scholarly journals Inhibition of Glutamine Synthetase by Phosphinothricin Leads to Transcriptome Reprograming in Root Nodules of Medicago truncatula

2012 ◽  
Vol 25 (7) ◽  
pp. 976-992 ◽  
Author(s):  
Ana R. Seabra ◽  
Patrícia A. Pereira ◽  
Jörg D. Becker ◽  
Helena G. Carvalho
Keyword(s):  
Glutamine Synthetase ◽  
Medicago Truncatula ◽  
Root Nodule ◽  
Higher Plants ◽  
Cell Metabolism ◽  
Root Nodules ◽  
Organic N ◽  
N Availability ◽  
Time Points ◽  
N Assimilation

Glutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.

Studies on possible routes of ammonium assimilation in soybean root nodule bacteroids

1973 ◽  
Vol 19 (12) ◽  
pp. 1493-1499 ◽  
Author(s):  
Stanley D. Dunn ◽  
Robert V. Klucas
Keyword(s):  
Glutamine Synthetase ◽  
Nitrogen Assimilation ◽  
Reductive Amination ◽  
Root Nodule ◽  
Root Nodules ◽  
Ammonium Assimilation ◽  
Kinetic Properties ◽  
Transferase Activity ◽  
Soybean Root

Glutamine amide–2-oxoglutarate aminotransferase NAD+ oxidoreductase (GOGAT), glutamine synthetase (GS), glutamate dehydrogenase (GD), and alanine dehydrogenase (AD) were studied in soybean root nodules. GS, GOGAT, and AD were present in bacteroids at levels that could account for ammonium assimilation, but GD activity was quite low. The total activities of GS and GD were higher in the cytosol than in the bacteroids by factors of 20 and 7, respectively, whereas GOGAT was not detected in the cytosol. GS (transferase activity) was inhibited by alanine, CTP, glycine, and tryptophan at 5 mM but was relatively unaffected by asparagine, aspartic acid, CMP, glucosamine, and histidine at 5 mM. GOGAT activity was unaffected by ATP, ADP, 8-hydroxyquinoline, and 1,10-phenanthroline but was inhibited by EDTA, citrate, and parachloromercuribenzoate. GOGAT activity (reductive amination) was also inhibited 97% by preincubation with 10−4 M azaserine for 30 min but GD activity was inhibited only 13%. The apparent Km values for NH4+ by AD was 7.4 × 10−3 M and by GD was 7.3 × 10−2 M while for glutamine by GOGAT it was 9.3 × 10−5 M. Activities and kinetic properties for these enzymes may suggest potential routes of nitrogen assimilation in vivo.

scholarly journals Rhizobium etli Mutant Modulates Carbon and Nitrogen Metabolism in Phaseolus vulgaris Nodules

2002 ◽  
Vol 15 (7) ◽  
pp. 728-733 ◽  
Author(s):  
Sonia Silvente ◽  
Lourdes Blanco ◽  
Alberto Camas ◽  
José-Luis Ortega ◽  
Mario Ramírez ◽  
...  
Keyword(s):  
Root Nodule ◽  
Root Nodules ◽  
Glutamate Synthase ◽  
Rhizobium Etli ◽  
Wild Type ◽  
Respiratory Capacity ◽  
Carbon And Nitrogen ◽  
N Assimilation ◽  
Mutant Strains ◽  
Carbon And Nitrogen Metabolism

The aim of this study was to evaluate the biochemical events in root nodules which lead to increased yield when bean is inoculated with a Rhizobium etli mutant (CFN037) having increased respiratory capacity. CFN037-inoculated plants had 22% more nitrogen (N) than did wild-type (CE3)-inoculated plants. Root nodule enzymes involved in nodule carbon and nitrogen assimilation as well as in ureides and amides synthesis were assessed in plants inoculated with CFN037 and the CE3. Our results show that the xylem ureides content was lower while that of amino acids was higher in CFN037- compared with CE3-inoculated plants. Supporting these results, enzymes involved in ureide synthesis were reduced while activity of aspartate aminotransferase, glutamate synthase, sucrose synthase, and glucose-6-P dehydrogenase were increased in CFN037- induced nodules. Glutamate synthase and phosphoenolpyruvate carboxylase transcripts were detected early in the development of nodules induced by CFN037 compared with CE3. However, plants inoculated with strain CE3-vhb, which express the Vitreoscilla sp. hemoglobin and also displays increased respiratory capacity, did not have altered ureide transport in N2-fixing plants. The data suggest that inoculation with special selected mutant strains of R. etli can modulate nodule N assimilation and N transport compounds.

scholarly journals A Novel Nuclear Protein Interacts With the Symbiotic DMI3 Calcium- and Calmodulin-Dependent Protein Kinase of Medicago truncatula

2007 ◽  
Vol 20 (8) ◽  
pp. 912-921 ◽  
Author(s):  
Elsa Messinese ◽  
Jeong-Hwan Mun ◽  
Li Huey Yeun ◽  
Dhileepkumar Jayaraman ◽  
Pierre Rougé ◽  
...  
Keyword(s):  
Medicago Truncatula ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Root Nodules ◽  
Yellow Fluorescent Protein ◽  
Coiled Coil ◽  
Am Fungi ◽  
Homologous Proteins ◽  
Root Cells ◽  
Am Symbiosis

Many higher plants establish symbiotic relationships with arbuscular mycorrhizal (AM) fungi that improve their ability to acquire nutrients from the soil. In addition to establishing AM symbiosis, legumes also enter into a nitrogen-fixing symbiosis with bacteria known as rhizobia that results in the formation of root nodules. Several genes involved in the perception and transduction of bacterial symbiotic signals named “Nod factors” have been cloned recently in model legumes through forward genetic approaches. Among them, DMI3(Doesn't Make Infections 3) is a calcium- and calmodulin-dependent kinase required for the establishment of both nodulation and AM symbiosis. We have identified, by a yeast two-hybrid system, a novel protein interacting with DMI3 named IPD3 (Interacting Protein of DMI3). IPD3 is predicted to interact with DMI3 through a C-terminal coiled-coil domain. Chimeric IPD3∷GFP is localized to the nucleus of transformed Medicago truncatula root cells, in which split yellow fluorescent protein assays suggest that IPD3 and DMI3 physically interact in Nicotiana benthamiana. Like DMI3, IPD3 is extremely well conserved among the angiosperms and is absent from Arabidopsis. Despite this high level of conservation, none of the homologous proteins have a demonstrated biological or biochemical function. This work provides the first evidence of the involvement of IPD3 in a nuclear interaction with DMI3.

scholarly journals Glutamine Synthetase Is a Molecular Target of Nitric Oxide in Root Nodules of Medicago truncatula and Is Regulated by Tyrosine Nitration

2011 ◽  
Vol 157 (3) ◽  
pp. 1505-1517 ◽  
Author(s):  
Paula M. Melo ◽  
Liliana S. Silva ◽  
Isa Ribeiro ◽  
Ana R. Seabra ◽  
Helena G. Carvalho
Keyword(s):  
Nitric Oxide ◽  
Glutamine Synthetase ◽  
Medicago Truncatula ◽  
Root Nodules ◽  
Molecular Target ◽  
Tyrosine Nitration

scholarly journals Expression Map for Genes Involved in Nitrogen and Carbon Metabolism in Alfalfa Root Nodules

1999 ◽  
Vol 12 (6) ◽  
pp. 526-535 ◽  
Author(s):  
Gian B. Trepp ◽  
Stephen J. Temple ◽  
Bruna Bucciarelli ◽  
Li Fang Shi ◽  
Carroll P. Vance
Keyword(s):  
Nitrogen Fixation ◽  
Glutamine Synthetase ◽  
Carbon Metabolism ◽  
Root Nodule ◽  
Root Nodules ◽  
Constitutive Expression ◽  
Nodule Development ◽  
Relative Distribution ◽  
Nitrogen Fixing ◽  
Alfalfa Root

During root nodule development several key genes involved in nitrogen fixation and assimilation exhibit enhanced levels of expression. However, little is known about the temporal and spatial distribution patterns of these transcripts. In a systematic study the transcripts for 13 of the essential enzymes involved in alfalfa (Medicago sativa) root nodule nitrogen and carbon metabolism were localized by in situ hybridization. A serial section approach allowed the construction of a map that reflects the relative distribution of these transcripts. In 33-day-old root nodules, the expression of nifH, NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) and a cytosolic isoform of glutamine synthetase (GS13; GS; EC 6.3.1.2) were localized predominantly in a 5- to 15-cell-wide region in the distal part of the nitrogen-fixing zone. This zone was also the region of high expression for leghemoglobin, a second cytosolic glutamine synthetase isoform (GS100), aspartate aminotransferase-2 (AAT-2; EC 2.6.1.1), asparagine synthetase (AS; 6.3.5.4), phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), and sucrose synthase (SuSy; EC 2.4.1.13). This suggests that, in 33-day-old alfalfa root nodules, nitrogen fixation is restricted to this 5- to 15-cell-wide area. The continued significant expression of the GS100 subclass of GS and AS in the proximal part of the nitrogen-fixing zone implicates these gene products in nitrogen remobilization. A low constitutive expression of NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) was observed throughout the nodule. The transcript distribution map will be used as a navigational tool to assist in developing strategies for the genetic engineering of alfalfa root nodules for enhanced nitrogen assimilation.

Asparagine synthetase from root nodules of alfalfa

1989 ◽  
Vol 67 (8) ◽  
pp. 455-459 ◽  
Author(s):  
T. C. Ta ◽  
F. D. H. Macdowall ◽  
M. A. Faris
Keyword(s):  
Glutamine Synthetase ◽  
Rhizobium Meliloti ◽  
Root Nodules ◽  
Subcellular Location ◽  
Asparagine Synthetase ◽  
Organic N ◽  
Synthetase Activity ◽  
Ph Optimum ◽  
Control Activity ◽  
Dependent Activity

Asparagine synthetase, the enzyme which catalyzes the formation of asparagine from aspartate and glutamine (the preferable N donor), was partially purified to 300-fold from root nodules of alfalfa (Medicago sauva L.). The enzyme has Km values for aspartate, glutamine, and ammonium of 1.25, 0.16, and 2.70 mM, respectively. The ratio of glutamine- to ammonium-dependent activity is 2.5 and the pH optimum is between 7.8 and 8.2. Its subcellular location is the cytoplasm. The activity of asparagine synthetase increased in parallel with that of glutamine synthetase and the amounts of organic N in the nodules during their development. Maximum levels were formed at about 3 weeks following innoculation with Rhizobium meliloti. These values decreased rapidly after removal of the shoots: asparagine synthetase reached its lowest value (10% of original) while glutamine synthetase retained 45% of its activity at day 7. Treatment with an argon atmosphere (80% Ar + 20% O2, v/v) to prevent N2 fixation by nodules, and the resulting inhibition of NH4+ production, caused a decrease in asparagine synthetase activity to 10% of control, while this treatment only slightly affected glutamine synthetase (80% of control activity remained).Key words: alfalfa nodules, asparagine synthetase.

scholarly journals The wheat cytosolic glutamine synthetase GS1.1 modulates N assimilation and spike development by characterizing CRISPR-edited mutants

2020 ◽  
Author(s):  
Yazhou Wang ◽  
Wan Teng ◽  
Yanpeng Wang ◽  
Xiang Ouyang ◽  
He Xue ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Field Experiments ◽  
Inorganic Nitrogen ◽  
Grain Filling ◽  
N Availability ◽  
Wild Type ◽  
Spike Development ◽  
N Assimilation ◽  
Cytosolic Glutamine Synthetase ◽  
Ammonium Stress

AbstractGlutamine synthetase (GS) mediates the first step in the assimilation of inorganic nitrogen (N) into amino acids, however the function of GS encoding genes is not well understood in wheat (Triticum aestivum). We found that the cytosolic TaGS1.1 was the major transcripted GS1 gene and was up-regulated by low-N availability. CRISPR/Cas9 mediated genome editing was employed to develop two gs1.1 mutants with mutated TaGS1.1-6A, −6B, and -6D. Both mutants had lower grains per spike and grain yield per plant than the wild type under both low-N and high-N conditions in field experiments. In a hydroponic culture treated with different N resources, the two mutants was more sensitive to low-N stress than the wild type, but showed similar sensitivity to high ammonium stress with the wild type. The growth deficiency and impaired spike development were associated with the imbalance of N metabolites in the mutant plants. During grain filling, TaGS1.1 mutation reduced N translocation efficiency and delayed leaf N loss and grain N filling. Our results suggested that TaGS1.1 is important for N assimilation and remobilization, and required for wheat adaptation to N-limited conditions and spike development.HighlightThe wheat cytosolic glutamine synthetase TaGS1.1 is important for N assimilation and remobilization, and is required for wheat adaptation to low-N stress and spike development.

scholarly journals Potassium content diminishes in infected cells of Medicago truncatula nodules due to the mislocation of channels MtAKT1 and MtSKOR/GORK

2020 ◽  
Author(s):  
Elena E Fedorova ◽  
Teodoro Coba de la Peña ◽  
Victoria Lara-Dampier ◽  
Natalia A Trifonova ◽  
Olga Kulikova ◽  
...  
Keyword(s):  
Medicago Truncatula ◽  
Root Nodule ◽  
Root Nodules ◽  
Phylogenetic Analyses ◽  
Gene Copy ◽  
Gene Localization ◽  
K Channel ◽  
Infected Cells ◽  
Plant Ion Channels ◽  
Mature Nodule

Root nodule-infected cells have defects in K+ balance, as compared with non-infected cells, probably due to variation in the location of K+ channel proteins MtAKT1 and MtSKOR/GORK. Abstract Rhizobia establish a symbiotic relationship with legumes that results in the formation of root nodules, where bacteria encapsulated by a membrane of plant origin (symbiosomes), convert atmospheric nitrogen into ammonia. Nodules are more sensitive to ionic stresses than the host plant itself. We hypothesize that such a high vulnerability might be due to defects in ion balance in the infected tissue. Low temperature SEM (LTSEM) and X-ray microanalysis of Medicago truncatula nodules revealed a potassium (K+) decrease in symbiosomes and vacuoles during the life span of infected cells. To clarify K+ homeostasis in the nodule, we performed phylogenetic and gene expression analyses, and confocal and electron microscopy localization of two key plant Shaker K+ channels, AKT1 and SKOR/GORK. Phylogenetic analyses showed that the genome of some legume species, including the Medicago genus, contained one SKOR/GORK and one AKT1 gene copy, while other species contained more than one copy of each gene. Localization studies revealed mistargeting and partial depletion of both channels from the plasma membrane of M. truncatula mature nodule-infected cells that might compromise ion transport. We propose that root nodule-infected cells have defects in K+ balance due to mislocation of some plant ion channels, as compared with non-infected cells. The putative consequences are discussed.

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