Metabolomic profiling of wild-type and mutant soybean root nodules using laser-ablation electrospray ionization mass spectrometry reveals altered metabolism.

The establishment of the nitrogen-fixing symbiosis between soybean and Bradyrhizobium japonicum is a complex process. To document the changes in plant metabolism as a result of symbiosis, we utilized laser ablation electrospray ionization-mass spectrometry (LAESI-MS) for in situ metabolic profiling of wild-type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl-acyl carrier protein desaturase (sacpd-c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix+ ) and ineffective (nifH mutant, fix- ) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd-c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI-MS for high-throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.

High-Throughput Analysis of Tissue-Embedded Single Cells by Mass Spectrometry with Bimodal Imaging and Object Recognition.

In biological tissues, cell-to-cell variations stem from the stochastic and modulated expression of genes and the varying abundances of corresponding proteins. These variations are then propagated to downstream metabolite products and result in cellular heterogeneity. Mass spectrometry imaging (MSI) is a promising tool to simultaneously provide spatial distributions for hundreds of biomolecules without the need for labels or stains. Technological advances in MSI instrumentation for the direct analysis of tissue-embedded single cells are dominated by improvements in sensitivity, sample pretreatment, and increased spatial resolution but are limited by low throughput. Herein, we introduce a bimodal microscopy imaging system combined with fiber-based laser ablation electrospray ionization (f-LAESI) MSI with improved throughput ambient analysis of tissue-embedded single cells (n > 1000) to provide insight into cellular heterogeneity. Based on automated image analysis, accurate single-cell sampling is achieved by f-LAESI leading to the discovery of cellular phenotypes characterized by differing metabolite levels.

In-Situ Metabolomic Analysis of Setaria viridis Roots Colonized by Beneficial Endophytic Bacteria.

Over the past decades, crop yields have risen in parallel with increasing use of fossil fuel-derived nitrogen (N) fertilizers but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic, and endophytic plant growth-promoting bacteria (PGPB) are known to stimulate plant growth. However, compared with the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae SmR1 (fix+) or a fix- mutant strain (SmR54) compared with uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole-root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized in-situ laser ablation electrospray ionization mass spectrometry to sample only those root segments at or adjacent to the sites of bacterial colonization. Metabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants, while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix- strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix- strain, perhaps reflecting a plant defense response.

Single-Cell Metabolic Profiling: Metabolite Formulas from Isotopic Fine Structures in Heterogeneous Plant Cell Populations.

Characterization of the metabolic heterogeneity in cell populations requires the analysis of single cells. Most current methods in single-cell analysis rely on cell manipulation, potentially altering the abundance of metabolites in individual cells. A small sample volume and the chemical diversity of metabolites are additional challenges in single-cell metabolomics. Here, we describe the combination of fiber-based laser ablation electrospray ionization (f-LAESI) with 21 T Fourier transform ion cyclotron resonance mass spectrometry (21TFTICR-MS) for in situ single-cell metabolic profiling in plant tissue. Single plant cells infected by bacteria were selected and sampled directly from the tissue without cell manipulation through mid-infrared ablation with a fine optical fiber tip for ionization by f-LAESI. Ultrahigh performance 21T-FTICR-MS enabled the simultaneous capture of isotopic fine structures (IFSs) for 47 known and 11 unknown compounds, thus elucidating their elemental compositions from single cells and providing information on metabolic heterogeneity in the cell population.

Ambient Metabolic Profiling and Imaging of Biological Samples with Ultrahigh Molecular Resolution Using Laser Ablation Electrospray Ionization 21 Tesla FTICR Mass Spectrometry.

Mass spectrometry (MS) is an indispensable analytical tool to capture the array of metabolites within complex biological systems. However, conventional MS-based metabolomic workflows require extensive sample processing and separation resulting in limited throughput and potential alteration of the native molecular states in these systems. Ambient ionization methods, capable of sampling directly from tissues, circumvent some of these issues but require high-performance MS to resolve the molecular complexity within these samples. Here, we demonstrate a unique combination of laser ablation electrospray ionization (LAESI) coupled with a 21 tesla Fourier transform ion cyclotron resonance (21T-FTICR) for direct MS analysis and imaging applications. This analytical platform provides isotopic fine structure information directly from biological tissues, enabling the rapid assignment of molecular formulas and delivering a higher degree of confidence for molecular identification.

Laser-ablation electrospray ionization mass spectrometry with ion mobility separation reveals metabolites in the symbiotic interactions of soybean roots and rhizobia.

Technologies enabling in situ metabolic profiling of living plant systems are invaluable for understanding physiological processes and could be used for rapid phenotypic screening (e.g., to produce plants with superior biological nitrogen-fixing ability). The symbiotic interaction between legumes and nitrogen-fixing soil bacteria results in a specialized plant organ (i.e., root nodule) where the exchange of nutrients between host and endosymbiont occurs. Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is a method that can be performed under ambient conditions requiring minimal sample preparation. Here, we employed LAESI-MS to explore the well characterized symbiosis between soybean (Glycine max L. Merr.) and its compatible symbiont, Bradyrhizobium japonicum. The utilization of ion mobility separation (IMS) improved the molecular coverage, selectivity, and identification of the detected biomolecules. Specifically, incorporation of IMS resulted in an increase of 153 differentially abundant spectral features in the nodule samples. The data presented demonstrate the advantages of using LAESI-IMS-MS for the rapid analysis of intact root nodules, uninfected root segments, and free-living rhizobia. Untargeted pathway analysis revealed several metabolic processes within the nodule (e.g., zeatin, riboflavin, and purine synthesis). Compounds specific to the uninfected root and bacteria were also detected. Lastly, we performed depth profiling of intact nodules to reveal the location of metabolites to the cortex and inside the infected region, and lateral profiling of sectioned nodules confirmed these molecular distributions. Our results established the feasibility of LAESI-IMS-MS for the analysis and spatial mapping of plant tissues, with its specific demonstration to improve our understanding of the soybean-rhizobial symbiosis.

Metabolic Noise and Distinct Subpopulations Observed by Single Cell LAESI Mass Spectrometry of Plant Cells in situ.

Phenotypic variations and stochastic expression of transcripts, proteins, and metabolites in biological tissues lead to cellular heterogeneity. As a result, distinct cellular subpopulations emerge. They are characterized by different metabolite expression levels and by associated metabolic noise distributions. To capture these biological variations unperturbed, highly sensitive in situ analytical techniques are needed that can sample tissue embedded single cells with minimum sample preparation. Optical fiber-based laser ablation electrospray ionization mass spectrometry (f-LAESI-MS) is a promising tool for metabolic profiling of single cells under ambient conditions. Integration of this MS-based platform with fluorescence and brightfield microscopy provides the ability to target single cells of specific type and allows for the selection of rare cells, e.g., excretory idioblasts. Analysis of individual Egeria densa leaf blade cells (n = 103) by f-LAESI-MS revealed significant differences between the prespecified subpopulations of epidermal cells (n = 97) and excretory idioblasts (n = 6) that otherwise would have been masked by the population average. Primary metabolites, e.g., malate, aspartate, and ascorbate, as well as several glucosides were detected in higher abundance in the epidermal cells. The idioblasts contained lipids, e.g., PG(16:0/18:2), and triterpene saponins, e.g., medicoside I and azukisaponin I, and their isomers. Metabolic noise for the epidermal cells were compared to results for soybean (Glycine max) root nodule cells (n = 60) infected by rhizobia (Bradyrhizobium japonicum). Whereas some primary metabolites showed lower noise in the latter, both cell types exhibited higher noise for secondary metabolites. Post hoc grouping of epidermal and root nodule cells, based on the abundance distributions for certain metabolites (e.g., malate), enabled the discovery of cellular subpopulations characterized by different mean abundance values, and the magnitudes of the corresponding metabolic noise. Comparison of prespecified populations from epidermal cells of the closely related E. densa (n = 20) and Elodea canadensis (n = 20) revealed significant differences, e.g., higher sugar content in the former and higher levels of ascorbate in the latter, and the presence of species-specific metabolites. These results demonstrate that the f-LAESI-MS single cell analysis platform has the potential to explore cellular heterogeneity and metabolic noise for hundreds of tissue-embedded cells.

Observed metabolic asymmetry within soybean root nodules reflects unexpected complexity in rhizobacteria-legume metabolite exchange.

In this study, the three-dimensional spatial distributions of a number of metabolites involved in regulating symbiosis and biological nitrogen fixation (BNF) within soybean root nodules were revealed using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While many metabolites exhibited distinct spatial compartmentalization, some metabolites were asymmetrically distributed throughout the nodule (e.g., S-adenosylmethionine). These results establish a more complex metabolic view of plant-bacteria symbiosis (and BNF) within soybean nodules than previously hypothesized. Collectively these findings suggest that spatial perspectives in metabolic regulation should be considered to unravel the overall complexity of interacting organisms, like those relating to associations of nitrogen-fixing bacteria with host plants.