Abstract
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.
Highlights
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
Most genetic studies on PA biosynthesis to date have been performed in Arabidopsis (Arabidopsis thaliana; Lepiniec et al, 2006) or barley (Hordeum vulgare; Jende-Strid, 1993)
Our results provide the background for further development of Medicago as a model system for studying PA biosynthesis and deposition in forage legumes
Summary
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. Because expression of many foreign flavonoid pathway genes (including ANR) gives a clear metabolic phenotype in solanaceous plants (Muir et al, 2001; Tian and Dixon, 2006; Xie et al, 2006), we predicted formation of catechin in tobacco tissues that overexpresses LAR and expresses the anthocyanin pathway (e.g. flowers), even if PAs themselves may not be made.
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