Metabolic engineering of isoflavonoid biosynthesis in alfalfa

Abstract

The potential health benefits of dietary isoflavones have generated considerable interest in engineering the synthesis of these phytoestrogens into plants. Genistein glucoside production (up to 50 nmol g(-1) fresh weight) was engineered in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A (4'-O-methylgenistein) and pratensein (3'-hydroxybiochanin A) also accumulated. Although MtIFS1 was highly expressed in all organs examined, genistein accumulation was limited to leaves. MtIFS1-expressing lines accumulated several additional isoflavones, including formononetin and daidzein, in response to UV-B or Phoma medicaginis, whereas the chalcone and flavanone precursors of these compounds accumulated in control lines. Enhanced accumulation of the phytoalexin medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing plants. Microarray profiling indicated that MtIFS1 expression does not significantly alter global gene expression in the leaves. Our results highlight some of the challenges associated with metabolic engineering of plant natural products, including tissue-specific accumulation, potential for further modification by endogenous enzyme activities (hydroxylation, methylation, and glycosylation), and the differential response of engineered plants to environmental factors.

Figure 1.

Schematic of biosynthetic pathways leading to flavonoid and isoflavonoid natural products. CHI, Chalcone isomerase; CHR, chalcone reductase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; FS, flavone synthase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; HI4′OMT, 2,7,4′-trihydroxyisoflavanone 4′-O-methyltransferase; I3′H, isoflavone 3′-hydroxylase; IFS, 2-hydroxyisoflavanone synthase.

Figure 2.

Vector construct for alfalfa transformation and molecular characterization of transgenic lines. A, The MtIFS1 cDNA was subcloned into the pRTL2 vector and the expression cassette containing MtIFS1 flanked by CaMV 35S promoter and terminator sequences was cloned into the HindIII site of the binary vector pCAMBIA 2300. B, DNA gel-blot analysis of MtIFS1-expressing lines C22 and B20, vector control lines VC11 and VB2, and nontransgenic alfalfa cv Regen SY-4D (NT). Genomic DNA was digested with either HindIII (H) or EcoRI (E) restriction enzymes and transferred to a nylon membrane. The membrane was hybridized with a PCR fragment of the NPTII gene. Molecular mass markers are indicated.

Figure 3.

Effect of MtIFS1 expression on the isoflavonoid composition of alfalfa leaf extracts. HPLC traces of unhydrolyzed leaf extracts of vector control line VC11 (A) and MtIFS1-expressing line C22 (B). Peaks with UV spectra similar to genistein and not present in the control extracts are numbered 1 to 5. Peaks 1 and 4 were identified as genistin and sissotrin, respectively. Leaf extracts of control line VC11 (C) and MtIFS1-expressing line C22 (D) after digestion with β-glucuronidase. Apigenin and tricin aglycone were detected in both C and D, peaks A and T, respectively. E, HPLC traces of leaf extracts of line C22 (top trace) and VC11 (bottom trace, offset by 25 mAu) after digestion with purified β-glucosidase. Residues were resuspended in anhydrous MeOH to minimize solubilization of flavone glucuronides. Peaks labeled G, P, and B were identified as genistein, pratensein, and biochanin A, respectively.

Figure 4.

Isoflavone levels in leaves of MtIFS1-expressing lines C22 and B20. Leaves were harvested from plants grown in a greenhouse over the course of 7 weeks. Isoflavone levels in hydrolyzed leaf extracts were quantified by HPLC. White bars, Genistein; black bars, biochanin A. No isoflavones were detected in vector control lines.

Figure 5.

Tissue-specific expression of IFS, HI4′OMT, and I3′H in MtIFS1-expressing line C22 and vector control line VC11. Total RNA from leaf (L), stem (S), and root (R) tissues was fractionated on a 1% formaldehyde-agarose gel and transferred to nylon membrane. Blots were hybridized with probes for IFS, HI4′OMT, I3′H, and 18S rRNA.

Figure 6.

Effect of UV-B on the flavonoid profile of MtIFS1-expressing and vector control plants. HPLC traces of hydrolyzed leaf extracts of MtIFS1-expressing line C22 (A and B) and vector control line VC11 (C and D) from control (A and C) and UV-B-treated (B and D) plants. Peaks labeled A, Af, B, D, DHF, F, G, I, and L were identified as apigenin, afromosin, biochanin A, daidzein, 7,4′-dihydroxyflavone, formononetin, genistein, isoliquiritigenin, and liquiritigenin, respectively. Peaks at 10 and 12 min represent unhydrolyzed conjugates of the flavone apigenin. Similar results were obtained for MtIFS1-expressing line B20 and vector control line VB2. Note that images A and B and images C and D are shown at different scales.

Figure 7.

Isoflavone levels in MtIFS1-expressing and vector control lines in response to UV-B. A, Effect of UV-B on levels of genistein and biochanin A in MtIFS1-expressing lines C22 and B20. Plants were exposed to UV-B for 6 h and isoflavone levels in hydrolyzed leaf extracts were quantified by HPLC. White bars, Genistein; gray bars, biochanin A. B, Levels of 5-deoxyflavonoids in UV-B-treated MtIFS1-expressing (C22 and B20) and vector control (VC11 and VB2) lines. White bars, Formononetin; diagonally striped bars, liquiritigenin; gray bars, isoliquiritigenin; vertically striped bars, 7,4′-dihydroxyflavone; stipled bars, daidzein; black bars, total (summed) levels of 5-deoxyflavonoids.

Figure 8.

Effect of P. medicaginis infection on the flavonoid profile of MtIFS1-expressing and vector control plants. HPLC traces of hydrolyzed leaf extracts of MtIFS1-expressing line C22 (A and B) and vector control line VC11 (C and D) from control (A and C) and spore-inoculated (B and D) plants at 24 h postinfection. Peaks labeled A, Af, B, D, DHF, F, G, I, L, and M were identified as apigenin, afromosin, biochanin A, daidzein, 7,4′-dihydroxyflavone, formononetin, genistein, isoliquiritigenin, liquiritigenin, and medicarpin, respectively. Similar results were obtained for MtIFS1-expressing line B20 and vector control line VB2. Note that A₂₈₀ is shown due to the low absorbance of medicarpin at 254 nm.

Figure 9.

Isoflavonoid phytoalexin levels in leaves of MtIFS1-expressing and vector control lines in response to P. medicaginis infection. Plants were sprayed with a spore suspension and leaves were harvested 0 to 72 h later. Isoflavone levels in hydrolyzed leaf extracts were quantified by HPLC. Formononetin and medicarpin were not detected at the start of the experiment (T = 0) in any of the transgenic lines (data not shown). Medicarpin concentration is presented as peak area (arbitrary units). White bars, Formononetin; gray bars, medicarpin.