Analysis Of Primary Metabolites Research Articles

Time-dependent behaviour of methyl jasmonate elicited cell suspension cultures of Ocimum species via untargeted mass spectrometry-based metabolomics

Ocimum (basil) is a widespread genus commonly consumed for culinary and medicinal purposes attributed to unique secondary metabolites . The present research investigated the metabolic profiles of two Ocimum suspension cultures derived from O. basilicum and O. americanum , following elicitation by methyl jasmonate at different time intervals (24, 48, and 72 h) compared to the baseline at T = 0 h identifying bioactive metabolites and the influence on their expressional behaviour. Based on comprehensive extraction and mass spectrometry (MS)-based analytical protocols, the results showed that phenolic acid abundances, particularly rosmarinic acid, were increased significantly in O. americanum culture after 72 h to a 2.0-fold increase ( p < 0.05). Similarly, its 3- O -methyl derivative was increased by 1.6-fold after 48 h in O. basilicum , and drastically (21.9-fold) after 72 h in O. americanum . Moreover, 23 out of the 56 identified bioactive secondary metabolites showed variable importance in the projection (VIP) scores ≥ 1, including isovitexin and 2,3-dihydroxybenzoic acid for O. basilicum . Furthermore, analysis of primary metabolites indicated that aromatic amino acids and sugars decreased indicating higher rates of consumption, while organic acids as end products of metabolisms increased significantly following elicitation for O. basilicum . Besides, the lipid profile showed a total of 107 metabolites. However, the effect of elicitation was variable between both species. Hence, the integration of untargeted metabolomics with chemical elicitation may improve our knowledge regarding the effects on various metabolites accumulation behaviour of different basil suspension cultures in a comparative and time-dependent approach. • A metabolomic-based study for Ocimum sp. following chemical elicitation was performed. • O. basilicum showed different accumulation behaviours of most metabolites following elicitaion. • Around 56 bioactive metabolites, in addition to other metabolites were identified in Ocimum suspension cultures. • Production increase of phenolic metabolites is notably observed. • The study results may be further employed for optimization of certain metabolites production.

Effect of desiccation damage on the seed viability of Hydnocarpus alpina Wight. of Western Ghats

Hydnocarpus alpina Wight is an evergreen endangered medicinal tree species of Western Ghats. Several local names are available based on the distribution among the state in India that is Torathi (Kannada), Maravetti/Marotti (Malayalam), Attuchankalai (Tamil), Kastel (Hindi). Hydnocarpus alpina has very appreciated value in various medicinal properties like anti-larvicidal, anti-feedant, antimicrobial etc. due to the presence of significant chemical constituents. The seeds of this tree species are characterized with high moisture content and are intolerant to desiccation. They are under recalcitrant type seeds.Fully mature H. alpina seeds have 20% moisture content on harvest and recorded 80% germination. After 2 weeks of desiccation at open laboratory conditions (28±2 °C and 65% RH) their moisture content and percentage of germination decreased to 10.2% and 20 % respectively. Complete loss of viability occurred at 10% moisture level. This clearly indicates the recalcitrant nature of seeds. Different biochemical parameters such as lipid peroxidation, leachate conductivity, assay of free radical scavenging enzymes, analysis of primary metabolites like total soluble sugars, starch, total proteins, amino acids, lipids, phenols etc. were recorded for finding the biochemical basis of desiccation induced damage on seed viability. The results of the study reveals the metabolic disturbances associated with the desiccation of seeds and such parameters can be considered as markers for assessing the seed viability. Hence, safest low moisture content for the long term storage of seeds can be identified for the conservation of this endangered species of Western Ghats.

BnaPPT1 is essential for chloroplast development and seed oil accumulation in Brassica napus

IntroductionPhosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis. ObjectivesThis study investigated PPTs’ functions in plant growth and seed oil biosynthesis in oilseed crop Brassica napus. MethodsWe created over-expression and mutant material of BnaPPT1. The plant development, oil content, lipids, metabolites and ultrastructure of seeds were compared to evaluate the gene function. ResultsThe plastid membrane localized BnaPPT1 was found to be required for normal growth of B. napus. The plants grew slower with yellowish leaves in BnaA08.PPT1 and BnaC08.PPT1 double mutant plants. The results of chloroplast ultrastructural observation and lipid analysis show that BnaPPT1 plays an essential role in membrane lipid synthesis and chloroplast development in leaves, thereby affecting photosynthesis. Moreover, the analysis of primary metabolites and lipids in developing seeds showed that BnaPPT1 could impact seed glycolytic metabolism and lipid level. Knockout of BnaA08.PPT1 and BnaC08.PPT1 resulted in decreasing of the seed oil content by 2.2 to 9.1%, while overexpression of BnaC08.PPT1 significantly promoted the seed oil content by 2.1 to 3.3%. ConclusionOur results suggest that BnaPPT1 is necessary for plant chloroplast development, and it plays an important role in maintaining plant growth and promoting seed oil accumulation in B. napus.

Herbivore-induced plant volatiles, not natural enemies, mediate a positive indirect interaction between insect herbivores.

Many insect herbivores engage in apparent competition whereby two species interact through shared natural enemies. Upon insect attack, plants release volatile blends that attract natural enemies, but whether these volatiles mediate apparent competition between herbivores is not yet known. We investigate the role of volatiles that are emitted by bean plants upon infestation by Acyrthosiphon pisum aphids on the population dynamics and fitness of Sitobion avenae aphids, and on wheat phloem sap metabolites. In a field experiment, the dynamics of S.avenae aphids on wheat were studied by crossing two treatments: exposure of aphid colonies to A.pisum-induced bean volatiles and exclusion of natural enemies. Glasshouse experiments and analyses of primary metabolites in wheat phloem exudates were performed to better understand the results from the field experiment. In the field, bean volatiles did not affect S.avenae dynamics or survival when aphids were exposed to natural enemies. When protected from them, however, volatiles led to larger aphid colonies. In agreement with this observation, in glasshouse experiments, aphid-induced bean volatiles increased the survival of S.avenae aphids on wheat plants, but not on an artificial diet. This suggests that volatiles may benefit S.avenae colonies via metabolic changes in wheat plants, although we did not find any effect on wheat phloem exudate composition. We report a potential case of associational susceptibility whereby plant volatiles weaken the defences of receiving plants, thus leading to increased herbivore performance.

Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica)

BackgroundSalt stress is one of the main constraints determining crop productivity, and therefore one of the main limitations for food production. The aim of this study was to characterize the salt stress response at the physiological and molecular level of different Broccoli (Brassica oleracea L. var. Italica Plenck) cultivars that were previously characterized in field and greenhouse trials as salt sensitive or salt tolerant. This study aimed to identify functional and molecular traits capable of predicting the ability of uncharacterized lines to cope with salt stress. For this purpose, this study measured different physiological parameters, hormones and metabolites under control and salt stress conditions.ResultsThis study found significant differences among cultivars for stomatal conductance, transpiration, methionine, proline, threonine, abscisic acid, jasmonic acid and indolacetic acid. Salt tolerant cultivars were shown to accumulate less sodium and potassium in leaves and have a lower sodium to potassium ratio under salt stress. Analysis of primary metabolites indicated that salt tolerant cultivars have higher concentrations of several intermediates of the Krebs cycle and the substrates of some anaplerotic reactions.ConclusionsThis study has found that the energetic status of the plant, the sodium extrusion and the proline content are the limiting factors for broccoli tolerance to salt stress. Our results establish physiological and molecular traits useful as distinctive markers to predict salt tolerance in Broccoli or to design novel biotechnological or breeding strategies for improving broccoli tolerance to salt stress.

INFLUENCE OF TEMPERATURE CONDITIONS ON THE ACCUMULATION OF PRIMARY AND SECONDARY METABOLITES BY LICHENS FLAVOCETRARIA CUCULLATA AND CETRARIA LAEVIGATA

The study of the effect of temperature on the accumulation of primary and secondary metabolites by lichens Flavocetraria cucullata (Bellardi) Kärnefelt &amp; Thell and Cetraria laevigata Rass was carried out. Lichen samples were taken out from under the snow (-20 °C) together with the soil substrate and transferred to the climatic chamber. Then the temperature in the climatic chamber was sequentially increased to +10 and +20 °C. The lichen exposure was carried out for 30 days for each temperature regime. The analysis of primary metabolites was performed by gas chromatography-mass spectrometry. It was shown that in lichens F. cucullata and C. laevigata at temperatures of +10 and +20 °C, an increase in the content of mannitol, ribitol, sucrose, and hydroxyproline was observed, as well as a decrease in the content of unsaturated fatty acids as compared to the initial samples. At the same time, the content of glycerol and arabitol in the thalli of the initial lichens (-20 °C) was higher than after exposure at +10 and +20 °C, which, apparently, is associated with the cryoprotective properties of these compounds. The content of secondary metabolites in lichens was determined by high performance liquid chromatography. It was shown that the content of usnic, allo-protolichesterinic, and protolichesterinic acids in F. cucullata increased after exposure in a climatic chamber, which may be associated with a general activation of metabolic processes with an increase in temperature. At the same time, the content of fumarprotocetraric acid in C. laevigata lichens decreased at temperatures of +10 and +20 °C compared to the initial samples, which may be associated with its protective properties under the action of low-temperature stress.

Exploiting targeted and untargeted approaches for the analysis of bacterial metabolites under altered growth conditions.

In the host, pathogenic microorganisms have developed stress responses to cope with constantly changing environments. Stress responses are directly related to changes in several metabolomic pathways, which could hamper microorganisms' unequivocal identification. We evaluated the effect of various in vitro stress conditions (acidic, basic, oxidative, ethanolic, and saline conditions) on the metabolism of Staphylococcus aureus, Bacillus cereus, and Pseudomonas aeruginosa, which are common lung pathogens. The metabolite profiles of the bacteria were analyzed using liquid chromatography coupled to triple quadrupole and quadrupole time-of-flight mass spectrometry. The advantages of targeted and untargeted analysis combined with univariate and multivariate statistical analysis (principal component analysis, hierarchical cluster analysis, partial least square discriminant analysis, random forest) were combined to unequivocally identify bacterial species. In normal in vitro conditions, the targeted methodology, based on the analysis of primary metabolites, enabled the rapid and efficient discrimination of the three bacteria. In changing in vitro conditions and specifically in presence of the various stressors, the untargeted methodology proved to be more valuable for the global and accurate differentiation of the three bacteria, also considering the type of stress environment within each species. In addition, species-specific metabolites (i.e., fatty acids, polysaccharides, peptides, and nucleotide bases derivatives) were putatively identified. Good intra-day repeatability and inter-day repeatability (< 10% RSD and < 15% RSD, respectively) were obtained for the targeted and the untargeted methods. This untargeted approach highlights its importance in unusual (and less known) bacterial growth environments, being a powerful tool for infectious disease diagnosis, where the accurate classification of microorganisms is sought.

Biotechnological Potential of Cephalaria uralensis (Murray) Roem. & Schult. and C. gigantea (Ledeb.) Bobrov-Comparative Analysis of Plant Anatomy and the Content of Biologically Active Substances.

Studies conducted to date have shown that Cephalaria uralensis and C. gigantea have high contents of substances with antibacterial, anti-inflammatory, and antioxidant properties; hence, they are attractive plants from the pharmaceutical point of view. However, despite their multifarious desirable biotechnological aspects, the knowledge of these plants is insufficient. The present study focused on the analysis of the morphological, anatomical, and histological structure of aboveground parts of the plants, the identification of the distribution of biologically active compounds in the tissues, and quantitative phytochemical analyses of polyphenolic compounds contained in their aboveground organs. Importantly, the phenological and morphological features of the aboveground organs in the analyzed species were maintained, as in the same plant species growing in different climatic conditions. The analysis of primary metabolites and phenolic compounds in the tissues revealed their distribution in the aboveground organs, which has never been described before. The comparative analyses of the content of total phenolics, total phenolic acids, and total flavonoids in the aboveground organs showed that the level of these substances differed not only between the species but also between the organs. It should be emphasized that the level of these compounds is higher than in many other medicinal plants.

Integrated transcriptomic and metabolomic analyses of yellow horn (Xanthoceras sorbifolia) in response to cold stress.

Xanthoceras sorbifolia, a medicinal and oil-rich woody plant, has great potential for biodiesel production. However, little study explores the link between gene expression level and metabolite accumulation of X. sorbifolia in response to cold stress. Herein, we performed both transcriptomic and metabolomic analyses of X. sorbifolia seedlings to investigate the regulatory mechanism of resistance to low temperature (4 °C) based on physiological profile analyses. Cold stress resulted in a significant increase in the malondialdehyde content, electrolyte leakage and activity of antioxidant enzymes. A total of 1,527 common differentially expressed genes (DEGs) were identified, of which 895 were upregulated and 632 were downregulated. Annotation of DEGs revealed that amino acid metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, galactose metabolism, fructose and mannose metabolism, and the citrate cycle (TCA) were strongly affected by cold stress. In addition, DEGs within the plant mitogen-activated protein kinase (MAPK) signaling pathway and TF families of ERF, WRKY, NAC, MYB, and bHLH were transcriptionally activated. Through metabolomic analysis, we found 51 significantly changed metabolites, particularly with the analysis of primary metabolites, such as sugars, amino acids, and organic acids. Moreover, there is an overlap between transcript and metabolite profiles. Association analysis between key genes and altered metabolites indicated that amino acid metabolism and sugar metabolism were enhanced. A large number of specific cold-responsive genes and metabolites highlight a comprehensive regulatory mechanism, which will contribute to a deeper understanding of the highly complex regulatory program under cold stress in X. sorbifolia.

Phytohormone supplementation significantly increases fatty acid content of Phaeodactylum tricornutum in two-phase culture

The culture of marine microalgae for biodiesel production has become a subject of interest in recent years. Environment stresses are effective at increasing lipid content at the cost of decreasing cell growth. In this study, we propose a two-phase culture where 2.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) was for the initial 5 days of culture and then 2 mg L−1 abscisic acid (ABA) was added for another 3 days of culture, allowing both biomass and lipid increase in Phaeodactylum tricornutum. Addition of only 2.5 mg L−1 2,4-D improved biomass accumulation by 60% and led to lipid increase nearly by 3-fold. Two-phase culture which was combined of 2.5 mg L−1 2,4-D with 2 mg L−1 ABA resulted in a considerable improvement both in growth and lipid production in P. tricornutum, and total lipid content was increased by 4-fold compared with no phytohormone treatment. GC-MS analysis showed two-phase culture has no significant effect on lipid components. The analysis of primary metabolites indicated that phytohormone treatment downregulated the content of carbohydrate and protein, but upregulated lipid accumulation. Gene transcriptional levels revealed that the GS/GOGAT and fatty acid synthesis pathways were induced by two-phase culture and thus accelerated fatty acid synthesis from carbohydrate and protein. This study suggests that two-phase culture combined with 2,4-D and ABA enhances the accumulation of biomass and lipid more than single phytohormone treatment in P. tricornutum.

Analysis of Primary Metabolites in Cabbage (Brassica oleracea var. capitata) Varieties Correlated with Antioxidant Activity and Taste Attributes by Metabolic Profiling.

Brassica vegetables, such as cabbage, have many health benefits arising from their antioxidant and anticancer properties. These properties are endowed by the metabolite composition of the plant, and it is therefore important to elucidate the metabolic profile and associated activities in this genus. This study objectively evaluated the characteristics of cabbage varieties using metabolic profiling to identify the primary metabolic components that correlate with antioxidant activity and taste attributes. GC-MS analysis was used to identify the primary metabolites. Antioxidant activity was measured by oxygen radical absorbance capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging assays, and an electronic tongue was used to quantitate nine taste attributes. Orthogonal projections to latent structures (OPLS) using SIMCA 14 correlated the metabolite components with the taste and antioxidant characteristics. We identified 4-aminobutyric acid, fructose 1-phosphate, adipic acid, 5-oxoproline, N-acetylglycine, O-phosphoethanolamine, and homovanillic acid as important determinants of DPPH scavenging activity and umami, sourness, acidic bitterness, irritant and saltiness, bitterness, astringency, and richness, respectively. These metabolites represent markers indicating breed differences and contribute to differential cabbage functionality. These studies could be extended to measure additional metabolites, as well as to understand the role of growth conditions on the metabolic profile and health benefits of plants.

Impact of light and sucrose supplementation on cellular differentiation, metabolic shift and modulation of gene expression in hairy roots of Daucus carota

Green hairy root cultures of Daucus carota were subjected to gradually lowered sucrose concentrations to understand the modulation of secondary metabolism upon carbon-starved conditions. An apparent negative correlation with the sucrose concentrations in the media was observed with chloroplast development, as confirmed by both histological studies and relevant biochemical assays. Expansion of relative vascular area and well-developed multi-layered endodermis were evidenced in green hairy roots with low sucrose concentrations as compared to normal hairy roots cultivated with 2% sucrose under dark condition. Thus, anatomical modulation was evident in photo-oxidatively stressed green hairy roots because of their maintenance under continuous illumination. This was demonstrated further by the expression analysis of glutathione peroxidase and NADPH reductase genes which showed upregulation in green hairy roots maintained in ½ MS media containing 0.5% sucrose as compared to normal hairy roots. The amount of phenolic marker compound p-hydroxybenzoic acid was reduced to half, while enhanced levels of emitted terpenoid volatiles were noticed in these carbon-starved green hairy roots. Analysis of primary metabolites in green hairy roots showed increased levels of amino acids and sugar alcohols accumulation. Further, analysis revealed the elevated accumulation of organic acids which have significant bearing towards redirection of primary metabolites leading to the formation of enhanced terpenoid volatiles in green hairy roots. Thus, our study revealed the impact of light and sucrose supplementation towards cellular differentiation and redirection of secondary metabolism in hairy roots of D. carota.

Effects of Different Pollens on Primary Metabolism and Lignin Biosynthesis in Pear.

To investigate the effect of pollination on the fruit quality of ‘Dangshan Su’ pear, ‘Dangshan Su’ was fertilized by the pollen of ‘Wonhwang’ (Pyrus pyrifolia Nakai.) (DW) and ‘Jingbaili’ (Pyrus ussuriensis Maxim.) (DJ). The analysis of primary metabolites was achieved through untargeted metabolomics, and the quantitative analysis of intermediate metabolites of lignin synthesis was undertaken using targeted metabolomics. The untargeted metabolomics analysis was performed via gas chromatography-mass spectrometry (GC-MS). The targeted metabolomics analysis was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) under the multiple reaction monitoring (MRM) mode. The results showed that the metabolite content was significantly different between DW and DJ. Compared with that in DJ, the sugar and amino acid content in DW was higher and the fatty acid content was lower at 47 days after pollination (DAPs), and the sugar, amino acid, and fatty acid content in DW was lower at 63 DAPs. The intermediate metabolites of lignin synthesis were analyzed using the orthogonal partial least squares discriminant analysis (OPLS-DA) model, and the differential metabolites at 47 DAPs were p-coumaric acid, ferulic acid, sinapaldehyde, coniferyl alcohol, and sinapyl alcohol. The differential significant metabolite at 63 DAPs was p-coumaric acid. At 47 DAPs and 63 DAPs, the p-coumaric acid level was significantly different, and the p-coumaric acid content was positively correlated with lignin synthesis. The pollination pollen affects the quality of ‘Dangshan Su’ pear fruit through regulation of the sugar, amino acid, and fatty acid content; at the same time, regulating the levels of intermediate metabolites of lignin synthesis, especially the p-coumaric acid content, to affect lignin synthesis ultimately affects the stone cell content and improves the quality of the pears.

Integrated transcriptomics and metabolomics analysis to characterize cold stress responses in Nicotiana tabacum

BackgroundCB-1 and K326 are closely related tobacco cultivars; however, their cold tolerance capacities are different. K326 is much more cold tolerant than CB-1.ResultsWe studied the transcriptomes and metabolomes of CB-1 and K326 leaf samples treated with cold stress. Totally, we have identified 14,590 differentially expressed genes (DEGs) in CB-1 and 14,605 DEGs in K326; there was also 200 differentially expressed metabolites in CB-1 and 194 in K326. Moreover, there were many overlapping genes (around 50%) that were cold-responsive in both plant cultivars, although there were also many differences in the cold responsive genes between the two cultivars. Importantly, for most of the overlapping cold responsive genes, the extent of the changes in expression were typically much more pronounced in K326 than in CB-1, which may help explain the superior cold tolerance of K326. Similar results were found in the metabolome analysis, particularly with the analysis of primary metabolites, including amino acids, organic acids, and sugars. The large number of specific responsive genes and metabolites highlight the complex regulatory mechanisms associated with cold stress in tobacco. In addition, our work implies that the energy metabolism and hormones may function distinctly between CB-1 and K326.ConclusionsDifferences in gene expression and metabolite levels following cold stress treatment seem likely to have contributed to the observed difference in the cold tolerance phenotype of these two tobacco cultivars.

Time-course metabolic profiling in Arabidopsis thaliana cell cultures after salt stress treatment*

Salt stress is one of the most important factors limiting plant cultivation. Many investigations of plant response to high salinity have been performed using conventional transcriptomics and/or proteomics approaches. However, transcriptomics and proteomics techniques are not all-encompassing methods that can achieve exclusive insights into the metabolite networks contributing to biochemical reactions. Hence, the functions of the complex stress response pathways are yet to be determined, especially at the metabolic level. A time-course metabolic profiling with Arabidopsis thaliana cell cultures after the imposition of salt stress is reported in this study. Analyses of primary metabolites, especially small polar metabolites such as amino acids, sugars, sugar alcohols, organic acids, and amines, was performed by GC/MS and LC/MS at 0.5, 1, 2, 4, 12, 24, 48, and 72 h after a salt-stress treatment with 100 mM NaCl being the final concentration. The mass chromatographic data were converted into matrix data sets, which were subjected to data mining processes, including principal component analysis (PCA) and batch-learning self-organizing mapping analysis (BL-SOM). The mining results suggest that the methylation cycle for the supply of methyl groups, the phenylpropanoid pathway for lignin production, and glycinebetaine biosynthesis are synergetically induced as a short-term response against salt-stress treatment. The results also suggest the the co-induction of glycolysis and sucrose metabolism as well as co-reduction of the methylation cycle as long-term responses to salt stress.