VapC10 toxin of the legume symbiont Sinorhizobium meliloti targets tRNASer and controls intracellular lifestyle.

The soil bacterium Sinorhizobium meliloti can establish a nitrogen-fixing symbiosis with the model legume Medicago truncatula. The rhizobia induce the formation of a specialized root organ called nodule, where they differentiate into bacteroids and reduce atmospheric nitrogen into ammonia. Little is known on the mechanisms involved in nodule senescence onset and in bacteroid survival inside the infected plant cells. Although toxin-antitoxin (TA) systems have been shown to promote intracellular survival within host cells in human pathogenic bacteria, their role in symbiotic bacteria was rarely investigated. S. meliloti encodes several TA systems, mainly of the VapBC family. Here we present the functional characterization, through a multidisciplinary approach, of the VapBC10 TA system of S. meliloti. Following a mapping by overexpression of an RNase in Escherichia coli (MORE) RNA-seq analysis, we demonstrated that the VapC10 toxin is an RNase that cleaves the anticodon loop of two tRNASer. Thereafter, a bioinformatics approach was used to predict VapC10 targets in bacteroids. This analysis suggests that toxin activation triggers a specific proteome reprogramming that could limit nitrogen fixation capability and viability of bacteroids. Accordingly, a vapC10 mutant induces a delayed senescence in nodules, associated to an enhanced bacteroid survival. VapBC10 TA system could contribute to S. meliloti adaptation to symbiotic lifestyle, in response to plant nitrogen status.

Dynamic nitrogen fixation in an aerobic endophyte of Populus.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

γ-Aminobutyric acid (GABA) in N2-fixing-legume symbiosis: Metabolic flux and carbon/nitrogen homeostasis in responses to abiotic constraints.

Nodule symbiosis is an energetic process that demands a tremendous carbon (C) cost, which massively increases in responses to environmental stresses. Notably, most common respiratory pathways (e.g., glycolysis and Krebs cycle) that sustain nitrogenase activity and subsequent nitrogen (N) assimilation (amino acid formation) display a noncyclic mode of C flux. In such circumstances, the nodule's energy charge could markedly decrease, leading to a lower symbiotic activity under stresses. The host plant then attempts to induce alternative robust metabolic pathways to minimize the C expenditure and compensate for the loss in respiratory substrates. GABA (γ-aminobutyric acid) shunt appears to be among the highly conserved metabolic bypass induced in responses to stresses. Thus, it can be suggested that GABA, via its primary biosynthetic pathway (GABA shunt), is simultaneously induced to circumvent stress-susceptible decarboxylating portion of the Krebs cycle and to replenish symbiosome with energy and C skeletons for enhancing nitrogenase activity and N assimilation besides the additional C costs expended in the metabolic stress acclimations (e.g., biosynthesis of secondary metabolites and excretion of anions). The GABA-mediated C/N balance is strongly associated with interrelated processes, including pH regulation, oxygen (O2) protection, osmoregulation, cellular redox control, and N storage. Furthermore, it has been anticipated that GABA could be implicated in other functions beyond its metabolic role (i.e., signaling and transport). GABA helps plants possess remarkable metabolic plasticity, which might thus assist nodules in attenuating stressful events.

IMA peptides regulate root nodulation and nitrogen homeostasis by providing iron according to internal nitrogen status.

Legumes control root nodule symbiosis (RNS) in response to environmental nitrogen availability. Despite the recent understanding of the molecular basis of external nitrate-mediated control of RNS, it remains mostly elusive how plants regulate physiological processes depending on internal nitrogen status. In addition, iron (Fe) acts as an essential element that enables symbiotic nitrogen fixation; however, the mechanism of Fe accumulation in nodules is poorly understood. Here, we focus on the transcriptome in response to internal nitrogen status during RNS in Lotus japonicus and identify that IRON MAN (IMA) peptide genes are expressed during symbiotic nitrogen fixation. We show that LjIMA1 and LjIMA2 expressed in the shoot and root play systemic and local roles in concentrating internal Fe to the nodule. Furthermore, IMA peptides have conserved roles in regulating nitrogen homeostasis by adjusting nitrogen-Fe balance in L. japonicus and Arabidopsis thaliana. These findings indicate that IMA-mediated Fe provision plays an essential role in regulating nitrogen-related physiological processes.

Phosphorus availability regulates nitrogen fixation rate through a key diazotrophic assembly: Evidence from a subtropical Moso bamboo forest subjected to nitrogen application.

Biological N fixation (BNF) is an important N input process for terrestrial ecosystems. Long-term N application increases the availability of N, but may also lead to phosphorus (P) deficiency or an imbalance between N and P. Here, we performed a 5-year N application experiment in a subtropical Phyllostachys heterocycla forest in site and a P application experiment in vitro to investigate the effect of N application on the BNF rate and its regulatory factor. The BNF rate, nifH gene, free-living diazotrophic community composition and plant properties were measured. We found that N application suppressed the BNF rate and nifH gene abundance, whereas the BNF rate in soils with added P was significantly higher overall than that in soils without added P. Moreover, we identified a key diazotrophic assembly (Mod#2), primarily comprising Bradyrhizobium, Geobacter, Desulfovibrio, Anaeromyxobacter, and Pseudodesulfovibrio, which explained 77 % of the BNF rate variation. There was a significant positive correlation between the Mod#2 abundance and soil available P, and the random forest results showed that soil available P is the most important factor affecting the Mod#2 abundance. Our findings highlight the importance of soil P availability in regulating the activities of key diazotrophs, and thus increasing P supply may help to promote N accumulation and primary productivity through facilitating the BNF process in forest ecosystems.

Strigolactones repress nodule development and senescence in pea.

Strigolactones are a class of phytohormones that are involved in many different plant developmental processes, including the rhizobium-legume nodule symbiosis. Although both positive and negative effects of strigolactones on the number of nodules have been reported, the influence of strigolactones on nodule development is still unknown. Here, by means of the ramosus (rms) mutants of Pisum sativum (pea) cv Terese, we investigated the impact of strigolactone biosynthesis (rms1 and rms5) and signaling (rms3 and rms4) mutants on nodule growth. The rms mutants had more red, that is, functional, and larger nodules than the wild-type plants. Additionally, the increased nitrogen fixation and senescence zones with consequently reduced meristematic and infection zones indicated that the rms nodules developed faster than the wild-type nodules. An enhanced expression of the nodule zone-specific molecular markers for meristem activity and senescence supported the enlarged, fast maturing nodules. Interestingly, the master nodulation regulator, NODULE INCEPTION, NIN, was strongly induced in nodules of all rms mutants but not prior to inoculation. Determination of sugar levels with both bulk and spatial metabolomics in roots and nodules, respectively, hints at slightly increased malic acid levels early during nodule primordia formation and reduced sugar levels at later stages, possibly the consequence of an increased carbon usage of the enlarged nodules, contributing to the enhanced senescence. Taken together, these results suggest that strigolactones regulate the development of nodules, which is probably mediated through NIN, and available plant sugars.

NCR343 is required to maintain the viability of differentiated bacteroids in nodule cells in Medicago truncatula.

Bacteroid (name for rhizobia inside nodule cells) differentiation is a prerequisite for successful nitrogen-fixing symbiosis. In certain legumes, under the regulation of host proteins, for example, a large group of NCR (nodule cysteine rich) peptides, bacteroids undergo irreversible terminal differentiation. This process causes them to lose the ability to propagate inside nodule cells while boosting their competency for nitrogen fixation. How host cells maintain the viability of differentiated bacteroids while maximizing their nitrogen-reducing activities remains elusive. Here, through mutant screen, map-based cloning, and genetic complementation, we find that NCR343 is required for the viability of differentiated bacteroids. In Medicago truncatula debino1 mutant, differentiated bacteroids decay prematurely, and NCR343 is proved to be the casual gene for debino1. NCR343 is mainly expressed in the nodule fixation zone, where bacteroids are differentiated. In nodule cells, mature NCR343 peptide is secreted into the symbiosomes. RNA-Seq assay shows that many stress-responsive genes are significantly induced in debino1 bacteroids. Additionally, a group of stress response-related rhizobium proteins are identified as putative interacting partners of NCR343. In summary, our findings demonstrate that beyond promoting bacteroid differentiation, NCR peptides are also required in maintaining the viability of differentiated bacteroids.

What determines symbiotic nitrogen fixation efficiency in rhizobium: recent insights into Rhizobium leguminosarum.

Symbiotic nitrogen fixation (SNF) by rhizobium, a Gram-negative soil bacterium, is an essential component in the nitrogen cycle and is a sustainable green way to maintain soil fertility without chemical energy consumption. SNF, which results from the processes of nodulation, rhizobial infection, bacteroid differentiation and nitrogen-fixing reaction, requires the expression of various genes from both symbionts with adaptation to the changing environment. To achieve successful nitrogen fixation, rhizobia and their hosts cooperate closely for precise regulation of symbiotic genes, metabolic processes and internal environment homeostasis. Many researches have progressed to reveal the ample information about regulatory aspects of SNF during recent decades, but the major bottlenecks regarding improvement of nitrogen-fixing efficiency has proven to be complex. In this mini-review, we summarize recent advances that have contributed to understanding the rhizobial regulatory aspects that determine SNF efficiency, focusing on the coordinated regulatory mechanism of symbiotic genes, oxygen, carbon metabolism, amino acid metabolism, combined nitrogen, non-coding RNAs and internal environment homeostasis. Unraveling regulatory determinants of SNF in the nitrogen-fixing protagonist rhizobium is expected to promote an improvement of nitrogen-fixing efficiency in crop production.

Nitrogen fixation associated with two cohabiting moss species expresses different patterns under Cu and Zn contamination.

Nitrogen (N2) fixation by moss-associated cyanobacteria is an important N source in pristine ecosystems. Previous studies have shown that moss-associated N2 fixation is sensitive to anthropogenic N pollution. However, we still lack understanding of the effects of other factors derived from anthropogenic sources, such as heavy metal pollution on N2 fixation. To test this, we collected two dominant mosses (Pleurozium schreberi and Spaghnum palustre) from a temperate bog in Denmark and assessed their N2 fixation responses to simulated heavy metal pollution by adding 5 levels (plus a control) of copper (Cu, 0-0.05 mg g dw-1) and zinc (Zn, 0-0.1 mg g dw-1). Metal concentrations in both mosses increased linearly with Cu and Zn addition, but N2 fixation activity associated with S. palustre was to a greater extent negatively affected by both Cu and Zn additions than that associated with P. schreberi. Copper additions even promoted N2 fixation in P. schreberi. Hence, the heavy metal sensitivity of N2-fixing cyanobacteria is dependent on the host moss-species, and the vulnerability of ecosystems towards heavy metal pollution could vary depending on the dominant moss species.

Rhizobial nitrogen fixation efficiency shapes endosphere bacterial communities and Medicago truncatula host growth.

BACKGROUND: Despite the knowledge that the soil-plant-microbiome nexus is shaped by interactions amongst its members, very little is known about how individual symbioses regulate this shaping. Even less is known about how the agriculturally important symbiosis of nitrogen-fixing rhizobia with legumes is impacted according to soil type, yet this knowledge is crucial if we are to harness or improve it. We asked how the plant, soil and microbiome are modulated by symbiosis between the model legume Medicago truncatula and different strains of Sinorhizobium meliloti or Sinorhizobium medicae whose nitrogen-fixing efficiency varies, in three distinct soil types that differ in nutrient fertility, to examine the role of the soil environment upon the plant-microbe interaction during nodulation. RESULTS: The outcome of symbiosis results in installment of a potentially beneficial microbiome that leads to increased nutrient uptake that is not simply proportional to soil nutrient abundance. A number of soil edaphic factors including Zn and Mo, and not just the classical N/P/K nutrients, group with microbial community changes, and alterations in the microbiome can be seen across different soil fertility types. Root endosphere emerged as the plant microhabitat more affected by this rhizobial efficiency-driven community reshaping, manifested by the accumulation of members of the phylum Actinobacteria. The plant in turn plays an active role in regulating its root community, including sanctioning low nitrogen efficiency rhizobial strains, leading to nodule senescence in particular plant-soil-rhizobia strain combinations. CONCLUSIONS: The microbiome-soil-rhizobial dynamic strongly influences plant nutrient uptake and growth, with the endosphere and rhizosphere shaped differentially according to plant-rhizobial interactions with strains that vary in nitrogen-fixing efficiency levels. These results open up the possibility to select inoculation partners best suited for plant, soil type and microbial community. Video Abstract.

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system.

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

Comparison of the seasonal and successional variation of asymbiotic and symbiotic nitrogen fixation along a glacial retreat chronosequence.

Climate change is resulting in accelerated retreat of glaciers worldwide and much nitrogen-poor debris is left after glacier retreats. Asymbiotic dinitrogen (N2) fixation (ANF) can be considered a 'hidden' source of nitrogen (N) for non-nodulating plants in N limited environments; however, seasonal variation and its relative importance in ecosystem N budgets, especially when compared with nodulating symbiotic N2-fixation (SNF), is not well-understood. In this study, seasonal and successional variations in nodulating SNF and non-nodulating ANF rates (nitrogenase activity) were compared along a glacial retreat chronosequence on the eastern edge of the Tibetan Plateau. Key factors regulating the N2-fixation rates as well as the contribution of ANF and SNF to ecosystem N budget were also examined. Significantly greater nitrogenase activity was observed in nodulating species (0.4-17,820.8 nmol C2H4 g-1 d-1) compared to non-nodulating species (0.0-9.9 nmol C2H4 g-1 d-1) and both peaked in June or July. Seasonal variation in acetylene reduction activity (ARA) rate in plant nodules (nodulating species) and roots (non-nodulating species) was correlated with soil temperature and moisture while ARA in non-nodulating leaves and twigs was correlated with air temperature and humidity. Stand age was not found to be a significant determinant of ARA rates in nodulating or non-nodulating plants. ANF and SNF contributed 0.3-51.5 % and 10.1-77.8 %, respectively, of total ecosystem N input in the successional chronosequence. In this instance, ANF exhibited an increasing trend with successional age while SNF increased only at stages younger than 29 yr and then decreased as succession proceeded. These findings help improve our understanding of ANF activity in non-nodulating plants and N budgets in post glacial primary succession.

H2 S works synergistically with rhizobia to modify photosynthetic carbon assimilation and metabolism in nitrogen-deficient soybeans.

Hydrogen sulfide (H2 S) performs a crucial role in plant development and abiotic stress responses by interacting with other signalling molecules. However, the synergistic involvement of H2 S and rhizobia in photosynthetic carbon (C) metabolism in soybean (Glycine max) under nitrogen (N) deficiency has been largely overlooked. Therefore, we scrutinised how H2 S drives photosynthetic C fixation, utilisation, and accumulation in soybean-rhizobia symbiotic systems. When soybeans encountered N deficiency, organ growth, grain output, and nodule N-fixation performance were considerably improved owing to H2 S and rhizobia. Furthermore, H2 S collaborated with rhizobia to actively govern assimilation product generation and transport, modulating C allocation, utilisation, and accumulation. Additionally, H2 S and rhizobia profoundly affected critical enzyme activities and coding gene expressions implicated in C fixation, transport, and metabolism. Furthermore, we observed substantial effects of H2 S and rhizobia on primary metabolism and C-N coupled metabolic networks in essential organs via C metabolic regulation. Consequently, H2 S synergy with rhizobia inspired complex primary metabolism and C-N coupled metabolic pathways by directing the expression of key enzymes and related coding genes involved in C metabolism, stimulating effective C fixation, transport, and distribution, and ultimately improving N fixation, growth, and grain yield in soybeans.

Distribution and survival strategies of endemic and cosmopolitan diazotrophs in the Arctic Ocean.

Dinitrogen (N2) fixation is the major source of reactive nitrogen in the ocean and has been considered to occur specifically in low-latitude oligotrophic oceans. Recent studies have shown that N2 fixation also occurs in the polar regions and thus is a global process, although the physiological and ecological characteristics of polar diazotrophs are not yet known. Here, we successfully reconstructed diazotroph genomes, including that of cyanobacterium UCYN-A (Candidatus 'Atelocyanobacterium thalassa'), from metagenome data corresponding to 111 samples isolated from the Arctic Ocean. These diazotrophs were highly abundant in the Arctic Ocean (max., 1.28% of the total microbial community), suggesting that they have important roles in the Arctic ecosystem and biogeochemical cycles. Further, we show that diazotrophs within genera Arcobacter, Psychromonas, and Oceanobacter are prevalent in the <0.2 µm fraction in the Arctic Ocean, indicating that current methods cannot capture their N2 fixation. Diazotrophs in the Arctic Ocean were either Arctic-endemic or cosmopolitan species from their global distribution patterns. Arctic-endemic diazotrophs, including Arctic UCYN-A, were similar to low-latitude-endemic and cosmopolitan diazotrophs in genome-wide function, however, they had unique gene sets (e.g., diverse aromatics degradation genes), suggesting adaptations to Arctic-specific conditions. Cosmopolitan diazotrophs were generally non-cyanobacteria and commonly had the gene that encodes the cold-inducible RNA chaperone, which presumably makes their survival possible even in deep, cold waters of global ocean and polar surface waters. This study shows global distribution pattern of diazotrophs with their genomes and provides clues to answering the question of how diazotrophs can inhabit polar waters.

Ecosystem carbon stocks and sequestration rates in white oak forests in the central Himalaya: Role of nitrogen-fixing Nepalese alder.

Nitrogen-fixing Nepalese alder (Alnus nepalensis D. Don.), a pioneer species and nurse tree species, forms pure stands, and sometimes occurs in mixed stands in areas affected by landslides. The objective of this study was to understand the influence of A. nepalensis on carbon stock in white oak (Quercus leucotrichophora A. Camus) forests. We investigated the differences in vegetation biomass carbon (tree, sapling, seedling, shrub and herbs, and forest floor mass), soil organic carbon stock, and sequestration rates in five naturally occurring oak mixed alder (OMA) forest stands and five naturally occurring oak without alder (OWA) forest stands along the basal area gradient in order to investigate the role of A. nepalensis on ecosystem carbon stock. The total basal area ranged from 61.20 to 89.51 m2 ha-1 in the OMA stands and from 38.02 to 53.54 m2 ha-1 in the OWA stands. The total tree density of the OMA stands (1120 to 1330 trees ha-1) was higher than that of the OWA stands (950 to 1230 trees ha-1). The total ecosystem carbon stock in the OMA stands was significantly (P<0.05) higher than that in the OWA stands, ranging from 485.3 to 635.6 Mg C ha-1 in the former and from 378.8 to 472 Mg C ha-1 in the latter. Soil was the second largest carbon pool in all the studied stands, with the values ranging from 238.1 to 254.1 Mg C ha-1 in the OMA and 185.5 to 215.8 Mg C ha-1 in the OWA stands. The soil organic carbon (SOC) stock was 1.19 to 1.28 times higher in the OMA than in the OWA stands. Of the total ecosystem carbon stock in different OMA stands, A. nepalensis stored 16.2 to 38.8%. Annual carbon sequestration rates (6.6 to 9.5 Mg C ha-1 yr-1) in the OMA stands were significantly (P<0.05) higher than in the OWA (2.5 to 5.4 Mg C ha-1 yr-1) stands. Among all the species and across the stands, the greatest carbon sequestration was exhibited by A. nepalensis (3.4 to 5.3 Mg C ha-1 yr-1). The present results show the role of A. nepalensis in ecosystem carbon stock and sequestration rates. Significantly higher rates of carbon sequestration by oak in OMA stands than OWA stands clearly indicate the facilitative role of co-occurring nitrogen-fixing A. nepalensis. The results imply that Q. leucotrichophora mixed with a A. nepalensis plantation may be a good option for enhancing ecosystem carbon stock, carbon sequestration, and habitat restoration in the central Himalaya.

Dancing to a different tune, can we switch from chemical to biological nitrogen fixation for sustainable food security?

Our current food production systems are unsustainable, driven in part through the application of chemically fixed nitrogen. We need alternatives to empower farmers to maximise their productivity sustainably. Therefore, we explore the potential for transferring the root nodule symbiosis from legumes to other crops. Studies over the last decades have shown that preexisting developmental and signal transduction processes were recruited during the evolution of legume nodulation. This allows us to utilise these preexisting processes to engineer nitrogen fixation in target crops. Here, we highlight our understanding of legume nodulation and future research directions that might help to overcome the barrier of achieving self-fertilising crops.

Significance of nitrogen-fixing actinorhizal symbioses for restoration of depleted, degraded, and contaminated soil.

Atmospheric nitrogen (N2)-fixing legume trees are frequently used for the restoration of depleted, degraded, and contaminated soils. However, biological N2 fixation (BNF) can also be performed by so-called actinorhizal plants. Actinorhizal plants include a high diversity of woody species and therefore can be applied in a broad spectrum of environments. In contrast to N2-fixing legumes, the potential of actinorhizal plants for soil restoration remains largely unexplored. In this Opinion, we propose related basic research requirements for the characterization of environmental stress responses that determine the restoration potential of actinorhizal plants for depleted, degraded, and contaminated soils. We identify advantages and unexplored processes of actinorhizal plants and describe a mainly uncharted avenue of future research for this important group of plant species.

Transcriptomic and metabolomic analysis reveals that symbiotic nitrogen fixation enhances drought resistance in common bean.

Common bean (Phaseolus vulgaris L.), one of the most important legume crops, uses atmospheric nitrogen through symbiosis with soil rhizobia, reducing the need for nitrogen fertilization. However, this legume is particularly sensitive to drought conditions, prevalent in arid regions where this crop is cultured. Therefore, studying the response to drought is important to sustain crop productivity. We have used integrated transcriptomic and metabolomic analysis to understand the molecular responses to water deficit in a marker-class common bean accession cultivated under N2 fixation or fertilized with nitrate (NO3-). RNA-seq revealed more transcriptional changes in the plants fertilized with NO3- than in the N2-fixing plants. However, changes in N2-fixing plants were more associated with drought tolerance than in those fertilized with NO3-. N2-fixing plants accumulated more ureides in response to drought, and GC/MS and LC/MS analysis of primary and secondary metabolite profiles revealed that N2-fixing plants also had higher levels of abscisic acid, proline, raffinose, amino acids, sphingolipids, and triacylglycerols than those fertilized with NO3-. Moreover, plants grown under nitrogen fixation recovered from drought better than plants fertilized with NO3-. Altogether we show that common bean plants grown under symbiotic nitrogen fixation were more protected against drought than the plants fertilized with nitrate.

Investigating the Unique Ability of Trichodesmium To Fix Carbon and Nitrogen Simultaneously Using MiMoSA.

The open ocean is an extremely competitive environment, partially due to the dearth of nutrients. Trichodesmium erythraeum, a marine diazotrophic cyanobacterium, is a keystone species in the ocean due to its ability to fix nitrogen and leak 30 to 50% into the surrounding environment, providing a valuable source of a necessary macronutrient to other species. While there are other diazotrophic cyanobacteria that play an important role in the marine nitrogen cycle, Trichodesmium is unique in its ability to fix both carbon and nitrogen simultaneously during the day without the use of specialized cells called heterocysts to protect nitrogenase from oxygen. Here, we use the advanced modeling framework called multiscale multiobjective systems analysis (MiMoSA) to investigate how Trichodesmium erythraeum can reduce dimolecular nitrogen to ammonium in the presence of oxygen. Our simulations indicate that nitrogenase inhibition is best modeled as Michealis-Menten competitive inhibition and that cells along the filament maintain microaerobia using high flux through Mehler reactions in order to protect nitrogenase from oxygen. We also examined the effect of location on metabolic flux and found that cells at the end of filaments operate in distinctly different metabolic modes than internal cells despite both operating in a photoautotrophic mode. These results give us important insight into how this species is able to operate photosynthesis and nitrogen fixation simultaneously, giving it a distinct advantage over other diazotrophic cyanobacteria because they can harvest light directly to fuel the energy demand of nitrogen fixation. IMPORTANCE Trichodesmium erythraeum is a marine cyanobacterium responsible for approximately half of all biologically fixed nitrogen, making it an integral part of the global nitrogen cycle. Interestingly, unlike other nitrogen-fixing cyanobacteria, Trichodesmium does not use temporal or spatial separation to protect nitrogenase from oxygen poisoning; instead, it operates photosynthesis and nitrogen fixation reactions simultaneously during the day. Unfortunately, the exact mechanism the cells utilize to operate carbon and nitrogen fixation simultaneously is unknown. Here, we use an advanced metabolic modeling framework to investigate and identify the most likely mechanisms Trichodesmium uses to protect nitrogenase from oxygen. The model predicts that cells operate in a microaerobic mode, using both respiratory and Mehler reactions to dramatically reduce intracellular oxygen concentrations.

The putative transporter MtUMAMIT14 participates in nodule formation in Medicago truncatula.

Transport systems are crucial in many plant processes, including plant-microbe interactions. Nodule formation and function in legumes involve the expression and regulation of multiple transport proteins, and many are still uncharacterized, particularly for nitrogen transport. Amino acids originating from the nitrogen-fixing process are an essential form of nitrogen for legumes. This work evaluates the role of MtN21 (henceforth MtUMAMIT14), a putative transport system from the MtN21/EamA-like/UMAMIT family, in nodule formation and nitrogen fixation in Medicago truncatula. To dissect this transporter's role, we assessed the expression of MtUMAMIT14 using GUS staining, localized the corresponding protein in M. truncatula root and tobacco leaf cells, and investigated two independent MtUMAMIT14 mutant lines. Our results indicate that MtUMAMIT14 is localized in endosomal structures and is expressed in both the infection zone and interzone of nodules. Comparison of mutant and wild-type M. truncatula indicates MtUMAMIT14, the expression of which is dependent on the presence of NIN, DNF1, and DNF2, plays a role in nodule formation and nitrogen-fixation. While the function of the transporter is still unclear, our results connect root nodule nitrogen fixation in legumes with the UMAMIT family.