Metabolic engineering of proanthocyanidin production by repressing the isoflavone pathways and redirecting anthocyanidin precursor flux in legume

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anglais. MtPAR is a proanthocyanidin (PA) biosynthesis regulator; the mechanism underlying itspromotion of PA biosynthesis is not fully understood. Here, we showed that MtPAR promotes PAproduction by a direct repression of biosynthesis of isoflavones, the major flavonoids in legume,and by redirecting immediate precursors, such as anthocyanidins, flux into PA pathway. Ectopicexpression of MtPAR repressed isoflavonoid production by directly binding and suppressingisoflavone biosynthetic genes such as isoflavone synthase (IFS). Meanwhile, MtPAR up-regulatedPA-specific genes and decreased the anthocyanin levels without altering the expression ofanthocyanin biosynthetic genes. MtPAR may shift the anthocyanidin precursor flux fromanthocyanin pathway to PA biosynthesis. MtPAR complemented PA-deficient phenotype ofArabidopsis tt2 mutant seeds, demonstrating their similar action on PA production. We showedthe direct interactions between MtPAR, MtTT8 and MtWD40-1 proteins from Medicagotruncatula and Glycine max, to form a ternary complex to trans-activate PA-specific ANR gene.Finally, MtPAR expression in alfalfa (Medicago sativa) hairy roots and whole plants only promotedthe production of small amount of PAs, which was significantly enhanced by co-expression ofMtPAR and MtLAP1. Transcriptomic and metabolite profiling showed an additive effect betweenMtPAR and MtLAP1 on the production of PAs, supporting that efficient PA production requiresmore anthocyanidin precursors. This study provides new insights into the role and mechanism ofMtPAR in partitioning precursors from isoflavone and anthocyanin pathways into PA pathwaysfor a specific promotion of PA production. Based on this, a strategy by combining MtPAR andMtLAP1 co-expression to effectively improve metabolic engineering performance of PAproduction in legume forage was developed.