scholarly journals Introduction of H2-Uptake Hydrogenase Genes Into Rhizobial Strains Improves Symbiotic Nitrogen Fixation in Vicia sativa and Lotus corniculatus Forage Legumes

2021 ◽  
Vol 3 ◽  
Author(s):  
Mariana Sotelo ◽  
Ana Claudia Ureta ◽  
Socorro Muñoz ◽  
Juan Sanjuán ◽  
Jorge Monza ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Rhizobium Leguminosarum ◽  
Symbiotic Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Root Nodules ◽  
Lotus Corniculatus ◽  
Vicia Sativa ◽  
Uptake Hydrogenase ◽  
Forage Crops ◽  
Mesorhizobium Loti

Biological nitrogen fixation by the Rhizobium-legume symbiosis allows the conversion of atmospheric nitrogen into ammonia within root nodules mediated by the nitrogenase enzyme. Nitrogenase activity results in the evolution of hydrogen as a result of a side reaction intrinsic to the activity of this enzyme. Some rhizobia, and also other nitrogen fixers, induce a NiFe uptake hydrogenase (Hup) to recycle hydrogen produced by nitrogenase, thus improving the efficiency of the nitrogen fixation process. In this work we report the generation and symbiotic behavior of hydrogenase-positive Rhizobium leguminosarum and Mesorhizobium loti strains effective in vetch (Vicia sativa) and birsfoot trefoil (Lotus corniculatus) forage crops, respectively. The ability of hydrogen recycling was transferred to these strains through the incorporation of hup minitransposon TnHB100, thus leading to full recycling of hydrogen in nodules. Inoculation of Vicia and Lotus plants with these engineered strains led to significant increases in the levels of nitrogen incorporated into the host legumes. The level of improvement of symbiotic performance was dependent on the recipient strain and also on the legume host. These results indicate that hydrogen recycling has the potential to improve symbiotic nitrogen fixation in forage plants.

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 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 The Biological fertilizer research results of Seabuckthorn (Hippophae rhamnoides)

2021 ◽  
Vol 31 (3) ◽  
pp. 116-119
Author(s):  
Galt Lantuu ◽  
Ninj Badam ◽  
Ankhtuya Mijidorj
Keyword(s):  
Nitrogen Fixation ◽  
Root Nodule ◽  
Symbiotic Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Root Nodules ◽  
Hippophae Rhamnoides ◽  
Fresh Weight ◽  
Hippophaë Rhamnoides ◽  
Air Temperatures ◽  
Biological Fertilizer

The seabuckthorn subspecies mongolica used in this research. Seabuckthorn (Нippophae ramnoides L.) root nodule research focused on symbiotic nitrogen fixation bacteria that could aid in the cultivation of this species. Seabuckthorn root nodules have Frankia actinorhizal microorganisms.Under nitrogen-free conditions, seabuckthorn seedlings inoculated with a homogenate of root nodules that grew normally and the fresh weight of root nodules had positively correlated with plant growth. In the field, nitrogenase activity in root nodules was high in the period from June to September,when air temperatures were high and photosynthesis was active. Also we investigated the effect of nitrate on nitrogenase activity in seabuckthorn root nodules. Root nodules with many lobes were found in mature seabuckthorn trees grown in the field. Чацаргана (Hippophae Rhamnoides) тарьсан талбайд биологийн бордоо хэрэглэсэн дүнгээс Чацарганы (Hippophae rhamnoides) үндэсний булцуунаас азот шингээгч бактерийг ялган өсгөвөрлөх, шингээх идэвхи нь өөрчлөгдөж буй эсэхийг лабораторийн нөхцөлд турших, биобордоо бэлтгэж чацарганы тарьц суулгацыг бордож туршин үр дүнг хяналтын ургамалтай харьцуулж гаргахад  VI,VII,VIII сард өссөн үзүүлэлттэй буюу 3,74-7,1 х 106 бактерийн  эсээр нэмэгдсэн байна.  Түлхүүр үг:  Булцуу, биологийн бордоо, эрдэс бордоо,бичил биетэн

scholarly journals Antioxidant ability of glutaredoxins and their role in symbiotic nitrogen fixation in Rhizobium leguminosarum bv. viciae 3841

2020 ◽  
Author(s):  
Qian Zou ◽  
Yanlin Zhou ◽  
Guojun Cheng ◽  
Yang Peng ◽  
Sha Luo ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Quantitative Proteomics ◽  
Rhizobium Leguminosarum ◽  
Symbiotic Nitrogen Fixation ◽  
Proteome Analysis ◽  
Wild Type ◽  
Triple Mutant ◽  
Antioxidant Proteins ◽  
Mixed Disulfides ◽  
Nodule Symbiosis

Glutaredoxins (Grx) are redoxin family proteins that reduce disulfides and mixed disulfides between glutathione and proteins. Rhizobium leguminosarum bv. Viciae 3841 contains three genes coding for glutaredoxins: RL4289 (grxA) codes for a dithiolic glutaredoxin, RL2615 (grxB) codes for a monothiol glutaredoxin, while RL4261 (grxC) codes for a glutaredoxin-like NrdH protein. We generated mutants interrupted in one, two, or three glutaredoxin genes. These mutants had no obvious differences in growth phenotypes from the wild type RL3841. However, while a mutant of grxC did not affect the antioxidant or symbiotic capacities of R. leguminosarum, grxA-derived or grxB mutants decreased antioxidant and nitrogen fixation capacities. Furthermore, grxA mutants were severely impaired in rhizosphere colonization, and formed smaller nodules with defects of bacteroid differentiation, whereas nodules induced by grxB mutants contained abnormally thick cortices and prematurely senescent bacteroids. The grx triple mutant had the greatest defect in antioxidant and symbiotic capacities of R. leguminosarum and quantitative proteomics revealed it had 56 up-regulated and 81 down-regulated proteins relative to wildtype. Of these proteins, twenty-eight are involved in transporter activity, twenty are related to stress response and virulence, and sixteen are involved in amino acid metabolism. Overall, R. leguminosarum glutaredoxins behave as antioxidant proteins mediating root nodule symbiosis. IMPORTANCE Glutaredoxin catalyzes glutathionylation/deglutathionylation reactions, protects SH-groups from oxidation and restores functionally active thiols. Three glutaredoxins exist in R. leguminosarum and their properties were investigated in free-living bacteria and during nitrogen-fixing symbiosis. All the glutaredoxins were necessary for oxidative stress defense. Dithiol GrxA affects nodulation and nitrogen fixation of bacteroids by altering deglutathionylation reactions, monothiol GrxB is involved in symbiotic nitrogen fixation by regulating Fe-S cluster biogenesis, and GrxC may participate in symbiosis by an unknown mechanism. Proteome analysis provides clues to explain the differences between the grx triple mutant and wild-type nodules.

scholarly journals Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Michael J. Mitsch ◽  
George C. diCenzo ◽  
Alison Cowie ◽  
Turlough M. Finan
Keyword(s):  
Nitrogen Fixation ◽  
Carbon Source ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Symbiotic Nitrogen Fixation ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Changes ◽  
Symbiotic Properties

ABSTRACTSymbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C4-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) inSinorhizobium melilotiRm1021dctmutants unable to transport C4-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF withMedicago sativa, with acetylene reduction rates of up to 50% those of plants inoculated with wild-typeS. meliloti.Rhizobium leguminosarumbv. viciae 3841dctmutants unable to transport C4-dicarboxylates but expressing themaePtransporter had strong symbiotic properties, withPisum sativumplants inoculated with these strains appearing similar to plants inoculated with wild-typeR. leguminosarum. This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combinedP. sativum-R. leguminosarumnodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses,l-malate is likely the primary C4-dicarboxylate provided to the bacterium.IMPORTANCESymbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.

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