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.     

Induction of phenylalanine ammonia lyase and pisatin by photosensitive psoralen compounds

The psoralen compounds, xanthotoxin and 4,5′, 8-trimethylpsoralen, when activated, increased phenylalanine ammonia lyase (PAL) activity and the synthesis of pisatin in excised pea pods. Pods presoaked 1 hr with 4,5′,8-trimethylpsoralen and then irradiated 4 minutes with 366 nanometer ultraviolet light had twice as much PAL activity 3 hours after irradiation and 12 times as much PAL activity 20 hours after irradiation as the pods of the water-treated control. Increases in PAL activity and pisatin synthesis were not obtained with 4,5′,8-trimethylpsoralen, xanthotoxin, or 366 nanometer light treatment alone. 4,5′,8-Trimethylpsoralen in combination with the irradiation treatment (366 nanometers) enhanced the rate at which l-leucine is incorporated into various fractions of soluble proteins in excised pods 8 hours after treatment. This treatment decreased the rate at which orotic acid is incorporated into RNA. The increase in PAL activity induced by irradiated psoralens was prevented when 6-methylpurine (0.5 milligram per milliliter) or cycloheximide (10 micrograms per milliliter) was applied immediately following the irradiation period. Possible functions of psoralen compounds in plants are discussed.     

Mode of Pisatin Induction: Increased Template Activity and Dye-binding Capacity of Chromatin Isolated from Polypeptide-treated Pea Pods

Increases in phenylalanine ammonia lyase activity and pisatin synthesis were induced in excised pea pods (a) by basic polypeptides such as protamine, histone, lysozyme, cytochrome c, and ribonuclease; (b) by the polyamines spermine, spermidine, cadaverine, and putrescine, and (c) by the synthetic oligopeptides poly-l-lysine, poly-dl-ornithine, and poly-l-arginine.Poly-l-lysine (1 milligram per milliliter, molecular weight 7,200) was utilized as a model inducer of pisatin and phenylalanine ammonia lyase. The poly-l-lysine-induced responses could be inhibited by adding the RNA synthesis inhibitors cordycepin or alpha-amanitin to the pods prior to or at the time of inducer application. Cordycepin added 1.5 hours after inducer no longer completely inhibited induction. The application of poly-l-lysine was shown to characteristically change the rate of RNA synthesis within 30 minutes. Ultrastructural changes in pea nuclei were detected within 3 hours, and gross changes in nuclear morphology were apparent at 14 hours after inducer application. The physical appearance of uranyl acetate-stained chromatin isolated from poly-l-lysine 2 hours after inducer application differed from that of water-treated tissues. The template properties of chromatin extracted from pods 3 hours after inducer application were consistently superior to control chromatin when assayed with Escherichia coli RNA polymerase (without sigma factor). Chromatin from poly-l-lysine-induced tissue also bound 49% more actinomycin D-(3)H.The DNA-complexing properties of inducer compounds and the induced changes in the template and dye-binding properties of pea chromatin formed the basis for a proposed mode of action for phytoalexin induction.     

The comparative cell cycle and metabolic effects of chemical treatments on root tip meristems. III. Chlorsulfuron

Intact and excised cultured pea roots (Pisum sativum L. cv Alaska) were treated with chlorsulfuron at concentrations ranging from 2.8 ×10^?4 M to 2.8×10^?6 M. At all concentrations this chemical was demonstrated to inhibit the progression of cells from G₂ to mitosis (M) and secondarily from G₁ to DNA synthesis (S). The S and M phases were not directly affected, but the transition steps into those phases were inhibited. Total protein synthesis was unaffected by treatment of intact roots with 2.8×10^?6 M chlorsulfuron. RNA synthesis was inhibited by 43% over a 24-h treatment period. It is hypothesized that chlorsulfuron inhibits cell cycle progression by blocking the G₂ and G₁ transition points through inhibition of cell cycle specific RNA synthesis.     

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.     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

An Endogenous Suppressor of the Defense Response in Pisum sativum

Healthy pea plants contain a substance, tentatively called "endogenoussuppressor", which specifically suppresses the accumulationof pisatin in pea plants that is induced by treatment with CuCl₂or an elicitor from Mycosphaerella pinodes. This suppressorelicits the accumulation of phytoalexins in other legumes, suchas kidney bean, soybean and cowpea. The endogenous suppressorfunctions to delay the accumulation of pisatin, the activationof phenylalanine ammonialyase (PAL) and the accumulation ofmRNAs for PAL and chalcone synthase induced by the elicitorfrom M. pinodes. The substance specifically induces susceptibilityto nonpathogens, such as Mycosphaerella ligulicola and M. melonis,in pea out of four species of legume tested, but the effectis not cultivar-specific. Thus, the endogenous suppressor inhealthy pea plants suppresses a series of self-defense reactionsand induces susceptibility in pea plants in a species-specificmanner, being similar to the exogenous fungal suppressor fromthe pea pathogen, M.pinodes. (Received February 19, 1992; Accepted May 11, 1992)     

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.     

Effect of Ca2+ on programmed death of guard and epidermal cells of pea leaves

The effect of Ca²⁺ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca²⁺ at concentrations of 1–100 μM increased and at a concentration of 1 mM prevented the CN—induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca²⁺ at concentrations of 0.1–1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN^? was suppressed by 2 mM Ca²⁺ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 μM concentration prevented GC death induced by CN^? or CN^? + 0.1 mM Ca²⁺ but had no influence on respiration and photosynthetic O₂ evolution in pea leaf slices. The generation of reactive oxygen species determined from 2′,7′-dichlorofluorescein fluorescence was promoted by Ca²⁺ in epidermal peels from pea leaves.     

Specificity of deoxyribonucleic Acid intercalating compounds in the control of phenylalanine ammonia lyase and pisatin levels

Compounds with planar triple ring systems such as acridine orange, 9-amino acridine, 9-amino-1,2,3,4-tetrahydroacridine (tacrine), 6,9-diamino-2-ethoxyacridine lactate monohydrate (DE-acridine), 6-chloro-9-(3′-diethylamino-2′-hydroxypropylamino) -2-methoxyacridine·2 HCl (CDM-acridine), quinacrine, 6-chloro-9-(4′-diethylamino-1′-methylbutylamino) -2-methoxy-1,10-diazaanthracene (CDM 1,10-diazaanthracene), thionine, azure A, methylene blue, and pyronine Y when applied to excised pea pods were potent inducers of phenylalanine ammonia lyase or of pisatin, or of both. Compounds with an array of structural variation around the planar three-ring system were tested for their ability to induce these responses in pea tissue. In general, dimethylamino, diethylamino, or amino substitutions at position 2 and 6 or an amino (with or without an aliphatic side chain) substitution at position 9 of the three-ring system augmented induction potential. Methyl green, methylene blue, 2,7-diaminofluorene, nile blue, neutral red, pyrogallol red, ethidium bromide, nogalamycin, quinine, chloroquine, spermine, 8-azaguanine, gliotoxin, chromomycin A₃, actinomycin D, and mitomycin C were also potent inducers. The inhibition of phenylalanine ammonia lyase induction by the application of actinomycin D (300 micrograms per milliliter) or 6-methylpurine (1 milligram per milliliter) within 1 hour after inducer application indicated that newly synthesized RNA is necessary for induction. Phenylalanine ammonia lyase induction was also inhibited by cycloheximide (150 micrograms per milliliter).     

(−)-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.     

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.     

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)     

Role of oxygenases in pisatin biosynthesis and in the fungal degradation of maackiain

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 ¹⁸O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under ¹⁸O₂, the product was labeled with 99% efficiency; no label was incorporated by metabolism in H₂¹⁸O. Pisatin synthesized by pea pods in the presence of ¹⁸O₂ or H₂¹⁸O 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 H₂O and not from O₂. 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.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures

Sodium selenite (Na₂Se0₃) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetyl aminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 × 10^-6 M Na₂SeO₃ resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na₂SeO₃ concentrations (7.90 × 10^?6 and 1.19 × 10^?5 M) resulted in a three-fold increase in the SCE frequency above background level (6–7 SCEs/cell). Exposure of lymphocytes to 1 × 10^?4 M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 ± 0.75 while a similar exposure to 2.7 × 10^?5 M N-OH-AAF resulted in 13.61 ± 0.43 SCEs/cell. Simultaneous addition of the high Na₂Se0₃ concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25–30% and 11–17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

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     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. K_I, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k₃, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. K_I values of 1.3 × 10^{? 4} M^{? 1}, 5.6 × 10^{? 6} M^{? 1} and 7.2 × 10^{? 6} M^{? 1} were obtained for malathion, malaoxon and isomalathion, respectively. The IC₅₀ values for free/immobilized AChE, (3.7 ± 0.2) × 10^{? 4} M/(1.6 ± 0.1) × 10^{? 4}, (2.4 ± 0.3) × 10^{? 6}/(3.4 ± 0.1) × 10^{? 6} M and (3.2 ± 0.3) × 10^{? 6} M/(2.7 ± 0.2) × 10^{? 6} M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10 mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 × 10^{? 7} M/2 × 10^{? 7} M, 2 × 10^{? 7} M/3 × 10^{? 7} M and 2 × 10^{? 7} M/4.5 × 10^{? 7} M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

Inhibition of the neutrophil oxidative response to a chemotactic peptide by inhibitors of arachidonic acid oxygenation

N-formylmethionylphenylalanine stimulates a short burst of antimycin A-insensitive O₂ uptake, O₂^? production and hexosemonophosphate shunt oxidation of glucose by guinea pig peritoneal neutrophils. The stimulated oxidative metabolism, as well as release of lysosomal enzymes ± cytochalasin B, are inhibited by 5,8,11,14-eicosatetraynoic acid (ID₅₀ 1.5 × 10^?5 M). High concentrations of indomethacin inhibit the peptide-stimulated oxidations (ID₅₀ 1.6 × 10^?4 M) while acetylsalicylic acid (2.5 × 10^?3 M) does not. Digitonin-stimulated oxidative metabolism and enzyme release are not inhibited by 5,8,11,14-eicosatetraynoic acid or indomethacin at concentrations that depress effects of the N-formylated peptide.     

Comparison of proteinase inhibitor-inducing activities and phytoalexin elicitor activities of a pure fungal endopolygalacturonase, pectic fragments, and chitosans

Rhizopus stolonifer endopolygalacturonase, an elicitor of casbene synthetase activity in castor bean seedlings, was found to be a potent elicitor of the phytoalexin pisatin in pea pods and of proteinase Inhibitor I in tomato leaves. The enzyme was an active elicitor or inducer only in its active native state; heat-denatured enzyme was inactive in all three systems. The activities of (a) the tomato pectic polysaccharide proteinase inhibitor-inducing factor, (b) a partially acid hydrolyzed proteinase inhibitor-inducing factor, (c) citrus pectic fragments, and (d) chitosan, were also compared in the three bioassay systems. The four oligosaccharide preparations were active in all three systems, but with different degrees of potency. In tomato leaves and pea pods, chitosans were most active, whereas in castor beans, the citrus pectic fragments were the best elicitors. The data presented support the hypothesis that plant and fungal cell wall fragments are important signals in mobilizing a wide variety of biochemically different types of plant defense responses, and that endopolygalacturonases play a key role in releasing the plant cell wall fragments during pest attacks.     

Synthesis of the phytoalexin pisatin by a methyltransferase from pea

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 CuCl₂, 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.