FtMYB18 acts as a negative regulator of anthocyanin/proanthocyanidin biosynthesis in Tartary buckwheat.

KEY MESSAGE: FtMYB18 plays a role in the repression of anthocyanins and proanthocyanidins accumulation by strongly down-regulating the CHS and DFR genes in Tartary buckwheat, and the C5 motif plays an important role in this process. Anthocyanins and proanthocyanidins (PAs) are important flavonoids in Tartary buckwheat (Fagopyrum tataricum Gaertn.), which provides various vibrant color and stronge abiotic stress resistance. Their synthesis is generally regulated by MYB transcription factors at transcription level. However, the negative regulations of MYB and their effects on flavonol metabolism are poorly understood. A SG4-like MYB subfamily TF, FtMYB18, containing C5 motif was identified from Tartary buckwheat. The expression of FtMYB18 was not only showed a negative correlation with anthocyanins and PAs content but also strongly respond to MeJA and ABA. As far as the transgenic lines with FtMYB18 overexpression, anthocyanins and PAs accumulations were decreased through down-regulating expression levels of NtCHS and NtDFR in tobacco, AtDFR and AtTT12 in Arabidopsis, FtCHS, FtDFR and FtANS in Tartary buckwheat hairy roots, respectively. However, FtMYB18 showed no effect on the FLS gene expression and the metabolites content in flavonol synthesis branch. The further molecular interaction analysis indicated FtMYB18 could mediate the inhibition of anthocyanins and PAs synthesis by forming MBW transcriptional complex with FtTT8 and FtTTG1, or MYB-JAZ complex with FtJAZ1/-3/-4/-7. Importantly, in FtMYB18 mutant lines with C5 motif deletion (FtMYB18-C), both of anthocyanins and PAs accumulations had recovered to the similar level as that in wild type, which was attributed to the weakened MBW complex activity or the deficient molecular interaction between FtMYB18ΔC5 with FtJAZ3/-4. The results showed that FtMYB18 could suppress anthocyanins and PAs synthesis at transcription level through the specific interaction of C5 motif with other proteins in Tartary buckwheat.

Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins.

The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants.

Ectopic expression of tea MYB genes alter spatial flavonoid accumulation in alfalfa (Medicago sativa).

Flavonoids are one of the largest secondary metabolite groups, which are widely present in plants. Flavonoids include anthocyanins, proanthocyanidins, flavonols and isoflavones. In particular, proanthocyanidins possess beneficial effects for ruminant animals in preventing lethal pasture bloat. As a major legume forage, alfalfa (Medicago sativa) contains little proanthocyanidins in foliage to combat bloat. In an attempt to improve proanthocyanidin content in alfalfa foliage, we over-expressed two MYB transcription factors (CsMYB5-1 and CsMYB5-2) from tea plant that is rich in proanthocyanidins. We showed that, via targeted metabolite and transcript analyses, the transgenic alfalfa plants accumulated higher levels of flavonoids in stems/leaves than the control, in particular anthocyanins and proanthocyanidins. Over-expression of CsMYB5-1 and CsMYB5-2 induced the expression levels of genes involved in flavonoid pathway, especially anthocyanin/proanthocyanidin-specific pathway genes DFR, ANS and ANR in stems/leaves. Both anthocyanin/proanthocyanidin content and the expression levels of several genes were conversely decreased in flowers of the transgenic lines than in control. Our results indicated that CsMYB5-1 and CsMYB5-2 differently regulate anthocyanins/proanthocyanidins in stems/leaves and flowers. Our study provides a guide for increasing anthocyanin/proanthocyanidin accumulation in foliage of legume forage corps by genetic engineering. These results also suggest that it is feasible to cultivate new varieties for forage production to potentially solve pasture bloat, by introducing transcription factors from typical plants with high proanthocyanidin level.

A Sucrose-Induced MYB (SIMYB) Transcription Factor Promoting Proanthocyanidin Accumulation in the Tea Plant ( Camellia sinensis).

Proanthocyanidins (PAs, also called condensed tannins), are an important class of secondary metabolites and exist widely in plants. Tea ( Camellia sinensis) is rich in PAs and their precursors, (-)-epicatechin (EC) and (+)-catechin (C). The biosynthesis of PAs is constantly regulated by many different MBW complexes, consisting of MYB transcription factors (TFs), basic-helix-loop-helix (bHLH) TFs, and WD-repeat (WDR) proteins. These regulatory factors can be environmentally affected, such as by biotic and abiotic stresses. In this study, we revalidated the effect of sucrose treatment on tea branches, and a sucrose-induced MYB (SIMYB) TF was screened and studied. Phylogenetic analysis indicted that this SIMYB TF belonged to MYB subgroup 5, named CsMYB5b. Heterologous expression of CsMYB5b in tobacco strongly induced PA accumulation, through up-regulating the key target genes LAR or ANRs. In addition, CsMYB5b restored PA production in the seed coat of A. thaliana tt2 mutant and rescued its phenotype. Yeast two-hybrid assay demonstrated CsMYB5b can interact directly with CsTT8 (an AtTT8 ortholog) and CsWD40 protein. Linking to the expression profiling of CsMYB5b and the PA accumulation pattern in tea plants suggest that the CsMYB5b acts as an important switch for the synthesis of monomeric catechins and PAs. Therefore, these data provide insight into the regulatory mechanisms controlling the biosynthesis of PAs.

[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.

Elicitation and treatment with precursors of phenolics synthesis improve low-molecular antioxidants and antioxidant capacity of buckwheat sprouts.

BACKGROUND: Recently, an increase of interest in the modification of food products on each step of production (breeding, production technology, storage condition) is observed. Nutritional properties as well as level and activity of bioactive compounds in plant-origin food may be modified using a range of technological and biotechnological practices and elicitation should be mentioned between them. METHODS: Elicitation with willow bark infusion supported by feeding with the phenylpropanoid pathway precursors were used for improving the quality of buckwheat sprouts. Special emphasis has been placed on the metabolomic and biochemical changes and the mechanism of overproduction of low-molecular antioxidants. RESULTS: The accumulation of phenolics is caused by stimulation of two main enzymes the phenylpropanoid pathway (tyrosine ammonia-lyase and phenylalanine ammonia-lyase). Tyrosine ammonia-lyase activities were effectively induced by feeding with tyrosine (about four times that of the control), whereas phenylalanine ammonia-lyase activity was the highest in the elicited control sprouts and those fed with shikimic acid (an increase by 60% compared to the control). Shikimic acid feeding (both elicited and non-elicited sprouts) effectively improved the total phenolics (by about 10% and 20%, respectively), condensed tannins (by about 30% and 28%, respectively), and flavonoids (by about 46% and 70%, respectively). Significant increase of vitexin, rutin, chlorogenic acid and isoorientin contents was also observed. The treatments increased the ascorbic acid content, too. Total antioxidant capacity of sprouts was most effectively increased by feeding with shikimic acid and further elicitation. CONCLUSIONS: The studies transfer biotechnology commonly used for the induction of overproduction of secondary metabolites in plant cell line systems to low-processed food production. The obtained results could be used for better understanding of the effect of elicitation and precursor feeding on antioxidants production and contribute to improving the buckwheat sprouts quality.

Analysis of accumulation patterns and preliminary study on the condensation mechanism of proanthocyanidins in the tea plant [Camellia sinensis].

In the present study, proanthocyanidins were qualitatively and quantitatively identified using hydrolysis and thiolysis assays, NP-HPLC, HPLC-ESI-MS, MALDI-TOF-MS, (1)H-NMR, and (13)C-NMR techniques in different organs of tea plants. The results showed that in leaves, the tri-hydroxyl, cis- and galloylated flavan-3-ols were the main monomeric catechins units, and (epi)catechin was found to be the major unit of polymeric flavan-3-ols when the degree of polymerization was greater than five. In roots, the PAs were found to be abundant, and epicatechin formed the predominant extension unit of oligomeric and polymeric PAs. In order to understand the mechanism of proanthocyanidins polymerization, auto-condensation of the flavan-3-ols was investigated. The results showed that the same trimers (m/z 865) were detected in the extracts of tea plants and in the non-enzymatic in vitro assay, in weak acid as well as weak alkaline solutions at room temperature, when the substrates used were either procyanidin B2 and monomeric flavan-3-ols (epicatechin or catechin), or only procyanidin B2. This suggested that procyanidin B2 not only released carbocation as electrophilic upper units, but also could be used as nucleophilic lower units directly itself, to form the procyanidin trimer in vitro or in vivo.

Variation in proanthocyanidin content and composition among commonly grown North American cranberry cultivars (Vaccinium macrocarpon).

BACKGROUND: Cranberry fruit (Vaccinium macrocarpon) is rich in polyphenols, particularly oligomeric proanthocyanidins (PACs) possessing antimicrobial and antioxidant properties. PACs may play a role in resistance to fruit rot. Although many cranberry cultivars are grown for use in foods, beverages and nutraceuticals, data on PAC content among cultivars is limited. Eight cultivars were sampled from four growing regions during the 2010 season and analyzed for PAC content and composition. RESULTS: MALDI-TOF MS showed that isolated PACs had similar oligomer profiles among cultivars. The major constituents were A-type (epi)catechin oligomers of two to eight degrees of polymerization. Total PAC content ranged between 18 and 92 g PAC kg⁻¹ dried fruit, quantified as procyanidin A2 by the dimethylaminocinnamaldehyde method. Among the cultivars sampled, Howes had the highest total PACs (76-92 g kg⁻¹), followed by Mullica Queen and Early Black (48-82 g kg⁻¹). Ben Lear, a disease-susceptible variety, was significantly lower in PACs than the other cultivars (P < 0.001). CONCLUSIONS: Several traditional and newer cultivars of cranberry from various growing regions in North America are excellent sources of PACs, particularly the Howes, Mullica Queen and Early Black cultivars. PAC content may play a role in keeping quality.

Premature and ectopic anthocyanin formation by silencing of anthocyanidin reductase in strawberry (Fragaria × ananassa).

Strawberry (Fragaria × ananassa) is a fruit crop with a distinct biphasic flavonoid biosynthesis. Whereas, in the immature receptacle, high levels of proanthocyanidins accumulate, which are associated with herbivore deterrence and pathogen defense, the prominent color-giving anthocyanins are primarily produced in ripe 'fruits' helping to attract herbivores for seed dispersal. Here, constitutive experimental down-regulation of one branch of proanthocyanidin biosynthesis was performed. As a result, the proportion of epicatechin monomeric units within the proanthocyanidin polymer chains was reduced, but this was not the case for the epicatechin starter unit. Shortened chain lengths of proanthocyanidins were also observed. All enzymatic activities for the production of color-giving anthocyanins were already present in unripe fruits at levels allowing a striking red anthocyanin phenotype in unripe fruits of the RNAi silencing lines. An immediately recognizable phenotype was also observed for the stigmata of flowers, which is another epicatechin-forming tissue. Thus, the down-regulation of anthocyanidin reductase (ANR) induced a redirection of the proanthocyanidin pathway, leading to premature and ectopic anthocyanin biosynthesis via enzymatic glycosylation as the alternative pathway. This redirection is also seen in flavonol biosynthesis, which is paralleled by higher pollen viability in silencing lines. ANRi transgenic lines of strawberry provide a versatile tool for the study of the biological functions of proanthocyanidins.

Polyphenol variability in the fruits and juices of a cider apple progeny.

BACKGROUND: Polyphenols have a favourable antioxidant potential on human health, suggesting that their high content in apple is responsible for the beneficial effects of apple consumption. They are also linked to the quality of apple juices and ciders since they are predominantly responsible for astringency, bitterness and colour. Major phenolic compounds were quantified by liquid chromatography in fruits and juices from a cider apple progeny harvested for 3 years. The total content of procyanidins and their average degree of polymerisation (DPn) were also determined in fruits by phloroglucinolysis. Variability and extraction yield of these compounds were determined. RESULTS: The variability observed in the progeny was representative of the variability observed in many cider apple varieties. Hydroxycinnamic acids were the most extractable group, with an average extraction yield of 67%, whereas flavonols and anthocyanins were the least. CONCLUSION: This study is the first to introduce variability and extraction yields of the main phenolic compounds in both fruits and juices of a cider apple progeny. This dataset will be used for an upcoming QTL mapping study, an original approach that has never been undertaken for cider apple.

Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering.

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia.

BACKGROUND: Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. FINDINGS: Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. CONCLUSION: To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

Revealing impaired pathways in the an11 mutant by high-throughput characterization of Petunia axillaris and Petunia inflata transcriptomes.

Petunia is an excellent model system, especially for genetic, physiological and molecular studies. Thus far, however, genome-wide expression analysis has been applied rarely because of the lack of sequence information. We applied next-generation sequencing to generate, through de novo read assembly, a large catalogue of transcripts for Petunia axillaris and Petunia inflata. On the basis of both transcriptomes, comprehensive microarray chips for gene expression analysis were established and used for the analysis of global- and organ-specific gene expression in Petunia axillaris and Petunia inflata and to explore the molecular basis of the seed coat defects in a Petunia hybrida mutant, anthocyanin 11 (an11), lacking a WD40-repeat (WDR) transcription regulator. Among the transcripts differentially expressed in an11 seeds compared with wild type, many expected targets of AN11 were found but also several interesting new candidates that might play a role in morphogenesis of the seed coat. Our results validate the combination of next-generation sequencing with microarray analyses strategies to identify the transcriptome of two petunia species without previous knowledge of their genome, and to develop comprehensive chips as useful tools for the analysis of gene expression in P. axillaris, P. inflata and P. hybrida.

Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula.

Oligomeric proanthocyanidins (PAs) composed primarily of epicatechin units accumulate in the seed coats of the model legume Medicago truncatula, reaching maximal levels at around 20 d after pollination. Genes encoding the single Medicago anthocyanidin synthase (ANS; EC 1.14.11.19) and leucoanthocyanidin reductase (LAR; EC 1.17.1.3) were cloned and the corresponding enzymes functionally identified. Recombinant MtANS converted leucocyanidin to cyanidin, and, more efficiently, dihydroquercetin to the flavonol quercetin. Levels of transcripts encoding dihydroflavonol reductase, ANS, and anthocyanidin reductase (ANR), the enzyme responsible for conversion of anthocyanidin to (-)-epicatechin, paralleled the accumulation of PAs in developing seeds, whereas LAR transcripts appeared to be more transiently expressed. LAR, ANS, and ANR proteins were localized to the cytosol in transfected tobacco (Nicotiana tabacum) leaves. Antisense down-regulation of ANS in M. truncatula resulted in reduced anthocyanin and PA levels, but had no impact on flavonol levels. Transgenic tobacco plants constitutively overexpressing MtLAR showed reduced anthocyanin content, but no catechin or increased levels of PAs were detected either in leaves or in flowers. Our results confirm previously ascribed in vivo functions for ANS and ANR. However, the apparent lack of catechin in M. truncatula PAs, the poor correlation between LAR expression and PA accumulation, and the lack of production of catechin monomers or oligomers in transgenic plants overexpressing MtLAR question the role of MtLAR in PA biosynthesis in Medicago.