PAs) are oligomers or polymers of plant flavan-3-ols and are important to plant adaptation in extreme environmental conditions. The characterization of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) has demonstrated the different biogenesis of four stereo-configurations of flavan-3-ols. It is important to understand whether ANR and the ANR pathway widely occur in the plant kingdom. Here, we report an integrated approach to demonstrate the ANR pathway in plants. This includes different methods to extract native ANR from different tissues of eight angiosperm plants (Lotus corniculatus, Desmodium uncinatum, Medicago sativa, Hordeum vulgare, Vitis vinifera, Vitis bellula, Parthenocissus heterophylla, and Cerasus serrulata) and one fern plant (Dryopteris pycnopteroides), a general enzymatic analysis approach to demonstrate the ANR activity, high-performance liquid chromatography-based fingerprinting to demonstrate (-)-epicatechin and other flavan-3-ol molecules, and phytochemical analysis of PAs. Results demonstrate that in addition to leaves of M. sativa, tissues of other eight plants contain an active ANR pathway. Particularly, the leaves, flowers and pods of D. uncinatum, which is a model plant to study LAR and the LAR pathways, are demonstrated to express an active ANR pathway. This finding suggests that the ANR pathway involves PA biosynthesis in D. uncinatum. In addition, a sequence BLAST analysis reveals that ANR homologs have been sequenced in plants from both gymnosperms and angiosperms. These data show that the ANR pathway to PA biosynthesis occurs in both seed and seedless vascular plants.     

Maize <Emphasis Type="Italic">Lc</Emphasis> transcription factor enhances biosynthesis of anthocyanins,distinct proanthocyanidins and phenylpropanoids in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Here, we report on metabolic engineering of the flavonoid biosynthetic pathways in apple (Malus domestica Borkh.) by overexpression of the maize (Zea mays L.) leaf colour (Lc) regulatory gene. The Lc gene was transferred into the M. domestica cultivar Holsteiner Cox via Agrobacterium tumefaciens-mediated transformation which resulted in enhanced anthocyanin accumulation in regenerated shoots. Five independent Lc lines were investigated for integration of Lc into the plant genome by Southern blot and PCR analyses. The Lc-transgenic lines contained one or two Lc gene copies and showed increased mRNA levels for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3 beta-hydroxylase (FHT), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductases (LAR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). HPLC-DAD and LC-MS analyses revealed higher levels of the anthocyanin idaein (12-fold), the flavan 3-ol epicatechin (14-fold), and especially the isomeric catechin (41-fold), and some distinct dimeric proanthocyanidins (7 to 134-fold) in leaf tissues of Lc-transgenic lines. The levels of phenylpropanoids and their derivatives were only slightly increased. Thus, Lc overexpression in Malus domestica resulted in enhanced biosynthesis of specific flavonoid classes, which play important roles in both phytopathology and human health.     

The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development

The anthocyanin and proanthocyanidin (PA) biosynthetic pathways share common intermediates until leucocyanidin, which may be used by leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanin, or the enzyme leucoanthocyanidin reductase (LAR) to produce catechin, a precursor of PA. The Arabidopsis mutant tannin deficient seed 4 (tds4-1) has a reduced PA level and altered pattern PA accumulation. We identified the TDS4 gene as LDOX by complementation of the tds4-1 mutation either with a cosmid encoding LDOX or a 35S:LDOX construct. Independent Arabidopsis lines with a T-DNA insertion in the LDOX gene had a similar phenotype, and one was allelic to tds4-1. The seed phenotype of ban tds4 double mutants showed that LDOX precedes BANYULS (BAN) in the PA pathway, confirming recent biochemical characterisation of BAN as an anthocyanidin reductase. Double mutant analysis was also used to order the other TDS genes. Analysis of the PA intermediates in tds4-1 revealed three dimethylaminocinnamaldehyde (DMACA) reacting compounds that accumulated in extracts from developing seeds. Analysis of Arabidopsis PA and its precursors indicates that Arabidopsis, unlike many other plants, exclusively uses the epicatechin and not the catechin pathway to PA. Transmission electron microscopy (TEM) showed that the pattern observed when seeds of tds4 were stained with DMACA was a result of the accumulation of PA intermediates in the cytoplasm of endothelial cells. Fluorescent marker dyes were used to show that tds4 endothelial cells had multiple small vacuoles, instead of a large central vacuole as observed in the wild types (WT). These results show that in addition to its established role in the formation of anthocyanin, LDOX is also part of the PA biosynthesis pathway.     

Decreased anthocyanidin reductase expression strongly decreases silver birch (Betula pendula) growth and alters accumulation of phenolics

Phenolics, formed via a complex phenylpropanoid pathway, are important defensive agents in plants and are strongly affected by nitrogen (N) fertilization. Proanthocyanidins (PAs) are one possible endpoint of the phenylpropanoid pathway, and anthocyanidin reductase (ANR) represents a key enzyme in PA biosynthesis. In this study, the expression of silver birch (Betula pendula) anthocyanidin reductase BpANR was inhibited using the RNA interference (RNAi) method, in three consequent BpANR RNAi (ANRi birches) lines. The growth, the metabolites of the phenylpropanoid pathway, and the number of resin glands of the ANRi birches were studied when grown at two N levels. ANRi birches showed decreased growth and reduction in PA content, while the accumulation of total phenolics in both stems and leaves increased. Moreover, ANRi birches produced more resin glands than did wild‐type (WT) birches. The response of ANRi birches to N depletion varied compared with that of WT birches, and in particular, the concentrations of some phenolics in stems increased in WT birches and decreased in ANRi birches. Because the inhibition of PAs biosynthesis via ANR seriously affected birch growth and resulted in accumulation of the precursors, the native level of PAs in plant tissues is assumed to be the prerequisite for normal plant growth. This draws attention to the real plant developmental importance of PAs in plant tissues.     

Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance

Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.). Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance.     

Biosynthesis of flavan 3-ols by leucoanthocyanidin 4-reductases and anthocyanidin reductases in leaves of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crops.

Catechin and epicatechin biosyntheses were studied of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crop leaves, since these monomers and the derived proanthocyanidins are important disease resistance factors. Grape and apple leucoanthocyanidin 4-reductase (LAR; EC 1.17.1.3) enzymes were characterized on basis of plant and recombinant enzymes. In case of grape, two LAR cDNAs were cloned by assembling available EST sequences. Grape and apple leaf anthocyanidin reductase (ANR; EC 1.3.1.77) cDNAs were also obtained and the respective plant and recombinant enzymes were characterized. Despite general low substrate specificity, within the respective flavonoid biosyntheses of grape and apple leaves, both enzyme types deliver differently hydroxylated catechins and epicatechins, due to substrate availability in vivo. Furthermore, for LAR enzymes conversion of 3-deoxyleucocyanidin was shown. Beside relevance for plant protection, this restricts the number of possible reaction mechanisms of LAR. ANR enzyme activity was demonstrated for a number of other crop plants and its correlation with (-)-epicatechin and obvious competition with UDP-glycosyl:flavonoid-3-O-glycosyltransferases was considered.     

Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana

Anthocyanidin reductase (ANR), encoded by the BANYULS gene, is a newly discovered enzyme of the flavonoid pathway involved in the biosynthesis of condensed tannins. ANR functions immediately downstream of anthocyanidin synthase to convert anthocyanidins into the corresponding 2,3-cis-flavan-3-ols. We report the biochemical properties of ANRs from the model legume Medicago truncatula (MtANR) and the model crucifer Arabidopsis thaliana (AtANR). Both enzymes have high temperature optima. MtANR uses both NADPH and NADH as reductant with slight preference for NADPH over NADH. In contrast, AtANR only uses NADPH and exhibits positive cooperativity for the co-substrate. MtANR shows preference for potential anthocyanidin substrates in the order cyanidin>pelargonidin>delphinidin, with typical Michaelis-Menten kinetics for each substrate. In contrast, AtANR exhibits the reverse preference, with substrate inhibition at high concentrations of cyanidin and pelargonidin. (+)-Catechin and (+/-)-dihydroquercetin inhibit AtANR but not MtANR, whereas quercetin inhibits both enzymes. Possible catalytic reaction sequences for ANRs are discussed.     

Proanthocyanidin biosynthesis in plants. Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA

Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and thus is the first committed step in PA biosynthesis. The enzyme was purified to near homogeneity from PA-rich leaves of the legume Desmodium uncinatum (Jacq.) DC, partially sequenced and the corresponding cDNA cloned. The identity of the enzyme was confirmed by expressing active recombinant LAR in Escherichia coli and in tobacco and white clover. The enzyme is a monomer of 43 kDa (382 amino acids) and is most active synthesizing catechin (specific activity of approximately 10 micromol min+1 mg of protein+1) but also synthesizes afzelechin and gallocatechin. LAR is most closely related to the isoflavone reductase group of plant enzymes that are part of the Reductase-Epimerase-Dehydrogenase (RED) family of proteins. Unlike all other plant isoflavone reductase homologues that are about 320 amino acids long, LAR has an additional 65-amino acid C-terminal extension whose function is not known. Curiously, although Arabidopsis makes PA, there is no obvious LAR orthologue in the Arabidopsis genome. This may be because Arabidopsis seems to produce only an epicatechin, rather than a dual catechin/epicatechin-based PA similar to many other plants.     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.