Phoma medicaginis colonizes Medicago truncatula root nodules and affects nitrogen fixation capacity

Abstract Background and Aims Nitrogen fixation in legumes requires tight control of carbon and nitrogen balance. Thus, legumes control nodule numbers via an autoregulation mechanism. ‘Autoregulation of nodulation’ mutants super-nodulate and are thought to be carbon-limited due to the high carbon-sink strength of excessive nodules. This study aimed to examine the effect of increasing carbon supply on the performance of super-nodulation mutants. Methods We compared the responses of Medicago truncatula super-nodulation mutants (sunn-4 and rdn1-1) and wild type to five CO2 levels (300-850 μmol mol -1). Nodule formation and N2 fixation were assessed in soil-grown plants at 18 and 42 days after sowing. Key results Shoot and root biomass, nodule number and biomass, nitrogenase activity and fixed-N per plant of all genotypes increased with increasing CO2 concentration and reached the maximum around 700 μmol mol -1. While the sunn-4 mutant showed strong growth-retardation compared to wild-type plants, elevated CO2 increased shoot biomass and total N content of rdn1-1 mutant up to two-fold. This was accompanied by a four-fold increase in nitrogen fixation capacity in the rdn1-1 mutant. Conclusions These results suggest that the super-nodulation phenotype per se did not limit growth. The additional nitrogen fixation capacity of the rdn1-1 mutant may enhance the benefit of elevated CO2 on plant growth and N2 fixation.

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Sinorhizobium meliloti Controls Nitric Oxide–Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-15-0118-r ◽

2015 ◽

Vol 28 (12) ◽

pp. 1353-1363 ◽

Cited By ~ 21

Author(s):

Pauline Blanquet ◽

Liliana Silva ◽

Olivier Catrice ◽

Claude Bruand ◽

Helena Carvalho ◽

...

Keyword(s):

Nitric Oxide ◽

Nitrogen Fixation ◽

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Molecular Mechanisms ◽

Root Nodules ◽

Plant Protein ◽

Post Translational Modification ◽

Model Legume ◽

Bacterial Mutants

Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction. Here, we investigate the contribution of the bacterial NO response to the modulation of a plant protein post-translational modification in nitrogen-fixing nodules. We made use of different bacterial mutants to finely modulate NO levels inside M. truncatula root nodules and to examine the consequence on tyrosine nitration of the plant glutamine synthetase, a protein responsible for assimilation of the ammonia released by nitrogen fixation. Our results reveal that S. meliloti possesses several proteins that limit inactivation of plant enzyme activity via NO-mediated post-translational modifications. This is the first demonstration that rhizobia can impact the course of nitrogen fixation by modulating the activity of a plant protein.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1500777112 ◽

2015 ◽

Vol 112 (49) ◽

pp. 15232-15237 ◽

Cited By ~ 77

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.

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Medicago truncatula Yellow Stripe-Like7 encodes a peptide transporter required for symbiotic nitrogen fixation

10.1101/2020.03.26.009159 ◽

2020 ◽

Author(s):

Rosario Castro-Rodríguez ◽

María Reguera ◽

Viviana Escudero ◽

Patricia Gil-Díez ◽

Julia Quintana ◽

...

Keyword(s):

Nitrogen Fixation ◽

Transition Metal ◽

Medicago Truncatula ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Metal Homeostasis ◽

Peptide Transporter ◽

Short Peptides ◽

Yellow Stripe ◽

Mutant Lines

ABSTRACTYellow Stripe-Like (YSL) proteins are a family of plant transporters typically involved in transition metal homeostasis. The substrate of three of the four YSL clades (clades I, II, and IV) are metal complexes with non-proteinogenic amino acid nicotianamine or its derivatives. No such transport capabilities have been shown for any member of the remaining clade (clade III), which is able to translocate short peptides across the membranes instead. The connection between clade III YSL members and metal homeostasis might have been masked by the functional redundancy characteristic of this family. This might have been circumvented in legumes through neofunctionalization of YSLs to ensure a steady supply of transition metals for symbiotic nitrogen fixation in root nodules. To test this possibility, Medicago truncatula clade III transporter MtYSL7 has been studied both when the plant was fertilized with ammonium nitrate or when nitrogen had to be provided by endosymbiotic rhizobia within the root nodules. MtYSL7 is a plasma membrane protein expressed in the vasculature and in the nodule cortex. This protein is able to transport short peptides into the cytosol, although none with known metal homeostasis roles. Reducing MtYSL7 expression levels resulted in diminished nitrogen fixation rates. In addition, nodules of mutant lines lacking YSL7 accumulated more copper and iron, the later the likely result of increased expression in roots of iron uptake and delivery genes. The available data is indicative of a role of MtYSL7, and likely other clade III YSLs, in transition metal homeostasis.ONE SENTENCE SUMMARYMedicago truncatula YSL7 is a peptide transporter required for symbiotic nitrogen fixation in legume nodules, likely controlling transition metal allocation to these organs.

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A comparison of the effect of straw incorporation and CO2 enrichment on the growth, nitrogen fixation and yield of soya beans

The Journal of Agricultural Science ◽

10.1017/s0021859600026733 ◽

1976 ◽

Vol 87 (1) ◽

pp. 181-185 ◽

Cited By ~ 13

Author(s):

K. Shivashankar ◽

K. Vlassak ◽

J. Livens

Keyword(s):

Nitrogen Fixation ◽

Grain Yield ◽

Root Nodules ◽

Co2 Enrichment ◽

Dry Weight ◽

Growth And Yield ◽

Culture Experiment ◽

Fixation Capacity ◽

The Mean ◽

Straw Incorporation

SUMMARYIn a glasshouse pot culture experiment, the effect of adding straw at 3 and 6 t/ha with and without CO2 enrichment treatments at 1000 mg/1 from flowering to the pod-filling stage in open top chambers was evaluated on the growth and yield of soya beans in relation to nitrogen fixation. N2-ase activity of the soya-bean root nodules as determined by the acetylene reduction technique indicated that (1) straw on average gave significantly 34 and 43% higher N2-ase activity at 3 and 6 t/ha respectively than the controls; (2) CO2 treatments on average increased the activity by 34% compared with the no CO2 treatments; and (3) the mean N2-ase activity nearly doubled from 9·7 μg/h/plant in the control to 18·7 and 19·7 μg/h/plant with straw incorporation in conjunction with CO2 enrichment. High correlations were observed between weight of nodules and dry weight of leaves, between dry weight of nodules and grain yield and between dry weight of leaves and grain yield. Incorporation of straw was found to be beneficial in increasing CO2 content of soil air and in improving the growth and development of the plants. This study lends support to a hypothesis that straw can be considered to provide a partial substitute for the expensive CO2 enrichment treatment for improving N2(C2H2) fixation capacity and thereby the general growth and yield of crops.

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Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation–Deficient Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-14-0407-r ◽

2015 ◽

Vol 28 (8) ◽

pp. 856-868 ◽

Cited By ~ 15

Author(s):

Claus Lang ◽

Sharon R. Long

Keyword(s):

Gene Expression ◽

Nitrogen Fixation ◽

Bacterial Infection ◽

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Cell Envelope ◽

Root Nodules ◽

Bacterial Cells ◽

Nitrogen Fixing ◽

Bacterial Genes

The bacterium Sinorhizobium meliloti interacts symbiotically with legume plant hosts such as Medicago truncatula to form nitrogen-fixing root nodules. During symbiosis, plant and bacterial cells differentiate in a coordinated manner, resulting in specialized plant cells that contain nitrogen-fixing bacteroids. Both plant and bacterial genes are required at each developmental stage of symbiosis. We analyzed gene expression in nodules formed by wild-type bacteria on six plant mutants with defects in nitrogen fixation. We observed differential expression of 482 S. meliloti genes with functions in cell envelope homeostasis, cell division, stress response, energy metabolism, and nitrogen fixation. We simultaneously analyzed gene expression in M. truncatula and observed differential regulation of host processes that may trigger bacteroid differentiation and control bacterial infection. Our analyses of developmentally arrested plant mutants indicate that plants use distinct means to control bacterial infection during early and late symbiotic stages.

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Structural Modeling and in planta Complementation Studies Link Mutated Residues of the Medicago truncatula Nitrate Transporter NPF1.7 to Functionality in Root Nodules

Frontiers in Plant Science ◽

10.3389/fpls.2021.685334 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yao-Chuan Yu ◽

Rebecca Dickstein ◽

Antonella Longo

Keyword(s):

Nitrogen Fixation ◽

Medicago Truncatula ◽

Conformational Changes ◽

Root Nodules ◽

Salt Bridge ◽

Peptide Transporter ◽

Nitrate Transport ◽

In Planta ◽

Transporter Family ◽

Major Facilitator

Symbiotic nitrogen fixation is a complex and regulated process that takes place in root nodules of legumes and allows legumes to grow in soils that lack nitrogen. Nitrogen is mostly acquired from the soil as nitrate and its level in the soil affects nodulation and nitrogen fixation. The mechanism(s) by which legumes modulate nitrate uptake to regulate nodule symbiosis remain unclear. In Medicago truncatula, the MtNPF1.7 transporter has been shown to control nodulation, symbiosis, and root architecture. MtNPF1.7 belongs to the nitrate/peptide transporter family and is a symporter with nitrate transport driven by proton(s). In this study we combined in silico structural predictions with in planta complementation of the severely defective mtnip-1 mutant plants to understand the role of a series of distinct amino acids in the transporter’s function. Our results support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. Results reveal that Motif A, a motif conserved among major facilitator transport (MFS) proteins, is essential for function. We hypothesize that it participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. Our results also question the existence of a putative TMH4-TMH10 salt bridge. These results are discussed in the context of potential nutrient transport functions for MtNPF1.7. Our findings add to the knowledge of the mechanism of alternative conformational changes as well as symport transport in NPFs and enhance our knowledge of the mechanisms for nitrate signaling.

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

10.1101/717983 ◽

2019 ◽

Cited By ~ 1

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.

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The Medicago truncatula Yellow Stripe1-Like3 gene is involved in vascular delivery of transition metals to root nodules

Journal of Experimental Botany ◽

10.1093/jxb/eraa390 ◽

2020 ◽

Vol 71 (22) ◽

pp. 7257-7269

Author(s):

Rosario Castro-Rodríguez ◽

Isidro Abreu ◽

María Reguera ◽

Lorena Novoa-Aponte ◽

Ana Mijovilovich ◽

...

Keyword(s):

Nitrogen Fixation ◽

Transition Metals ◽

Medicago Truncatula ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Apical Region ◽

Long Distance ◽

Metal Transporters ◽

Model Legume ◽

Iron And Zinc

Abstract Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living within the nodule cells, a process in which vascular transport is essential. As members of the Yellow Stripe-Like (YSL) family of metal transporters are involved in root to shoot transport, they should also be required for root to nodule metal delivery. The genome of the model legume Medicago truncatula encodes eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules and by cortical nodule cells. Reducing the expression level of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role for MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation.

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Medicago truncatula Yellow Stripe1-Like3 gene is involved in symbiotic nitrogen fixation

10.1101/2019.12.23.887448 ◽

2019 ◽

Author(s):

Rosario Castro-Rodríguez ◽

Isidro Abreu ◽

María Reguera ◽

Lorena Novoa-Aponte ◽

Ana Mijovilovich ◽

...

Keyword(s):

Nitrogen Fixation ◽

Medicago Truncatula ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Major Effect ◽

Apical Region ◽

Long Distance ◽

Metal Transporters ◽

Model Legume ◽

Iron And Zinc

AbstractSymbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living with the nodule cells, a process in which vascular transport is essential. As occurs in root-to-shoot transport, members of the Yellow Stripe-Like (YSL) family of metal transporters should also be required for root-to-nodule metal delivery. The genome of the model legume Medicago truncatula encodes for eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules, and by cortical nodule cells. Reducing expression levels of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role of MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation.HighlightMedicago truncatula YSL3 transporter is required for optimal nitrogen fixation in root nodules, mediating iron and zinc distribution in these organs.

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