Symbiotic Nitrogen Fixation Activity Research Articles

A VIT‐like transporter facilitates iron transport into nodule symbiosomes for nitrogen fixation in soybean

Effective legume-rhizobia symbiosis depends on efficient nutrient exchange. Rhizobia need to synthesize iron-containing proteins for symbiotic nitrogen fixation (SNF) in nodules, which depends on host plant-mediated iron uptake into the symbiosome. We functionally investigated a pair of vacuolar iron transporter like (VTL) genes, GmVTL1a/b, in soybean (Glycine max) and evaluated their contributions to SNF, including investigations of gene expression patterns, subcellular localization, and mutant phenotypes. Though both GmVTL1a/b genes were specifically expressed in the fixation zone of the nodule, GmVTL1a was the lone member to be localized at the tonoplast of tobacco protoplasts, and shown to facilitate ferrous iron transport in yeast. GmVTL1a targets the symbiosome in infected cells, as verified by in situ immunostaining. Two vtl1 knockout mutants had lower iron concentrations in nodule cell sap and peribacteroid units than in wild-type plants, suggesting that GmVTL1 knockout inhibited iron import into symbiosomes. Furthermore, GmVTL1 knockout minimally affected soybean growth under nonsymbiotic conditions, but dramatically impaired nodule development and SNF activity under nitrogen-limited and rhizobia-inoculation conditions, which eventually led to growth retardation. Taken together, these results demonstrate that GmVTL1a is indispensable for SNF in nodules as a transporter of ferrous iron from the infected root cell cytosol to the symbiosome.

Digalactosyldiacylglycerol Synthase Gene MtDGD1 Plays an Essential Role in Nodule Development and Nitrogen Fixation.

Little is known about the genes participating in digalactosyldiacylglycerol (DGDG) synthesis during nodule symbiosis. Here, we identified full-length MtDGD1, a synthase of DGDG, and characterized its effect on symbiotic nitrogen fixation in Medicago truncatula. Immunofluorescence and immunoelectron microscopy showed that MtDGD1 was located on the symbiosome membranes in the infected cells. β-Glucuronidase histochemical staining revealed that MtDGD1 was highly expressed in the infection zone of young nodules as well as in the whole mature nodules. Compared with the control, MtDGD1-RNA interference transgenic plants exhibited significant decreases in nodule number, symbiotic nitrogen fixation activity, and DGDG abundance in the nodules, as well as abnormal nodule and symbiosome development. Overexpression of MtDGD1 resulted in enhancement of nodule number and nitrogen fixation activity. In response to phosphorus starvation, the MtDGD1 expression level was substantially upregulated and the abundance of nonphospholipid DGDG was significantly increased in the roots and nodules, accompanied by corresponding decreases in the abundance of phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Overall, our results indicate that DGD1 contributes to effective nodule organogenesis and nitrogen fixation by affecting the synthesis and content of DGDG during symbiosis.

Light regulates tropical symbiotic nitrogen fixation more strongly than soil nitrogen.

Nitrogen limits primary production in almost every biome on Earth1,2. Symbiotic nitrogen fixation, conducted by certain angiosperms and their endosymbiotic bacteria, is the largest potential natural source of new nitrogen into the biosphere3, influencing global primary production, carbon sequestration and element cycling. Because symbiotic nitrogen fixation represents an alternative to soil nitrogen uptake, much of the work on symbiotic nitrogen fixation regulation has focused on soil nitrogen availability4-8. However, because symbiotic nitrogen fixation is an energetically expensive process9, light availability to the plant may also regulate symbiotic nitrogen fixation rates10,11. Despite the importance of symbiotic nitrogen fixation to biosphere functioning, the environmental factors that most strongly regulate this process remain unresolved. Here we show that light regulates symbiotic nitrogen fixation more strongly than does soil nitrogen and that light mediates the response of symbiotic nitrogen fixation to soil nitrogen availability. In a shadehouse experiment, low light levels (comparable with forest understories) completely shut down symbiotic nitrogen fixation, whereas soil nitrogen levels that far exceeded plant demand did not fully downregulate symbiotic nitrogen fixation at high light. For in situ forest seedlings, light was a notable predictor of symbiotic nitrogen fixation activity, but soil-extractable nitrogen was not. Light as a primary regulator of symbiotic nitrogen fixation is a departure from decades of focus on soil nitrogen availability. This shift in our understanding of symbiotic nitrogen fixation regulation can resolve a long-standing biogeochemical paradox12, and it will improve our ability to predict how symbiotic nitrogen fixation will fuel the global forest carbon sink and respond to human alteration of the global nitrogen cycle.

Challenges in Using Precision Agriculture to Optimize Symbiotic Nitrogen Fixation in Legumes: Progress, Limitations, and Future Improvements Needed in Diagnostic Testing

Precision agriculture (PA) has been used for ≥25 years to optimize inputs, maximize profit, and minimize negative environmental impacts. Legumes play an important role in cropping systems, by associating with rhizobia microbes that convert plant-unavailable atmospheric nitrogen into usable nitrogen through symbiotic nitrogen fixation (SNF). However, there can be field-level spatial variability for SNF activity, as well as underlying soil factors that influence SNF (e.g., macro/micronutrients, pH, and rhizobia). There is a need for PA tools that can diagnose spatial variability in SNF activity, as well as the relevant environmental factors that influence SNF. Little information is available in the literature concerning the potential of PA to diagnose/optimize SNF. Here, we critically analyze SNF/soil diagnostic methods that hold promise as PA tools in the short–medium term. We also review the challenges facing additional diagnostics currently used for research, and describe the innovations needed to move them forward as PA tools. Our analysis suggests that the nitrogen difference method, isotope methods, and proximal and remote sensing techniques hold promise for diagnosing field-level variability in SNF. With respect to soil diagnostics, soil sensors and remote sensing techniques for nitrogen, phosphorus, pH, and salinity have short–medium term potential to optimize legume SNF under field conditions.

A Biosensor-Based Leaf Punch Assay for Glutamine Correlates to Symbiotic Nitrogen Fixation Measurements in Legumes to Permit Rapid Screening of Rhizobia Inoculants under Controlled Conditions.

Legumes are protein sources for billions of humans and livestock. These traits are enabled by symbiotic nitrogen fixation (SNF), whereby root nodule-inhabiting rhizobia bacteria convert atmospheric nitrogen (N) into usable N. Unfortunately, SNF rates in legume crops suffer from undiagnosed incompatible/suboptimal interactions between crop varieties and rhizobia strains. There are opportunities to test much large numbers of rhizobia strains if cost/labor-effective diagnostic tests become available which may especially benefit researchers in developing countries. Inside root nodules, fixed N from rhizobia is assimilated into amino acids including glutamine (Gln) for export to shoots as the major fraction (amide-exporting legumes) or as the minor fraction (ureide-exporting legumes). Here, we have developed a new leaf punch based technique to screen rhizobia inoculants for SNF activity following inoculation of both amide exporting and ureide exporting legumes. The assay is based on measuring Gln output using the GlnLux biosensor, which consists of Escherichia coli cells auxotrophic for Gln and expressing a constitutive lux operon. Subsistence farmer varieties of an amide exporter (lentil) and two ureide exporters (cowpea and soybean) were inoculated with different strains of rhizobia under controlled conditions, then extracts of single leaf punches were incubated with GlnLux cells, and light-output was measured using a 96-well luminometer. In the absence of external N and under controlled conditions, the results from the leaf punch assay correlated with 15N-based measurements, shoot N percentage, and shoot total fixed N in all three crops. The technology is rapid, inexpensive, high-throughput, requires minimum technical expertise and very little tissue, and hence is relatively non-destructive. We compared and contrasted the benefits and limitations of this novel diagnostic assay to methods.

Disparate role of rhizobial ACC deaminase in root-nodule symbioses

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase converts ACC, a precursor of the plant hormone ethylene, into ammonia and α-ketobutyrate. ACC deaminase is widespread among the rhizobia in which it might play a crucial role in protecting rhizobia against inhibitory effects of ethylene synthesized by the host plant in response to the nodulation process. The beneficial action of this enzyme was demonstrated in several rhizobia such as Mesorhizobium loti and Rhizobium leguminosarum where knock-out mutants of the ACC deaminase gene showed nodulation defects. The genome of the slow-growing rhizobial species Bradyrhizobium japonicum also carries an annotated gene for a putative ACC deaminase (blr0241). Here, we tested the possible importance of this enzyme in B. japonicum by constructing an insertion mutant of blr0241 and studying its phenotype. First, the activity of ACC deaminase itself was measured. Unlike the B. japonicum wild type, the blr0241 mutant did not show any enzymatic activity. By contrast, the mutant was not impaired in its ability to nodulate soybean, cowpea, siratro, and mungbean. Likewise, symbiotic nitrogen fixation activity remained unaffected. Furthermore, a co-inoculation assay with the B. japonicum wild type and the blr0241 mutant for soybean and siratro nodulation revealed that the mutant was not affected in its competitiveness for nodulation and nodule occupation. The results show that the role previously ascribed to ACC deaminase in the rhizobia cannot be generalized, and species-specific differences may exist.

Quantitative trait locus analysis of symbiotic nitrogen fixation activity in the model legume Lotus japonicus

Many legumes form nitrogen-fixing root nodules. An elevation of nitrogen fixation in such legumes would have significant implications for plant growth and biomass production in agriculture. To identify the genetic basis for the regulation of nitrogen fixation, quantitative trait locus (QTL) analysis was conducted with recombinant inbred lines derived from the cross Miyakojima MG-20×Gifu B-129 in the model legume Lotus japonicus. This population was inoculated with Mesorhizobium loti MAFF303099 and grown for 14days in pods containing vermiculite. Phenotypic data were collected for acetylene reduction activity (ARA) per plant (ARA/P), ARA per nodule weight (ARA/NW), ARA per nodule number (ARA/NN), NN per plant, NW per plant, stem length (SL), SL without inoculation (SLbac-), shoot dry weight without inoculation (SWbac-), root length without inoculation (RLbac-), and root dry weight (RWbac-), and finally 34 QTLs were identified. ARA/P, ARA/NN, NW, and SL showed strong correlations and QTL co-localization, suggesting that several plant characteristics important for symbiotic nitrogen fixation are controlled by the same locus. QTLs for ARA/P, ARA/NN, NW, and SL, co-localized around marker TM0832 on chromosome 4, were also co-localized with previously reported QTLs for seed mass. This is the first report of QTL analysis for symbiotic nitrogen fixation activity traits.

FUNCTIONING OF SYMBIOTIC SYSTEMS OF COWPEA – NODULE BACTERIA

The nodule bacteria were isolated from the nodules of cowpea. On the basis of phenotypical properties these rhizobia were referred to slow growing bacteria of Bradyrhizobium genus. Interaction features of cowpea with the nodule bacteria of cowpea (Bradyrhizobium sp. (Vigna)) and soybean (Bradyrhizobium japonicum) on nitrogen-free substrate and in soil culture were studied. It was established that the cowpea rhizobia strains possess high specificity to the host plant, promote symbiotic nitrogen fixation activity in 1,8-2,6 times and increase plants aboveground mass yield in 1,4-3,4 times, in comparison with control. The significant positive influence of the active soybean microsymbiont Bradyrhizobium japonicum 46 on the growth and development of cowpea was shown.

Disparate Pathways for the Biogenesis of Cytochrome Oxidases in Bradyrhizobium japonicum

This work addresses the biogenesis of heme-copper terminal oxidases in Bradyrhizobium japonicum, the nitrogen-fixing root nodule symbiont of soybean. B. japonicum has four quinol oxidases and four cytochrome oxidases. The latter include the aa(3)- and cbb(3)-type oxidases. Although both have a Cu(B) center in subunit I, the subunit II proteins differ in having either a Cu(A) center (in aa(3)) or a covalently bound heme c (in cbb(3)). Two biogenesis factors were genetically studied here, the periplasmically exposed CoxG and ScoI proteins, which are the respective homologs of the mitochondrial copper-trafficking chaperones Cox11 and Sco1 for the formation of the Cu(B) center in subunit I and the Cu(A) center in subunit II of cytochrome aa(3). We could demonstrate copper binding to ScoI in vitro, a process for which the thiols of cysteine residues 74 and 78 in the ScoI polypeptide were shown to be essential. Knock-out mutations in the B. japonicum coxG and scoI genes led to loss of cytochrome aa(3) assembly and activity in the cytoplasmic membrane, whereas the cbb(3)-type cytochrome oxidase apparently remained unaffected. This suggests that subunit I of the cbb(3)-type oxidase obtains its copper cofactor via a different pathway than cytochrome aa(3). In contrast to the coxG mutation, the scoI mutation caused a decreased symbiotic nitrogen fixation activity. We hypothesize that a periplasmic B. japonicum protein other than any of the identified Cu(A) proteins depends on ScoI and is required for an effective symbiosis.

Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus

Plant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma, respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1- or AfHb1-overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1- and AfHb1-overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodules, whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.

Evidence for functional differentiation of duplicatednifHgenes inAzorhizobium caulinodans

Azorhizobium caulinodans is a symbiotic diazotroph that contains duplicated nifH genes. This study focused on the biological sense behind the duplication. In-frame deletion mutants of nifH1 and nifH2 were constructed in order to analyze nitrogen fixation activity, both in symbiosis and in free-living conditions. Symbiotic nitrogen fixation activity was not affected by deletion of nifH1 or nifH2, while free-living nitrogen fixation activity was significantly decreased. Deletion of nifH1 had a significant effect in semi-aerobic condition, while deletion of nifH2 was significant in microaerobic condition, suggesting functional differences between nifH1 and nifH2. Transcriptional activity of nifH1 was higher than nifH2, both in microaerobic and semi-aerobic conditions.

Growth media effects on shoot physiology, nodule numbers and symbiotic nitrogen fixation in soybean

Several research groups (both in South Africa and other countries) are currently involved in research aimed at improving symbiotic nitrogen fixation (SNF) and root nodule sustainability in soybean [ Glycine max (L.) Merr.]. In many of these experiments potted plants are used, and in this paper the importance of careful selection of growth media is demonstrated. Bradyrhizobium japonicum-inoculated soybean seedlings were cultivated in pots containing N-free growth media (sand, fine vermiculite or coarse vermiculite) or a growth medium containing low concentrations of water-soluble nitrogen predominantly in the form of ammonium (mixture of potting soil, sand and vermiculite). The effects of growth media on shoot physiology were assessed by measurement of plastochron index, chlorophyll content and CO 2 assimilation rates. Nodule numbers, nitrogenase activity and nodule ureide content were also determined. Although similar source–sink relationships were maintained in plants cultured in the various growth media, large effects on nodule numbers and SNF were observed. Shoot phenotype and physiology did not provide any insight into these belowground effects. The presence of mineral N, or sand as culture medium, led to the formation of more abundant nodules but with low SNF activity. Vermiculite, irrespective of particle size, resulted in plants with root systems housing nodules with high SNF activity. It is concluded that choice of growth media for cultivating soybean plants under controlled growth conditions is an important consideration, especially in multi-institution collaborations where comparability between experiments is a pre-requisite.

Characterization of an rpoN mutant of Mesorhizobium ciceri

To study the genetic basis of C(4)-dicarboxylate transport (Dct) in relation to symbiotic nitrogen fixation in Mesorhizobium ciceri. A Tn5-induced mutant strain (TL16) of M. ciceri, unable to grow on C(4)-dicarboxylates, was isolated from the wild-type strain TAL 620. The mutant lacked activities of the enzymes, which use C(4)-dicarboxylates as substrate. The sequencing of the 3.2kb EcoRI fragment, which was the site of Tn5 insertion, revealed three complete and two partial open reading frames. In the mutant, Tn5 interrupted the rpoN gene, of which only one copy was there. Complementation and biochemical studies suggest that the M. ciceri rpoN activity is required for C(4)-Dct, maturation of bacteroids and symbiotic nitrogen fixation. The fine structure of the ineffective nodules produced by TL16 on Cicer arietinum L changed in comparison with those produced by the wild type. The mutant strain TL16 suffered a disruption in the rpoN gene. Only one copy of rpoN gene is present in M. ciceri. The mutation abolishes Dct activity. It additionally abolishes the symbiotic nitrogen fixation activity of the bacteroids in the nodules. This first document in M. ciceri shows that a functional rpoN gene is essential for the transport of dicarboxylic acids and symbiotic nitrogen fixation.

Genetic Determinants of Swarming in Rhizobium etli

Swarming motility is considered to be a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer, and the phenomenon has previously been linked with biofilm formation and virulence. The gram-negative nitrogen-fixing soil bacterium Rhizobium etli CNPAF512 was previously shown to display swarming behavior on soft agar plates. In a search for novel genetic determinants of swarming, a detailed analysis of the swarming behavior of 700 miniTn5 mutants of R. etli was performed. Twenty-four mutants defective in swarming or displaying abnormal swarming patterns were identified and could be divided into three groups based on their swarming pattern. Fourteen mutants were completely swarming deficient, five mutants showed an atypical swarming pattern with no completely smooth edge and local extrusions, and five mutants displayed an intermediate swarming phenotype. Sequence analysis of the targeted genes indicated that the mutants were likely affected in quorum-sensing, polysaccharide composition or export, motility, and amino acid and polyamines metabolism. Several of the identified mutants displayed a reduced symbiotic nitrogen fixation activity.

The Rhizobium etli opt operon is required for symbiosis and stress resistance

Rhizobium etli is a Gram-negative root-colonizing soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. A genome-wide screening for R. etli symbiotic mutants revealed a R. etli operon encoding an oligopeptide ABC-transporter (Opt), two redA homologous genes and one redB gene. Expression analysis showed this opt operon to be transcribed both under free-living and symbiotic conditions and expression levels were demonstrated to be growth-phase-dependent. Plants nodulated by R. etli opt mutants showed a reduced symbiotic nitrogen fixation activity (approximately 50% reduction). Growth experiments with opt mutants in the presence of oligopeptides as the sole nitrogen source confirmed the involvement of the opt genes in oligopeptide uptake. Further phenotypic analysis of the opt mutants revealed them to display an enhanced resistance to the oligopeptide antibiotic bacitracin, an increased susceptibility to the beta-lactam antibiotic ampicillin and a decreased osmotolerance. In conclusion, our results demonstrate that the opt operon plays a crucial role during symbiosis and stress resistance.

Symbiotic N2 fixation activity in relation to C economy of Pisum sativum L. as a function of plant phenology.

The relationships between symbiotic nitrogen fixation (SNF) activity and C fluxes were investigated in pea plants (Pisum sativum L. cv. Baccara) using simultaneous 13C and 15N labelling. Analysis of the dynamics of labelled CO2 efflux from the nodulated roots allowed the different components associated with SNF activity to be calculated, together with root and nodule synthetic and maintenance processes. The carbon costs for the synthesis of roots and nodules were similar and decreased with time. Carbon lost by turnover, associated with maintenance processes, decreased with time for nodules while it increased in the roots. Nodule turnover remained higher than root turnover until flowering. The effect of the N source on SNF was investigated using plants supplied with nitrate or plants only fixing N2. SNF per unit nodule biomass (nodule specific activity) was linearly related to the amount of carbon allocated to the nodulated roots regardless of the N source, with regression slopes decreasing across the growth cycle. These regression slopes permitted potential values of SNF specific activity to be defined. SNF activity decreased as the plants aged, presumably because of the combined effects of both increasing C costs of SNF (from 4.0 to 6.7 g C g-1 N) and the limitation of C supply to the nodules. SNF activity competed for C against synthesis and maintenance processes within the nodulated roots. Synthesis was the main limiting factor of SNF, but its importance decreased as the plant aged. At seed-filling, SNF was probably more limited by nodule age than by C supply to the nodulated roots.

GroEL chaperonins are required for the formation of a functional nitrogenase in Bradyrhizobium japonicum

At least five highly conserved, but disparately regulated groESL operons are present in Bradyrhizobium japonicum. Expression of groESL 3 is coregulated with symbiotic nitrogen fixation genes, implying a role of GroESL chaperonins in the nitrogen fixation process. Null mutants of individual groEL genes, however, were not impaired in symbiotic nitrogen fixation activity. By contrast, the groEL 3-plus-groEL 4 double mutant strain D4, which is mutated in those groEL genes that contribute most to the GroEL pool under symbiotic conditions, exhibited less than 5% Fix activity as compared to the wild-type. Expression of lacZ fusions made to several representative nif and fix genes was not, or only marginally, reduced in mutant D4, indicating that the requirement of chaperonins for nitrogen fixation does not occur at the level of RegSR-NifA-σ54- or FixLJ-FixK2-dependent gene regulation. Instead, immunoblot analyses revealed that the level of NifH and NifDK nitrogenase proteins was drastically decreased in extracts prepared from D4 bacteroids and from free-living cells grown anaerobically. Transcriptional fusions of the anaerobically induced groESL 3 promoter (P3) to all five B. japonicum groESL operons and also to groESL from Escherichia coli were integrated into the chromosome of mutant D4. Strains harboring P3 fused to groESL 1, groESL 2, groESL 5, or E. coli groESL partially complemented the symbiotic defect of mutant D4, whereas the wild-type phenotype was completely restored in strains complemented with P3 fused to groESL 3 (control) or groESL 4. Likewise, the growth defect of an E. coli groEL mutant could be corrected at least partially by individual B. japonicum groESL operons. In conclusion, both series of complementation analyses were not indicative of a strict substrate specificity of any of the B. japonicum groESL gene products, which is in good agreement with their high degree of sequence conservation.

A new Bradyrhizobium japonicum gene required for free-living growth and bacteroid development is conserved in other bacteria and in plants.

In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, a new DNA region, orf74, was discovered which is required for optimal free-living growth and, by consequence, also necessary for the formation of an effective symbiosis. A Tn5-233 insertion of orf14 resulted in a mutant, strain 74, that has a reduced growth rate in free-living cultures under all conditions tested and less than 1% residual symbiotic nitrogen fixation activity as compared with the wild type. Nodule distribution and nodule morphology are severely affected similarly as in a previously characterized B. japonicum nifA mutant. Protein databank searches revealed that the 142-amino-acid protein encoded by orf74 is homologous to a 161-amino-acid protein encoded by orf17 of Bacillus subtilis (approximately 46% identity; J. C. R. Struck, R. Kretschmer-Kazemi Far, W. Schröder, F. Hucho, H. Y. Toschka, and V. A. Erdmann, Biochim. Biophys. Acta, 1050:80-83, 1990) as well as to a 178-amino-acid protein encoded by orf178 of Escherichia coli (approximately 45% identity; K. L. Poulsen, N. W. Larsen, S. Molin, and P. Andersson, Mol. Microbiol., 6:895-905, 1992). Significant similarity was also found with unknown ORFs of two plant species. When expressed from a strong constitutive promoter, orf17 of B. subtilis could partially complement B. japonicum mutant 74. By contrast, orf74 of B. japonicum was unable to functionally complement an E. coli orf178 mutant. The conservation of this DNA region in gram-negative and gram-positive bacteria suggests that the gene is essential for a fundamental cellular process which is required in B. japonicum for both free-living and symbiotic growth.

Soybean nitrogen fixation and growth as affected by drought stress and potassium fertilization

Abstract Several studies have identified drought stress as having particularly deleterious effects on symbiotic nitrogen fixation activity in soybean. Potassium is important in the growth of soybean and an important ion in the physiology of plant water relations. Therefore, this study was undertaken to examine the interactive effects of various levels of potassium and water in the soil on the nitrogen fixation activity of soybean. The experiment was done under greenhouse conditions to provide the plants with well‐defined levels of potassium and to control the soil water availability. No difference in vegetative growth or acetylene reductions rates was detected among the potassium treatments. However, seed yield showed an optimum for the middle potassium treatment (50 mg K2O/kg soil) and the total accumulated nitrogen was lowest for the highest potassium treatment. As expected, dramatic differences as a result of the drought treatments were detected in nitrogen fixation rates and plant growth. Overall, the...

Heterosis for symbiotic nitrogen fixation in Phaseolus interspecific hybrids

Interspecific hybrids of Phaseolus vulgaris and Phaseolus acutifolius or Phaseolus filiformis have been evaluated for evidence of heterosis for symbiotic nitrogen fixation activity. Hybrids showing composite leghemoglobin profiles were selected for comparison. Rooted cuttings from hybrids and their respective parents were used to estimate nitrogen fixation (acetylene reduction) rates and to determine nitrogen accumulation during the growth period. Some hybrids of Phasolus vulgaris × Phaseolus acutifolius and Phaseolus vulgaris × Phaseolus filiformis had significantly (P<0.01) higher nitrogen fixation rates and in the latter case, accumulated more total nitrogen, than either parent. The interspecific hybrids also showed more complex leghemoglobin profiles than either parent.