Alfalfa genetically transformed to produce resveratrol,
a foreign antifungal compound
N.L. Paiva and J.D. Hipskind
Plant Biology Division, The Samuel Roberts Noble Foundation,
P.O. Box 2180, Ardmore, OK 73402
Alfalfa naturally produces antifungal compounds (phytoalexins) such as medicarpin as part of its defense response to invading pathogenic fungi. However, many alfalfa pathogens have co-evolved mechanisms for avoiding the toxic effects of their host’s native phytoalexins. We have therefore introduced into alfalfa a gene encoding resveratrol synthase (RS) from peanut, which converts coumaroyl-CoA and malonyl-CoA to 3,5,4’-trihydroxystilbene, commonly known as resveratrol. Resveratrol is a phytoalexin found in a wide range of plants including peanut and grape, but is not found in alfalfa. The main goals of this work are to determine if this foreign metabolite accumulates to high levels or is rapidly degraded by alfalfa, to test whether or not accumulation of this foreign phytoalexin improves the resistance of alfalfa to any pathogens, and to check for any negative impact of resveratrol accumulation on plant health. Other labs have indicated that introduction of the RS gene from grape into transgenic tobacco conferred resistance to Botrytis, a weak tobacco pathogen (1). Strong expression of grape RS under the CaMV 35S (cauliflower mosaic virus 35S ribosomal subunit) promoter resulted in high levels of resveratrol (via ELISA assay) in transgenic tobacco, but the plants had non-viable pollen and lost their pink flower color(2).
To date we have analyzed transgenic alfalfa plants expressing a resveratrol synthase cDNA clone from peanut, under the control of a very strong CaMV 35S promoter with a duplicated enhancer region. We do not observe the accumulation of any free resveratrol, but do detect very high levels of a resveratrol-glucose conjugate (resveratrol-3-O-beta-D-glucopyranoside, or "piceid"), using HPLC analysis. The concentration varies with the age of the tissues. For example, in higher accumulating lines, levels decrease from 150-170 ug/g.fr.wt. in younger leaves to 50 ug/g.fr.wt. in older leaves. On a molar basis, these levels equal or exceed the maximum levels observed for the natural alfalfa phytoalexins, and alfalfa pathogens were inhibited by resveratrol at these levels in agar-plate bioassays. Pathogen challenges of the transgenic plants are still in progress.
Unlike the reports for tobacco, high resveratrol-accumulating transgenic alfalfa plants show no visible differences compared to parent RegenSY and "empty vector" control plants, with respect to flower color, seed set following self-pollination, and nodulation by Rhizobium meliloti. A field test is currently underway to compare the levels of resveratrol observed in transgenics under greenhouse conditions with levels under field conditions. High levels of resveratrol and its glycosides are found naturally in wine and grapes, and may have beneficial effects on human health, including tumor-suppressing activities(3). We are pursuing animal tests to determine if a diet containing high resveratrol alfalfa is more beneficial than a diet supplemented with wild-type alfalfa.
References:
(1) Hain et al., 1993. Nature 361: 153-156.
(2) Fischer et al., 1997. Plant Journal 11:489-498.
(3) Jang et al., 1997. Science 275: 218-220.