3-Phosphoglycerate dehydrogenase in Mesorhizobioum loti is essential for maintaining symbiotic nitrogen fixation of Lotus japonicus root nodules

Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix− mutant, apn1 (aspartic peptidase nodule-induced 1), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix− phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix− nodules on apn1 roots, and identified TONO mutants that formed Fix+ nodules on apn1. The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix+ nodules on apn1 roots, resulted in Fix− nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules.

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The Sulfate Transporter SST1 Is Crucial for Symbiotic Nitrogen Fixation in Lotus japonicus Root Nodules

The Plant Cell ◽

10.1105/tpc.104.030106 ◽

2005 ◽

Vol 17 (5) ◽

pp. 1625-1636 ◽

Cited By ~ 134

Author(s):

Lene Krusell ◽

Katja Krause ◽

Thomas Ott ◽

Guilhem Desbrosses ◽

Ute Krämer ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Lotus Japonicus ◽

Sulfate Transporter

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

10.12681/eadd/37826 ◽

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.

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CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

New Phytologist ◽

10.1111/nph.16077 ◽

2019 ◽

Vol 224 (2) ◽

pp. 818-832 ◽

Cited By ~ 13

Author(s):

Longlong Wang ◽

Maria Carmen Rubio ◽

Xian Xin ◽

Baoli Zhang ◽

Qiuling Fan ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Lotus Japonicus

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The SNARE Protein SYP71 Expressed in Vascular Tissues Is Involved in Symbiotic Nitrogen Fixation in Lotus japonicus Nodules

PLANT PHYSIOLOGY ◽

10.1104/pp.112.200782 ◽

2012 ◽

Vol 160 (2) ◽

pp. 897-905 ◽

Cited By ~ 24

Author(s):

Tsuneo Hakoyama ◽

Ryo Oi ◽

Kazuya Hazuma ◽

Eri Suga ◽

Yuka Adachi ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Lotus Japonicus ◽

Snare Protein ◽

Vascular Tissues

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Absence of Symbiotic Leghemoglobins Alters Bacteroid and Plant Cell Differentiation During Development of Lotus japonicus Root Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-7-0800 ◽

2009 ◽

Vol 22 (7) ◽

pp. 800-808 ◽

Cited By ~ 36

Author(s):

Thomas Ott ◽

John Sullivan ◽

Euan K. James ◽

Emmanouil Flemetakis ◽

Catrin Günther ◽

...

Keyword(s):

Gene Expression ◽

Nitrogen Fixation ◽

Cell Differentiation ◽

Root Nodules ◽

Lotus Japonicus ◽

Nodule Development ◽

Primary And Secondary Metabolism ◽

Bacterial Differentiation ◽

Plant Genes ◽

Bacterial Genes

During development of legume root nodules, rhizobia and their host plant cells undergo profound differentiation, which is underpinned by massive changes in gene expression in both symbiotic partners. Oxygen concentrations in infected and surrounding uninfected cells drop precipitously during nodule development. To assess what effects this has on plant and bacterial cell differentiation and gene expression, we used a leghemoglobin-RNA-interference (LbRNAi) line of Lotus japonicus, which is devoid of leghemoglobins and has elevated levels of free-oxygen in its nodules. Bacteroids in LbRNAi nodules showed altered ultrastructure indicating changes in bacterial differentiation. Transcript analysis of 189 plant and 192 bacterial genes uncovered many genes in both the plant and bacteria that were differentially regulated during nodulation of LbRNAi plants compared with the wild type (containing Lb and able to fix nitrogen). These included fix and nif genes of the bacteria, which are involved in microaerobic respiration and nitrogen fixation, respectively, and plant genes involved in primary and secondary metabolism. Metabolite analysis revealed decreased levels of many amino acids in nodules of LbRNAi plants, consistent with the defect in symbiotic nitrogen fixation of this line.

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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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Induction and Spatial Organization of Polyamine Biosynthesis During Nodule Development in Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.12.1283 ◽

2004 ◽

Vol 17 (12) ◽

pp. 1283-1293 ◽

Cited By ~ 24

Author(s):

Emmanouil Flemetakis ◽

Rodica C. Efrose ◽

Guilhem Desbrosses ◽

Maria Dimou ◽

Costas Delis ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Expression Patterns ◽

Lotus Japonicus ◽

Cyclin D3 ◽

Nodule Development ◽

Cdna Clones ◽

Long Distance ◽

Polyamine Biosynthesis ◽

Transcript Levels

Putrescine and other polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase (ADC; EC 4.1.1.19), the other with ornithine decarboxylase (ODC; EC 4.1.1.17). Metabolite profiling of nitrogen-fixing Lotus japonicus nodules, using gas chromatography coupled to mass spectrometry, revealed a two- to sixfold increase in putrescine levels in mature nodules compared with other organs. Genes involved in polyamine biosynthesis in L. japonicus nodules were identified by isolating cDNA clones encoding ADC (LjADC1) and ODC (LjODC) from a nodule library. Searches of the public expressed sequence tag databases revealed the presence of a second gene encoding ADC (LjADC2). Real-time reverse-transcription-polymerase chain reaction analysis showed that LjADC1 and LjADC2 were expressed throughout the plant, while LjODC transcripts were detected only in nodules and roots. Induction of LjODC and LjADC gene expression during nodule development preceded symbiotic nitrogen fixation. Transcripts accumulation was maximal at 10 days postinfection, when a 6.5-fold increase in the transcript levels of LjODC was observed in comparison with the uninfected roots, while a twofold increase in the transcript levels of LjADC1 and LjADC2 was detected. At later stages of nodule development, transcripts for ADC drastically declined, while in the case of ODC, transcript accumulation was higher than that in roots until after 21 days postinfection. The expression profile of genes involved in putrescine biosynthesis correlated well with the expression patterns of genes involved in cell division and expansion, including a L. japonicus Cyclin D3 and an α-expansin gene. Spatial localization of LjODC and LjADC1 gene transcripts in developing nodules revealed that both transcripts were expressed in nodule inner cortical cells and in the central tissue. High levels of LjADC1 transcripts were also observed in both nodule and connecting root vascular tissue, suggesting that putrescine and other polyamines may be subject to long-distance transport. Our results indicate that polyamines are primarily involved in physiological and cellular processes involved in nodule development, rather than in processes that support directly symbiotic nitrogen fixation and assimilation.

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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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Recurrent mutualism breakdown events in a legume rhizobia metapopulation

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2019.2549 ◽

2020 ◽

Vol 287 (1919) ◽

pp. 20192549 ◽

Cited By ~ 3

Author(s):

Kelsey A. Gano-Cohen ◽

Camille E. Wendlandt ◽

Khadija Al Moussawi ◽

Peter J. Stokes ◽

Kenjiro W. Quides ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Nitrogen Concentration ◽

Phylogenetic Reconstruction ◽

Nodulation Capacity ◽

Host Growth ◽

Native Legume ◽

Fitness Benefits ◽

Mutualism Breakdown

Bacterial mutualists generate major fitness benefits for eukaryotes, reshaping the host phenotype and its interactions with the environment. Yet, microbial mutualist populations are predicted to generate mutants that defect from providing costly services to hosts while maintaining the capacity to exploit host resources. Here, we examined the mutualist service of symbiotic nitrogen fixation in a metapopulation of root-nodulating Bradyrhizobium spp . that associate with the native legume Acmispon strigosus . We quantified mutualism traits of 85 Bradyrhizobium isolates gathered from a 700 km transect in California spanning 10 sampled A. strigosus populations. We clonally inoculated each Bradyrhizobium isolate onto A. strigosus hosts and quantified nodulation capacity and net effects of infection, including host growth and isotopic nitrogen concentration. Six Bradyrhizobium isolates from five populations were categorized as ineffective because they formed nodules but did not enhance host growth via nitrogen fixation. Six additional isolates from three populations failed to form root nodules. Phylogenetic reconstruction inferred two types of mutualism breakdown, including three to four independent losses of effectiveness and five losses of nodulation capacity on A. strigosus . The evolutionary and genomic drivers of these mutualism breakdown events remain poorly understood.

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