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.     

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.     

Composition and functional analysis of low-molecular-weight glutenin alleles with Aroona near-isogenic lines of bread wheat

Background

The polyphenolic products of the phenylpropanoid pathway, including proanthocyanidins, anthocyanins and flavonols, possess antioxidant properties that may provide health benefits. To investigate the genetic architecture of control of their biosynthesis in apple fruit, various polyphenolic compounds were quantified in progeny from a 'Royal Gala' × 'Braeburn' apple population segregating for antioxidant content, using ultra high performance liquid chromatography of extracts derived from fruit cortex and skin.

Results

Construction of genetic maps for 'Royal Gala' and 'Braeburn' enabled detection of 79 quantitative trait loci (QTL) for content of 17 fruit polyphenolic compounds. Seven QTL clusters were stable across two years of harvest and included QTLs for content of flavanols, flavonols, anthocyanins and hydroxycinnamic acids. Alignment of the parental genetic maps with the apple whole genome sequence in silico enabled screening for co-segregation with the QTLs of a range of candidate genes coding for enzymes in the polyphenolic biosynthetic pathway. This co-location was confirmed by genetic mapping of markers derived from the gene sequences. Leucoanthocyanidin reductase (LAR1) co-located with a QTL cluster for the fruit flavanols catechin, epicatechin, procyanidin dimer and five unknown procyanidin oligomers identified near the top of linkage group (LG) 16, while hydroxy cinnamate/quinate transferase (HCT/HQT) co-located with a QTL for chlorogenic acid concentration mapping near the bottom of LG 17.

Conclusion

We conclude that LAR1 and HCT/HQT are likely to influence the concentration of these compounds in apple fruit and provide useful allele-specific markers for marker assisted selection of trees bearing fruit with healthy attributes.     

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.     

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.     

Genetic characterization of the Ma locus with pH and titratable acidity in apple

Apple fruit flavor is greatly affected by the level of malic acid, which is the major organic acid in mature apple fruit. To understand the genetic and molecular basis of apple fruit acidity, fruit juice pH and/or titratable acidity (TA) were measured in two half-sib populations GMAL 4595 [Royal Gala?×?PI (Plant Introduction) 613988] and GMAL 4590 (Royal Gala?×?PI 613971) of 438 trees in total. The maternal parent Royal Gala is a commercial variety and the paternal parents are two M. sieversii (the progenitor species of domestic apple) elite accessions. The low-acid trait segregates recessively and the overall acidity variations in the two populations were primarily controlled by the Ma (malic acid) locus, a major gene discovered in the 1950s (Nybom in Hereditas 45:332?C350, 1959) and later mapped to linkage group 16 (Maliepaard et al. in Theor Appl Genet 97:60?C73, 1998). The allele Ma has a strong additive effect in increasing fruit acidity and is incompletely dominant over ma. QTL (quantitative trait locus) analyses in GMAL 4595 mapped the major QTL Ma in both Royal Gala and PI 613988, the effects of which explained 17.0?C42.3% of the variation in fruit pH and TA. In addition, two minor QTL, tentatively designated M2 and M3, were also detected for fruit acidity, with M2 on linkage group 6 of Royal Gala and M3 on linkage group 1 of PI 613988. By exploring the genome sequences of apple, eight new simple sequence repeat markers tightly linked to Ma were developed, leading to construction of a fine genetic map of the Ma locus that defines it to a physical region no larger than 150?kb in the Golden Delicious genome.     

Water relations,mineral nutrition,growth, and13C discrimination in two apple cultivars under daily episodes of high root-medium temperature

Although high soil temperatures can occur in apple orchards throughout the world, there is little information on their effect. This investigation was conducted to determine the influence of various durations of root exposure to 34 °C on the growth and physiology of the apple plant. Roots of Royal Gala and McIntosh cultivars were exposed to 34 °C for 0, 8, 16, and 24 hours/day for seven weeks. Royal Gala grown at the 24 hours/day treatment exhibited significant decreases in leaf, shoot, and root growth; chlorophyll concentration of the older leaves; transpiration; discrimination against¹³C in leaves; and an increase in leaf temperature. In McIntosh, root growth and chlorophyll concentration of leaves were not affected. For both cultivars compared to the control treatment, the continuous high temperature treatment resulted in lower levels of P, Mg, and Mn in leaves. Royal Gala at this treatment showed significantly higher values of foliar N and K and lower values of Ca, Fe, and Zn. For McIntosh the levels of Cu and B decreased significantly in this treatment as compared to the control treatment. We conclude that 34 °C in the root-zone does not stress these cultivars unless it persists throughout the day/night cycle.