FT-ICR-MS-based metabolomics: A deep dive into plant metabolism.

Metabolomics involves the identification and quantification of metabolites to unravel the chemical footprints behind cellular regulatory processes and to decipher metabolic networks, opening new insights to understand the correlation between genes and metabolites. In plants, it is estimated the existence of hundreds of thousands of metabolites and the majority is still unknown. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a powerful analytical technique to tackle such challenges. The resolving power and sensitivity of this ultrahigh mass accuracy mass analyzer is such that a complex mixture, such as plant extracts, can be analyzed and thousands of metabolite signals can be detected simultaneously and distinguished based on the naturally abundant elemental isotopes. In this review, FT-ICR-MS-based plant metabolomics studies are described, emphasizing FT-ICR-MS increasing applications in plant science through targeted and untargeted approaches, allowing for a better understanding of plant development, responses to biotic and abiotic stresses, and the discovery of new natural nutraceutical compounds. Improved metabolite extraction protocols compatible with FT-ICR-MS, metabolite analysis methods and metabolite identification platforms are also explored as well as new in silico approaches. Most recent advances in MS imaging are also discussed.

An apoplastic fluid extraction method for the characterization of grapevine leaves proteome and metabolome from a single sample.

The analysis of complex biological systems keeps challenging researchers. The main goal of systems biology is to decipher interactions within cells, by integrating datasets from large scale analytical approaches including transcriptomics, proteomics and metabolomics and more specialized 'OMICS' such as epigenomics and lipidomics. Studying different cellular compartments allows a broader understanding of cell dynamics. Plant apoplast, the cellular compartment external to the plasma membrane including the cell wall, is particularly demanding to analyze. Despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Analyzing woody plants such as grapevine raises even more challenges. Grapevine is among the most important fruit crops worldwide and a wider characterization of its apoplast is essential for a deeper understanding of its physiology and cellular mechanisms. Here, we describe, for the first time, a vacuum-infiltration-centrifugation method that allows a simultaneous extraction of grapevine apoplastic proteins and metabolites from leaves on a single sample, compatible with high-throughput mass spectrometry analyses. The extracted apoplast from two grapevine cultivars, Vitis vinifera cv 'Trincadeira' and 'Regent', was directly used for proteomics and metabolomics analysis. The proteome was analyzed by nanoLC-MS/MS and more than 700 common proteins were identified, with highly diverse biological functions. The metabolome profile through FT-ICR-MS allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes.

The glyoxalase pathway: the first hundred years... and beyond.

The discovery of the enzymatic formation of lactic acid from methylglyoxal dates back to 1913 and was believed to be associated with one enzyme termed ketonaldehydemutase or glyoxalase, the latter designation prevailed. However, in 1951 it was shown that two enzymes were needed and that glutathione was the required catalytic co-factor. The concept of a metabolic pathway defined by two enzymes emerged at this time. Its association to detoxification and anti-glycation defence are its presently accepted roles, since methylglyoxal exerts irreversible effects on protein structure and function, associated with misfolding. This functional defence role has been the rationale behind the possible use of the glyoxalase pathway as a therapeutic target, since its inhibition might lead to an increased methylglyoxal concentration and cellular damage. However, metabolic pathway analysis showed that glyoxalase effects on methylglyoxal concentration are likely to be negligible and several organisms, from mammals to yeast and protozoan parasites, show no phenotype in the absence of one or both glyoxalase enzymes. The aim of the present review is to show the evolution of thought regarding the glyoxalase pathway since its discovery 100 years ago, the current knowledge on the glyoxalase enzymes and their recognized role in the control of glycation processes.

From the grapevine to the glass: A wine metabolomics tale by FT-ICR-MS.

Wine is one of the most consumed beverages around the world. Its unique characteristics arise from numerous processes, from the selection of grapevine varieties and grapes, the effect of the terroir and geographical origin, through the biochemical process of fermentation by microorganisms, until its aging. All molecules found in wine define its chemical fingerprint and can be used to tell the story of its origin, production, authenticity and quality. Wine's chemical composition can be characterized using an untargeted metabolomics approach based on extreme resolution mass spectrometry. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is currently the most powerful analytical technique to analyse such complex sample, providing the most comprehensive analysis of the chemical fingerprint of wine.

Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains.

Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g L⁻¹ for L. acidophilus, with a cell mass yield (Y (X/S)) of 0.37 g g⁻¹. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g L⁻¹. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis-Menten constant (K(m)) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U mg protein⁻¹, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K(m) for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP+ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.

Cultivation of Melilotus officinalis as a source of bioactive compounds in association with soil recovery practices.

Introduction: Melilotus officinalis is a Leguminosae with relevant applications in medicine and soil recovery. This study reports the application of Melilotus officinalis plants in soil recovery and as a source of bioactive compounds. Methods: Plants were cultivated in semiarid soil under four different fertilizer treatments, urban waste compost at 10 t/ha and 20 t/ha, inorganic fertilizer and a control (no fertilizer). Agronomic properties of soil (pH, EC, soil respiration, C content, macro- and microelements) were analyzed before and after treatment. Also, germination, biomass, element contents, and physiological response were evaluated. Metabolite composition of plants was analyzed through Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Results and discussion: Results showed a significant enhancement of the soil microbial activity in planted soils amended with compost, though there were no other clear effects on the soil physicochemical and chemical characteristics during the short experimental period. An improvement in M. officinalis germination and growth was observed in soils with compost amendment. Metabolite composition of plants was analyzed through Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Principal Component and Agglomerative Hierarchical Clustering models suggest that there is a clear separation of the metabolome of four groups of plants grown under different soil treatments. The five most important discriminative metabolites (annotated) were oleamide, palmitic acid, stearic acid, 3-hydroxy-cis-5-octenoylcarnitine, and 6-hydroxynon-7- enoylcarnitine. This study provides information on how the metabolome of Melilotus might be altered by fertilizer application in poor soil regions. These metabolome changes might have repercussions for the application of this plant in medicine and pharmacology. The results support the profitability of Melilotus officinalis cultivation for bioactive compounds production in association with soil recovery practices.

A Multimethodological Approach for the Chemical Characterization of Edible Insects: The Case Study of Acheta domesticus.

Acheta domesticus (house cricket) has been recently introduced into the official European list of novel foods, representing an alternative and sustainable food source. Up to now, the chemical characterization of this edible insect has been focused only on specific classes of compounds. Here, three production batches of an A. domesticus powder were investigated by means of a multimethodological approach based on NMR, FT-ICR MS, and GC-MS methodologies. The applied analytical protocol, proposed for the first time in the study of an edible insect, allowed us to identify and quantify compounds not previously reported in crickets. In particular, methyl-branched hydrocarbons, previously identified in other insects, together with other compounds such as citrulline, formate, γ-terpinene, p-cymene, α-thujene, ß-thujene, and 4-carene were detected. Amino acids, organic acids, and fatty acids were also identified and quantified. The improved knowledge of the chemical profile of this novel food opens new horizons both for the use of crickets as a food ingredient and for the use of extracts for the production of new formulations. In order to achieve this objective, studies regarding safety, biological activity, bioaccessibility, and bioavailability are needed as future perspectives in this field.

The glyoxalase pathway in protozoan parasites.

The glyoxalase system is the main catabolic route for methylglyoxal, a non-enzymatic glycolytic byproduct with toxic and mutagenic effects. This pathway includes two enzymes, glyoxalase I and glyoxalase II, which convert methylglyoxal to d-lactate by using glutathione as a catalytic cofactor. In protozoan parasites the glyoxalase system shows marked deviations from this model. For example, the functional replacement of glutathione by trypanothione (a spermidine-glutathione conjugate) is a characteristic of trypanosomatids. Also interesting are the lack of glyoxalase I and the presence of two glyoxalase II enzymes in Trypanosoma brucei. In Plasmodium falciparum the glyoxalase pathway is glutathione-dependent, and glyoxalase I is an atypical monomeric enzyme with two active sites. Although it is tempting to exploit these differences for their potential therapeutic value, they provide invaluable clues regarding methylglyoxal metabolism and the evolution of protozoan parasites. Glyoxalase enzymes have been characterized in only a few protozoan parasites, namely Plasmodium falciparum and the trypanosomatids Leishmania and Trypanosoma. In this review, we will focus on the key features of the glyoxalase pathway in major human protozoan parasites, with particular emphasis on the characterized systems in Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. We will also search for genes encoding glyoxalase I and II in Toxoplasma gondii, Entamoeba histolytica, and Giardia lamblia.

Graph Properties of Mass-Difference Networks for Profiling and Discrimination in Untargeted Metabolomics.

Untargeted metabolomics seeks to identify and quantify most metabolites in a biological system. In general, metabolomics results are represented by numerical matrices containing data that represent the intensities of the detected variables. These matrices are subsequently analyzed by methods that seek to extract significant biological information from the data. In mass spectrometry-based metabolomics, if mass is detected with sufficient accuracy, below 1 ppm, it is possible to derive mass-difference networks, which have spectral features as nodes and chemical changes as edges. These networks have previously been used as means to assist formula annotation and to rank the importance of chemical transformations. In this work, we propose a novel role for such networks in untargeted metabolomics data analysis: we demonstrate that their properties as graphs can also be used as signatures for metabolic profiling and class discrimination. For several benchmark examples, we computed six graph properties and we found that the degree profile was consistently the property that allowed for the best performance of several clustering and classification methods, reaching levels that are competitive with the performance using intensity data matrices and traditional pretreatment procedures. Furthermore, we propose two new metrics for the ranking of chemical transformations derived from network properties, which can be applied to sample comparison or clustering. These metrics illustrate how the graph properties of mass-difference networks can highlight the aspects of the information contained in data that are complementary to the information extracted from intensity-based data analysis.

Grapevine leaf MALDI-MS imaging reveals the localisation of a putatively identified sucrose metabolite associated to Plasmopara viticola development.

Despite well-established pathways and metabolites involved in grapevine-Plasmopara viticola interaction, information on the molecules involved in the first moments of pathogen contact with the leaf surface and their specific location is still missing. To understand and localise these molecules, we analysed grapevine leaf discs infected with P. viticola with MSI. Plant material preparation was optimised, and different matrices and solvents were tested. Our data shows that trichomes hamper matrix deposition and the ion signal. Results show that putatively identified sucrose presents a higher accumulation and a non-homogeneous distribution in the infected leaf discs in comparison with the controls. This accumulation was mainly on the veins, leading to the hypothesis that sucrose metabolism is being manipulated by the development structures of P. viticola. Up to our knowledge this is the first time that the localisation of a putatively identified sucrose metabolite was shown to be associated to P. viticola infection sites.

Enlightening the molecular basis of trypanothione specificity in trypanosomatids: mutagenesis of Leishmania infantum glyoxalase II.

Leishmania infantum glyoxalase II shows absolute specificity towards its trypanothione thioester substrate. In the previous work, we performed a comparative analysis of glyoxalase II structures determined by X-ray crystallography which revealed that Tyr291 and Cys294, absent in the human homologue, are essential for substrate binding. To validate this trypanothione specificity hypothesis we produced a mutant L. infantum GLO2 enzyme by replacing Tyr291 and Cys294 by arginine and lysine, respectively. This new enzyme is capable to use the glutathione thioester substrate, with kinetic parameters similar to the ones from the human enzyme. Substrate specificity is likely to be mediated by spermidine moiety binding, providing a primer for understanding the molecular basis of trypanothione specificity.

Binary Simplification as an Effective Tool in Metabolomics Data Analysis.

Metabolomics aims to perform a comprehensive identification and quantification of the small molecules present in a biological system. Due to metabolite diversity in concentration, structure, and chemical characteristics, the use of high-resolution methodologies, such as mass spectrometry (MS) or nuclear magnetic resonance (NMR), is required. In metabolomics data analysis, suitable data pre-processing, and pre-treatment procedures are fundamental, with subsequent steps aiming at highlighting the significant biological variation between samples over background noise. Traditional data analysis focuses primarily on the comparison of the features' intensity values. However, intensity data are highly variable between experimental batches, instruments, and pre-processing methods or parameters. The aim of this work was to develop a new pre-treatment method for MS-based metabolomics data, in the context of sample profiling and discrimination, considering only the occurrence of spectral features, encoding feature presence as 1 and absence as 0. This "Binary Simplification" encoding (BinSim) was used to transform several benchmark datasets before the application of clustering and classification methods. The performance of these methods after the BinSim pre-treatment was consistently as good as and often better than after different combinations of traditional, intensity-based, pre-treatments. Binary Simplification is, therefore, a viable pre-treatment procedure that effectively simplifies metabolomics data-analysis pipelines.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyoxalase I from Leishmania infantum.

Glyoxalase I (GLO1) is the first of the two glyoxalase-pathway enzymes. It catalyzes the formation of S-D-lactoyltrypanothione from the non-enzymatically formed hemithioacetal of methylglyoxal and reduced trypanothione. In order to understand its substrate binding and catalytic mechanism, GLO1 from Leishmania infantum was cloned, overexpressed in Escherichia coli, purified and crystallized. Two crystal forms were obtained: a cube-shaped form and a rod-shaped form. While the cube-shaped form did not diffract X-rays at all, the rod-shaped form exhibited diffraction to about 2.0 A resolution. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 130.03, b = 148.51, c = 50.63 A and three dimers of the enzyme per asymmetric unit.

Integrating metabolomics and targeted gene expression to uncover potential biomarkers of fungal/oomycetes-associated disease susceptibility in grapevine.

Vitis vinifera, one of the most cultivated fruit crops, is susceptible to several diseases particularly caused by fungus and oomycete pathogens. In contrast, other Vitis species (American, Asian) display different degrees of tolerance/resistance to these pathogens, being widely used in breeding programs to introgress resistance traits in elite V. vinifera cultivars. Secondary metabolites are important players in plant defence responses. Therefore, the characterization of the metabolic profiles associated with disease resistance and susceptibility traits in grapevine is a promising approach to identify trait-related biomarkers. In this work, the leaf metabolic composition of eleven Vitis genotypes was analysed using an untargeted metabolomics approach. A total of 190 putative metabolites were found to discriminate resistant/partial resistant from susceptible genotypes. The biological relevance of discriminative compounds was assessed by pathway analysis. Several compounds were selected as promising biomarkers and the expression of genes coding for enzymes associated with their metabolic pathways was analysed. Reference genes for these grapevine genotypes were established for normalisation of candidate gene expression. The leucoanthocyanidin reductase 2 gene (LAR2) presented a significant increase of expression in susceptible genotypes, in accordance with catechin accumulation in this analysis group. Up to our knowledge this is the first time that metabolic constitutive biomarkers are proposed, opening new insights into plant selection on breeding programs.

Protein glycation in vivo: functional and structural effects on yeast enolase.

Protein glycation is involved in structure and stability changes that impair protein functionality, which is associated with several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer's disease, Parkinson's disease and Andrade's syndrome). To understand the relationship of protein glycation with protein dysfunction, unfolding and beta-fibre formation, numerous studies have been carried out in vitro. All of these previous experiments were conducted in non-physiological or pseudo-physiological conditions that bear little to no resemblance to what may happen in a living cell. In vivo, glycation occurs in a crowded and organized environment, where proteins are exposed to a steady-state of glycation agents, namely methylglyoxal, whereas in vitro, a bolus of a suitable glycation agent is added to diluted protein samples. In the present study, yeast was shown to be an ideal model to investigate glycation in vivo since it shows different glycation phenotypes and presents specific protein glycation targets. A comparison between in vivo glycated enolase and purified enolase glycated in vitro revealed marked differences. All effects regarding structure and stability changes were enhanced when the protein was glycated in vitro. The same applies to enzyme activity loss, dimer dissociation and unfolding. However, the major difference lies in the nature and location of specific advanced glycation end-products. In vivo, glycation appears to be a specific process, where the same residues are consistently modified in the same way, whereas in vitro several residues are modified with different advanced glycation end-products.

Vitis vinifera 'Pinot noir' leaves as a source of bioactive nutraceutical compounds.

Agricultural by-products are often hidden sources of healthy plant ingredients. The investigation of the nutritional values of these by-products is essential towards sustainable agriculture and improved food systems. In the vine industry, grape leaves are a bulky side product which is strategically removed and treated as waste in the process of wine production. In this work we performed an untargeted metabolomic profiling of the methanol extract of the leaves of Vitis vinifera cultivar 'Pinot noir', analysed their fatty acid content, and estimated their antioxidative capacity, with the purpose of investigating its nutritional and medicinal value. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) analysis identified the presence of numerous compounds which are known to possess diverse nutritional and pharmacological properties, particularly polyphenols and phenolic compounds (e.g. caffeic acid, catechin, kaempferol and quercetin), several phytosterols and fatty acids. Fatty acids were the most represented lipids' secondary class, with the essential alpha-linolenic acid being the most abundant in 'Pinot noir' leaves, with a relative content of 42%. Also, we have found that 'Pinot noir' leaves present a high antioxidant capacity, putting grapevine leaves at the top of the list of foods with the highest antioxidative activity. Our findings scientifically confirmed that 'Pinot noir' leaves have a high content and diversity of biologically active phytochemical compounds which make it of exceptional interest for pharmaceutical and food industries.

Author Correction: The interplay between membrane lipids and phospholipase A family members in grapevine resistance against Plasmopara viticola.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Subtilisin-like proteases in plant defence: the past, the present and beyond.

Subtilisin-like proteases (or subtilases) are a very diverse family of serine peptidases present in many organisms, but mostly in plants. With a broad spectrum of biological functions, ranging from protein turnover and plant development to interactions with the environment, subtilases have been gaining increasing attention with regard to their involvement in plant defence responses against the most diverse pathogens. Over the last 5 years, the number of published studies associating plant subtilases with pathogen resistance and plant immunity has increased tremendously. In addition, the observation of subtilases and serine protease inhibitors secreted by pathogens has also gained prominence. In this review, we focus on the active participation of subtilases in the interactions established by plants with the environment, highlighting their role in plant-pathogen communication.

The interplay between membrane lipids and phospholipase A family members in grapevine resistance against Plasmopara viticola.

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most important diseases in modern viticulture. The search for sustainable disease control measure is of extreme importance, thus becoming imperative to fully characterize the mechanisms leading to an incompatible interaction. We have previously shown that lipid signalling events play an important role in grapevine's response to this pathogen, namely through changes in linolenic acid content, lipid peroxidation and jasmonic acid synthesis. Here, we have characterized the modulation of lipid metabolism in leaves from two V. vinifera cultivars (resistant and susceptible to P. viticola) in the first hours after pathogen inoculation. Prior to pathogen inoculation both genotypes present an inherently different fatty acid composition that is highly modulated in the resistant genotype after pathogen challenge. Such changes involve modulation of phospholipase A activity suggesting that the source of lipids mobilized upon pathogen infection are the chloroplast membranes. This work thus provides original evidence on the involvement of lipid signalling and phospholipases in grapevine immune responses to pathogen infection. The results are discussed considering the implications on the plant's physiological status and the use of discriminating lipid/fatty acids pattern in future selection procedures of cultivars.