Synthesis of the Phytoalexin Pisatin by a Methyltransferase from Pea

Abstract

Previous labeling studies in vivo suggest that the terminal step of (+)pisatin biosynthesis in Pisum sativum L. is methylation of the phenol (+)6a-hydroxymaackiain (HMK). We have found that extracts from pea seedlings perform this reaction, using S-adenosylmethionine as the methyl donor. The enzyme activity was induced by microbial infection or treatment with CuC12, which elicit pisatin synthesis, though some activity was also present in healthy tissues. It has been reported that CuCl2-treated pea tissue provided with (-)HMK or (-)maackiain can synthesize (-)pisatin. Our extract showed no methyltransferase activity dependent on either of these substrates. Methylation of (+)maackiain was detectable, but much slower than that of (+)HMK.Pisatin is one of a large group of low mol wt toxic compounds known to be synthesized de novo by plant tissues in response to certain kinds of stress, including microbial infection. Such compounds, called phytoalexins, are of interest in part because of their possible role as a determinant ofdisease resistance in plants. In addition, phytoalexin synthesis has been a useful subject for studies on the regulation of gene expression in higher plants. These studies have shown that the accumulation of isoflavonoid phytoalexins in legumes is preceded by increases in the levels of some of the biosynthetic enzymes (4, 10); where examined, the increases have been found to be mediated primarily by higher concentrations of the corresponding mRNAs (15,16).The enzymes whose regulation has been examined in most detail are phenylalanine ammonia-lyase and chalcone synthase, enzymes required not only for phytoalexin synthesis but also for other important pathways of secondary metabolism. These enzymes are also inducible by some stimuli which do not elicit phytoalexin synthesis (8, 12), so it seems likely that steps in the later, committed portion of the phytoalexin pathway are important in controlling the plant's phytoalexin response. Unfortunately, the enzymes catalyzing these late reactions are largely unknown. Recently two such enzymes were obtained from soybean. Levels of these enzyme activities, a monooxygenase (9) and a dimethylallyltransferase (11), were also increased by conditions which elicited phytoalexin production.We now report that Pisum sativum possesses an inducible methyltransferase which catalyzes the terminal step in the biosynthesis of (+)pisatin, the major phytoalexin produced by this plant. MATERIALS AND METHODS Plant Material. Pea seeds (cv Alaska) were treated for 15 min with a 1:20 dilution of commercial bleach (5.25% NaOCl) in 80% ethanol, and maintained under sterile conditions until harvested. The seeds were rinsed with H20, spread on paper toweling, and imbibed overnight with 4 ml H20/g seeds at 18°C in the dark. Excess water was removed and germination was continued for 3 d, at which time the radicles were 2 to 4 cm long. The seedlings were covered with 5 mM CuCl2 (control seedlings with H20) for 1 h, drained, and incubated further until used for enzyme preparati...

Purification and Characterization of S-Adenosyl-l-methionine:6a-Hydroxymaackiain 3-O-Methyltransferase from Pisum sativum

“…Furthermore, HMKMT exhibited a low Km for (+)HMK. These facts, combined with the observation that it is induced by treatments which elicit pisatin synthesis (23), suggest that the enzyme we purified is induced specifically as part of the phytoalexin response.…”

“…The reaction was stopped with 350 ,uL 0.3 M sodium citrate (pH 4) and 4.5 mL of 0.55% PPO in toluene. The two phase system was agitated in a vortex mixer, and the content of nonpolar radioactivity was measured directly by scintillation counting (23). One unit of enzyme activity (1 kat) was the amount producing 1 mol of pisatin per s under these conditions.…”

“…the 3-0-methylation of HMK. 3 Recently, we reported the detection in pea of a methyltransferase, inducible by infection or treatment with CuCl2, which catalyzes this step (23).…”

“…The accumulation of these compounds in legumes is preceded by increases in the extractable activities of many of the biosynthetic enzymes and their corresponding mRNAs, which are in turn newly transcribed (19). Although several enzymes specific for the isoflavonoid branch of the phenylpropanoid pathway have now been detected (2,8,12,17,(22)(23)(24), studies at the nucleic acid level have been reported only for earlier enzymes which provide the precursors of lignin and flavonoid pigments as well (5). Purification of the later enzymes and cloning of their genes will allow comparison of their regulation with that of the enzymes acting earlier in the phenylpropanoid pathway.…”

Identification and genetic regulation of the chalcone synthase multigene family in pea.

“…Severa1 lines of evidence suggested that CHS was synthesized in root tissue: root tissue treated with CuCI, synthesizes pisatin (Banks and Dewick, 1982;Sweigard, Matthews, and van Etten, 1986), and the expression of nodulation-specific genes in Rhizobium is induced by flavones and flavanones extracted from pea roots (Firmin et al, 1986;Peters et al, 1986;Redmond et al, 1986). Preliminary induction time course experiments were carried out to optimize for CHS mRNA expression in CuCI,-induced roots.…”