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Genomic and transcriptomic characterization of genes expressed at 20 MPa by the marine actinobacterium Kocuria flava.

A marine hydrocarbonoclastic actinobacterium Kocuria flava IOS11 was isolated from 3500 m deep-sea water of the Indian Ocean. The isolate efficiently degraded phenanthrene (250 mg/L) achieving 82 and 98% of degradation at 0.1 MPa and 20 MPa, respectively within a period of 5 days. Whole genome, transcriptomee and metabolomic analysis elucidated its phenanthrene biodegradation efficiency under in situ deep-sea conditions. The genome sequence comprises 3.47 Mb distributed across 88 scaffolds with a high GC content of 74.30%. The genome analysis encoded 3126 genes including 3052 protein coding sequences with functional annotation identifying a broad array of genes associated with PAHs degradation, environmental stress adaptation, biosurfactant and siderophore synthesis. Transcriptome profiling under 0.1 and 20 MPa conditions with phenanthrene as a sole carbon source revealed enhanced expression of hydrocarbon degrading genes, transporters, biosurfactant associated enzymes and stress responsive genes including integrases, DNA repair protein Rad, alanine ligase, heat and cold shock proteins under high pressure conditions underscoring the deep-sea adaptation capabilities of the strain. The degradation pathway of phenanthrene was proposed through integrated genome, transcriptome and metabolomic analysis. These studies provided K. flava IOS11 as a metabolically versatile and pressure adapted bacterium with promising potential for bioremediation application in extreme marine environment.

Transcriptome↗

Unveiling tumor heterogeneity by single cell RNA-sequencing: From basic considerations to clinical applications.

Tumor heterogeneity-encompassing diverse cellular phenotypes, genomic alterations, and microenvironmental contexts-is a principal barrier to effective cancer therapy. Single-cell RNA sequencing (scRNA-seq) has transformed our ability to resolve this complexity by capturing transcriptomes at single-cell resolution. Here, we review the technical foundations required for high-quality scRNA-seq studies. We then trace the evolution of scRNA-seq platforms from manual micromanipulation to high-throughput systems, and describe the computational pipelines that enable reliable data interpretation. The application of scRNA-seq is exemplarily shown in the context of lung cancer, where single-cell profiling has revealed (i) the clonal and sub-clonal architecture of tumors, (ii) extensive remodeling of the immune microenvironment, iii) key mechanisms underlying resistance to targeted agents and immune-checkpoint blockade, and (iv) the dynamics of neo-antigen-specific T-cell responses. Integrating machine-learning techniques-such as deep-learning classifiers and graph-based models-with single-cell transcriptomic data has markedly sped up biomarker discovery, produced more accurate risk-stratification scores, and enabled the generation of patient-specific therapeutic predictions. We surveyed the major trial registry ClinicalTrials.gov and identified ∼380 ongoing or completed studies that explicitly incorporate scRNA-seq as a correlative or pharmacodynamic endpoint. Overall, the analysis shows that scRNA-seq becomes an increasingly important component of modern trials, providing high-resolution cellular and molecular readouts that complement conventional imaging and bulk-omics endpoints. While key challenges remain, ranging from costs, scalability and need for rigorous validation before routine clinical deployment, ongoing technological advances continue to expand the potential of scRNA-seq as a cornerstone of precision medicine.

Humans↗

Comparison of sampling techniques for parallel analysis of transcript and metabolite levels in Saccharomyces cerevisiae.

Mathematical modelling of cellular processes is crucial for the understanding of the cell or organism as a whole. Genome-wide observations, at the levels of the transcriptome, proteome and metabolome, provide a high coverage of the molecular constituents of the system in study. Time-course experiments are important for gaining insight into a system's dynamics and are needed for mathematical modelling. In time-course experiments it is crucial to use efficient and fast sampling techniques. We evaluated several techniques to sample and process yeast cultures for parallel analysis of the transcriptome and metabolome. The evaluation was made by measuring the quality of the RNA obtained with UV-spectroscopy, capillary electrophoresis and microarray hybridization. The protocol developed involves rapid collection by spraying the sample into -40 degrees C tricine-buffered methanol (as previously described for yeast metabolome analysis), followed by the separation of cells from the culture medium in low-temperature rapid centrifugation. Removal of the residual methanol is carried out by freeze-drying the pellet at -35 degrees C. RNA and metabolites can then be extracted from the same freeze-dried sample obtained with this procedure.

Centrifugation↗

An Immunosenescent CD8+ T Cell Subset in Patients with Axial Spondyloarthritis and Psoriatic Arthritis Links Spontaneous Motility to Telomere Shortening and Dysfunction.

OBJECTIVE: A pathogenetic role of CD8+ T lymphocytes in radiographic axial spondyloarthritis (r-axSpA) and other spondyloarthritis (SpA) is sustained by genome-wide association studies and by the expansion of public T cell clonotypes in the target tissues. This study investigates the migration of CD8+ T cells along with their phenotype and functions in patients with r-axSpA and psoriatic arthritis (PsA). METHODS: Peripheral blood CD8+ and CD4+ T cells were isolated from patients with r-axSpA (n = 128), PsA (n = 60), and rheumatoid arthritis (RA) (n = 74) and healthy donors (HDs) (n = 79). Transwell migration assay was performed in the presence of different chemokines. CD8+ T cell immunoprofiling and effector functions were assessed by multiparametric flow cytometry. Transcriptome signature was evaluated by RNA sequencing analysis, whereas telomere length and dysfunction were measured by reverse transcriptase-polymerase chain reaction and immunofluorescence-fluorescence in situ hybridization, respectively. RESULTS: A significantly higher number of CD8+ T cells migrating in the absence of chemokine stimuli was found in patients with SpA compared with HDs and patients with RA. This subset, producing cytotoxic (granzyme B, perforin, granulysin) and proinflammatory molecules (tumor necrosis factor), was significantly enriched in terminally differentiated (CCR7-CD45RA+) and senescent (CD28-CD57+) cells having a gene expression profile characterized by cytolytic signature and natural killer markers. Remarkably, these spontaneously migrating CD8+ T cells showed DNA damage response activation, telomere shortening, and dysfunction. CONCLUSION: These data describe a terminally differentiated CD8+ T cell subset with a senescent and cytotoxic/proinflammatory profile and an intrinsic invasive potential enriched in patients with SpA that represents a possible player in disease pathogenesis.

Humans↗

Functional genomics strategies to identify susceptibility genes and treatment targets in alcohol dependence.

Genetic factors contribute to alcohol dependence through two main categories of mechanisms. The 50-60% heritability observed in this disorder is presumably conferred by polymorphic variants, encoding functionally altered proteins, or leading to differential transcriptional activity. Secondly, long term changes during the process of developing dependence are likely encoded by persistent changes in gene expression. Thus, genetic and environmental factors interact at the level of the transcriptome, making this an attractive level of analysis. For this purpose, we have applied differential display and more recently Affymetrix oligonucleotide gene arrays to models of genetic susceptibility and alcohol-induced neuroadaptation.

Adaptation, Physiological↗

Clinical and molecular prognostic factors in follicular lymphoma.

The observed variability in the clinical course of follicular lymphoma (FL), along with the diverse range of therapeutic options available, necessitates accurate prognostic stratification of the individual patient. A number of clinical, laboratory, and pathologic parameters have been associated with both good and poor risk disease; in some instances these have been incorporated into readily calculable prognostic indices. With new insights into disease biology and the resulting identification of biomarkers that have arisen from the analysis of both the genome and the transcriptome, more accurate individualization of prognosis will be realized. At present the clinical application of such biomarkers, however, remains largely in its infancy. This review examines the clinical and molecular prognostic features that have been identified as of value in FL.

Biomarkers, Tumor↗

REG3α is a Predictive Biomarker of Complicated Disease from Preclinical through Established Crohn's Disease.

BACKGROUND: Regenerating islet-derived 3-alpha (REG3&#x3b1;) is a serum biomarker in patients with graft-versus-host disease (GVHD) linked to 6-month mortality. REG3&#x3b1; is produced by intestinal Paneth cells, which are implicated in Crohn's disease (CD) pathophysiology. OBJECTIVE: To assess associations between serum REG3&#x3b1; and progressive CD DESIGN: Serum REG3&#x3b1; was measured in two cross-sectional (M: Mount Sinai, L: Leuven) and a pre-diagnostic cohort (P: PREDICTS) with serial samples up to 10 years before CD diagnosis. Tissue REG3&#x3b1; expression was assessed via bulk RNA sequencing from paired ileal and colonic biopsies. Serum REG3&#x3b1; and tissue REG3&#x3b1; were associated with CD progression (hospitalization, surgery, steroid course, or new advanced therapy). Single-cell RNA sequencing data explored associations between REG3&#x3b1; expression, Paneth cell phenotypes, and CD. RESULTS: In 394 patients, high serum REG3&#x3b1; associated with CD progression, independent of C-reactive protein and endoscopic activity (M: HR 1.9 (95%CI 1.3-2.8); L: HR 2.9 (95%CI 1.9-4.6), both p<0.001). The association persisted in patients with mild or inactive CD. In the pre-diagnostic cohort, high serum REG3&#x3b1; predicted the development of CD, particularly complicated (B2/3) and surgical presentations, up to 10 years before diagnosis (P). Analysis of REG3&#x3b1; expression and Paneth cell transcriptomes suggested that CD is associated with loss of regenerative Paneth cell populations and enrichment in REG3&#x3b1;-expressing populations, suggesting a mechanism through which changes in serum REG3&#x3b1; associate with complicated CD. CONCLUSION: Serum REG3&#x3b1; holds potential as a non-invasive, prognostic biomarker in CD, independent of disease activity. High serum REG3&#x3b1;, even years before diagnosis, is linked to a complicated disease course.

Crohn&#x2019;s Disease↗

Mapping the Immune cell-specific gene regulatory network in bipolar disorder: A framework from scTWMR to exploratory drug-target annotation.

BACKGROUND: Although the involvement of the immune system in the genetic susceptibility of bipolar disorder (BD) is widely acknowledged, the causal relationship between gene expression in specific immune cell subtypes and BD requires systematic elucidation. METHODS: We implemented an analytical framework integrating single-cell transcriptome-wide Mendelian randomization (scTWMR) with colocalization analysis. This approach utilized cis-expression quantitative trait loci (cis-eQTLs) derived from 14 distinct immune cell types as instrumental variables to interrogate BD genome-wide association study (GWAS) summary statistics (comprising 41,917 cases and 371,549 controls). Subsequent investigations encompassed functional enrichment analysis, protein-protein interaction (PPI) network construction, phenome-wide association study (PheWAS), and performed an exploratory drug-target annotation. RESULTS: Our analysis identified 33 gene-immune cell associations. Colocalization analysis provided robust evidence (PPH4 > 90%) for shared causal variants implicating the MAD1L1, APOM, and NFKBIL1 loci. Significantly enriched biological pathways included cell cycle regulation, circadian rhythm entrainment, and neuroinflammation. The PPI network revealed a core regulatory module centered on histone-encoding and immune-related genes. Exploratory drug-target annotation nominated compounds for further investigation for compounds targeting APOM, TMEM258, and NFKBIL1. CONCLUSION: This study systematically delineates a genetically supported regulatory network of immune cell-specific gene expression in BD, predominantly implicating CD8&#x207a; effector T cells, plasma cells, and B cells. The findings corroborate established pathological pathways while uncovering novel cell type-specific therapeutic targets, thereby providing a genetic framework for prioritizing candidate targets for future investigation.

Bipolar disorder↗

UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN.

The emergence of SARS-CoV-2 has posed significant threats to global health, particularly for the older population. Similarly, common human coronaviruses, such as HCoV-229E, which typically cause mild cold-like symptoms, can lead to severe diseases, underscoring the need to understand virus-host interactions and identify host factors contributing to viral pathogenesis and disease progression. In this study, we perform a genome-wide CRISPR knockout screen using HCoV-229E and identify UHRF1 as a potent restriction factor. Mechanistically, UHRF1 suppresses HCoV-229E infection by downregulating the expression of its cell entry receptor, APN, through promoter hypermethylation. Focused CRISPR activation screens of UHRF1-downregulated genes confirm the critical role of APN in HCoV-229E infection and identify additional genes (e.g., SIGLEC1, PLAC8, and heparan sulfate biosynthesis genes) contributing to the restrictive functions of UHRF1. Transcriptomic and single-cell RNA sequencing analysis reveal that UHRF1 expression decreases with age, negatively correlating with increased APN expression. This age-related decline in UHRF1 is validated in primary alveolar macrophages from elderly individuals, which exhibit heightened susceptibility to HCoV-229E compared to those from younger individuals. Our findings highlight UHRF1 as a key age-related host defense factor against coronavirus and provide insights into the epigenetic regulation of viral entry receptors.

Animals↗

PSEUDO-RESPONSE REGULATOR 3b and transcription factor ABF3 modulate abscisic acid-dependent drought stress response in soybean.

The circadian system plays a pivotal role in facilitating the ability of crop plants to respond and adapt to fluctuations in their immediate environment effectively. Despite the increasing comprehension of PSEUDO-RESPONSE REGULATORs and their involvement in the regulation of diverse biological processes, including circadian rhythms, photoperiodic control of flowering, and responses to abiotic stress, the transcriptional networks associated with these factors in soybean (Glycine max (L.) Merr.) remain incompletely characterized. In this study, we provide empirical evidence highlighting the significance of GmPRR3b as a crucial mediator in regulating the circadian clock, drought stress response, and abscisic acid (ABA) signaling pathway in soybeans. A comprehensive analysis of DNA affinity purification sequencing and transcriptome data identified 795 putative target genes directly regulated by GmPRR3b. Among them, a total of 570 exhibited a significant correlation with the response to drought, and eight genes were involved in both the biosynthesis and signaling pathways of ABA. Notably, GmPRR3b played a pivotal role in the negative regulation of the drought response in soybeans by suppressing the expression of abscisic acid-responsive element-binding factor 3 (GmABF3). Additionally, the overexpression of GmABF3 exhibited an increased ability to tolerate drought conditions, and it also restored the hypersensitive phenotype of the GmPRR3b overexpressor. Consistently, studies on the manipulation of GmPRR3b gene expression and genome editing in plants revealed contrasting reactions to drought stress. The findings of our study collectively provide compelling evidence that emphasizes the significant contribution of the GmPRR3b-GmABF3 module in enhancing drought tolerance in soybean plants. Moreover, the transcriptional network of GmPRR3b provides valuable insights into the intricate interactions between this gene and the fundamental biological processes associated with plant adaptation to diverse environmental conditions.

Glycine max↗

A non-EST-based method for exon-skipping prediction.

It is estimated that between 35% and 74% of all human genes can undergo alternative splicing. Currently, the most efficient methods for large-scale detection of alternative splicing use expressed sequence tags (ESTs) or microarray analysis. As these methods merely sample the transcriptome, splice variants that do not appear in deeply sampled tissues have a low probability of being detected. We present a new method by which we can predict that an internal exon is skipped (namely whether it is a cassette-exon) merely based on its naked genomic sequence and on the sequence of its mouse ortholog. No other data, such as ESTs, are required for the prediction. Using our method, which was experimentally validated, we detected hundreds of novel splice variants that were not detectable using ESTs. We show that a substantial fraction of the splice variants in the human genome could not be identified through current human EST or cDNA data.

Alternative Splicing↗

Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis.

Reactive oxygen species (ROS) are key players in the regulation of plant development, stress responses, and programmed cell death. Previous studies indicated that depending on the type of ROS (hydrogen peroxide, superoxide, or singlet oxygen) or its subcellular production site (plastidic, cytosolic, peroxisomal, or apoplastic), a different physiological, biochemical, and molecular response is provoked. We used transcriptome data generated from ROS-related microarray experiments to assess the specificity of ROS-driven transcript expression. Data sets obtained by exogenous application of oxidative stress-causing agents (methyl viologen, Alternaria alternata toxin, 3-aminotriazole, and ozone) and from a mutant (fluorescent) and transgenic plants, in which the activity of an individual antioxidant enzyme was perturbed (catalase, cytosolic ascorbate peroxidase, and copper/zinc superoxide dismutase), were compared. In total, the abundance of nearly 26,000 transcripts of Arabidopsis (Arabidopsis thaliana) was monitored in response to different ROS. Overall, 8,056, 5,312, and 3,925 transcripts showed at least a 3-, 4-, or 5-fold change in expression, respectively. In addition to marker transcripts that were specifically regulated by hydrogen peroxide, superoxide, or singlet oxygen, several transcripts were identified as general oxidative stress response markers because their steady-state levels were at least 5-fold elevated in most experiments. We also assessed the expression characteristics of all annotated transcription factors and inferred new candidate regulatory transcripts that could be responsible for orchestrating the specific transcriptomic signatures triggered by different ROS. Our analysis provides a framework that will assist future efforts to address the impact of ROS signals within environmental stress conditions and elucidate the molecular mechanisms of the oxidative stress response in plants.

Arabidopsis↗

Serial analysis of gene expression reveals conserved links between protein kinase A, ribosome biogenesis, and phosphate metabolism in Ustilago maydis.

The switch from budding to filamentous growth is a key aspect of invasive growth and virulence for the fungal phytopathogen Ustilago maydis. The cyclic AMP (cAMP) signaling pathway regulates dimorphism in U. maydis, as demonstrated by the phenotypes of mutants with defects in protein kinase A (PKA). Specifically, a mutant lacking the regulatory subunit of PKA encoded by the ubc1 gene displays a multiple-budded phenotype and fails to incite disease symptoms, although proliferation does occur in the plant host. A mutant with a defect in a catalytic subunit of PKA, encoded by adr1, has a constitutively filamentous phenotype and is nonpathogenic. We employed serial analysis of gene expression to examine the transcriptomes of a wild-type strain and the ubc1 and adr1 mutants to further define the role of PKA in U. maydis. The mutants displayed changes in the transcript levels for genes encoding ribosomal proteins, genes regulated by the b mating-type proteins, and genes for metabolic functions. Importantly, the ubc1 mutant displayed elevated transcript levels for genes involved in phosphate acquisition and storage, thus revealing a connection between cAMP and phosphate metabolism. Further experimentation indicated a phosphate storage defect and elevated acid phosphatase activity for the ubc1 mutant. Elevated phosphate levels in culture media also enhanced the filamentous growth of wild-type cells in response to lipids, a finding consistent with PKA regulation of morphogenesis in U. maydis. Overall, these findings extend our understanding of cAMP signaling in U. maydis and reveal a link between phosphate metabolism and morphogenesis.

Acid Phosphatase↗

Kidney collecting duct acid-base "regulon".

Kidneys are essential for acid-base homeostasis, especially when organisms cope with changes in acid or base dietary intake. Because collecting ducts constitute the final site for regulating urine acid-base balance, we undertook to identify the gene network involved in acid-base transport and regulation in the mouse outer medullary collecting duct (OMCD). For this purpose, we combined kidney functional studies and quantitative analysis of gene expression in OMCDs, by transcriptome and candidate gene approaches, during metabolic acidosis. Furthermore, to better delineate the set of genes concerned with acid-base disturbance, the OMCD transcriptome of acidotic mice was compared with that of both normal mice and mice undergoing an adaptative response through potassium depletion. Metabolic acidosis, achieved through an NH4Cl-supplemented diet for 3 days, not only induced acid secretion but also stimulated the aldosterone and vasopressin systems and triggered cell proliferation. Accordingly, metabolic acidosis increased the expression of genes involved in acid-base transport, sodium transport, water transport, and cell proliferation. In particular, >25 transcripts encoding proteins involved in urine acidification (subunits of H-ATPase, kidney anion exchanger, chloride channel Clcka, carbonic anhydrase-2, aldolase) were co-regulated during acidosis. These transcripts, which cooperate to achieve a similar function and are co-regulated during acidosis, constitute a functional unit that we propose to call a "regulon".

Acid-Base Equilibrium↗

Meningioma transcript profiles reveal deregulated Notch signaling pathway.

Meningiomas constitute the second most common central nervous system tumor, and yet relatively little is known about the molecular events that are important for the pathogenesis and malignant progression of these tumors. We have used serial analysis of gene expression to compare the transcriptomes of nonneoplastic meninges and meningiomas of all malignancy grades. A novel finding from this screen is the induction of three components of the Notch signaling pathway: the transcription factor, hairy and enhancer of Split1 (HES1) and two members of the Groucho/transducin-like enhancer of Split family of corepressors, TLE2 and TLE3. TLE corepressors interact and modulate the activity of a wide range of transcriptional regulatory systems, one of which is HES1. We have shown that the transcript and protein levels of HES1, the Notch2 and Notch1 receptors and the Jagged1 ligand are induced in meningiomas of all grades, whereas induction of TLE2 and TLE3 occurs specifically in higher-grade meningiomas. Meningioma cell lines express components of the Notch signaling pathway and an inhibitor of this pathway suppresses meningioma cell survival. These results suggest that deregulated expression of the Notch pathway is a critical event in meningioma pathogenesis and that modulation of this and potentially other signaling pathways by TLE corepressors leads to a more malignant phenotype.

Basic Helix-Loop-Helix Proteins↗

IBAS: Interaction-bridged association studies discovering novel genes underlying complex traits.

Genetic contributions to complex traits are often mediated through coordinated gene-gene interaction networks, yet most existing association frameworks focus on marginal single-gene effects and overlook higher-order dependency structures. Direct modeling of interactions remains challenging due to combinatorial complexity and statistical instability. We introduce Interaction-Bridged Association Study (IBAS), a general framework that incorporates pathway-level interaction patterns into genotype-phenotype association analysis without explicitly enumerating interactions. IBAS leverages transcriptomic reference data to construct low-dimensional representations of pathway activity, which guide SNP-weighting and gene-level association testing within a kernel-based framework. In perturbation-based simulations, IBAS demonstrates improved stability and reproducibility compared to conventional TWAS and gene-based methods, while maintaining well-calibrated Type I error under phenotype permutation. Application to the WTCCC datasets identifies both known and novel genes across multiple complex diseases, including candidates with modest marginal effects missed by standard approaches. These findings are supported by replication in an independent cohort, and analyses across multiple reference tissues revealing both shared and tissue-specific signals. Overall, IBAS provides a statistically robust and computationally tractable framework for incorporating interaction effects into association mapping, extending beyond the single-gene paradigm and enabling more comprehensive characterization of complex trait. IBAS is available on GitHub at: https://github.com/QingrunZhangLab/IBAS.

Polymorphism, Single Nucleotide↗

Genome-Wide Identification and Characterization of the TBL Gene Family and Temporal Expression Dynamics During Powdery Mildew Infection in Cucumber (Cucumis sativus).

Cell-wall polysaccharide O-acetylation contributes to cell-wall assembly, organ development, and plant-pathogen interactions, but the cucumber TBL gene family remains poorly characterized. Here, 37 CsTBL genes were identified genome-wide and analyzed using phylogenetic, syntenic, conserved-motif, gene-structure, promoter, protein-structure, Gene Ontology, and transcriptome approaches, followed by RT-qPCR analysis after powdery mildew inoculation. All CsTBL proteins contained the conserved GDS and DxxH motifs, whereas accessory motifs and predicted structural features varied among clades. Intraspecific analysis identified dispersed, WGD/segmental, and tandem duplication categories, and cross-species synteny was more extensive with melon than with Arabidopsis. Homology-derived annotations associated CsTBL genes with cell-wall polysaccharide metabolism, Golgi/endomembrane compartments, and O-acetyltransferase activity, including six genes assigned to xylan O-acetyltransferase-related annotations. Expression profiling revealed tissue- and developmental-stage-dependent patterns, whereas the publicly available powdery mildew RNA-seq dataset provided descriptive temporal expression profiles in Podosphaera xanthii-inoculated samples. Independent RT-qPCR analysis using time-matched mock controls revealed distinct post-inoculation responses among six selected genes. Relative to the corresponding mock controls, CsTBL2 was consistently repressed; CsTBL15 showed transient induction at 1 dpi followed by repression; CsTBL24 exhibited a biphasic response; CsTBL25 was induced at all sampled post-inoculation time points; CsTBL26 showed progressive induction; and CsTBL30 reached its highest observed expression level at 3 dpi. Integrated functional annotation and expression evidence highlighted CsTBL26 as a priority candidate for further functional characterization, while CsTBL24 and CsTBL25 represented fruit-associated candidates with distinct powdery mildew responses; CsTBL30 remained an additional strongly infection-responsive candidate. These findings provide an evolutionary and expression-based framework for the functional characterization of the cucumber TBL gene family.

O-acetylation↗

Lotus japonicus metabolic profiling. Development of gas chromatography-mass spectrometry resources for the study of plant-microbe interactions.

Symbiotic nitrogen fixation (SNF) in legume root nodules requires differentiation and integration of both plant and bacterial metabolism. Classical approaches of biochemistry, molecular biology, and genetics have revealed many aspects of primary metabolism in legume nodules that underpin SNF. Functional genomics approaches, especially transcriptomics and proteomics, are beginning to provide a more holistic picture of the metabolic potential of nodules in model legumes like Medicago truncatula and Lotus japonicus. To extend these approaches, we have established protocols for nonbiased measurement and analysis of hundreds of metabolites from L. japonicus, using gas chromatography coupled with mass spectrometry. Following creation of mass spectral tag libraries, which represent both known and unknown metabolites, we measured and compared relative metabolite levels in nodules, roots, leaves, and flowers of symbiotic plants. Principal component analysis of the data revealed distinct metabolic phenotypes for the different organs and led to the identification of marker metabolites for each. Metabolites that were enriched in nodules included: octadecanoic acid, asparagine, glutamate, homoserine, cysteine, putrescine, mannitol, threonic acid, gluconic acid, glyceric acid-3-P, and glycerol-3-P. Hierarchical cluster analysis enabled discrimination of 10 groups of metabolites, based on distribution patterns in diverse Lotus organs. The resources and tools described here, together with ongoing efforts in the areas of genome sequencing, and transcriptome and proteome analysis of L. japonicus and Mesorhizobium loti, should lead to a better understanding of nodule metabolism that underpins SNF.

Gas Chromatography-Mass Spectrometry↗