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The aim of this study was to demonstrate the metabolic profile of post-culture medium as an expression of cell suspension metabolic activity of the tree fern Cyathea delgadii Sternb. The molecular profile of the tree fern’s cell culture has been never described, according to our knowledge. The cell suspension was established using ½ MS medium supplemented with various concentrations of 2,4-D and BAP. The optimal concentrations were 2.0 mg·L−1 and 0.2 mg·L−1, respectively. The cell suspension initially showed an organized system of cell division and later unorganized cell proliferation. LC-MS and GC-MS were used to identify the chemical composition of the post-culture medium. The LC-MS analysis results suggested that the color of liquid medium could be due to the presence of flavonoid derivatives, as this group of compounds was represented by eight compounds. After GC-MS analysis based on retention indexes and thanks to mass spectra comparison, 130 natural products were recognized, belonging to various classes of primary and secondary metabolites.

The aim of this study was to evaluate the utility of a hydroalcoholic extract of Hippocastani flos (HF) in wound healing, especially in regards to cell viability, proliferation, reactive oxygen species (ROS) generation and proinflammatory cytokine production using an endothelial cell line [human umbilical vein endothelial cell (HUVEC) line EAhy926]. Antibacterial and anti-hyaluronidase activity was also evaluated. The phytochemical profile was determined using an the UPLC–MS. The UPLC–MS analysis confirmed the presence of nine flavonoids, mainly kaempferol and quercetin derivatives. Specifically, the HF extract contained a high content of polyphenols (88.84 ± 4.13 mg chlorogenic acid equivalents/g dw), including flavonoids (15.68 ± 0.04 mg/quercetin equivalents/g dw). Antioxidant properties were demonstrated using FRAP assays (IC0.5 = 159.82 μg/mL). The HF extract exhibited a level of anti-hyaluronidase activity (IC50 = 0.86 mg/mL) similar to the positive control kaempferol (IC50 = 0.78 mg/mL). The antimicrobial properties of HF were shown against Staphylococcus aureus (MIC, MBC = 12.5 mg/mL). After exposing the endothelial cells to HF extract in vitro, it did not observed any change in the cell viability, proliferation, wound healing or proinflammatory cytokine production, but there was a dose dependent-reduction in ROS generation. In conclusion, HF extract exhibits anti-hyaluronidase activity, antimicrobial properties, and reduces ROS production. These observations may partly justify the use of HF in supporting wound healing.

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.

Dimers of phenylpropanoid acids play an important role in cross-linking of polysaccharide chains present in the cell wall. These compounds are responsible not only for mechanical properties of cell walls, but also for plant tolerance to both biotic and abiotic stresses. Due to conjugated double bonds system these molecules are efficient radicals scavengers and UV light shield. Moreover, the presence of these natural products in many plant-derived foods provides a variety of health benefits and lowers disease risk. Therefore development of efficient methods of phenylpropanoid cyclobutane dimers and dehydrodimers analysis is of the highest interest. Liquid chromatography coupled to mass spectrometry with electrospray ionization is an analytical technique of choice for identification and – within certain limits – structural characterization of low molecular weight secondary metabolites in samples of plant origin. Utilization of high resolution analyzers in mass spectrometers enables accurate mass measurements, which allows to determine the elemental composition of these compounds with at least 5 ppm accuracy. The ions produced with electrospray ionization have low internal energy and their fragmentation can be generated by collision induced dissociation (CID MS/MS). LC/MS/MS system was used to profile phenylpropanoid dimers in barley leave cell wall extracts. Fifteen natural products were recognized after structural analysis of recorded CID MS/MS spectra, some of them were isomeric. We were able to detect: four isomers of two phenylpropanoid acids, three cyclobutane ferulic acid dimers, six isomeric ferulic acid dehydrodimers, one mixed dehydrodimer of sinapic and coumaric acid and one decarboxylated dehydrodimer of ferulic acid. Differentiation of isomeric compounds belonging to both classes of dimers, and their further quantitative analysis, will have influence on research directed to biological role of the dimers in plant responses to environmental biotic and abiotic factors.

Abiotic and biotic stresses are the main reasons of substantial crop yield losses worldwide. Research devoted to reveal mechanisms of plant reactions during their interactions with the environment are conducted on the level of genome, transcriptome, proteome, and metabolome. Data obtained during these studies would permit to define biochemical and physiological mechanisms of plant resistance or susceptibility to affecting factors/stresses. Metabolomics based on mass spectrometric techniques is an important part of research conducted in the direction of breeding new varieties of crop plants tolerant to the affecting stresses and possessing good agronomical features. Studies of this kind are carried out on model, crop and resurrection plants. Metabolites profiling yields large sets of data and due to this fact numerous advanced statistical and bioinformatic methods permitting to obtain qualitative and quantitative evaluation of the results have been developed. Moreover, advanced integration of metabolomics data with these obtained on other omics levels: genome, transcriptome and proteome should be carried out. Such a holistic approach would bring us closer to understanding biochemical and physiological processes of the cell and whole plant interacting with the environment and further apply these observations in successful breeding of stress tolerant or resistant crop plants.

Drought is a major abiotic stress that negatively influences crop yield. Breeding strategies for improved drought resistance require an improved knowledge of plant drought responses. We therefore applied drought to barley recombinant inbred lines and their parental genotypes shortly before tillering. A large-scale proteomic analysis of leaf and root tissue revealed proteins that respond to drought in a genotype-specific manner. Of these, Rubisco activase in chloroplast, luminal binding protein in endoplasmic reticulum, phosphoglycerate mutase, glutathione S-transferase, heat shock proteins and enzymes involved in phenylpropanoid biosynthesis showed strong genotype×environment interactions. These data were subjected to genetic linkage analysis and the identification of proteomic QTLs that have potential value in marker-assisted breeding programs.

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.

Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of plant secondary metabolite mixtures isolated from biological material. Plant tissues contain thousands of natural products fulfilling different roles in plant physiology and biochemistry. These natural products have various biological activities in respect to plants synthesizing them, in their responses to different environmental stresses and are also active principles of food supplements and pharmaceuticals of plant origin. Flavonoids constitute a large group of phenolic secondary metabolites and are probably produced by all terrestrial plant species. More than 9000 glycoconjugates of flavonoids are presently known in the plant kingdom and more than 50 of them may be present in a single plant. For this reason methods of identification and analysis of this group of compounds are particularly demanded. Due to a high number of metabolites present in plant extracts, the isolation and purification of most compounds in amounts suitable for unambiguous characterization with NMR methods is often impossible. For these reasons elaboration of strategies for sufficiently precise structural characterization of compounds present in mixture samples is currently a primary task. Mass spectrometry, thanks to application of different physical phenomena for ionization, separation and detection of analyzed molecules, became the method of choice among analytical methods applied for identification, structural characterization and quantitative analysis of the natural products. Methods of analysis of differently substituted flavonoids (O- and C-glycosides, differentiation of various oligosaccharidic substituents, detection of acylated compounds) are presented in the paper. A proper application of mass spectrometric methods in well-defined and strictly controlled technical parameters of analysis permits obtaining important structural information. Among others, recording collision induced dissociation mass spectra allows identification of compounds after comparison of the registered MS spectra with these present in the existing databases.

Drought stress is perhaps one of the most common abiotic factors which crop plants have to cope with. To survive, plants have to adapt to periods of water deficit that may occur during their vegetation. This can be achieved by triggering various changes in the plant genome, transcriptome, proteome, and metabolome, leading to different physiological and biochemical reactions of plants. We have compared changes in barley leaf and root metabolomes in response to drought in recombinant inbred line (RIL) population derived from hybrids between two spring genotypes: German variety Maresi and Syrian breeding line Cam/B1//CI08887/CI05761. Response of plants to drought of the studied barley lines was rather conservative; most barley genotypes changed their metabolome composition independently in leaf and root. Based on analysis of variance, metabolites were classified with respect to significance of difference between lines, drought effect (understood as the difference between metabolite level in drought and control plants), and line × drought interaction. The revealed changes in accumulation of some metabolites, e.g., proline and other amino acids, carbohydrates or carboxylic acids have been regarded to be a basic plant strategy for acquiring drought stress tolerance. It was possible to draw some general inferences from obtained results: changes of metabolites involved in barley response to drought were rather similar qualitatively but varied quantitatively among the studied RILs. Compatible solutes and osmolytes were the major group of compounds accumulated under drought. We have also observed significant organ specificity between leaf and root response to drought at the metabolome level in all recognized metabolites classes. Moreover, we have found metabolites which differentiated tested genotypes under drought—and these compounds might be considered as potential biomarkers associated with drought tolerance in barley.

Liquid chromatography coupled to mass spectrometry (MS) with electrospray ionization (ESI) is one of analytical techniques to obtain accurate results of low molecular weight aromatic compounds in biological samples of different origin. The interpretations of mass spectra of these aromatic compounds in the negative spectra registered in the full scan MS mode may be uneasy due to presence of deprotonated molecules [M-H](-) from different co-eluting entities, fragment ions created after the break-up of precursor ions and also ions representing modified molecules clusters. Thus, the first aim of this study was to evaluate general parameters during analysis performed in the full scan MS or MS/MS mode. Secondly, to set general fragmentation rules for aromatic compounds and entities in a complex biological matrix. We established that different groups of low molecular weight phenolic acids form unique adduct ions and additionally registration LC/MS/MS spectra with two different collision energies may allow for differentiating isomeric or isobaric molecules. These findings together with some general fragmentation rules can facilitate identifications of aromatic acids as we outlined in the sample of cold-pressed rose-hip oil and lupine leaves extract.

Every year since 2007, the Central and Eastern European Proteomic Conference (CEEPC) has excelled in representing state-of-the-art proteomics in and around Central and Eastern Europe, and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including traditional and often-revisited methodologies as well as the latest technological achievements in clinical, quantitative and structural proteomics with a view to systems biology of a variety of processes. The 9th CEEPC was held from June 15th to 18th, 2015, at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznan, Poland. The scientific program stimulated exchange of proteomic knowledge whilst the spectacular venue of the conference allowed participants to enjoy the cobblestoned historical city of Poznan.

Plant responses to abiotic stresses are very complex phenomena with individual characteristics for various species. Abiotic stresses (e.g. drought, salinity, flooding, cold, heat, UV radiation, heavy metals, etc.) strongly affect plant growth and development. It is estimated that they are the cause of more than 50 % of crop yield losses. Abiotic stresses are known to activate a multigene response resulting in the changes in various proteins and primary and secondary metabolite accumulation. Therefore, proteomic and metabolomic approaches are becoming very important and powerful tools used in studying plants’ reaction to various stimuli. Precise analysis of proteome and metabolome is essential for understanding the fundamentals of stress physiology and biochemistry. In this review, we focus on recent reports concerned to the influence of abiotic stresses on changes in the level of different protein groups and metabolite classes. Basic information about physicochemical methods applied to qualitative and quantitative analyses of biopolymers and natural products is also briefly presented.

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.

The aim of this study was to present integrated mass spectrometric methods for the structural characterization and identification of flavonoid glycoconjugates. During the liquid chromatography/mass spectrometry analyses, TriVersa NanoMate chip-based system with nanoelectrospray ionization and fraction collection was combined to a quadrupole time-of-flight mass spectrometer. In the extract samples prepared from green leaves of wheat plantlets, 41 flavonoid derivatives were recognized. Part of the target natural products had the full structure being characterized after the registration of mass spectra, where m/z values for protonated [M + H](+) and deprotonated molecules [M - H](-) were annotated. MS(2) and pseudo-MS(3) experiments were performed for [M + H](+) or [M - H](-) and aglycone ions (Y0(+/-)-type), respectively. It should be underlined that pseudo-MS(3) mass spectra were registered for aglycone product ions in the mass spectra of O-glycosides present in the extract samples. In many cases, only tentative structural identification of aglycones was possible, mainly because of the presence of numerous C-monoglycoside or C-diglycoside in the samples. Acylation of the sugar moiety and/or methylation of the aglycone in the flavonoid glycosides under study was observed. The existence of isobaric and/or isomeric compounds was demonstrated in the extract studied. The collision-induced dissociation mass spectra registered for C,O-diglycosides and C,C-diglycosides did not permit to draw complete structural conclusions about the compounds studied. For the investigated class of natural products, unambiguous classification of sugar moieties linked to the aglycones from the recorded mass spectra was not possible. Registration of the positive and negative ion mass spectra did not lead to the precise conclusion about the glycosylation position at C-6 or C-8, and O-4' or O-7 atoms. It was possible, on the basis of the collected MS(2) spectra, to differentiate between O-glycosides and C-glycosides present in the samples analyzed.

The major cause of mortality in patients with chronic kidney disease (CKD) is atherosclerosis related to traditional and non-traditional risk factors. However, the understanding of the molecular specificity that distinguishes the risk factors for classical cardiovascular disease (CVD) and CKD-related atherosclerosis (CKD-A) is far from complete. In this study we investigated the disease-related differences in the proteomes of patients with atherosclerosis related and non-related to CKD. Plasma collected from patients in various stages of CKD, CVD patients without symptoms of kidney dysfunction, and healthy volunteers (HVs), were analyzed by a coupled label-free and mass spectrometry approach. Dysregulated proteins were confirmed by an enzyme-linked immunosorbent assay (ELISA). All proteomic data were correlated with kidney disease development and were subjected to bioinformatics analysis. One hundred sixty-two differentially expressed proteins were identified. By directly comparing the plasma proteomes from HVs, CKD, and CVD patients in one study, we demonstrated that proteins involved in inflammation, blood coagulation, oxidative stress, vascular damage, and calcification process exhibited greater alterations in patients with atherosclerosis related with CKD. These data indicate that the above nontraditional risk factors are strongly specific for CKD-A and appear to be less essential for the development of “classical” CVD.

Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency.

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.

Elevated levels of homocysteine (Hcy) are associated with cardiovascular and neurodegenerative diseases in humans. Hcy becomes a component of human proteins as a result of N-homocysteinylation of protein lysine residues by Hcy-thiolactone, which affects the protein's structure and function, and contributes to Hcy-related pathology. Albumin is the major target for N-homocysteinylation in human blood in vivo. Previous work has identified Lys-525 as a predominant site of N-homocysteinylation in vitro and in vivo. Here we show that Lys-4, Lys-12, Lys-137, Lys-159, Lys-205, and Lys-212 of human albumin are susceptible to N-homocysteinylation in vitro and provide evidence that two of those residues, Lys-137 and Lys-212, in addition to Lys-525, are N-homocysteinylated in vivo in human plasma.

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.

Profiles of flavonoid conjugates present in the root and leaf tissues of the Mexican wild lupine, Lupinus reflexus, were established using two LC-MSMS systems in the positive and negative ion modes. The ion trap mass spectrometer and quadrupole time-of flight instrument provided sequential MS(n) spectra and MSMS spectra with accurate m/z values of [M + H](+) and [M - H] (-) ions, respectively. Sixty-two flavone and isoflavone glycoconjugates were found and tentatively identified. Numerous isomeric or isobaric compounds with the same molecular mass could be differentiated. Isomeric di- and mono glucosides of biochanin A, genistein, 2'-hydroxygenistein, luteone, and 2,3-didehydrokievitone were distinguished on the basis of relative abundances of product ions. The studied flavonoid glycoconjugates were acylated with dicarboxylic aliphatic acids and their methyl esters at either the aglycone or glycosidic moiety.

Introduction – Flavonoids are important plant compounds occurring in tissues mostly in the form of glycoconjugates. Most frequently the sugar moiety is comprised of mono- or oligosaccharides consisting of common sugars like glucose, rhamnose or galactose. In some plant species the glycosidic moiety contains glucuronic acid and may be acylated by phenylpropenoic acids. Methodology – Flavonoid glyconjugates were extracted from leaves of Medicago truncatula ecotype R108 and submitted to analysis using high-performance liquid chromatography combined with high-resolution tandem (quadrupole-time of flight, QToF) mass spectrometry. Results – The studied leaf extracts contained 26 different flavonoid glycosides among which 22 compounds were flavone (apigenin, luteolin, chrysoeriol and tricin) glucuronides and 13 were acylated with aromatic acids (p-coumaric, ferulic or sinapic). The fragmentation pathways observed in positive and negative ion mass spectra differed substantially between each other and from these of flavonoid glycosides which did not contain acidic sugars. The application of high-resolution MS techniques allowed unequivocal differentiation between ions with the same nominal m/z values containing different substituents (e.g. ferulic acid or glucuronic acid). Eleven of the identified flavonoids have not been reported previously in this species. Perspectives – The presented unique fragmentation pathways of flavonoid glucuronates enable detection of these compounds in tissue extracts from different plant species. Copyright © 2009 John Wiley & Sons, Ltd.

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.

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.

Ionically bound cell wall peroxidases (POXs) were liberated to intercellular washing fluids (IWFs) and isolated together with other proteins and metabolites present in the apoplast of white lupine (Lupinus albus L. var. Bac) root. After separation of proteins from low molecular weight compounds, activity of peroxidases was monitored in in vitro experiments. Oxidation of methyl ferulate with H2O2 was studied in multi-component mixtures of plant metabolites. Secondary metabolites identified in IWFs or other natural products playing important roles in different physiological processes were applied as modifiers of the dehydrodimerization process during oxidation reactions performed in vitro. These were isoflavones and their conjugates, lupanine representing quinolizidine alkaloids synthesized in lupine, or other natural products such as quercetin, ascorbic, and salicylic acid. The influence of these substances on the oxidation kinetics of methyl ferulate was monitored with liquid chromatography with ultraviolet detection (LC/UV), and identification of compounds was confirmed with the liquid chromatography/mass spectroscopy (LC/MS) system. On the basis of data collected, it was possible to reveal changes in the activities of cell wall POXs. Application of the LC system permitted us to monitor, independently, quantitative changes of two or more reaction products in the mixtures. In multi-component combinations, oxidation yields of methyl ferulate by POXs were modified depending on the actual composition of the reaction mixture. We conclude that various classes of plant secondary metabolites can modify the yield of methyl ferulate oxidation by hydrogen peroxide in the presence of POX, due to interactions with the enzyme's active site (genistein) or radical scavenging properties of metabolites present in the reaction mixture.

Flavonoid conjugates constitute several classes of plant phenolic secondary metabolites including many isomeric compounds differing in the hydroxylation pattern and substitution of their rings with different groups such as alkyls, acyls or sugars. These compounds occur in plant tissues mainly as glycosides and in many cases it is necessary to have reliable and detailed information concerning the structure of these natural products. Our results were obtained using leaf extracts of Arabidopsis thaliana and Lupinus angustifolius in which different glycosides of flavones, flavonols and isoflavones are present. Analysis of collision-induced dissociation (CID)/MS/MS spectra of protonated [M + H](+), sodiated [M + Na](+) or deprotonated [M - H](-) molecules recorded during HPLC runs may bring needed information in this respect. However, registration of mass spectra of [M + Na](+) ions with a good efficiency is possible only after post-column addition of a sodium acetate solution to the LC column eluate. The retention of sodium cation on the saccharidic parts of the molecule is observed after the CID fragmentation. In many cases, the location of this cation on the glycan attached to C-3 hydroxyl group of flavonol led to assignment of its structure. Additionally, the determination of the structure of the aglycone and of the sequence of the glycan part was made possible through the CID data obtained from the [M + H](+) and [M - H](-) ions. CID spectra show a different order of sugar elimination from hydroxyl groups at C-3 and C-7 in flavonol glycosides isolated from A. thaliana leaves and give sufficient information to discriminate flavonoid O-diglycosides from flavonoid di-O-glycosides.

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.

Deciphering of the plant metabolome is one of the most difficult analytical tasks in functional genomic research. Studies directed at the gene or protein expression are well established, sequencing analyses of these kinds of biopolymers on genome or proteome level are possible. This is not the case for metabolites, where identification in single sample of many chemical entities of different elemental composition and structures and various physicochemical properties is necessary. Different instrumental methods are applied for identification of metabolites but none of them allows obtaining unambiguous structural information about more than 500 compounds in single mixture (metabolite profiling). This is a much smaller number of metabolites than is predicted for single plant metabolome. However, instrumental approaches were proposed (metabolite fingerprinting) in which biochemical phenotype of an organism may be estimated, but identification of individual compounds is not possible.

A mass spectrometric method based on the combined use of electrospray ionization, collision-induced dissociation and tandem mass spectrometry at high mass resolution has been applied to an investigation of the structural characterization of genistein-7-O-β- d -glucoside (5,7,4′-trihydroxyisoflavone). The product ion mass spectrum of [M−H]− ions shows neutral losses of the glycan residue (162 Da) and of the glycan residue + H (163 Da) by rearrangement and scission, respectively, where the latter loss dominates at higher collision energies. The genistein moiety remained intact and only minor fragmentation of the glucose moiety was observed. The low-energy product ion mass spectrum of [M+H]+ ions shows extensive fragmentation of the glucose moiety, though at low ion signal intensity, loss of the glycan residue, and simple fragmentation of the genistein moiety that permits characterization of the substituents in the A and B rings. The use of elevated cone voltages permitted observation of product ion mass spectra of selected primary fragment ions. Product ion mass spectra examined at high mass resolution allowed unambiguous determination of the elemental composition of fragment ions. Fragmentation mechanisms and ion structures have been proposed.

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.

Mass spectrometric fragmentation patterns of herbacetin 3-O-glucopyranoside-8-O-glucuronopyranoside (1), gossypetin 3-O-glucopyranoside-8-O-glucuronopyranoside (2) and takakin 7-O-glucopyranoside-8-O-glucuronopyranoside (3) were elucidated from mass spectra obtained with electrospray ionisation. The usefulness of the fragmentation patterns observed in the positive and negative mode for structural elucidation of the studied compounds is discussed. The fragmentation of 3 was substantially different from 1 and 2, especially in the negative mode. In order to explain these differences, theoretical calculations were performed. Copyright © 2003 John Wiley & Sons, Ltd.

In this paper we show that transfer ribonucleic acids (tRNAs) catalyze the Diels-Alder cycloaddition reaction. A new DNA oxidative damage product, 6-furfuryladenine (kinetin) or its riboside (diene), was transformed with dimethyl acetylenedicarboxylate or maleic anhydride (dienophile). The reaction proceeds in the presence of tRNA at high pressure but not at ambient condition. If so tRNA in prebiotic conditions (RNA world) had at least two functions: catalytic and a carrier of genetic information. It means that tRNA at high pressure shows catalytic properties and is a true Diels-Alderase.

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.

The aim of this study was to determine the flavonoid profile of Lupinus mexicanus germinated seed extract (PE) and to evaluate its effect as a phytoestrogen on the morphometric parameters of CA3 hippocampal neurons of ovariectomized rats (OVX). L. mexicanus seeds, germinated for 48 h, were homogenized and macerated using an 80% ethanol solution. The extract was analyzed by HPLC/MS-MS. Thirty young Wistar strain female rats (200±10 g) were randomly distributed into four groups: sham operated (S) treated with dimethyl sulfoxide (vehicle); ovariectomized and treated with 1250 μg of PE extract (OVX-PE); ovariectomized and treated with 5 μg estradiol benzoate (OVX-EB); and ovariectomized and vehicle treated (OVX). All substances were injected subcutaneously daily for 28 days. On day 29, the animals were sacrificed, perfused, and fixed to obtain the brains for histological processing. Each brain was cut and stained with hematoxylin and eosin. The thickness of the stratum oriens (SO), the nuclear diameter, and the neuronal density were measured in the hippocampus CA3 area. Nine different flavonoids and one non-identified compound were detected. The histological analysis demonstrated that the thickness of the SO was higher in the OVX-EB and S groups than in the OVX-PE and OVX groups (p⟨0.05); in addition, the nuclear diameters of the neurons in the OVX-EB and S groups were higher compared with the other groups (p⟨0.05). The OVX group had the highest cellular density among groups (p⟨0.05). Based on our results, the PE obtained did not have beneficial effects on CA3 hippocampal neurons.

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.