Biochemical Changes in Two Barley Genotypes Inoculated With a Beneficial Fungus Trichoderma harzianum Rifai T-22 Grown in Saline Soil.

One of the most important environmental factors impacting crop plant productivity is soil salinity. Fungal endophytes have been characterised as biocontrol agents that help in plant productivity and induce resistance responses to several abiotic stresses, including salinity. In the salt-tolerant cereal crop barley (Hordeum vulgare L.), there is limited information about the metabolites and lipids that change in response to inoculation with fungal endophytes in saline conditions. In this study, gas chromatography coupled to mass spectrometry (GC-MS) and LC-electrospray ionisation (ESI)-quadrupole-quadrupole time of flight (QqTOF)-MS were used to determine the metabolite and lipid changes in two fungal inoculated barley genotypes with differing tolerance levels to saline conditions. The more salt-tolerant cultivar was Vlamingh and less salt tolerant was Gairdner. Trichoderma harzianum strain T-22 was used to treat these plants grown in soil under control and saline (200 mM NaCl) conditions. For both genotypes, fungus-colonised plants exposed to NaCl had greater root and shoot biomass, and better chlorophyll content than non-colonised plants, with colonised-Vlamingh performing better than uninoculated control plants. The metabolome dataset using GC-MS consisted of a total of 93 metabolites of which 74 were identified in roots of both barley genotypes as organic acids, sugars, sugar acids, sugar alcohols, amino acids, amines, and a small number of fatty acids. LC-QqTOF-MS analysis resulted in the detection of 186 lipid molecular species, classified into three major lipid classes-glycerophospholipids, glycerolipids, and sphingolipids, from roots of both genotypes. In Cultivar Vlamingh both metabolites and lipids increased with fungus and salt treatment while in Gairdner they decreased. The results from this study suggest that the metabolic pathways by which the fungus imparts salt tolerance is different for the different genotypes.

Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia.

The interaction of germline variation and somatic cancer driver mutations is under-investigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondrially-encoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 (IDH1), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1-mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.

Characterization of epidermal bladder cells in Chenopodium quinoa.

Chenopodium quinoa (quinoa) is considered a superfood with its favourable nutrient composition and being gluten free. Quinoa has high tolerance to abiotic stresses, such as salinity, water deficit (drought) and cold. The tolerance mechanisms are yet to be elucidated. Quinoa has epidermal bladder cells (EBCs) that densely cover the shoot surface, particularly the younger parts of the plant. Here, we report on the EBC's primary and secondary metabolomes, as well as the lipidome in control conditions and in response to abiotic stresses. EBCs were isolated from plants after cold, heat, high-light, water deficit and salt treatments. We used untargeted gas chromatography-mass spectrometry (GC-MS) to analyse metabolites and untargeted and targeted liquid chromatography-MS (LC-MS) for lipids and secondary metabolite analyses. We identified 64 primary metabolites, including sugars, organic acids and amino acids, 19 secondary metabolites, including phenolic compounds, betanin and saponins and 240 lipids categorized in five groups including glycerolipids and phospholipids. We found only few changes in the metabolic composition of EBCs in response to abiotic stresses; these were metabolites related with heat, cold and high-light treatments but not salt stress. Na+ concentrations were low in EBCs with all treatments and approximately two orders of magnitude lower than K+ concentrations.

Inoculation of barley with Trichoderma harzianum T-22 modifies lipids and metabolites to improve salt tolerance.

Soil salinity has a serious impact on plant growth and agricultural yield. Inoculation of crop plants with fungal endophytes is a cost-effective way to improve salt tolerance. We used metabolomics to study how Trichoderma harzianum T-22 alleviates NaCl-induced stress in two barley (Hordeum vulgare L.) cultivars, Gairdner and Vlamingh, with contrasting salinity tolerance. GC-MS was used to analyse polar metabolites and LC-MS to analyse lipids in roots during the early stages of interaction with Trichoderma. Inoculation reversed the severe effects of salt on root length in sensitive cv. Gairdner and, to a lesser extent, improved root growth in more tolerance cv. Vlamingh. Biochemical changes showed a similar pattern in inoculated roots after salt treatment. Sugars increased in both cultivars, with ribulose, ribose, and rhamnose specifically increased by inoculation. Salt stress caused large changes in lipids in roots but inoculation with fungus greatly reduced the extent of these changes. Many of the metabolic changes in inoculated cv. Gairdner after salt treatment mirror the response of uninoculated cv. Vlamingh, but there are some metabolites that changed in both cultivars only after fungal inoculation. Further study is required to determine how these metabolic changes are induced by fungal inoculation.

Comparative metabolomics implicates threitol as a fungal signal supporting colonization of Armillaria luteobubalina on eucalypt roots.

Armillaria root rot is a fungal disease that affects a wide range of trees and crops around the world. Despite being a widespread disease, little is known about the plant molecular responses towards the pathogenic fungi at the early phase of their interaction. With recent research highlighting the vital roles of metabolites in plant root-microbe interactions, we sought to explore the presymbiotic metabolite responses of Eucalyptus grandis seedlings towards Armillaria luteobuablina, a necrotrophic pathogen native to Australia. Using a metabolite profiling approach, we have identified threitol as one of the key metabolite responses in E. grandis root tips specific to A. luteobubalina that were not induced by three other species of soil-borne microbes of different lifestyle strategies (a mutualist, a commensalist, and a hemi-biotrophic pathogen). Using isotope labelling, threitol detected in the Armillaria-treated root tips was found to be largely derived from the fungal pathogen. Exogenous application of d-threitol promoted microbial colonization of E. grandis and triggered hormonal responses in root cells. Together, our results support a role of threitol as an important metabolite signal during eucalypt-Armillaria interaction prior to infection thus advancing our mechanistic understanding on the earliest stage of Armillaria disease development. Comparative metabolomics of eucalypt roots interacting with a range of fungal lifestyles identified threitol enrichment as a specific characteristic of Armillaria pathogenesis. Our findings suggest that threitol acts as one of the earliest fungal signals promoting Armillaria colonization of roots.

Spatio-Temporal Metabolite and Elemental Profiling of Salt Stressed Barley Seeds During Initial Stages of Germination by MALDI-MSI and µ-XRF Spectrometry.

Seed germination is the essential first step in crop establishment, and can be severely affected by salinity stress which can inhibit essential metabolic processes during the germination process. Salt stress during seed germination can trigger lipid-dependent signalling cascades that activate plant adaptation processes, lead to changes in membrane fluidity to help resist the stress, and cause secondary metabolite responses due to increased oxidative stress. In germinating barley (Hordeum vulgare), knowledge of the changes in spatial distribution of lipids and other small molecules at a cellular level in response to salt stress is limited. In this study, mass spectrometry imaging (MSI), liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS), inductively coupled plasma mass spectrometry (ICP-MS), and X-ray fluorescence (XRF) were used to determine the spatial distribution of metabolites, lipids and a range of elements, such as K+ and Na+, in seeds of two barley genotypes with contrasting germination phenology (Australian barley varieties Mundah and Keel). We detected and tentatively identified more than 200 lipid species belonging to seven major lipid classes (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, prenol lipids, sterol lipids, and polyketides) that differed in their spatial distribution based on genotype (Mundah or Keel), time post-imbibition (0 to 72 h), or treatment (control or salt). We found a tentative flavonoid was discriminant in post-imbibed Mundah embryos under saline conditions, and a delayed flavonoid response in Keel relative to Mundah. We further employed MSI-MS/MS and LC-QToF-MS/MS to explore the identity of the discriminant flavonoid and study the temporal pattern in five additional barley genotypes. ICP-MS was used to quantify the elemental composition of both Mundah and Keel seeds, showing a significant increase in Na+ in salt treated samples. Spatial mapping of elements using µ-XRF localized the elements within the seeds. This study integrates data obtained from three mass spectrometry platforms together with µ-XRF to yield information on the localization of lipids, metabolites and elements improving our understanding of the germination process under salt stress at a molecular level.

Morphological and metabolic responses to salt stress of rice (Oryza sativa L.) cultivars which differ in salinity tolerance.

Salinization is one of the most important abiotic stressors for crop growth and productivity. Rice (Oryza sativa L.), as the major food source around the world, is very sensitive to salt, especially at seedling stage. In order to examine how salt stress influences the metabolism of rice, we compared the levels of a range of sugars and organic acids in three rice cultivars with different tolerance under salt stress over time. According to the morphological result, the shoot length and root fresh weight were only affected by salinity in the salt sensitive cultivar (Nipponbare). The responses of metabolites to salinity were time-, tissue- and cultivar-dependent. Shikimate and quinate, involved in the shikimate pathway, were dramatically decreased in the leaves of all three cultivars, which was regarded as a response to salinity. Many sugars in the leaves of the salt tolerant cultivar (Dendang and Fatmawati) showed earlier increases to salt stress compared to Nipponbare leaves. Moreover, only in the leaves of tolerant cultivars (Dendang and Fatimawati), malate was significantly decreased while sucrose was significantly increased. In Dendang roots, mannitol levels were significantly higher than in Nipponbare roots after 14 days of salt treatment, which may be attributed to its higher salt tolerance. It is proposed that these responses in the more tolerant cultivars are involved in their resistance to high salt stress which may lay the foundation for breeding tolerant rice cultivars.

Genome-wide association studies of 74 plasma metabolites of German shepherd dogs reveal two metabolites associated with genes encoding their enzymes.

INTRODUCTION: German shepherd dogs (GSDs) are a popular breed affected by numerous disorders. Few studies have explored genetic variations that influence canine blood metabolite levels. OBJECTIVES: To investigate genetic variants affecting the natural metabolite variation in GSDs. METHODS: A total of 82 healthy GSDs were genotyped on the Illumina CanineHD Beadchip, assaying 173,650 markers. For each dog, 74 metabolites were measured through liquid and gas chromatography mass spectrometry (LC-MS and GC-MS) and were used as phenotypes for genome-wide association analyses (GWAS). Sliding window and homozygosity analyses were conducted to fine-map regions of interest, and to identify haplotypes and gene dosage effects. RESULTS: Summary statistics for 74 metabolites in this population of GSDs are reported. Forty-one metabolites had significant associations at a false discovery rate of 0.05. Two associations were located around genes which encode for enzymes for the relevant metabolites: 4-hydroxyproline was significantly associated to D-amino acid oxidase (DAO), and threonine to L-threonine 3-dehydrogenase (LOC477365). Three of the top ten haplotypes associated to 4-hydroxyproline included at least one SNP on DAO. These haplotypes occurred only in dogs with the highest 15 measurements of 4-hydroxyproline, ranging in frequency from 16.67 to 20%. None of the dogs were homozygous for these haplotypes. The top two haplotypes associated to threonine included SNPs on LOC477365 and were also overrepresented in dogs with the highest 15 measurements of threonine. These haplotypes occurred at a frequency of 90%, with 80% of these dogs homozygous for the haplotypes. In dogs with the lowest 15 measurements of threonine, the haplotypes occurred at a frequency of 26.67% and 0% homozygosity. CONCLUSION: DAO and LOC477365 were identified as candidate genes affecting the natural plasma concentration of 4-hydroxyproline and threonine, respectively. Further investigations are needed to validate the effects of the variants on these genes.

A high-resolution HPLC-QqTOF platform using parallel reaction monitoring for in-depth lipid discovery and rapid profiling.

Here, we developed a robust lipidomics workflow merging both targeted and untargeted approaches on a single liquid chromatography coupled to quadrupole-time of flight (LC-QqTOF) mass spectrometry platform with parallel reaction monitoring (PRM). PRM assays integrate both untargeted profiling from MS1 scans and targeted profiling obtained from MS/MS data. This workflow enabled the discovery of more than 2300 unidentified features and identification of more than 600 lipid species from 23 lipid classes at the level of fatty acid/long chain base/sterol composition in a barley root extracts. We detected the presence of 142 glycosyl inositol phosphorylceramides (GIPC) with HN(Ac)-HA as the core structure of the polar head, 12 cardiolipins and 17 glucuronosyl diacylglycerols (GlcADG) which have been rarely reported previously for cereal crops. Using a scheduled algorithm with up to 100 precursors multiplexed per duty cycle, the PRM assay was able to achieve a rapid profiling of 291 species based on MS/MS data by a single injection. We used this novel approach to demonstrate the applicability and efficiency of the workflow to study salt stress induced changes in the barley root lipidome. Results show that 221 targeted lipids and 888 unknown features were found to have changed significantly in response to salt stress. This combined targeted and untargeted single workflow approach provides novel applications of lipidomics addressing biological questions.

Structural and functional measures of marine microbial communities: An experiment to assess implications for oil spill management.

Microbial communities are ecologically important in aquatic environments and impacts on microbes have the potential to affect a number of functional processes. We have amended seawater with a crude oil and assessed changes in species composition as well as a measure of functional diversity (the ability of the community to utilise different carbon sources) and the community level metabolic signature. We found that there was a degree of functional redundancy in the community we tested. Oiled assemblages became less diverse and more dominated by specialist hydrocarbon degraders, carbon source utilisation increased initially but there was no change in metabolic signature in this small scale laboratory experiment. This study supports the decision framework around management of oil spills. This package of methods has the potential to be used in the testing and selection of new dispersants for use in oil spill response.

From common to rare Zingiberaceae plants - A metabolomics study using GC-MS.

Zingiberaceae plants, commonly known as gingers, have been popular for their medicinal and culinary uses since time immemorial. In spite of their numerous health-promoting applications, many Zingiberaceae plants still receive no scientific attention. Moreover, existing reports mostly focused only on the Zingiberaceae rhizomes. Here, untargeted metabolite profiling using Gas Chromatography - Mass Spectrometry (GC-MS) was used to compare the metabolic composition of leaves and rhizomes of the more common gingers, Zingiber officinale Rosc. (ZO), Curcuma longa L. (CL), and Etlingera elatior (Jack) R.M. Smith (EE), and the rare gingers, Amomum muricarpum Elmer (AM), Etlingera philippinensis (Ridl.) R.M. Smith (EP), and Hornstedtia conoidea Ridl. (HC). Principal component analysis (PCA) demonstrated that different species show substantial chemical differentiation and revealed potential markers among the different Zingiberaceae plants. Interestingly, the leaves of AM, CL, EE, EP, and HC had significantly higher levels of chlorogenic acid than ZO. Moreover, rhizomes of EP and HC were found to contain significantly higher levels of amino acids than ZO. Sugars and organic acids were generally less abundant in ZO leaves and rhizomes than in the other gingers. The leaves of EP and rhizomes of AM were found most similar to the leaves and rhizomes of common gingers, respectively. Results of this study provide significant baseline information on assessing the possible usage of the leaves of common gingers and further propagation and exploration of EP and AM. This study, being the first metabolomics report on rare plants such as AM, EP and HC, affirms the usefulness of untargeted metabolite profiling in exploring under-investigated plants.

Salt-stress induced alterations in the root lipidome of two barley genotypes with contrasting responses to salinity.

Changes in lipid metabolism and composition as well as in distinct lipid species have been linked with altered plant growth, development and responses to environmental stresses including salinity. However, there is little information available in the literature focusing on lipids in roots under soil-related stresses such as salinity. Barley (Hordeum vulgare L.) is a major cereal grain and, as a glycophyte, suffers substantial yield loss when grown under saline conditions. Relatively little is understood of adaptation and tolerance mechanisms involving lipids and lipid metabolism in barley roots during development and under exposure to salinity stress. In this study we investigated the lipid composition of barley roots of Clipper and Sahara - two genotypes with contrasting responses to salinity - before and after salinity stress using a combination of three lipidomics techniques: Fatty acid compositional analysis, untargeted lipid profiling, and targeted analysis to profile quantitatively the individual molecular species of key plant lipid classes. Our results provide new insight into the effect of salinity on fatty acid profiles and key lipid classes within barley roots of two different genotypes, which is discussed in the context of current knowledge of the root metabolic responses of cereal crops to salinity stress.

Insights into lipidomic perturbations in zebrafish tissues upon exposure to microcystin-LR and microcystin-RR.

This work represents the first study of its kind that was conducted to evaluate changes in lipid metabolic networks following a balneation exposure of adult zebrafish to MCLR (microcystin-leucine-arginine) and MCRR (microcystin-arginine-arginine) at a sublethal dose (10 µg L(-1)) for a period of 30 days. Following the exposure to MCLR and MCRR, gills, liver, intestine, and brain tissues were harvested for metabolite extraction. Extracted metabolites were detected using qTOF-LC-MS (time-of-flight-liquid chromatography-mass spectrometry). Metabolites were identified using Kegg pathways. The identified metabolites are shown on lipid biochemical maps to demonstrate major perturbations in the metabolic machinery. Results showed that most of the metabolic pathways under the lipid class were affected in different tissues of zebrafish following the exposure to MCLR and MCRR (10 µg L(-1) for 30 days). The kind and flux of metabolic perturbations varied among different tissues of the organs after the exposure to MCLR and MCRR with the tissues of gills being the most affected. Among the various lipid pathways, cholesterol synthesis was affected significantly as observed from the highest number of perturbed metabolites in that pathway. Cholesterol is responsible for synthesis of steroid hormones and bile acids, which have been recognized as endocrine signaling molecules. Disruption in the synthesis of these compounds following MCLR/MCRR exposure suggests that MCs are capable of causing endocrine disruption among aquatic organisms even under sublethal conditions. Apart from cholesterol synthesis, various other metabolic pathways belonging to the class of essential fatty acids and lipid oxidation were also observed to be perturbed following a balneation exposure of zebrafish to MCLR/MCRR.

Rice suspension cultured cells are evaluated as a model system to study salt responsive networks in plants using a combined proteomic and metabolomic profiling approach.

Salinity is one of the major abiotic stresses affecting plant productivity but surprisingly, a thorough understanding of the salt-responsive networks responsible for sustaining growth and maintaining crop yield remains a significant challenge. Rice suspension culture cells (SCCs), a single cell type, were evaluated as a model system as they provide a ready source of a homogenous cell type and avoid the complications of multicellular tissue types in planta. A combination of growth performance, and transcriptional analyses using known salt-induced genes was performed on control and 100 mM NaCl cultured cells to validate the biological system. Protein profiling was conducted using both DIGE- and iTRAQ-based proteomics approaches. In total, 106 proteins were identified in DIGE experiments and 521 proteins in iTRAQ experiments with 58 proteins common to both approaches. Metabolomic analysis provided insights into both developmental changes and salt-induced changes of rice SCCs at the metabolite level; 134 known metabolites were identified, including 30 amines and amides, 40 organic acids, 40 sugars, sugar acids and sugar alcohols, 21 fatty acids and sterols, and 3 miscellaneous compounds. Our results from proteomic and metabolomic studies indicate that the salt-responsive networks of rice SCCs are extremely complex and share some similarities with thee cellular responses observed in planta. For instance, carbohydrate and energy metabolism pathways, redox signaling pathways, auxin/indole-3-acetic acid pathways and biosynthesis pathways for osmoprotectants are all salt responsive in SCCs enabling cells to maintain cellular function under stress condition. These data are discussed in the context of our understanding of in planta salt-responses.

Analysis of the Oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry.

To identify integral and peripheral plasma membrane (PM) proteins from Oryza sativa (rice), highly enriched PM fractions from rice suspension cultured cells were analyzed using two complementary approaches. The PM was enriched using aqueous two-phase partitioning and high pH carbonate washing to remove soluble, contaminating proteins and characterized using enzymatic and immunological analyses. Proteins from the carbonate-washed PM (WPM) were analyzed by either one-dimensional gel electrophoresis (1D-SDS-PAGE) followed by tryptic proteolysis or proteolysis followed by strong cation exchange liquid chromatography (LC) with subsequent analysis of the tryptic peptides by LC-MS/MS (termed Gel-LC-MS/MS and 2D-LC-MS/MS, respectively). Combining the results of these two approaches, 438 proteins were identified on the basis of two or more matching peptides, and a further 367 proteins were identified on the basis of single peptide matches after data analysis with two independent search algorithms. Of these 805 proteins, 350 were predicted to be PM or PM-associated proteins. Four hundred and twenty-five proteins (53%) were predicted to be integrally associated with a membrane, via either one or many (up to 16) transmembrane domains, a GPI-anchor, or membrane-spanning beta-barrels. Approximately 80% of the 805 identified proteins were assigned a predicted function, based on similarity to proteins of known function or the presence of functional domains. Proteins involved in PM-related activities such as signaling (21% of the 805 proteins), transporters and ATPases (14%), and cellular trafficking (8%), such as via vesicles involved in endo- and exocytosis, were identified. Proteins that are involved in cell wall biosynthesis were also identified (5%) and included three cellulose synthase (CESA) proteins, a cellulose synthase-like D (CSLD) protein, cellulases, and several callose synthases. Approximately 20% of the proteins identified in this study remained functionally unclassified despite being predicted to be membrane proteins.

Increased abundance of proteins involved in phytosiderophore production in boron-tolerant barley.

Boron (B) phytotoxicity affects cereal-growing regions worldwide. Although B-tolerant barley (Hordeum vulgare) germplasm is available, molecules responsible for this tolerance mechanism have not been defined. We describe and use a new comparative proteomic technique, iTRAQ peptide tagging (iTRAQ), to compare the abundances of proteins from B-tolerant and -intolerant barley plants from a 'Clipper' x 'Sahara' doubled-haploid population selected on the basis of a presence or absence of two B-tolerance quantitative trait loci. iTRAQ was used to identify three enzymes involved in siderophore production (Iron Deficiency Sensitive2 [IDS2], IDS3, and a methylthio-ribose kinase) as being elevated in abundance in the B-tolerant plants. Following from this result, we report a potential link between iron, B, and the siderophore hydroxymugineic acid. We believe that this study highlights the potency of the iTRAQ approach to better understand mechanisms of abiotic stress tolerance in cereals, particularly when applied in conjunction with bulked segregant analysis.

Proteomics as a functional genomics tool.

To understand the function of all the genes in an organism, one needs to know not only which genes are expressed, when, and where, but also what the protein end products are and under which conditions they accumulate in certain tissues. Proteomics aims at describing the whole protein output of the genome and complements transcriptomic and metabolomic studies. Proteomics depends on extracting, separating, visualizing, identifying, and quantifying the proteins and their interactions present in an organism or tissue at any one time. All of these stages have limitations. Therefore, it is, at present, impossible to describe the whole proteome of any organism. Plants might synthesize many thousands of proteins at one time, and the whole potentially synthesized proteome certainly exceeds the number of estimated genes for that genome. This occurs because the gene products of one gene can differ due to alternative splicing and a variety of possible posttranslational modifications. It is, therefore, essential to optimize every step towards detecting the whole proteome while realizing the limitations. We concentrate here on the most commonly used steps in high-throughput plant proteomics with the techniques we have found most reproducible and with the highest resolution and quality.

A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

Evaluation of proteome reference maps for cross-species identification of proteins by peptide mass fingerprinting.

We tested whether proteome reference maps established for one species can be used for cross-species protein identification by comparing two-dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species. First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability. Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross-species similarities. In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities. However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species. Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species-specific protein identification than N-terminal sequencing. While peptide masses were found to be unreliable for cross-species protein identification, we present useful criteria to determine confident matching against species-specific expressed sequence tag databases. In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protein identification.