Regulation of nodule formation in soybean-Bradyrhizobium symbiosis is controlled by shoot or/and root signals

Two strains of Bradyrhizobium japonicum wereevaluated with five commercial cultivars of soybean(Clark, Crauford, Davis, Centaur, and Nessen) and onehypernodulating mutant NOD1-3. The hypernodulatingNOD1-3 produced 30–50 times more nodules thancommercial cultivars either inoculated with B.japonicum strain USDA 123 or RCR 3409. The currentexperiments were extended to determine if therestricted nodulation of commercial cultivars could be overcome by grafting them to a hypernodulated shoot (NOD1-3). Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. The shoots of Clark, Davis, Centaur and Nessen significantlyinhibited nodule formation on the root of NOD1-3,while Crauford shoots did not alter nodule formationon the roots of NOD1-3 as compared with self-grafts ofNOD1-3. It appears that the shoot of NOD1-3 has theability to alter autoregulatory control of nodulationof Clark and Davis cultivars, but did not withCrauford, Centaur and Nessen. The results suggestedthat the regulation of nodulation in soybean cultivarsClark and Davis is controlled by the shoot factors,while the Crauford was root controlled.Reciprocal-grafts between NOD1-3 and Centaur or Nessenindicate that both shoot and root factors involved inregulation of nodulation and the regulation ofnodulation did not depend on bradyrhizobial strains. Isoflavonoid analyses from extracts of grafted plantsshowed that NOD1-3 shoots had markedly higher rootisoflavonoid concentrations in roots of both Clark andNOD1-3. The shoot control of hypernodulation may becausally related to differential root isoflavonoidlevels, which are also controlled by the shoot. Thecurrent work was extended to investigate the effect ofapplication of an isoflavonoid (daidzein) on nodulationand nitrogen fixation of soybean cultivars Clark andCentaur as well as in vitro growth of Bradyrhizobium japonicum. Application of theisoflavonoid (daidzein) significantly enhanced thenodulation and nitrogenase activity of Clark but notof Centaur indicating that this character is notrelated to isoflavonoids. Therefore, autoregulationin Clark and Centaur plants may be separate events inlegume-rhizobia symbiosis and regulated by differentkinds of signals. Addition of daidzein to yeastmannitol broth medium promoted the growth of B.japonicum strain USDA 123 and RCR 3409. It seemsthat this compound is able to help the nodulation ofsoybean cv Clark by a Bradyrhizobium strain. Understanding the signaling pathways between rhizobiaand their host plants may allow modifications of thisinteraction to improve symbiotic performance.     

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O₂ (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 g· ml^-1) or chloramphenicol (50 g·ml^-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N₂ fixation.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Characterization of a Rhizobium etli chromosomal gene required for nodule development on Phaseolus vulgaris L.

A chromosomal gene, required for nodule development on Phaseolus bean, was characterized from Rhizobium etli strain TAL182. MLC640 is a Tn5 insertion mutant of TAL182 which shows decreased motility in soft TY agar and is defective in nodule development. The site of Tn5 insertion in MLC640 mapped to a 3.6-kb EcoRI chromosomal fragment. The 3.6-kb fragment was subcloned from the cosmid pUHR80 which complemented MLC640. Further subcloning and site-directed Tn5 mutagenesis localized the gene for nodule development to a 1.7-kb region within the 3.6-kb EcoRI fragment. Southern hybridization using the 3.6-kb EcoRI fragment as the probe against genomic DNA of several Rhizobium spp. indicated that this gene is conserved in different rhizobia.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gimore 402, Honolulu, Hawaii 96822. USA;     

The regulation of symbiotic N2 fixation: a conceptual model of N feedback from the ecosystem to the gene expression level

Symbiotic nitrogen (N₂) fixation in legumes may give the host plant a distinct competitive advantage; at the same time it is mainly responsible for introducing N into terrestrial ecosystems which may ultimately benefit all organisms. Depending on environmental conditions, symbiotic N₂ fixation may be tuned to the plant's N demand or specifically inhibited (a disadvantage for plants which depend mainly on symbiotic N₂ fixation), or even prevented. Thus, the ecological range for symbiotic N₂ fixation can be narrower than that of the host plants. A shortage of mineral N is the only case in which adverse environmental conditions clearly favour symbiotic N₂ fixation. Variations in number or mass of nodules or nodule morphology are persistent features, that may represent one kind of regulation of N₂ fixation. In addition, varying O₂ permeability of nodules functions as a rapid and reversible control of N₂ fixation which may compensate partially or fully for poor nodulation. The plant's demand for symbiotically fixed N is thought to play a central role in modulating both nodulation and N₂ fixation activity; an N feedback mechanism is assumed. The control of symbiotic N₂ fixation operates through a series of ecophysiological triggers which are also influenced by complex interactions between legume plants and other organisms in the ecosystem. The proportion of legume biomass and the performance of symbiotic N₂ fixation in each individual legume are the main parameters which determine the amount of symbiotically fixed N introduced into a terrestrial ecosystem. The various triggers and N feedback mechanisms from the whole ecosystem to the gene expression level which regulate symbiotic N₂ fixation in terrestrial ecosystems are reviewed and discussed in terms of a conceptual model. Although the presented model is based primarily on our knowledge about the physiology of a few leguminous crop species and of ecosystem processes in managed, perennial grassland in temperate climatic conditions, it may stimulate thinking about functional relationships between symbiotic N₂ fixation and terrestrial ecosystems at various system levels.     

Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N₂-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O₂ flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N₂-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Host genetic control of symbiosis in soybean (Glycine max L.)

Genes controlling nitrogen-fixing symbioses of legumes with specialized bacteria known as rhizobia are presumably the products of many millions of years of evolution. Different adaptative solutions evolved in response to the challenge of survival in highly divergent complexes of symbionts. Whereas efficiency of nitrogen fixation appears to be controlled by quantitative inheritance, genes controlling nodulation are qualitatively inherited. Genes controlling nodulation include those for non-nodulation, those that restrict certain microsymbionts, and those conditioning hypernodulation, or supernodulation. Some genes are naturally occurring polymorphisms, while others were induced or were the result of spontaneous mutations. The geographic patterns of particular alleles indicate the role of coevolution in determining symbiont specificites and compatibilities. For example, the Rj4 allele occurs with higher frequency (over 50%) among the soybean (G. max) from Southeast Asia. DNA homology studies of strains of Bradyrhizobium that nodulate soybean indicated two groups so distinct as to warrant classification as two species. Strains producing rhizobitoxine-induced chlorosis occur only in Group II, now classified as B. elkanii. Unlike B. japonicum, B. elkanii strains are characterized by (1) the ability to nodulate the rj1 genotype, (2) the formation of nodule-like structures on peanut, (3) a relatively high degree of ex planta nitrogenase activity, (4) distinct extracellular polysaccharide composition, (5) distinct fatty acid composition, (6) distinct antibiotic resistance profiles, and (7) low DNA homology with B. japonicum. Analysis with soybean lines near isogenic for the Rj4 versus rj4 alleles indicated that the Rj4 allele excludes a high proportion of B. elkanii strains and certain strains of B. japonicum such as strain USDA62 and three serogroup 123 strains. These groups, relatively inefficient in nitrogen fixation with soybean, tend to predominate in soybean nodules from many US soils. The Rj4 allele, the most common allelic form in the wild species, has a positive value for the host plants in protecting them from nodulation by rhizobia poorly adapted for symbiosis.     

Bradyrhizobium strains and the nodulation,nodule efficiency and growth of soybean (Glycine max L.) in Egyptian soils

Six strains and a commercial inoculant ofBradyrhizobium japonicum were evaluated in association withGlycine max (L.) cultivar Clark. Inoculated and uninoculated plants were grown in pot and field experiments. Nodules were counted and weighed and roots and shoots were separated and analysed for total nitrogen. In pot experiments, two of six bacterial strains were superior to the other four, and to the commercial inoculant (Nitragin) in promoting greater root and top growth and plant nitrogen accumulation. In the field experiment, there were indications that environmental conditions may have affected nodulation by the bacteria. The strains could be divided into three groups according to nodule efficiencies, accumulation of plant dry matter, and total nitrogen content. The greater variations in nodule efficiencies of the tested strains could be attributed to the quantities of bacteroid, cytosol protein and leghaemoglobin in the nodules.     

Viability of Rhizobium bacteroids isolated from soybean nodule protoplasts

Bacteriods isolated from protoplasts taken from Rhizobium japonicum induced root nodule of Glycine max L. showed complete viability when plated onto a conventional rhizobial growth medium supplemented with 0.2 M Mannitol. The same medium but without extra mannitol resulted in the absence of colony formation. The protoplast isolation method eliminated the possibility of contaminant bacteria from infection threads to be scored. The redifferentiated bacteroid clones have the same genetical characteristics as the orginal inoculum strain. This and other recent findings of bacteroid viability are discussed in the light of the existing belief that bacteroids are non-viable.     

Comparison between parental and variant soybeanBradyrhizobium strains with regard to the production of lipo-chitin nodulation signals,early stages of root infection,nodule occupancy,and N2 fixation rates

Soybean is the most important leguminous crop in Brazil and the nitrogen required for plant growth is supplied byBradyrhizobium bacteria through the symbiotic relation established by the inoculation process. Since 1992, two new strains, CPAC 7 and CPAC 15, which have been shown to increase yields in several field experiments, have been recommended in Brazilian commercial inoculants. CPAC 15 is a natural variant of theB. elkanii SEMIA 566 strain, and was isolated after several years of adaptation to a Brazilian Cerrado soil, while CPAC 7 is a variant ofB. japonicum strain CB 1809, selected under laboratory conditions for higher nodulation and yield. The comparison between parental and variant strains, under greenhouse conditions, showed that both CPAC 15 and CPAC 7 increased N₂ fixation rates in relation to the parental strains. The better performance of CPAC 15 was related to an increase in nodule efficiency (mg N₂ fixed mg^-1 nodule) while with CPAC 7 the higher N₂ fixation rates were due to increased nodulation. Both CPAC 15 and CPAC 7 increased nodule occupancy, when co-inoculated at a ratio of 1:1 withB. elkanii 29w, in relation to their parental strains. Variant strains also differed from parental in their ability to increase numbers of root hairs (Hai phenotype) either when inoculated onto plants, or when supernatants of bacteria exposed to seed exudates were used as inoculants. This results lead to the hypothesis that a modification in some of the “common” nodulation genes had occurred. However, the increase in Hai phenotype with CPAC 7 was dependent on the soybean cultivar, indicating a possible alteration in some genotypic specific nodulation gene. Apparently, there were no differences in Nod metabolites produced by strains CPAC 15 and SEMIA 566, but a more detailed chemical analysis would be required to rule out subtle differences. On the contrary, significant differences were found between CPAC 7 and the parental strain CP 1809, in the profile of Nod metabolites. Consequently, it may be possible that diffusable molecules, responsible for Hai phenotype, would be related to nodulation ability, competiviveness, and N₂ fixation, resulting in the higher yields that have been associated with CPAC 7 and CPAC 15. For the CPAC 7 strain, the increase in Hai phenotype could be atributed to the differences found in the Nod molecules. Consequently, a high degree of physiological and genetic variability can result from the adaptation of rhizobial strains to the soil. Also, this variability can be found under laboratory conditions, when searching single colonies with specific properties. ei]Section editor: R O D Dixon     

The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis

A study was conducted to investigate the effects of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis. The effects of P on growth and survival of Frankia in the rhizosphere was assessed by examing Frankia growth and survival in flasks of basal nutrient solution. There was no growth in the nutrient solution during the experimental period. However, the viability of Frankia in the nutrient solution without P supply was half that of the initial level, whereas, with P supply, there was only a minor decline during the first week. In a growth pouch experiment, supplying P increased plant and nodule growth, irrespective of P status of the inoculant Frankia culture. There were no effects of P status on any growth or nodulation parameters measured when the inoculants had been standardized on the basis of viability. In a split root experiment, Frankia inoculation and application of P together or separately did not cause any significant difference. This suggests that growth and nodulation respond only to total P supply. Increasing P from 0.1 to 10 M significantly increased plant growth but not N concentrations. Both nitrogen-fixation and nitrate supported growth were strongly increased as P increased from 0.1 to 1.0 M. This study indicates that P deficiency limits the growth of host plants more severely than nitrogen fixation processes and P deficiency on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana operated indirectly via reducing host plant growth.     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA     

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 M added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2–5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron.All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 M added iron).Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 M added iron) during the stationary phase of growth.Abbreviations CAS chrome azurol S - HDTMA hexadecyltrime-thylammonium bromide     

Nodulation and N2 fixation of guar at high root temperature

Guar (Cyamopsis tetregonoloba (L.) may be grown when soil temperatures are potentially high enough at the time of planting to inhibit nodulation and N₂ fixation. An experiment was conducted using controlled conditions to determine the influence of high root temperature on growth and N₂ fixation of guar. The experiment included two strains of rhizobia, two varieties of guar, two mineral N treatments, and root temperatures of 34, 37, and 40°C. Plants were grown for 44 days. The root temperature of 40°C reduced N fixation by at least 80% and nodule weight by more than 50%. Significant interactions occurred between most factors in influencing nodulation, N₂ fixation and dry matter production. Guar, nodulated by rhizobial strain GAR022-1 and fully dependent on N₂ fixation or provided with starter mineral N (25 mg pot^–1), was not influenced by the root temperature of 37°C as compared to 34°C. Nodulation and N₂ fixation by strain 32H1 was reduced by at least 40% when no starter mineral N was provided and the root temperature was 37°C. Providing starter mineral N to one variety of guar doubled the quantity of N₂ fixed by strain 32H1 at both 34 and 37°C but N₂ fixation was lower at the higher root temperature. It appears that root temperatures between 37° and 40°C bracketed the critical root temperature for N₂ fixation by nodulated guar and that the critical root temperature for guar dependent on mineral N was above 40°C.     

Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia)

Summary Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif ^r showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.     

The relationship between the state of adenylylation of glutamine synthetase I and the export of ammonium in free-living,N2-fixing Rhizobium

The relationship between ammonium assimilation and ammonium export has been studied in free-living, N₂-fixing Rhizobium sp. 32H1. After 55 to 67 h of microaerobic growth under a gas phase of 0.2% O₂ – 1.0% CO₂ – 98.8% Ar high levels of nitrogenase were observed concomitant with a slightly adenylylated glutamine synthetase (GSI) and some glutamine synthetase (GSII) activity. However, after growth of 89 h, or longer, GSI became adenylylated and the level of GSII had decreased. When the gas phase was shifted to 0.2% O₂ – 1.0% CO₂ – 98.8% N₂, a lag was observed before ammonium export could be detected in the 55 to 67 h cultures. No lag in ammonium export was observed in the cultures previously grown for 89 h. The onset of ammonium export in the 55 to 67 h cultures was found to correlate with the adenylylation state of GSI. There appeared to be no correlation between the level of GSII and the export of ammonium. Neither an increase in the adenylylation level of GSI nor ammonium export was observed when the 55 to 67 h cultures were maintained under the Ar gas mixture.Abbreviations GOGAT Glutamate synthase - GS glutamine synthetase - BES [N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid] - CTAB cetyltrimethylammonium bromide - MES [2-(N-morpholino)-ethane sulfonic acid]     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

Effects of water deficit on N2(C2H2) fixation in cowpea and groundnut

The effect of water deficit on nodulation, N₂ fixation, photosynthesis, and total soluble sugars and leghemoglobin in nodules was investigated in cowpea and groundnut. Nitrogenase activity completely ceased in cowpea with a decrease in leaf water potential ( _leaf) from –0.4 MPa to –0.9 MPa, while in groundnut it continued down to –1.7 MPa. With increasing water stress, the acetylene reduction activity (ARA) declined very sharply in cowpea, but ARA gradually decreased in groundnut. Even with mild water stress ( _leaf of 0.2 MPa), nodule fresh weight declined 50% in cowpea partly due to a severe nodule shedding whereas nodule fresh weight declined in groundnut only when _leaf decreased by 1.0 MPa. No nodule shedding was noticed even at a higher stress level in groundnut. Photosynthesis and stomatal conductance were also more stable in groundnut than in cowpea under water stress. There was a sharp increase in total soluble sugars and leghemoglobin in the nodules of groundut with water stress, but no definite trend could be found in cowpea.     

Hopanoid lipids in Bradyrhizobium and other plant-associated bacteria and cloning of the Bradyrhizobium japonicum squalene-hopene cyclase gene

Hopanoid lipids have been discovered recently in a number of nitrogen-fixing soil bacteria and in Bradyrhizobium bacteria which fix nitrogen in association with legume plants. We report here an investigation of the hopanoid content in an additional number of soil bacteria capable of living in close association with plants. Of the strains investigated, hopanoids were discovered in phototrophic, nitrogen-fixing bacteria and in an extended number of Bradyrhizobium strains. Strains in which hopanoids so far have not been found belong to the following genera: Rhizobium, Sinorhizobium, Phyllobacterium, Agrobacterium, and Azoarcus. To address the function of hopanoids in Bradyrhizobium, we cloned the gene coding for a key enzyme of hopanoid biosynthesis, the squalene-hopene cyclase, and expressed the gene in E. coli. The recombinant enzyme catalyzed in vitro the cyclization of squalene to hopanoid derivatives.Abbreviations SHC squalene-hopene cyclase - shc squalene-hopene cyclase gene     

Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2 fixation

The ¹⁵N methods are potentially accurate for measuring N₂ fixation in plants. The only problem with those methods is, how to ensure that the ¹⁵N/¹⁴N ratio in the plant accurately reflects the integrated ¹⁵N/¹⁴N ratio (R) in soil which is variable in time and with soil depth. However, the consequences of using an inappropriate reference plant vary with the level of N₂ fixation and the conditions under which the study was made. For example, the errors introduced into the values of N₂ fixation are higher at low levels of fixation, and decrease with increasing rates of fixation. At very high N₂ fixation rates, the errors are often insignificant. Also, the magnitude of error is proportional to the rate of decline of the ¹⁵N/¹⁴N ratio with time. Since N₂ fixation in most plants would be expected to below 60%, the question of how to select a good reference plant is still pertinent. In this paper, we have discussed some of the criteria to adopt in selecting reference plants, e.g. how to ensure that the reference plant is not fixing N₂, is absorbing most of its N from the same zone as the fixing plant, and in the same pattern with time, etc. In addition, we have discussed ¹⁵N labelling materials and methods that are likely to minimize any errors even when the fixing and reference plants don't match well in certain important criteria. The use of slow release ¹⁵N fertilizer or ¹⁵N labelled plant materials results in slow changes in the ¹⁵N/¹⁴N ratio of soil, and is strongly recommended. Where ¹⁵N inorganic fertilizers are used, the application of the fertilizer in small splits at various intervals is recommended over a one-time application. The problem with the reference crop, which has sometimes discouraged potential users of the ¹⁵N methods, is surmountable, as discussed in this paper.