Molecular cloning and characterization of a cDNA for pterocarpan 4-dimethylallyltransferase catalyzing the key prenylation step in the biosynthesis of glyceollin, a soybean phytoalexin

Abstract

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C5 prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(-)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-(13)C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.

Figure 1.

Biosynthesis of glyceollin isomers in soybean. Abbreviations not defined in the text: HID, 2-hydroxyisoflavanone dehydratase; IFS, 2-hydroxyisoflavanone synthase; P6aH, pterocarpan 6a-hydroxylase; G2DT, dimethylallyl diphosphate: (−)-glycinol 2-dimethylallyltransferase.

Figure 2.

Properties of PT2 (G4DT). A, Structural features of PT2. Conserved amino acids for this prenyltransferase family are also shown. TP, Transit peptide; TM, transmembrane α-helix; L2, loop number 2; L6, loop number 6. B, HPLC chromatogram of the enzymatic reaction mixture of PT2. The assay mixture contained DMAPP, (−)-glycinol, and the microsome fraction of yeast expressing PT2. For the negative control, the microsomal fraction of yeast cells transformed with pYES2 without a cDNA insert was used with the same substrates. The peak before the substrate glycinol is a solvent peak and not an enzymatic reaction product.

Figure 3.

Time course of isoflavonoid formation in suspension-cultured soybean cells treated with yeast extract (0.3% w/v). A, Formation of daidzein and daidzin. B, Formation of glycinol and glyceollins. The values are the averages from four independent experiments. Vertical bars represent the sd.

Figure 4.

Semiquantitative RT-PCR analysis of gene expression of isoflavonoid biosynthetic enzyme in soybean cells upon yeast extract treatment. Transcripts levels were normalized against those of actin in the respective cells. The basal level of the control cells (time 0) was set at 1.0. The values are the averages of three independent experiments. Vertical bars represent the sd. Abbreviations not defined in the text: HID, 2-hydroxyisoflavanone dehydratase; IFS, 2-hydroxyisoflavanone synthase; P6aH, pterocarpan 6a-hydroxylase.

Figure 5.

Transient expression of G4DT(TP)-GFP fusion protein in onion epidermal peels. A, G4DT(TP)-GFP and WxTP-DsRed plasmids were cotransformed into onion epidermal peels by particle bombardment. B, For the control, pGWB5 (CaMV35S promoter + GFP) and WxTP-DsRed plasmids were double transformed into onion epidermal peels. The images were obtained at 24 h after bombardment. WxTP-DsRed (red fluorescence) was used as a control for plastid targeting. All scale bars = 100 μm.

Figure 6.

Incorporation of [1-¹³C]Glc into glyceollin I. The labeled carbon (¹³C) shown in black dot in Glc is incorporated into each metabolite. Expected labeling patterns of DMAPP via the MEP (black triangles) and mevalonate pathways (gray dots) and of p-coumaroyl-CoA (black squares) and malonyl-CoA (gray squares) are also shown.

Figure 7.

The phylogenetic relationship among soybean G4DT and related prenyltransferase proteins of plants. A neighbor-joining tree was produced from the results of 1,000 bootstrap replicates. Numbers at the branch points indicate bootstrap fraction (maximum 100). Abbreviations used are: ApVTE2-1 (DQ231057), homogentisate phytyltransferase of Allium porrum; AtVTE2-1 (AY089963), homogentisate phytyltransferase of Arabidopsis; AtVTE2-2 (DQ231060), homogentisate prenyltransferase of Arabidopsis; CpVTE2-1 (DQ231058), homogentisate phytyltransferase of Cuphea pulcherrima; GmVTE2-2 (DQ231061), homogentisate prenyltransferase of soybean; HvHGGT (AY222860), homogentisate geranylgeranyltransferase of Hordeum vulgare; OsHGGT (AY222862), homogentisate geranylgeranyltransferase of Oryza sativa; SfN8DT-1 (AB325579) and SfN8DT-2 (AB370330), naringenin 8-dimethylallyltransferases of S. flavescens; SfL17a (AB371287) and SfL17b (AB370329), prenyltransferase homologs of S. flavescens; TaHGGT (AY222861), homogentisate geranylgeranyltransferase of Triticum aestivum; TaVTE2-1 (DQ231056), homogentisate phytyltransferase of T. aestivum; ZmVTE2-1 (DQ231055), homogentisate phytyltransferase of Zea mays.