(−)-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.     

Chromatographic analysis of isoflavonoid accumulation in stressed Pisum sativum

High-performance liquid chromatography has been used to study isoflavonoid accumulation in copper(II) chloride stressed Pisum sativum. Liquiritigenin, isoliquiritigenin, formononetin, pseudobaptigenin, afrormosin and anhydropisatin have been identified in addition to the pterocarpan phytoalexin pisatin. The relationships of these metabolites to isoflavonoid biosynthesis and stress response in pea are discussed.     

Degradation of the isoflavone biochanin a by isolates of Nectria haematococca (Fusarium solani)

Twelve isolates of Nectria haematococca, mating population VI (Fusarium solani) previously characterized for their virulence on pea plants and their ability to degrade the phytoalexin pisatin were assayed for the catabolism of the isoflavone biochanin A (5,7-dihydroxy-4′-methoxyisoflavone). Eleven isolates catabolized the isoflavone along the pathway: biochanin A → dihydrobiochanin A → 3-(p-methoxyphenyl)-6-hydroxy-γ-pyrone → p-methoxyphenylacetic acid → p-hydroxyphenylacetic acid → 3,4-dihydroxyphenylacetic acid.     

Metabolism of the phytoalexins medicarpin and maackiain by Fusarium solani

Non-inhibitory concentrations of the pterocarpan phytoalexin medicarpin were completely metabolized by isolates of Fusarium solani f. sp. pisi, f. sp. cucurbitae, f. sp. phaseoli and two other F. solani isolates genetically related to f. sp. pisi during 24 hr of growth in liquid medium. The major metabolic products accumulated without significant further degradation. Medicarpin was modified at one of three adjacent carbon atoms to form either an isoflavanone derivative, a 1a-hydroxydienone derivative or 6a-hydroxymedicarpin. Whereas each isolate degraded medicarpin to one or more metabolises, the isolates varied as to which metabolise they produced. Maackiain, another pterocarpan phytoalexin, was also metabolized by all the isolates to products analogous to those formed from medicarpin. The ability to metabolize medicarpin and maackiain was not always associated with the ability to metabolize pisatin and phaseollin, two other pterocarpan phytoalexins that were degraded by several of the isolates. Tolerance of medicarpin and maackiain was similarly not always associated with tolerance to pisatin.     

An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition.

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.     

An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition.

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.     

Detoxification of the phytoalexin pisatin by a fungal cytochrome P-450

The fungus Nectria haematococca, a pathogen of garden pea (Pisum sativum), can demethylate pisatin, an antimicrobial compound synthesized by infected pea tissue. The phenolic product is less toxic than pisatin to many microorganisms. Cell extracts catalyzing pisatin demethylation were obtained from N. haematococca, and the properties of the reaction were examined. The enzyme activity was greatest in the high-speed pellet fraction, in which rates up to 20 nmol/min/mg protein were observed. The K_m for pisatin was relatively low, less than 5 μm. The reaction was dependent on NADPH, which could not be replaced by any other cofactor tested. However, in the presence of NADPH, NADH increased the rate of demethylation. Oxygen uptake by the enzyme was stimulated by addition of pisatin, the increment of oxygen utilization being approximately equimolar with pisatin added. Formaldehyde was a product of the reaction. The effects of various inhibitors were tested to determine whether this reaction is mediated by cytochrome P-450. The respiratory inhibitors KCN (1 mm) and antimycin A strongly inhibited the demethylation of pisatin by intact cells of the fungus, but not by the NADPH-supplemented enzyme. The cytochrome P-450 inhibitors SKF 525-A and 1-(2-isopropylphenyl)imidazole inhibited demethylation both in whole cells and in the enzyme preparation, though the latter compound was effective only at high concentrations. Most other cytochrome P-450 inhibitors tested had little effect. However the reaction was quite sensitive to CO, and this inhibition was readily reversed by light at wavelengths near 450 nm. It is concluded that pisatin demethylase is a cytochrome P-450 monooxygenase.     

Expression of pisatin demethylating ability in Nectria haematococca

A mycelial suspension of Nectria haematococca completely demethylated 0.1 mM pisatin in 2 h in a medium free of other carbon sources while no demethylation of pisatin by the fungus occurred in 6 h when 2% glucose was in the medium. Prior exposure of the fungal cells to pisatin in glucosefree medium markedly enhanced the rate of pisatin demethylation, with maximum stimulation occurring 5–9h after the initial exposure. The stimulation of pisatin demethylating ability was relatively specific for pisatin as the inducer. Out of a large variety of isoflavonoids tested the only compounds other than pisatin that stimulated the activity significantly were pterocarpan or isoflavan derivatives. Protoplasts with pisatin demethylating ability were isolated from pisatin-treated mycelium. Attempts to obtain a cell-free system with pisatin demethylating ability from these protoplasts were unsuccessful.     

3-Hydroxymaackiainisoflavan,a pisatin metabolite produced by Fusarium oxysporum f. sp. pisi

3,7,2′-Trihydroxy-4′,5′-methylenedioxyisoflavan, a novel fungal metabolite of pisatin, was identified on the basis of NMR and MS data. The isoflavan arises by reductive opening of the dihydrofuran ring in the pterocarpan 6a-hydroxymaackiain, the first breakdown product of pisatin. The trivial name 3-hydroxymaackiainisoflavan is proposed for this substance.     

Phytoalexin production by leaves of Pisum sativum in relation to senescence

A sterile culture nitrate of Penicillium expansum was shown to induce pisatin synthesis in pea leaf discs. The amount of pisatin produced by pea leaves was shown to decrease as they underwent senescence. N⁶-benzyladenine delayed senescence, and at the same time maintained the production of pisatin at a high level. In darkness, leaf discs maintained on either benzyl-adenine solution or distilled water produced greater amounts of pisatin than leaf discs which were not treated in this way. Benzyladenine also increased pisatin production by leaf discs kept in the light. The relevance of these results to disease resistance and possible mechanisms involved are discussed.     

Dynamics of Free Pisatin Elicitor Accumulation in Infection-droplets of Monilinia fructicola on Pea Endocarp*

Rapid elicitor accumulation in the infection-droplet of the pea-M. fructicola interaction began between 2 and 3 h after inoculation. Rapid accumulation of pisatin began between 2 and 3 h, however, low levels (0.06–0.1 μg/ml) were detected in the infection-droplet as early as 1–2 h following inoculation with the fungus. The pisatin concentration reached levels inhibitory to the fungus between 6 and 12 h (ca. 1–5 μg/ml) and the ED₅₀ value of 10 μg pisatin/ml for mycelial growth of M. fructicola was attained after 14 h. Elicitor activity in infection-droplets after 4 and 18 h was a function of inoculum concentration and pisatin accumulation in diffusate after 40 h was a function of elicitor concentration (linear doseresponse curve). However, when elicitor was applied at zero time, the rate of pisatin accumulation over the first 12 h was indistinguishable from that which occurred when M. fructicola conidia were applied to the endocarp and elicitor accumulated with time. The initial rate of pisatin accumulation therefore appears to be dependent on the pea pod and independent of any time delays associated with conidial germination and elicitor accumulation. However, the final pisatin concentration which accumulated in the infection-droplet was dependent on the, dose' of elicitor irrespective of the nature and timing of the elicitor treatment. The presence of elicitor activity was demonstrated in the interaction in space and time where and when these molecules could function as elicitors of pisatin in vivo.     

An ABC transporter and a cytochrome P450 of Nectria haematococca MPVI are virulence factors on pea and are the major tolerance mechanisms to the phytoalexin pisatin

The fungal plant pathogen Nectria haematococca MPVI produces a cytochrome P450 that is responsible for detoxifying the phytoalexin pisatin, produced as a defense mechanism by its host, garden pea. In this study, we demonstrate that this fungus also produces a specific ATP-binding cassette (ABC) transporter, NhABC1, that enhances its tolerance to pisatin. In addition, although both mechanisms individually contribute to the tolerance of pisatin and act as host-specific virulence factors, mutations in both genes render the fungus even more sensitive to pisatin and essentially nonpathogenic on pea. NhABC1 is rapidly induced after treatment with pisatin in vitro and during infection of pea plants. Furthermore, NhABC1 was able to confer tolerance to the phytoalexin rishitin, produced by potato. NhABC1 appears to be orthologous to GpABC1 of the potato pathogen Gibberella pulicaris and, along with MoABC1 from Magnaporthe oryzae, resides in a phylogenetically related clade enriched with ABC transorters involved in virulence. We propose that NhABC1 and the cytochrome P450 may function in a sequential manner in which the energy expense from pisatin efflux by NhABC1 releases the repression of the cytochrome P450, ultimately allowing pisatin tolerance by two mechanisms. These results demonstrate that a successful pathogen has evolved multiple mechanisms to overcome these plant antimicrobial compounds.     

Effects of pisatin on Dictyostelium discoideum: its relationship to inducible resistance to nystatin and extension to other isoflavonoid phytoalexins

Dictyostelium discoideum amoebae can acquire resistance to otherwise inhibitory concentrations of pisatin, an isoflavonoid phytoalexin of pea, and nystatin, a polyene antibiotic, following pretreatment with sublethal concentrations of these compounds. Additionally, growth on medium containing pisatin can induce nystatin resistance. We show here that distinct mechanisms mediate the inducible resistance to these two compounds because it is possible to isolate mutations that specifically block the induction of nystatin resistance but do not affect the induction of pisatin resistance. Pisatin did not affect wild-type sterol biosynthesis; therefore, the induction of nystatin resistance by pisatin is probably not via an alteration of membrane sterols. The inducible pisatin resistance phenotype was shown to extend to the isoflavonoid phytoalexins maackiain and biochanin A, and all three compounds inhibited the aggregation of amoebae that is normally triggered by starvation. Received: 23 February 1998 / Accepted: 26 June 1998     

Plant Lectins Induce the Production of a Phytoalexin in Pisum sativum

The effects of several plant lectins on the production of apea phytoalexin, pisatin, were examined. Con A, PHA, PNA andPSA each induced the production of pisatin in pea epicotyl tissues,demonstrating that plant lectins can act as elicitors. The productionof pisatin in response to PHA, PNA or PSA was not affected bythe simultaneous presence of the respective hapten sugars, whereashaptens specific for Con A, such as -D-mannose and methyl--D-mannoside,abolished the induction of pisatin by Con A. These results indicatethat the elicitor effect of Con A is attributable to its abilityto bind to specific carbohydrates in pea cells. Induction ofthe production of pisatin by Con A was markedly inhibited bythe suppressor derived from a pea pathogen, Mycosphaerella pinodes,and by several inhibitors related to signal-transduction pathways.It is suggested, therefore, that the Con A-induced productionof pisatin in pea tissues might be associated with activationof a signal-transduction pathway. An additive effect on theaccumulation of pisatin was observed when Con A was presentwith a polysaccharide elicitor from M. pinodes, suggesting thatexogenous Con A does not compete with the recognition site(s)for the fungal elicitor in pea cells. The present data alsoindicate that Con A may be useful for characterization of thesignal-transduction system that leads to the synthesis of phytoalexinin pea epicotyl tissues. (Received November 16, 1994; Accepted April 20, 1995)     

Dictyostelium caveatum mutants selected for a nystatin-resistant phenotype are cross-resistant to pisatin and other isoflavonoid phytoalexins

Cellular slime mould amoebae can be induced to become resistant to an otherwise inhibitory concentration of pisatin, an isoflavonoid phytoalexin of pea, if they are first treated with a subinhibitory concentration. We report here the serendipitous isolation of pisatin-resistant mutants in the cellular slime mouldDictyostelium caveatum. However, the pisatin resistance phenotype of the mutants appears to have a different basis than the inducible pisatin resistance phenotype of the wild type.     

12-O-tetradecanoylphorbol-13-acetate stimulates release of arachidonic acid, prostaglandin E2 and prostaglandin F2 alpha from TPA nonproliferative variants of 3T3 cells

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, β-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

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.     

L-Phenylalanine ammonia-lyase and pisatin induction by 5-bromodeoxyuridine in Pisum sativum.

The substitution of the base analogue 5-bromodeoxyuridine (BrdU) for thymidine in the DNA of pea pods (Pisum sativum) induces or enhances the level of phenylalanine ammonia-lyase (PAL) and also induces the phytoalexin, pisatin, a product of the same metabolic pathway. Cordycepin, a polyadenylate inhibitor at the RNA level, is a potent inhibitor of pisatin synthesis. Kinetic studies on the inhibition of the PAL-pisatin production by hydroxyurea indicate that BrdU must be incorporated into DNA before any induction takes place. 5-Iododeoxyuridine is also an inducer while 5-fluorodeoxyuridine is ineffective when applied alone. BrdU is incorporated into the DNA of pea cells and the nuclei undergoes condensation just prior to the detection of the induced responses.