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PubMed2003

Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants.

Legumes can obtain nitrogen from symbiotic nitrogen fixation in root nodules. The glutamine synthetase/glutamate synthase cycle is responsible for the initial nitrogen assimilation. This work reports the analysis of transgenic Lotus japonicus plants with the chimeric gene containing the alfalfa cytosolic glutamine synthetase (GS1) (EC 6.3.1.2) gene controlled by the Sesbania rostrata leghemoglobin gene promoter (Srglb3p). Surprisingly, all of the transgenic primary transformants analysed were sterile. Two transformants designated GS39 and GS44 were further analysed. GS in nodules of GS39 and GS44 plants was upregulated, at the level of transcript and protein. The transgenic plants had 2-fold higher nodule GS activity and similar root GS activity compared to control plants. The GS39 and GS44 sterile plants showed morphological alterations in pollen grains and in ovules. An increase in GS transcript abundance and enzyme activity was measured during early and late stages of flower development of GS plants. Flowers of GS plants showed higher glutamine content, resulting in an increased glutamine/glutamate ratio. The GS transcript and protein were detected in ovules. These data indicate that overexpression of GS1 in reproductive organs critically affects their development and might be a reason for sterility of L. japonicus plants.

Journal Article↗
PubMed2003

Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.).

Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.

Amino Acid Oxidoreductases↗
PubMed2003

The soybean NRAMP homologue, GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport.

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.

Amino Acid Sequence↗
PubMed1983

Soybean nodulin genes: Analysis of cDNA clones reveals several major tissue-specific sequences in nitrogen-fixing root nodules.

Plant gene products that could play a role in the process of symbiotic nitrogen fixation in leguminous plants were detected by screening a cDNA library prepared from soybean nodule poly(A)(+) RNA. About 13% of the 5,700 clones screened contained sequences detectable with a root cDNA probe while about 2,100 clones (37% of the library) contained sequences that were detectable only with nodule cDNA. Five unique sequence species, accounting for more than half of the 2,100 nodule-specific clones, were identified by cross-hybridization experiments. The most abundant species, represented by 860 clones, encodes the well-characterized protein, leghemoglobin (Lb). The other four species, designated NodA, NodB, NodC, and NodD, are represented by 350, 55, 61, and 6 clones, respectively. Each of these four species was found to be encoded by the plant nuclear genome at low copy number. The transcripts corresponding to the nodule-specific clones represented 12-15% (Lb), 6% (NodA), and 0.5-1.1% (NodB, NodC, and NodD) mole fraction of nodule polysomal mRNAs but could not be detected in root polysomal RNA. Hybrid-selection of nodule mRNAs by representative clones and in vitro translation indicated that polypeptides of M(r) 44,000, 27,000, 24,000, and 100,000-120,000 are encoded by NodA, NodB, NodC, and NodD sequences, respectively. These polypeptides reacted with antiserum prepared against total soluble nodule proteins suggesting that the cloned sequences encode nodule-specific proteins, nodulins.

Journal Article↗
PubMed1989

Early nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport inhibitors.

Rhizobium nod genes are essential for root hair deformation and cortical cell division, early stages in the development of nitrogen-fixing root nodules. Nod(-) mutants are unable to initiate nodules on legume roots. We observed that N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid, compounds known to function as auxin transport inhibitors, induced nodule-like structures on alfalfa roots. The nodule-like structures (pseudonodules) were white, devoid of bacteria, and resembled nodules elicited by Rhizobium meliloti exopolysaccharide (exo) mutants at both the histological and molecular level. Two nodulin genes, ENOD2 and Nms-30, were expressed. RNA isolated from the nodule-like structures hybridized to pGmENOD2, a soybean early nodulin cDNA clone. RNA isolated from roots did not hybridize. We determined by in vitro translations of total RNA that the alfalfa nodulin transcript Nms-30 was also expressed in the nodule-like structures. The late expressed nodulin genes, such as the leghemoglobin genes, were not transcribed. Because N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid induce the development of nodules on alfalfa roots, we suggest that the auxin transport inhibitors mimic the activity of compound(s) made upon the induction of the Rhizobium nod genes.

Journal Article↗
PubMed1999

A protein phosphatase 2C gene, LjNPP2C1, from Lotus japonicus induced during root nodule development.

Symbiotic interactions between legumes and compatible strains of rhizobia result in root nodule formation. This new plant organ provides the unique physiological environment required for symbiotic nitrogen fixation by the bacterial endosymbiont and assimilation of this nitrogen by the plant partner. We have isolated two related genes (LjNPP2C1 and LjPP2C2) from the model legume Lotus japonicus that encode protein phosphatase type 2C (PP2C). Expression of the LjNPP2C1 gene was found to be enhanced specifically in L. japonicus nodules, whereas the LjPP2C2 gene was expressed at a similar level in nodules and roots. A glutathione S-transferase-LjNPP2C1 fusion protein was shown to have Mg2+- or Mn2+-dependent and okadaic acid-insensitive PP2C activity in vitro. A chimeric construct containing the full-length LjNPP2C1 cDNA, under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter, was found to be able to complement a yeast PP2C-deficient mutant (pct1Delta). The transcript level of the LjNPP2C1 gene was found to increase significantly in mature nodules, and its highest expression level occurred after leghemoglobin (lb) gene induction, a molecular marker for late developmental events in nodule organogenesis. Expression of the LjNPP2C1 gene was found to be drastically altered in specific L. japonicus lines carrying monogenic-recessive mutations in symbiosis-related loci, suggesting that the product of the LjNPP2C1 gene may function at both early and late stages of nodule development.

Amino Acid Sequence↗
PubMed1978

Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules.

The membrane envelope enclosing the bacteroids in soybean root nodules is shown by ultrastructural and biochemical studies to be derived from, and to retain the characteristics of, the host cell plasma membrane. During the early stages of the infection process, which occurs through an invagination, Rhizobium becomes surrounded by the host cell wall and plasma membrane, forming the infection thread. The cell wall of the infection thread is degraded by cellulolytic enzyme(s), leaving behind the enclosed plasma membrane, the membrane envelope. Cellulase activity in young nodules increases two- to threefold as compared to uninfected roots, and this activity is localized in the cell wall matrix of the infection threads. Membrane envelopes were isolated by first preparing bacteroids enclosed in the envelopes on a discontinuous sucrose gradient followed by passage through a hypodermic needle, which released the bacteroids from the membranes. This membrane then sedimented at the interface of 34--45% sucrose (mean density of 1.14 g/cm3). Membranes were characterized by phosphotungstic acid (PTA)-chromic acid staining. ATPase activity, and localization, sensitivity to nonionic detergent Nonidet P-40 (NP-40) and sodium dodecyl sulfate (SDS) gel electrophoresis. These analyses revealed a close similarity between plasma membrane and the membrane envelope. Incorporation of radioactive amino acids into the membrane envelope proteins was sensitive to cycloheximide, suggesting that the biosynthesis of these proteins is primarily under host-cell control. No immunoreactive material to leghemoglobin antibodies was found inside or associated with the isolated bacteroids enclosed in the membrane envelope, and its location is confined to the host cell cytoplasmic matrix.

Adenosine Triphosphatases↗
PubMed1988

Developmental regulation of nodule-specific genes in alfalfa root nodules.

We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins. Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa. One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa. The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel. Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.

Base Sequence↗
PubMed2005

Comparison of the transcript profiles from the root and the nodulating root of the model legume Lotus japonicus by serial analysis of gene expression.

We performed a comprehensive transcript analysis on the early stage of root nodulation in the model legume Lotus japonicus by serial analysis of gene expression (SAGE). SAGE libraries were made from uninfected roots and nodulating roots abundant in nodule primordia, and 85,482 and 80,233 SAGE tags were recovered, respectively. Comparison of the tag frequency identified 407 tag species that appeared in significantly greater numbers in the nodulating root than in the uninfected root, and the converse was found for 428 tag species. Gene identification of the tags was performed by matching them to L. japonicus expressed sequence tag sequences. We made several novel findings by applying SAGE to transcript analysis of legume root nodulation. A gene that showed the most significant increase in tag number upon nodulation has not been described previously. Different levels of transcription induction among leghemoglobin gene paralogs were found, indicating the effectiveness of SAGE in discriminating different gene family members. We identified genes for 44 unknown tags by means of reverse SAGE. We found 11 antisense tags that increased during nodulation, indicating that regulation of gene expression by antisense transcripts may occur in an organ-dependent manner.

Expressed Sequence Tags↗
PubMed2005

An iron uptake operon required for proper nodule development in the Bradyrhizobium japonicum-soybean symbiosis.

Rhizobia live in the soil or enter into a nitrogen-fixing symbiosis with a suitable host plant. Each environment presents different challenges with respect to iron acquisition. The soybean symbiont Bradyrhizobium japonicum 61A152 can utilize a variety of siderophores (Fe[III]-specific ligands). Purification of iron-regulated outer membrane proteins had previously allowed the cloning of a gene, fegA, from B. japonicum 61A152, whose predicted protein shares significant amino acid similarity with known TonB-dependent siderophore receptors. Here, we show that fegA is in an operon with a gene, fegB, that is predicted to encode an inner membrane protein. Characterization of fegAB and fegB mutants shows that bothfegA and fegB are required for utilization of the siderophore ferrichrome. Whereas thefegB mutant forms a normal symbiosis, the fegAB mutant has a dramatic phenotype in planta. Six weeks after inoculation with a fegAB strain, soybean nodules do not contain leghemoglobin and do not fix nitrogen. Infected cells contain few symbiosomes and are filled with vesicles. As ferrichrome is a fungal siderophore not likely to be available in nodules, the symbiotic defect suggests that the fegAB operon is serving a different function in planta, possibly one involved in signaling between the two partners.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

Cells expressing ENOD2 show differential spatial organization during the development of alfalfa root nodules.

We have used in situ hybridization to examine the spatial organization of cells expressing the early nodulin gene (ENOD2) during the development of alfalfa root nodules. ENOD2 gene expression was found in the nodule parenchyma, uninfected cells surrounding the symbiotic region of both effective and ineffective nodules. However, in empty nodules, ENOD2 gene expression was found in a mass of parenchyma cells at the base of the nodule. Similar results were also observed in 11-day-old nodules that contained infected cells but that had not yet begun to express leghemoglobin. Although early events of nodulation result in the induction of ENOD2 expression in cells at the nodule base, the pattern of cells expressing ENOD2 during nodule growth appears to be correlated with the development of other peripheral tissues.

Fabaceae↗
PubMedDate not supplied

GmN56, a novel nodule-specific cDNA from soybean root nodules encodes a protein homologous to isopropylmalate synthase and homocitrate synthase.

We have isolated a novel nodule-specific cDNA clone, GmN56, from soybean root nodules. The expression of GmN56 was induced almost concomitantly with the onset of nitrogen fixation, together with leghemoglobin and other late nodulin genes. In situ hybridization studies demonstrated the localization of GmN56 mRNA in the bacterial infected cells of mature nodules. The predicted amino acid sequence of the GmN56 protein exhibits significant homology to those of LeuA (isopropylmalate synthase) of several microorganisms and NifV (putative homocitrate synthase) of nitrogen-fixing bacteria, suggesting that GmN56 encodes an enzyme catalyzing a reaction involving acetyl-CoA and alpha-keto acid as substrates.

2-Isopropylmalate Synthase↗
PubMedDate not supplied

Use of differential display to identify novel Sesbania rostrata genes enhanced by Azorhizobium caulinodans infection.

Upon infection of the tropical legume Sesbania rostrata with Azorhizobium caulinodans ORS571, nodules are formed on the roots as well as on the stems. Stem nodules appear at multiple predetermined sites consisting of dormant root primordia, which are positioned in vertical rows along the stem of the plant. We used the differential display method to isolate and characterize three cDNA clones (differential display; didi-2, didi-13, and didi-20), corresponding to genes whose expression is enhanced in the dormant root primordia after inoculation. Database searches revealed that the deduced (partial) didi-2 gene product shares significant similarity with hydroxyproline-rich cell wall proteins. The (partial) didi-13 and didi-20 products are similar to chitinases and chalcone reductases, respectively. Transcripts corresponding to the cDNA clones didi-2 and didi-13 were first detectable 1 day after inoculation. In contrast, didi-20 transcripts were found at low levels in uninfected root primordia and were enhanced significantly around 3 days after inoculation. In addition, a cDNA was isolated (didi-42) that corresponds to the previously identified leghemoglobin gene Srlb6. These studies show that differential display is a useful method for the isolation of infection-related genes.

Amino Acid Sequence↗
PubMed2000

Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis.

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Knockout of an azorhizobial dTDP-L-rhamnose synthase affects lipopolysaccharide and extracellular polysaccharide production and disables symbiosis with Sesbania rostrata.

A nonpolar mutation was made in the oac2 gene of Azorhizobium caulinodans. oac2 is an ortholog of the Salmonella typhimurium rfbD gene that encodes a dTDP-L-rhamnose synthase. The knockout of oac2 changed the lipopolysaccharide (LPS) pattern and affected the extracellular polysaccharide production but had no effect on bacterial hydrophobicity. Upon hot phenol extraction, the wild-type LPS partitioned in the phenol phase. The LPS fraction of ORS571-oac2 partitioned in the water phase and had a reduced rhamnose content and truncated LPS molecules on the basis of faster migration in detergent gel electrophoresis. Strain ORS571-oac2 induced ineffective nodule-like structures on Sesbania rostrata. There was no clear demarcation between central and peripheral tissues, and neither leghemoglobin nor bacteroids were present. Light and electron microscopy revealed that the mutant bacteria were retained in enlarged, thick-walled infection threads. Infection centers emitted a blue autofluorescence under UV light. The data indicate that rhamnose synthesis is important for the production of surface carbohydrates that are required to sustain the compatible interaction between A. caulinodans and S. rostrata.

Azorhizobium caulinodans↗
PubMed2002

Expression of genes encoding late nodulins characterized by a putative signal peptide and conserved cysteine residues is reduced in ineffective pea nodules.

Five nodulin genes, PsN1, PsN6, PsN314, PsN335, and PsN466, with reduced expression in ineffective nodules on the pea (Pisum sativum) mutant E135 (sym13) were characterized. They encode small polypeptides containing a putative signal peptide and conserved cysteine residues and show homology to the nodulins PsENOD3/14 and PsNOD6. For each gene, multiple bands were detected by genomic Southern analysis. Northern analysis showed that all five genes were expressed exclusively in nodules and that their temporal expression patterns were similar to that of the leghemoglobin (Lb) gene during nodule development. Their transcripts were localized predominantly from the interzone II-III to the distal part of nitrogen-fixing zone III in effective nodules, resembling the Lb gene. However, transcripts in ineffective E135 nodules were localized in narrower regions than those in the effective nodules. These results indicate that these nodulins are abundant in pea nodules and that their successive expression during nodule development is associated with nitrogen-fixing activity.

Amino Acid Sequence↗
PubMed1992

Reversible o(2) inhibition of nitrogenase activity in attached soybean nodules.

Various forms of stress result in decreased O(2) permeability or decreased capacity to consume O(2) in legume root nodules. These changes alter the nodule interior O(2) concentration (O(i)). To determine the relationship between O(i) and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled O(i) by varying external pO(2) while monitoring internal H(2) concentration (H(i)) with microelectrodes. O(i) was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in O(i), H(i) approached a new steady state, with a time constant averaging 23 s. The rate of H(2) production by nitrogenase was calculated as the product of H(i), nodule surface area, and nodule H(2) permeability. H(2) permeability was estimated from O(2) permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. O(i) was nearly optimal for nitrogenase activity (H(2) production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO(2) (40-100 kPa) reduced H(i) to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO(2). The mechanism of nitrogenase inhibition may differ between brief and prolonged O(2) exposures.

Journal Article↗
PubMed2003

Nodule-specific modulation of glutamine synthetase in transgenic Medicago truncatula leads to inverse alterations in asparagine synthetase expression.

Transgenic Medicago truncatula plants were produced harboring chimeric gene constructs of the glutamine synthetase (GS) cDNA clones (MtGS1a or MtGS1b) fused in sense or antisense orientation to the nodule-specific leghemoglobin promoter Mtlb1. A series of transgenic plants were obtained showing a 2- to 4-fold alteration in nodule GS activity when compared with control plants. Western and northern analyses revealed that the increased or decreased levels of GS activity correlate with the amount of cytosolic GS polypeptides and transcripts present in the nodule extracts. An analysis of the isoenzyme composition showed that the increased or decreased levels of GS activity were attributable to major changes in the homo-octameric isoenzyme GS1a. Nodules of plants transformed with antisense GS constructs showed an increase in the levels of both asparagine synthetase (AS) polypeptides and transcripts when compared with untransformed control plants, whereas the sense GS transformants showed decreased AS transcript levels but polypeptide levels similar to control plants. The polypeptide abundance of other nitrogen metabolic enzymes NADH-glutamic acid synthase and aspartic acid amino-transferase as well as those of major carbon metabolic enzymes phosphoenolpyruvate carboxylase, carbonic anhydrase, and sucrose synthase were not affected by the GS-gene manipulations. Increased levels of AS polypeptides and transcripts were also transiently observed in nodules by inhibiting GS activity with phosphinothricin. Taken together, the results presented here suggest that GS activity negatively regulates the level of AS in root nodules of M. truncatula. The potential role of AS in assimilating ammonium when GS becomes limiting is discussed.

Aminobutyrates↗