Elevation of Condensed Tannins in the Leaves of Ta-MYB14-1 White Clover (Trifolium repens L.) Outcrossed with High Anthocyanin Lines.

Condensed tannins (CT) are highly desirable in forage as they sequester dietary protein and reduce bloat and methane emissions in ruminants. However, the widely used forage legume white clover (Trifolium repens) only produces CTs in flowers and trichomes and at levels too low to achieve therapeutic effects. Genetic transformation with transcription factor Ta-MYB14-1 from Trifolium arvense was effective in inducing CTs to 0.6% of leaf dry matter. CT synthesis has been elevated further by crossing the primary white clover transgenic line with wild type genotypes producing the related phenylpropanoids, anthocyanins. CT levels in leaves were highest under the anthocyanin leaf marks associated with the "red midrib" trait; however, there was no evidence for CT accumulation in leaf sections with the "red V" anthocyanin marking. Ta-MYB14-1 was stably inherited in two generations of crosses, and T2 progeny produced up to 3.6-fold higher CTs than the T0 parent. The profile of small CT oligomers such as dimers and trimers was consistent in T0, T1, T2, and BC2 progeny and consisted predominantly of prodelphinidins (PD), with lesser amounts of procyanidins (PC) and mixed PC:PD oligomers.

Characterization of Proanthocyanidins from Seeds of Perennial Ryegrass (Lolium perenne L.) and Tall Fescue (Festuca arundinacea) by Liquid Chromatography-Mass Spectrometry.

Perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) are forage species of the grass family (Poaceae) that are key components of temperate pasture-based agricultural systems. Proanthocyanidins (PAs) are oligomeric flavonoids that, when provided as part of a farm animal's diet, have been reported to improve animal production and health. Up to now, forage grasses have been deemed not to produce PAs. This paper reports for the first time the detection of polymerized PAs in aqueous methanolic extracts of seed tissue of both perennial ryegrass and tall fescue, using LC-MS/MS. We have determined the structure of the PAs to be trans-flavan-3-ol-based, consisting predominately of afzelechin and catechin and linked primarily by B-type bonds. Investigations into the leaf tissue of both species failed to detect any PAs. This discovery opens the possibility of using genetic engineering tools to achieve tannin accumulation in leaf tissue of perennial ryegrass and tall fescue.

Expression of the R2R3-MYB transcription factor TaMYB14 from Trifolium arvense activates proanthocyanidin biosynthesis in the legumes Trifolium repens and Medicago sativa.

Proanthocyanidins (PAs) are oligomeric flavonoids and one group of end products of the phenylpropanoid pathway. PAs have been reported to be beneficial for human and animal health and are particularly important in pastoral agricultural systems for improved animal production and reduced greenhouse gas emissions. However, the main forage legumes grown in these systems, such as Trifolium repens and Medicago sativa, do not contain any substantial amounts of PAs in leaves. We have identified from the foliar PA-accumulating legume Trifolium arvense an R2R3-MYB transcription factor, TaMYB14, and provide evidence that this transcription factor is involved in the regulation of PA biosynthesis in legumes. TaMYB14 expression is necessary and sufficient to up-regulate late steps of the phenylpropanoid pathway and to induce PA biosynthesis. RNA interference silencing of TaMYB14 resulted in almost complete cessation of PA biosynthesis in T. arvense, whereas Nicotiana tabacum, M. sativa, and T. repens plants constitutively expressing TaMYB14 synthesized and accumulated PAs in leaves up to 1.8% dry matter. Targeted liquid chromatography-multistage tandem mass spectrometry analysis identified foliar PAs up to degree of polymerization 6 in leaf extracts. Hence, genetically modified M. sativa and T. repens plants expressing TaMYB14 provide a viable option for improving animal health and mitigating the negative environmental impacts of pastoral animal production systems.