Constitutive Accumulation of a Resveratrol-Glucoside in Transgenic Alfalfa Increases Resistance to Phoma medicaginis

Abstract

Alfalfa (Medicago sativa) was transformed with a peanut (Arachis hypogaea) cDNA encoding resveratrol synthase (RS) transcriptionally regulated by an enhanced Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated a new compound, not present in wild-type or vector-transformed alfalfa, that was identified as trans-resveratrol-3-O-beta-D-glucopyranoside (RGluc) by high-pressure liquid chromatography (HPLC), UV, 1H- and 13C-nuclear magnetic resonance (NMR) analyses. RGluc concentration was highest in the youngest leaves (>15 microg per g fresh weight) and oldest stem internode segments (>10 microg per g fresh weight) while roots contained only trace amounts (<0.2 microg per g fresh weight). RS transcript levels were highest in leaves and stems, with comparatively little transcript accumulation in the roots, while an inverse pattern was observed for chalcone synthase (CHS) transcript levels. CHS directly competes with RS for the metabolic precursors p-coumaroyl CoA and malonyl CoA, and may also contribute to the developmental variations in RGluc levels by limiting the availability of substrates. Agar-plate bioassays indicated that both RGluc and resveratrol greatly inhibit hyphal growth of the alfalfa fungal pathogen Phoma medicaginis. Subsequently, RGluc-containing leaves were wound inoculated and showed a significant reduction (relative to control leaves) in the size of necrotic lesions, intensity of adjacent chlorosis, and number of fungal reproductive structures (pycnidia). Decreasing sporulation of this pathogen may greatly reduce disease spread and severity throughout the field.

The R2R3-MYB Transcription Factors MYB14 and MYB15 Regulate Stilbene Biosynthesis in Vitis vinifera

“…Similar results were observed in MYB15 overexpressing hairy roots ( Figure 5B; see Supplemental Figure 6 online). It has been demonstrated that stilbenes, and in particular d-viniferin, an oxidative resveratrol dimer, and pterostilbene, the 3,5-dimethoxy analog of resveratrol, have fungitoxic properties (Pezet et al, 2003(Pezet et al, , 2004Schmidlin et al, 2008) and that the glycosylation of stilbenes may occur in plant cells both to protect cells from their potential toxic effects and to protect resveratrol from degradation Hipskind and Paiva, 2000). This might explain the high amount of trans-piceid in the MYB15 transgenic hairy roots in comparison with the relatively low amounts of other modified stilbenes (see Supplemental Figure 6 online).…”

A Stress-Inducible Resveratrol O-Methyltransferase Involved in the Biosynthesis of Pterostilbene in Grapevine

“…Transgenic expression of VST1 in various plant species, including tobacco (Nicotiana spp. ), tomato (Solanum lycopersicum), papaya (Carica papaya), and grapevine, has been shown to result in resveratrol and resveratrol derivative accumulation in transformed plants (Hain et al, 1990;Thomzik et al, 1997;Hipskind and Paiva, 2000;Coutos-Thévenot et al, 2001). As a control, tobacco leaves were infiltrated with Agrobacterium harboring a 35S-GFP construct (Haseloff et al, 1997).…”

“…In consequence, the ability to synthesize stilbenes can be conferred to many plant species by expressing an STS gene (Hain et al, 1990;Hipskind and Paiva, 2000;Yu et al, 2006). In this work, we used tobacco leaves capable of stilbene biosynthesis due to the transient expression of the grapevine STS VST1 cDNA to characterize the activity of ROMT in planta.…”

“…Given the antibiotic properties of stilbenes, STS genes have been used as targets for transgenic crop enhancement. STS genes from peanut (Arachis hypogaea) and grape have been expressed in tobacco and alfalfa (Medicago sativa), respectively, and in both cases, enhanced resistance to pathogenic fungi was conferred to the transgenic host plants (Hain et al, 1990;Hipskind and Paiva, 2000). Due to the higher fungitoxicity of pterostilbene compared to resveratrol (Pezet and Pont, 1990;Pezet et al, 2004), the combination of STS and ROMT genes in transgenic plants may lead to improved resistance characteristics.…”

Engineering disease resistance in plants

“…Attempts to engineer resistance using this strategy have worked in tobacco and alfalfa He and Dixon, 2000;Hipskind and Paiva, 2000), but the number of successes has remained low, and the level of resistance relatively modest. It is questionable whether this strategy will ever be widely used.…”