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

Summary Mt PAR is a proanthocyanidin ( PA ) biosynthesis regulator; the mechanism underlying its promotion of PA biosynthesis is not fully understood. Here, we showed that Mt PAR promotes PA production 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. Ectopic expression of Mt PAR repressed isoflavonoid production by directly binding and suppressing isoflavone biosynthetic genes such as isoflavone synthase ( IFS ). Meanwhile, Mt PAR up‐regulated PA ‐specific genes and decreased the anthocyanin levels without altering the expression of anthocyanin biosynthetic genes. Mt PAR may shift the anthocyanidin precursor flux from anthocyanin pathway to PA biosynthesis. Mt PAR complemented PA ‐deficient phenotype of Arabidopsis tt2 mutant seeds, demonstrating their similar action on PA production. We showed the direct interactions between Mt PAR , Mt TT 8 and Mt WD 40‐1 proteins from Medicago truncatula and Glycine max , to form a ternary complex to trans ‐activate PA ‐specific ANR gene. Finally, Mt PAR expression in alfalfa ( Medicago sativa ) hairy roots and whole plants only promoted the production of small amount of PA s, which was significantly enhanced by co‐expression of Mt PAR and Mt LAP 1 . Transcriptomic and metabolite profiling showed an additive effect between Mt PAR and Mt LAP 1 on the production of PA s, supporting that efficient PA production requires more anthocyanidin precursors. This study provides new insights into the role and mechanism of Mt PAR in partitioning precursors from isoflavone and anthocyanin pathways into PA pathways for a specific promotion of PA production. Based on this, a strategy by combining Mt PAR and Mt LAP 1 co‐expression to effectively improve metabolic engineering performance of PA production in legume forage was developed.