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.