Comparative analysis of wild and cultivated Lathyrus L. species to assess their content of sugars, polyols, free fatty acids, and phytosterols

Under climate change, the need for crops resistant to abiotic and biotic stresses increases. Lathyrus spp. Are characterized by a high nutritional value of their green biomass. The grass pea is one of the most resistant to drought, aterlogging, cold, salinity, diseases and pests among cultivated legumes, and it is grown at minimal cost. The development of new Lathyrus cultivars with an improved composition of nutrients will make it possible to produce high-quality animal feed in areas with extremely unstable weather conditions. With this in view, the patterns of variability in the parameters of the carbohydrate complex (sugars, their lactone and methyl forms), polyols (including phenol-containing alcohols), phytosterols, free fatty acids (FFA) and acylglycerols were studied in the green biomass of 32 accessions representing Lathyrus sativus L., L. tuberosus L., L. sylvestris L., L. vernus (L.) Bernh., L. latifolius L. and L. linifolius (Reichard) Bassler. from the VIR collection, reproduced in Leningrad oblast under contrasting conditions of 2012 and 2013. The content of identified compounds varied depending on the genotype, species, and weather. High temperatures and high precipitation in 2013 contributed to the accumulation of monosaccharides, and the colder and drier conditions of 2012 to an increase in oligosaccharides, most polyols, and FFA. The cultivated species (L. sativus) was distinguished by its high sugar content, and the wild species as follows: L. latifolius by FFA; L. linifolius by ononitol, myo-inositol, and glycerol 3-phosphate; L. vernus by MAG and methylpentofuranoside. The resulting data showed that the Lathyrus accessions studied are promising for breeding stress-resistant cultivars of high nutritional quality

Сomparative analysis of wild and cultivated Lathyrus L. spp. according to their primary and secondary metabolite contents

Species of the genus  Lathyrus L. are known as forage and medicinal plants, widely used in traditional medicine  and homeopathy. The content of protein,  essential  amino  acids and  carotene in their green  biomass  is higher  than  in other  annual  leguminous plants  traditionally  cultivated  in Russia. Until now, the requirements for the crop’s quality were reduced to a high content of protein  and dry matter in seeds  and herbage. In-depth biochemical  analysis of accessions  from the collection of plant genetic resources  will significantly improve  selection  of source materials  for breeding. Such tasks can be solved  using  gas chromatography with mass spectrometry in plant  diversity studies. In view of the above,  our goal was to analyze organic  acids, free amino  acids and secondary metabolites in green biomass  of Lathyrus to facilitate comprehensive assessment of its forage  and  pharmacological value. We analyzed 32 accessions  of Lathyrus sativus L., L. tuberosus L., L. sylvestris L., L. vernus (L.) Bernh., L. latifolius L. and  L. linifolius (Reichard) Bassler from the collection of the Vavilov Institute (VIR). The studied Lathyrus accessions had significant interspecific and intraspecific variability both in the composition (presence)  and number of the identified compounds. The analysis of plants across different years confirmed that biochemical parameters depended on weather conditions. The colder and drier conditions of 2012 contributed to the accumulation of organic acids (mean: 890 mg/100 g), free amino acids (mean: 201.59 mg/100 g), and secondary metabolites (mean: 84.14 mg/100 g). The range of variability for organic acids ranged from 140 to 2140, for free amino acids from 11.8 to 610, and for secondary metabolites from 4.4 to 224.6 mg/100  g. Grass pea accessions  with high organic acid, free amino acid and secondary metabolite contents were identified: k-900 (Colombia) for organic acids (2140, 610 and 178 mg/100  g); k-51 (Georgia) and k-959 (Afghanistan) for free amino acids (401.29 and 540.63 mg/100 g); k-893 (Eritrea) for secondary metabolites (199.39 mg/100 g), etc. They can serve as source material for the development of cultivars for different uses (forage and medicinal)

A simple and efficient method to extract polar metabolites from guar leaves (Cyamopsis tetragonoloba (L.) Taub.) for GC-MS metabolome analysis

Guar (Cyamopsis tetragonoloba (L.) Taub.) is an agricultural crop species new to Russia and is in demand by the gas, oil and food industries. Due to the progress of “omics” technologies and the marker-assisted selection, there is a huge interest in the studies that compare the metabolites of various guar varieties, employing metabolomics as a method of functional genomics. For a large-scale screening of guar germplasm from the VIR collection, it is important to choose an efficient method to extract metabolites from samples. The accuracy of the assessment of the content of metabolites in samples is crucial for distinguishing varieties within the crop, since the metabolome profiles of plants within the same species differ mainly in the quantitative ratio of metabolites, and not in their qualitative composition. In metabolome practice, two methods of extracting polar compounds are usually employed in the preparation of samples for GC-MS analysis. One of the widely used methods of sample preparation is the long-term extraction of metabolites from whole tissues with the aid of a methanol solvent. Another method of sample preparation is based on the short-term methanol extraction of metabolites from frozen and homogenized material. The advantages and disadvantages of these two methods revealed in the course of our work have prompted us to develop a new approach that avoids some difficulties in analyzing the metabolic profiles of leaves of various guar varieties. The method we suggested combines the advantages of the two above-mentioned approaches of sample preparation, namely eliminates the loss of metabolites due to centrifugation and ensures the complete destruction of all cell walls, ensuring the maximum extraction level of polar metabolites. The essence of the new method is that the leaf is rapidly frozen in liquid nitrogen with subsequent thawing in cold methanol. Thus, leaf tissues retain morphological integrity, and subsequent centrifugation, necessary for homogenization, is skipped. We have checked the effectiveness of this improved method by experiments with leaf samples of three guar genotypes. It has been shown that the amount of extracted metabolites increases more than 5-fold compared to extraction with methanol from fresh unfrozen leaf tissues and more than 2-fold compared to extraction with methanol after freezing and homogenization. Extraction of metabolites using the new method allows the GC-MS analysis of guar samples to be conducted with the least loss and high accuracy required to distinguish varieties

The metabolomic approach to the assessment of cultivar specificity of Brassica napus L. seeds

Recent biomolecular studies tend to involve combinations of different methods and approaches that allow analyzing organisms on the genomic and proteomic levels, as well as on the level of metabolomics. However, in order to justify the use of the metabolomics techniques in plant breeding, it is important to perform comprehensive analysis of a broad range of species and varieties. In this study, we evaluated the contents of low-molecular-weight substances in seeds of different rapeseed cultivars by the gas chromatography–mass spectrometry (GC-MS) technique. For every metabolomic profile, we estimated 168 target substances, and 52 of them were unambiguously identified. These compounds included amino acids, organic and fatty acids, tocopherols, and phytosterols. In order to keep the data assay within the context of multivariate statistics, we used principal component analysis (PCA), partial least square discriminant analysis (PLS-DA), and partial least square regression (PLS-R). Subsequent analysis revealed a significant difference between the metabolomic profiles of the investigated rapeseed cultivars, with the primary role of the amino acids and organic acids. Noticeably, the PLS-DA model showed 65% of the explained variance and, according to the Venetian blinds cross- validation test, 91.67 % of the accuracy. Thus, we demonstrate the effectiveness of the metabolomics approach to the varietal identification of seeds. This strategy can be further improved with a continuously updated database of the metabolomic profiles of different species and cultivars. Application of the PLS-DA method will allow comparison of the metabolites of unknown samples with the existing profiles and, subsequently, identification of new seed samples

The metabolomic approach to the comparative analysis of wild and cultivated species of oats (Avena L.)

Seed metabolomic profiles have been investigated in wild and cultivated forms (cultivars) of oat (Avena L.). Seed accessions from the VIR oat collection were used for the research. Metabolomic analysis employed gas liquid chromatography-mass spectrometry (GLC- MS) using an Agilent 6850 chromatographer (USA). The analysis covered the composition and content of organic and fatty acids, amino acids, polyatomic spirits and sugars. The content fluctuation range for the studied groups of compounds was found to be narrower (significantly in some cases) in cultivars than in the wild species. Along with a sharp increase in oleic acid content, cultivars demonstrated a decrease in that of linoleic acid. The general conclusions from the comparison of seed metabolomic profiles in wild species and cultivars are presented below. A number of wild species can be recommended as a potential source of biochemical quality traits for breeding purposes. A series of metabolites (compounds), the content of which changes during domestication or which differentiate wild oat species from cultivars has been identified was found. Along with such well-known healthy food chemical factors as oleic acid, glucose and fructose, etc., differences concerning monoacylglycerol compounds (MAG 16 : 0 and MAG-2 18 : 2, etc.) have been found. The latter have been proposed to be related to the formation of adaptive traits, in particular, resistance to diseases and pests, and to environmental abiotic stresses

Selection of an optimal method for screening the collection of narrow-leaved lupine held by the Vavilov Institute for the qualitative and quantitative composition of seed alkaloids

Narrow-leaved lupine (Lupinus аngustifolius L.) is a widely cultivated leguminous forage and green manure crop with a potential for human nutrition. However, the presence of secondary metabolites – alkaloids – in lupine seeds considerably affects the quality of raw produce, reducing its nutritive value; in addition, high concentrations of alkaloids are toxic to humans and animals. Therefore, plant breeders working with lupine need to gain knowledge about the variability of alkaloid content in seeds of different genotypes and search for the sources of their low concentrations in the crop’s gene pool. The collection of narrow-leaved lupine genetic resources held by the N.I. Vavilov Institute of Plant Genetic Resources (VIR) offers wide opportunities for such search by means of mass screening. For its part, largescale gene pool screening requires the selection of an optimal technique to measure alkaloid content in seeds, so that it would be easily reproducible and as little labor-, time- and fund-consuming as possible. The results of the search for such method are presented. Qualitative and quantitative indices were compared when target compounds had been extracted with multicomponent mixtures and individual reagents (chloroform, methanol, etc.) and the extracts analyzed using gas chromatography-mass spectrometry. High-performance liquid chromatography combined with mass spectrometry was also employed. Five major alkaloids were found to be present in all types of extracts: lupanine, 13-hydroxylupanine (dominant ones), angustifoline, sparteine, and isolupanine. The fullest extraction of alkaloids was observed when the extractant with an added alkaline agent was used (425 mg/100 g). The lowest level of extraction was registered with chloroform (216 mg/100 g). The significance of the differences was confirmed statistically

Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication

Structural lipids and carbohydrates of the deep mycelium of phoma-like micromycetes, potential mycoherbicides

The work is devoted to the mycelium biochemical composition of Stagonospora cirsii C-211, Calophoma complanata 32.121, Didymella macrostoma 32.52. These phylogenetically distant species of phoma-like micromycetes are the potential mycoherbicides of Cirsium arvense, Heracleum sosnowskyi, and Convolvulus arvensis, respectively. The S. cirsii C-211, C. complanata 32.121, D. macrostoma 32.52 mycelium in the early stationary growth phase was obtained on sucrose-soybean nutrient medium. It was shown that the lipid and carbohydrate (polyols, sugars) profiles of these strains have much in common. We suppose that levels of arabitol and trehalose influence to the stress-resistant of phoma-like micromycetes. In particularly, these carbohydrates serve structural and protective roles in the cell walls during osmotic and temperatures stress. The ratio of phosphatidylcholine to phosphatidylethanolamine and the proportion of phosphatidylserine among structural lipids also determine the properties of mycelium, and can be used to assess its quality

Metabolomic Profiling of Biolayers on the Surface of Marble in Nature and Urban Environment. Case Study of Karelia and St. Petersburg

The formation of biolayers of various taxonomic and biochemical composition occurs on the rock surfaces under various environmental conditions. The composition of metabolites in various types of biolayers on the marble surface in natural outcrops and urban environment was studied. Metabolome profiling was fulfilled by GC-MS. It was found that communities in urban environment are much less biochemically diverse than in a quarry. The seasonal differences in metabolite network between samples dominate over taxonomic ones in biolayers with predomination of algae and cyanobacteria and in biolayers with predomination of fungi. The biolayers of different stage of soil formation are less susceptible to seasonal variability

Fullerenol changes metabolite responses differently depending on the iron status of cucumber plants.

The unique properties of carbon-based nanomaterials, including fullerenol, have attracted great interest in agricultural and environmental applications. Iron (Fe) is an essential micronutrient for major metabolic processes, for which a shortage causes chlorosis and reduces the yield of many crops cultivated worldwide. In the current study, the metabolic responses of Cucumis sativus (a Strategy I plant) to fullerenol treatments were investigated depending on the Fe status of plants. Cucumber plants were grown hydroponically, either with [+FeII (ferrous) and +FeIII (ferric)] or in Fe-free (-FeII and -FeIII) nutrient solution, with (+F) or without (-F) a fullerenol supply. Iron species-dependent effects were observed in either Fe-fed or Fe-starved plants, with alteration of metabolites involved in the metabolism of carbohydrates, amino acids, organic acids, lipophilic compounds. Metabolic perturbations triggered by fullerenol in the FeIII-treated plants were in the opposite kind from those in the FeII-treated plants. Whereas in the FeIII-fed plants, fullerenol activated the metabolisation of carbohydrates and amino acids, in the FeII-fed plants, fullerenol activated the metabolisation of lipophilic compounds and repressed the metabolisation of carbohydrates and amino acids. In FeIII-deficient plants, fullerenol stimulated the metabolism of C3 carboxylates and lipophilic compounds while repressing the metabolism of amino acids, hexoses and dicarboxylates, while in FeII-deficient plants, activations of the metabolism of amino acids and dicarboxylates and repression of sterol metabolism by fullerenol were observed. The results indicated that the valence state of Fe sources is of importance for re-programming metabolome responses in cucumber to fullerenol either in Fe-sufficient or Fe-deficient conditions. These investigations are significant for understanding fullerenol interactions and risk assessment in plants with different Fe statuses