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Auxin response factor 2 acts as a positive regulator to fine-tune the spatial and temporal accumulation of flavonoid compounds, mainly flavonols and proanthocyanidins in Arabidopsis. Auxin response factor (ARF) proteins are reported to involve in auxin-mediated regulation of flavonoid biosynthesis. However, the detailed regulation mechanism of ARF remains still unknown. Here, we provide genetic and molecular evidence that one of the twenty-three ARF members-ARF2-positively regulates flavonoid biosynthesis at multi-level in tissue-specific manner in Arabidopsis thaliana. Loss-of-function mutation of ARF2 led to significant reduction in flavonoid content (e.g., flavonols and proanthocyanidins) in the seedlings and seeds of the Arabidopsis arf2 mutants. Over-expression of ARF2 increased flavonols and proanthocyanidins content in Arabidopsis. Additionally, the changes of flavonoid content correlate well with the transcript abundance of several regulatory genes (e.g., MYB11, MYB12, MYB111, TT2, and GL3), and key biosynthetic genes (e.g., CHS, F3'H, FLS, ANS, ANR, TT12, TT19, and TT15), in the arf2 mutant and ARF2 over-expression lines. Transient transactivation assays with site-directed mutagenesis confirmed that ARF2 directly regulates the expression of MYB12 and FLS genes in the flavonol pathway and ANR in the proanthocyanidin pathway, and indirectly regulates MYB11 and MYB111 genes in the flavonol pathway, and ANS, TT12, TT19 and TT15 genes in the proanthocyanidin pathway. Further genetic results indicated that ARF2 acts upstream of MYB12 to regulate flavonol accumulation, and of TT2 to regulate proanthocyanidins accumulation. In particular, yeast two-hybrid assays revealed that ARF2 physically interacts with TT2, a master regulator of proanthocyanidins biosynthesis. Combined together, these results indicated that ARF2 functions as a positive regulator for the fine-tuned spatial and temporal regulation of flavonoids (mainly flavonols and proanthocyanidins) accumulation in Arabidopsis.

Kudzu, Pueraria lobata, is a traditional Chinese food and medicinal herb that has been commonly used since ancient times. Kudzu roots are rich sources of isoflavonoids, e.g., puerarin, with beneficial effects on human health. To gain global information on the isoflavonoid biosynthetic regulation network in kudzu, de novo transcriptome sequencings were performed using two genotypes of kudzu with and without puerarin accumulation in roots. RNAseq data showed that the genes of the isoflavonoid biosynthetic pathway were significantly represented in the upregulated genes in the kudzu with puerarin. To discover regulatory genes, 105, 112, and 143 genes encoding MYB, bHLH, and WD40 transcription regulators were identified and classified, respectively. Among them, three MYB, four bHLHs, and one WD40 gene were found to be highly identical to their orthologs involved in flavonoid biosynthesis in other plants. Notably, the expression profiles of PlMYB1, PlHLH3-4, and PlWD40-1 genes were closely correlated with isoflavonoid accumulation profiles in different tissues and cell cultures of kudzu. Over-expression of PlMYB1 in Arabidopsis thaliana significantly increased the accumulation of anthocyanins in leaves and proanthocyanidins in seeds, by activating AtDFR, AtANR, and AtANS genes. Our study provided valuable comparative transcriptome information for further identification of regulatory or structural genes involved in the isoflavonoid pathway in P. lobata, as well as for bioengineering of bioactive isoflavonoid compounds.

Kudzu, Pueraria lobata, is a traditional Chinese food and medicinal herb that has been commonly used since ancient times. Kudzu roots are rich sources of isoflavonoids, e.g., puerarin, with beneficial effects on human health. To gain global information on the isoflavonoid biosynthetic regulation network in kudzu, de novo transcriptome sequencings were performed using two genotypes of kudzu with and without puerarin accumulation in roots. RNAseq data showed that the genes of the isoflavonoid biosynthetic pathway were significantly represented in the upregulated genes in the kudzu with puerarin. To discover regulatory genes, 105, 112, and 143 genes encoding MYB, bHLH, and WD40 transcription regulators were identified and classified, respectively. Among them, three MYB, four bHLHs, and one WD40 gene were found to be highly identical to their orthologs involved in flavonoid biosynthesis in other plants. Notably, the expression profiles of PlMYB1, PlHLH3-4, and PlWD40-1 genes were closely correlated with isoflavonoid accumulation profiles in different tissues and cell cultures of kudzu. Over-expression of PlMYB1 in Arabidopsis thaliana significantly increased the accumulation of anthocyanins in leaves and proanthocyanidins in seeds, by activating AtDFR, AtANR, and AtANS genes. Our study provided valuable comparative transcriptome information for further identification of regulatory or structural genes involved in the isoflavonoid pathway in P. lobata, as well as for bioengineering of bioactive isoflavonoid compounds.

Legume plants are rich in prenylated flavonoid compounds, which play an important role in plant defense and human health. In the present study, we identified a prenyltransferase (PT) gene, named LaPT2, in white lupin (Lupinus albus), which shows a high identity and close relationship with the other known PT genes involved in flavonoid prenylation in planta. The recombinant LaPT2 protein expressed in yeast cells exhibited a relatively strong activity toward several flavonols (e.g., kaempferol, quercetin, and myricetin) and a relatively weak activity toward flavanone (naringenin). In addition, the recombinant LaPT2 protein was also active toward several other types of flavonoids, including galangin, morin, 5-deoxyquercetin, 4'-O-methylkaempferol, taxifolin, and aromadendrin, with distinct enzymatic affinities. The LaPT2 gene was preferentially expressed in the roots, which is consistent with the presence of prenylated flavonoid kaempferol in the roots. Moreover, we found that the expression level of LaPT2 paralleled with those of LaF3H1 and LaFLS2 genes that were relatively higher in roots and lower in leaves, suggesting that they were essential for the accumulation of prenylated flavonoid kaempferol in roots. The deduced full-length LaPT2 protein and its signal peptide fused with a green fluorescent protein (GFP) are targeted to plastids in the Arabidopsis thaliana protoplast. Our study demonstrated that LaPT2 from white lupin is responsible for the biosynthesis of prenylated flavonoids, in particular flavonols, which could be utilized as phytoalexin for plant defense and bioactive flavonoid compounds for human health.

Prenylated isoflavonoids have been found in several legume plants, and they possess various biological activities that play important roles in both plant defense and human health. However, it is still unknown whether prenylated isoflavonoids are present in the model legume plant Lotus japonicus. In the present study, we found that the prenylated isoflavonoid wighteone was produced in L. japonicus when leaf was supplemented with genistein. Furthermore, a novel prenyltransferase gene, LjG6DT, was identified, which shared high similarity with and was closely related to several known prenyltransferase genes involved in isoflavonoid biosynthesis. The recombinant LjG6DT protein expressed in yeast exhibited prenylation activity toward genistein as an exclusive substrate, which produced wighteone, a prenylated genistein at the C-6 position that occurs normally in legume plants. The LjG6DT-green fluorescent protein (GFP) fusion protein is targeted to plastids. The transcript level of LjG6DT is induced by glutathione, methyl jasmonate and salicylic acid, implying that LjG6DT is involved in stress response. Overexpression of LjG6DT in L. japonicus hairy roots led to increased accumulation of wighteone when genistein was supplied, indicating that LjG6DT is functional in vivo. Feeding assays with the upstream intermediate naringenin revealed that accumulation of wighteone in L. japonicus was dependent on genistein supplementation, and accumulation of wighteone is competed by genistein methylation. This study demonstrated that phytoalexin wighteone is inducibly produced in L. japonicus, and it provides new insight into the biosynthesis and accumulation of prenylated isoflavonoids in legume plants.