Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentrationand structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not containsubstantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in theiredible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulatorsand their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background,we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus,polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression ofthe above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatusorgans with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that,when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase andleucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp.Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotusas a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findingsare crucial to engineering this trait in pasture legumes.