Anthocyanidin Reductase Genes Research Articles

Genome-Wide Identification and Analysis of Anthocyanidin Reductase Gene Family in Lychee (Litchi chinensis Sonn.).

Anthocyanidin reductase (ANR) is a key enzyme regulating anthocyanin synthesis and accumulation in plants. Here, lychee ANR genes were globally identified, their sequence and phylogenetic characteristics were analyzed, and their spatiotemporal expression patterns were characterized. A total of 51 ANR family members were identified in the lychee genome. The length of the encoded amino acid residues ranged from 87 aa to 289 aa, the molecular weight ranged from 9.49 KD to 32.40 KD, and the isoelectric point (pI) ranged from 4.83 to 9.33. Most of the members were acidic proteins. Most members of the LcANR family were located in the cytoplasm. The 51 LcANR family members were unevenly distributed in 11 chromosomes, and their exons and motif conserved structures were significantly different from each other. Promoters in over 90% of LcANR members contained anaerobically induced response elements, and 88% contained photoresponsive elements. Most LcANR family members had low expression in nine lychee tissues and organs (root, young leaf, bud, female flower, male flower, pericarp, pulp, seed, and calli), and some members showed tissue-specific expression patterns. The expression of one gene, LITCHI029356.m1, decreased with the increase of anthocyanin accumulation in 'Feizixiao' and 'Ziniangxi' pericarp, which was negatively correlated with pericarp coloring. The identified LcANR gene was heterologously expressed in tobacco K326, and the function of the LcANR gene was verified. This study provides a basis for the further study of LcANR function, particularly the role in lychee pericarp coloration.

Simultaneous Application of Several Exogenous dsRNAs for the Regulation of Anthocyanin Biosynthesis in Arabidopsis thaliana.

Plant surface treatment with double-stranded RNAs (dsRNAs) has gained recognition as a promising method for inducing gene silencing and combating plant pathogens. However, the regulation of endogenous plant genes by external dsRNAs has not been sufficiently investigated. Also, the effect of the simultaneous application of multiple gene-specific dsRNAs has not been analyzed. The aim of this study was to exogenously target five genes in Arabidopsis thaliana, namely, three transcription factor genes (AtCPC, AtMybL2, AtANAC032), a calmodulin-binding protein gene (AtCBP60g), and an anthocyanidin reductase gene (AtBAN), which are known as negative regulators of anthocyanin accumulation. Exogenous dsRNAs encoding these genes were applied to the leaf surface of A. thaliana either individually or in mixtures. The mRNA levels of the five targets were analyzed using qRT-PCR, and anthocyanin content was evaluated through HPLC-MS. The results demonstrated significant downregulation of all five target genes by the exogenous dsRNAs, resulting in enhanced expression of chalcone synthase (AtCHS) gene and increased anthocyanin content. The simultaneous foliar application of the five dsRNAs proved to be more efficient in activating anthocyanin accumulation compared to the application of individual dsRNAs. These findings hold considerable importance in plant biotechnology and gene function studies.

Establishment of TRV-mediated Gene Silencing and Application for Elucidating Functions of Anthocyanidin Reductase Gene HcANR in Kenaf (Hibiscus cannabinus L.)

Establishment of TRV-mediated Gene Silencing and Application for Elucidating Functions of Anthocyanidin Reductase Gene HcANR in Kenaf (Hibiscus cannabinus L.)

Interplay between R2R3 MYB-type activators and repressors regulates proanthocyanidin biosynthesis in banana (Musa acuminata).

Proanthocyanidins are oligomeric flavonoids that promote plant disease resistance and benefit human health. Banana is one of the world's most extensively farmed crops and its fruit pulp contain proanthocyanidins. However, the transcriptional regulatory network that fine tunes proanthocyanidin biosynthesis in banana remains poorly understood. We characterised two proanthocyanidin-specific R2R3 MYB activators (MaMYBPA1-MaMYBPA2) and four repressors (MaMYBPR1-MaMYBPR4) to elucidate the mechanisms underlying the transcriptional regulation of proanthocyanidin biosynthesis in banana. Heterologous expression of MaMYBPA1 and MaMYBPA2 partially complemented the Arabidopsis thaliana proanthocyanidin-deficient transparent testa2 mutant. MaMYBPA1 and MaMYBPA2 interacted physically with MaMYCs to transactivate anthocyanin synthase, leucoanthocyanidin reductase, and anthocyanidin reductase genes in vitro and form functional MYB-bHLH-WD Repeat (MBW) complexes with MaTTG1 to transactivate these promoters in vivo. Overexpression of MaMYBPAs alone or with MaMYC in banana fruits induced proanthocyanidin accumulation and transcription of proanthocyanidin biosynthesis-related genes. MaMYBPR repressors are also shown to interact with MaMYCs forming repressing MBW complexes, and diminished proanthocyanidin accumulation. Interestingly overexpression of MaMYBPA induces the expression of MaMYBPR, indicating an agile regulation of proanthocyanidin biosynthesis through the formation of competitive MBW complexes. Our results reveal regulatory modules of R2R3 MYB- that fine tune proanthocyanidin biosynthesis and offer possible targets for genetic manipulation for nutritional improvement of banana.

Molecular characterization of polyphenol oxidase between small and large leaf tea cultivars

Tea is a widely consumed beverage prepared using the fresh leaves of Camellia sinensis L. Tea plants are classified into small- and large-leaf varieties. Polyphenol oxidase (PPO), a crucial enzyme in tea manufacturing, catalyzes essential phenolic metabolites into different derivatives. To compare the molecular characteristics of CsPPO between cultivars, we cloned the full-length sequence of CsPPO cDNA from four representative tea cultivars in Taiwan. Amino acid sequence alignment analyses indicated that CsPPO is highly conserved. PPO exhibited similar enzymatic activity in different tea cultivars. Quantitative real-time polymerase chain reaction revealed no significant differences in the CsPPO transcript level between the small- and large-leaf varieties. However, tea harvested in summer and from low-altitude areas had a higher CsPPO transcript level than that harvested in winter and from high-altitude areas. Regulation of CsPPO by temperature was more significant in the small-leaf variety than in the large-leaf variety. The content of flavonoids and the expression of chalcone synthase, anthocyanidin synthase, and anthocyanidin reductase genes involved in the tea flavonoid biosynthesis pathway were higher in the large-leaf than in the small-leaf varieties, suggesting that the large-leaf variety had a higher antioxidative capacity than did the small-leaf variety. Our study compared the molecular properties of CsPPO between two tea varieties and clarified the physiological role of PPO in tea.

Gb_ANR-47 Enhances the Resistance of Gossypium barbadense to Fusarium oxysporum f. sp. vasinfectum (FOV) by Regulating the Content of Proanthocyanidins.

Anthocyanidin reductase (ANR) is an important regulator of flavonoid metabolism, and proanthocyanidins, the secondary metabolites of flavonoids, play an important role in the response of plants to pathogenic stress. Therefore, in this study, the expression analysis of the ANR gene family of Gossypium barbadense after inoculation with Fusarium oxysporum f. sp. vasinfectum (FOV) was performed at different time points. It was found that Gb_ANR-47 showed significant differences in the disease-resistant cultivar 06-146 and the susceptible cultivar Xinhai 14, as well as in the highest root expression. It was found that the expression of Gb_ANR-47 in the resistant cultivar was significantly higher than that in the susceptible cultivar by MeJA and SA, and different amounts of methyl jasmonate (MeJA) and salicylic acid (SA) response elements were found in the promoter region of Gb_ANR-47. After silencing GbANR-47 in 06-146 material by VIGS technology, its resistance to FOV decreased significantly. The disease severity index (DSI) was significantly increased, and the anthocyanin content was significantly decreased in silenced plants, compared to controls. Our findings suggest that GbANR-47 is a positive regulator of FOV resistance in Gossypium barbadense. The research results provide an important theoretical basis for in-depth analysis of the molecular mechanism of GbANR-47 and improving the anti-FOV of Gossypium barbadense.

Fine Mapping of the “black” Peel Color in Pomegranate (Punica granatum L.) Strongly Suggests That a Mutation in the Anthocyanidin Reductase (ANR) Gene Is Responsible for the Trait

Anthocyanins are important dietary and health-promoting substances present in high quantities in the peel and arils of the pomegranate (Punica granatum L.) fruit. Yet, there is a high variation in the content of anthocyanin among different pomegranate varieties. The ‘Black’ pomegranate variety (P.G.127-28) found in Israel contains exceptionally high levels of anthocyanins in its fruit peel which can reach up to two orders of magnitude higher content as compared to that of other pomegranate varieties’ peel anthocyanins. Biochemical analysis reveals that delphinidin is highly abundant in the peel of ‘Black’ variety. The pattern of anthocyanin accumulation in the fruit peel during fruit development of ‘Black’ variety differs from that of other pomegranates. High anthocyanin levels are maintained during all developmental stages. Moreover, the accumulation of anthocyanin in the fruit peel of ‘Black’ variety is not dependent on light. Genetic analysis of an F2 population segregating for the “black” phenotype reveals that it is determined by a single recessive gene. Genetic mapping of the F2 population using single nucleotide polymorphism (SNP) markers identified few markers tightly linked to the “black” phenotype. Recombination analysis of the F2 population and F3 populations narrowed the “black” trait to an area of 178.5 kb on the draft genome sequence of pomegranate cv. ‘Dabenzi.’ A putative anthocyanidin reductase (ANR) gene is located in this area. Only pomegranate varieties displaying the “black” trait carry a base pair deletion toward the end of the gene, causing a frame shift resulting in a shorter protein. We propose that this mutation in the ANR gene is responsible for the different anthocyanin composition and high anthocyanin levels of the “black” trait in pomegranate.

Characterization, expression and function analysis of anthocyanidin reductase gene from Vitis vinifera L. cv. Cabernet Sauvignon under UV-C irradiation

Flavonoids are important polyphenols, and widely distributed in plants. Anthocyanidin reductase (ANR), a precursor enzyme in (-)- epicatechin synthesis, could play an important role in anthocyanins biosynthesis. In the present study, the ANR gene was cloned from Vitis vinifera L. cv. Cabernet Sauvignon. Compared with VvANR (GenBank Accession No. NM_001280956.1), they differed in only 4 bp and shared similarity of 99.6%. The ANR protein belongs to the cl33424 Superfamily. In addition, many light-responsive elements and abiotic stress-related elements (ABRE, MYB, WRKY, etc.) were found in the promoter region of the ANR gene. STRING analysis predicted that VvMYBPA1 and VvMYBPAR may be involved in regulating the expression of the ANR gene. Different UV-C irradiation times were used for Cabernet Sauvignon berries during grape berry development, and the comparison analysis showed that UV-C irradiation improved significantly the accumulation of catechin and epicatechin during 20–50 days after full bloom, especially, under UV-C irradiation for 5 min. Furthermore, UV-C irradiation for 5 min had a greater effect on the expression of the ANR gene, the accumulation of catechin and epicatechin, and the enzymatic activity of ANR than UV-C irradiation for 10 min. Through the virus-induced gene silencing system, the ANR-silenced grape leaves were chlorotic, in which the contents of catechin and epicatechin were both significantly lower than those in the control, showing a very significant difference, respectively. In summary, our results demonstrated that the ANR gene played a crucial role in the accumulation of epicatechin in grapes, and its expression was upregulated by UV-C irradiation.

MBW complexes impinge on anthocyanidin reductase gene regulation for proanthocyanidin biosynthesis in persimmon fruit

MBW protein complexes containing MYB, bHLH and WD40 repeat factors are known transcriptional regulators of secondary metabolites production such as proanthocyanidins and anthocyanins, and developmental processes such as trichome formation in many plant species. DkMYB2 and DkMYB4 (MYB-type), DkMYC1 (bHLH-type) and DkWDR1 (WD40-type) factors have been proposed by different authors to take part of persimmon MBW complexes for proanthocyanidin accumulation in immature fruit, leading to its characteristic astringent flavour with important agronomical and ecological effects. We have confirmed the nuclear localization of these proteins and their mutual physical interaction by bimolecular fluorescence complementation analysis. In addition, transient expression of DkMYB2, DkMYB4 and DkMYC1 cooperatively increase the expression of a persimmon anthocyanidin reductase gene (ANR), involved in the biosynthesis of cis-flavan-3-ols, the structural units of proanthocyanidin compounds. Collectively, these data support the presence of MBW complexes in persimmon fruit and suggest their coordinated participation in ANR regulation for proanthocyanidin production.

Dissection of Chemical Composition and Associated Gene Expression in the Pigment-Deficient Tea Cultivar 'Xiaoxueya' Reveals an Albino Phenotype and Metabolite Formation.

The tea cultivar ‘Xiaoxueya’, a temperature-sensitive albino mutant, is a rare tea germplasm because of its highly enriched amino acid content and brisk flavour. In comparison with green leaf tissues of ‘Xiaoxueya’, albino leaves show significant deficiency in chlorophylls and carotenoids and severely disrupted chloroplasts. Furthermore, the accumulation of quality-related secondary metabolites is altered in ‘Xiaoxueya’ albino leaf, with significantly increased contents of total amino acids, theanine, and glutamic acid and significantly decreased contents of alkaloids, catechins, and polyphenols. To uncover the molecular mechanisms underlying albinism and quality-related constituent variation in ‘Xiaoxueya’ leaves, expression profiles of pivotal genes involved in the biosynthetic pathways of pigments, caffeine, theanine, and catechins were investigated by quantitative real-time PCR technology. The results revealed that suppressed expression of the chloroplast-localized 1-deoxy-D-xylulose-5-phosphate synthase genes DXS1 and DXS2 involved in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and protochlorophyllide oxidoreductase genes POR1 and POR2 involved in the chlorophyll biosynthetic pathway is responsible for the pigment deficiency in ‘Xiaoxueya’ albino leaf. Additionally, the low expression of the tea caffeine synthase gene (TCS) involved in caffeine biosynthesis and the chalcone synthase genes CHS1, CHS2, and CHS3, the chalcone isomerase gene CHI, the flavonoid 3’,5’-hydroxylase genes F3’5’H1 and F3’5’H2, and the anthocyanidin reductase genes ANR1 and ANR2 involved in the flavonoid pathway is related to the reduction in alkaloid and catechin levels in ‘Xiaoxueya’ albino leaves.

Identification of leucoanthocyanidin reductase and anthocyanidin reductase genes involved in proanthocyanidin biosynthesis in Malus crabapple plants

Proanthocyanidins (PAs) from plants are a nutritionally valuable component of the human diet and play important roles in defense against pests and diseases. PAs are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. The enzymes leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) are involved in PA biosynthesis. The PA biosynthetic pathway has been characterized in several plant species, but the relationship between its expression and PA accumulation in Malus crabapple remains unclear. Here, we cloned the LAR genes MrLAR1, 2, and the ANR genes MrANR1, 2, from the red leaved Malus crabapple cultivar ‘Royalty’. The contents of PAs and the expression levels of the LAR and ANR genes were investigated in different organs of the two crabapple cultivars. The transcript levels of two LAR genes and two ANR genes correlated with the contents of the catechin and epicatechin, which are proanthocyanidin precursors. Over-expression of the MrLAR1, 2 and MrANR1, 2 in tobacco (Nicotiana tabacum) promoted the accumulation of PAs, while transient silencing of their expression in crabapple resulted in reduced PA levels. In addition, a negative correlation between quercetin, anthocyanin, and PA biosynthesis was also found during crabapple leaf and fruit peel development. We also found that MrLAR1 and 2 may contribute to epicatechin biosynthesis. In summary, the LAR and ANR genes are critical factors in PA biosynthesis, and there is competition between the quercetin, anthocyanin, and PA biosynthetic pathways during leaf and fruit peel development in Malus crabapple.

Influence of light on gene expression of key synthesis enzyme genes FtANR and FtLAR about proanthocyanidin in seeds of homologous plant of food and medicine Fagopyrum tataricum

Tartary buckwheat Fagopyrum tataricum is an important medicinal and functional herb due to its rich content of flavonoids in the seeds. F.tataricum exhibited good functions for free radicals scavenging， anti-oxidation， anti-aging activities. Although much genetic knowledge of the synthesis， regulation， accumulation of rutin， the genetic basis of proanthocyanidins(PAs) in tartary buckwheat and their related gene expression changes under different lights(blue， red， far red， ultraviolet light) remain largely unexplored. In this study， we cloned one anthocyanidin reductase gene(ANR) and two leucocyanidin reductase gene(LAR) named FtANR，FtLAR1，FtLAR3 involved in formation of(+)-catechin and(-)-epicatechin precusor proanthocyanidin by digging out F. tataricum seed transcriptome data. The expression data showed that the opposite influence of red light on these gene transcript level compared to others lights. The expression levels of FtANR and FtLAR1 decreased and FtLAR3 appeared increment after exposed in the red light， while the expression levels of those genes appeared opposite result after exposed in the blue and far red light.

Cloning, expression and molecular modeling of the anthocyanidin reductase (FaANR) gene during strawberry fruit development

Introduction - Proanthocyanidins (PAs) are a group of polyphenolic secondary metabolites synthesized in plants via flavonoid pathway. Strawberry (Fragaria x ananassa Duch.) is a rich source of flavonoids and proanthocyanins, which are known to have multiple health benefits. The anthocyanidin reductase (ANR) is an interesting gene to study within the flavonoid biosynthesis pathway, since it diverts the anthocyanin pathway to flavonol synthesis. Materials and methods - The present study describes cloning, semi-quantitative expression analysis and molecular modeling of the strawberry (cv. Sweet Charlie) anthocyanidin reductase (FaANR) gene during the progressive stages of fruit development. Results and discussion - The FaANR gene was 1,020 bp long with an open reading frame encoding a protein of 308 amino acid residues. The estimated molecular mass and isoelectric point of the protein were 32.89 kD and 5.54, respectively. The expression of FaANR was only seen during early stages of fruit development indicating its early involvement in PA accumulation, well before ripening onset. Analysis of the FaANR sequence showed 98% similarity to ANR from diploid strawberry Fragaria vesca. The cladistic analysis indicated that the FaANR was phylogenetically similar to Pyrus and Prunus ANR genes. Protein modeling suggested protein-ligand interactions at active site with NADP binding as the plausible mechanism of action. Conclusion - This is the first report on cloning, expression study and in silico modeling of an anthocyanidin reductase of strawberry. The conditions of in vivo modulation of ANR expression open applied perspectives for commercial production of PAs in strawberry and other berries.

Expression of Key Structural Genes of the Phenylpropanoid Pathway Associated with Catechin Epimerization in Tea Cultivars.

Catechin epimerization is an important factor affecting tea catechin compositions and thereby tea quality. However, a lack of tea germplasms with high non-epicatechins limits relative research. Here, a tea cultivar Y510 with high non-epicatechins was firstly reported and used for catechin and RNA sequencing (RNA-Seq) analysis. Results showed that the (-)-gallocatechin gallate and (+)-catechin (C) contents in Y510 were at least 136 and 6 times higher than those in Fudingdabaicha and 0306I, but the epicatechins (-)-epigallocatechin and (-)-epicatechin (EC) were significantly lower. Eleven unigenes potentially involved in catechin epimerization were identified by RNA-Seq analysis. Based on a combination of catechin and gene expression analysis, it was hypothesized that two anthocyanidin reductase genes (CsANR1, CsANR2) and an anthocyanidin synthase gene (CsANS) are the key genes affecting catechin epimerization in tea. Non-epicatechin formations were hypothesized to be mainly influenced by the expression ratio of CsANR2 to CsANR1 and the expression of CsANS. Overexpression of CsANS in an Arabidopsis mutant tds4-2 led to a significant increase of EC accumulation in seeds, revealing CsANS is important for catechin epimerization. These results shed new light on breeding tea cultivars with special catechin compositions.

Functional characterization of an anthocyanidin reductase gene from the fibers of upland cotton (Gossypium hirsutum).

Metabolic profiling, gene cloning, enzymatic analysis, ectopic expression, and gene silencing experiments demonstrate that the anthocyanidin reductase (ANR) pathway is involved in the biosynthesis of proanthocyanidins in upland cotton. Proanthocyanidins (PAs) are oligomeric or polymeric flavan-3-ols, however, the biosynthetic pathway of PAs in cotton remains to be elucidated. Here, we report on an anthocyanidin reductase (ANR) gene from cotton fibers and the ANR pathway of PAs. Phytochemical analysis demonstrated that leaves, stems, roots, and early developing fibers produced PAs and their monomers, including (-)-epicatechin, (-)-catechin, (-)-epigallocatechin, and (-)-gallocatechin. Crude PA extractions from different tissues were boiled in Butanol:HCl. Cyanidin, delphinidin, and pelargonidin were produced, indicating that cotton PAs include diverse extension unit structures. An ANR cDNA homolog (named GhANR1) was cloned from developing fibers. The open reading frame, composed of 1,011 bp nucleotides, was expressed in E. coli to obtain a recombinant protein. In the presence of NADPH, the recombinant enzyme catalyzed cyanidin, delphinidin, and pelargonidin to (-)-epicatechin and (-)-catechin, (-)-epigallocatechin and (-)-gallocatechin, and (-)-epiafzelechin and (-)-afzelechin, respectively. The ectopic expression of GhANR11 in an Arabidopsis ban mutant allowed for the reconstruction of the ANR pathway and PA biosynthesis in the seed coat. Virus-induced gene silencing (VIGS) of GhANR11 led to a significant increase in anthocyanins and a decrease in the PAs, (-)-epicatechin, and (-)-catechin in the stems and leaves of VIGS-infected plants. Taken together, these data demonstrate that the ANR pathway contributes to the biosynthesis of flavan-3-ols and PAs in cotton.

Characterization of proanthocyanin-related leucoanthocyanidin reductase and anthocyanidin reductase genes in Lycium ruthenicum Murr.

Characterization of proanthocyanin-related leucoanthocyanidin reductase and anthocyanidin reductase genes in Lycium ruthenicum Murr.

Molecular cloning and functional characterization of the anthocyanidin reductase gene from Vitis bellula.

Anthocyanidin reductase (ANR) is an NADPH-/NADH-dependent enzyme that transfers two hydrides to anthocyanidins to produce three types of isomeric flavan-3-ols. This reductase forms the ANR pathway toward the biosynthesis of proanthocyanidins (PAs, which are also called condensed tannins). Here, we report cloning and functional characterization of an ANR (called VbANR) homolog from the leaves of Vitis bellula, a newly developed grape crop in southern China. The open reading frame (ORF) of VbANR is 1,017 bp in length and encodes 339 amino acids. A phylogenetic analysis and an alignment using 17 sequences revealed that VbANR is approximately 99.9 % identical to the ANR homolog from Vitis vinifera. The VbANR ORF is fused to the Trx gene containing a His-tag in the pET32a(+) vector to obtain a pET32a(+)-VbANR construct for expressing the recombinant VbANR. In vitro enzyme assays show that VbANR converts cyanidin, delphinidin, and pelargonidin to their corresponding flavan-3-ols. Enzymatic products include 2S,3R-trans- and 2R,3R-cis-flavan-3-ols isomers, such as (-)-catechin and (-)-epicatechin. In addition, the third compound that is observed from the enzymatic products is most likely a 2S,3S-cis-flavan-3-ol. To analyze the kinetics and optimize pH and temperature values, a UV spectrometry method was developed to quantify the concentrations of total enzymatic products. The optimum pH and temperature values are 4.0 and 40 °C, respectively. The K m , K cat, V max, and K cat/K m values for pelargonidin and delphinidin were similar. In comparison, VbANR exhibits a slightly lower affinity to cyanidin. VbANR uses both NADPH and NADH but prefers to employ NADPH. GFP fusion and confocal microscopy analyses revealed the cytosolic localization of VbANR. The overexpression of VbANR in ban mutants reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate that VbANR forms the ANR pathway, leading to the formation of three types of isomeric flavan-3-ols and PAs in the leaves of V. bellula.

Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin.

Three genes encoding anthocyanidin reductase (ANR) in apple (Malus×domestica Borkh.), designated MdANR1, MdANR2a, and MdANR2b, have been cloned and characterized. MdANR1 shows 91% identity in coding DNA sequences with MdANR2a and MdANR2b, while MdANR2a and MdANR2b are allelic and share 99% nucleotide sequence identity in the coding region. MdANR1 and MdANR2 genes are located on linkage groups 10 and 5, respectively. Expression levels of both MdANR1 and MdANR2 genes are generally higher in yellow-skinned cv. Golden Delicious than in red-skinned cv. Red Delicious. Transcript accumulation of MdANR1 and MdANR2 genes in fruits gradually decreased throughout fruit development. Ectopic expression of apple MdANR genes in tobacco positively and negatively regulates the biosynthesis of proanthocyanidins (PAs) and anthocyanin, respectively, resulting in white, pale pink-coloured, and white/red variegated flowers. The accumulation of anthocyanin is significantly reduced in all tobacco transgenic flowers, while catechin and epicatechin contents in transgenic flowers are significantly higher than those in flowers of wild-type plants. The inhibition of anthocyanin synthesis in tobacco transgenic flowers overexpressing MdANR genes is probably attributed to down-regulation of CHALCONE ISOMERASE (CHI) and DIHYDROFLAVONOL-4-REDUCTASE (DFR) genes involved in the anthocyanin pathway. Interestingly, several transgenic lines show no detectable transcripts of the gene encoding leucoanthocyanidin reductase (LAR) in flowers, but accumulate higher levels of catechin in flowers of transgenic plants than those of wild-type plants. This finding suggests that the ANR gene may be capable of generating catechin via an alternative route, although this mechanism is yet to be further elucidated.

Identification of Two Anthocyanidin Reductase Genes and Three Red-Brown Soybean Accessions with Reduced Anthocyanidin Reductase 1 mRNA, Activity, and Seed Coat Proanthocyanidin Amounts

Anthocyanidin reductase (ANR; EC 1.3.1.77) catalyzes a key step in the biosynthesis of proanthocyanidins (PAs; also known as condensed tannins), flavonoid metabolites responsible for the brown pigmentation of seeds. Here, two ANR genes (ANR1 and ANR2) from the seed coat of brown soybean (Glycine max (L.) Merr.) have been isolated and their enzymatic function confirmed for the reduction of cyanidin to (-)-epicatechin in vitro. Biochemical and genetic comparisons of soybean lines differing in seed coat color revealed three red-brown lines to exhibit major reductions in the amounts of soluble PAs in the seed coat compared to brown soybean lines. Two spontaneous mutants with red-brown grain color had reduced ANR1 gene expression in the seed coat, and an EMS-mutagenized red-brown mutant had nonsynonymous substitutions that resulted in slightly reduced ANR1 activity in vitro. These results suggest that defects in the ANR1 gene can be associated with red-brown soybean grain color. These results suggest that suppressing ANR1 gene expression or activity may be a rational approach toward engineering seed coat color to enable the visual identification of genetically modified soybean grains.