MtPAR (Medicago truncatula proanthocyanidin regulator) is an MYB family transcription factor that functions as a key regulator of proanthocyanidin (PA) biosynthesis in the model legume Medicago truncatula. MtPAR expression is confined to the seed coat, the site of PA accumulation. Loss-of-function par mutants contained substantially less PA in the seed coat than the wild type, whereas levels of anthocyanin and other specialized metabolites were normal in the mutants. In contrast, massive accumulation of PAs occurred when MtPAR was expressed ectopically in transformed hairy roots of Medicago. Transcriptome analysis of par mutants and MtPAR-expressing hairy roots, coupled with yeast one-hybrid analysis, revealed that MtPAR positively regulates genes encoding enzymes of the flavonoid-PA pathway via a probable activation of WD40-1. Expression of MtPAR in the forage legume alfalfa (Medicago sativa) resulted in detectable levels of PA in shoots, highlighting the potential of this gene for biotechnological strategies to increase PAs in forage legumes for reduction of pasture bloat in ruminant animals.condensed tannin | forage quality | metabolic engineering
Summary MtPAR is a proanthocyanidin (PA) biosynthesis regulator; the mechanism underlying its promotion of PA biosynthesis is not fully understood. Here, we showed that MtPAR 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 MtPAR repressed isoflavonoid production by directly binding and suppressing isoflavone biosynthetic genes such as isoflavone synthase (IFS). Meanwhile, MtPAR up‐regulated PA‐specific genes and decreased the anthocyanin levels without altering the expression of anthocyanin biosynthetic genes. MtPAR may shift the anthocyanidin precursor flux from anthocyanin pathway to PA biosynthesis. MtPAR complemented PA‐deficient phenotype of Arabidopsis tt2 mutant seeds, demonstrating their similar action on PA production. We showed the direct interactions between MtPAR, MtTT8 and MtWD40‐1 proteins from Medicago truncatula 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 promoted the production of small amount of PAs, which was significantly enhanced by co‐expression of MtPAR and MtLAP1. Transcriptomic and metabolite profiling showed an additive effect between MtPAR and MtLAP1 on the production of PAs, supporting that efficient PA production requires more anthocyanidin precursors. This study provides new insights into the role and mechanism of MtPAR 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 MtPAR and MtLAP1 co‐expression to effectively improve metabolic engineering performance of PA production in legume forage was developed.
Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6'-deoxy- and 6'-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.
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