Search

The development of an in vitro model system to study the process of dimerization of hydroxycinnamic acids is reported. Model compounds ferulic acid (FA) or methyl ferulate were reacted with H2O2 in the presence of horseradish peroxidase (HRP) or peroxidases from Lupinus albus L. apoplastic fluids. Following solid-phase extraction, the reaction products were analysed using gas chromatography-mass spectrometry. Major analytical and biochemical parameters of model reactions were determined and optimized. Six ferulic dimers were identified and quantified. With the use of this model system we found methyl ferulate to be a better substrate for lupin and horseradish peroxidases than FA. Use of various peroxidases did not influence the qualitative composition of reaction products although it affected the rate of substrate utilization and the quantitative output of reactions. Various isoforms of lupin apoplastic peroxidases revealed differentiated specificity towards hydroxycinnamic acids or their methyl esters. The potential of isoflavonoids (major phenolic components in white lupin cell walls) to influence peroxidase-catalysed formation of ferulic dehydrodimers was also tested. Genistein (5,7,4'-trihydroxyisoflavone) and genistin (genistein-7-O-β-glucoside) did not affect the qualitative composition of the reaction products. However, genistein inhibited the rate of ferulic substrate oxidation, while genistin showed the opposite effect, stimulating both utilization of ferulic substrate by HRP and subsequent polymerization and / or degradation of dehydrodimers formed. To our knowledge, this is the first indirect evidence that isoflavonoids might play a regulatory role in the oxidative formation of intermolecular cross-links in cell walls.

The metabolomics analysis was used in this study to examine the changes in metabolite profile of Moldavian balm under water-deficit stress, symbiosis with arbuscular mycorrhizal fungus, plant growth-promoting rhizobacteria, and their interactions. This project was realized by applying a completely randomized model considering three factors, the arbuscular mycorrhizal fungus Claroideoglomus etunicatum, the rhizobacteria Micrococcus yunnanensis, and irrigation regimes (100, 70, and 40% field capacity). The results showed that the sugar metabolisms are the most affected metabolic pathways under drought stress. Moreover, the increase of some amino acids, citric acid cycle intermediates, and phenolic acid metabolites could contribute to the tolerance mechanisms of non-inoculated plants against water-deficit stress. These results suggested that soil microorganisms could be used to mitigate the negative impacts of drought stress by inducing the accumulation of some of the intermediary metabolites that act as osmolytes or their precursors. Overall, these results indicated that symbiotic association with soil microorganisms leads to metabolic and physiological adaptation which can increase the performance of this important medicinal plant under drought conditions.

A wide range of compounds that occur in the genus Hypericum are listed as effective drugs of natural origin. The main biological activities of several Hypericum representatives are due to the presence of naphthodianthrones, phloroglucinols, and other diverse groups of secondary metabolites that synergistically contribute to their therapeutic effects. The regulation of biosynthesis of hypericin as the key bioactive naphthodianthrone remains uncertain. Here, we present liquid chromatography mass spectrometry-based phenotyping of 17 Hypericum species, the results of which suggest an important role for skyrin and its derivatives in the polyketide pathway that leads to hypericin formation. Moreover, we report for the first time the presence of new metabolites in the genus Hypericum that are related to classes of anthraquinones, their derivatives, and phloroglucinols. As skyrin and other species of anthraquinones are rarely found in higher plants but frequently occur in fungal microorganisms, the obtained results suggest that further research on the synthesis pathways of hypericin and the role of anthraquinone derivatives in plant metabolism should be carried out. The fact that these compounds are commonly synthesized in endophytic fungi and perhaps there is some similarity in the metabolic pathways between these organisms should also be investigated. Electronic supplementary material The online version of this article (10.1007/s00216-018-1384-0) contains supplementary material, which is available to authorized users.

Flavonoid glycoconjugates from roots and leaves of eight North America lupine species (Lupinus elegans, Lupinus exaltatus, Lupinus hintonii, Lupinus mexicanus, Lupinus montanus, Lupinus rotundiflorus, Lupinus stipulatus, Lupinus sp.), three Mediterranean species (Lupinus albus, Lupinus angustifolius, Lupinus luteus) and one species from South America domesticated in Europe (Lupinus mutabilis) were analyzed using two LC/MS systems: low-resolution ion trap instrument and high-resolution quadrupole-time-of-flight spectrometer. As a result of the LC/MS profiling using the CID/MS(n) experiments structures of 175 flavonoid glycoconjugates found in 12 lupine species were identified at three confidence levels according to the Metabolomic Standard Initiative, mainly at level 2 and 3, some of them were classified to the level 1. Among the flavonoid derivatives recognized in the plant extracts were isomeric or isobaric compounds, differing in the degree of hydroxylation of the aglycones and the presence of glycosidic, acyl or alkyl groups in the molecules. The elemental composition of the glycoconjugate molecules was established from the exact m/z values of the protonated/deprotonated molecules ([M+H](+)/[M-H](-)) measured with the accuracy better than 5 ppm. Information concerning structures of the aglycones, the type of sugar moieties (hexose, deoxyhexose or pentose) and, in some cases, their placement on the aglycones as well as the acyl substituents of the flavonoid glycoconjugates was achieved in experiments, in which collision-induced dissociation was applied. Flavonoid aglycones present in the studied O-glycoconjugates were unambiguously identified after the comparison of the pseudo-MS(3) spectra with the spectra registered for the standards. Isomers of flavonoid glycoconjugates, in which one or two sugar moieties were attached to 4'- or 7-hydroxyl groups or directly to the C-6 or C-8 of the aglycones, could be distinguished on the basis of the MS(2) spectra. However, the collision energy applied in the CID experiments had to be optimized for each group of the compounds and there were no universal settings that allowed the acquisition of structural information for all the compounds present in the sample. Information obtained from the flavonoid conjugate profiling was used for the chemotaxonomic comparison of the studied lupine species. A clear-cut discrimination of the Mediterranean and North American lupines was obtained as a result of this analysis.

Three research problems were addressed in this work: an extent of root damage caused by carrot fly (Psila rosae) in a collection of carrot breeding accessions as well as correlation between resistance to this pest and accumulation of certain phenolics and mRNAs of the genes coding for phenylalanine ammonia-lyase (PAL). Differences in susceptibility between the studied accessions were statistically significant. The most resistant were lines 7262A and DC 96367. Moreover, no correlation was observed between accumulation of the specific metabolites and the extent of the root damage. However, proportion of certain phenolics exhibited positive correlation with the resistant phenotype. This was shown for the ratios of luteolin 7-O-glucoside to kaempferol 3-O-glucoside and methyluteolin 7-O-glucoside to kaempferol 3-O-glucoside. Susceptibility of roots to larval damage also correlated with semi-quantitatively estimated accumulation of PAL1 and PAL3 mRNAs in leaves. Both PAL genes were expressed at the highest level in line DC 96367 and less strongly in line 7262A. Distinctly lower accumulation of the PAL transcripts was observed for the susceptible varieties: Dolanka and Karotan.

Ascochyta pisi is a necrotrophic pathogenic fungus, which mainly survives between seasons through infected seeds. Defence responses of pea embryo axes to A. pisi were investigated in the heterotrophic phase of seed germination and during the transition from the heterotrophic to the autotrophic phase. Germinated pea seeds, both non-inoculated and inoculated with A. pisi, were cultured in perlite for 96 h. Polarographic studies performed on intact embryo axes of germinating pea seeds infected with A. pisi showed a high respiratory intensity in time from 48 to 96 h after inoculation. Forty-eight-hour embryo axes of germinating pea seeds exhibited the highest respiration rate, which in infected axes was maintained at the following time points after inoculation. Moreover, at 72 and 96 h after inoculation, respiratory intensity was by 64% and 73% higher than in the control. Electron paramagnetic resonance analysis revealed a higher concentration of semiquinone free radicals with g values of g || = 2.0031 ± 0.0004 and g ⊥ = 2.0048 ± 0.0004 in infected axes than in the control. Generation of superoxide anion radical was also higher in infected axes than in the control but stronger at 72 and 96 h after inoculation. Starting from 72 h after infection, the level of Mn2+ ions in infected axes decreased in relation to the control. At the same time, the highest activity of superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) was observed in 72-h infected axes. In turn, the activity of peroxidase (EC 1.11.1.7) up to 72 h after infection was lower than in the control. In 48-h infected embryo axes, a very high level of pterocarpan pisatin was observed. Infection of germinating pea seeds with A. pisi restricted mainly the growth of the epicotyl, but did not inhibit the increase in length and fresh weight of root embryo axes versus cultivation time. These results indicate that in pea during the stages of seed germination and early seedling growth, protective mechanisms are induced in embryo axes against A. pisi.

Little biochemical information is available regarding the responses of embryo axes to Fusarium oxysporum during the stages of seed germination and early seedling growth of yellow lupine, i.e. in the heterotrophic phase and during the transition from the heterotrophic to the autotrophic phase [1] . In the experimental protocol used in this study germinated yellow lupine seeds, both non-inoculated and inoculated with F. oxysporum , were cultured for 96 h in perlite. An analysis of soluble carbohydrates, between 48 and 96 h of culture, shows a decrease in sugar content, i.e. that of sucrose and fructose in infected embryo axes of germinated seeds and that of sucrose and glucose in the controls at 72 h. After infection, lower levels of sucrose, glucose and fructose were observed in infected axes than in controls. Invertase activity (EC 3.2.1.26) in infected tissues was higher than in the controls with the exception of acid invertase activity at 96 h. The highest post-infection alkaline and acid invertase activity was recorded in 48 h embryo axes of germinated seeds. An HPLC analysis in the period between 48 and 96 h after infection showed a decrease in the level of glycosylated isoflavonoids, i.e. genistein 7-O-glucoside, 2′OH genistein 7-O-glucoside, genistein 4′,7-O-diglucoside, 2′OH genistein malonyl glucoside and 2′OH genistein malonyl glucoside, which was particularly marked at 72 h after infection. Concentrations of these glucosides in 72 h and 96 h infected tissues were lower than in controls. We need to stress here a very high isoflavonoid content observed in intact embryo axes of germinating seeds at 48 h after inoculation, i.e. shortly after the embryo axes penetrated the seed coat. At this time point in infected tissues the concentrations of genistein glucosides and that of a free aglycone, genistein, were many times higher than in the controls. At later time points after inoculation genistein content was also higher than in the controls. A different tendency to that observed in infected axes was seen in the control specimens, where the level of flavonoid glucosides rose in the period between 48 and 72 h of culture, followed by a subsequent decrease. The activity of phenylalanine ammonialyase (PAL, EC. 4.3.1.5) in infected embryo axes of germinating seeds was higher than in the controls and the highest post-infection activity of this enzyme was observed in 48 h axes. At this time point, a considerable post-infection accumulation was observed using semi-quantitative RT-PCR for transcripts of phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and isoflavone synthase (IFS), which was consistent with the high level of isoflavonoids. These results indicate that in yellow lupine during the stages of seed germination and early seedling growth changes in carbohydrate and isoflavonoid metabolism occur in embryo axes in response to infection with F. oxysporum f.sp. lupini . These changes may be included in the active protection mechanism in these axes.

Flavones and isoflavones are a major group of phenolic secondary metabolites which occur in leaves of narrow leafed lupine (Lupinus angustifolius) either as free aglycones or in a form of glycosides and malonyl-glycosides. Profiles of phenolic compounds in leaves of seedlings infected with anthracnose causing fungus Colletotrichum lupini were compared to those of healthy plants. A HPLC with diode array UV detector was used as the analytical method and identification of these secondary metabolites was confirmed with a HPLC/MSn instrument. Isomers of several target compounds differing in the glycosilation and/or malonylation pattern were detected in the studied samples. However, the application of standard HPLC with C18 columns resulted in the co-elution of several glyconjugates in single chromatographic peaks whereas for isoflavonoid aglycones complete resolution was achieved. Lupine plants grown in a greenhouse were either sprayed with the C. lupini spore suspension or the suspension was spotted on to wounded leaves. Profiles of the isoflavones were altered in result to infection with both methods. In particular, the concentration of isoflavone free aglycones detected in extracts from diseased plants was substantially increased in all of the studied samples. However, the pattern of these compounds depended on the age of lupine leaves as well as on the method of infection. Synthesis of luteone and 2′-hydroxygenistein was enhanced in the youngest leaves of plants sprayed with spores as well as in wound-infected leaves. Wighteone synthesis was induced mainly in older leaves of plants sprayed with the spore suspension.

Lupine (Lupinus spp.) is one of the crop plants from the Fabaceae family cultivated on a moderate scale in Europe, Australia and South America; however, its cultivation suffers from a severe fungal disease anthracnose caused by Colletotrichum lupini fungus. The search for resistant plant genotypes as well as methods of plant immunization against such infections is of importance. Plant interaction with pathogenic microorganisms results in complex regulation of many biochemical and physiological processes. Activation of expression of defence genes that leads to the induction of bioactive secondary metabolites biosynthesis is among them. The aim of the presented work was to investigate changes in the isoflavonoids quantities as the reaction of narrow leaf lupine (Lupinus angustifolius) plants growing in the field conditions to infection with the pathogenic fungus (C. lupini) or treatment with its phytotoxic metabolites followed after 48 h with infection with the fungus. The metabolic profiling after field experiments revealed variety-specific changes of these compounds. Elicitation of plants with fungal phytotoxin prior to infection resulted in higher levels of prenylated isoflavones, especially phytoalexins luteone and wighteone and their various glycoconjugates in comparison to those observed in plants infected only with the fungal spores. The metabolomic analyses were supported by the transcriptomic view of genes involved in isoflavonoids biosynthesis. Infection of Lupinus angustifolius by Colletotrichum lupini combined with former elicitation of plants results in accumulation of prenylated isoflavonoids and change in the isoflavone prenyltransferase gene expression pattern.

In wheat (Triticum aestivum L.), treatment with herbicide safeners enhances the expression of enzymes involved in pesticide detoxification and reduces crop sensitivity to herbicides. Since these same enzymes are involved in plant secondary metabolism, it was of interest to determine whether or not the safener cloquintocet mexyl perturbed phenolic metabolism in wheat seedlings. LC/ESI/MS analysis identified 14 phenolic substrates in the shoots of young wheat plants. Fragmentation imposed by collision induced dissociation identified specific C-glycosidic conjugates of 4',5,7-trihydroxflavone (apigenin), 3',4',5,7-tetrahydroxyflavone (luteolin) and 3'-O-methylluteolin. Treatment of 7-day-old wheat shoots with cloquintocet mexyl resulted in an accelerated depletion of the conjugates of all three flavones, most notably with the glycosides of luteolin. In contrast, safener treatment caused the selective accumulation of 4',5,7-trihydroxy-3',5'-dimethoxyflavone (tricin) and the phenylpropanoid ferulic acid. Changes in phenolic content were associated with an increase in O-methyltransferase and C-glucosyltransferase activity toward flavonoid substrates as well as the classic enhancement of detoxifying glutathione transferases. Our results suggest that in addition to altering the capacity of wheat to metabolise herbicides and other xenobiotics, safeners can also cause a selective shift in the metabolism of endogenous phenolics.

The profiles of isoflavone conjugates in extracts obtained from different parts of Lupinus exaltatus Zucc. grown in Mexico were compared using HPLC-UV and HPLC-ESI/MSn. Collision-induced dissociation–MSn experiments were performed using an ion trap analyser during HPLC-ESI/MS analyses. Nineteen isoflavone conjugates were identified in samples obtained from air-dried roots, leaves, stems and inflorescences of lupin plants. It was possible to determine the structures of the studied compounds on the basis of the MS recorded. The compounds identified were di- and mono-glucosides of genistein and 2′-hydroxygenistein with a different pattern of C- and O-glycosylation. Some glucosides were acylated with malonic acid. It was not possible to establish the glycosylation sites on the basis of MS alone; however, it was possible to differentiate isoflavone C- and O-glucosides. The highest levels of isoflavones and their conjugates were detected in roots and the lowest in stems. Free aglycones were identified in roots and inflorescences but they were not found in stems and leaves. Copyright © 2006 John Wiley & Sons, Ltd.

Extracts from plantlets of different species of the genus Drosera, grown as in vitro cultures, were evaluated for the level of phenolic secondary metabolites from the group of naphthoquinones and flavonols. The profiles of natural products in the extracts obtained from different species were monitored by HPLC with UV detection at 260 and 330 nm. On the basis of the data obtained, Drosera binata, the species with the highest amount of plumbagin, was selected for further studies. The most effective method of extraction of quinones was established and the composition of phenolic secondary metabolites in the tissues was determined. For the identification of phenolic compounds, HPLC-UV and HPLC-ESI/MS were applied. Copyright © 2005 John Wiley & Sons, Ltd.

Defense responses to inoculation with Fusarium oxysporum SCHLECHT f. sp. lupini were studied in embryo axes of Lupinus luteus L. cv. Polo cultured on a medium with sucrose (60 mM) or without it. Exogenous sucrose caused a marked endogenous increase in concentrations of sucrose, glucose and fructose in embryo axes. In axes cultured with sucrose, high performance liquid chromatography (HPLC) revealed generally higher levels of isoflavone glycosides (particularly until 48 h of culture) and free aglycones (genistein, wighteone, luteone). Inoculation resulted in a considerable decline in soluble carbohydrates between 24 and 72 h of culture. Simultaneously, the infection stimulated an increase in the level of free isoflavone aglycones in inoculated embryo axes, as compared to non-inoculated ones. Concentrations of free aglycones (i.e. genistein, wighteone and luteone) after infection were particularly high in inoculated embryo axes fed with sucrose. Genistein was a better inhibitor to F. oxysporum growth than genistein 7-O-glucoside tested. Exogenous sucrose also stimulated the activity of phenylalanine ammonialyase (PAL, EC 4.3.1.5)--an important enzyme initiating phenylpropanoid metabolism. After infection of tissues, a strong increase was observed in the activity of PAL and beta-glucosidase (EC 3.2.1.21)--an enzyme hydrolyzing isoflavone glycosides. Furthermore, the growth of inoculated embryo axes cultured with sucrose was less inhibited as a result of infection than inoculated axes cultured under carbohydrate deficiency conditions. Additionally, it had been reported previously that disease symptoms of embryo axes growing in the presence of sucrose were less intensive [30]. These results suggest that soluble sugars are involved in the mechanism of resistance, as they can stimulate phenylpropanoid metabolism and contribute to the increase in concentration of isoflavonoids, which are important elements of the defense system of legumes.

Transgenic potato plants overexpressing and repressing enzymes of flavonoids biosynthesis were created and analyzed. The selected plants clearly showed the expected changes in anthocyanins synthesis level. Overexpression of a DNA encoding dihydroflavonol 4-reductase (DFR) in sense orientation resulted in an increase in tuber anthocyanins, a 4-fold increase in petunidin and pelargonidin derivatives. A significant decrease in anthocyanin level was observed when the plant was transformed with a corresponding antisense construct. The transformation of potato plants was also accompanied by significant changes in steroid alkaloid glycosides (SAG) level in transgenic potato tuber. The changes in SAGs content was not dependent on flavonoid composition in transgenic potato. However, in an extreme situation where the highest (DFR11) or the lowest (DFRa3) anthocyanin level was detected the positive correlation with steroid alkaloid content was clearly visible. It is suggested that the changes in SAGs content resulted from chromatin stressed upon transformation. A liquid chromatography/mass spectrometry (LC/MS) system with electrospray ionization was applied for profiling qualitative and quantitative changes of steroid alkaloid glycosides in tubers of twelve lines of transgenic potato plants. Except alpha-chaconine and alpha-solanine, in the extracts from dried tuber skin alpha-solamargine and alpha-solasonine, triglycosides of solasonine, were identified in minor amounts, triglycosides of solanidine dehydrodimers were also recognized.

Qualitative and quantitative composition of flavonoid and isoflavo- noid glycosides as well as free aglycones in lupin seedlings (roots and aerial parts) grown under different light conditions or responding to infection with Pleiochaeta setosa, a fungus causing brown leaf spot, were monitored by liquid chromatography with UV and/or mass spectrometric detection. Both physical and biotic factors affected flavonoid and isoflavonoid levels in lupin tissues. Fungal infection evoked significant increase in the amounts of genistein, 2′-hydroxygenistein, and their prenylated derivatives that are thought to function as lupin phytoalexins. Effect on quantitative changes of glycosylated flavonoids and isoflavonoids in the roots and aerial parts was less significant. Moreover, different light conditions applied during seedling growth caused relative changes of flavonoid and isoflavonoid conjugates composition, especially in the leaves of white lupin plants. The chemical structures of flavonoid and isoflavonoid conjugates present in Lupinus angustifolius were elucidated. In addition to genistein and2′-hydroxygenistein glycosides, flavonol conjugates were identified in leaves, while the composition of root isoflavonoids was similar to that of L. albus reported earlier.

Plants respond to various biotic or abiotic stimuli with different mechanisms and the mobilization of phenolic compounds is one of them. In white lupin (Lupinus albus L.) plants, isoflavones and their glucosides were localized in cell walls where the high constitutive activity of β-glucosidase (EC 3·2·1·21) was also identified. The enzyme was partially purified from root cell walls. Its polymeric active form has 180 or 200 kDa as determined by non-denaturing electrophoresis and gel filtration, respectively, and the isoelectric point is at pH 6·9. The enzyme is an exoglucosidase, preferentially hydrolysing conjugates of phenolic compounds with β anomers of glucose. It is not active against purified lupin cell walls. The specific β-glucosidase activity varies in different tissues with the highest one in roots, and always higher in cell walls than in protoplast. The cell wall location of the enzyme was confirmed biochemically by its activity in intercellular washing fluids. Both aglycones and glycosides were also present in these fluids. The specific β-glucosidase activity correlated well with the isoflavonoid aglycone/glycoside ratios in various tissues: the higher β-glucosidase activity, the higher relative aglycone content. β-glucosidase activity was found to be inducible under conditions of yeast elicitor treatment. Induction of the enzyme was accompanied by changes in the isoflavone secretion and accumulation, suggesting the regulatory role of β-glucosidase activity in root exudation of isoflavonoids. No correlation however, was found between changes in β-glucosidase activity and the presence of isoflavonoids in root exudates of plants grown under various nitrogen nutrition regimes.

Two flavonoid sulphates, i.e. quercetin 3-O-sulphate-7-O-α-arabinopyranoside and kaempferol 3-O-sulphate-7-O-α-arabinopyranoside, were isolated from leaves of Atriplex hortensis L. The structures of these compounds were established by UV, 1H and 13C NMR, 2D NMR and MS spectra. The compounds were isolated for the first time from plant material.

Solid phase extraction on strong cation exchanger (benzene sulphonic groups) and reverse phase (octadecyl groups) cartridges connected in series was used for a one-step isolation of quinolizidine alkaloids and phenolic compounds present in extracts from lupin seedlings. Both classes of compounds were further analysed by gas chromatography-mass spectrometry. This technique was used to analyse changes in the alkaloid and isoflavonoid profiles during the defence reaction of lupin plants evoked by abiotic (CuCl2) and biotic (elicitor preparation from Colletotrichum lindemuthianumcell walls) factors. © 1997 John Wiley & Sons, Ltd.

Among many aspects of plant defence responses to pathogenic infection are changes in the composition of the exocellular matrix. To study potential defences in white lupin (Lupinus albus L.), suspension-cultured cells were treated for 24 h with one of three different elicitors : CuCl 2 , and two fungal elicitor preparations from purified cell walls of yeast and Colletotrichum lindemuthianum. Two subsets of exocellular proteins: ionicallybound wall proteins and proteins secreted into culture medium, were isolated, and their patterns compared following electrophoretic separation. Only a few proteins were observed in culture filtrates with dominating bands at 27, 33, and 42 kDa. About 30 proteins were observed in cell wall extracts. Changes in protein intensities evoked by elicitor treatments depended on the type of elicitor used, the age and composition of lupin cell culture, and concentration of applied fungal elicitor. Based on these observations, ten proteins were chosen for N-terminal sequencing, and sequences 5-30 amino acids long for nine proteins were obtained. Three of the major proteins sequenced were identified as acidic exocellular chitinase, polygalacturonase-inhibiting protein, and germin/oxalate oxidase.

Flavan-3-ols and an A-type proanthocyanidin have been isolated from seeds of Lupinus angustifolius . The structures of these compounds were establis

Summary Two isoflavonoid glycosides (7-O-β-glucopyranosides of 2′-hydroxygenistein and genistein) and up to six aglycones (2′-hydroxygenistein, genistein, wighteone, luteone, lupinisoflavone A, parvisoflavone B) were identified in root exudates and/or root extracts from white lupin ( Lupinus albus L. cv. Bac) plants. Three-d-old seedlings were cultivated for 14d on 1/2 Knop's nutrient solutions. Various treatments, differing in the form (No 3 − , NH 4 + , NH 4 N0 3 , urea) and concentration of nitrogen, and the presence of plant growth regulators in the medium were tested. Nitrogen, in all forms applied, affects both the exudation and accumulation of phenolics with the inhibitory activites arranged in the order: N0 3 − > NH 4 N0 3 > urea > NH 4 + . With higher concentration of N in the medium, the exudation of phenolics decreases. Processes of isoflavone exudation and accumulation respond differentially to nitrogen status. Exogenously applied plant growth regulators modify inhibitory effects of nitrate on the exudation and accumulation of isoflavones. Their activity depend on the type of growth regulator used (NAA, BAP, GA 3 ), its chemical structure (IAA, IBA, NAA) and concentration in the medium. Lupin isoflavones might be potential signals in legume — Rhizobium interactions. These data show a correlation between plant nutrient status and the accumulation and exudation of putative phenolic signal molecules, thus indicating the existence of possible additional controls in the regulation of nodulation process.

The phenolic acids, including 4-hydroxybenzoic and 4-hydroxycinnamic acids, and their derivatives, such as 6,7-dihydroxycoumarin and 1,2-dihydroxybenzene, were isolated from bitter lupine seeds and were identified using gas chromatography-mass spectrometry. These compounds inhibited lettuce seed germination in the first 24 hr after sowing, but after 72 hr germination was comparable with that of the control. However, very strong suppression of seedling growth, especially the roots, was observed for higher concentrations of the lupine seed fractions containing phenolic acids. Effects observed in the lettuce germination bioassays were compared with those produced by nine pure phenolic acids previously identified in the mixture. The ethyl acetate fraction from lupine seed extract inhibited seedling growth as effectively as pure 1,2-dihydroxybenzene, the strongest inhibitor of the pure phenolic compounds studied. The possible reason for this could be the synergistic effect created in the mixture of phenolic compounds isolated from the extract.

Medicago truncatula is a model species for the study of the unique secondary metabolism in legumes. LC/MS/MS analysis was used to identify and profile flavonoid glycoconjugates and free aglycones in leaves of M. truncatula (ecotype R108-1) infected with the fungal pathogen Phoma medicaginis. Use of a high resolution analyzer with a collision induced dissociation tandem mass spectrometer (CID MS/MS) permitted structural elucidation of target secondary metabolites and four new acylated flavone glycosides have been identified. Changes in the phytoalexin medicarpin and its isoflavone precursors were quantitatively monitored at various time points after fungal spore application. Application of spores induced disease symptoms in the leaves of infected plants and resulted in an increase in the medicarpin precursors formononetin 7-O-glucoside and malonylated formononetin 7-O-glucoside between one and three days post-infection. Relative concentrations of medicarpin were highest five days post-infection. The rapid increase of these molecules was clearly positively correlated to the infection process as certain of them were absent in uninfected leaves, suggesting that the relative rate of their synthesis is tightly related with the infection process.

HPLC combined with electrospray ionization MS (LC–ESI-MS) was used for the identification of isoflavonoids and their glycosides in extracts from white lupin (Lupinus albus) tissues. The applicability of LC–ESI-MS for the simultaneous analysis of secondary metabolites of different polarities (free aglycones and their glycosides) is demonstrated. For optimization of the chromatographic and MS conditions, genistin (genistein 7-O-β-glucoside) and rutin [quercetin 3-O-β-(6″-O-α-rhamnosyl glucoside)] were used as standards. The detection limit for the analysis of genistin by HPLC was determined to be 20 ng (ca. 50 pmol). The analytical conditions established were employed to analyse isoflavonoids and their glycosidic conjugates obtained from lupin root extracts by solid phase extraction. Four glycosides and seven isoflavonoid aglycones were detected in the analysed samples. Copyright © 1999 John Wiley & Sons, Ltd.

Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H](+). Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS(3) experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0287-2) contains supplementary material, which is available to authorized users.

Plant interactions with environmental factors cause changes in the metabolism and regulation of biochemical and physiological processes. Plant defense against pathogenic microorganisms depends on an innate immunity system that is activated as a result of infection. There are two mechanisms of triggering this system: basal immunity activated as a result of a perception of microbe-associated molecular patterns through pattern recognition receptors situated on the cell surface and effector-triggered immunity (ETI). An induced biosynthesis of bioactive secondary metabolites, in particular phytoalexins, is one of the mechanisms of plant defense to fungal infection. Results of the study on narrow leaf lupin (Lupinus angustifolius L.) plants infected with the anthracnose fungus Colletotrichum lupini and treated with fungal phytotoxic metabolites are described in the paper. The C. lupini phytotoxins were isolated from liquid cultures, purified and partially characterized with physicochemical methods. Accumulation of secondary metabolites on leaf surface and within the tissues of plants either infected, treated with the fungal phytotoxin or submitted to both treatments was studied using GC-MS and LC-MS, respectively. Substantial differences in isoflavone aglycones and glycoconjugate profiles occurred in response to different ways of plant treatment.

A suspension culture of white lupin cells has been established, and proteins of the exocellular matrix analysed. Based on homologies of N-terminal amino-acid sequences, three stress- or defence-related proteins: acidic class III chitinase, polygalacturonase-inhibiting protein, and germin/oxalate oxidase, secreted by lupin cell culture, were identified.