Azolla ferns testify: seed plants and ferns share a common ancestor for leucoanthocyanidin reductase enzymes.

Questions about in vivo substrates for proanthocyanidin (PA) biosynthesis and condensation have not been resolved and wide gaps in the understanding of transport and biogenesis in 'tannosomes' persist. Here we examined the evolution of PA biosynthesis in ferns not previously reported, asking what PAs are synthesised and how. Chemical and gene-expression analyses were combined to characterise PA biosynthesis, leveraging genome annotation from the floating fern Azolla filiculoides. In vitro assay and phylogenomics of PIP-dehydrogenases served to infer the evolution of leucoanthocyanidin reductase (LAR). Sporophyte-synthesised (epi)catechin polymers, averaging only seven subunits, accumulated to 5.3% in A. filiculoides, and 8% in A. pinnata biomass dry weight. Consistently, a LAR active in vitro was highly expressed in A. filiculoides. LAR, and paralogous fern WLAR-enzymes with differing substrate binding sites, represent an evolutionary innovation of the common ancestor of fern and seed plants. The specific ecological niche of Azolla ferns, a floating plant-microbe mat massively fixing CO2 and N2 , shaped their metabolism in which PA biosynthesis predominates and employs novel fern LAR enzymes. Characterisation of in vivo substrates of these LAR, will help to shed light on the recently assigned and surprising dual catalysis of LAR from seed plants.

Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development.

BACKGROUND: The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. RESULTS: The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. CONCLUSIONS: PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.

Characterization of two TT2-type MYB transcription factors regulating proanthocyanidin biosynthesis in tetraploid cotton, Gossypium hirsutum.

MAIN CONCLUSION: Two TT2-type MYB transcription factors identified from tetraploid cotton are involved in regulating proanthocyanidin biosynthesis, providing new strategies for engineering condensed tannins in crops. Proanthocyanidins (PAs), also known as condensed tannins, are important secondary metabolites involved in stress resistance in plants, and are health supplements that help to reduce cholesterol levels. As one of the most widely grown crops in the world, cotton provides the majority of natural fabrics and is a supplemental food for ruminant animals. The previous studies have suggested that PAs present in cotton are a major contributor to fiber color. However, the biosynthesis of PAs in cotton still remains to be elucidated. AtTT2 (transparent testa 2) is a MYB family transcription factor from Arabidopsis that initiates the biosynthesis of PAs by inducing the expression of multiple genes in the pathway. In this study, we isolated two R2R3-type MYB transcription factors from Gossypium hirsutum that are homologous to AtTT2. Expression analysis showed that both genes were expressed at different levels in various cotton tissues, including leaf, seed coat, and fiber. Protoplast transactivation assays revealed that these two GhMYBs were able to activate promoters of genes encoding enzymes in the PA biosynthesis pathway, namely anthocyanidin reductase and leucoanthocyanidin reductase. Complementation experiments showed that both of the GhMYBs were able to recover the transparent testa seed coat phenotype of the Arabidopsis tt2 mutant by restoring PA biosynthesis. Ectopic expression of either of the two GhMYBs in Medicago truncatula hairy roots increased the contents of anthocyanins and PAs compared to control lines expressing the GUS gene, and expression levels of MtDFR, MtLAR, and MtANR were also elevated in lines expressing GhMYBs. Together, these data provide new insights into engineering condensed tannins in cotton.

Transcription factor VvibHLH93 negatively regulates proanthocyanidin biosynthesis in grapevine.

Proanthocyanidins (PAs) derived from grape berries determine the astringency and bitterness of red wines. The two leucoanthocyanidin reductases (VviLAR1 and VviLAR2) are crucial for PA accumulation in grapevine. Our previous studies show that the promoter of VviLAR1 contains multiple proposed bHLH transcription factor binding sites, but the corresponding bHLH family regulators remain unknown. Here we identified and functionally characterized VvibHLH93 as a new bHLH transcription factor in PA pathway. Yeast one-hybrid and electrophoretic mobility shift assays showed that VvibHLH93 bound the E/G-box in VviLAR1 promoter. And VvibHLH93 gene was mainly expressed in grape flowers, tendrils, stems and berries at PA active stages. Overexpression of VvibHLH93 suppressed PA accumulation in grape callus, which was linked to the repression of the transcript levels of two VviLARs. The gene expression analysis in transgenic grape callus and the dual-luciferase assay in tobacco leaves together revealed that VvibHLH93 targeted a broad set of structural genes and transcription factors in flavonoid pathway. This research enriches the regulatory mechanism of the two VviLAR genes, and provides new insights into regulating PA content in grape berries.

Overexpression of leucoanthocyanidin reductase or anthocyanidin reductase elevates tannins content and confers cassava resistance to two-spotted spider mite.

The two-spotted spider mite (TSSM) is a destructive cassava pest. Intensive demonstration of resistance mechanism greatly facilitates the creation of TSSM-resistant cassava germplasm. Gene to metabolite network plays a crucial role in modulating plant resistance, but little is known about the genes and related metabolites which are responsible for cassava resistance to TSSM. Here, a highly resistant (HR) and a highly susceptible (HS) cassava cultivar were used, integrative and comparative transcriptomic and metabolomic analyses between these two cultivars after TSSM infestation revealed that several genes and metabolites were closely related and significantly different in abundance. In particular, the expression of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes showed a high positive correlation with most of the metabolites in the tannin biosynthesis pathway. Furthermore, transgenic cassava lines overexpressing either of the genes elevated tannin concentrations and conferred cassava resistance to TSSM. Additionally, different forms of tannins possessed distinct bioactivity on TSSM, of which total condensed tannins (LC50 = 375.68 mg/l) showed maximum lethal effects followed by procyanidin B1 (LC50 = 3537.10 mg/l). This study accurately targets LAR, ANR and specific tannin compounds as critical genes and metabolites in shaping cassava resistance to TSSM, which could be considered as biomarkers for evaluation and creation of pest-resistant cassava germplasm.

Virtual screening and docking analysis of novel ligands for selective enhancement of tea (Camellia sinensis) flavonoids.

Flavour of tea is mainly contributed by a group of polyphenols - flavonoids. However, the content of flavonoid fluctuates seasonally and is found to be higher in the first flush of tea, when compared to the second flush. This disparity in the flavonoid content, and hence taste, incurs heavy economic losses to the tea plantation industry each harvest season. For our present study, four key product-specific enzymes (PAL, FNS, FLS and ANS) of the tea-specific flavonoid pathway were selected to perform molecular docking studies with specific virtually screened allosteric modulators. Results of docking analyses showed Naringenin, 2-Morpholin-4-ium-4-ylethanesulfonate, 6-C-Glucosylquercetin, 2-Oxoglutaric acid, 3,5,7,3',4'-pentahydroxyflavone to be capable of improving the spontaneity of the enzyme-substrate reactions in terms of docking score, RMSD values, and non-covalent interactions (H-bond,hydrophobic interaction, Π-stacking, salt bridge, etc.). Further, the evolutionary relationship of tea flavonoid pathway enzymes was constructed and compared with related taxa. The codon usage-based of tea flavonoid biosynthetic genes indicated the non-biasness of their nucleotide composition. Overall this study will provide a direction towards putative ligand-dependent enhancement of flavonoid content, irrespective of seasonal variation.

Molecular characterization of Leucoanthocyanidin reductase and Flavonol synthase gene in Arachis hypogaea.

Arachis hypogaea (peanut) is a potential source of bioactive compounds including flavonols and proanthocyanidins, which have gained particular interest of metabolic engineering owing to their significance in the growth, development and defense responses in plants. To gain insight of proanthocyanidins and flavonols production in A. hypogaea, Leucoanthocyanidin reductase (AhLAR) and Flavonol synthase (AhFLS) enzymes responsible for their production, have been structurally, transcriptionally and functionally characterized. Structural and functional analysis of putative protein sequence of AhFLS indicated two functional motifs 2OG-FeII_Oxy and DIOX_N, while six functional motifs belonging to the families of NAD-dependent dehydratase, 3, ß hydroxysteroid dehydrogenase and NmrA-like family were observed in case of AhLAR. Promoter sequence analysis unraveled several promoter elements related to the development regulation, environmental stress responses and hormonal signaling. Furthermore, the expression analysis of AhFLS and AhLAR and accumulation pattern analysis of proanthocyanidins and flavonols in three selected cultivars of A. hypogaea under saline environment confirmed their role against salinity in genotype-dependent and stress level-dependent manner. Correlation studies revealed that AhFLS and AhLAR expression is not directly dependent on the antioxidant enzymes activity, biochemical and growth parameters but higher Pearson r value depicted some level of dependency. This detailed study of AhLAR and AhFLS can assist in the metabolic engineering of flavonoid biosynthetic pathway to produce stress tolerant varieties and production of proanthocyanidins and flavonols at an industrial scale.

Transcription Factor VviMYB86 Oppositely Regulates Proanthocyanidin and Anthocyanin Biosynthesis in Grape Berries.

Proanthocyanidins (PAs) and anthocyanins are two vital groups of flavonoid compounds for grape berries and red wines. Several transcription factors (TFs) have been identified to be involved in regulating PA and anthocyanin biosynthesis in grape berries. However, research on TFs with different regulatory mechanisms for these two biosynthesis branches in grapes remains limited. In this study, we identified an R2R3-MYB TF, VviMYB86, whose spatiotemporal gene expression pattern in grape berries coincided well with PA accumulation but contrasted with anthocyanin synthesis. Both in vivo and in vitro experiments verified that VviMYB86 positively regulated PA biosynthesis, primarily by upregulating the expression of the two leucoanthocyanidin reductase (LAR) genes in the Arabidopsis protoplast system, as well as in VviMYB86-overexpressing grape callus cultured under 24 h of darkness. Moreover, VviMYB86 was observed to repress the anthocyanin biosynthesis branch in grapes by downregulating the transcript levels of VviANS and VviUFGT. Overall, VviMYB86 is indicated to have a broad effect on flavonoid synthesis in grape berries. The results of this study will help elucidate the regulatory mechanism governing the expression of the two LAR genes in grape berries and provide new insights into the regulation of PA and anthocyanin biosynthesis in grape berries.

The Effect of Light Intensity on the Expression of Leucoanthocyanidin Reductase in Grapevine Calluses and Analysis of Its Promoter Activity.

To investigate the effect of light intensity on flavonoid biosynthesis, grapevine calluses were subjected to high light (HL, 250 µmol m-2 s-1) and dark (0 µmol m-2 s-1) in comparison to 125 µmol m-2 s-1 under controlled conditions (NL). The alteration of flavonoid profiles was determined and was integrated with RNA sequencing (RNA-seq)-based transcriptional changes of the flavonoid pathway genes. Results revealed that dark conditions inhibited flavonoid biosynthesis. Increasing light intensity affected flavonoids differently-the concentrations of flavonols and anthocyanins as well as the expressions of corresponding genes were less affected, whereas flavan-3-ol concentrations were predominantly increased, which caused enhanced trans-flavan-3-ol concentrations. Moreover, genes encoding leucoanthocyanidin reductase (LAR) exhibited different response patterns to light intensity changes-VviLAR1 expression increased with an increased light intensity, whereas VviLAR2 expression was insensitive. We further confirmed that the known transcription factors (TFs) involved in regulating flavan-3-ol biosynthesis utilized VviLAR1 as a target gene in grapevine calluses. In addition, VviLAR1 promoter activity was more sensitive to light intensity changes than that of VviLAR2 as determined using a transgenic Arabidopsis leaf system. These results suggested that light intensity had the most prominent effect on trans-flavan-3-ols in grapevine calluses and demonstrated that the two LAR genes had different response patterns to light intensity changes.

Production of 3,4-cis- and 3,4-trans-Leucocyanidin and Their Distinct MS/MS Fragmentation Patterns.

(+)-2,3-trans-3,4-cis-Leucocyanidin was produced by acidic epimerization of (+)-2,3-trans-3,4-trans-leucocyanidin synthesized by reduction of (+)-dihydroquercetin with NaBH4, and structures of the two stereoisomers purified by C18- and phenyl-reverse-phase high-performance liquid chromatography (HPLC) were confirmed by NMR spectroscopy. We confirm that only 3,4-cis-leucocyanidin is used by leucoanthocyanidin reductase as substrate. The two stereoisomers are quite stable in aqueous solution at -20 °C. Characterization of the two stereoisomers was also performed using electrospray ionization tandem mass spectrometry (ESI-MS/MS), and we discuss here for the first time the corresponding MS/MS fragmentation pathways, which are clearly distinct. The main difference is that of the mode of dehydration of the 3,4-diol in positive ionization mode, which involves a loss of hydroxyl group at either C3 or C4 for the 3,4-cis isomer but only at C3 for the 3,4-trans isomer. Tandem mass spectrometry therefore proves useful as a complementary methodology to NMR to identify each of the two stereoisomers.

Proanthocyanidin Synthesis in Chinese Bayberry (Myrica rubra Sieb. et Zucc.) Fruits.

Proanthocyanidins (PAs) are distributed widely in Chinese bayberry fruit and have been associated with human health benefits, but molecular and biochemical characterization of PA biosynthesis remains unclear. Here, two genes encoding key PA biosynthetic enzymes, anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) were isolated in bayberry fruit. MrANR was highly expressed at the early stage of fruit development when soluble PAs accumulated at high levels. Meanwhile, the transcript abundance of both MrANR and MrLAR observed at the late stage was paralleled with the high amounts of insoluble PAs. LC-MS/MS showed that PAs in developing Chinese bayberry fruits were comprised predominantly of epigallocatechin-3-O-gallate terminal subunits, while the extension subunits were a mixture of epigallocatechin-3-O-gallate, epigallocatechin and catechin. Recombinant MrANR protein converted cyanidin to a mixture of epicatechin and catechin, and delphinidin to a mixture of epigallocatechin and gallocatechin in vitro. Recombinant MrLAR was active with leucocyanidin as substrate to produce catechin. Ectopic expression of MrANR in tobacco reduced anthocyanin levels but increased PA accumulation. The catechin and epicatechin contents in transgenic flowers overexpressed MrANR were significantly higher than those of wild-type. However, overexpression of MrLAR in tobacco led to an increase in catechin levels but had no impact on PA contents. Quantitative real time PCR revealed that the loss of anthocyanin in transgenic flowers overexpressed MrANR or MrLAR is probably attributed to decreased expression of tobacco chalcone isomerase (CHI) gene. Our results not only reveal in vivo and in vitro functions for ANR and LAR but also provide a resource for understanding the mechanism of PA biosynthesis in Chinese bayberry fruit.

Chlorogenic acid, anthocyanin and flavan-3-ol biosynthesis in flesh and skin of Andean potato tubers (Solanum tuberosum subsp. andigena).

Natural variation of Andean potato was used to study the biosynthesis of phenolic compounds. Levels of phenolic compounds and corresponding structural gene transcripts were examined in flesh and skin of tubers. Phenolic acids, mainly chlorogenic acid (CGA), represent the major compounds, followed by anthocyanins and flavan-3-ols. High-anthocyanin varieties have high levels of CGA. Both metabolite and transcript levels were higher in skin than in flesh and showed a good correspondence. Two hydroxycinnamoyl-CoA transferases (HCT/HQT) have been involved in CGA production, of which HCT reflects CGA levels. Catechin was found in pigmented tissues whereas epicatechin was restricted to tuber skin. Transcripts of leucoanthocyanidin reductase (LCR), which generates catechin, could not be detected. Anthocyanidin reductase (ANR) transcripts, the enzyme responsible for epicatechin production, showed similar levels among samples. These data suggest that the biosynthesis of flavan-3-ols in potato tuber would require ANR but not LCR and that an epimerization process is involved.

Accumulation of catechins and expression of catechin synthetic genes in Camellia sinensis at different developmental stages.

BACKGROUND: Catechins are the main polyphenol compounds in tea (Camellia sinensis). To understand the relationship between gene expression and product accumulation, the levels of catechins and relative expressions of key genes in tea leaves of different developmental stages were analyzed. RESULTS: The amounts of catechins differed significantly in leaves of different stages, except for gallocatechin gallate. Close correlations between the expression of synthesis genes and the accumulation of catechins were identified. Correlation analysis showed that the expressions of chalcone synthase 1, chalcone synthase 3, anthocyanidin reductase 1, anthocyanidin reductase 2 and leucoanthocyanidin reductase genes were significantly and positively correlated with total catechin contents, suggesting their expression may largely affect total catechin accumulation. Anthocyanidin synthase was significantly correlated with catechin. While both ANRs and LAR were significantly and positively correlated with the contents of (-)-epigallocatechin gallate and (-)-epicatechin gallate. CONCLUSION: Our results suggest synergistic changes between the expression of synthetic genes and the accumulation of catechins. Based on our findings, anthocyanidin synthase may regulate earlier steps in the conversion of catechin, while the anthocyanidin reductase and leucoanthocyanidin reductase genes may both play important roles in the biosynthesis of galloylated catechins.

Peach MYB7 activates transcription of the proanthocyanidin pathway gene encoding leucoanthocyanidin reductase, but not anthocyanidin reductase.

Proanthocyanidins (PAs) are a group of natural phenolic compounds that have a great effect on both flavor and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs) via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase and anthocyanidin reductase. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5) via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants.

Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple.

Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

Transcriptome profiling reveals differential gene expression in proanthocyanidin biosynthesis associated with red/green skin color mutant of pear (Pyrus communis L.).

Anthocyanin concentration is the key determinant for red skin color in pear fruit. However, the molecular basis for development of red skin is complicated and has not been well-understood thus far. "Starkrimson" (Pyrus communis L.), an introduced red pear cultivated in the north of China and its green mutant provides a desirable red/green pair for identification of candidate genes involved in color variation. Here, we sequenced and annotated the transcriptome for the red/green color mutant at three stages of development using Illumina RNA-seq technology. The total number of mapped reads ranged from 26 to 46 million in six libraries. About 70.11-71.95% of clean reads could be mapped to the reference genome. Compared with green colored fruit, a total of 2230 differentially expressed genes (DEGs) were identified in red fruit. Gene Ontology (GO) terms were defined for 4886 differential transcripts involved in 15 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Three DEGs were identified as candidate genes in the flavonoid pathway, LAR, ANR, and C3H. Tellingly, higher expression was found for genes encoding ANR and LAR in the green color mutant, promoting the proanthocyanidin (PA) pathway and leading to lower anthocyanin. MYB-binding cis-motifs were identified in the promoter region of LAR and ANR. Based on these findings, we speculate that the regulation of PA biosynthesis might be a key factor for this red/green color mutant. Besides the known MYB and MADS transcription families, two new families, AP2 and WRKY, were identified as having high correlation with anthocyanin biosynthesis in red skinned pear. In addition, qRT-PCR was used to confirm the transcriptome results for 17 DEGs, high correlation of gene expression, further proved that AP2 and WARK regulated the anthocyanin biosynthesis in red skinned "Starkrimson," and ANR and LAR promote PA biosynthesis and contribute to the green skinned variant. This study can serve as a valuable new resource laying a solid foundation for functional gene identification in the anthocyanin pathway of red-skinned pear and provide a good reference for relevant research on molecular mechanisms of color variation in other pear species.

Erwinia amylovora affects the phenylpropanoid-flavonoid pathway in mature leaves of Pyrus communis cv. Conférence.

Flavonoids, which are synthesized by the phenylpropanoid-flavonoid pathway, not only contribute to fruit colour and photoprotection, they also may provide antimicrobial and structural components during interaction with micro-organisms. A possible response of this pathway was assessed in both mature and immature leaves of shoots of 2-year-old pear trees cv. Conférence, which were inoculated with the gram-negative bacterium Erwinia amylovora strain SGB 225/12, were mock-inoculated or were left untreated. The phenylpropanoid-flavonoid pathway was analysed by histological studies, by gene expression using RT-qPCR and by HPLC analyses of the metabolites at different time intervals after infection. Transcription patterns of two key genes anthocyanidin reductase (ANR) and chalcone synthase (CHS) related to the phenylpropanoid-flavonoid pathway showed differences between control, mock-inoculated and E. amylovora-inoculated mature leaves, with the strongest reaction 48 h after inoculation. The impact of E. amylovora was also visualised in histological sections, and confirmed by HPLC, as epicatechin -which is produced via ANR- augmented 72 h after inoculation in infected leaf tissue. Besides the effect of treatments, ontogenesis-related differences were found as well. The increase of certain key genes, the rise in epicatechin and the visualisation in several histological sections in this study suggest a non-negligible impact on the phenylpropanoid-flavonoid pathway in Pyrus communis due to inoculation with E. amylovora. In this study, we propose a potential role of this pathway in defence mechanisms, providing a detailed analysis of the response of this system attributable to inoculation with E. amylovora.