Biosynthesis of the 6a-hydroxypterocarpan phytoalexin pisatin in Pisum sativum

Comparative feeding experiments in cupric chloride-treated Pisum sativum pods and seedlings have demonstrated excellent incorporation into the 6a-h     

(−)-Pisatin,an induced pterocarpan metabolite of abnormal configuration from Pisum sativum

Pea (Pisum sativum) tissues, on treatment with aqueous CuCl₂ synthesize the 6a-hydroxypterocarpan phytoalexin (+) - (6aR, 11aR) - pisatin. By supplying (?) - (6aR, 11aR) - maackiain during this induction process, sigruficant quantities of ( ? ) - (6aS, 11aS) - pisatin are produced, immature pods being most effective. Pisatin levels are considerably reduced when compared with the normal induction process, but may contain as much as 92% (?)-pisatin. This confirms that the 6a-hydroxylation of maackiain during the biosynthesis of pisatin must proceed with retention of configuration at C-6a.     

Biosynthesis of pterocarpan phytoalexins in Trifolium pratense

Feeding experiments have demonstrated that 7,2′-dihydroxy-4′-methoxy-isoflavone-[¹⁴C-Me] and -isoflavanone-[¹⁴C-Me] are extremely efficient precursors of the phytoalexin demethylhomopterocarpin in Cu²⁺-treated red clover seedlings. Neither of these compounds, nor demethylhomopterocarpin-[¹⁴C-Me], was incorporated into a second pterocarpan phytoalexin, maackiain. 3-Hydroxy-9-methoxypterocarp-6a-ene-[¹⁴C-Me] was a poor precursor of both pterocarpans. A biosynthetic pathway to demethylhomopterocarpin via 2′-hydroxylation of formononetin (7-hydroxy-4′-methoxyisoflavone) and subsequent reduction to the isoflavanone is proposed. The conversion of this isoflavanone into the pterocarpan may involve the corresponding isoflavanol and a carbonium ion intermediate. The branch-point to maackiain is probably at the formononetin stage. The presence of two coumestans, 9-O-methylcoumestrol and medicagol, previously unreported in red clover, is demonstrated. Biosynthetic implications are discussed.     

Biosynthesis of the isoflavan isomucronulatol: Origin of the 2′,3′,4′-oxygenation pattern

Feeding experiments with ¹⁴C-labelled isoflavones in seedlings and pods of bladder senna (Colutea arborescens) have demonstrated that 7-hydroxy-4′-methoxyisoflavone (formononetin), 7,3′-dihydroxy-4′-methoxyisoflavone (calycosin), 7,2′,3′-trihydroxy-4′-methoxyisoflavone (koparin) and 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone are excellent precursors of (3R)-isomucronulatol (7,2′-dihydroxy-3′,4′-dimethoxyisoflavan). 7,2′-Dihydroxy- 4′-methoxyisoflavone (2′-hydroxyformononetin) and 7-hydroxy-3′,4′-dimethoxyisoflavone (cladrin) were, however, poor substrates. Thus, the biosynthetic sequence to isomucronulatol from formononetin involves 3′-hydroxylation, 2′-hydroxylation and then 3′-O-methylation, followed presumably by stereospecific reduction of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone. Treatment of 2′,3′,4′-trimethoxyisoflavones with aluminium chloride in acetonitrile gives modest yields of 2′,3′-dihydroxy derivatives rather than 2′-monohydroxyisoflavones, and thus provides a convenient access to 2′,3′-dihydroxyisoflavones and related pterocarpans.     

Biosynthesis of the phytoalexin phaseollin in Phaseolus vulgaris

Feeding experiments in CuCl₂-treated French bean (Phaseolus vulgaris) seedlings have demonstrated that labelled 2′,4′,4-trihydroxychalcone, daidzein, 7,2′,4′-trihydroxyisoflavone, 3,9-dihydroxypterocarpan and phaseollidin are all good precursors of the pterocarpan phytoalexin phaseollin. These compounds represent a logical sequence in the biosynthetic pathway to phaseollin.     

Isoflavone precursors of the pterocarpan phytoalexin maackiain in Trifolium pratense

Feeding experiments in Cu²⁺-treated red clover seedlings have demonstrated that ¹⁴C-labelled isoflavones formononetin, 7,3′-dihydroxy-4′-me     

Biosynthesis of the 2-arylbenzofuran phytoalexin vignafuran in Vigna unguiculata

Feeding experiments using l-phenylalanine-[U-¹⁴C], dl-phenylalanine-[1-¹⁴C] and -[2-¹⁴C] together with degradative studies have been used to investigate the biosynthesis of the 2-arylbenzofuran phytoalexin vignafuran in UV-treated seedlings of cowpea (Vigna unguiculata). During the biosynthetic process, C-3 of phenylalanine appears to be lost, and the resulting labelling pattern is consistent with vignafuran being derived from an isoflavonoid precursor, but the phenylalanine-derived aromatic ring becomes the 2-aryl substituent and not part of the benzofuran system. A previously proposed pathway to 2-arylbenzofurans by loss of C-6 from a coumestan is thus excluded. Alternative routes are suggested.     

Isolation of the cDNAs encoding (+)6a-hydroxymaackiain 3-O-methyltransferase,the terminal step for the synthesis of the phytoalexin pisatin in Pisum satvium

Pisatin is the major phytoalexin produced by pea upon microbial infection. The enzyme that catalyzes the terminal step in the pisatin biosynthetic pathway is (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM). We report here the isolation and characterization of two HMM cDNA clones (pHMM1 and pHMM2) made from RNA obtained from Nectria haematococca-infected pea tissue. The two clones were confirmed to encode HMM activity by heterologous expression in Escherichia coli/. The substrate specificity of the methyltransferases in E. coli was similar to the activity detected in CuCl₂-treated pea tissue. Nucleotide sequence analysis of Hmm1 and Hmm2 revealed an open reading frame of 1080 bp and 360 amino acid residues which would encode 40.36 kda and 40.41 kDa polypeptides, respectively. The deduced amino acid sequence of HMM1 has 95.8% identity to HMM2, 40.6% identity to Zrp4, a putative O-methyltransferase (OMT) in maize root, and 39.1% to pBH72-F1, a putative OMT induced in barley by fungal pathogens or UV light. Comparison of the deduced amino acid sequences of the cDNA clones to OMTs from other higher plants identified the binding sites of S-adenosylmethionine (AdoMet). Southern blot analysis showed two closely linked genes with strong homology to Hmm in the pea genome.     

Biosynthesis of pterocarpan,isoflavan and coumestan metabolites of Medicago sativa: chalcone,isoflavone and isoflavanone precursors

Comparative feeding experiments in CuCl₂,- and UV-treated lucerne (Medicago sativa) seedlings have shown that 2′,4,4′-trihydroxychalcone-[carbonyl-¹⁴C] and formononetin-[Me-¹⁴C] but not 2′,4′-dihydroxy-4-methoxychalcone-[carbonyl- ¹⁴C] or daidzein-[4-¹⁴C] were incorporated into the phytoalexins demethylhomopterocarpin, sativan and vestitol, and also into 9-O-methylcoumestrol. The synthesis of 9-O-methylcoumestrol is greatly stimulated by this abiotic treatment but coumestrol production is not noticeably affected. Daidzein and the trihydroxychalcone were precursors of coumestrol. The results are interpreted in favour of a mechanism in which methylation is an integral part of the aryl migration process associated with the biosynthesisof 4′-methoxyisoflavonoids. Formononetin, 2′,7-dihydroxy-4′-methoxyisoflavone-[Me-¹⁴C], 7-hydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] and 2′,7-dihydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] were all excellent precursors of demethylhomopterocarpin, sativan, vestitol and 9-O-methylcoumestrol, and thus a metabolic grid may be involved in their biosynthetic origin.     

Pterocarpan biosynthesis: Chalone and isoflavone precursors of demethylhomopterocarpin and maackiain in Trifolium pratense

Feeding experiments with dl-phenylalanine-[1-¹⁴C] have demonstrated the de novo synthesis of the pterocarpan phytoalexins demethylhomopterocarpin and maackiain in CuCl₂-treated Trifolium pratense L. seedlings. 2′,4′,4-Trihydroxychalcone-[carbonyl-¹⁴C] and 7-hydroxy-4′-methoxyisoflavone-[methyl-¹⁴C] (formononetin) were readily incorporated into demethylhomopterocarpin and maackiain, but 2′,4′-dihydroxy-4-methoxychalcone-[carbonyl-¹⁴C] and 7,4′-dihydroxyisoflavone-[T] (daidzein) proved inefficient precursors. The trihydroxychalcone was also an excellent precursor of formononetin in T. pratense, but the trihydroxymethoxychalcone and daidzen were poorly incorporated. These observations offer further evidence that methylation may be an associated part of the mechanism for aryl migration in the biosynthesis of formononetin.     

Biosynthesis of pterocarpan and isoflavan phytoalexins in Medicago sativa: the biochemical interconversion of pterocarpans and 2′-hydroxyisoflavans

Feeding experiments have shown that 2′-7-dihydroxy-4′-methoxy-isoflavone-[Me-¹⁴C] and -isoflavanone-[Me-¹⁴C] are efficient precursors of the phytoalexins demethylhomopterocarpin, sativan and vesitol in CuCl₂-treated lucerne (Medicago sativa) seedlings. Demethylhomopterocarpin-[Me-¹⁴C] was also incorporated into sativan and vestitol, and vestitol-[Me-¹⁴C] was incorporated into demethylhomopterocarpin and sativan. Thus, the pterocarpan demethylhomopterocarpin and the 2′-hydroxy-isoflavan vestitol are interconvertible in M. sativa, but incorporation data, and the results of kinetic feeding experiments with l-phenylalanine-[U-¹⁴C] suggest that these compounds are synthesized simultaneously from a common intermediate, which could be involved in the interconversion. A carbonium ion, derived from an isoflavanol, a likely intermediate in the biosynthetic reductive sequence from 2′,7-dihydroxy-4′-methoxy-isoflavone and -isoflavanone, is proposed as this common intermediate. 7-Hydroxy-2′,4′-dimethoxyisoflavone-[4′-Me-¹⁴C] was a very poor precursor of all three phytoalexins. Sativan, then, is most probably derived by methylation of vestitol. The incorporation of vestitol-[Me-¹⁴C] into demethylhomopterocarpin, but not into maackiain, pterocarpan phytoalexins of red clover (Trifolium pratense), is also demonstrated.     

Effect of pisatin inducing fungi on pea polyamines

The spermine and spermidine content of pea pod tissue is not significantly altered by inoculation with the pisatin-inducing fungi, Fusarium solani. Although these polyamines induce pisatin, it appears that they do not accumulate in levels sufficient to serve as internal mediators of pisatin production in infected tissues.     

Biosynthesis of pterocarpan,isoflavan and coumestan metabolites of Medicago sativa: The role of an isoflav-3-ene

Feeding experiments in CUCl₂- and UV-treated lucerne (Medicago sativa) seedlings have shown that demethylhomopterocarpin- [6a-³H, Me- ¹⁴C] is incorporated into vestitol and sativan without any loss of ³H label, and vestitol-[3-³H, Me- ¹⁴C] is similarly incorporated into demethylhomopterocarpin and sativan with retention of the ³H: ¹⁴C ratio. Thus, an isoflav-3-ene intermediate in the interconversion of demethylhomopterocarpin and vestitol is excluded. 7,2′-Dihydroxy-4′-methoxyisoflav-3-ene- [Me-¹⁴C] was not incorporated into the three phytoalexins, but was an excellent precursor of 9-O-methylcoumestrol, as also was 7,2′-dihydroxy-4′-methoxyisoflav-3-en-2-one- [Me-¹⁴C]. A biosynthetic pathway to coumestans via isoflav-3-enes and 3-arylcoumarins is proposed. A metabolic scheme in M. sativa interrelating eight classes of naturally occurring isoflavonoids is presented.     

Identification of 1 a-hydroxy phaseollone,a phaseollin metabolite produced by Fusarium solani

A structure for the phaseollin metabolite of Fusarium solani f. sp phaseoli has been proposed and assigned the name 1 a-hydroxyphaseollone.     

Biosynthesis of the phytoalexin wyerone in Vicia faba

In contrast to earlier results [1-¹⁴C] acetate, [2-¹⁴C] malonate and [n 9,10-³H] oleate show significant incorporations into wyerone and related Vicia faba phytoalexins, following infection by Botrytis cinerea.     

Biosynthesis of Se-Methylselenocysteine in lima beans

It has been shown that maturing seeds of lima beans synthesize Se-methylselenocysteine, the selenium analogue of S-methyleysteine. The latter amino acid is a natural constituent of these seeds. The leaves of lima beans do not contain detectable amounts of S-methylcysteine, and in this tissue Se-methionine appears to be the principal product of selenate assimilation.     

Biosynthesis of triterpenoid sapogenols in soybean and alfalfa seedlings

By incubation of germinating soybeans with mevalonate-[2-¹⁴C] (MVA), radioactivity was incorporated into four sapogenols which were identified by TLC. Unequivocal evidence for the identity of three of the four sapogenols was provided by co-crystallization to constant specific radioactivity. The partition of incorporated radioactivity into lipid- and water-soluble fractions and the pattern of radioactivity of individual sapogenols varied with the mode of administering labeled substrates to soybean seedlings, such as incubation of germinating soybeans with MVA-[2-¹⁴C], immersion of roots into MVA-[2-¹⁴C] or foliar application of squalene-[¹⁴C]. When alfalfa seedlings were incubated with MVA-[2-¹⁴C], about two-thirds of the radioactivity incorporated into the sapogenols was associated with medicagenic acid.     

Phaseollin formation and metabolism in Phaseolus vulgaris

Following fungal-inoculation, P. vulgaris was found to produce small amounts of 7,4′-dihydroxyisoflavone (daidzein), 7,2′,4′-trihydroxyisoflavone, 7,2′,4′-trihydroxyisoflavanone, (6aR, 11aR)-3,9-dihydroxypterocarpan, and (3R)-7,2′,4′-trihydroxyisoflavan. The structures of the latter four compounds were confirmed by synthesis. The principal pterocarpans isolated were phaseollidin and phaseollin and ORD spectra indicate that these compounds have the same (6aR, 11aR)-configuration as 3,9-dihydroxypterocarpan. A pathway leading to phaseollidin and phaseollin is proposed involving 2′-hydroxylation of daidzein, reduction to the isoflavanone, further reduction, dehydration and cyclization to the pterocarpan, and prenylation to give phaseollidin and then cyclization and dehydrogenation to give phaseollin. No evidence of prenylation at the isoflavone or isoflavanone stage was obtained. The phaseollin metabolite, (6aS, 11aS)-6a-hydroxyphaseollin, was also detected.     

Antifungal activity of pterocarpans and other selected isoflavonoids

Six pterocarpans and 11 structurally related isoflavonoids were tested for antifungal activity against Fusarium solani f. sp. cucurbitae and Aphanomyces euteiches. Representatives from the pterocarpan, isoflavan, and 6a, 11a-dehydropterocarpan classes of isoflavonoids were found that were antifungal. The activity of the antifungal isoflavonoids does not appear to be dependent on a common 3-dimensional shape.