Nodule Metabolism Research Articles

Metabolic Responses to Excess Manganese in Legumes: Variations in Nitrogen Compounds in Canavalia ensiformis (L.) DC and Cajanus cajan (L.) Millsp

ABSTRACTManganese (Mn) is crucial as a trace element for plant metabolism, but high concentrations in soil can induce symptoms of toxicity. This study aimed to evaluate the metabolism of nitrogen compounds and biomass production in Canavalia ensiformis and Cajanus cajan, clarifying the effects of this metal on nitrogen metabolism. The soil was treated with Mn concentrations of 80, 100, 120, 140, and 160 mg kg−1. Differential responses were observed in the metabolism of nitrogenous compounds between species. C. cajan affected nitrogen metabolism in shoots, roots, and nodules, with significant variations in amino acids, total soluble proteins, ureides, and root biomass concentration. In contrast, C. ensiformis showed stability in the concentrations of compounds, mainly ureides, and proteins, even with increasing doses of Mn in the soil. These findings highlight the importance of nitrogen metabolism in legumes studied as a key aspect for understanding their Mn tolerance mechanisms in soil.

Nitrogen inhibition of nitrogenase activity involves the modulation of cytosolic invertase in soybean nodule

Legume symbiotic nitrogen fixation (SNF) is suppressed by inorganic N in the soil. High N inhibition of nitrogenase activity is associated with the deprivation of carbon allocation and metabolism in nodules. However, the underlying molecular mechanisms remain unclear. Here, we identify GmCIN1 which encodes a cytosolic invertase, as a gateway for the N-tuning of sucrose utilization in nodules. GmCIN1 is enriched in mature soybean nodules and its expression is regulated by nitrogen status. The knockout of GmCIN1 using genome editing partially mimics the inhibitory effects of N on nitrogenase activity and sugar content and the impact of high N on nodule transcriptomes. This indicates that GmCIN1 partially mediates the high N inhibition of nodule activity. Moreover, ChIP-qPCR and EMSA reveal that SNAP1/2 transcription factors directly bind to the GmCIN1 promoter. In addition, SNAP1/2 may be involved in the repression of GmCIN1 expression in mature nodules at high N concentrations. Our findings provide insights into the involvement of the transcriptional tuning of C metabolism genes by N-signaling modulators in the N-induced inhibition of nitrogenase activity.

Mineral and Carbon Metabolic Adjustments in Nodules of Symbiotically Grown Faba Bean (Vicia faba L.) Varieties in Response to Organic Phosphorus Supplementation.

Phosphorus (P) is a major limiting factor for legume and symbiotic nitrogen fixation (SNF). Although overall adaptations of legumes to P supplementation have been extensively studied in connection with inorganic P, little information is currently available regarding nodulation or SNF responses to organic P (Po) in hydroponics. We investigated the mineral and carbon metabolism of Po-induced nodules of two contrasting faba bean varieties grown hydroponically under inorganic P (Pi), viz., in P-deficient (2 µM KH2PO4, -Pi), sufficient-P (200 µM KH2PO4, +Pi), and phytic acid (200 µM, Po) conditions, and were inoculated with Rhizobium leguminosarum bv. viciae 3841 and grown for 30 days. The results consistently reveal similar growth and biomass partitioning patterns between +Pi and Po, with both varying substantially from -Pi. In comparison, +Pi and Po observed equivalent accumulations of overall elemental P concentrations, with both increasing by 114 and 119%, respectively, relative to -Pi. A principal component analysis on metabolites showed a clear separation of the -Pi treatment from the others, with +Pi and Po correlating closely together, highlighting the nonsignificant differences between them. Additionally, the δ15N abundance of shoots, roots, and nodules was not significantly different between treatments and varieties and exhibited negative δ15N signatures for all tissues. Our study provides a novel perspective on mineral and carbon metabolism and their regulation of the growth, functioning, and reprogramming of nodules upon phytate supply.

A genome-scale metabolic reconstruction of soybean and Bradyrhizobium diazoefficiens reveals the cost-benefit of nitrogen fixation.

Nitrogen-fixing symbioses allow legumes to thrive in nitrogen-poor soils at the cost of diverting some photoassimilate to their microsymbionts. Effort is being made to bioengineer nitrogen fixation into nonleguminous crops. This requires a quantitative understanding of its energetic costs and the links between metabolic variations and symbiotic efficiency. A whole-plant metabolic model for soybean (Glycine max) with its associated microsymbiont Bradyrhizobium diazoefficiens was developed and applied to predict the cost-benefit of nitrogen fixation with varying soil nitrogen availability. The model predicted a nitrogen-fixation cost of c. 4.13 g C g-1 N, which when implemented into a crop scale model, translated to a grain yield reduction of 27% compared with a non-nodulating plant receiving its nitrogen from the soil. Considering the lower nitrogen content of cereals, the yield cost to a hypothetical N-fixing cereal is predicted to be less than half that of soybean. Soybean growth was predicted to be c. 5% greater when the nodule nitrogen export products were amides versus ureides. This is the first metabolic reconstruction in a tropical crop species that simulates the entire plant and nodule metabolism. Going forward, this model will serve as a tool to investigate carbon use efficiency and key mechanisms within N-fixing symbiosis in a tropical species forming determinate nodules.

Cow manure compost‐based products as alternative rhizobial carrier materials

AbstractCow manure compost (CMC) has been used widely as soil amendment to improve soil fertility and health, and to minimize environmental impacts. However, direct use of commercial CMC‐based products as alternative rhizobial carrier materials is rare, it could promote inoculant commercialization. This research aimed to explore the potential of four CMC‐based products (CMC powder, CMC granular, CMC + 50% gypsum, and CMC + 30% biochar) as a rhizobial carrier. Peat was used as a standard (control) material. All the carriers were directly mixed with Bradyrhizobium japonicum CB 1809 strain to prepare four CMC‐based inoculants. Shelf life of inoculants was evaluated up to 120 days of storage and survival rate in soil was determined after 30 days under both optimum moisture (55% WHC—water‐holding capacity) and drought stress (15% WHC) conditions. Results revealed that overall, CMC + 50% gypsum product showed better physicochemical characteristics such as WHC (126.9 ± 6.87%), Ca (134.15 ± 5.78 mg kg−1), and S (104.9 ± 6.52 mg kg−1) with the best shelf life during storage at 28°C and 35°C temperatures. The difference in shelf life between the CMC + 50% gypsum and other CMC‐based products became more apparent at higher temperature (35°C) and longer‐term storage (120 days). Additionally, when introduced into the soil, the CMC + 50% gypsum carrier outperformed the peat (control), showing the best strain survival rate of 95% (under optimum moisture) and 87% (under drought) after 30 days of storage. The strain population of CMC + 30% biochar as carrier was the lowest with values 8.66 Log 10 CFU g−1 and 7.52 Log 10 CFU g−1 in the shelf‐life test at both storage temperatures 28°C and 35°C, respectively, likely due to its alkaline pH. Multivariate analysis revealed that the carriers' WHC, Ca (involved in nodule formation and stability), and S (essential for nodule metabolism and nitrogenase activity) contents had a strong positive correlation with the long‐term shelf life and the survival rate in soil, confirming the importance of carrier materials' moisture retention and availability of specific elements (Ca, S). Hence, CMC + 50% gypsum showed the highest values of WHC, Ca and S, and optimum pH, reflecting its highest rhizobial population density and survival rate as compared to other carriers. This work has demonstrated the great potential of CMC‐based commercial products as alternative carrier materials, opening an alternative avenue for rapid commercial development of inoculants.

3-Pentanol glycosides from root nodules of the actinorhizal plant Alnus cremastogyne

Alnus cremastogyne Burkill (Betulaceae), an actinorhizal plant, can enter a mutualistic symbiosis with Frankia species that leads to the formation of nitrogen fixing root nodules. Some primary metabolites (carbohydrates, dicarboxylic acids, amino acids, citrulline and amides) involved in carbon and nitrogen metabolism in actinorhizal nodules have been identified, while specialized metabolites in A. cremastogyne root nodules are yet to be characterized. In this study, we isolated and identified three undescribed 3-pentanol glycosides, i.e., 3-pentyl α-l-arabinofuranosyl-(1′′→6′)-β-d-glucopyranoside, 3-pentyl α-l-rhamnopyranosyl-(1′′→6′)-β-d-glucopyranoside, and 3-pentyl 6′-(3-hydroxy3-methylglutaryl)-β-d-glucopyranoside, as well as seventeen known compounds from A. cremastogyne root nodules. 3-Pentanol glycosides are abundantly distributed in root nodules, while they are distributed in stems, roots, leaves and fruits at low/zero levels. A. cremastogyne plants treated by root nodule suspension emit 3-pentanol. This study enriches the knowledge about specialized metabolites in the actinorhizal host, and provides preliminarily information on the signal exchange in the actinorhizal symbiosis between A. cremastogyne and Frankia.

18F]FDG Uptake and Expression of Immunohistochemical Markers Related to Glycolysis, Hypoxia, and Proliferation in Indeterminate Thyroid Nodules

PurposeThe current study explored the association between 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) uptake and the quantitative expression of immunohistochemical markers related to glucose metabolism, hypoxia, and cell proliferation in benign and malignant thyroid nodules of indeterminate cytology.ProceduresUsing a case–control design, 24 patients were selected from participants of a randomized controlled multicenter trial (NCT02208544) in which [18F]FDG-PET/CT and thyroid surgery were performed for Bethesda III and IV nodules. Three equally sized groups of [18F]FDG-positive malignant, [18F]FDG-positive benign, and [18F]FDG-negative benign nodules were included. Immunohistochemical staining was performed for glucose transporters (GLUT) 1, 3, and 4; hexokinases (HK) 1 and 2; hypoxia-inducible factor-1 alpha (HIF1α; monocarboxylate transporter 4 (MCT4); carbonic anhydrase IX (CA-IX); vascular endothelial growth factor (VEGF); sodium-iodide symporter (NIS); and Ki-67. Marker expression was scored using an immunoreactive score. Unsupervised cluster analysis was performed. The immunoreactive score was correlated to the maximum and peak standardized uptake values (SUVmax, SUVpeak) and SUVmax ratio (SUVmax of nodule/background SUVmax of contralateral, normal thyroid) of the [18F]FDG-PET/CT using the Spearman’s rank correlation coefficient and compared between the three groups using Kruskal–Wallis tests.ResultsThe expression of GLUT1, GLUT3, HK2, and MCT4 was strongly positively correlated with the SUVmax, SUVpeak, and SUVmax ratio. The expression of GLUT1 (p = 0.009), HK2 (p = 0.02), MCT4 (p = 0.01), and VEGF (p = 0.007) was statistically significantly different between [18F]FDG-positive benign nodules, [18F]FDG-positive thyroid carcinomas, and [18F]FDG-negative benign nodules. In both [18F]FDG-positive benign nodules and [18F]FDG-positive thyroid carcinomas, the expression of GLUT1, HK2, and MCT4 was increased as compared to [18F]FDG-negative benign nodules. VEGF expression was higher in [18F]FDG-positive thyroid carcinomas as compared to [18F]FDG-negative and [18F]FDG-positive benign nodules.ConclusionsOur results suggest that [18F]FDG-positive benign thyroid nodules undergo changes in protein expression similar to those in thyroid carcinomas. To expand the understanding of the metabolic changes in benign and malignant thyroid nodules, further research is required, including correlation with underlying genetic alterations.

Malate Transport and Metabolism in Nitrogen-Fixing Legume Nodules.

Legumes form a symbiosis with rhizobia, a soil bacterium that allows them to access atmospheric nitrogen and deliver it to the plant for growth. Biological nitrogen fixation occurs in specialized organs, termed nodules, that develop on the legume root system and house nitrogen-fixing rhizobial bacteroids in organelle-like structures termed symbiosomes. The process is highly energetic and there is a large demand for carbon by the bacteroids. This carbon is supplied to the nodule as sucrose, which is broken down in nodule cells to organic acids, principally malate, that can then be assimilated by bacteroids. Sucrose may move through apoplastic and/or symplastic routes to the uninfected cells of the nodule or be directly metabolised at the site of import within the vascular parenchyma cells. Malate must be transported to the infected cells and then across the symbiosome membrane, where it is taken up by bacteroids through a well-characterized dct system. The dicarboxylate transporters on the infected cell and symbiosome membranes have been functionally characterized but remain unidentified. Proteomic and transcriptomic studies have revealed numerous candidates, but more work is required to characterize their function and localise the proteins in planta. GABA, which is present at high concentrations in nodules, may play a regulatory role, but this remains to be explored.

Dissecting Metabolism of Leaf Nodules in Ardisia crenata and Psychotria punctata

Root-microbe interaction and its specialized root nodule structures and functions are well studied. In contrast, leaf nodules harboring microbial endophytes in special glandular leaf structures have only recently gained increased interest as plant-microbe phyllosphere interactions. Here, we applied a comprehensive metabolomics platform in combination with natural product isolation and characterization to dissect leaf and leaf nodule metabolism and functions in Ardisia crenata (Primulaceae) and Psychotria punctata (Rubiaceae). The results indicate that abiotic stress resilience plays an important part within the leaf nodule symbiosis of both species. Both species showed metabolic signatures of enhanced nitrogen assimilation/dissimilation pattern and increased polyamine levels in nodules compared to leaf lamina tissue potentially involved in senescence processes and photosynthesis. Multiple links to cytokinin and REDOX-active pathways were found. Our results further demonstrate that secondary metabolite production by endophytes is a key feature of this symbiotic system. Multiple anhydromuropeptides (AhMP) and their derivatives were identified as highly characteristic biomarkers for nodulation within both species. A novel epicatechin derivative was structurally elucidated with NMR and shown to be enriched within the leaf nodules of A. crenata. This enrichment within nodulated tissues was also observed for catechin and other flavonoids indicating that flavonoid metabolism may play an important role for leaf nodule symbiosis of A. crenata. In contrast, pavettamine was only detected in P. punctata and showed no nodule specific enrichment but a developmental effect. Further natural products were detected, including three putative unknown depsipeptide structures in A. crenata leaf nodules. The analysis presents a first metabolomics reference data set for the intimate interaction of microbes and plants in leaf nodules, reveals novel metabolic processes of plant-microbe interaction as well as the potential of natural product discovery in these systems.

Nitrogen and Phosphorus Interplay in Lupin Root Nodules and Cluster Roots.

Nitrogen (N) and phosphorus (P) are two major plant nutrients, and their deficiencies often limit plant growth and crop yield. The uptakes of N or P affect each other, and consequently, understanding N–P interactions is fundamental. Their signaling mechanisms have been studied mostly separately, and integrating N–P interactive regulation is becoming the aim of some recent works. Lupins are singular plants, as, under N and P deficiencies, they are capable to develop new organs, the N2-fixing symbiotic nodules, and some species can also transform their root architecture to form cluster roots, hundreds of short rootlets that alter their metabolism to induce a high-affinity P transport system and enhance synthesis and secretion of organic acids, flavonoids, proteases, acid phosphatases, and proton efflux. These modifications lead to mobilization in the soil of, otherwise unavailable, P. White lupin (Lupinus albus) represents a model plant to study cluster roots and for understanding plant acclimation to nutrient deficiency. It tolerates simultaneous P and N deficiencies and also enhances uptake of additional nutrients. Here, we present the structural and functional modifications that occur in conditions of P and N deficiencies and lead to the organogenesis and altered metabolism of nodules and cluster roots. Some known N and P signaling mechanisms include different factors, including phytohormones and miRNAs. The combination of the individual N and P mechanisms uncovers interactive regulation pathways that concur in nodules and cluster roots. L. albus interlinks N and P recycling processes both in the plant itself and in nature.

The influence of ethylene, gibberellins and brassinosteroids on energy and nitrogen-fixation metabolites in nodule tissue

Legume nodules are a unique plant organ that contain nitrogen-fixing rhizobial bacteria. For this interaction to be mutually beneficial, plant and bacterial metabolism must be precisely co-ordinated. Plant hormones are known to play essential roles during the establishment of legume-rhizobial symbioses but their role in subsequent nodule metabolism has not been explored in any depth. The plant hormones brassinosteroids, ethylene and gibberellins influence legume infection, nodule number and in some cases nodule function. In this paper, the influence of these hormones on nodule metabolism was examined in a series of well characterised pea mutants with altered hormone biosynthesis or response. A targeted set of metabolites involved in nutrient exchange and nitrogen fixation was examined in nodule tissue of mutant and wild type plants. Gibberellin-deficiency had a major negative impact on the level of several major dicarboxylates supplied to rhizobia by the plant and also led to a significant deficit in the amino acids involved in glutamine-aspartate transamination, consistent with the limited bacteroid development and low fixation rate of gibberellin-deficient na mutant nodules. In contrast, no major effects of brassinosteroid-deficiency or ethylene-insensitivity on the key metabolites in these pathways were found. Therefore, although all three hormones influence infection and nodule number, only gibberellin is important for the establishment of a functional nodule metabolome.

Soybean Yellow Stripe-like 7 is a symbiosome membrane peptide transporter important for nitrogen fixation.

Legumes form a symbiosis with rhizobia that convert atmospheric nitrogen (N2) to ammonia and provide it to the plant in return for a carbon and nutrient supply. Nodules, developed as part of the symbiosis, harbor rhizobia that are enclosed in a plant-derived symbiosome membrane (SM) to form an organelle-like structure called the symbiosome. In mature nodules exchanges between the symbionts occur across the SM. Here we characterize Yellow Stripe-like 7 (GmYSL7), a Yellow stripe-like family member localized on the SM in soybean (Glycine max) nodules. It is expressed specifically in infected cells with expression peaking soon after nitrogenase becomes active. Unlike most YSL family members, GmYSL7 does not transport metals complexed with phytosiderophores. Rather, it transports oligopeptides of between four and 12 amino acids. Silencing GmYSL7 reduces nitrogenase activity and blocks infected cell development so that symbiosomes contain only a single bacteroid. This indicates the substrate of YSL7 is required for proper nodule development, either by promoting symbiosome development directly or by preventing inhibition of development by the plant. RNAseq of nodules where GmYSL7 was silenced suggests that the plant initiates a defense response against rhizobia with genes encoding proteins involved in amino acid export downregulated and some transcripts associated with metal homeostasis altered. These changes may result from the decrease in nitrogen fixation upon GmYSL7 silencing and suggest that the peptide(s) transported by GmYSL7 monitor the functional state of the bacteroids and regulate nodule metabolism and transport processes accordingly. Further work to identify the physiological substrate for GmYSL7 will allow clarification of this role.

A Novel OmpR-Type Response Regulator Controls Multiple Stages of the Rhizobium etli - Phaseolus vulgaris N2-Fixing Symbiosis.

OmpR, is one of the best characterized response regulators families, which includes transcriptional regulators with a variety of physiological roles including the control of symbiotic nitrogen fixation (SNF). The Rhizobium etli CE3 genome encodes 18 OmpR-type regulators; the function of the majority of these regulators during the SNF in common bean, remains elusive. In this work, we demonstrated that a R. etli mutant strain lacking the OmpR-type regulator RetPC57 (ΔRetPC57), formed less nodules when used as inoculum for common bean. Furthermore, we observed reduced expression level of bacterial genes involved in Nod Factors production (nodA and nodB) and of plant early-nodulation genes (NSP2, NIN, NF-YA and ENOD40), in plants inoculated with ΔRetPC57. RetPC57 also contributes to the appropriate expression of genes which products are part of the multidrug efflux pumps family (MDR). Interestingly, nodules elicited by ΔRetPC57 showed increased expression of genes relevant for Carbon/Nitrogen nodule metabolism (PEPC and GOGAT) and ΔRetPC57 bacteroids showed higher nitrogen fixation activity as well as increased expression of key genes directly involved in SNF (hfixL, fixKf, fnrN, fixN, nifA and nifH). Taken together, our data show that the previously uncharacterized regulator RetPC57 is a key player in the development of the R. etli - P. vulgaris symbiosis.

GmPAP12 Is Required for Nodule Development and Nitrogen Fixation Under Phosphorus Starvation in Soybean.

Nodulation process in legume plants is essential for biological nitrogen fixation during which process a large amount of phosphorus (P) is required. Under P deficiency, nodule formation is greatly affected, and induction of purple acid phosphatases (PAPs) is an adaptive strategy for nodules to acquire more P. However, regulation roles of PAPs in nodules remain largely understood. In this study, by transcriptome sequencing technology, five PAP genes were found to be differentially expressed, which led to the greatly increased acid phosphatase (APase) and phytase activities in soybean mature nodules under P starvation conditions; and among the five PAP genes, GmPAP12 had the highest transcript level, and RT-PCR indicated expression of GmPAP12 was gradually increasing during nodule development. GUS activity driven by GmPAP12 promoter was also significantly induced in low phosphorus conditions. Further functional analysis showed that under low phosphorus stress, overexpression of GmPAP12 resulted in higher nodule number, fresh weight, and nitrogenase activity as well as the APase activity than those of control plant nodules, whereas the growth performance and APase activity of nodules on hairy roots were greatly lower when GmPAP12 was suppressed, indicating that GmPAP12 may promote P utilization in soybean nodules under low P stress, which thus played an important role in nodulation and biological nitrogen fixation. Moreover, P1BS elements were found in the promoter of GmPAP12, and yeast one-hybrid experiment further proved the binding of P1BS by transcription factor GmPHR1 in the promoter of GmPAP12. At last, overexpression and suppression of GmPHR1 in nodules indeed caused highly increased and decreased expression of GmPAP12, respectively, indicating that GmPAP12 is regulated by GmPHR1 in soybean nodules. Taken together, these data suggested that GmPAP12 was a novel soybean PAP involved in the P utilization and metabolism in soybean root nodules and played an important role in the growth and development of root nodules and biological nitrogen fixation.

Responses of mature symbiotic nodules to the whole-plant systemic nitrogen signaling.

In symbiotic root nodules of legumes, terminally differentiated rhizobia fix atmospheric N2 producing an NH4+ influx that is assimilated by the plant. The plant, in return, provides photosynthates that fuel the symbiotic nitrogen acquisition. Mechanisms responsible for the adjustment of the symbiotic capacity to the plant N demand remain poorly understood. We have investigated the role of systemic signaling of whole-plant N demand on the mature N2-fixing nodules of the model symbiotic association Medicago truncatula/Sinorhizobium using split-root systems. The whole-plant N-satiety signaling rapidly triggers reductions of both N2 fixation and allocation of sugars to the nodule. These responses are associated with the induction of nodule senescence and the activation of plant defenses against microbes, as well as variations in sugars transport and nodule metabolism. The whole-plant N-deficit responses mirror these changes: a rapid increase of sucrose allocation in response to N-deficit is associated with a stimulation of nodule functioning and development resulting in nodule expansion in the long term. Physiological, transcriptomic, and metabolomic data together provide evidence for strong integration of symbiotic nodules into whole-plant nitrogen demand by systemic signaling and suggest roles for sugar allocation and hormones in the signaling mechanisms.

Sulfate is transported at significant rates through the symbiosome membrane and is crucial for nitrogenase biosynthesis.

Legume–rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced (“fixed”) to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20‐fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant–microbe interaction that is most relevant for agriculture and soil fertility.

Involvement of Glutaredoxin and Thioredoxin Systems in the Nitrogen-Fixing Symbiosis between Legumes and Rhizobia.

Leguminous plants can form a symbiotic relationship with Rhizobium bacteria, during which plants provide bacteria with carbohydrates and an environment appropriate to their metabolism, in return for fixed atmospheric nitrogen. The symbiotic interaction leads to the formation of a new organ, the root nodule, where a coordinated differentiation of plant cells and bacteria occurs. The establishment and functioning of nitrogen-fixing symbiosis involves a redox control important for both the plant-bacteria crosstalk and the regulation of nodule metabolism. In this review, we discuss the involvement of thioredoxin and glutaredoxin systems in the two symbiotic partners during symbiosis. The crucial role of glutathione in redox balance and S-metabolism is presented. We also highlight the specific role of some thioredoxin and glutaredoxin systems in bacterial differentiation. Transcriptomics data concerning genes encoding components and targets of thioredoxin and glutaredoxin systems in connection with the developmental step of the nodule are also considered in the model system Medicago truncatula–Sinorhizobium meliloti.

Comparative Analysis of the Nodule Transcriptomes of Ceanothus thyrsiflorus (Rhamnaceae, Rosales) and Datisca glomerata (Datiscaceae, Cucurbitales).

Two types of nitrogen-fixing root nodule symbioses are known, rhizobial and actinorhizal symbioses. The latter involve plants of three orders, Fagales, Rosales, and Cucurbitales. To understand the diversity of plant symbiotic adaptation, we compared the nodule transcriptomes of Datisca glomerata (Datiscaceae, Cucurbitales) and Ceanothus thyrsiflorus (Rhamnaceae, Rosales); both species are nodulated by members of the uncultured Frankia clade, cluster II. The analysis focused on various features. In both species, the expression of orthologs of legume Nod factor receptor genes was elevated in nodules compared to roots. Since arginine has been postulated as export form of fixed nitrogen from symbiotic Frankia in nodules of D. glomerata, the question was whether the nitrogen metabolism was similar in nodules of C. thyrsiflorus. Analysis of the expression levels of key genes encoding enzymes involved in arginine metabolism revealed up-regulation of arginine catabolism, but no up-regulation of arginine biosynthesis, in nodules compared to roots of D. glomerata, while arginine degradation was not upregulated in nodules of C. thyrsiflorus. This new information corroborated an arginine-based metabolic exchange between host and microsymbiont for D. glomerata, but not for C. thyrsiflorus. Oxygen protection systems for nitrogenase differ dramatically between both species. Analysis of the antioxidant system suggested that the system in the nodules of D. glomerata leads to greater oxidative stress than the one in the nodules of C. thyrsiflorus, while no differences were found for the defense against nitrosative stress. However, induction of nitrite reductase in nodules of C. thyrsiflorus indicated that here, nitrite produced from nitric oxide had to be detoxified. Additional shared features were identified: genes encoding enzymes involved in thiamine biosynthesis were found to be upregulated in the nodules of both species. Orthologous nodule-specific subtilisin-like proteases that have been linked to the infection process in actinorhizal Fagales, were also upregulated in the nodules of D. glomerata and C. thyrsiflorus. Nodule-specific defensin genes known from actinorhizal Fagales and Cucurbitales, were also found in C. thyrsiflorus. In summary, the results underline the variability of nodule metabolism in different groups of symbiotic plants while pointing at conserved features involved in the infection process.

Sulfur Transport and Metabolism in Legume Root Nodules.

Sulfur is an essential nutrient in plants as a constituent element of some amino acids, metal cofactors, coenzymes, and secondary metabolites. Not surprisingly, sulfur deficiency decreases plant growth, photosynthesis, and seed yield in both legumes and non-legumes. In nodulated legumes, sulfur supply is positively linked to symbiotic nitrogen fixation (SNF) and sulfur starvation causes three additional major effects: decrease of nodulation, inhibition of SNF, and slowing down of nodule metabolism. These effects are due, at least in part, to the impairment of nitrogenase biosynthesis and activity, the accumulation of nitrogen-rich amino acids, and the decline in leghemoglobin, ferredoxin, ATP, and glucose in nodules. During the last decade, some major advances have been made about the uptake and metabolism of sulfur in nodules. These include the identification of the sulfate transporter SST1 in the symbiosomal membrane, the finding that glutathione produced in the bacteroids and host cells is essential for nodule activity, and the demonstration that sulfur assimilation in the whole plant is reprogrammed during symbiosis. However, many crucial questions still remain and some examples follow. In the first place, it is of paramount importance to elucidate the mechanism by which sulfur deficiency limits SNF. It is unknown why homoglutahione replaces glutathione as a major water-soluble antioxidant, redox buffer, and sulfur reservoir, among other relevant functions, only in certain legumes and also in different tissues of the same legume species. Much more work is required to identify oxidative post-translational modifications entailing cysteine and methionine residues and to determine how these modifications affect protein function and metabolism in nodules. Likewise, most interactions of antioxidant metabolites and enzymes bearing redox-active sulfur with transcription factors need to be defined. Solving these questions will pave the way to decipher sulfur-dependent mechanisms that regulate SNF, thereby gaining a deep insight into how nodulated legumes adapt to the fluctuating availability of nutrients in the soil.

The intertwined metabolism during symbiotic nitrogen fixation elucidated by metabolic modelling

Genome-scale metabolic network models can be used for various analyses including the prediction of metabolic responses to changes in the environment. Legumes are well known for their rhizobial symbiosis that introduces nitrogen into the global nutrient cycle. Here, we describe a fully compartmentalised, mass and charge-balanced, genome-scale model of the clover Medicago truncatula, which has been adopted as a model organism for legumes. We employed flux balance analysis to demonstrate that the network is capable of producing biomass components in experimentally observed proportions, during day and night. By connecting the plant model to a model of its rhizobial symbiont, Sinorhizobium meliloti, we were able to investigate the effects of the symbiosis on metabolic fluxes and plant growth and could demonstrate how oxygen availability influences metabolic exchanges between plant and symbiont, thus elucidating potential benefits of inter organism amino acid cycling. We thus provide a modelling framework, in which the interlinked metabolism of plants and nodules can be studied from a theoretical perspective.