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.

Expression of the Arabidopsis TRANSPARENT TESTA 2 (TT2) MYB family transcription factor leads to massive accumulation of proanthocyanidins (PAs) in hairy roots of Medicago truncatula. Microarray analysis showed that TT2 induces genes for flavonoid/PA biosynthesis, transcription factors, and a large number of genes of unknown function. A second microarray dataset identified genes that were preferentially expressed in the M. truncatula seed coat. Comparison of the two datasets defines target genes for steps that are yet unidentified in PA biosynthesis and accumulation. Of these genes, a glycosyltransferase, UGT72L1, was active specifically toward the PA precursor (؊)؊epicatechin, and its expression pattern in developing seeds correlated with the presence of epicatechin glucoside and accumulation of PAs. UGT72L1 may be involved in the production of epicatechin 3 -O-glucoside in the seed coat as a key step in PA biosynthesis or its regulation.glucosyltransferase ͉ model legume ͉ proanthocyanidin

HighlightThe identification of three UDP-glycosyltransferases involved in the biosynthesis of galloylated catechins and glycosylated flavonols which are astringent taste compounds in tea.

WD40 repeat proteins regulate biosynthesis of anthocyanins, proanthocyanidins (PAs), and mucilage in the seed and the development of trichomes and root hairs. We have cloned and characterized a WD40 repeat protein gene from Medicago truncatula (MtWD40-1) via a retrotransposon-tagging approach. Deficiency of MtWD40-1 expression blocks accumulation of mucilage and a range of phenolic compounds, including PAs, epicatechin, other flavonoids, and benzoic acids, in the seed, reduces epicatechin levels without corresponding effects on other flavonoids in flowers, reduces isoflavone levels in roots, but does not impair trichome or root hair development. MtWD40-1 is expressed constitutively, with highest expression in the seed coat, where its transcript profile temporally parallels those of PA biosynthetic genes. Transcript profile analysis revealed that many genes of flavonoid biosynthesis were down-regulated in a tissue-specific manner in M. truncatula lines harboring retrotransposon insertions in the MtWD40-1 gene. MtWD40-1 complemented the anthocyanin, PA, and trichome phenotypes of the Arabidopsis (Arabidopsis thaliana) transparent testa glabrous1 mutant. We discuss the function of MtWD40-1 in natural product formation in M. truncatula and the potential use of the gene for engineering PAs in the forage legume alfalfa (Medicago sativa).

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SUMMARYMYB transcription factors help to control anthocyanin biosynthesis in plants, and ectopic expression of the Arabidopsis Production of Anthocyanin Pigment 1 (PAP1) transcription factor activates the anthocyanin pathway in tobacco, suggesting the general utility of such factors for metabolic engineering of anthocyanins and anthocyanin-derived compounds such as proanthocyanidins (condensed tannins). However, PAP1 does not activate anthocyanin biosynthesis in the model legume Medicago truncatula or in alfalfa (Medicago sativa). A related Legume Anthocyanin Production 1 (LAP1) gene was identified from the genome of M. truncatula. When constitutively expressed in transgenic alfalfa, M. truncatula or white clover, LAP1 induced massive accumulation of anthocyanin pigments comprising multiple glycosidic conjugates of cyanidin. Oligomeric/ polymeric compounds with some diagnostic characteristics of proanthocyanidins also accumulated in LAP1-expressing plants, but these compounds were not composed of (epi)catechin units. Over 260 and 70 genes were up-regulated in leaves of alfalfa or M. truncatula, respectively, in response to constitutive expression of LAP1, many of which are involved in anthocyanin biosynthesis. In particular, the glucosyltransferase UGT78G1, previously identified as showing preference for isoflavonoid substrates in vitro, was strongly up-regulated by LAP1, and appears to function as an anthocyanin glycosyltransferase in vivo. Over-expression of UGT78G1 in transgenic alfalfa resulted in increased anthocyanin accumulation when plants were exposed to abiotic stress.

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 blightresistant 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 (2)-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 (2)-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.

HighlightCHIL is essential for the accumulation of proanthocyanidins and flavonols in seeds of Arabidopsis and it functions as a unique enhancer in the flavonoid pathway.