Pterocarpan Phytoalexins Research Articles

Effect of sodium selenite on isoflavonoid contents and antioxidant capacity of chickpea (Cicer arietinum L.) sprouts.

Isoflavonoid compositions, phenylalanine ammonia lyase (PAL) activity and antioxidant capacity were evaluated in chickpea (Cicer arietinum L.) sprouts germinated after soaking with different sodium selenite (Na2SeO3) concentrations (0, 1 and 2mg/100g seeds). Chickpea seeds were germinated during four days at 24°C and the isoflavonoid profiles and concentrations evaluated by HPLC-UV daily during four days of germination. Eleven isoflavones and two pterocarpan phytoalexins forms were identified in sprouts, being malonylated formononetin glycoside, formononetin, isoformononetin glycoside and malonylated biochanin A glycoside the major compounds. Compared to untreated sprouts, total isoflavonoid, PAL activity and antioxidant capacity showed a remarkable increase of 83%, 56%, and 33%, respectively in chickpea sprouts that were treated with a high sodium selenite content (2mg/100g seeds). Results suggest that Se-enriched chickpea sprouts could represent a good source of dietary Se and as an upgraded source of isoflavonoids.

The inducible soybean glyceollin phytoalexins with multifunctional health-promoting properties

Abstract The glyceollins are soybean-derived phytoalexins that accumulate in the seeds in response to various extrinsic factors. Several elicitors such as fungi, UV irradiation and physical injury have been demonstrated to alter the secondary metabolome of soybeans via induction of glyceollin synthesis and accumulation. The pterocarpan phytoalexins have exhibited antiestrogenic activity against estrogen receptor-mediated cellular processes. As a result, the glyceollins display anticancer activities against breast and ovarian cancers through inhibition of tumor growth and progression, and also by inducing cell cycle arrest in the cancer cells. Moreover, glyceollins were found to exhibit antimicrobial activity against pathogenic fungi, cellular antioxidant activities against endotoxin-induced oxidative stress, and regulatory effects on glucose metabolism and endotoxin-induced inflammatory response. The soybean-derived compounds warrant further studies to evaluate detailed molecular mechanisms during aberrant cellular processes, safety and efficacy in animal disease models and human subjects, and potential use for food preservation. The bioactivity of glyceollins can result in the enrichment of soybean with these phytoalexins for improved health functions and other novel applications.

Germination Dramatically Increases Isoflavonoid Content and Diversity in Chickpea (Cicer arietinum L.) Seeds

The effect of germination on bioactive components in legume seeds was investigated in terms of the antioxidant capacity and total phenolic contents. Germination increased the total phenolic content and antioxidant capacity of most seeds. Particularly in chickpea seeds, the isoflavone contents increased by over 100 fold, mainly due to the increase of formononetin and biochanin A level. As a result, these two compounds were conveniently isolated from the germinated seeds in preparative scale and structurally confirmed by UV-vis, ESI-MS, and (1)H NMR spectroscopies. Isoflavonoid fingerprints analyzed by HPLC-PDA and LC-ESI-MS demonstrated that germination could significantly increase isoflavonoids diversity. Twenty-five isoflavonoids were detected and identified tentatively. These include 20 isoflavones, 2 isoflavanones, and 3 pterocarpan phytoalexins. Total isoflavonoid content of germinated chickpea was approximately 5-fold of that of germinated soybean. Our findings suggest that the germinated chickpea seeds could serve as a promising functional food rich in isoflavonoids.

Premature T cell senescence in Ovx mice is inhibited by repletion of estrogen and medicarpin: a possible mechanism for alleviating bone loss

Presently the relationship between CD28, biological marker of senescence, and ovariectomy is not well understood. We show that ovariectomy leads to CD28 loss on T cells and estrogen (E2) repletion and medicarpin (Med) inhibits this effect. We thus propose that Med/E2 prevents bone loss by delaying premature T cell senescence. Estrogen deficiency triggers reproductive aging by accelerating the amplification of TNF-α-producing T cells, thereby leading to bone loss. To date, no study has been carried out to explain the relationship between CD4(+)CD28null T cells and ovariectomy or osteoporosis. We aim to determine the effect of Ovx on CD28 expression on T cells and effects of E2 and medicarpin (a pterocarpan phytoalexin) with proven osteoprotective effect on altered T cell responses. Adult, female Balb/c mice were taken for the study. The groups were: sham, Ovx, Ovx + Med or E2. Treatments were given daily by oral gavage. At autopsy bone marrow and spleen were flushed out and cells labelled with antibodies for FACS analysis. Serum was collected for ELISA. In Ovx mice, Med/E2 at their respective osteoprotective doses resulted in thymus involution and lowered Ovx-induced increase in serum TNF-α level and its mRNA levels in the BM T cells. Med/E2 reduced BM and spleen CD4(+) T cell proliferation and prevented CD28 loss on CD4(+) T cells. Further, Med abrogated TNF-α-induced loss of CD28 expression in the BM T cells. To our knowledge this is the first report to determine the mechanism of CD28 loss on T cells as a result of ovariectomy. Our study demonstrates that Ovx leads to the generation of premature senescent CD4(+)CD28null T cells, an effect inhibited by E2 and Med. We propose that one of the mechanisms by which Med/E2 alleviates Ovx-induced bone loss is by delaying T cell senescence and enhancing CD28 expression.

Biosynthesis of the 3-Ethylchromone Phytoalexin Lathodoratin in Lathyrus Odoratus

Abstract Feeding experiments with 14C- and 13C-labelled precursors have demonstrated that the phloroglucinol ring of the 3-ethylchromone phytoalexin lathodoratin from cupric sulphate-induced pods of sweet pea (Lathyrus odoratus) has a polyketide origin. The remaining five carbon ‘isoprene unit’ is derived from the amino acid isoleucine which provides the starter unit, probably 2-methylbutanoate, for acetate-malonate chain extension. The pterocarpan phytoalexin pisatin is produced along with lathodoratin, and precursors were incorporated in agreement with earlier studies. Two minor 3-ethylchromones were also synthesized on cupric sulphate treatment of the pods. These were the previously reported 7-O-methyl ether of lathodoratin (methyl-lathodoratin) and a new compound, the 5,7-di-O-methyl ether (dimethyl-lathodoratin). Dimethyl-lathodoratin is the most fungitoxic of the 3-ethylchromone phytoalexins.

Glyceollin I enantiomers distinctly regulate ER-mediated gene expression

Glyceollins are pterocarpan phytoalexins elicited in high concentrations when soybeans are stressed. We have previously reported that the three glyceollin isomers (GLY I-III) exhibit antiestrogenic properties, which may have significant biological effects upon human exposure. Of the three isomers, we have recently shown that glyceollin I is the most potent antiestrogen. Natural (-)-glyceollin I recently was synthesized along with its racemate and unnatural (+) enantiomer. In this study, we compared the glyceollin I enantiomers' ER binding affinity, ability to inhibit estrogen responsive element transcriptional (ERE) activity and endogenous gene expression in MCF-7 cells. The results demonstrated similar binding affinities for both ERalpha and ERbeta. Reporter gene assays in MCF-7 cells revealed that while (+)-glyceollin I slightly stimulated ERE transcriptional activity, (-)-glyceollin I decreased activity induced by estrogen. Co-transfection reporter assays performed in HEK 293 cells demonstrated that (+)-glyceollin I increased ERE transcriptional activity of ERalpha and ERbeta with and without estrogen with no antiestrogenic activity observed. Conversely, (-)-glyceollin I decreased the activity of both ER subtypes stimulated by estradiol demonstrating potent antiestrogenic properties. Additionally, each Gly I enantiomer induced unique gene expression profiles in a PCR array panel of genes commonly altered in breast cancer.

Transcriptome changes in the phenylpropanoid pathway of Glycine max in response to Pseudomonas syringaeinfection

BackgroundReports of plant molecular responses to pathogenic infections have pinpointed increases in activity of several genes of the phenylpropanoid pathway leading to the synthesis of lignin and flavonoids. The majority of those findings were derived from single gene studies and more recently from several global gene expression analyses. We undertook a global transcriptional analysis focused on the response of genes of the multiple branches of the phenylpropanoid pathway to infection by the Pseudomonas syringae pv. glycinea with or without the avirulence gene avrB to characterize more broadly the contribution of the multiple branches of the pathway to the resistance response in soybean. Transcript abundance in leaves was determined from analysis of soybean cDNA microarray data and hybridizations to RNA blots with specific gene probes.ResultsThe majority of the genes surveyed presented patterns of increased transcript accumulation. Some increased rapidly, 2 and 4 hours after inoculation, while others started to accumulate slowly by 8 – 12 hours. In contrast, transcripts of a few genes decreased in abundance 2 hours post inoculation. Most interestingly was the opposite temporal fluctuation in transcript abundance between early responsive genes in defense (CHS and IFS1) and F3H, the gene encoding a pivotal enzyme in the synthesis of anthocyanins, proanthocyanidins and flavonols. F3H transcripts decreased rapidly 2 hours post inoculation and increased during periods when CHS and IFS transcripts decreased. It was also determined that all but one (CHS4) family member genes (CHS1, CHS2, CHS3, CHS5, CHS6 and CHS7/8) accumulated higher transcript levels during the defense response provoked by the avirulent pathogen challenge.ConclusionBased on the mRNA profiles, these results show the strong bias that soybean has towards increasing the synthesis of isoflavonoid phytoalexins concomitant with the down regulation of genes required for the synthesis of anthocyanins and proanthocyanins. Although proanthocyanins are known to be toxic compounds, the cells in the soybean leaves seem to be programmed to prioritize the synthesis and accumulation of isoflavonoid and pterocarpan phytoalexins during the resistance response. It was known that CHS transcripts accumulate in great abundance rapidly after inoculation of the soybean plants but our results have demonstrated that all but one (CHS4) member of the gene family member genes accumulated higher transcript levels during the defense response.

Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean

Lactofen, the active ingredient of the soybean disease resistance-inducing herbicide, Cobra, induces large accumulations of isoflavone conjugates and aglycones in soybean tissues. The predominant isoflavones induced in cotyledon tissues are daidzein (and its conjugates) and formononetin and glycitein aglycones. The latter two isoflavones are usually present only at very low levels in soybean seedling tissues. In leaves, the predominant lactofen-induced isoflavones are daidzein and formononetin aglycones and the malonyl-glucosyl conjugate of genistein. Isoflavone induction also occurs in cells distal to the point of treatment, but is only weakly systemic. Lactofen also induces elicitation competency, the capacity of soybean cells to accumulate the pterocarpan phytoalexin glyceollin in response to glucan elicitors from the cell wall of the pathogen Phytophthora sojae. Comparison of the activity of a series of diphenyl ether herbicides demonstrated that while all diphenyl ethers tested induced some degree of elicitation competency, only certain ones induced isoflavone accumulation in the absence of glucan elicitor. As a group the diphenyl ethers are thought to inhibit protoporhyrinogen oxidase, eventually leading to singlet oxygen generation. Another singlet oxygen generator, rose bengal, also induced elicitation competency, but little isoflavone accumulation. It is hypothesized that diphenyl ether-induced activated oxygen species mimic some aspects of hypersensitive cell death, which leads to elicitation competency in infected tissues.

Release of phytoalexins and related isoflavonoids from intact chickpea seedlings elicited with reduced glutathione at root level

Abstract Induction of phytoalexin formation in roots of chickpea (Cicer arietinum L.) seedlings treated with the reduced form of glutathione (GSH) was investigated. Accumulation of phytoalexins and constitutive isoflavonoids upon GSH treatment was assessed by high performance liquid chromatography analysis of extracts from roots and liquid incubating media of seedlings, whereas the requirement for concurrent de novo synthesis of induced compounds was assessed in elicitation studies conducted in the presence of key enzyme inhibitors of the phenylpropanoid pathway and/or 〚U-14C〛-L-phenylalanine. The results indicate that: (a) GSH elicits the formation not only of the pterocarpan phytoalexins medicarpin and maackiain, but also that of the constitutive isoflavones biochanin A and formononetin and, in seedlings older than 4 d, that of the isoflavanones homoferreirin and cicerin. (b) GSH-induced secondary metabolites do not accumulate in plant root tissue; rather, they are released into the surrounding external medium. (c) Phytoalexins and isoflavonoids induced by GSH are de novo synthesized from early phenylpropanoid pathway precursors rather than derived from pre-existing conjugate forms or immediate precursors. (d) Because of metabolic competence and/or proximity, only a small proportion of root cells is responsive to GSH elicitation, hence the very small amounts or absence of induced compounds detected in root tissue samples.

Age-related regulation of induced isoflavonoid responses in soybean lines differing in inherent elicitation competency

The accumulation of the pterocarpan phytoalexin glyceollin in soybean tissues, in response to the general resistance wall glucan elicitor from Phytophthora sojae, requires both the synthesis (or release from pre-formed conjugates) of the glyceollin precursor daidzein and the pre-activation of a state of glyceollin elicitation competence in responding cells by factors associated with wounded or hypersensitively dying cells. While many soybean cultivars (e.g. Williams) do not produce glyceollin in response to elicitor in the absence of competence activation, the cultivar Harosoy is partially inherently elicitation competent, although its capacity for elicitation competence can be further induced. To gain some insight into the fundamental differences in these lines, developmental and age-related effects were compared on various isoflavonoid responses in the two lines. The activation of glyceollin elicitation competency in Williams (and its enhancement in Harosoy) are both dramatically affected by the developmental age of tissues, being maximal in cotyledons of intermediate age. In both cultivars these effects are similar and appear to relate to the capacity of the tissues to generate the endogenous competency factors. Parallel developmental age effects were also observed in hypocotyl and root tissues. However, very clear differences in the light and glucan induction of the isoflavones genistein and daidzein, respectively, were observed in the two cultivars depending on tissue age. Thus, the differences in glyceollin elicitation competency in Williams and Harosoy may relate more to inherent differences in the net accumulations of isoflavone precursor pools than to the generation of the elicitation competent state per se.

Functional expression and subcellular localization of the Nectria haematococca Mak1 phytoalexin detoxification enzyme in transgenic tobacco.

Medicarpin and maackiain are antifungal pterocarpan phytoalexins produced by many legumes, and are thought to be important components of the defense response of these legumes to certain fungal pathogens. The Mak1 gene from the fungal pathogen Nectria haematococca encodes an FAD-dependent mono-oxygenase, known to specifically hydroxylate the phytoalexins medicarpin and maackiain, converting them to less fungitoxic derivatives. Two binary vector constructs were made containing the coding regions from two fungal clones, a Mak1 cDNA (intronless) and a genomic (including three fungal introns) clone, regulated by an enhanced cauliflower mosaic virus 35S promoter. The constructs were introduced into tobacco to check for expression of active fungal enzyme in plant cells and for splicing of fungal introns. Leaves of tobacco plants transformed with the Mak1 cDNA construct readily metabolized infiltrated medicarpin to 1a-hydroxymedicarpin, indicating high levels of active enzyme. RT-PCR analysis of tobacco plants transformed with the Mak1 genomic construct indicated no processing of Mak1 introns, and no Mak1 activity was detected in these plants. When using plants containing the Mak1 cDNA construct, immunolocalization with a Mak1-specific antibody together with cellular fractionation indicated that Mak1 protein accumulated in the plant cytoplasm, associated with endoplasmic reticulum membranes; medicarpin biosynthetic enzymes have been localized to the same subcellular region. The Mak1 cDNA construct is therefore suitable for use in studies to selectively eliminate medicarpin accumulation to assess the relative importance of medicarpin in the antifungal defense mechanisms of alfalfa and other legumes.

Effect of spores of saprophytic fungi on phytoalexin accumulation in seeds of frog-eye leaf spot and stem canker-resistant and -susceptible soybean (Glycine max L.) cultivars.

Two saprophytic fungi (Mucor ramosissimus and Rhizopus sp.) were tested for their ability to induce phytoalexin production by seeds of frog-eye leaf spot and stem canker-resistant and -susceptible soybean (Glycine max L.) cultivars. Only M. ramosissimus was shown to elicit a response and qualitative differences in phytoalexin accumulation were found between the susceptible and resistant cultivars. Glyceollins I, II, and III and glycinol were isolated from the susceptible cultivar, whereas Glyceollins I, II, and III, glycinol, glyceocarpin, genistein, isoformononetin, and N-acetyltyramine accumulated in the resistant cultivar in response to the same fungal elicitor. Genistein was found to be an inducibly formed isoflavonoid instead of a constitutive metabolite in the resistant cultivar, whereas N-acetyltyramine is described for the first time as a soybean phytoalexin. All the compounds, except genistein, showed fungitoxic activity against Cladosporium sphaerospermum. Spectral data of the pterocarpan phytoalexins, genistein, and N-acetyltyramine are also given in this work.

The effect of rishitin on potato tonoplast vesicle and vacuole proton transport

Rishitin, a known potato phytoalexin, was tested for its effects on proton transport. Like the pterocarpan phytoalexins, glyceollin and phaseollin, rishitin was found to inhibit proton transport. At 100 microM rishitin, proton transport in potato tonoplast vesicles was inhibited by > 95%. This inhibition appears to be due to an increase in proton conductance and not to inhibition of the tonoplast ATPase. Potato vacuoles were also shown to have increased proton leakage in the presence of rishitin.

Phytoalexin-like Activity of Abietic Acid and Its Derivatives

Abietic acid and some of its derivatives, not known to be naturally occurring phytoalexins, were examined for their ability to promote H + conductance across membranes and to inhibit growth of two fungal species-Aphanomyces euteiches and Fusarium moniliforme. These results were compared with those of phaseollin, a phytoalexin isolated from kidney beans. Ion leakage and fungal growth inhibition were shown to be a function of structure, results that suggest that pterocarpan phytoalexin activity requires the presence of a polar, protic functional group situated at the end of a large, rigid hydrophobic moiety. Log P values for phaseollin, abietic acid, and some derivatives of abietic acid were also determined.

Characterization of binding proteins that recognize oligoglucoside elicitors of phytoalexin synthesis in soybean

We are studying the cellular signaling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta‐β‐glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete Phytophthora sojae. Our research has focused on the specific recognition of the hepta‐β‐glucoside elicitor by binding proteins in soybean cells. Elicitor‐binding proteins with properties expected of physiological receptors for the hepta‐β‐glucoside elicitor have been identified in soybean root membranes. These elicitor‐binding proteins co‐migrate with a plasma membrane marker (vanadate‐sensitive H+‐ATPase) on linear sucrose density gradients. Binding of a radio‐iodinated derivative of the hepta‐β‐glucoside elicitor by membrane‐localized elicitor‐binding proteins is specific, reversible, saturable, and of high affinity (Kd≅ 1 nM). After solubilization with the nonionic detergent, n‐dodecylsucrose, the elicitor‐binding proteins retain their high affinity (Kd= 1.8 nM) for the radiolabeled elicitor and their binding specificity for elicitor‐active oligoglucosides. A direct correlation is observed between the ability of oligoglucosides to displace labeled elicitor from the elicitor‐binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor‐binding proteins recognize the same structural elements of the hepta‐β‐glucoside elicitor that are essential for its phytoalexin‐inducing activity, suggesting that the binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification of the hepta‐β‐glucoside elicitor‐binding proteins by using ligand affinity chromatography. Purification and characterization of the hepta‐β‐glucoside binding proteins are among the first steps toward elucidating how the hepta‐β‐glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.

The isoflavonoid phytoalexin pathway: From enzymes to genes to transcription factors

The pterocarpan phytoalexins of the Leguminosae are synthesized from L‐phenyl‐alanine via a minimum of 11 enzymatic steps involving the central phenylpropanoid pathway, three reactions of flavonoid biosynthesis, and the isoflavonoid branch pathway. The extractable activities of all these enyzmes, and of enzymes supplying precursors from primary metabolism, increase in response to fungal infection or exposure of plant cells to elicitor macromolecules isolated from the cell walls of yeast or plant pathogenic fungi. The involvement of reductases and cytochrome P450 hydroxylases places a high demand for NADPH on elicited cells. The NADPH is most likely supplied by activation of the pentose phosphate pathway. Genes or cDNAs encoding 7 of the enzymes involved in the synthesis of the phytoalexin medicarpin have been cloned from alfalfa and/or other species. Induction of enzyme activity results from transcriptional activation of the corresponding genes, leading to increased steady state levels of translatable mRNAs. This transcriptional activation is programmed through the interaction of sets of elicitor/infection‐modulated transcription factors with their cognate cis elements in the promoters of the phytoalexin biosynthetic genes. Gene activation occurs through generation of intracellular signals which lead to modulation of transcription factor activity, through either increased synthesis of the factor(s), activation via reversible post‐translational modification (e.g. phosphorylation/dephos‐phorylation), translocation of factors from cytoplasm to nucleus, or combinations of these. Coordinated induction of the enzymes of phytoalexin synthesis may involve multiple signals and factors for transcriptional activation, as well as feedback and feed‐forward fine controls at both transcriptional and post‐transcriptional levels. In beneficial mycorrhizal interactions, induction of early pathway genes is uncoupled from that of later, phytoalexin‐specific genes.

Conversion of vestitone to medicarpin in alfalfa (Medicago sativa L.) is catalyzed by two independent enzymes. Identification, purification, and characterization of vestitone reductase and 7,2‘-dihydroxy-4‘-methoxyisoflavanol dehydratase.

Pterocarpan phytoalexins are antimicrobial compounds in leguminous plants. The final step of pterocarpan biosynthesis, conversion of vestitone to medicarpin, was thought to be catalyzed by a single enzyme "pterocarpan synthase." We have shown that the pterocarpan synthase activity observed in crude extracts of alfalfa suspension cell cultures is the sum of two independent enzymatic activities: vestitone reductase, which catalyzes the NADPH-dependent reduction of vestitone to 7,2'-dihydroxy-4'-methoxyisoflavanol (DMI), and DMI dehydratase, which catalyzes loss of water and closure of an ether ring to form medicarpin. The first enzyme, vestitone reductase, was purified 1,840-fold to homogeneity by a 5-step procedure. Purified vestitone reductase showed a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 38 kDa. The native molecular mass measured by gel filtration was shown to be 34 kDa, indicating that vestitone reductase is a monomer. Vestitone reductase has strict substrate stereo specificity for (3R)-vestitone with a Km value of 45 microM. The second enzyme, DMI dehydratase, was partially purified 962-fold. DMI dehydratase had a native molecular mass of 38 kDa as estimated by gel filtration and a Km value of 5 microM for DMI. Both enzymes have a temperature optimum of 30 degrees C and a pH optimum of 6.0. The discovery of vestitone reductase and DMI dehydratase will facilitate future genetic manipulation of pterocarpan biosynthesis.

Molecular Cloning of Isoflavone Reductase from Pea ( Pisum sativum L): Evidence for a 3R-Isoflavanone Intermediate in (+)-Pisatin Biosynthesis

Isoflavone reductase (IFR) reduces achiral isoflavones to chiral isoflavanones during the biosynthesis of chiral pterocarpan phytoalexins. A cDNA clone for IFR from pea (Pisum sativum) was isolated using the polymerase chain reaction and expressed in Escherichia coli. Analysis of circular dichroism (CD) spectra of the reduction product sophorol obtained using the recombinant enzyme indicated that the isoflavanone possessed the 3R stereochemistry, in contrast to previous reports indicating a 3S-isoflavanone as the product of the pea IFR. Analysis of CD spectra of sophorol produced using enzyme extracts of CuCl2-treated pea seedlings confirmed the 3R stereochemistry. Thus, the stereochemistry of the isoflavanone intermediate in (+)-pisatin biosynthesis in pea is the same as that in (-)-medicarpin biosynthesis in alfalfa, although the final pterocarpans have the opposite stereochemistry. At the amino acid level the pea IFR cDNA was 91.8 and 85.2% identical to the IFRs from alfalfa and chickpea, respectively. IFR appears to be encoded by a single gene in pea. Its transcripts are highly induced in CuCl2-treated seedlings, consistent with the appearance of IFR enzyme activity and pisatin accumulation.

Characterization of metabolic changes involved in hypersensitive-like browning reactions of chickpea ( Cicer arietinum L.) cell cultures following challenge by Ascochyta rabiei culture filtrate

A putative protein in the culture filtrate of Ascochyta rabiei induced a rapid browning response in cell cultures derived from an A. rabiei resistant cultivar of chickpea ( Cicer arietinum ). Toluidine blue O staining showed massive deposition of phenolic material in the cell wall. Reduced fluorescence after fluorescein diacetate treatment revealed impaired cells, although the cells still showed plasmolysis and deplasmolysis indicating an intact plasma membrane and therefore viability. These reactions could not be detected in cell cultures derived from a susceptible chickpea cultivar. The cultivar-specific cell browning strictly correlated with rapid metabolism of several constitutive isoflavonoid malonylglucoside conjugates. The fate of the pterocarpan phytoalexin medicarpin was closely analysed. The level of medicarpin malonylglucoside decreased rapidly after culture filtrate application and simultaneously, medicarpin was found in alkaline hydrolysable form in cell wall components. Evidence that the active component in the culture filtrate is a protein was based on the observations that the browning inducing activity of the culture filtrate was destroyed by proteinase K and heat treatment.

Compatible and Incompatible Responses in Alfalfa Cotyledons to Races 1 and 2 ofColletotrichum trifolii

Specific race interactions between Colletotrichum trifolii and cultivars of resistant and susceptible alfalfa were examined in nonwounded excised cotyledons. Cotyledons in incompatible and compatible interactions exhibited visible differential reactions within 7-10 days after inoculation. C. trifolii infected alfalfa by directly penetrating the epidermis via a penetration peg from the appressorium. Appressoria matured within 20 h on resistant and susceptible cotyledons, but primary hyphae did not develop on resistant cotyledons. Phenylalanine ammonia-lyase and the pterocarpan phytoalexin medicarpin began to increase in resistant tissues after 24 h. Cotyledon tissues apparently became resistant prior to significant increases in medicarpin [...]