Major Phytoalexin Research Articles

Functional Analysis of the Promoters of Phenylalanine Ammonia-Lyase Genes in Pea

Pycnospore germination fluid of Mycosphaerella pinodes, a fungus pathogenic on pea, contains both an elicitor and a suppressor of the accumulation of pisatin, a major phytoalexin of pea. Transcription of the genes encoding key enzymes in the biosynthesis of pisatin, namely PAL (a gene encoding phenylalanine ammonia-lyase) and CHS (a gene encoding chalcone synthase), was shown to be activated upon the treatment of pea epicotyl tissues with the fungal elicitor and suppressed upon treatment with the fungal suppressor. To investigate the mechanisms underlying activation and suppression of plant defense genes by signal molecules secreted by a fungal pathogen and other stresses, such as ultraviolet (UV) light, we constructed chimeric genes composed of the 5'-flanking regions of two members of the PSPAL family (the genes encoding phenylalanine ammonia-lyase in Pisum sativum) fused to a bacterial gene for chloramphenicol acetyltransferase. Then, the cis-regulatory elements necessary for elicitor-mediated activation and suppressor-mediated suppression were examined in pea protoplasts. Functional analysis of 5' nested deletions of PSPAL1 and PSPAL2 suggested that an enhancer-like element is located in the TATA-distal region (-2,196 to -406) in PSPAL2. A cis-acting element(s) responsible for elicitor-mediated activation was found in the TATA-proximal region (-340 to -95 in PSPAL1; -406 to -158 in PSPAL2), in which the consensus sequence motifs known as box 1, box 2 and box 4 [Yamada et al. (1992) Plant Cell Physiol. 33: 715, Lois et al. (1989) EMBO J. 8: 1641] were present in close proximity. Furthermore, both promoters were activated by UV light but were partially suppressed in response to the fungal suppressor.

Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis

The major phytoalexin in alfalfa is the isoflavonoid (-)-medicarpin (or 6aR, 11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in medicarpin biosynthesis, is responsible for introducing one of two chiral centers in (-)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1) which, when expressed in Escherichia coli, converts 2'-hydroxyformononetin stereospecifically to (3R)-vestitone, as would be predicted for IFR from alfalfa. The calculated molecular weight of the polypeptide (35,400) derived from the 954 bp open reading frame compares favorably to estimated Mrs determined for IFR proteins purified from other legumes. The transcript (1.4 kb) is highly induced in elicited alfalfa cell cultures. The kinetics of induction are consistent with the appearance of IFR activity, the accumulation of medicarpin, and the observed induction of other enzymes in the pathway. Low levels of IFR transcripts were found in healthy plant parts (roots and nodules) which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded by a single gene in alfalfa. The cloning of IFR opens up the possibility of genetic manipulation of phytoalexin biosynthesis in alfalfa by altering isoflavonoid stereochemistry.

Separation and identification of phytoalexins from leaves of groundnut ( Arachis hypogaea) and development of a method for their determination by reversed-phase high-performance liquid chromatography

Leaves of groundnut, Arachis hypogaea, infected with the early leaf spot fungus, Cercospora arachidicola, were extracted in aqueous ethanol and the phytoalexins partitioned into ethyl acetate. Flash chromatography of the ethyl acetate extract on silica gel yielded fractions with one to five compounds from which the phytoalexins could be isolated by semipreparative reversed-phase high-performance liquid chromatography (HPLC). The major phytoalexins were demethylmedicarpin, formononetin, 7,4′-dimethoxy-2′-hydroxyisoflavanone and medicarpin. Minor components were 7,2′-dihydroxy-4′-methoxyisoflavanone and daidzein. Compounds were identified by cochromatography and comparison of their ultraviolet and mass spectra with authentic samples using an HPLC system equipped with a diode-array detector, HPLC mass spectrometry and gas chromatography—mass spectrometry of their trimethylsilyl derivatives. A solid-phase extraction method was developed for processing large numbers of samples. Acetonitrile eluates from C 18 cartridges were separated by reversed-phase HPLC and the phytoalexins quantified by reference to external standards of the authentic compounds.

Variation in Phytoalexin Production by Peanut Seed from Several Genotypes1

Abstract Evaluation of twenty peanut (Arachis hypogaea L.) genotypes for their phytoalexin producing ability showed wide variation in the amount and composition of phytoalexins produced. Some genotypes produced one major phytoalexin component while the other genotypes produced seven major phytoalexin components. In addition, high phytoalexin producing genotypes utilized more methionine-rich protein than the low phytoalexin producing genotypes suggesting that methionine-rich protein or its breakdown products may have a role in phytoalexin production.

Induction and purification of kievitone hydratase from Fusarium solani f. sp. phaseoli

Fusarium solani f. sp. phaseoli is capable of detoxifying the major isoflavonoid phytoalexins produced by its host plant Phaseolus vulgaris. One of the enzymic activities involved is kievitone hydratase (KHase), a secreted glycoprotein which catalyses the conversion of kievitone to the less fungitoxic derivative, kievitone hydrate. Even under conditions of substrate induction, the enzyme is expressed at levels that are too low for satisfactory purification. Therefore, several other isoflavonoids were tested as inducers in culture. Among the phytoalexins produced by the host plant, phaseollinisoflavan was the best inducer, elevating the level of secreted enzyme eight-fold. Treatment with biochanin A, a product of chickpea, resulted in a 16-fold increase of secreted activity. The maximum rate of induction was observed 9–24 hr after addition of biochanin A, during which time several metabolises of the inducer were also present. KHase was purified from filtrates of biochanin A-induced cultures. Denaturing gel electrophoresis indicated that two species of M r 47 000 and 49 000 copurified with the activity. N-Terminal sequence analysis indicated that the two species possessed related, or identical, polypeptide moieties. Comparison with the size of the non-denatured enzyme, previously determined to be ca 100000, indicates that its native state is a dimer.

The phytoalexins of oat leaves: 4 [formula omitted]-3,1-benzoxazin-4-ones or amides?

Synthetic evidence is presented that the major phytoalexin of oat leaves is not the 4-H-3,1-benzoxazin-4-one previously reported, but the corresponding amide. Other oat and carnation phytoalexins are prepared.

Dihydrostilbene phytoalexins from Dioscorea bulbifera and D. dumentorum

Analysis of the ethyl acetate extract of the buibil of Dioscorea bulbifera and the tuber of Dioscorea dumentorum infected with Botryodiplodia theobromae gave demethylbatatasin IV and a new dihydrostilbene, 3,5,4′-trihydroxybibenzyl (dihydroresveratrol) respectively, as their major phytoalexins.

A possible mechanism of nondegradative tolerance of pisatin in Nectria haematococca MP VI

Nectria haematococca MP VI, a pathogen of pea, is tolerant of pisatin, the major phytoalexin that accumulates in pea during pathogenesis. One mode of pisatin tolerance is the detoxification of this phytoalexin. In previous research, we identified and partially characterized a second mode of tolerance that occurs independently of detoxification (“nondegradative” tolerance). In the current study, we attempted to determine a likely mechanism for nondegradative pisatin tolerance. Pisatin was taken up and retained by mycelium of isolate 126-80 during the time that growth was inhibited by the phytoalexin, but a decrease in the amount of pisatin retained was associated with development of nondegradative tolerance. The evidence was consistent with nondegradative tolerance being the result of a mechanism that excludes pisatin from its site of action, although other mechanisms could not be ruled out. The initial accumulation of pisatin did not appear to require metabolic energy, but energy and protein synthesis were needed for the expression of reduced pisatin retention. However, it was unclear whether metabolic energy was necessary for the maintenance of reduced levels of pisatin. In the absence of any significant changes in pisatin efflux, we deduced that reduced pisatin retention probably occurs when the rate of pisatin influx decreases, and that membrane modifications may be occurring simultaneously.

Synthesis of the phytoalexin (±)-phaseollin: 3-phenylthiochromans as masked 2H-chromenes and o-prenyl phenols

Phenylthiyl radicals are shown to add regiospecifically to 2H-chromenes to afford 3-phenylthiochromans (8), (10), (13), (14), and (15). The sulphide (15), as equivalent to a chromene protected against acid and oxidation, has been used in two syntheses of (±)-phaseollin, a major phytoalexin of beans and other legumes, via the sequence (17)→(18)→(19)→(20)→(21)→(±)-(1) or (19)→(22)→(23)→-(±)-(1). Also the 3-phenylthiochromans, on electron transfer from metal naphthalenide or a mercury cathode, open to o-prenylphenols, providing a two step route to biogenetically important phenols from chromenes which is tolerant of free phenol and carbonyl functions and trisubstituted double bonds.

Isolation of three novel sulphur-containing phytoalexins from the chinese cabbage Brassica campestris L. ssp. pekinensis(cruciferae)

Inoculation of Chinese cabbage heads with the bacterium Pseudomonas cichorii induced the production of three major phytoalexins named methoxybrassinin (1), brassinin (2), and cyclobrassinin (3), whose structures have been elucidated on the basis of spectroscopic studies and synthesis.

Isolation and Identification of Isoflavanone Phytoalexins from Leaflets of Diphysa robinioides

A major phytoalexin produced by the fungus-inoculated leaflets of Diphysa robinioides has been identified as the new isoflavonoid 5,7,2′,4′-tetrahydroxy-6-(3,3-dimethylallyl)isoflavanone (diphysolone). It is accompanied by smaller quantities of diphysolone-4′ (or 2′)-O-methyl ether (diphysolidone), and the known isoflavonoids 5,7,2′4′-tetrahydroxy-8-(3,3-dimethylallyljiso- flavanone (kievitone) and 5,7,2′-trihydroxy-4′-methoxyisoflavanone (ferreirin)

Development of a Radioimmunoassay for the Soybean Phytoalexin Glyceollin I

A radioimmunoassay for glyceollin I, the major phytoalexin produced by soybean (Glycine max [L.] Merr.), has been developed. Antibodies were raised in rabbits against a glyceollin I-bovine serum albumin conjugate. The antisera were used to establish a radioimmunoassay for glyceollin I using [(125)I]glyceollin I as the tracer. A logit plot of a standard concentration series yielded a straight line in the range of 1 to 100 picomoles (0.34-34 nanograms) of glyceollin I. The structurally related pterocarpan phytoalexins, glyceollins II and III, glyceollidin II and glycinol, which also accumulate in infected soybean tissue, show a low cross-reactivity in the radioimmunoassay (0.5-5% at 50% displacement of the tracer). Two related isoflavones present constitutively in soybean tissue, daidzein and genistein, have cross-reactivities of less than 0.84% and 1.1%, respectively. The radioimmunoassay permitted the quantitative determination of glyceollin I in 15-micrometer microtome sections of soybean hypocotyl tissue infected with zoospores of Phytophthora megasperma f. sp. glycinea.

Tolerance of Nectria haematococca MP VI to the Phytoalexin Pisatin in the Absence of Detoxification

Most isolates of Nectria haematococca mating population (MP) VI detoxify pisatin, the major phytoalexin produced by pea. However, there is evidence of another pisatin tolerance mechanism that does not depend on pisatin degradation. To facilitate further studies on nondegradative tolerance, a bioassay was developed in which culture turbidity was used as a measure of mycelial growth. Under conditions of this assay, four N. haematococca MP VI isolates degraded 0·4 mm-pisatin within 15 h, but two other isolates required more than 24 h. A remaining group of four isolates did not degrade pisatin. These three groups were typified by the isolates 126–71. T-77 and 126–80, respectively. Regardless of their ability to degrade pisatin, however, all 10 isolates grew equally well after addition of 0·4 mm-pisatin, suggesting that detoxification may not be needed for pisatin tolerance during the short time interval of this assay. The existence of a nondegradative tolerance in isolate 126–71 was confirmed when additional experiments showed that growth could occur (i) before degradation had reduced pisatin below initially noninhibitory levels, and (ii) when pisatin degradation was inhibited by 2 % ethanol. When tested in growth medium, pretreatment with a noninhibitory concentration of pisatin for 3 h enhanced equally the ability of isolates 126–71 and 126–80 to tolerate pisatin. After cultures were pretreated with pisatin while temporarily suspended in buffer, isolates 126–71 and T-77 were stimulated to degrade pisatin, and they grew better than did isolate 126–80. Again, however, growth began before there was significant detoxification, especially for isolate T-77. Thus, these three isolates all appeared to have an inducible, nondegradative tolerance to pisatin. Nectria haematococca MP I isolate T-145 lacked an adaptive tolerance to pisatin and was more sensitive to pisatin than any of the MP VI isolates.

Dianthalexine, nouvelle phytoalexine, de type benzoxazinone, isolee de l'oillet dianthus caryophyllus l. (caryophyllacees).

Resume The major phytoalexin from carnation has been identified as 7-hydroxy 2-phenyl 4H-3, 1-benzoxazin-4-one, and named dianthalexin ( 1 ).

Differential patterns of phytoalexin accumulation and enzyme induction in wounded and elicitor-treated tissues ofPhaseolus vulgaris

In wounded cotyledons ofPhaseolus vulgaris L. the accumulation of the 5-hydroxy isoflavonoids kievitone and 2'-hydroxygenistein precedes the major increases in the levels of the 5-deoxy compounds phaseollin and coumestrol. Increased phytoalexin levels are preceded by transient increases in the extractable activities of L-phenylalanine ammonia-lyase (EC 4.3.1.5.), chalcone synthase and chalcone isomerase (EC 5.5.1.6.). Accumulation of phytoalexins, above wounded control levels, is observed following treatment of excised cotyledons or hypocotyls with crude or fractionated elicitor preparations heat-released from the cell walls ofColletotrichum lindemuthianum. Chalcone synthase levels are also induced in cotyledons, although crude elicitor and all fractions suppress L-phenylalanine ammonia-lyase activity in both tissues. Kievitone is the major phytoalexin induced in cotyledons, whereas in hypocotyls phaseollin predominates. Patterns of phytoalexin accumulation have been studied in response to varying concentrations of the crude and fractionated elicitor; 5-hydroxy isoflavonoid accumulation is highly dependent upon elicitor concentration, the dose-response curves for kievitone accumulation showing maxima at around 1 μg glucose equivalents per cotyledon, minima at 2-3 μg equivalents and increasing induction at higher concentrations. Similar patterns are observed for L-phenylalanine ammonia-lyase and chalcone synthase levels, although the overall extent of these changes is masked by the high wound response. Accumulation of 5-deoxy isoflavonoids above control levels requires high elicitor concentrations; no experimental conditions were found under which phaseollin accumulated to higher levels than kievitone in cotyledons during the first 48 h after elicitation.

Isolation and structure elucidation of genuine oat phytoalexin, avenalumin I

The major phytoalexin from oat leaves has been identified as 2-[2-(4-hydroxyphenyDethenyl] -6-hydroxy-4H-3,1-benzoxazin-4-one ( A ).

A chemical dichotomy in phytoalexin induction within the tribe vicieae of the leguminosae

Using either leaf or cotyledon tissues, the phytoalexin response of over 60 representative members of the genera Lathyrus, Lens, Pisum and Vicia was determined. Twenty-nine Vicia species produced furanoacetylenes, as did the two Lens species examined. By contrast, Pisum and Lathyrus failed to produce any of these phytoalexins, but formed pisatin instead. Thus, pisatin was formed as the major phytoalexin in 29 of 31 Lathyrus species tested, representing 10 sections within the genus. This dichotomy in phytoalexin response is discussed in relation to the taxonomy of the tribe Vicieae.

Identification of two chromone phytoalexins in the sweet pea, Lathyrus odoratus

A major phytoalexin isolated from the Helminthosporium Carbonum-inoculated leaflets and pods of Lathyrus odoratus has been identified by spectroscopic procedures as 5,7-dihydroxy-3-ethylchromone (lathodoratin). Small amounts of the corresponding 7- O-methyl ether (methyl-lathodoratin) are also formed by this plant. Both compounds similarly occur as phytoalexins in the closely related legume L. hirsutus but are absent from the other Lathyrus species examined. The unusual 3-substitution of the chromone nucleus appears to be essential for fungitoxicity since the synthetic isomer 5,7-dihydroxy-2-ethylchromone is apparently inactive.

Isoflavonoid Phytoalexins of Yam Bean (Pachyrrhizus erosus)

Abstract The furanopterocarpan, neodunol, has been isolated as a major phytoalexin from the fungus-inoculated stems of Pachyrrhizus erosus. Neodunol is accompanied by small quantities of demethylmedicarpin and a third compound provisionally identified as the prenylated pterocarpan, homoedudiol; traces of a neodunol isomer were also isolated from P. erosus although its precise structure could not be determined. The apparently close chemical relationship between Pachyrrhizus and Neorautanenia is discussed in the light of a recent study which allocates these genera to differ­ent sections of the legume tribe Phaseoleae.

Notizen: A Revised Structure for the Phytoalexin Cajanol

Abstract Peroxide oxidation of cajanol ethyl ether afforded the B ring derived product, 2-methoxy-4-ethoxybenzoic acid. For­ mation of this compound means that the structure of cajanol, a major phytoalexin from Cajanus cajan, must be revised to 5,4ʹ-dihydroxy-7,2ʹ-dimethoxyisoflavanone.