Accumulation Of Proanthocyanidins Research Articles

Anthocyanidin Reductase Gene Expression and Accumulation of Flavan-3-ols in Grape Berry

Proanthocyanidins, which are polymers and oligomers of flavan-3-ol units, are important components of grape for red winemaking and accumulate in the berry skins and seeds. Major flavan-3-ol units of grape proanthocyanidins are (-)-epicatechin and (-)-epigallocatechin. It was recently reported that (-)-epicatechin and (-)-epigallocatechin were biosynthesized from corresponding anthocyanidins by anthocyanidin reductase (ANR). In the current study, we obtained a genomic sequence of the ANR gene of <i>Vitis vinifera</i> cv. Cabernet Sauvignon (<i>VvANR</i>). The deduced amino acid sequence of <i>VvANR</i> conserved the characteristic sequence of ANR of other plants. Southern blot analysis showed that grape ANR is probably encoded by a single copy gene in its haploid genome. Real-time quantitative-PCR using berry skins and seeds showed that the mRNA of <i>VvANR</i> accumulates at the early stage of berry development and then decreases toward the ripening stage. Our analysis and other reports show that proanthocyanidins accumulate in the berry skins and seeds before veraison, especially during the early stage of development, and then decrease in concentration during ripening. Thus, the change of mRNA accumulation substantially coincides with the change of proanthocyanidin accumulation in the berry skins and seeds. These results indicate that at least a part of (-)-epicatechin and (-)-epigallocatechin biosynthesis is probably controlled by the transcription of <i>VvANR</i>.

Metabolic engineering of proanthocyanidins by ectopic expression of transcription factors in Arabidopsis thaliana

Genetic transformation of Arabidopsis thaliana with the Arabidopsis TT2 MYB transcription factor resulted in ectopic expression of the BANYULS gene, encoding anthocyanidin reductase, AHA10 encoding a P-type proton-pump and TT12 encoding a transporter involved in proanthocyanidin biosynthesis. When coupled with constitutive expression of PAP1, a positive regulator of anthocyanin biosynthesis, TT2 expression in Arabidopsis led to accumulation of proanthocyanidins, but only in a subset of cells in which the BANYULS promoter is naturally expressed. Ectopic expression of the maize Lc MYC transcription factor weakly induced AHA10 but did not induce BANYULS, TT12 or accumulation of proanthocyanidins. However, high-level combined expression of TT2, PAP1 and Lc resulted in proanthocyanidin synthesis throughout young leaves and cotyledons, followed by death of the plants 1 to 2 weeks after germination. We discuss these results in relation to engineering proanthocyanidins to improve the quality of food and forage plants.

Suppression of the biosynthesis of proanthocyanidin in Arabidopsis by a chimeric PAP1 repressor

Flavonoids are secondary metabolites that are specific to higher plants. PAP1, a member of the family of MYB domain transcription factors in Arabidopsis, is a positive regulator of the biosynthesis of anthocyanin. We show here that a chimeric PAP1 repressor, in which the EAR-motif repression domain from SUPERMAN was fused to PAP1, suppressed the expression of four flavonoid biosynthetic genes, namely CHS, DFR, LDOX, and BAN, in siliques, and inhibited the accumulation of proanthocyanidin, even in the presence of an endogenous positive regulator, such as TT2. This suppression resulted in the formation of light yellow seeds in 35S::PAP1SRDX transgenic plants. Our results indicate that PAP1 has the potential ability to regulate the biosynthesis not only of anthocyanin but also of proanthocyanidin. Our gene silencing system, using chimeric repressors, appears to be a useful tool for the manipulation of the biosynthesis of secondary metabolites in plants.

The Role of the Barley Testa Layer and its Flavonoid Content in Resistance to Fusarium Infections

Developing testa layers of the barley proanthocyanidin-free mutants ant 13-152, anf17-148, ant 18-159, ant19-109, ant22-1508, ant25-264, ant26-485, ant27-489, ant28-484 and ant29-2110 and their mother varieties, were analysed for accumulation of proanthocyanidins and their flavonoid precursors. In vitro infection of developing barley caryopses of wild type and mutants with Fusarium poae, F. culmorum and F. graminearum revealed all mutants except ant18-159 to be more sensitive to Fusarium attack than wildtype. Mutant ant 18-159 showed extreme resistance. Histological investigations of the infection process revealed that the hyphae were unable to penetrate the testa of this mutant. The testa layer of ant 18-159 accumulates small amounts of dihydroquercetin as a result of nonsense mutations in the structural gene for dihydroflavonol reductase. Authentic dihydroquercetin and an autographic assay proves this flavonoid to be a strong inhibitor of Fusarium growth and macrospore formation. Mutant ant 17-148, which accumulates the flavone chrysoeriol as a consequence of a mutation in the step catalysed by flavanone 3-hydroxylase, demonstrates that this flavone is not an inhibitor of Fusariwn growth in vivo. Comparison of flavonoid standards and monomers and polymers from other plants reveal monomeric flavonoids generally to be potent inhibitors of Fusarium.

TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis.

Flavonoid compounds such as anthocyanins and proanthocyanidins (PAs; so-called condensed tannins) have a multitude of functions in plants. They must be transported from the site of synthesis in the cytosol to their final destination, the vacuoles. Three models have been proposed for sequestering anthocyanins in vacuoles, but the transport machinery for PAs is poorly understood. Novel Arabidopsis mutants, transparent testa 19 (tt19), which were induced by ion beam irradiation, showed a great reduction of anthocyanin pigments in the vegetative parts as well as brown pigments in the seed coat. The TT19 gene was isolated by chromosome walking and a candidate gene approach, and was shown to be a member of the Arabidopsis glutathione S-transferase (GST) gene family. Heterologous expression of a putative ortholog, petunia anthocyanin 9 (AN9), in tt19 complemented the anthocyanin accumulation but not the brown pigmentation in the seed coat. This suggests that the TT19 gene is required for vacuolar uptake of anthocyanins into vacuoles, but that it has also a function different from that of AN9. The depositional pattern of PA precursors in the mutant was different from that in the wild type. These results indicate that TT19 participates in the PA pathway as well as the anthocyanin pathway of Arabidopsis. As involvement of GST in the PA pathway was previously considered unlikely, the function of TT19 in the PA pathway is also discussed in the context of the putative transporter for PA precursors.

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.

Analysis of tannins in seeds and skins of Shiraz grapes throughout berry development

The flavan-3-ol and proanthocyanidin composition of both seeds and skin of Vitis vinifera L. cv. Shiraz grapes was determined by reversed-phase HPLC after acetone extraction and acid-catalysis in the presence of excess phloroglucinol. Samples were taken at weekly intervals from fruit-set until commercial harvest. The main period of proanthocyanidin accumulation in grape seeds occurred immediately after fruit-set with maximum levels observed around veraison. Over two seasons there was variation in both the timing and content of proanthocyanidins in seeds. In skin, proanthocyanidin accumulation occurred from fruit set until 1–2 weeks after veraison. Proanthocyanidin subunit composition was different in seeds and skin and changed during berry development but the mean degree of polymerisation of the tannin polymers in skins was higher than in the seeds at all stages of berry development. Proanthocyanidin levels in both seeds and skin decreased between veraison and harvest. Additional proanthocyanidin subunits were released when the residues remaining after acetone extraction were subjected to direct acid-catalysis in the presence of phloroglucinol. In the seeds, these accounted for much of the post-veraison decrease, but not in grape skin. At harvest, 75% of extractable berry proanthocyanidin was in the seeds. Accumulation of proanthocyanidins in the seeds appears to be independent of that in the skins, but in both tissues synthesis occurs early in berry development and maximum levels are reached around veraison.

The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed.

In Arabidopsis, proanthocyanidins specifically accumulate in the endothelium during early seed development. At least three TRANSPARENT TESTA (TT) genes, TT2, TT8, and TTG1, are necessary for the normal expression of several flavonoid structural genes in immature seed, such as DIHYDROFLAVONOL-4-REDUCTASE and BANYULS (BAN). TT8 and TTG1 were characterized recently and found to code for a basic helix-loop-helix domain transcription factor and a WD-repeat-containing protein, respectively. Here the molecular cloning of the TT2 gene was achieved by T-DNA tagging. TT2 encoded an R2R3 MYB domain protein with high similarity to the rice OsMYB3 protein and the maize COLORLESS1 factor. A TT2-green fluorescent protein fusion protein was located mostly in the nucleus, in agreement with the regulatory function of the native TT2 protein. TT2 expression was restricted to the seed during early embryogenesis, consistent with BAN expression and the proanthocyanidin deposition profile. Finally, in gain-of-function experiments, TT2 was able to induce ectopic expression of BAN in young seedlings and roots in the presence of a functional TT8 protein. Therefore, our results strongly suggest that stringent spatial and temporal BAN expression, and thus proanthocyanidin accumulation, are determined at least partially by TT2.

The Arabidopsis TT2 Gene Encodes an R2R3 MYB Domain Protein That Acts as a Key Determinant for Proanthocyanidin Accumulation in Developing Seed

The Arabidopsis TT2 Gene Encodes an R2R3 MYB Domain Protein That Acts as a Key Determinant for Proanthocyanidin Accumulation in Developing Seed

The Arabidopsis TT2 Gene Encodes and R2R3 MYB Domain Protein That Acts as a Key Determinant for Proanthocyanidin Accumulation in Developing Seed

The Arabidopsis TT2 Gene Encodes and R2R3 MYB Domain Protein That Acts as a Key Determinant for Proanthocyanidin Accumulation in Developing Seed

The maize transcription factor Sn alters proanthocyanidin synthesis in transgenic Lotus corniculatus plants

Lotus corniculatus L. plants were transformed with Agrobacterium rhizogenes binary vector carrying the maize Sn regulatory gene driven by the 35S promoter. These plants showed modifications in the pattern of accumulation of proanthocyanidin (PA). All the transformed plants but one showed an increase in PA content in the root relative to control untransformed and control gus gene transformed plants (C). With respect to the PAaccumulation in leaves, Sn transgenic plants were grouped in two classes: suppressed (S), that showed a consistent reduction of foliar PAcontent, and unsuppressed (U) that did not differ significantly from controls. Dihydroflavanol reductase (DFR) and leucocyanidin reductase (LAR) enzyme activities in S and U plant leaves mirrored the changes seen with foliar PA accumulation. LAR activity in the roots was consistent with the root PA levels. Mature Sn mRNA accumulated in the leaves of U plants, but not in leaves of S plants; however, leaves of both S and U plants were able to initiate Sn transcription. All Sn-transformed plants accumulated Sn message in root tissue. A possible negative interaction of Sn and an unidentified homologous endogene is proposed for explaining the behaviour of S plants.

Presence of proanthocyanidins in mutant green Sarracenia indicate blockage in late anthocyanin biosynthesis between leucocyanidin and pseudobase

The insectivorous pitcher plant genus Sarracenia contains several species with mutants which lack red pigment in leaves, flowers and growth point. Mutants and wild-type contain various glycosides of the flavonols quercetin and kaempferol and we hypothesized that the blockage in the mutant occurred between either dihydroflavonols and leucoanthocyanidin or between leucoanthocyanidin and the end product anthocyanin. Acid hydrolysis and acid addition were used to quantify and qualify spectral characteristics of intermediates. Aqueous phase extracts of leaves demonstrated the end product was most likely an anthocyanin because of red color, absorption at 530 nm and miscibility with amyl alcohol. Mutants and wild-type plants in Sarracenia purpurea and S. rubra ssp. jonesii are capable of producing proanthocyanidins while only wild-type contain pseudobase. Sarracenia purpurea mutants had a significantly greater accumulation of proanthocyanidins than wild-type while there was no such significant difference in S. rubra ssp. jonesii. These results suggest that the mutant is blocked in a late stage of anthocyanin biosynthesis between leucocyanidin and pseudobase. While mutants may accumulate precursors, additional investigation is needed. Sarracenia may serve as useful plants for research since the highly modified carnivorous and absorptive leaves may be fed suspected intermediates in this last step of anthocyanin synthesis.

Proanthocyanidin synthesis in the forage legume Onobrychis viciifolia. A study of chalcone synthase, dihydroflavonol 4-reductase and leucoanthocyanidin 4-reductase in developing leaves

The timing of proanthocyanidin (PA) biosynthesis in leaves of Onobrychis viciifolia (sainfoin) was examined using nucleotide probes, enzyme assays and immunochemical detection. To achieve this, a chalcone synthase (CHS) cDNA clone was isolated and a PCR-amplified DNA fragment corresponding to part of a dihydroflavonol reductase (DFR) gene was obtained from sainfoin. The high sequence homology of the CHS clone, both at the nucleotide and amino acid levels, with CHS sequences from other species, confirmed the identity of the clone. The identity of the PCR fragment was confirmed by sequence homology to known DFR sequences. The level of DFR mRNA, DFR and leucoanthocyanidin reductase (LAR) enzyme activities, CHS mRNA and CHS protein were at a maximum in young, unexpanded leaves of sainfoin. The levels of DFR mRNA, and DFR and LAR enzyme activity declined rapidly and were absent in older tissues. PA content of leaves declined slightly on a fresh and dry weight basis in the more mature phases of leaf development; however, PA content on a per leaf basis continued to increase throughout development. The apparent anomaly between the activity of critical enzymes (LAR and DFR) and the accumulation of PA indicates that the process of proanthocyanidin biosynthesis is still poorly understood.

Leucocyanidin reductase activity and accumulation of proanthocyanidins in developing legume tissues

Proanthocyanidin (PA) and anthocyanin accumulation and location in developing leaves, flowers, and seeds of the legumes Medicago sativa, Lotus japonicus, Lotus uliginosus, Hedysarum sulfurescens, and Robinia pseudacacia were investigated by quantitative measurements and by histological analysis after staining with 1% vanillin/HCl, butanol/HCl, or 50% HCl. M. sativa leaves and flowers, L. japonicus leaves, and R. pseudacacia flowers do not contain PAs, but seeds of all investigated species contain PAs. Anthocyanins are absent in the seed coats of all five species and in leaves of L. japonicus. PA content generally increases as a function of development in leaves, but declines in flowers. With the exception of H. sulfurescens, flower PAs are synthesized in the parenchyma cells of the standard petal, while anthocyanins are located in the neighboring epidermal cells. Leucocyanidin reductase (LCR) catalyzes the conversion of 2,3‐trans‐3,4‐cis‐leucocyanidin to (+)‐catechin and is the first enzyme in the PA‐specific pathway. LCR activity was only detected in PA‐containing tissues and generally declined during tissue development.

Anthocyanin pathway in rice (Oryza sativa L): identification of a mutant showing dominant inhibition of anthocyanins in leaf and accumulation of proanthocyanidins in pericarp

The present study has surveyed a collection of indica rice (Oryza sativa) lines for tissue-specific anthocyanin pigmentation pattern, which has also been used for a genetically meaningful classification. This classification helped predict probable genotypes of rice lines and, in the process, a leaf blade-specific dominant inhibitor of pigmentation (Ilb) was predicted and its presence later confirmed in two lines. We ascribe most tissue-specific accumulation of anthocyanins to the presence of a different set of Pl alleles. Cyanidin, as a major pigment, and peonidin, as a minor pigment, were detected in purple-pigmented tissues. Further, the floral organ-derived tissues always contained a higher level of anthocyanins and, correspondingly, a relatively increased proportion of peonidin. One line, N22B, with a brown pericarp was identified and shown to accumulate proanthocyanidins, but with no anthocyanins, in the pericarp. We propose that the accumulation of proanthocyanidins is due to a block in the anthocyanin biosynthetic pathway in rice at the anthocyanidin synthase-mediated conversion of leucoanthocyanidin to anthocyanidin.

Expression of Anthocyanins in Callus Cultures of Cranberry (Vaccinium macrocarpon Ait)

ABSTRACTExpression of anthocyanins and other flavonoids in callus cultures established from different parts of cranberry plant was investigated and the effect of explant source on the in vitro product was determined. Callus cultures were initiated from different parts of the plant in a modified Gamborg's medium with 5.37 μM α‐naphthaleneacetic acid, 0.45 μM 2,4‐dichlorophenoxyacetic acid, and 2.32 μM kinetin in the dark at 25°C. Callus cultures accumulated anthocyanins only on exposure to light and maximum concentration was observed by day 12. The cultures had lower levels of anthocyanins and only cyanidin 3‐galactoside, cyanidin 3‐glucoside, and cyanidin 3‐arabinoside were identified in all cultures regardless of source of explant. Proanthocyanidin accumulation in cultures was independent of light, and levels were higher than in mature fruit. Exposure to light induced accumulation of flavonols and enhanced activity of phenylalanine ammonia‐lyase in the cultures.

The formation of flavonoids in cell suspension cultures ofPrunus x yedoensis matsum

Two lines of the red and pale yellow cell suspension cultures, prepared fromPrunus x yedoensis Matsum. callus induced by Murashige and Skoog's (1962) basal medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D, 1.0 mg/l), kinetin (0.1 mg/l) and sucrose (30 g/l), were maintained on Schenk and Hildebrandt medium as modified by Mitchell and Gildow (1975). The red cell suspension culture produced cyanidin 3-monoglucoside, 5, 4′-dihydroxy-7-methoxyisoflavone 4′-glucoside (prunetrin), isoquercitrin, catechin, epicatechin, and procyanidin B-1, B-2, B-3 and B-4, while the pale yellow cells produced only a small amount of catechin and epicatechin as main flavonoids. These flavonoid compounds found in the red cell culture were present also in maturePrunus leaves. Maximum growth and maximum amount of total phenol and proanthocyanidin (procyanidins) were obtained with 0.3 mg/l of both 2,4-D and kinetin. Maximum concentration of anthocyanin was also obtained with 0.3 mg/l 2, 4-D regardless of kinetin concentration. Accumulation of proanthocyanidin was markedly stimulated by low concentrations of phosphate, which reduced growth by about half, and also by high concentrations of inorganic nitrogen. Production of both anthocyanin and proanthocyanidin was reduced by lowered nitrogen levels. Cell growth and production of all phenolics were inhibited when ammonium ion replaced nitrate in the medium.

Variability in accumulation of proanthocyanidins (condensed tannins) in needles of Douglas-fir (Pseudotsuga menziesii) following long-term budworm defoliation

Long-term defoliation by budworms was associated with higher levels of soluble proanthocyanidins in the current year needles of Douglas-fir trees. The proanthocyanidin contents of needles from defoliated Douglas-fir trees were considerably more variable than those levels of undefoliated ones. The increased mean and variability of proanthocyanidin levels following defoliation may have interesting ecological consequences for Douglas-fir and its defoliators.