The isoflavonoid phytoalexin pisatin is synthesized by Pisum sativum in response to microbial infection and certain other forms of stress. An enzyme which synthesizes pisatin by methylating the 3-hydroxyl of (+)6a-hydroxymaackiain (HMK) was extracted from CuCI2-stressed pea seedlings. The enzyme was enriched 370-fold by (NH4)2SO4 precipitation, DEAE chromatography, chromatofocusing, and hydrophobic interaction chromatography (HIC), to a specific activity of 8.2 microkatals per gram protein.Enzyme activity profiles from chromatofocusing and HIC columns suggested the presence of two isozymes, of pi 5.2 and 4.9. Nondenaturing gel filtration of the HIC-purified enzyme gave a single peak of activity at the same elution volume as BSA (66 kilodaltons); the active fractions showed two proteins upon SDS-PAGE, of M, 66,000 and 43,000. The smaller protein was most abundant in chromatographic fractions containing peak enzyme activity throughout purification. In a partially purified preparation, this 43 kilodalton protein was the only one photoaffinity labelled by [3H]S-adenosyl-L-methionine. The purified enzyme preferred the (+) over the (-) stereoisomer of HMK and other pterocarpans; overall, (+)HMK was the best substrate. Km values were 2.3 micromolar for (+)HMK and 35 micromolar for S-adenosyl-L-methionine. The methyltransferase had a pH optimum of 7.9 and no apparent divalent cation requirement.
The hypothesis that arachidonic acid (AA) induction of sesquiterpene accumulation and browning in potato (Solanum tuberosum) is mediated by a lipoxygenase metabolite of AA was tested using lipoxygenase inhibitors. Salicylhydroxamic acid (SHAM) and 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) delayed the response to AA. Inhibition by eicosatetraynoic acid (ETYA) and decreased steroid glycoalkaloid synthesis are observed. These changes are ascribed roles in the defense of potato against P. infestans, since they occur during attenuation of hyphal growth in an incompatible interaction but are not observed in a compatible interaction until the fungus has already colonized the host tissue.Induction of components of HR in potato requires specific elicitors. Two polyunsaturated fatty acids, AA and eicosapentaenoic acid (3, 4), present in P. infestans but not in potato, elicit browning and sesquiterpene accumulation. In addition to these fatty acids, certain polyamines, such as PLL, although not native to P. infestans or to potato, are active as elicitors of sesquiterpene accumulation and browning (M Zook, JA Kuc, unpublished data). Specific activities of the polyamines as elicitors are severalfold lower than those of the fatty acids; nevertheless, the polyamines are useful tools for elucidation of the sequence of events which induce these components of HR in potato.A survey of lipids structurally related to AA indicated that the 20-carbon chain ofAA was necessary and the free carboxyl group was optimum for elicitor activity (19). Among the free, 20-carbon polyunsaturated fatty acids, those with a minimum double bond configuration of A5,8, 11 were by far the most active elicitors. The significance of this fatty acid structure in plants has not been reported previously. In animals, the same kinds of 20-carbon fatty acids are precursors of the highly physiologically active leukotrienes, which are formed via the action of lipoxygenase (12)
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