Light-driven, bias-free nitrogenase-based bioelectrochemical cell for ammonia generation.

Nitrogen fixation is a key process that sustains life on Earth. Nitrogenase is the sole enzyme capable of fixing nitrogen under ambient conditions. Extensive research efforts have been dedicated to elucidating the enzyme mechanism and its artificial activation through high applied voltage, photochemistry, or strong reducing agents. Harnessing light irradiation to minimize the required external bias can lower the process's high energy investment. Herein, we present the development of photo-bioelectrochemical cells (PBECs) utilizing BiVO4/CoP or CdS/NiO photoanodes for nitrogenase activation toward N2 fixation. The constructed PBEC based on BiVO4/CoP photoanode requires minimal external bias (200 mV) and suppresses O2 generation that allows efficient activation of the nitrogenase enzyme, using glucose as an electron donor. In a second developed PBEC configuration, CdS/NiO photoanode was used, enabling bias-free activation of the nitrogenase-based cathode to produce 100 µM of ammonia at a faradaic efficiency (FE) of 12%. The ammonia production was determined by a commonly used fluorescence probe and further validated using 1H-NMR spectroscopy. The presented PBECs lay the foundation for biotic-abiotic systems to directly activate enzymes toward value-added chemicals by light-driven reactions.

Actomonas aquatica gen. nov., sp. nov., a marine lineage in the phylum Verrucomicrobiota with genetic potential for carbon and nitrogen fixation.

A novel bacterial strain, designated WL0086T, was isolated from a marine sediment sample collected in Lianyungang city, Jiangsu province, PR China. This strain showed the highest 16S rRNA gene sequence similarity to Geminisphaera colitermitum TAV2T (92.7 %) of the family Opitutaceae, and all the unclassified cultured and uncultured isolates with similarities >95 % were from marine environments. Cells were Gram-stain-negative, aerobic, non-motile cocci with a size of 0.6-0.8 µm in diameter. Strain WL0086T was positive for both oxidase and catalase, and grew at 20-37 °C (optimum, 28 °C), with 1.5-11.0 % NaCl (w/v; optimum, 2.5-4.0 %) and at pH 5.0-9.0 (optimum, pH 7.0). The major polar lipid profile of strain WL0086T consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylcholine. The major isoprenoid quinone was menaquinone-7 and the predominant fatty acids were iso-C14 : 0, anteiso-C15 : 0, C16 : 0 and C16 : 1 ω9c. The complete genome consisted of a chromosome with 6 109 182 bp. The G+C content of genomic DNA was 64.0%. Results of phylogenomic analysis based on the 16S rRNA gene sequence and the whole genome suggested that strain WL0086T formed a distinct clade closely neighbouring the members of the family Opitutaceae. On the basis of phylogenetic, phenotypic, and chemotaxonomic evidences, strain WL0086T should represent a novel genus of the family Opitutaceae, for which the name Actomonas aquatica gen. nov., sp. nov. is proposed. The type strain is WL0086T (=MCCC 1K05844T=JCM 34677T=GDMCC 1.2411T).

Haplotype-resolved genome of Mimosa bimucronata revealed insights into leaf movement and nitrogen fixation.

BACKGROUND: Mimosa bimucronata originates from tropical America and exhibits distinctive leaf movement characterized by a relative slow speed. Additionally, this species possesses the ability to fix nitrogen. Despite these intriguing traits, comprehensive studies have been hindered by the lack of genomic resources for M. bimucronata. RESULTS: To unravel the intricacies of leaf movement and nitrogen fixation, we successfully assembled a high-quality, haplotype-resolved, reference genome at the chromosome level, spanning 648 Mb and anchored in 13 pseudochromosomes. A total of 32,146 protein-coding genes were annotated. In particular, haplotype A was annotated with 31,035 protein-coding genes, and haplotype B with 31,440 protein-coding genes. Structural variations (SVs) and allele specific expression (ASE) analyses uncovered the potential role of structural variants in leaf movement and nitrogen fixation in M. bimucronata. Two whole-genome duplication (WGD) events were detected, that occurred ~ 2.9 and ~ 73.5 million years ago. Transcriptome and co-expression network analyses revealed the involvement of aquaporins (AQPs) and Ca2+-related ion channel genes in leaf movement. Moreover, we also identified nodulation-related genes and analyzed the structure and evolution of the key gene NIN in the process of symbiotic nitrogen fixation (SNF). CONCLUSION: The detailed comparative genomic and transcriptomic analyses provided insights into the mechanisms governing leaf movement and nitrogen fixation in M. bimucronata. This research yielded genomic resources and provided an important reference for functional genomic studies of M. bimucronata and other legume species.

Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification.

Biological nitrogen fixation by free-living bacteria and rhizobial symbiosis with legumes plays a key role in sustainable crop production. Here, we study how different crop combinations influence the interaction between peanut plants and their rhizosphere microbiota via metabolite deposition and functional responses of free-living and symbiotic nitrogen-fixing bacteria. Based on a long-term (8 year) diversified cropping field experiment, we find that peanut co-cultured with maize and oilseed rape lead to specific changes in peanut rhizosphere metabolite profiles and bacterial functions and nodulation. Flavonoids and coumarins accumulate due to the activation of phenylpropanoid biosynthesis pathways in peanuts. These changes enhance the growth and nitrogen fixation activity of free-living bacterial isolates, and root nodulation by symbiotic Bradyrhizobium isolates. Peanut plant root metabolites interact with Bradyrhizobium isolates contributing to initiate nodulation. Our findings demonstrate that tailored intercropping could be used to improve soil nitrogen availability through changes in the rhizosphere microbiome and its functions.

Nitrogen-fixing organelle in a marine alga.

Symbiotic interactions were key to the evolution of chloroplast and mitochondria organelles, which mediate carbon and energy metabolism in eukaryotes. Biological nitrogen fixation, the reduction of abundant atmospheric nitrogen gas (N2) to biologically available ammonia, is a key metabolic process performed exclusively by prokaryotes. Candidatus Atelocyanobacterium thalassa, or UCYN-A, is a metabolically streamlined N2-fixing cyanobacterium previously reported to be an endosymbiont of a marine unicellular alga. Here we show that UCYN-A has been tightly integrated into algal cell architecture and organellar division and that it imports proteins encoded by the algal genome. These are characteristics of organelles and show that UCYN-A has evolved beyond endosymbiosis and functions as an early evolutionary stage N2-fixing organelle, or "nitroplast."

Overexpression of GmPAP4 Enhances Symbiotic Nitrogen Fixation and Seed Yield in Soybean under Phosphorus-Deficient Condition.

Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean.

Nitrogen and Nod factor signaling determine Lotus japonicus root exudate composition and bacterial assembly.

Symbiosis with soil-dwelling bacteria that fix atmospheric nitrogen allows legume plants to grow in nitrogen-depleted soil. Symbiosis impacts the assembly of root microbiota, but it is unknown how the interaction between the legume host and rhizobia impacts the remaining microbiota and whether it depends on nitrogen nutrition. Here, we use plant and bacterial mutants to address the role of Nod factor signaling on Lotus japonicus root microbiota assembly. We find that Nod factors are produced by symbionts to activate Nod factor signaling in the host and that this modulates the root exudate profile and the assembly of a symbiotic root microbiota. Lotus plants with different symbiotic abilities, grown in unfertilized or nitrate-supplemented soils, display three nitrogen-dependent nutritional states: starved, symbiotic, or inorganic. We find that root and rhizosphere microbiomes associated with these states differ in composition and connectivity, demonstrating that symbiosis and inorganic nitrogen impact the legume root microbiota differently. Finally, we demonstrate that selected bacterial genera characterizing state-dependent microbiomes have a high level of accurate prediction.

Ecological aspects and relationships of the emblematic Vachellia spp. exposed to anthropic pressures and parasitism in natural hyper-arid ecosystems: ethnobotanical elements, morphology, and biological nitrogen fixation.

MAIN CONCLUSION: Emblematic Vachellia spp. naturally exposed to hyper-arid conditions, intensive grazing, and parasitism maintain a high nitrogen content and functional mutualistic nitrogen-fixing symbioses. AlUla region in Saudi Arabia has a rich history regarding mankind, local wildlife, and fertility islands suitable for leguminous species, such as the emblematic Vachellia spp. desert trees. In this region, we investigated the characteristics of desert legumes in two nature reserves (Sharaan and Madakhil), at one archaeological site (Hegra), and in open public domains et al. Ward and Jabal Abu Oud. Biological nitrogen fixation (BNF), isotopes, and N and C contents were investigated through multiple lenses, including parasitism, plant tissues, species identification, plant maturity, health status, and plant growth. The average BNF rates of 19 Vachellia gerrardii and 21 Vachellia tortilis trees were respectively 39 and 67%, with low signs of inner N content fluctuations (2.10-2.63% N) compared to other co-occurring plants. The BNF of 23 R. raetam was just as high, with an average of 65% and steady inner N contents of 2.25 ± 0.30%. Regarding parasitism, infected Vachellia trees were unfazed compared to uninfected trees, thereby challenging the commonly accepted detrimental role of parasites. Overall, these results suggest that Vachellia trees and R. raetam shrubs exploit BNF in hyper-arid environments to maintain a high N content when exposed to parasitism and grazing. These findings underline the pivotal role of plant-bacteria mutualistic symbioses in desert environments. All ecological traits and relationships mentioned are further arguments in favor of these legumes serving as keystone species for ecological restoration and agro-silvo-pastoralism in the AlUla region.

Fulvic Acid Promotes Legume-Rhizobium Symbiosis by Stimulating Endogenous Flavonoids Synthesis and Secretion.

Fulvic acid (FA) promotes symbiosis between legumes and rhizobia. To elucidate from the aspect of symbiosis, the effects of root irrigation of water-soluble humic materials (WSHM) or foliar spraying of its highly active component, FA, on soybean root exudates and on rhizosphere microorganisms were investigated. As a result, WSHM/FA treatments significantly altered root exudate metabolite composition, and isoflavonoids were identified as key contributors in both treatments compared to the control. Increased expression of genes related to the isoflavonoid biosynthesis were validated by RT-qPCR in both treatments, which notably elevated the synthesis of symbiotic signals genistein, daidzin, coumestrol, and biochanin A. Moreover, the WSHM/FA treatments induced a change in rhizosphere microbial community, coupled with an increase in the relative abundance of rhizobia. Our findings showed that WSHM/FA promotes symbiosis by stimulating the endogenous flavonoid synthesis and leads to rhizobia accumulation in the rhizosphere. This study provides new insights into mechanisms underlying the FA-mediated promotion of symbiosis.

Rice N-biofertilization by inoculation with an engineered photosynthetic diazotroph.

Photosynthetic diazotrophs expressing iron-only (Fe-only) nitrogenase can be developed into a promising biofertilizer, as it is independent on the molybdenum availability in the soil. However, the expression of Fe-only nitrogenase in diazotrophs is repressed by the fixed nitrogen of the soil, limiting the efficiency of nitrogen fixation in farmland with low ammonium concentrations that are inadequate for sustainable crop growth. Here, we succeeded in constitutively expressing the Fe-only nitrogenase even in the presence of ammonium by controlling the transcription of Fe-only nitrogenase gene cluster (anfHDGK) with the transcriptional activator of Mo nitrogenase (NifA*) in several different ways, indicating that the engineered NifA* strains can be used as promising chassis cells for efficient expression of different types of nitrogenases. When applied as a biofertilizer, the engineered Rhodopseudomonas palustris effectively stimulated rice growth, contributing to the reduced use of chemical fertilizer and the development of sustainable agriculture.

Metabolic trade-offs constrain the cell size ratio in a nitrogen-fixing symbiosis.

Biological dinitrogen (N2) fixation is a key metabolic process exclusively performed by prokaryotes, some of which are symbiotic with eukaryotes. Species of the marine haptophyte algae Braarudosphaera bigelowii harbor the N2-fixing endosymbiotic cyanobacteria UCYN-A, which might be evolving organelle-like characteristics. We found that the size ratio between UCYN-A and their hosts is strikingly conserved across sublineages/species, which is consistent with the size relationships of organelles in this symbiosis and other species. Metabolic modeling showed that this size relationship maximizes the coordinated growth rate based on trade-offs between resource acquisition and exchange. Our findings show that the size relationships of N2-fixing endosymbionts and organelles in unicellular eukaryotes are constrained by predictable metabolic underpinnings and that UCYN-A is, in many regards, functioning like a hypothetical N2-fixing organelle (or nitroplast).

Phototrophic Nitrogen Fixation, a Neglected Biogeochemical Process in Mine Tailings?

Biological nitrogen fixation (BNF) has important ecological significance in mine tailing by contributing to the initial accumulation of nitrogen. In addition to chemolithotrophic and heterotrophic BNF, light may also fuel BNF in oligotrophic mine tailings. However, knowledge regarding the occurrence and ecological significance of this biogeochemical process in mine tailings remains ambiguous. The current study observed phototrophic BNF in enrichment cultures established from three primary successional stages (i.e., original tailings, biological crusts, and pioneer plants) of tailings. Notably, phototrophic BNF in tailings may be more active at vegetation stages (i.e., biological crusts and pioneering plants) than in bare tailings. DNA-stable isotope probing identified Roseomonas species as potential aerobic anoxygenic phototrophs responsible for phototrophic BNF. Furthermore, metagenomic binning as well as genome mining revealed that Roseomonas spp. contained essential genes involved in nitrogen fixation, anoxygenic photosynthesis, and carbon fixation, suggesting their genetic potential to mediate phototrophic BNF. A causal inference framework equipped with the structural causal model suggested that the enrichment of putative phototrophic diazotrophic Roseomonas may contribute to an elevated total nitrogen content during primary succession in these mine tailings. Collectively, our findings suggest that phototrophic diazotrophs may play important roles in nutrient accumulation and hold the potential to facilitate ecological succession in tailings.

Hopanoid lipids promote soybean-Bradyrhizobium symbiosis.

The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

Identification of QTLs for symbiotic nitrogen fixation and related traits in a soybean recombinant inbred line population.

KEY MESSAGE: The genetic architecture of symbiotic N fixation and related traits was investigated in the field. QTLs were identified for percent N derived from the atmosphere, shoot [N] and C to N ratio. Soybean [Glycine max (L.) Merr.] is cultivated worldwide and is the most abundant source of plant-based protein. Symbiotic N2 fixation (SNF) in legumes such as soybean is of great importance; however, yields may still be limited by N in both high yielding and stressful environments. To better understand the genetic architecture of SNF and facilitate the development of high yielding cultivars and sustainable soybean production in stressful environments, a recombinant inbred line population consisting of 190 lines, developed from a cross between PI 442012A and PI 404199, was evaluated for N derived from the atmosphere (Ndfa), N concentration ([N]), and C to N ratio (C/N) in three environments. Significant genotype, environment and genotype × environment effects were observed for all three traits. A linkage map was constructed containing 3309 single nucleotide polymorphism (SNP) markers. QTL analysis was performed for additive effects of QTLs, QTL × environment interactions, and QTL × QTL interactions. Ten unique additive QTLs were identified across all traits and environments. Of these, two QTLs were detected for Ndfa and eight for C/N. Of the eight QTLs for C/N, four were also detected for [N]. Using QTL × environment analysis, six QTLs were detected, of which five were also identified in the additive QTL analysis. The QTL × QTL analysis identified four unique epistatic interactions. The results of this study may be used for genomic selection and introgression of favorable alleles for increased SNF, [N], and C/N via marker-assisted selection.

Exploring the role of symbiotic modifier peptidases in the legume - rhizobium symbiosis.

Legumes can establish a mutual association with soil-derived nitrogen-fixing bacteria called 'rhizobia' forming lateral root organs called root nodules. Rhizobia inside the root nodules get transformed into 'bacteroids' that can fix atmospheric nitrogen to ammonia for host plants in return for nutrients and shelter. A substantial 200 million tons of nitrogen is fixed annually through biological nitrogen fixation. Consequently, the symbiotic mechanism of nitrogen fixation is utilized worldwide for sustainable agriculture and plays a crucial role in the Earth's ecosystem. The development of effective nitrogen-fixing symbiosis between legumes and rhizobia is very specialized and requires coordinated signaling. A plethora of plant-derived nodule-specific cysteine-rich (NCR or NCR-like) peptides get actively involved in this complex and tightly regulated signaling process of symbiosis between some legumes of the IRLC (Inverted Repeat-Lacking Clade) and Dalbergioid clades and nitrogen-fixing rhizobia. Recent progress has been made in identifying two such peptidases that actively prevent bacterial differentiation, leading to symbiotic incompatibility. In this review, we outlined the functions of NCRs and two nitrogen-fixing blocking peptidases: HrrP (host range restriction peptidase) and SapA (symbiosis-associated peptidase A). SapA was identified through an overexpression screen from the Sinorhizobium meliloti 1021 core genome, whereas HrrP is inherited extra-chromosomally. Interestingly, both peptidases affect the symbiotic outcome by degrading the NCR peptides generated from the host plants. These NCR-degrading peptidases can shed light on symbiotic incompatibility, helping to elucidate the reasons behind the inefficiency of nitrogen fixation observed in certain groups of rhizobia with specific legumes.

Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe4S] Fd activity.

Ferredoxins (Fds) are small proteins which shuttle electrons to pathways like biological nitrogen fixation. Physical properties tune the reactivity of Fds with different pathways, but knowledge on how these properties can be manipulated to engineer new electron transfer pathways is lacking. Recently, we showed that an evolved strain of Rhodopseudomonas palustris uses a new electron transfer pathway for nitrogen fixation. This pathway involves a variant of the primary Fd of nitrogen fixation in R. palustris, Fer1, in which threonine at position 11 is substituted for isoleucine (Fer1T11I). To understand why this substitution in Fer1 enables more efficient electron transfer, we used in vivo and in vitro methods to characterize Fer1 and Fer1T11I. Electrochemical characterization revealed both Fer1 and Fer1T11I have similar redox transitions (-480 mV and - 550 mV), indicating the reduction potential was unaffected despite the proximity of T11 to an iron­sulfur (FeS) cluster of Fer1. Additionally, disruption of hydrogen bonding around an FeS cluster in Fer1 by substituting threonine with alanine (T11A) or valine (T11V) did not increase nitrogenase activity, indicating that disruption of hydrogen bonding does not explain the difference in activity observed for Fer1T11I. Electron paramagnetic resonance spectroscopy studies revealed key differences in the electronic structure of Fer1 and Fer1T11I, which indicate changes to the high spin states and/or spin-spin coupling between the FeS clusters of Fer1. Our data implicates these electronic structure differences in facilitating electron flow and sets a foundation for further investigations to understand the connection between these properties and intermolecular electron transfer.

Label-free functional analysis of root-associated microbes with dynamic quantitative oblique back-illumination microscopy.

The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable agricultural practices. Nitrogen-fixing bacteria, known as diazotrophs, offer a potential solution by converting atmospheric nitrogen into bioavailable forms, reducing the reliance on synthetic fertilizers. However, a deeper understanding of their interactions with plants and other microbes is needed. In this study, we introduce a recently developed label-free 3D quantitative phase imaging technology called dynamic quantitative oblique back-illumination microscopy (DqOBM) to assess the functional dynamic activity of diazotrophs in vitro and in situ. Our experiments involved three different diazotrophs (Sinorhizobium meliloti, Azotobacter vinelandii, and Rahnella aquatilis) cultured on media with amendments of carbon and nitrogen sources. Over 5 days, we observed increased dynamics in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize microbial dynamics within the root cap and elongation zone of Arabidopsis thaliana primary roots. This allowed us to identify distinct areas of microbial infiltration in plant roots without the need for fluorescent markers. Our findings demonstrate that DqOBM can effectively characterize microbial dynamics and provide insights into plant-microbe interactions in situ, offering a valuable tool for advancing our understanding of sustainable agriculture.

Identification and characterization of the TetR family transcriptional regulator NffT in Rhizobium johnstonii.

Symbiotic nitrogen fixation (SNF) by rhizobia is not only the main natural bionitrogen-source for organisms but also a green process leveraged to increase the fertility of soil for agricultural production. However, an insufficient understanding of the regulatory mechanism of SNF hinders its practical application. During SNF, nifA-fixA signaling is essential for the biosynthesis of nitrogenases and electron transfer chain proteins. In the present study, the TetR regulator NffT, whose mutation increased fixA expression, was discovered through a fixA-promoter-ß-glucuronidase fusion assay performed with Rhizobium johnstonii. Real-time quantitative PCR analysis showed that nffT deletion increased the expression of symbiotic genes including nifA and fixA in nifA-fixA signaling, and fixL, fixK, fnrN, and fixN9 in fixL-fixN signaling. nffT overexpression resulted in disordered nodules and reduced nitrogen-fixing efficiency. Electrophoretic mobility shift assays revealed that NffT directly regulated the transcription of RL0091-93, which encode an ATP-binding ABC transporter predicted to be involved in carbohydrate transport. Purified His-tagged NffT bound to a 68 bp DNA sequence located -32 to -99 bp upstream of RL0091-93 and NffT deletion significantly increased the expression of RL0091-93. nffT-promoter-ß-glucuronidase fusion assay indicated that nffT expression was regulated by the cobNTS genes and cobalamin. Mutations in cobNTS significantly decreased the expression of nffT, and cobalamin restored its expression. These results revealed that NffT affects nodule development and nitrogen-fixing reaction by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes and, thus, plays a pivotal regulatory role during symbiosis of R. johnstonii-Pisum sativum.IMPORTANCESymbiotic nitrogen fixation (SNF) by rhizobia is a green way to maintain soil fertility without causing environmental pollution or consuming chemical energy. A detailed understanding of the regulatory mechanism of this complex process is essential for promoting sustainable agriculture. In this study, we discovered the TetR-type regulator NffT, which suppressed the expression of fixA in Rhizobium johnstonii. Furthermore, NffT was confirmed to play pleiotropic roles in R. johnstonii-Pisum sativum symbiosis; specifically, it inhibited rhizobial growth, nodule differentiation, and nitrogen-fixing reactions. We revealed that NffT indirectly affected R. johnstonii-P. sativum symbiosis by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes. Furthermore, cobalamin, a chemical molecule, was reported for the first time to be involved in TetR-type protein transcription during symbiosis. Thus, NffT identification connects SNF regulation with genetic, metabolic, and chemical signals and provides new insights into the complex regulation of SNF, laying an experimental basis for the targeted construction of rhizobial strains with highly efficient nitrogen-fixing capacity.

Molybdenum-mediated nitrogen accumulation and assimilation in legumes stepwise boosted by the coexistence of arbuscular mycorrhizal fungi and earthworms.

Molybdenum (Mo) is a critical micronutrient for nitrogen (N) metabolism in legumes, yet the impact of Mo on legume N metabolism in the context of natural coexistence with soil microorganisms remains poorly understood. This study investigated the dose-dependent effect of Mo on soil N biogeochemical cycling, N accumulation, and assimilation in alfalfa under conditions simulating the coexistence of arbuscular mycorrhizal fungi (AMF) and earthworms. The findings indicated that Mo exerted a hormetic effect on alfalfa N accumulation, facilitating it at low concentrations (below 29.98 mg/kg) and inhibiting it at higher levels. This inhibition was attributed to Mo-induced constraints on C supply for nitrogen fixation. Concurrently, AMF colonization enhanced C assimilation in Mo-treated alfalfas by promoting nutrients uptake, particularly Mg, which is crucial for chlorophyll synthesis. This effect was further amplified by earthworms, which improved AMF colonization (p < 0.05). In the soil N cycle, these organisms exerted opposing effects: AMF enhanced soil nitrification and earthworms reduced soil nitrate (NO3--N) reduction to jointly increase soil phyto-available N content (p < 0.05). Their combined action improved alfalfa N assimilation by restoring the protein synthesis pathway that is compromised by high Mo concentrations, specifically the activity of glutamine synthetase. These findings underscored the potential for soil microorganisms to mitigate N metabolic stress in legumes exposed to elevated Mo levels.