scholarly journals Medicago truncatulaFerroportin2 mediates iron import into nodule symbiosomes

2019 ◽  
Author(s):  
Viviana Escudero ◽  
Isidro Abreu ◽  
Manuel Tejada-Jiménez ◽  
Elena Rosa-Núñez ◽  
Julia Quintana ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Symbiotic Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Specific Gene ◽  
Nitrogen Fixing ◽  
Loss Of Function ◽  
Model Legume ◽  
Endosymbiotic Bacteria ◽  
Signal Transduction Proteins ◽  
Iron Delivery

ABSTRACTIron is an essential cofactor for symbiotic nitrogen fixation. It is required by many of the enzymes facilitating the conversion of N2into NH4+by endosymbiotic bacteria living within root nodule cells, including signal transduction proteins, O2homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen-fixing nodule cells. Model legumeMedicago truncatula Ferroportin2(MtFPN2) is a nodule-specific gene that encodes an iron-efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature, and in the symbiosome membranes that contain the nitrogen-fixing bacteria in the differentiation and early-fixation zones of the nodules. Loss-of-function ofMtFPN2leads to altered iron distribution and speciation in nodules, which causes a reduction in nitrogenase activity and in biomass production. Using promoters with different tissular activity to driveMtFPN2expression inMtFPN2mutants, we determined that MtFPN2-facilitated iron delivery across symbiosomes is essential for symbiotic nitrogen fixation, while its presence in the vasculature does not seem to play a major role in in the conditions tested.

scholarly journals Nicotianamine synthase 2 is required for symbiotic nitrogen fixation in Medicago truncatula nodules

2019 ◽  
Author(s):  
Viviana Escudero ◽  
Isidro Abreu ◽  
Eric del Sastre ◽  
Manuel Tejada-Jiménez ◽  
Camile Larue ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Medicago Truncatula ◽  
Symbiotic Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Root Nodules ◽  
Long Distance ◽  
Metal Transporters ◽  
Model Legume ◽  
Fluorescence Studies ◽  
Iron Delivery

SUMMARYSymbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires of relatively large levels of transition metals. These elements act as cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process participated by a number of metal transporters and small organic molecules that mediate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this non-proteinogenic amino acid forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, a phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would be the result of the altered iron distribution in nas2-1 nodules, as indicated by X-ray fluorescence studies. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation.Significance StatementNicotianamine synthesis mediated by MtNAS2 is important for iron allocation for symbiotic nitrogen fixation by rhizobia in Medicago truncatula root nodules.

scholarly journals Αναπρογραμματισμός του μεταβολισμού του Lotus japonicus σε μεταγραφικό, βιοχημικό και μεταβολομικό επίπεδο κατά τη συμβιωτική αζωτοδέσμευση

2016 ◽  
Author(s):  
Χρυσάνθη Καλλονιάτη
Keyword(s):  
Nitrogen Fixation ◽  
Metabolite Profiling ◽  
Symbiotic Nitrogen Fixation ◽  
Sulfur Metabolism ◽  
Lotus Japonicus ◽  
Nitrogen Fixing ◽  
Model Legume ◽  
Whole Plant ◽  
Plant Level ◽  
Molecular And Biochemical Mechanisms

Symbiotic nitrogen fixation in legumes takes place in specialized organs called nodules,which become the main source of assimilated nitrogen for the whole plant. Symbiotic nitro‐gen fixation requires exquisite integration of plant and bacterial metabolism and involvesglobal changes in gene expression and metabolite accumulation in both rhizobia and thehost plant. In order to study the metabolic changes mediated by symbiotic nitrogen fixationon a whole‐plant level, metabolite levels were profiled by gas chromatography–mass spec‐trometry in nodules and non‐symbiotic organs of Lotus japonicus plants uninoculated or in‐oculated with M. loti wt,  ΔnifA or  ΔnifH fix‐ strains. Furthermore, transcriptomic andbiochemical approaches were combined to study sulfur metabolism in nodules, its link tosymbiotic nitrogen fixation, and the effect of nodules on whole‐plant sulfur partitioning andmetabolism. It is well established that nitrogen and sulfur (S) metabolism are tightly en‐twined and sulfur is required for symbiotic nitrogen fixation, however, little is known aboutthe molecular and biochemical mechanisms governing sulfur uptake and assimilation duringsymbiotic nitrogen fixation. Transcript profiling in Lotus japonicus was combined with quan‐tification of S‐metabolite contents and APR activity in nodules and in non‐symbiotic organsof plants uninoculated or inoculated with M. loti wt, ΔnifA or ΔnifH fix‐ strains. Moreover,sulfate uptake and its distribution into different plant organs were analyzed and 35S‐flux intodifferent S‐pools was monitored. Metabolite profiling revealed that symbiotic nitrogen fixa‐tion results in dramatic changes of many aspects of primary and secondary metabolism innodules which leads to global reprogramming of metabolism of the model legume on awhole‐plant level. Moreover, our data revealed that nitrogen fixing nodules represent athiol‐rich organ. Their high APR activity and 35S‐flux into cysteine and its metabolites in com‐bination with the transcriptional up‐regulation of several genes involved in sulfur assimila‐tion highlight the function of nodules as a new site of sulfur assimilation. The higher thiolcontent observed in non‐symbiotic organs of nitrogen fixing plants in comparison touninoculated plants cannot be attributed to local biosynthesis, indicating that nodules couldserve as a novel source of reduced sulfur for the plant, which triggers whole‐plant repro‐gramming of sulfur metabolism. Interestingly, the changes in metabolite profiling and theenhanced thiol biosynthesis in nodules and their impact on the whole‐plant sulfur, carbonand nitrogen economy are dampened in fix‐ plants, which in most respects metabolically re‐sembled uninoculated plants, indicating a strong interaction between nitrogen fixation andsulfur and carbon metabolism.

scholarly journals Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

2009 ◽  
Vol 191 (8) ◽  
pp. 2593-2600 ◽  
Author(s):  
Chrysanthi Kalloniati ◽  
Daniela Tsikou ◽  
Vasiliki Lampiri ◽  
Mariangela N. Fotelli ◽  
Heinz Rennenberg ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Carbonic Anhydrase ◽  
Mutant Strain ◽  
Symbiotic Nitrogen Fixation ◽  
Plant Traits ◽  
Nitrogenase Activity ◽  
Nodule Development ◽  
Free Living ◽  
Model Legume ◽  
Mesorhizobium Loti

ABSTRACT Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active α-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and δ13C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH3 protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule δ13C abundance suggest the recycling of at least part of the HCO3 − produced by CAA1.

scholarly journals MtMOT1.2 is responsible for molybdate supply toMedicago truncatulanodules

2018 ◽  
Author(s):  
Patricia Gil-Díez ◽  
Manuel Tejada-Jiménez ◽  
Javier León-Mediavilla ◽  
Jiangqi Wen ◽  
Kirankumar S. Mysore ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Symbiotic Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Reductase Activity ◽  
Root Nodules ◽  
Loss Of Function ◽  
Intracellular Membrane ◽  
Wild Type ◽  
Endodermal Cells ◽  
Iron Molybdenum Cofactor

ABSTRACTSymbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. MtMOT1.2 is aMedicago truncatulaMOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. A loss-of-functionmot1.2-1mutant showed reduced growth compared to wild-type plants when nitrogen fixation was required, but not when nitrogen was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with a wild-typeMtMOT1.2gene or molybdate-fortification of the nutrient solution, both restored wild-type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.

scholarly journals Medicago truncatulacopper transporter 1 (MtCOPT1) delivers copper for symbiotic nitrogen fixation

2017 ◽  
Author(s):  
Marta Senovilla ◽  
Rosario Castro-Rodríguez ◽  
Isidro Abreu ◽  
Viviana Escudero ◽  
Igor Kryvoruchko ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Medicago Truncatula ◽  
Symbiotic Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Yeast Complementation ◽  
Copper Transporter ◽  
Model Legume ◽  
Essential Nutrient ◽  
Early Fixation

Summary• Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper transporter would introduce it into the cell to synthesize cupro-proteins.• COPT family members in model legumeMedicago truncatulawere identified and their expression determined. Yeast complementation assays, confocal microscopy, and phenotypical characterization of aTnt1insertional mutant line were carried out in the nodule-specificM.truncatulaCOPT family member.•Medicago truncatulagenome encodes eight COPT transporters.MtCOPT1(Medtr4g019870) is the only nodule-specificCOPTgene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a copper-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation ofMtCOPT1results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a copper-dependent function, such as cytochrome oxidase activity incopt1-1bacteroids.• These data are consistent with a model in which MtCOPT1 transports copper from the apoplast into nodule cells to provide copper for essential metabolic processes associated with symbiotic nitrogen fixation.

scholarly journals Plasmids Related to the Symbiotic Nitrogen Fixation Are Not Only Cooperated Functionally but Also May Have Evolved over a Time Span in Family Rhizobiaceae

2020 ◽  
Vol 12 (11) ◽  
pp. 2002-2014
Author(s):  
Ling-Ling Yang ◽  
Zhao Jiang ◽  
Yan Li ◽  
En-Tao Wang ◽  
Xiao-Yang Zhi
Keyword(s):  
Nitrogen Fixation ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Symbiotic Nitrogen Fixation ◽  
Gene Families ◽  
Time Span ◽  
Soil Conditions ◽  
Gene Gain ◽  
Nitrogen Fixing ◽  
Pan Genome

Abstract Rhizobia are soil bacteria capable of forming symbiotic nitrogen-fixing nodules associated with leguminous plants. In fast-growing legume-nodulating rhizobia, such as the species in the family Rhizobiaceae, the symbiotic plasmid is the main genetic basis for nitrogen-fixing symbiosis, and is susceptible to horizontal gene transfer. To further understand the symbioses evolution in Rhizobiaceae, we analyzed the pan-genome of this family based on 92 genomes of type/reference strains and reconstructed its phylogeny using a phylogenomics approach. Intriguingly, although the genetic expansion that occurred in chromosomal regions was the main reason for the high proportion of low-frequency flexible gene families in the pan-genome, gene gain events associated with accessory plasmids introduced more genes into the genomes of nitrogen-fixing species. For symbiotic plasmids, although horizontal gene transfer frequently occurred, transfer may be impeded by, such as, the host’s physical isolation and soil conditions, even among phylogenetically close species. During coevolution with leguminous hosts, the plasmid system, including accessory and symbiotic plasmids, may have evolved over a time span, and provided rhizobial species with the ability to adapt to various environmental conditions and helped them achieve nitrogen fixation. These findings provide new insights into the phylogeny of Rhizobiaceae and advance our understanding of the evolution of symbiotic nitrogen fixation.

Nitrogen fixation

1976 ◽  
Vol 274 (934) ◽  
pp. 341-358 ◽  
Keyword(s):  
Nitrogen Fixation ◽  
Metabolic Regulation ◽  
Higher Plants ◽  
Focal Point ◽  
Nitrogenase Activity ◽  
Nitrogen Fixing ◽  
Blue Green Algae ◽  
International Biological Programme ◽  
The 1960S ◽  
Biological Programme

The International Biological Programme served as a focal point for studies on biological nitrogen fixation during the 1960s. The introduction of the acetylene reduction technique for measuring nitrogenase activity in the field led to estimates becoming available of the contribution of lichens, blue-green algae, nodulated non-legumes and bacterial-grass associations, as well as of legumes. Other studies carried out on the physiology and biochemistry of the process led to the eventual purification and characterization of the nitrogenase enzyme. These studies, collectively, provided the springboard for current work, so essential in view of the present energy crisis, on how to increase the use and efficiency of nitrogen-fixing plants, on the metabolic regulation of the nitrogenase enzyme and on the genetics of the nitrogen-fixing process, both in higher plants and in free-living micro-organisms.

Phenotypic reversion of nitrogenase in pleiotropic mutants of Rhizobium meliloti

1979 ◽  
Vol 25 (3) ◽  
pp. 298-301 ◽  
Author(s):  
Ilona Barabás ◽  
Tibor Sik
Keyword(s):  
Amino Acids ◽  
Nitrogen Fixation ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Symbiotic Nitrogen Fixation ◽  
Rhizobium Meliloti ◽  
Nitrogenase Activity ◽  
Reductase Activity ◽  
Amino Acid Utilization ◽  
Phenotypic Reversion

In two out of three pleiotropic mutants of Rhizobium meliloti, defective in nitrate reductase induced by amino acid utilization in vegetative bacteria and in symbiotic nitrogen fixation, nitrogenase activity could be restored completely by purines and partially by the amino acids L-glutamate, L-aspartate, L-glutamine, and L-asparagine. The compounds restoring effectiveness in nitrogen fixation did not restore nitrate reductase activity in vegetative bacteria. The restoration of effectiveness supports our earlier conclusion that the mutation is not in the structural gene for a suggested common subunit of nitrogenase and nitrate reductase.

scholarly journals Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

2015 ◽  
Vol 112 (49) ◽  
pp. 15232-15237 ◽  
Author(s):  
Beatrix Horváth ◽  
Ágota Domonkos ◽  
Attila Kereszt ◽  
Attila Szűcs ◽  
Edit Ábrahám ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Medicago Truncatula ◽  
Symbiotic Nitrogen Fixation ◽  
Root Nodules ◽  
Nitrogen Fixing ◽  
In Planta ◽  
Functional Roles ◽  
Absolute Requirement ◽  
Cell Layers

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

NON-SYMBIOTIC NITROGEN FIXATION IN SOME QUEBEC SOILS

1965 ◽  
Vol 11 (1) ◽  
pp. 29-38 ◽  
Author(s):  
P-C. Chang ◽  
R. Knowles
Keyword(s):  
Nitrogen Fixation ◽  
Symbiotic Nitrogen Fixation ◽  
Soil Types ◽  
Anaerobic Incubation ◽  
Nitrogen Fixing ◽  
Free Living ◽  
Aerobic Conditions ◽  
Bacterial Counts ◽  
Facultatively Anaerobic ◽  
Nitrogen Fixers

The occurrence of free-living nitrogen fixers, the potential for nitrogen fixation, and the correlation between the nitrogen-fixing capacities of the soils and bacterial counts were studied using representative Quebec soils.Clostridium occurred more frequently than did Azotobacter. Studies with N15showed that nitrogen fixation was more frequent under anaerobic than under aerobic conditions in all the soil types studied in their unamended state. The addition of glucose stimulated nitrogen fixation. During anaerobic incubation, nitrogen fixation was found to be correlated significantly with the increase in numbers of both total aerobes and Clostridia. The results suggested that facultatively anaerobic nitrogen fixers, and aerobic nitrogen fixers other than Azotobacter, were present.

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