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Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus.

Research on legume nodule metabolism has contributed greatly to our knowledge of primary carbon and nitrogen metabolism in plants in general, and in symbiotic nitrogen fixation in particular. However, most previous studies focused on one or a few genes/enzymes involved in selected metabolic pathways in many different legume species. We utilized the tools of transcriptomics and metabolomics to obtain an unprecedented overview of the metabolic differentiation that results from nodule development in the model legume, Lotus japonicus. Using an array of more than 5000 nodule cDNA clones, representing 2500 different genes, we identified approximately 860 genes that were more highly expressed in nodules than in roots. One-third of these are involved in metabolism and transport, and over 100 encode proteins that are likely to be involved in signalling, or regulation of gene expression at the transcriptional or post-transcriptional level. Several metabolic pathways appeared to be co-ordinately upregulated in nodules, including glycolysis, CO(2) fixation, amino acid biosynthesis, and purine, haem, and redox metabolism. Insight into the physiological conditions that prevail within nodules was obtained from specific sets of induced genes. In addition to the expected signs of hypoxia, numerous indications were obtained that nodule cells also experience P-limitation and osmotic stress. Several potential regulators of these stress responses were identified. Metabolite profiling by gas chromatography coupled to mass spectrometry revealed a distinct metabolic phenotype for nodules that reflected the global changes in metabolism inferred from transcriptome analysis.

Expressed Sequence Tags↗

Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants.

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.

Carbon-Carbon Lyases↗

Absence of uniform progressive growth of long-term transplants of rat liver neoplastic nodules.

Fragments of normal rat liver and neoplastic nodules induced by feeding 0.02% N-2-fluorenylacetamide for 20 weeks were transplanted into the inguinal mammary fat pads of syngeneic rats. All animals were Charles River inbred male albino rats of the CDF strain derived from the F344 strain. Normal liver transplants and neoplastic nudule transplants were detected histologically in the transplant site up to 24 and 66 weeks of survival, respectively. Of 30 nodule transplants followed up to 121 weeks, only 1 neoplastic nodule developed progressive neoplastic growth. The tumor that arose in the transplant site from this nodule was a well-differentiated adenocarcinoma with mucin production. Thus rat liver neoplastic nodules lack the high frequency of progressive growth previously demonstrated for carcinomas.

2-Acetylaminofluorene↗

[Malignant tendency of basophilic liver cells in experimental rat hepatocarcinogenesis].

OBJECTIVE: To investigate the malignant tendency of liver basophilic cells in rats by examining the liver dynamic pathological changes during the hepatocarcinogenesis induced by diethylnitrosamine (DEN). METHODS: One hundred forty male Sprague-Dawley rats, about 200 g each, were randomly divided into a normal group and a model group. The model group rats were administered 1% DEN intragastrically once a week for 14 weeks. The normal control group rats were given saline instead of DEN. Seven to ten rats of the model group were sacrificed at 2, 3, 5, 8, 10, 12, 14, 18 weeks. The remaining rats were followed to the end of the experiment at 26 weeks. Histopathological changes of the livers were analyzed, and the localization of GST-P and PCNA in the livers were detected in situ by immunohistochemistry. RESULTS: According to the characteristics of the lesions in the model group, histological liver change patterns were categorized into three phases: (1) liver injury phase (2 to 5 weeks) with centrilobular necrosis, a small amount of collagen deposition in the necrotic regions with fibrous septa development and cell proliferation; (2) the cirrhosis phase (8 to 12 weeks) with significant hepatocellular regeneration and collagen deposition. As the regenerative nodules and fibrous septa formed, cirrhosis with uniform sized nodules developed in all the rats at 12 weeks. In the regenerative nodules, significant hepatocellular metamorphosis was seen; (3) In the carcinomatous transformation and nodular remodeling phase (after 14 weeks), two types of cancer, namely hepatocellular carcinoma and cholangiocarcinoma were found. Incidence of the cancer was 62.5% at 18 weeks. Basophilic cell lesions appeared beginning at 10 weeks. Pale bodies were seen in some basophilic cells. Small cell changes appeared starting at 12 weeks. Some of these cells containing droplets like lipid vacuoles, invaded into the surrounding liver tissues. Both basophilic cell lesions and small cell changes were all positive for proliferating cell nuclear antigen (PCNA). CONCLUSION: Development of foci of basophilic small cells, with cells containing lipid vacuoles and pale bodies, and invading into the surrounding liver tissues are the changes highly suggestive of an early hepatocellular carcinoma transformation.

Animals↗

The pseudomonas hot-foot syndrome.

BACKGROUND: Between March and May 1998, there was an outbreak of a clinically distinct skin eruption on the soles of the feet of children who used a community wading pool. METHODS: We reviewed the medical records of 40 children in whom this syndrome developed between March and May 1998. We treated 17 children and advised the attending physicians on the care of the other 23. Follow-up data were obtained for up to one year. RESULTS: Exquisitely painful erythematous plantar nodules developed in 40 children (age, 2 to 15 years) within 40 hours after they had used a wading pool whose floor was coated with abrasive grit. Culture of the plantar pustules from one child yielded Pseudomonas aeruginosa with a pattern on pulsed-field gel electrophoresis that was identical to that of a strain of P. aeruginosa cultured from the pool water. A skin-biopsy specimen from this patient showed a perivascular and perieccrine neutrophilic infiltrate, and a specimen from another patient showed a dermal microabscess. Thirty-seven patients were treated symptomatically; three others were treated with cephalexin. All patients recovered within 14 days, but three children had recurrences of the painful plantar nodules within 24 hours after using the pool again. Folliculitis developed in one patient. CONCLUSIONS: The "pseudomonas hot-foot syndrome" is characterized by the acute onset in children of exquisitely tender plantar nodules and a benign, self-limited course. This community outbreak developed after exposure to pool water containing high concentrations of P. aeruginosa.

Adolescent↗

The variations of S-adenosyl-L-methionine content modulate hepatocyte growth during phenobarbital promotion of diethylnitrosamine-induced rat liver carcinogenesis.

A decrease in liver S-adenosyl-L-methionine (SAM) content and an increase in ornithine decarboxylase (ODC) activity occurred between the 2nd and the 5th week after starting 2-acetylaminofluorene (AAF) feeding in diethylnitrosamine (DENA)-initiated rats. These rats then received a 0.05% phenobarbital (PB)-containing diet for 18 weeks after the end of AAF feeding. Two weeks after starting AAF, an increase in the hepatocyte labeling index (LI) also occurred in gamma-glutamyl-transpeptidase (GGT)-positive foci and surrounding tissue. LI returned to control values in a few days in surrounding tissue, while it remained high for at least 4 weeks in the foci. Analogous changes were observed, but for a shorter period of time, in the rats subjected to partial hepatectomy (PH) plus AAF, in which no GGT-positive foci developed. Twenty-four weeks after starting AAF, 30% of the liver was occupied by visible nodules. ODC activity and LI were high and SAM was low in nodules, but they were near to control values in surrounding liver. SAM administration reconstituted the liver SAM pool, inhibited ODC activity, and prevented visible nodule development. SAM inhibition of ODC activity occurred in vitro only after preincubation with liver homogenate and was enhanced by adenine, an inhibitor of methylthioadenosine (MTA) phosphorylase. MTA addition to the reaction of mixture for ODC determination was inhibitory. The SAM decrease in both liver and nodules was coupled with a decrease of MTA content. SAM administration caused MTA accumulation in the liver. It is suggested that liver SAM content by influencing MTA level, could be a rate-limiting factor for growth and promotion, through a modulation of polyamine synthesis.

2-Acetylaminofluorene↗

Histopathology and gene expression changes in rat liver during feeding of fumonisin B1, a carcinogenic mycotoxin produced by Fusarium moniliforme.

Fumonisin B1 (FB1) is a carcinogenic mycotoxin produced by the fungus Fusarium moniliforme in corn. Feeding of FB1 to rats causes acute liver injury, chronic liver injury progressing to cirrhosis, and sometimes terminates in hepatocellular carcinoma or cholangiocarcinoma. This study describes the histolopathology and changes in gene expression in the rat liver during short-term feeding of FB1. Male Fischer rats were fed either FB1 250 mg/kg or control diet, and were killed weekly for 5 weeks. FB1 caused a predominantly zone 3 'toxic' liver injury, with hepatocyte death due to necrosis and apoptosis. Hepatocyte injury and death were mirrored by hepatic stellate cell proliferation and marked fibrosis, with progressive disturbance of architecture and formation of regenerative nodules. Despite ongoing hepatocyte mitotic activity, oval cell proliferation was noted from week 2, glutathione S-transferase pi-positive hepatic foci and nodules developed and, at later time points, oval cells were noted inside some of the 'atypical' nodules. Northern blot (mRNA) analysis of liver specimens from weeks 3 to 5 showed a progressive increase in gene expression for alpha-fetoprotein, hepatocyte growth factor, transforming growth factor alpha (TGF-alpha) and especially TGF-beta1 and c-myc. Immunostaining with LC(1-30) antibody demonstrated a progressive increase in expression of mature TGF-beta1 protein by hepatocytes over the 5 week feeding period. The overexpression of TGF-beta1 may be causally related to the prominent apoptosis and fibrosis seen with FB1-induced liver injury. Increased expression of c-myc may be involved in the cancer promoting effects of FB1.

Animals↗

Overlapping plant signal transduction pathways induced by a parasitic nematode and a rhizobial endosymbiont.

Root-knot nematodes and rhizobia establish interactions with roots characterized by the de novo induction of host structures, termed giant cells and nodules, respectively. Two transcription regulators, PHAN and KNOX, required for the establishment of meristems were previously shown to be expressed in tomato giant cells. We isolated the orthologues of PHAN and KNOX (Mt-phan and Mt-knox-1) from the model legume Medicago truncatula, and established the spatial distribution of their expression in situ. We confirmed that Mt-phan and Mt-knox-1 are expressed in lateral root initials and in nematode-induced giant cells and showed that they are expressed in nodules induced by Sinorhizobium meliloti. Expression of both genes becomes spatially restricted as the nodules develop. We further examined nematode feeding sites for the expression of two genes involved in nodule formation, ccs52 (encodes a mitotic inhibitor) and ENOD40 (encodes an early, nodulation mitogen), and found transcripts of both genes to be present in and around giant cells induced in Medicago. Collectively, these results reveal common elements of host responses to mutualistic and parasitic plant endosymbionts and imply that overlapping regulatory pathways lead to giant cells and nodules. We discuss these pathways in the context of phytohormones and parallels between beneficial symbiosis and disease.

Amino Acid Sequence↗

Isolation and characterization of mutants of Rhizobium leguminosarum bv. viciae 248 with altered lipopolysaccharides: possible role of surface charge or hydrophobicity in bacterial release from the infection thread.

Effects of alterations in lipopolysaccharide (LPS) structure of Rhizobium leguminosarum bv. viciae on effective symbiosis and on a number of cell surface characteristics were studied. Tn5 mutants with altered LPSs were screened for their inability to bind monoclonal antibody 3, one of three monoclonal antibodies to the tentative O-antigenic part of the wild-type LPS of strain 248. Ten class I LPS mutants completely lacked the O-antigen-containing LPS species. The class II LPS mutant had a severely diminished amount of an antigenically altered O-antigen-containing LPS. The class III LPS mutant had normal amounts of an altered, O-antigen-containing LPS. Class I and II mutants, but not the class III mutant, showed abnormal nodule development (i.e., blocked in the stage of bacterial release from the infection thread) resulting in nodules in which very few, at the most, plant cells contained bacteroids and which were unable to fix nitrogen. Class I and II mutants were nonmotile and were more sensitive to hydrophobic compounds than the parent strain. The most striking difference between the symbiotically defective class I and II LPS mutants on one hand and the wild-type strain and the class III mutant on the other hand was that the class I and II mutants have a more hydrophobic cell surface and a higher electrophoretic mobility. A role for an O-antigen-containing LPS in bacterial release from the infection thread, through its effects on general physicochemical cell surface characteristics, is proposed.

Antibodies, Monoclonal↗

Atypical large well-differentiated hepatocellular carcinoma with benign nature: a new clinical entity.

Based on intranodular hemodynamic studies of nodular lesions associated with liver cirrhosis using computed tomography (CT) during arteriography, CT during arterial portography (CTAP) and angiography under ultrasonography, relatively large hepatocellular nodules with portal perfusion have been depicted. We report 17 such nodules in a total of 663 patients who underwent CTAP. Large well-differentiated hepatocellular carcinomas (HCCs) are characterized by the presence of intranodular portal perfusion even if they measure more than 2.5 cm in diameter. Such nodules display a benign nature clinically as well as pathologically. These nodules, called 'atypical large well-differentiated HCC or dysplastic nodules', show extremely slow growth. Pathological study showed extremely peculiar findings; the nodules consisted of homogeneously hyperplastic or well-differentiated HCC without the component of moderately or poorly differentiated HCC, and without fibrous capsules, vessel invasion or intrahepatic metastasis, all of which are commonly found in typical overt HCC. The incidence of such nodules is not rare; it was 2.6% (17/663) in our HCC patients who underwent CTAP over a period of 7 years. During the 7-year observation period, none of 10 such nodules developed into arterial supply-dominant overt HCC. The recognition of these benign-nature nodules (or large low-grade malignant nodules) is extremely important for the following three reasons. First, the knowledge that such large atypical nodular lesions exist is important for clinicians. Second, regarding the treatment strategy for such nodular lesions, it should be kept in mind that these nodules show a benign nature, and rarely cause death. Finally, considering the etiology and clinical significance of such atypical large benign-nature nodules, the recognition of these nodules provides an important suggestion regarding the pathogenesis of progression from dysplastic nodule to early HCC and finally to overt HCC during multistep human hepatocarcinogenesis. These atypical large benign-nature nodules do not seem to be the nodules in a sequence of multistep progression. The early step of hepatocarcinogenesis includes mainly hyperplastic change, whereas the late step includes mainly an angiogenic process. Although the detailed molecular pathogenesis of both the early and late steps of hepatocarcinogenesis is not clear, it is speculated that these atypical benign-nature nodules do not undergo the phenotypic change of late-step hepatocarcinogenesis. In that sense, these atypical benign-nature nodules are suggestive for the clarification of the mechanism of multistep human hepatocarcinogenesis based on the intranodular vascular supply or hemodynamics. From the viewpoint of clinical practice as well as pathogenesis of human hepatocarcinogenesis, these nodular lesions should be managed as a different disease from overt HCCs; atypical large HCCs or dysplastic nodules with benign nature should be regarded as a new clinical entity.

Angiography↗

[Valvulopathy in primary antiphospholipid syndrome. Prospective echocardiography study].

Echocardiographic studies have demonstrated a high prevalence of valvular disease in patients with primary antiphospholipid syndrome (PAPS). However, there are no studies assessing changes over time in valvular abnormalities. We conducted a study to determine whether there are changes over time in valvular lesions as detected by serial transesophageal echocardiography (TEE). Twelve patients with a first TEE had a second evaluation after a mean period of 13.5 months. There were 10 women and two men with a mean age of 38 years. Two patients had normal TEE on both initial and follow-up studies. Ten patients (83%) had valvular abnormalities, predominantly of the mitral and aortic valves in both studies. Abnormalities consisted of thickening, nodules, regurgitation, regurgitation and stenosis, and calcification. The type and frequency of lesions changed over time. As an example, one mitral valve nodule disappeared on follow up but three new aortic nodules developed even though all patients were receiving anticoagulant therapy. Two patients with mitral and aortic nodules presented cerebral ischemia. Mitral valvuloplasty was performed in one case. These results show that valvular abnormalities in patients with PAPS resolve, appear, or persist irrespective of anticoagulant therapy. Regurgitation is often mild or moderate, but stenosis may appear.

Adult↗

Enhancement of pancreatic tumorigenesis of 4-hydroxyaminoquinoline 1-oxide by ethionine in rats.

The effect of ethionine on pancreatic tumorigenesis of 4-hydroxyaminoquinoline 1-oxide (4-HAQO) in rats was studied. The high incidence of hyperplastic nodules developed in the exocrine pancreas of animals receiving a single injection of 4-HAQO only or protein-deficient diet containing 0.5% ethionine for 4 days followed by 4-HAQO. The incidence of adenoma was higher than that of hyperplastic nodules and well-differentiated adenocarcinoma developed in 1 out of 12 animals receiving protein-deficient diet containing ethionine for 18 days followed by 4-HAQO. No tumor was found in the pancreas of animals that received 0.005N HCl without 4-HAQO. These results suggest that the pancreatic tumorigenesis of 4-HAQO is enhanced by ethionine.

4-Hydroxyaminoquinoline-1-oxide↗

Rheumatoid nodules on amputation stumps: report of three cases.

We describe three patients, all of them lower limb amputees who also had seropositive, nodular rheumatoid arthritis. In each case, rheumatoid nodules developed on the amputation stump, causing difficulties with prosthesis fit and comfort and impairment of ambulation. Nodules were successfully excised in two cases and the prosthesis modified in the third. The patients were able to resume walking or transferring with their prostheses.

Aged↗

Efficacy of injectable doramectin in the therapy and control of Dermatobia hominis infestations in Latin America.

Three studies were conducted in Latin America, one in Brazil, one in Venezuela and one in Argentina, using a common protocol to investigate the efficacy of a single subcutaneous injection of doramectin at 200 micrograms kg-1 (1 ml per 50 kg) for the treatment and control of Dermatobia hominis infestations in cattle raised under commercial conditions. In each study, two groups of 20 animals each were allocated on the basis of D. hominis nodules present 24 h before treatment to a treated group (T1), or to a control group (T2) which received saline solution at 1 ml per 50 kg of live weight. All cattle were injected in the mid-dorsal cervical region and examined on treatment day and 2, 7, 15 and 30 days post-treatment (p.t.). At each observation day the number of D. hominis nodules was counted and the viability of the larvae inside them was assessed and recorded. Treatment with doramectin was 100% effective in eliminating D. hominis larvae and in controlling the re-establishment of the parasite under field conditions of continuous reinfestation. During the first 48 h p.t., dead larvae of different ages were found outside or partially outside the nodules in animals of the doramectin groups. After 48 h p.t., no live larvae were found inside existing nodules, nor did new nodules develop in animals of the doramectin groups in any of the three studies. When compared with nodule counts in the control group on the same observation day or with parasite burdens on the same animal before treatment, doramectin-treated animals began to eliminate parasitic nodules at 48 h p.t. Efficacy reached 100% at 7 days p.t. and remained at 100% on subsequent observation days (P < 0.05). None of the doramectin-treated animals exhibited any clinical signs of adverse reaction to medication.

Animals↗

Influence of host cultivars and Rhizobium species on the growth and symbiotic performance of Phaseolus vulgaris under salt stress.

In order to study the effect of salt stress on the Rhizobium-common bean symbiosis, we investigated the response of both partners, separately and in symbiosis. The comparison of the behaviour of five cultivars of Phaseolus vulgaris differing in seed colour, growing on nitrates and different concentrations of NaCl, showed genotypic variation with respect to salt tolerance. Coco Blanc was the most sensitive cultivar, whereas SMV 29-21 was the most tolerant one. At the Rhizobium level, two strains previously selected for their salt tolerance were used: Rhizobium tropici strain RP163 and Rhizobium giardinii strain RP161. Their relative growth was moderately decreased at 250mM NaCl, but they were able to grow at a low rate in the presence of 342 mM NaCl. Their viability at the minimal inhibitory concentration was slightly affected. The effect of salinity on Rhizobium-plant association was studied by using the tolerant variety SMV 29-21 and the sensitive one Coco Blanc inoculated separately with both strains. In the absence of salinity, the strains induced a significantly higher number of nodules on the roots of the cultivar SMV 29-21 compared to those of Coco Blanc. Concerning effectiveness, both strains were similarly effective with SMV 29-21, but not with Coco Blanc. In the presence of salinity, Coco Blanc was more severely affected when associated with RP163 than with RP161. Salinity affected the nodulation development more than it affected the infection steps. Neither of the two strains was able to nodulate SMV 29-21 under saline conditions, in spite of the fact that this was considered the most salt-tolerant variety. The unsuccessful nodulation of SMV 29-21 could be related to the inhibition by salt of one or more steps of the early events of the infection process. In conclusion, N-fixing plants were found to be more sensitive to salt stress than those depending on mineral nitrogen. Evidence presented here suggests that a best symbiotic N2 fixation under salinity conditions could be achieved if both symbiotic partners, as well as the different steps of their interaction (early events, nodule formation, activity, etc.), are all tolerant to this stress.

Adaptation, Physiological↗

Inhalation of NO2 and blood borne cancer cell spread to the lungs.

An experimental model was designed where the frequency of blood-borne cancer cell metastases to the lungs of animals was used as an indicator to detect adverse effects of inhaled nitrogen dioxide (NO2). Animals were exposed to air containing 0.40 +/- 0.05 ppm or 0.80 +/- 0.05 ppm of NO2. After the appropriate exposure periods, the animals were infused intravenously with B16 mouse melanoma cells. At 3 wk post-infusion the animals were killed and the lungs were examined for melanoma nodule development. The lungs of the NO2-exposed animals contained a significantly higher number of melanoma nodules than the lungs of control animals (P less than .0025). These results indicate that inhalation of ambient or near ambient levels of NO2 influences the metastasis of blood-borne cancer cells. This raises the possibility that similar events may occur in the human population.

Animals↗

Fluorescent-antibody approach to study of rhizobia in soil.

Application of fluorescent-antibody (FA) techniques to the study of rhizobia as free-living soil bacteria was explored. Antiserum to a particular strain of Rhizobium japonicum proved specific in both agglutination and FA tests. Within the R. japonicum group, 2 of 12 strains were stained by the conjugate and these fluoresced brightly; all others were entirely negative. FA tests were negative for 7 strains of R. meliloti, 9 strains of R. leguminosarum, 9 strains of R. trifolii, 6 strains of R. phaseoli, and 65 unidentified bacteria isolated from 12 soils. R. japonicum grew in autoclaved soil and was readily detectable by FA examination of contact slides. The FA technique also detected antibody-reacting bacteria in a field soil whose rhizobial content was unknown. Fluorescent cells were probably R. japonicum, since nodules developed on soybean plants grown in the same soil sample and FA preparations of the crushed nodules proved positive. Autofluorescence was not a problem, but nonspecific adsorption of conjugate restricted observations to microscopic fields free from soil particles. Nonspecific adsorption was substantial, irrespective of the soil used.

Agglutination Tests↗

Genotypic variation of N2-fixing common bean (Phaseolus vulgaris L.) in response to iron deficiency.

In calcareous soils, the yield of grain legumes is often limited by the lower availability of iron (Fe), especially when they depend upon symbiosis with root nodule bacteria for their N nutrition. In order to explore the variability of responses of N(2)-fixing common bean to Fe deficiency the common bean white-seeded lines Striker and Coco blanc, and coloured-seeded lines SVM-29-21 and ARA14 were inoculated with Rhizobium tropici (CIAT 899) and cultivated hydroaeroponically with a N-free nutrient solution supplied or not with 45microM Fe. Differences among lines were observed: Fe-deficiency-induced-chlorosis on young leaves was earlier and more severe in some lines than others. Nodule development and N(2)-fixing capacity was less affected in line ARA14 which preferentially allocated Fe towards nodules. Results suggest that Fe use efficiency for symbiotic nitrogen fixation (FeUE SNF) could be used to screen tolerant bean lines to Fe deficiency in condition of symbiotic nitrogen fixation.

Genetic Variation↗