Effect of nodulation on assimilate remobilization in soybean

The objectives of this work were to determine the effect of nodulation on dry matter, reduced-N, and phosphorus accumulation and partitioning in above-ground vegetative parts and pods of field-grown soybean (Glycine max [L.] Merr. cv Harosoy).

From comparison of nodulated and nonnodulated isolines, it was estimated that nodulated plants attained 81 and 71% of total-plant (above ground) N from uptake of soil N in 1981 and 1982, respectively. These data, along with visibly greener leaves of nodulated plants, led us to assume that nonnodulated plants were under a moderate N stress relative to nodulated plants. Nonnodulated plants accumulated less total-plant N and partitioned less dry matter and N to the pods, compared with nodulated plants. This occurred even though net photosynthesis, as estimated by rate and amount of dry matter accumulation, was the same for both nonnodulated and nodulated plants. Rate of dry matter and reduced-N accumulation in pods was less for nonnodulated than for nodulated plants while duration of podfill was similar for both isolines. From these data we concluded that moderate N stress affected partitioning of photosynthate rather than net photosynthesis, and that N played a role in translocation of photosynthate to the pods. Total plants (above-ground portion) and pods of both nodulated and nonnodulated plants accumulated similar amounts of phosphorus, which indicated that phosphorus and N accumulation were independent.

Remobilization of nitrogen and phosphorus from vegetation to pods preceded dry matter remobilization. It appeared that either more nitrogen accumulation prior to podfill, or continued nitrogen assimilation during podfill would increase nitrogen and dry matter partitioning to pods, but that increasing photosynthesis without concomitantly increasing nitrogen input may not necessarily result in enhanced seed production.

     

Efficiency of Nitrogen Assimilation by N(2)-Fixing and Nitrate-Grown Soybean Plants (Glycine max [L.] Merr.)

Nodulated and non-nodulated (not inoculated) soybeans (Glycine max [L.] Merr. cv Wells) were grown in controlled environments with N₂ or nonlimiting levels of NO₃⁻, respectively, serving as sole source of nitrogen. The efficiency of the N₂-fixing plants was compared with that of the nitrate-supplied plants on the basis of both plant age and plant size. Efficiency evaluations of the plants were expressed as the ratio of moles of carbon respired by the whole plant to the moles of nitrogen incorporated into plant material.

Continuous 24-hour CO₂ exchange measurements on shoot and root systems made at the beginning of flowering (28 days after planting) indicated that N₂-fixing plants respired 8.28 moles of carbon per mole of N, fixed from dinitrogen, while nitrate-supplied plants respired only 4.99 moles of carbon per mole of nitrate reduced. Twenty-one-day-old nitrate-supplied plants were even more efficient, respiring only 3.18 moles of carbon per mole of nitrate reduced. The decreased efficiency of the N₂-fixing plants was not due to plant size since, on a dry weight basis, the 28-day-old N₂-fixing plants were intermediate between the 28- and 21-day-old nitrate-supplied plants.

The calculated efficiencies were predominantly a reflection of root-system respiration. N₂-fixing plants lost 25% of their daily net photosynthetic input of carbon through root-system respiration, compared with 16% for 28-day-old nitrate-supplied plants and 12% for 21-day-old nitrate-supplied plants. Shoot dark respiration was similar for all three plant groups, varying between 7.9% and 9.0% of the apparent photosynthate.

The increased respiratory loss by the roots of the N₂-fixing plants was not compensated for by increased net photosynthetic effectiveness. Canopy photosynthesis expressed on a leaf area basis was similar for 28-day-old N₂-fixing plants (15.5 milligrams CO₂ square decimeter per hour) and 21-day-old nitrate-supplied plants (14.5 milligrams CO₂ square decimeter per hour). Both were similar in total canopy leaf area. The larger nitrate-supplied plants (28-day-old) had lower photosynthetic rates (12.5 milligrams CO₂ square decimeter per hour), presumably due to self-shading of the leaves.

These data indicate that, during the early stages of plant development, dependence solely on N₂-fixation is an expensive process compared to nitrate reduction in nitrate-supplied plants, since the N₂-fixing plants retained 8% to 12% less of their photosynthate as dry matter.

     

The Role of Cotyledons in Vegetative Growth and Nitrogen Assimilation by Soya Bean

The removal of both cotyledons from soya bean seedlings 10 daysafter sowing, when the primary leaves were unfolded, reducedtheir stem height, branching, leaf production and dry weightat flowering by a similar proportion whether they were nodulatedor nitrate-dependent. Nitrogen assimilation by the shoots ofnitrate-dependent plants was increased by the removal of onecotyledon and reduced by the removal of both cotyledons althoughthese effects were not significant. Both these treatments significantlyincreased the amount of nitrogen in the shoots of nodulatedplants at flowering, mainly by more than doubling the nitrogencontent of their leaves. In contrast, the proportion of thetotal plant nitrogen in the leaves of nitrate-dependent plantswas almost constant. These results suggest that the cotyledonsmarkedly inhibit nitrogen assimilation by nodulated plants butdo not appreciably affect nitrogen assimilation by plants dependentsolely on inorganic nitrogen for their nitrogen supply. Glycine mux (L) Merr., soya bean, cotyledons, nitrogen assimilation, growth     

Nitrogen fixation and assimilation efficiency in Ethiopian and German pea varieties

Dinitrogen (N₂) fixation and assimilation efficiency in a German and two Ethiopian varieties of Pisum sativum L. was studied in a pot experiment during vegetative and reproductive growth. The objective of the study was to assess whether genotypes having contrasting growth habits showed differences in physiological processes that affect the efficiency of N₂ fixation and assimilation. Dry matter formation, nodulation and nitrogen assimilation were compared between two treatments where one depended solely on N₂ fixation while the other was nourished with nitrate. Moreover, carbon (C) costs of N₂ fixation and the capacity of different respiratory chains in roots and nodules were determined at vegetative and reproductive growth. As compared to the Ethiopian cultivars, the German variety displayed a more rapid vegetative growth with intensive N₂ fixation and assimilation and highly efficient individual nodules. However, during reproductive growth, N₂ fixation in the German variety declined sharply, while continuing in the Ethiopian varieties. Lowest C costs of N₂ fixation coincided with most efficient individual nodules in both growth intervals. C costs of N₂ fixation were lower during reproductive growth in all varieties which was accompanied by a shift in root/nodule respiratory capacity towards more C efficient respiratory pathways. The results provide further evidence that unreliable nitrogen fixation rates during reproductive growth of pea can be connected with restricted C supply to nodules. One strategy of pea plants to adapt to critical C availability is an increase in capacity of more C efficient root/nodule respiration.     

Studies on Genetic Male-Sterile Soybeans : II. Effect of Nodulation on Photosynthesis and Carbon Partitioning in Leaves

Soybean (Glycine max L. Merr.) germplasm, essentially isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁), were developed to study the effects of reproductive development and nitrogen source on certain aspects of photosynthesis. Plants were sampled from flowering (77 days after transplanting) until maturity (150 days after transplanting). With all four genotypes, net carbon exchange rates were highest at flowering and declined thereafter. Photosynthetic rates of the sterile genotypes (nodulated and non-nodulated) declined more rapidly than the fertile genotypes, and after 105 days, both sterile genotypes maintained low but relatively constant carbon exchange rates (<3 milligrams CO₂/gram fresh weight per hour). Photosynthetic rates and starch accumulation (difference between afternoon and morning levels) declined with time. The sterile genotypes attained the highest morning starch levels, which reflected reduced starch mobilization. After 92 days, the proportion of photosynthetically fixed carbon that was partitioning into starch (relative leaf starch accumulation) in the sterile genotypes increased dramatically. In contrast, relative leaf starch accumulation in the fertile genotypes remained relatively constant with time. Throughout the test period, all four genotypes maintained leaf sucrose levels between 5 and 15 micromoles glucose equivalents per gram fresh weight.

The activities of sucrose phosphate synthase (SPS) in leaf extracts of the four genotypes declined from 77 to 147 days. Nodulated genotypes tended to maintain higher activities (leaf fresh weight basis) than did the non-nodulated genotypes. In general, relative leaf starch accumulation was correlated negatively with the activity of SPS (normalized with leaf net carbon exchange rate) in leaf extracts for all four genotypes during early reproductive development, and for the fertile genotypes at all sampling dates. In contrast, leaf sucrose content was correlated positively with SPS activity during early reproductive development. These results suggested that a direct relation existed between the activity of SPS and starch/sucrose levels in soybean leaves. However, the interaction between these processes also may be influenced by other factors, particularly when leaf photosynthetic rates and plant demand for assimilates is low, as in the sterile genotypes.

     

Photosynthate metabolism in the source leaves of n(2)-fixing soybean plants

Soybean plants (Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N₂ fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N₂-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO₃⁻ and NH₄⁺ (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N₂-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6-bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N₂-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N₂-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose-6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the K_m values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N₂-fixing as well as N-supplemented plants, and G1,6BP levels in N₂-fixing plant leaves was twice that of levels in N-supplied treatments. However the concentration of chloroplastic G1,6BP in illuminated leaves was computed to be saturating with respect to PGM in both N₂-fixing and N-supplemented plants. This suggested that the higher level of this cofactor in N₂-fixing plant leaves did not confer any higher PGM activation and was not a factor in higher starch synthesis rates. Relative to plants supplied with NO₃⁻ and NH₄⁺, the source leaf glycerate-3-phosphate (3-PGA) and orthophosphate (Pi) concentrations in leaves of N₂-fixing plants were two to four times higher. Although Pi is a physiological competitive inhibitor of leaf chloroplast ADPG-PPiase, and hence, starch synthesis, elevated chloroplast 3-PGA levels in N₂-fixing plant leaves apparently prevented interference of Pi with ADPG-PPiase catalysis and starch synthesis.     

Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by ¹⁴CO₂ assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total ¹⁴CO₂ fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = −0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.     

Nitrogen Nutrition and Xylem Transport of Nitrogen in Ureide-producing Grain Legumes

Xylem sap composition was examined in nodulated and nonnodulated cowpea (Vigna unguiculata [L.] Walp.) plants receiving a range of levels of NO₃ and in eight other ureide-forming legumes utilizing NO₃ or N₂ as sole source of nitrogen. A ¹⁵N dilution technique determined the proportions of plant nitrogen derived from N₂ in the nodulated cowpeas fed NO₃. Xylem sap composition of NO₃-fed, nodulated cowpea varied predictably with the relative extents to which N₂ and NO₃ were being utilized. The ratios of asparagine to glutamine (N/N) and of NO₃ to ureide (N/N) in xylem sap increased with increasing dependence on NO₃ whereas per cent of xylem nitrogen as ureide and the ratio of ureide plus glutamine to asparagine plus NO₃ (N/N) in xylem sap increased with increasing dependence on N₂ fixation. The amounts of NO₃ and ureides stored in leaflets, stems plus petioles, and roots of cowpea varied in a complex manner with level of NO₃ and the presence or absence of N₂ fixation. All species showed higher proportions of organic nitrogen as ureide and several-fold lower ratios of asparagine to glutamine in their xylem sap when relying on N₂ than when utilizing NO₃. In nodulated (minus nitrate) cowpea and mung bean (Vigna radiata [L.] Wilczek) the percentage of xylem nitrogen as ureide remained constant during growth but the ratio of asparagine to glutamine varied considerably. The biochemical significance of the above differences in xylem sap composition was discussed.     

Burkholderia, a Genus Rich in Plant-Associated Nitrogen Fixers with Wide Environmental and Geographic Distribution

The genus Burkholderia comprises 19 species, including Burkholderia vietnamiensis which is the only known N₂-fixing species of this bacterial genus. The first isolates of B. vietnamiensis were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N₂-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as B. cepacia using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of B. vietnamiensis TVV75^T. These features include nitrogenase activity with 10 different carbon sources, identical or very similar nifHDK hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75^T. Although the ability to fix N₂ is not reported to be a common feature among the known species of the genus Burkholderia, the results obtained show that many diazotrophic Burkholderia isolates analyzed showed phenotypic and genotypic features different from those of the known N₂-fixing species B. vietnamiensis as well as from those of B. kururiensis, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N₂-fixing Burkholderia species different from B. vietnamiensis. In addition, this study shows the wide geographic distribution and substantial capability of N₂-fixing Burkholderia spp. for colonizing diverse host plants in distantly separated environments.     

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization,Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.     

Nitrogen and phosphorus retranslocation and N:P ratios of litterfall in three tropical plantations: luxurious N and efficient P use by Acacia mangium

Some tropical N₂-fixing trees exhibit specific characteristics for phosphorus (P) acquisition and utilisation that contrast with the large nitrogen (N) fluxes in their litterfall. To investigate differences in N and P cycling in N₂-fixing plantations, litterfall and fresh leaf quality of a N₂-fixing Acacia mangium plantation were compared with that of a non-N₂-fixing Swietenia macrophylla plantation and a coniferous Araucaria cunninghamii plantation. The N concentration in the A. mangium litterfall was higher than that in the litterfall of the two other species, whereas the P concentration in the A. mangium leaf litterfall was 0.16 mg g^–1, which was only 12–22% of that of the other species. The P concentration in the reproductive parts of A. mangium was markedly higher (16.1 mg g^–1) than those in the other fractions. The N:P ratio was higher in the leaf fall (81) compared to the fresh leaves (29) of A. mangium, in contrast to the N:P ratios in the leaf samples of the other two species. An analysis of a global litterfall dataset of tropical plantations indicated that N:P ratios in litterfall were significantly higher in N₂-fixers than in non-N₂-fixers, and those of A. mangium were high among species in the N₂-fixer group. These results indicated that A. mangium efficiently retranslocated P in contrast to very large N cycling, under field conditions. These differences may be related to other physiological characteristics of A. mangium.     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

Effect of localized nitrogen availability to soybean half-root systems on photosynthate partitioning to roots and nodules

Soybean (Glycine max [L.] Merr. cv Davis) was grown in a split-root growth system designed to maintain control of the root atmosphere. Two experiments were conducted to examine how 80% Ar:20% O₂ (Ar:O₂) and air (Air) atmospheres affected N assimilation (NH₄NO₃ and N₂ fixation) and the partitioning of photosynthate to roots and nodules. Application of NH₄NO₃ to nonnodulated half-root systems enhanced root growth and root respiration at the site of application. A second experiment applied Ar:O₂ or air to the two sides of nodulated soybean half-root systems for 11 days in the following combinations: (a) Air to both sides (Air/Air); (b) Air to one side, Ar:O₂ to the other (Air/Ar:O₂), and (c) Ar:O₂ to both sides (Ar:O₂/Ar:O₂). Results indicated that dry matter and current photosynthate (¹⁴C) were selectively partitioned to nodules and roots where N₂ was available. Both root and nodule growth on the Air side of Air/Ar:O₂ plants was significantly greater than the Ar:O₂ side. The relative partitioning of carbon and current photosynthate between roots and nodules on a half-root system was also affected by N₂ availability. The Ar:O₂ sides partitioned relatively more current photosynthate to roots (57%) than nodules (43%), while N₂-fixing root systems partitioned 36 and 64% of the carbon to roots and nodules, respectively. The Ar:O₂ atmosphere decreased root and nodule respiration by 80% and nitrogenase activity by 85% compared to half-root systems in Air while specific nitrogenase activity of nodules in Ar:O₂ was 50% of nodules supplied Air. Results indicated that nitrogen assimilation, whether from N₂ fixation or inorganic sources, had a localized effect on root development. Nodule development accounted for the major decrease in total photosynthate partitioning to non-N₂-fixing nodules. Soybean compensates for ineffective nodulation by controlling the flux of carbon to ineffective nodules and their associated roots.     

Effects of pod removal on metabolism and senescence of nodulating and nonnodulating soybean isolines: I. Metabolic constituents

Field studies were conducted in 1981 and 1982 to ascertain the effects of pod removal on senescence of nodulating and nonnodulating isolines of soybean (Glycine max [L.] Merr. cv Harosoy) plants. Specifically, the test hypothesis was that nodules act as a nitrogen source and a carbohydrate sink which would in turn prevent or delay senescence in the absence of pods. Senescence was judged by changes in metabolite levels, in dry matter accumulation, and by visual observation.

For both nodulated and nonnodulated plants, pod removal had no effect on the magnitude or rate of dry matter and reduced-N accumulation by whole plants. Phosphorus accumulation was significantly less in both nodulated- and nonnodulated-depodded plants, compared with respective control plants with pods. These data suggested a role for pods in phosphorus uptake. Accumulation of dry matter, reduced N, and phosphorus ceased at approximately the same time for all treatments.

Pod removal did affect partitioning of plant constitments, with leaves and stems of depodded plants serving as a major alternate sink for accumulation of dry matter, reduced N, phosphorus, and nonstructural carbohydrates (primarily starch). While depodded plants eventually lost a significant amount of leaves, leaf drop was delayed relative to plants with pods; and depodded plants still retained some green leaves at 2 weeks past grain maturity of control (podded) plants.

The results indicated that senescence patterns of soybean plants were the same for nodulated and nonnodulated plants, and that pods did not control the initiation of senescence, but rather altered the partitioning of plant constituents and the visual manifestations of senescence.

     

Free-Living Rhizobium Strain Able To Grow on N2 as the Sole Nitrogen Source

A Rhizobium strain isolated from stem nodules of the legume Sesbania rostrata was shown to grow on atmospheric nitrogen (N₂) as the sole nitrogen source. Non-N₂-fixing mutants isolated directly on agar plates formed nodules that did not fix N₂ when inoculated into the host plant.     

Effect of Bentazon, a Hill Reaction Inhibitor, on Symbiotic Nitrogen-fixing Capability and Apparent Photosynthesis

Symbiotic associations of bean plants (Phaseolus vulgaris L. cv. Blue Lake) and Rhizobium phaseoli strain 127K17 were treated with the Hill reaction inhibitor bentazon (3-isopropyl-1 H-2,1,3-benzothiadiazin-4-(3H)-one-2,2-dioxide). Plants receiving foliar and root treatments of 1.8 kilograms per hectare bentazon were assayed at 6 hour intervals for N₂-fixing capacity by measuring C₂H₂-dependent C₂H₄ production and H₂ evolution and for CO₂ exchange rates. In foliar treated plants greatest measured inhibition of CO₂ exchange rates and N₂-fixing capacity occurred 6 and 12 hours after treatment, respectively. In root-treated plants maximum inhibition of both processes was delayed by 6 hours, and was less severe than in foliar treated plants. Nitrogen-fixing capacity and CO₂ exchange rate recovered to control levels in all plants. Application of higher rates of bentazon resulted in greater inhibition of CO₂ exchange rate and N₂-fixing capacity. Inhibition of the two processes was positively correlated (r = 0.985). The results indicate that inhibition of N₂-fixing capacity was not caused by bentazon directly, but indirectly through limiting the availability of photosynthate to support root nodule activity.     

Effect of Inoculation with N2-Fixing Spirilla and Azotobacter on Nitrogenase Activity on Roots of Maize Grown Under Subtropical Conditions

Inoculated and non-inoculated seedlings of maize were grown in fertile clayloam soils of Egypt and Belgium under subtropical conditions provided in a greenhouse. Acetylene-reducing activity and microbial counts were determined during a period ranging from 6 to 12 weeks after sowing. Irrespective of soil origin, N₂-fixing spirilla and Azotobacter were common under maize cultivation. Inoculation resulted in a transitional increase in their numbers at early stages of growth. Nitrogenase activity was not detected in the rhizosphere of young plants. The maximum activities measured (81 to 1,436 nmol of C₂H₄ g⁻¹ h⁻¹) occurred close to the 50 to 70% silking stage. Inoculation with N₂-fixing spirilla, particularly in Nile Delta soil, doubled the amount of N₂ fixed in a late period of growth (12 weeks), whereas inoculation with Azotobacter had no noticeable effect.     

Interactions between Symbiotic Nitrogen Fixation, Combined-N Application, and Photosynthesis in Pisum sativum

The effect of photosynthetic photon flux density (PPFD) on nitrogen utilization was determined in peas (Pisum sativum L. cv. Alaska) inoculated with Rhizobium leguminosarum and treated with nutrient solutions containing no combined nitrogen, 16 mM NO₃^?, or 16 mM NH₄⁺. Plants were grown under controlled conditions at three PPFD values ranging from severely limiting to nearly saturating. Carboxylation efficiencies and CO₂-exchange rates were highest in the N₂-fixing plants and lowest in plants supplied with NH₄⁺, and they generally increased with increasing PPFD. Photoefficiencies increased with PPFD but did not differ appreciably with the form of nitrogen applied. Nitrogen fixation, calculated from C₂H₂-reduction and H₂-evolution data, was inhibited more by NH₄⁺ than by NO₃^?application. Inhibition was counteracted by increasing PPFD. Percentage nitrogen decreased with increasing PPFD in plants treated with combined nitrogen and increased in the plants dependent on N₂ fixation. The data reveal that photosynthetic efficiency and the capacity to fix N₂ in peas are functions of PPFD and the availability of combined nitrogen and that these two factors are interrelated.     

Significance of nitrogen-fixing actinorhizal symbioses for restoration of depleted,degraded, and contaminated soil

Atmospheric nitrogen (N₂)-fixing legume trees are frequently used for the restoration of depleted, degraded, and contaminated soils. However, biological N₂ fixation (BNF) can also be performed by so-called actinorhizal plants. Actinorhizal plants include a high diversity of woody species and therefore can be applied in a broad spectrum of environments. In contrast to N₂-fixing legumes, the potential of actinorhizal plants for soil restoration remains largely unexplored. In this Opinion, we propose related basic research requirements for the characterization of environmental stress responses that determine the restoration potential of actinorhizal plants for depleted, degraded, and contaminated soils. We identify advantages and unexplored processes of actinorhizal plants and describe a mainly uncharted avenue of future research for this important group of plant species.