Induction of phytoalexin synthesis in soybean. Stereospecific 3,9‐dihydroxypterocarpan 6a‐hydroxylase from elicitor‐induced soybean cell cultures

Hagmann, Marie-Luise; Heller, Werner; Grisebach, Hans

doi:10.1111/j.1432-1033.1984.tb08259.x

Cited by 74 publications

(20 citation statements)

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“…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.…”

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confidence: 99%

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

Preisig

Matthews²,

VanEtten³

1989

Plant Physiol.

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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.

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confidence: 99%

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

Preisig

Matthews²,

VanEtten³

1989

Plant Physiol.

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“…Harosoy 63) and treatment with glucan elicitor from Phytophthora megasperma f.sp. glycinea was carried out as described previously [6]. Five-day-old seedlings of soybean (c.v. Harosoy 63) were treated with elicitor as described El.…”

Section: Plant Materialsmentioning

confidence: 99%

Enzymatic rearrangement of flavanone to isoflavone

Hagmann

Grisebach

1984

FEBS Letters

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A microsomal preparation from elicitor-challenged soybean cell suspension cultures catalyzes an NADPHdependent and dioxygen-dependent rearrangement of (2S)-naringenin to genistein. The conversion of (2S)-5,4'-dihydroxyflavanone to daidzein was also catalyzed by this preparation. The 'isoflavone synthase' was found in elicitor-challenged soybean seedlings as well. Biosynthesis Zsofavone Monooxygenase

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“…Glycinol (3,6a,9-trihydroxypterocarpan) and the glyceollin isomers are thought to be synthesized via 6a-hydroxylation of (-)3,9-dihydroxypterocarpan, based on evidence from incorporation of specific precursors (5) similar to the evidence supporting the pisatin pathway of Figure 1. In addition, however, an enzyme catalyzing this reaction has been obtained from elicitor-treated soybean, and it appears to be a monooxygenase since it was found to be dependent on NADPH and 02 (14).…”

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Role of Oxygenases in Pisatin Biosynthesis and in the Fungal Degradation of Maackiain

Matthews¹,

Weiner²,

Matthews³

et al. 1987

Plant Physiol.

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Some isolates of the plant pathogen Nectria haematococca detoxify the isoflavonoid phytoalexin (-)maackiain by hydroxylation at carbon 6a. Precursor feeding studies strongly suggest that the penultimate step in (+)pisatin biosynthesis by Pisum sativum is 6a-hydroxylation of (+)maackiain. We have used "O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under '"02, the product was labeled with 99% efficiency; no label was incorporated by metabolism in H210. Pisatin synthesized by pea pods in the presence of 1802 or H2180 was a mixture of molecules containing up to three labeled oxygen atoms. Primary mass spectra of such mixtures were complex but were greatly simplified by tandem MS. This analysis indicated that the 6a oxygen of pisatin was derived from H20 and not from 02. Labeling patterns for the other five oxygen atoms were consistent with the proposed pathway for biosynthesis of pisatin and related isoflavonoids. We conclude that the fungal hydroxylation of maackiain is catalyzed by an oxygenase, but the biosynthetic route to the 6a hydroxyl of pisatin is unknown.Several pterocarpan phytoalexins are known to be detoxified by Nectria haematococca or by related fungi with the same imperfect state, Fusarium solani. Pisatin is demethylated by a Cyt P-450 monooxygenase (16). Phaseollin is hydroxylated to la-hydroxyphaseollone by a reaction which also appears to be oxygenase-mediated, since 180 labeling has shown that the new oxygen atom is derived from 02 (15). Maackiain and medicarpin are metabolized by one or more of three different initial reactions, depending on the fungal isolate; all three involve the net addition of one oxygen atom (I1, 12). One of these reactions produces the la-hydroxydienone analogous to the phaseollin metabolite. Another, the hydroxylation at carbon 6a, also involves incorporation of an atom from 02, as reported below.Oxygenases are also thought to be responsible for several reactions in the biosynthesis of these phytoalexins. In particular, the 6a-hydroxylation of maackiain has been proposed to be a step in pisatin biosynthesis by Pisum sativum (Fig. 1) (2-4). This reaction is not identical with the fungal conversion, since the fungus metabolized only (-)maackiain (23) whereas the expected precursor of (+)pisatin, the isomer normally produced by pea, would be the enantiomer (+)maackiain. larity between these two reactions, which appear to play such opposed roles in the interaction between host and parasite, prompted us to examine both of them in this study.MATERIALS AND METHODS Biological Materials. Nectria haematococca Berk. and Br. mating population VI isolate T-95, from our culture collection (22, 23), was maintained on V-8 juice agar and grown in glucoseasparagine (GA) liquid medium as described (25). Pisum sativum L. cultivar Alaska was grown in a greenhouse.6a-Hydroxylation of Maackiain. A 3-d culture of N. haematococca in GA medium was filtered, weighed, and suspended in double strength GA medium at 60 m...

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Induction of phytoalexin synthesis in soybean. Stereospecific 3,9‐dihydroxypterocarpan 6a‐hydroxylase from elicitor‐induced soybean cell cultures

Cited by 74 publications

References 20 publications

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

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

Enzymatic rearrangement of flavanone to isoflavone

Role of Oxygenases in Pisatin Biosynthesis and in the Fungal Degradation of Maackiain

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