Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process.     

Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.) fruit

Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3′5′ hydroxylase (F3′5′H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3′ hydroxylation (F3′H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3′5′H to F3′H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3′5′H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.     

Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation

Potassium accumulation is essential for grapevine (Vitis vinifera L.) growth and development, but excessive levels in berries at harvest may reduce wine quality particularly for red wines. In addition to decreasing the free acid levels, potassium also combines with tartaric acid to form largely insoluble potassium bitartrate. This precipitates during winemaking and storage, resulting in an increase in wine pH that is associated with negative impacts on wine colour, flavour, and microbiological stability. For these reasons, a better understanding of potassium transport and accumulation within the vine and berries is important for producing fruit with improved winemaking characteristics. Here two genes encoding KUP/KT/HAK-type potassium transporters that are expressed in grape berries are described. Their function as potassium transporters was demonstrated by complementation of an Escherichia coli mutant. The two transporters are expressed most highly in the berry skin during the first phase of berry development (pre-veraison), with similar patterns in two grapevine varieties. The timing and location of expression of these transporters are consistent with an involvement in potassium accumulation in grape berries.     

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.     

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.     

Treatment of Grape Berries,a Nonclimacteric Fruit with a Synthetic Auxin,Retards Ripening and Alters the Expression of Developmentally Regulated Genes

Treatment of grape (Vitis vinifera L.) berries with the synthetic auxin-like compound benzothiazole-2-oxyacetic acid (BTOA) caused a delay in the onset of ripening of approximately 2 weeks. This was manifested as a retardation of the increases in berry weight, color, deformability, and hexose concentration. BTOA treatment also delayed by 2 weeks the increase in abscisic acid level that normally accompanies ripening and altered the expression of a number of developmentally regulated genes. A putative vacuolar invertase, which is normally expressed from berry set until ripening and turned off after ripening commences, remained expressed throughout development in BTOA-treated grape berries. This elevated expression resulted in increased levels of invertase activity. In contrast, the up-regulation of four other genes normally switched on at the time of ripening was delayed in BTOA-treated fruit. These included chalcone synthase and UDP-glucose-flavonoid 3-O-glucosyl transferase, both of which are involved in anthocyanin synthesis, a chitinase, and a ripening-related gene of an unknown function. These observations support the view that auxins (perhaps in conjunction with abscisic acid) may have a role in the control of grape berry ripening by affecting the expression of genes involved in the ripening process.     

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.