1.9.27
PHYTOALEXIN ENGINEERING IN ALFALFA: INTRODUCTION OF A RESVERATROL SYNTHASE FROM ARACHIS HYPOGAEA

JD HIPSKIND and NL PAIVA

Samuel Roberts Noble Foundation, Box 2180, Ardmore, OK 73402, USA

Background and objectives
Stilbenes are a widely distributed class of phenolic compounds which exhibit a broad spectrum of anti-fungal activity [1]. Some stilbenes are present constitutively as part of the plants preformed defenses or are synthesized as phytoalexins in response to biotic or abiotic stress. The biosynthesis of these compounds is initiated by a resveratrol synthase (E.C. 2.3.1) through the condensation of one molecule of p-coumaroyl CoA and three molecules of malonyl CoA to form trans-3, 5, 4'-trihydroxystilbene (resveratrol). Because only one gene is required for the synthesis of this compound from precursors commonly found in plants, it has been proposed that the introduction of a resveratrol synthase might offer a strategy to engineer increased resistance to fungal pathogens [1]. This has already been demonstrated by the expression of a grapevine resveratrol synthase in transgenic tobacco plants which resulted in a significant increase in resistance to Botrytis cinerea [1]. In this presentation, we have introduced a resveratrol synthase (RS) cDNA from groundnut (Arachis hypogaea ) into alfalfa ( Medicago sativa, lucerne), a legume which does not naturally accumulate stilbenes. In separate experiments, the cDNA will be transcriptionally regulated by the constitutively active CaMV-35S dual enhancer promoter or a pathogen-inducible promoter from the alfalfa isoflavone reductase (IFR) gene [2]. Transformants will be analyzed for the biosynthesis of resveratrol and for altered plant-microbe interaction.

Results and conclusions
Southern blot analysis of genomic DNA extracted from selected transformants identified a minimum of 14 independent lines harboring the CaMV-35S:RS construct and 13 lines harboring the IFR:RS construct. PCR reactions using these same DNA extracts as template and resveratrol synthase specific primers confirmed that several of the independent transformants did contain the full length RS coding region. Acetone extracts from CaMV-35S driven lines were analyzed by high pressure liquid chromatography (HPLC) for resveratrol. Preliminary experiments identified a new peak in the transgenic plants with an absorbance spectrum nearly identical to a resveratrol standard. However, the HPLC retention time for this unknown compound suggested a resveratrol glyco-conjugate. The purified compound was digested with a O-glucosidase to release resveratrol. The concentration of resveratrol conjugate in the leaves and stems CaMV-35S:RS lines ranged from 50 to 175 mg/g fw. Agar plate bioassays suggest that the concentrations of resveratrol extracted from the transgenic lines are sufficient to inhibit mycelial growth of several alfalfa fungal pathogens. As expected, however, no resveratrol could be recovered from leaves and stems of the IFR:RS plants. This promoter is constitutively active in alfalfa roots but not in the leaves and stems [2]. Currently, IFR:RS plants are being examine for pathogen-induced accumulation of resveratrol. In addition, the roots of both lines will be tested for the constitutive accumulation of the resveratrol conjugate. Subsequent experiments will be performed in order to determine if the synthesis of resveratrol in alfalfa increases resistance to fungal pathogens or alters interactions with beneficial microbes.

References
1. Hain R, Reif H-J, Krause E et al., 1993. Nature 361, 153-156.
2. Oommen A, Dixon R, Paiva NL, 1994. Plant Cell 6, 1789-1803.