Accumulation of isoflavones and pterocarpan phytoalexins in cell suspension cultures of different cultivars of chickpea (Cicer arietinum)

Cell suspension cultures of chickpea (Cicer arietinum L.) were established from cultivars ILC 3279 and ILC 1929, resistant and susceptible towards the chickpea pathogenic fungus Ascochyta rabiei. The two cell culture lines possess identical growth properties and show high accumulation of the isoflavones biochanin A and formononetin together with their glucoside and malonylglucoside conjugates. The cultures of the two cultivars, however, significantly differ in their accumulation of the phytoalexins medicarpin and maackiain essentially as previously demonstrated for the plant genotypes. Phytoalexin formation was elicited by using yeast extract as an inducing agent.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Elicitation and Suppression of Phytoalexin and Isoflavone Accumulation in Cotyledons of Cicer arietinum L. as Caused by Wounding and by Polymeric Components from the Fungus Ascochyta rabiei

Shortly after sowing cotyledons of chickpea (Cicer arietinum) start to accumulate the isoflavones biochanin A and formononetin together with their 7-0-glucosides and their 7-0-glucoside-6″-malonates. The additional accumulation of the pterocarpan phytoalexins medicarpin and maackiain can be induced by wounding of the cotyledons. Treatment of sliced cotyledons with a crude elicitor fraction obtained from the growth medium or the mycelium of the chickpea pathogenic fungus Ascochyta rabiei (Pass.) Lab. leads to a dramatic increase in the level of numerous aromatic compounds, especially of the isoflavone aglyca and the phytoalexins. Accumulation of isoflavone conjugates is not altered by elicitor treatment as shown by time course studies, and dose-response curves. A protein preparation (“suppressor”) isolated from the culture filtrate of the same fungus was shown to inhibit the accumulation of isoflavone aglyca, isoflavone conjugates and phytoalexins in the sliced cotyledons. The possible relevance of elicitor-suppressor counteraction with regard to the defence mechanisms of the host plant is discussed.     

Isolation of Solanapyrones A, B and C from Culture Filture and Spore Germination Fluids of Ascochyta rabiei and Aspects of Phytotoxin Action

Nine isolates of the fungus Ascochyta rabiei have been assayed for their ability to produce solanapyrone toxins. All isolates formed solanapyrone A, B and C which were secreted into the culture medium. Pronounced production of the toxins only occurred after onset of sporulation. The identification of the fungal products was achieved by cochromatography (TLC, HPLC), ¹H-NMR (solanapyrone A and B) and mass spectrometry (solanapyrone B). Work with A. rabiei isolate X showed that cultivation in chickpea seed extract medium in a surface culture provided best conditions for maximal toxin production. The accumulation of solanapyrones over the growth cycle was monitored. Germinating spores produced solanapyrones C and B whereas solanapyrone A was formed from the 6th day of the culture period on. Application of a mixture of solanapyrones A, B and C to leaflets of intact plants from an A. rabiei resistant cultivar (ILC 3279) and a susceptible cultivar (ILC 1929) led to characteristic changes in leaf morphology which had earlier been obsevad in susceptible plants following infection with spores of A. rabiei. Attempts to demonstrate the occurrence of toxins in the infected leaf were unsuccessful. Application of solanapyrones to solanapyrones to chickpea cell suspension cultures (derived from both cultivars) led to pronounced losses in viability and to plasmolysis of cells.     

Medicarpin and maackiain 3-O-glucoside-6′-O-malonate conjugates are constitutive compounds in chickpea (Cicer arietinum L.) cell cultures

Summary The pterocarpan phytoalexin conjugates medicarpin 3-O-glucoside-6-O-malonate and maackiain 3-O-glucoside-6-O-malonate were isolated from cell suspension cultures of chickpea (Cicer arietinum L.) cultivar ILC 3279 and structurally elucidated. Both pterocarpan conjugates are constitutive metabolites of the chickpea cell cultures. Upon application of an elicitor from yeast to the cell cultures a substantial increase in the level of the phytoalexin aglycones medicarpin and maackiain was observed although a delayed but significantly higher rise of the conjugates also occurred. The significance of the pterocarpan conjugates for phytoalexin production is discussed.Abbreviations MeGM medicarpin 3-O-glucoside-6-O-malonate - MaGM maackiain 3-O-glucoside-6-O-malonate - MeG medicarpin 3-O-glucoside - MaG maackiain 3-O-glucoside - FGM formononetin 7-O-glucoside-6-O-malonate - BGM biochanin A 7-O-glucoside-6-O-malonate - IFR NADPH: 2-hydroxyisoflavone oxidoreductase - PTS pterocarpan synthase - IGT UDP-glucose: isoflavone 7-O-glucosyltransferase - IMT malonyl-coA: isoflavone 7-O-glucoside-6 -O-malonyltransferase - RT retention time - sh shoulder - d duplette - m multiplette - s singulette     

Drought effect on photosynthetic activity,osmolyte accumulation and membrane integrity of two <Emphasis Type="Italic">Cicer arietinum</Emphasis> genotypes

Drought was induced in chickpea (Cicer arietinum L.) genotypes (ChK 3226 and ILC 3279) differing in yield capacity. Water stress (S1, RWC around 55–50%; S2, RWC ≤ 40%) drastically reduced stomatal conductance (g _s) and net photosynthetic rate (P _N) in both genotypes. ILC 3279 showed greater photosynthetic capacity (A _max) decreases. Maximum PSII photochemical efficiency (F_v/F_m), photochemical quenching (q_P), total chlorophylls (Chls) and carotenoids (Cars) content showed stability in both genotypes under stress, but in S2 ILC 3279 presented an increase in basal fluorescence (F₀) and a greater reduction in estimation of quantum yield of linear electron transport (Φ_e) than ChK 3226. Membrane damage evaluated by electrolyte leakage occurred earlier and was greater in ILC 3279. It also presented a decrease of total fatty acids (TFA) along drought, while in ChK 3226 greater amounts of TFA were observed in S1. In rehydration, P _N of S1 plants completely recovered (ILC 3279) or remained slightly below control (ChK 3226). As regards S2 plants, ILC 3279 showed stronger P _N and g _s reductions than ChK 3226, despite both genotypes totally recovered A _max and chlorophyll (Chl) a fluorescence. ChK 3226 recovered more efficiently from membrane damage. Under control conditions, greater amounts of most of the studied soluble metabolites occurred in ChK 3226 plants. Malate and citrate decreased with water stress (S2) in both genotypes. Sucrose and pinitol (that had a higher concentration than sucrose in both genotypes) increased in ILC 3279 (S1 and S2), and decreased in ChK 3226 (S2). In ILC 3279 proline and asparagine followed similar patterns. Genotypes showed a similar shoot dry mass (DM) in control plants, but root DM was higher in ChK 3226. Drought reduced root and shoot DM in ChK 3226 already under S1, while in ILC 3279 root DM was unaffected by drought and shoot biomass decreased only in S2. Root/shoot ratio was always higher in ChK 3226 but tended to decrease under stress, while the opposite was observed in ILC 3279. No pods were obtained from control plants of both genotypes, or droughted ILC 3279 plants. ChK 3226 produced pods under S1 (higher yield) and S2. Under stress conditions, ChK 3226 was less affected in photosynthetic activity and membrane integrity, showing a better tolerance to drought. This agrees with the better yield of this genotype under water stress. Distinct strategies seem to underlie the different physiological responses of the two genotypes to water deficit. In spite of its significant solutes accumulation, ILC 3279 was more affected in photosynthetic activity and membrane integrity during water stress than ChK 3226, which showed better yield under drought. A relation could not be established between solutes accumulation of ILC 3279 and yield.     

Differential proline metabolism in vegetative and reproductive tissues determine drought tolerance in chickpea

Proline is emerging as a critical component of drought tolerance and fine tuning of its metabolism under stress affects the plants sensitivity and response to stress. Thus the study was carried out to analyse the effect of water deficit on the proline content and principal enzymes involved in its synthesis (Δ¹-pyrolline-carboxylate synthetase) and catabolism (proline dehydrogenase) at different developmental stages and in different organs (roots, nodules, leaves, pod wall, and seeds) of two chickpea (Cicer arietinum L.) cultivars differing in drought tolerance (drought tolerant ICC4958 and drought sensitive ILC3279). It was observed that increased Δ¹-pyrolline-carboxylate synthetase activity under moderate stress in roots and nodules of ICC4958 caused an increase in proline content during initiation of reproductive development whereas increased proline dehydrogenase activity in nodules and leaves at this period helped to maintain reducing power and energy supply in tissues and proper seed development as seed biomass increased consistently up to maturity. On the other hand, roots and nodules of ILC3279 responded to stress by increasing proline content after the developmental phase of reproductive organs was over (near maturity) which negatively affected the response of pod wall to stress. Concurrent increase in activities of Δ¹-pyrolline-carboxylate synthetase and proline dehydrogenase in pod wall of ILC3279 aggravated the oxidative stress and affected seed development as seed biomass initially increased rapidly under stress but was unaffected near maturity.     

Purification,characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one -1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The -1,3-glucanase (M_r = 36 kDa) and one of the chitinases (M_r = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M_r = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic -1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of -1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.Abbreviations AC acidic chitinase - BC basic chitinase - BG = basic -1,3-glucanase - CM-Chitin-RBV carboxymethylated-chitin-remazol brilliant violet - 2,4-D 2,4-dichlorophenoxyacetic acid - ILC international legume chickpea - M_r relative molecular mass - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.     

Histology of Disease Development in Resistant and Susceptible Cultivars of Chickpea (Cicer arietinum L.) Inoculated with Spores of Ascochyta rabiei

Abstract Histological studies were performed on a compatible and an incompatible interaction between chickpea ( Cicer arietinum L.) plants and the fungus Ascochyta rabiei (Pass.) Labr. The time course of infection, development on leaflets and stems of susceptible (ILC 1929) and resistant (ILC 3279) plants was monitored by light or scanning electron microscopy with the aim to compare histological changes as the basis for further work on biochemical changes in this plant-pathogen interaction.
Spores of A. rabiei began to germinate from 12 hpi on and developed a polar germ tube; fungal colonization, secretion of a mucilaginous exudate and appressoria formation (1–3 dpi) were identical on both cultivars. Leaves of susceptible plants were invaded by the fungus directly through the cuticle, the fungus then spread subepidermally followed by a rapid collapse of the leaf tissue (4–6 dpi). Development of leaf spots and fungal pycnidia could be observed 6–8 dpi. The resistant cultivarrapidly responded (24–48 hpi) to fungal infection and cells of the palisade parenchyma exhibited autofluorescence. In later stages of the infection (4–5 dpi) fluorescent areas developed to small necrotic spots all over the leaflet. These necrotic areas, were the result of cell death and a subsequent change in the leaf structure and were characterized by the accumulation of phenolic compounds. Leaves of the resistant cultivar were invaded by the fungus to less than 5%.     

Genetic Analysis of the Role of Phytoalexin Detoxification in Virulence of the Fungus Nectria haematococca on Chickpea (Cicer arietinum)

Chickpea (Cicer arietium L.) produces the antimicrobial compounds (phytoalexins) medicarpin and maackiain in response to infection by microorganisms. Nectria haematococca mating population (MP) VI, a fungus pathogenic on chickpea, can metabolize maackiain and medicarpin to less toxic products. These reactions are thought to be detoxification mechanisms in N. haematococca MP VI and required for pathogenesis by this fungus on chickpea. In the present study, these hypotheses were tested by examining the phenotypes of progeny from crosses of the fungus that segregated for genes (Mak genes) controlling phytoalexin metabolism. Mak1 and Mak2, two genes that individually confer the ability to convert maackiain to its 1a-hydroxydienone derivative, were linked to higher tolerance of the phytoalexins and high virulence on chickpea. These results indicate that this metabolic reaction is a mechanism for increased phytoalexin tolerance in the fungus, which thereby allows a higher virulence on chickpea. Mak3, a gene conferring the ability to convert maackiain to its 6a-hydroxypterocarpan derivative, also increased tolerance to maackiain in strains which carried it; however, the contribution of Mak3 to the overall level of pathogenesis could not be evaluated because most progeny from the cross segregating for this gene were low in virulence. Thus, metabolic detoxification of phytoalexins appeared to be necessary, as demonstrated in the Mak1 and Mak2 crosses, but not sufficient by itself, as in the Mak3 cross, for high virulence of N. haematococca MP VI on chickpea.     

Degradation of the pterocarpan phytoalexin medicarpin by Ascochyta rabiei

Four strains of Ascochyta rabiei pathogenic to chickpea (Cicer arietinum L.) were shown to efficiently degrade medicarpin (3-hydroxy-9-methoxypterocarpan), the main phytoalexin of this plant. Degradative studies were performed with mycelium preparations or with crude protein extracts of the fungus. Isolation and structural elucidation of 10 catabolites by chromatographic and spectroscopic techniques revealed that medicarpin degradation involves 1. reductive conversion to a 2-hydroxyisoflavan, 2. O-demethylation, 3. aromatic hydroxylation in ring A and 4. formation of a 1a-hydroxy-pterocarp-1,4-diene-3-one. As terminal aromatic catabolite 2,4-dihydroxybenzoic acid was found. A catabolic sequence for medicarpin is postulated and the results are discussed with regard to pterocarpan dissimilation by other phytopathogenic fungi.     

Some physiological responses of chickpea cultivars to arbuscular mycorrhiza under drought stress

A factorial experiment based on RCB design with three replicates was conducted to investigate changes in some physiological responses of two chickpea (Cicer arietinum L.) cultivars (Pirouz from Desi type and ILC482 from Kabuli type) to arbuscular mycorrhiza (Glomus etunicatum Becker and Gerdman) under different irrigation treatments. The experiment was carried out in the greenhouse of the Agricultural Faculty of Kurdistan University from April to August 2009. The results showed that leaf chlorophyll content of chickpea cultivars was significantly increased by arbuscular mycorrhiza (AM) under both well and limited irrigation conditions. Proline accumulation in chickpea leaves under moderate and severe drought stresses was significantly stronger than that under optimum irrigation. Inoculation of chickpea with mycorrhizal fungi caused an increase in the activities of polyphenol oxidase and peroxidase, but a decrease in the activity of catalase. Comparisons among different irrigation levels showed that chickpea plants under drought stress had the most active lipid peroxidation. Non-AM plants showed stronger lipid peroxidation under moderate and severe water stresses than AM plants. Lipid peroxidation was more active in Pirouz leaves than in ILC₄₈₂ leaves. It seems that Kabuli-type cultivar responded better to mycorrhizal symbiosis under drought stress than Desitype cultivar.     

Biotic elicitors as a means of increasing isoflavone concentration of soybean seeds

Soybean (Glycine max) seeds contain isoflavones that have positive impacts on human health. Four greenhouse experiments were conducted to determine if isoflavone concentration of mature soybean seeds could be increased using elicitor compounds. The effects on soybean seed isoflavone concentrations following foliar applications of two lipo‐chitooligosaccharides (LCO) [Bj V (C_18:1 MeFuc) and Bj V (Ac, C¹⁶, MeFuc)], chitosan, actinomycetes spores (Streptomyces melanosporofaciens strain EF‐76) and yeast extract at different concentrations and growth stages were evaluated. Combined chitosan seed treatment and foliar applications were also evaluated. Concentrations of daidzein, genistein, glycitein, and total isoflavones were determined by HPLC. Foliar applications of LCOs, chitosan, and actinomycetes caused a marked increase in individual and total isoflavone concentration (ranging between 21% and 84%) of mature seeds when compared to untreated control plants. There were limited differences between the different concentrations and stages of application tested for chitosan and actinomycetes; however, response to LCOs was greatest at higher concentrations (i.e. 10^‐6 M) when applied at the early podding stage. Compared to untreated plants, combined seed treatment and foliar applications of chitosan increased individual and total isoflavone concentration of mature soybean seeds by 16% to 93%. Trends were similar for different cultivars, however, the magnitude of the response varied. Finally, response to foliar applications of yeast extract was highly concentration dependent with increases of up to 56% in total isoflavone observed with 2 mg mL^‐1. Results indicate that elicitors hold promise as a way of increasing isoflavone concentration of mature soybean seeds.     

Exploring Crop–Microbiome Interactions Towards Improving Symbiotic Performance of Chickpea (Cicer arietinum) Cultivars Using Cyanobacterial Inoculants

The significance of integrated nutrient management practices is well established in improving the productivity of chickpea (Cicer arietinum); however, the effects of the inoculation of cyanobacterial inoculants on nodule metabolism, microbiome and associated genes are less explored. In the present investigation, cyanobacterium Anabaena laxa (A. laxa) and biofilm developed using Anabaena torulosa, with Mesorhizobium ciceri as a partner (An-M. ciceri), were evaluated along with Mesorhizobium ciceri (M. ciceri) alone, in three chickpea cultivars. Microbial inoculation led to 40–70% enhancement in nitrogen fixation, leghaemoglobin and ureide content, and two- to threefold increment in nitrate reductase and phosphoenolpyruvate carboxylase activity of the nodules. An enhancement of 30–50% in the soil available macro- and micronutrients and plant growth attributes was also observed. A significant correlation between the soil microbiological and plant parameters was recorded, particularly in relation to the nitrogen dynamics. Increases in the leghaemoglobin content in nodules due to An-M. ciceri, A. laxa and M. ciceri ranged from 18 to 40%, particularly in chickpea cv. BG372 in which 60–80% enhancement was recorded. Whereas the nifH gene copies in the nodule tissues ranged from 5.00 × 10⁶ to 3.35 × 10⁷ g⁻¹, the application of A. laxa led to higher abundances of nifH gene copies in desi chickpea cv. BG372 and kabuli BG1053 cultivars. An-M. ciceri, followed closely by A. laxa, was the top-ranking treatment, and chickpea cv. BG372 was the best performing cultivar; An-M. ciceri—chickpea cv. BG372 proved to be the superior combination for higher plant growth and soil nutrient-related traits.

     

Involvement of Epicatechin in Cultivar Susceptibility of Avocado Fruits to Colletotrichum gloeosporioides after Harvest1

Avocado cultivars were defined as susceptible and resistant to Colletotrichum gloeosporioides depending upon the length of the incubation period of the disease after fruit softening. In the susceptible cultivars Fuerte, Horshim, Vurtz, Rincon, and Benik, epicatechin concentration of the peel decreased to 60-130 μg.g^?1, fr. wt. at fruit softening and symptoms appeared on the same or one day later. In the resistant cultivars Hass, Nabal, Netaim and Pinkerton, epicatechin concentration was still 632–1740 μg.g^?1 fr. wt. when fruit softening and symptoms appeared only 4-10 days later. When susceptible Fuerte fruits became soft the concentration of the antifungal compound 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15 diene, had decreased to 120 μg.g^?1 fr. wt. and symptoms appeared. In resistant Hass fruits, the antifungal diene was still 238 μg.g^?1 fr. wt. at fruit softening; and it had further decreased to 159 μg.g^?1 fr. wt. when symptoms appeared, four days later. A modified atmosphere and 0.2 M CaCl₂ infiltration both delayed softening of Fuerte fruits; but symptom appearance on these fruits was related to diene decrease and not to fruit softening. The results are discussed in relation to the hypothesis that the susceptibility of avocado cultivars to post-harvest decay by C. gloeosporioides is related to the degradation of the antifungal diene, catalyzed by avocado lipoxygenase, the activity of which is regulated by the decline of its inhibitor epicatechin.     

Interrelationship between Nutrients, Pathogenicity and Phytoalexin Metabolism of Botrytis cinerea on Clover Leaves

Botrytis cinerea spores suspended in 0.28 M glucose solution caused limited lesions on clover leaves, on which the clover phytoalexins maackiain and medicarpin accumulated to 1028 μg and 856 μg/g fresh wt respectively after 4 days incubation. During this time, little or none of the phytoalexin degradation products were detected. On the other hand, B. cinerea spores in sucrose casamino acids (SCA) liquid medium caused larger lesions than spores in glucose, and less maackiain and medicarpin (298 μg and 95 μg/g fresh wt respectively) and high concentrations of the degradation products were detected. B. cinerea mycelium in SCA also caused large lesions and both the phytoalexins and their degradation products were detected.,Sclerotinia laxa spores in 0.28 M glucose or its mycelium in SCA liquid medium did not cause any lesions apart from a minute necrotic fleck, and although phytoalexins were recovered from leaves inoculated with spores (67 μg and 78 μg/g fresh weight of maackiain and medicarpin respectively after 4 days) and leaves inoculated with mycelium (150 μg and 167 μg/g fresh wt maackiain and medicarpin respectively after 3 days), no phytoalexin degradation products were detected. The implications of, these results in understanding the interrelationship between nutrients, pathogenicity and phytoalexin metabolism are discussed.     

Constitutive and elicitation induced metabolism of isoflavones and pterocarpans in chickpea (Cicer arietinum) cell suspension cultures

Constitutive phenolics of chickpea cell suspension cultures are the isoflavones formononetin and biochanin A, the isoflavanones homoferreirin and cicerin and the pterocarpans medicarpin and maackiain. They accumulate as vacuolar malonylglucosides. The biosynthetic pathways to isoflavones, pterocarpans and malonylglucoside conjugates together with their enzymes are explained. Elicitation of cell cultures leads to pronounced increases in the activities of biosynthetic enzymes with differential effects on the enzymes involved in conjugate metabolism. Low elicitor doses favour pterocarpan conjugate formation whereas high doses lead to pterocarpan aglycone accumulation accompanied by vacuolar efflux of formononetin and pterocarpan malonylglucosides. Elicitor-induced changes in enzyme activities and vacuolar efflux of conjugates are prevented by application of 10^-3M concentrations of cinnamic acid. Cinnamate is alternatively metabolized to a glucose ester, a S-glutathionyl conjugate and to cell wall bounds forms; these reactions are intensified by elicitation. Isoflavone and pterocarpan biosynthesis and conjugate metabolism as regulated by elicitation and cinnamate is depicted in a metabolic grid to explain the complex regulatory pattern of phenolic accumulation in chickpea cell cultures.Abbreviations AOPP L--aminooxy--phenylpropionic acid - BGM biochanin A 7-0-glucoside-6-0-malonate - FGM formononetin 7-0-glucoside-6-0-malonate - HPLC high performance liquid chromatography - MaGM maackianin 3-0-glucoside-6-0-malonate - MeGM medicarpin 3-0-glucoside-6-0-malonate