Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens

Abstract

Prenylated flavonoids are natural compounds that often represent the active components in various medicinal plants and exhibit beneficial effects on human health. Prenylated flavonoids are hybrid products composed of a flavonoid core mainly attached to either 5-carbon (dimethylallyl) or 10-carbon (geranyl) prenyl groups derived from isoprenoid (terpenoid) metabolism, and the prenyl groups are crucial for their biological activity. Prenylation reactions in vivo are crucial coupling processes of two major metabolic pathways, the shikimate-acetate and isoprenoid pathways, in which these reactions are also known as a rate-limiting step. However, none of the genes responsible for the prenylation of flavonoids has been identified despite more than 30 years of research in this field. We have isolated a prenyltransferase gene from Sophora flavescens, SfN8DT-1, responsible for the prenylation of the flavonoid naringenin at the 8-position, which is specific for flavanones and dimethylallyl diphosphate as substrates. Phylogenetic analysis shows that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. The gene expression of SfN8DT-1 is strictly limited to the root bark where prenylated flavonoids are solely accumulated in planta. The ectopic expression of SfN8DT-1 in Arabidopsis thaliana resulted in the formation of prenylated apigenin, quercetin, and kaempferol, as well as 8-prenylnaringenin. SfN8DT-1 represents the first flavonoid-specific prenyltransferase identified in plants and paves the way for the identification and characterization of further genes responsible for the production of this large and important class of secondary metabolites.

Figure 1.

Biosynthetic pathway from naringenin to SFG in S. flavescens. Two dimethylallylations and a 2′-hydroxylation are involved. Nari, Naringenin; 2′OH, 8DN 2′-hydroxylase; LGDT, LG 2″-dimethylallyltransferase.

Figure 2.

Enzymatic characterization of recombinant SfN8DT-1 expressed in yeast. A, Structural features of the SfN8DT-1 polypeptide. B, HPLC chromatograms of standard specimens, 8DN, 3′-dimethylallyl naringenin (3′DN), and 6-dimethylallyl naringenin (6DN; a); ethyl acetate extract of incubation mixture of naringenin and DMAPP with empty-vector transformed cells (b); and recombinant SfN8DT-1 (c). Arrowheads indicate the retention time of 8DN (9.5 min). d, UV spectrum of 8DN formed from naringenin by recombinant SfN8DT-1. C, LC/ESI-MS analysis of enzymatic reaction products. a, Total ion chromatogram (top) and selected ion monitoring for specific m/z 341 for 8DN [M + H]⁺ (bottom) and mass spectrum of 8DN (b).

Figure 3.

Substrate specificity of recombinant SfN8DT-1. A, Relative enzyme activity with various aromatic substrates as prenyl acceptors, naringenin (nari), liquiritigenin (liq), hesperetin (hes), sakuranetin (sak), flavanone (flava), taxifolin (tax), kaempferol (kae), quercetin (que), apigenin (api), flavone (flavo), 2-hydroxy chalcone (2-hyd), 2′-hydroxy chalcone (2′-hyd), isoliquiritigenin (isoliq), genistein (geni), maackiain (maa), LG, and HG. B, Relative enzyme activity with various prenyl diphosphates of different chain length as substrates, DMAPP, GPP, FPP, GGPP, and PDP. C, Chemical structures of flavonoids used for substrate specificity analysis.

Figure 4.

Transient expression of the SfN8DT1-GFP fusion protein. The plasmid containing SfN8DT1-GFP was introduced into onion peels (A–D) and cultured S. flavescens cells (E–H) by particle bombardment. Scale bars show 100 μm for onion and 10 μm for S. flavescens cells. A, SfN8DT1∷GFP; B, PaIspS-TP∷GFP, as a plastid-targeted control; D, 35S-GFP (control); E, SfN8DT1-TP∷GFP; F, 35S-GFP (control). C, G, and H show bright field images of A, E, and F, respectively.

Figure 5.

Accumulation of SfN8DT-1 mRNA and prenylated flavonoids in S. flavescens. A, Effects of yeast extract, MJ, and salicylic acid on SfN8DT-1 expression in cultured S. flavescens cells monitored by RNA gel-blot analysis. B, Organ-specific accumulation of SfN8DT-1 mRNA in intact S. flavescens plants detected by RNA gel-blot analysis. C, Organ-specific accumulation of flavonoids in S. flavescens. a, Contents of prenylated flavonoids in each plant organ. b, Contents of flavone monoglucosides in each plant organ. Values are the mean ± sd of three replicates. D, Correlation of accumulation pattern of prenylated flavonoids with SfN8DT-1 gene expression. a, Content of prenylated flavonoids in the roots of S. flavescens. b, SfN8DT-1 mRNA expression level in the roots of S. flavescens monitored by RT-PCR analysis.

Figure 6.

Phylogenetic relationship of prenyltransferases accepting aromatic substrates. A rooted phylogram was generated using a ClustalW alignment. Ap, Allium porrum; At, Arabidopsis; Cp, Cuphea pulcherrima; Gm, Glycine max; Hv, Hordeum vulgare; Os, Oryza sativa; Ta, Triticum aestivum; Zm, Zea mays. HG phytyltransferases (VTE2-1) are involved in tocopherol biosynthesis, HG geranylgeranyltransferases (HGGT) are involved in tocotrienol biosynthesis, and HG solanesyltransferases (VTE2-2) are involved in plastoquinone biosynthesis. Accession numbers: ApVTE2-1, DQ231057; AtVTE2-1, AY089963; AtVTE2-2, DQ231060; CpVTE2-1, DQ231058; GmVTE2-1, DQ231059; GmVTE2-2, DQ231061; HvHGGT, AY222860; OsHGGT, AY222862; SfN8DT-1, AB325579; SfN8DT-2, AB370330; SfL17a, AB371287; SfL17b, AB370329; TaHGGT, AY222861; TaVTE2-1, DQ231056; ZmVTE2-1, DQ231055.