Phylogeny and symbiotic effectiveness of indigenous rhizobial microsymbionts of common bean (Phaseolus vulgaris L.) in Malkerns, Eswatini.

In most legumes, the rhizobial symbionts exhibit diversity across different environments. Although common bean (Phaseolus vulgaris L.) is one of the important legumes in southern Africa, there is no available information on the genetic diversity and N2-fixing effectiveness of its symbionts in Malkerns, Eswatini. In this study, we assessed the phylogenetic positions of rhizobial microsymbionts of common bean from Malkerns in Eswatini. The isolates obtained showed differences in morpho-physiology and N2-fixing efficiency. A dendrogram constructed from the ERIC-PCR banding patterns, grouped a total of 88 tested isolates into 80 ERIC-PCR types if considered at a 70% similarity cut-off point. Multilocus sequence analysis using 16S rRNA, rpoB, dnaK, gyrB, and glnII and symbiotic (nifH and nodC) gene sequences closely aligned the test isolates to the type strains of Rhizobium muluonense, R. paranaense, R. pusense, R. phaseoli and R. etli. Subjecting the isolates in this study to further description can potentially reveal novel species. Most of the isolates tested were efficient in fixing nitrogen and elicited greater stomatal conductance and photosynthetic rates in the common bean. Relative effectiveness (RE) varied from 18 to 433%, with 75 (85%) out of the 88 tested isolates being more effective than the nitrate fed control plants.

Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules.

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization-mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization-mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

Metabolic reprogramming in nodules, roots, and leaves of symbiotic soybean in response to iron deficiency.

To elucidate the mechanism of adaptation of leguminous plants to iron (Fe)-deficient environment, comprehensive analyses of soybean (Glycine max) plants (sampled at anthesis) were conducted under Fe-sufficient control and Fe-deficient treatment using metabolomic and physiological approach. Our results show that soybeans grown under Fe-deficient conditions showed lower nitrogen (N) fixation efficiency; however, ureides increased in different tissues, indicating potential N-feedback inhibition. N assimilation was inhibited as observed in the repressed amino acids biosynthesis and reduced proteins in roots and nodules. In Fe-deficient leaves, many amino acids increased, accompanied by the reduction of malate, fumarate, succinate, and α-ketoglutarate, which implies the N reprogramming was stimulated by the anaplerotic pathway. Accordingly, many organic acids increased in roots and nodules; however, enzymes involved in the related metabolic pathway (e.g., Krebs cycle) showed opposite activity between roots and nodules, indicative of different mechanisms. Sugars increased or maintained at constant level in different tissues under Fe deficiency, which probably relates to oxidative stress, cell wall damage, and feedback regulation. Increased ascorbate, nicotinate, raffinose, galactinol, and proline in different tissues possibly helped resist the oxidative stress induced by Fe deficiency. Overall, Fe deficiency induced the coordinated metabolic reprogramming in different tissues of symbiotic soybean plants.

Antitumor activity and ability to prevent acrylamide formation in fried foods of asparaginase from soybean root nodules.

A novel asparaginase (designated srnASNase) has been purified from soybean root nodules and identified by MALDI-TOF/TOF-MS. And the enzymatic properties, antitumor activity and the ability to prevent acrylamide formation in fried foods of srnASNase were evaluated. SrnASNase had high specific activity (531.37 U/mg) toward L-asparagine under optimum conditions (pH 8.0 and 40°C), no activity toward L-glutamine and D-glutamine, but trace activity toward D-asparagine. It was stable in the pH range of 7.0-9.0 and up to 40°C. The Km and Vmax of srnASNase were 0.36 mM and 51.64 mM/min, respectively. Further, in vitro anti-proliferative activity on human cancer cells assay showed that srnASNase was superior to commercial asparaginase in solution by controlling the tumor cell growth with time. In addition, srnASNase showed more effective acrylamide mitigation than commercial asparaginase in fried foods. These results indicate that srnASNase is a potential candidate for applications in the food processing and pharmaceutical industry. PRACTICAL APPLICATIONS: L-asparaginase (L-asparagine amidohydrolase; EC 3.5.1.1) is an enzyme that catalyzes the hydrolysis of the amide group of the side-chain of L-asparagine into aspartic acid and ammonia. It has long been used as a primary component in the treatment of acute lymphoblastic leukemia (All) and other related blood cancers. Apart from its clinical usage, L-asparaginase has attracted more attention in the food processing industries as a promising acrylamide-mitigating agent in recent years. This research revealed that soybean root nodules might be good sources of novel asparaginase.

Biochemical and Anatomical Investigation of Sesbania herbacea (Mill.) McVaugh Nodules Grown under Flooded and Non-Flooded Conditions.

Sesbania herbacea, a native North American fast-growing legume, thrives in wet and waterlogged conditions. This legume enters into symbiotic association with rhizobia, resulting in the formation of nitrogen-fixing nodules on the roots. A flooding-induced anaerobic environment imposes a challenge for the survival of rhizobia and negatively impacts nodulation. Very little information is available on how S. herbacea is able to thrive and efficiently fix N2 in flooded conditions. In this study, we found that Sesbania plants grown under flooded conditions were significantly taller, produced more biomass, and formed more nodules when compared to plants grown on dry land. Transmission electron microscopy of Sesbania nodules revealed bacteroids from flooded nodules contained prominent polyhydroxybutyrate crystals, which were absent in non-flooded nodules. Gas and ion chromatography mass spectrometry analysis of nodule metabolites revealed a marked decrease in asparagine and an increase in the levels of gamma aminobutyric acid in flooded nodules. 2-D gel electrophoresis of nodule bacteroid proteins revealed flooding-induced changes in their protein profiles. Several of the bacteroid proteins that were prominent in flooded nodules were identified by mass spectrometry to be members of the ABC transporter family. The activities of several key enzymes involved in nitrogen metabolism was altered in Sesbania flooded nodules. Aspartate aminotransferase (AspAT), an enzyme with a vital role in the assimilation of reduced nitrogen, was dramatically elevated in flooded nodules. The results of our study highlight the potential of S. herbacea as a green manure and sheds light on the morphological, structural, and biochemical adaptations that enable S. herbacea to thrive and efficiently fix N2 in flooded conditions.

Impact of overexpression of cytosolic isoform of O-acetylserine sulfhydrylase on soybean nodulation and nodule metabolome.

Nitrogen-fixing nodules, which are also major sites of sulfur assimilation, contribute significantly to the sulfur needs of whole soybean plants. Nodules are the predominant sites for cysteine accumulation and the activity of O-acetylserine(thiol)lyase (OASS) is central to the sulfur assimilation process in plants. Here, we examined the impact of overexpressing OASS on soybean nodulation and nodule metabolome. Overexpression of OASS did not affect the nodule number, but negatively impacted plant growth. HPLC measurement of antioxidant metabolites demonstrated that levels of cysteine, glutathione, and homoglutathione nearly doubled in OASS overexpressing nodules when compared to control nodules. Metabolite profiling by LC-MS and GC-MS demonstrated that several metabolites related to serine, aspartate, glutamate, and branched-chain amino acid pathways were significantly elevated in OASS overexpressing nodules. Striking differences were also observed in the flavonoid levels between the OASS overexpressing and control soybean nodules. Our results suggest that OASS overexpressing plants compensate for the increase in carbon requirement for sulfur assimilation by reducing the biosynthesis of some amino acids, and by replenishing the TCA cycle through fatty acid hydrolysis. These data may indicate that in OASS overexpressing soybean nodules there is a moderate decease in the supply of energy metabolites to the nodule, which is then compensated by the degradation of cellular components to meet the needs of the nodule energy metabolism.

Nodule-Specific Cysteine-Rich Peptides Negatively Regulate Nitrogen-Fixing Symbiosis in a Strain-Specific Manner in Medicago truncatula.

Medicago truncatula shows a high level of specificity when interacting with its symbiotic partner Sinorhizobium meliloti. This specificity is mainly manifested at the nitrogen-fixing stage of nodule development, such that a particular bacterial strain forms nitrogen-fixing nodules (Nod+/Fix+) on one plant genotype but ineffective nodules (Nod+/Fix-) on another. Recent studies have just begun to reveal the underlying molecular mechanisms that control this specificity. The S. meliloti strain A145 induces the formation of Fix+ nodules on the accession DZA315.16 but Fix- nodules on Jemalong A17. A previous study reported that the formation of Fix- nodules on Jemalong A17 by S. meliloti A145 was conditioned by a single recessive allele named Mtsym6. Here we demonstrate that the specificity associated with S. meliloti A145 is controlled by multiple genes in M. truncatula, including NFS1 and NFS2 that encode nodule-specific cysteine-rich (NCR) peptides. The two NCR peptides acted dominantly to block rather than promote nitrogen fixation by S. meliloti A145. These two NCR peptides are the same ones that negatively regulate nitrogen-fixing symbiosis associated with S. meliloti Rm41.

Associative nitrogen fixation in nodules of the conifer Lepidothamnus fonkii (Podocarpaceae) inhabiting ombrotrophic bogs in southern Patagonia.

Biological N2 fixation (BNF) in the rhizosphere of Podocarpaceae is currently attributed to unspecific diazotrophs with negligible impact on N acquisition. Here, we report specific and high associative BNF in dead cells of root nodules of Lepidothamnus fonkii distributed in ombrotrophic peatlands of Patagonia. BNF of nodulated roots, intact plants of L. fonkii and rhizospheric peat was assessed by 15N2 and acetylene reduction. Diazotrophs were identified by electron microscopy, analysis of nitrogenase encoding genes (nifH) and transcripts, and 16S rRNA. Nitrogenase encoding nifH transcripts from root nodules point to Beijerinckiaceae (Rhizobiales), known as free-living diazotrophs. Electron microscopy and 16S rRNA analysis likewise identified active Beijerinckiaceae in outer dead cells of root nodules. NifH transcripts from the rhizopshere peat revealed diverse active diazotrophs including Beijerinckiaceae. Both methods revealed high activity of nitrogenase rates in cut roots of L. fonkii (2.5 µmol N g-1 d.w. d-1 based on 15N2 assay; 2.4 µmol C2H4 g-1 d.w. d-1 based on acetylene reduction assay). The data suggest that (i) nodules recruit diazotrophic Beijerinckiaceae from peat, (ii) dead nodule cells provide an exclusive habitat for Beijerinckiaceae, and (iii) BNF in L. fonkii is one potent pathway to overcome N deficiency in ombrotrophic peatlands of Patagonia.

Metabolomic Profiling of Bradyrhizobium diazoefficiens-Induced Root Nodules Reveals Both Host Plant-Specific and Developmental Signatures.

Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection-time-of-flight mass spectrometry analysis the metabolome of (i) nodules and roots from four different B. diazoefficiens host plants; (ii) soybean nodules harvested at different time points during nodule development; and (iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by independent transcriptomics data. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.

Induction of nodD Gene in a Betarhizobium Isolate, Cupriavidus sp. of Mimosa pudica, by Root Nodule Phenolic Acids.

A range of phenolic acids, viz., p-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, caffeic acid, ferulic acid, and cinnamic acid have been isolated and identified by LC-MS analysis in the roots and root nodules of Mimosa pudica. The effects of identified phenolic acids on the regulation of nodulation (nod) genes have been evaluated in a betarhizobium isolate of M. pudica root nodule. Protocatechuic acid and p-hydroxybenzoic acid were most effective in inducing nod gene, whereas caffeic acid had no significant effect. Phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activities were estimated, indicating regulation and metabolism of phenolic acids in root nodules. These results showed that nodD gene expression of betarhizobium is regulated by simple phenolic acids such as protocatechuic acid and p-hydroxybenzoic acid present in host root nodule and sustains nodule organogenesis.

Phosphorylation of Leghemoglobin at S45 is Most Effective to Disrupt the Molecular Environment of Its Oxygen Binding Pocket.

In leguminous plants, nitrogenase that catalyzes anaerobic symbiotic nitrogen fixation is protected by the sequestration of O2 by Leghemoglobin (LegH). The modulation of the oxygen binding capacity of Hemoglobin (Hb) by different post-translational modifications is well studied; whereas similar studies on LegH's O2 binding are not yet benchmarked. Our results show that in vitro serine phosphorylation of recombinant LegH from Lotus japonicus, a model legume, by a homologous kinase caused a reduction in its oxygen consumption as determined by Clark type electrode. Although mass spectrometry revealed a few phosphorylated serine residues in the LegH, molecular modeling study showed that particularly S45 is the most critical one, along with S55, however the latter with lesser impact on its molecular environment responsible for oxygen consumption. Separate S45D and S55D mutants of recombinant LegH also corroborated the results obtained from molecular modeling study. Thus, this work lays groundwork for further investigation of structural and functional role of serine phosphorylation as one of the mechanisms by which oxygen consumption by LegH may possibly be regulated during nodulation.

Proteomic analysis of the soybean symbiosome identifies new symbiotic proteins.

Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis.

Multifaceted investigation of metabolites during nitrogen fixation in Medicago via high resolution MALDI-MS imaging and ESI-MS.

Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Owing to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula-Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula-Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional versus nonfunctional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology.

Identification of effective Pb resistant bacteria isolated from Lens culinaris growing in lead contaminated soils.

Soil bacteria are a new phytoremediation system for the removal of heavy metals from soils. In this study, fifteen soil bacteria were isolated from root nodules of lentil growing in heavy metals contaminated soils, particularly by lead. Molecular characterization of the collection showed a large diversity, including Agrobacterium tumefaciens, Rahnella aquatilis, Pseudomonas, and Rhizobium sp. These soil bacteria had a wide range of tolerance to heavy metals. Among them, strains of A. tumefaciens and R. aquatilis tolerated up to 3.35 mM Pb; whereas Pseudomonas tolerated up to 3.24 mM Pb. The inoculation of lentil grown hydroponically with inoculums formed by these efficient and Pb resistant bacteria enhanced plant biomass. The treatment of this symbiosis by 1 mM Pb for 10 days or by 2 mM Pb for 3 days demonstrated that lentil had Pb accumulation capacity and can be considered a Pb accumulator plant, elsewhere, roots accumulated more Pb than shoots, and the inoculation decreased the Pb up take by the plants, suggesting that this symbiosis should be investigated for use in phytostabilization of Pb-contaminated soils. At the same time, a modulation in the antioxidant enzyme activity and a specific duration was required for the induction of the superoxide dismutase (SOD), peroxidase (POX), and ascorbate peroxidase (APX) response and to adapt to Pb stress. These results suggested that these enzymes may be involved in the main mechanism of antioxidative defense in lentil exposed to Pb oxidative stress.

Accumulation and localization of extensin protein in apoplast of pea root nodule under aluminum stress.

Cell wall components such as hydroxyproline-rich glycoproteins (HRGPs, extensins) have been proposed to be involved in aluminum (Al) resistance mechanisms in plants. We have characterized the distribution of extensin in pea (Pisum sativum L.) root nodules apoplast under short (for 2 and 24h) Al stress. Monoclonal antibodie LM1 have been used to locate extensin protein epitope by immunofluorescence and immunogold labeling. The nodules were shown to respond to Al stress by thickening of plant and infection thread (IT) walls and disturbances in threads growth and bacteria endocytosis. Immunoblot results indicated the presence of a 17-kDa band specific for LM1. Irrespective of the time of Al stress, extensin content increased in root nodules. Further observation utilizing fluorescence and transmission electron microscope showed that LM1 epitope was localized in walls and intercellular spaces of nodule cortex tissues and in the infection threads matrix. Al stress in nodules appears to be associated with higher extensin accumulation in matrix of enlarged thick-walled ITs. In addition to ITs, thickened walls and intercellular spaces of nodule cortex were also associated with intense extensin accumulation. These data suggest that Al-induced extensin accumulation in plant cell walls and ITs matrix may have influence on the process of IT growth and tissue and cell colonization by Rhizobium bacteria.

MALDI-mass spectrometric imaging for the investigation of metabolites in Medicago truncatula root nodules.

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied(1). Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples(1-5). This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

Phenolphthalein false-positive reactions from legume root nodules.

Presumptive tests for blood play a critical role in the examination of physical evidence and in the determination of subsequent analysis. The catalytic power of hemoglobin allows colorimetric reactions employing phenolphthalein (Kastle-Meyer test) to indicate "whether" blood is present. Consequently, DNA profiles extracted from phenolphthalein-positive stains are presumed to be from blood on the evidentiary item and can lead to the identification of "whose" blood is present. Crushed nodules from a variety of legumes yielded phenolphthalein false-positive reactions that were indistinguishable from true bloodstains both in color quality and in developmental time frame. Clothing and other materials stained by nodules also yielded phenolphthalein false-positive reactivity for several years after nodule exposure. Nodules from leguminous plants contain a protein (leghemoglobin) which is structurally and functionally similar to hemoglobin. Testing of purified leghemoglobin confirmed this protein as a source of phenolphthalein reactivity. A scenario is presented showing how the presence of leghemoglobin from nodule staining can mislead investigators.

Localization of hydrogen peroxide accumulation and diamine oxidase activity in pea root nodules under aluminum stress.

Aluminum (Al) is one of the environmental stressors that induces formation of reactive oxygen species (ROS) in plants. Hydrogen peroxide (H2O2) and H2O2-generated apoplast diamine oxidase (DAO) activity were detected cytochemically via transmission electron microscopy (TEM), in pea (Pisum sativum L.) root nodules exposed to high (50 µM AlCl3, for 2 and 24h) Al stress. The nodules were shown to respond to Al stress by disturbances in infection thread (IT) growth, bacteria endocytosis, premature degeneration of bacteroidal tissue and generation of H2O2 in nodule apoplast. Large amounts of peroxide were found at the same sites as high DAO activity under Al stress, suggesting that DAO is a major source of Al-induced peroxide accumulation in the nodules. Peroxide distribution and DAO activity in the nodules of both control plants and Al-treated ones were typically found in the plant cell walls, intercellular spaces and infection threads. However, 2 h Al treatment increased DAO activity and peroxide accumulation in the nodule apoplast and bacteria within threads. A prolonged Al treatment (24 h) increased the H2O2 content and DAO activity in the nodule apoplast, especially in the thread walls, matrix and bacteria within infection threads. In addition to ITs, prematurely degenerated bacteroids, which occurred in response to Al, were associated with intense staining for H2O2 and DAO activity. These results suggest the involvement of DAO in the production of a large amount of H2O2 in the nodule apoplast under Al stress. The role of reactive oxygen species in pea-Rhizobium symbiosis under Al stress is discussed.

Proteome reference maps of the Lotus japonicus nodule and root.

Legume symbiosis with rhizobia results in the formation of a specialized organ, the root nodule, where atmospheric dinitrogen is reduced to ammonia. In Lotus japonicus (Lotus), several genes involved in nodule development or nodule function have been defined using biochemistry, genetic approaches, and high-throughput transcriptomics. We have employed proteomics to further understand nodule development. Two developmental stages representing nodules prior to nitrogen fixation (white) and mature nitrogen fixing nodules (red) were compared with roots. In addition, the proteome of a spontaneous nodule formation mutant (snf1) was determined. From nodules and roots, 780 and 790 protein spots from 2D gels were identified and approximately 45% of the corresponding unique gene accessions were common. Including a previous proteomics set from Lotus pod and seed, the common gene accessions were decreased to 7%. Interestingly, an indication of more pronounced PTMs in nodules than in roots was determined. Between the two nodule developmental stages, higher levels of pathogen-related 10 proteins, HSPs, and proteins involved in redox processes were found in white nodules, suggesting a higher stress level at this developmental stage. In contrast, protein spots corresponding to nodulins such as leghemoglobin, asparagine synthetase, sucrose synthase, and glutamine synthetase were prevalent in red nodules. The distinct biochemical state of nodules was further highlighted by the conspicuous presence of several nitrilases, ascorbate metabolic enzymes, and putative rhizobial effectors.