A stress-inducible resveratrol O-methyltransferase involved in the biosynthesis of pterostilbene in grapevine

Abstract

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl(3) treatment.

Figure 1.

Stilbene contents in control, P. viticola-infected, and UV-treated Cabernet Sauvignon leaves. Stilbenes were analyzed with HPLC-DAD, 72 h posttreatment. Note that, for better readability, the stilbene content scale is different in UV-treated leaves. Standard errors were calculated based on three replicates.

Figure 2.

Biosynthesis of 3,5-dimethoxytoluene in Rosa and proposed biosynthesis of pterostilbene in grapevine.

Figure 3.

Comparison of grapevine ROMT with other OMTs. A, The predicted amino acid sequence of grapevine ROMT was aligned with Chinese rose cv Old Blush OOMT1 and Scots pine PMT with ClustalW. Residues shaded in gray indicate identical amino acids. B, Phylogenetic tree of selected OMT cDNA sequences: grapevine ROMT (VvROMT, accession no. FM178870), grapevine caffeic acid OMT (VvCOMT, AF239740), Chinese rose OOMT1 (RcOOMT1, AJ439741), basil chavicol OMT (ObCVOMT, AF435007), basil eugenol OMT (ObEOMT, AF435008), Clarkia breweri (iso)eugenol OMT (CbIEMT, U86760), C. breweri caffeic acid OMT (CbCOMT, AF006009), Nicotiana tabacum caffeic acid OMT (NtCOMT, AF484252), and Scots pine pinosylvin OMT (PsPMT; Chiron et al., 2000b). The numbers beside the branches represent bootstrap values based on 500 replicates.

Figure 4.

Analysis of ROMT reaction products. A, TLC analysis of reaction products produced following incubation of recombinant ROMT with 200 μm resveratrol (1), 200 μm RME (2), and 200 μm pterostilbene (3) in the presence of [¹⁴C]SAM (50 μm). Reactions were carried out in a total volume of 25 μL with 200 ng of purified protein and were allowed to proceed for 15 min. The positions of the origin (O) and the reaction products RME and pterostilbene (P) are indicated. B, GC-MS analysis of ROMT reaction products. Recombinant ROMT was incubated with resveratrol (500 μm) in the presence of [¹⁴C]SAM (1 mm). Peak 1, Pterostilbene; peak 2, resveratrol. A total ion chromatogram is shown and mass spectra of the peaks matched those of the corresponding authentic standards. The reaction was carried out in a total volume of 200 μL with 2 μg of purified protein and was allowed to proceed for 60 min.

Figure 5.

Characterization of ROMT activity in planta using Agrobacterium-mediated transient transformation. Tobacco leaf sectors (150 mg fresh weight) expressing GFP (A), grapevine STS (B), or coexpressing STS and ROMT (C) were excised 48 h after Agrobacterium-mediated transformation. Stilbene content was analyzed using HPLC-DAD. Peak 1, Piceid; peak 2, resveratrol; peak 3, pterostilbene. Identity of peak 3 was confirmed by GC-MS analysis (data not shown).

Figure 6.

RT-PCR analysis of ROMT expression in grapevine leaf discs submitted to different stresses. Sets of leaf discs were immersed in sterile ultrapure water (mock) or infected by immersion in a suspension of P. viticola sporangia (2 × 10⁴ mL⁻¹) in sterile ultrapure water. UVC irradiation (λ = 254 nm, 90 μW cm⁻²) was applied for 7 min. AlCl₃ solution (1% [w/v]) was applied directly on the discs. Leaf discs were collected at t = 0 (control), 6, 24, and 48 h after treatments and frozen before RNA extraction. Selected genes are actin, ROMT, PAL, and STS. M, DNA M_r marker. The data shown are representative of three independent experiments.

Figure 7.

Quantitative RT-PCR analysis of ROMT expression in grapevine leaf discs infected by P. viticola. Transcript accumulation of ROMT (accession no. FM178870), STS (accession no. X76892), and PAL (accession no. CF511227) genes was monitored in mock-infected leaf discs (white squares) and P. viticola-infected leaf discs (black squares). Pathogen development was evaluated by monitoring P. viticola actin transcript accumulation (P. viticola actin). Analyses were performed by real-time quantitative RT-PCR. Transcript levels of grapevine genes are expressed as relative values normalized to the transcript level of actin gene, used as an internal reference (accession no. AF369525). Absolute copy number of mRNA for each target gene in the t = 0 control sample was 0 (OMT), 24 × 10³ (STS), and 571 × 10³ (PAL) molecules/μg of plant total RNAs. Results are means of duplicate experiments; bars indicate ± ses.