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Utilization of net photosynthate for nitrogen fixation and protein production in an annual legume.

The economy of C and N in nodulated cowpea (Vigna unguiculata [L.] Walp.) was described in terms of fixation of CO(2) and N(2), respiratory losses of C, and the production of dry matter and protein.Net daytime gain of C by the shoot (net photosynthesis) rose to a maximum at flowering and then declined sharply due to abscission of leaves. Maximum N fixation occurred 10 days prior to maximum net photosynthesis. Shedding of nodules reduced fixation to zero by midfruiting. Fifty per cent of the plant's N and 37% of its net photosynthate were assimilated before flowering; 39% of plant N was incorporated into seed dry matter.Respiration of nodules and roots utilized 24% of the C from net photosynthate assimilated over the growth cycle; night respiration of shoots, 20%; dry matter production in seeds, 17%; and dry matter production in other plant parts, 39%. The proportion of net photosynthate translocated to the nodulated root decreased from 41 to 14% during growth. Developing fruits were major competitors for translocate. Nodules consumed 9% of the C from the plant's total net photosynthate, 43% of which was respired, 6% made into dry matter, and 51% returned to the shoot with N fixation products.For every 1 g N fixed, net photosynthate equivalent to 6.8 g carbohydrate was consumed by nodules, 25.7 g carbohydrate by the nodulated root. Translocate was used most efficiently for N fixation in late vegetative growth when nodules were most active and their carbohydrate supply still adequate.During vegetative growth and early flowering (0 to 78 days after sowing) cowpea consumed 17.2 g net photosynthate (as carbohydrate) for every gram of protein synthesized in its shoot. The comparable conversion in seed production was 32.5 g net photosynthate/g seed protein or 6.6 g/g seed dry matter.

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Respiration and the energy requirement for nitrogen fixation in nodulated pea roots.

Pisum sativum L. cv. Trapper plants were inoculated and grown in a controlled environment on N-free nutrient solution. After 4 weeks N was supplied to treatment plants as NH(4)NO(3), KNO(3), or NH(4)Cl and rates of C(2)H(2) reduction, root + nodule respiration, and leaf photosynthesis were determined 1 week later. The increase in respiration per unit of C(2)H(2) reduction was not affected by either the form of N added or the light conditions during growth, although the basal respiration rate with no C(2)H(2) reduction increased with irradiance level. The mean regression coefficient from plots of respiration versus C(2)H(2) reduction was 0.23 + 0.04 (P [unk] .01) mg of CO(2) (mumol of C(2)H(2) reduced)(-1) which was very similar to the value for the coefficient of respiration associated with nitrogenase activity estimated by subtracting growth and maintenance respiration. Since the rate of N accumulation in N-free nutrient conditions was proportional to the rate of C(2)H(2) reduction, it appears that the method gives a true estimate of the energy requirements for N fixation which for these conditions was equivalent to 17 grams of carbohydrate consumed per gram of N fixed.

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Interdependence of Nitrogen Nutrition and Photosynthesis in Pisum sativum L: I. Effect of Combined Nitrogen on Symbiotic Nitrogen Fixation and Photosynthesis.

Photosynthesis, primary productivity, N content, and N(2) fixation were determined as a function of applied NH(4) (+) in peas (Pisum sativum L. cv. Alaska) which were inoculated or not inoculated with Rhizobium leguminosarum. Cabon dioxide exchange rate (CER) increased 10-fold, total N content 7-fold, and total dry weight 3-fold in 26-day-old uninoculated plants as applied NH(4) (+) was increased from 0 to 16 millimolar. In inoculated plants of the same age CER and dry weight were maximal at 2 millimolar NH(4) (+), and total N content increased between 0 and 2 millimolar NH(4) (+) but did not change significantly with higher NH(4) (+) applications. Per cent N content of uninoculated plants was significantly lower than that of inoculated plants except at the highest NH(4) (+) concentration (16 millimolar). Symbiotic N(2) fixation by inoculated plants was maximal in peas grown with 2 millimolar NH(4) (+); and apparent relative efficiency of N(2) fixation, calculated from C(2)H(2) reduction and H(2) evolution, was maximal in the 2 to 4 millimolar NH(4) (+) concentration range. The capacity to fix N(2) through the Rhizobium-legume symbiosis significantly enhanced the rate and efficiency of photosynthesis and plant N content when NH(4) (+) concentration in the nutrient solution was below 8 millimolar. Above 8 millimolar NH(4) (+) concentration uninoculated plants had greater CER, N content, and dry weight.

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Interdependence of Nitrogen Nutrition and Photosynthesis in Pisum sativum L: II. Host Plant Response to Nitrogen Fixation by Rhizobium Strains.

Physiological responses to infection by strains of Rhizobium leguminosarum which differed in their capacity to reduce N(2) were determined in 26-day-old pea plants (Pisum sativum L. cv. Alaska) grown under uniform environmental conditions in the absence of combined N. The highest N(2) reduction rates, calculated from H(2) evolution and C(2)H(2)-dependent C(2)H(4) production measurements, were approximately 6-fold greater than the lowest. Higher N(2) fixation rates were associated with greater CO(2) exchange rates (R(2) = 0.92) and carboxylation efficiency (R(2) = 0.99). Increases in the apparent relative efficiency of N(2) fixation [1-(H(2) evolved in air/C(2)H(2) reduced)] (acteroid efficiency) were associated with increases in whole-plant N(2) fixation efficiency (N(2)/CO(2) reduction ratio) (R(2) = 0.95). Whole-plant dry weight and total N content were related by regression analysis (R(2) = 0.98); both parameters were enhanced by increased N(2) fixation in a manner analogous to previously reported increases caused by greater external applications of NH(4) (+). These data reveal that photosynthetic parameters in genetically uniform host plants grown under identical environmental conditions are affected by N(2) fixation characteristics of the rhizobial symbiont. The measured efficiencies of micro- and macrosymbiont are directly related under uniform environments.

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Effect of irradiance on development of apparent nitrogen fixation and photosynthesis in soybean.

Soybeans grown with 2 millimolar NO(3) (-), which optimized apparent N(2) fixation by Rhizobium symbionts, showed significantly different rates of apparent photosynthesis and C(2)H(2) reduction during seedling development at two irradiances. Those physiological processes were lower for several weeks in plants grown at 1,500 microeinsteins per meter(2) per second than in those exposed to 700 microeinsteins per meter(2) per second. The irradiance-induced retardation was evident in short-term rates of apparent photosynthesis and N(2) fixation, as well as in measures of dry matter and total N accumulation. In spite of their previously inhibited development, plants grown at 1,500 microeinsteins per meter(2) per second were indistinguishable by day 28 from those exposed to 700 microeinsteins per meter(2) per second in terms of whole-shoot CO(2)-exchange rate; by day 35 they were identical in terms of whole-plant C(2)H(2)-reduction rate. On day 38 there was no significant difference in dry weight or N content between treatments. Shifting plants between irradiance treatments on day 21 showed that the higher irradiance also had a short-term inhibitory effect on C(2)H(2) reduction. The fact that 16 millimolar NO(3) (-) prevented the continuous exposure to 1,500 microeinsteins per meter(2) per second from inhibiting apparent photosynthesis suggested that seedlings grown on 2 millimolar NO(3) (-) with Rhizobium were N-limited. Although rates of apparent photosynthesis were similar by day 28, the additional week required to produce equal rates of apparent N(2) fixation between irradiance treatments showed that physiological adaptations of shoots, as well as photosynthesis per se, can affect root nodule activity.

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Inhibition of nitrogen fixation in alfalfa by arsenate, heavy metals, fluoride, and simulated Acid rain.

The acute effects of aqueous solutions of As, Cd, Cu, Pb, F, and Zn ions at concentrations from 0.01 to 100 micrograms per milliliter and solutions adjusted to pH 2 to 6 with nitric or sulfuric acid were studied with respect to acetylene reduction, net photosynthesis, respiration rate, and chlorophyll content in Vernal alfalfa (Medicago sativa L. cv. Vernal). The effects of the various treatments on acetylene reduction varied from no demonstrable effect by any concentration of F(-) and 42% inhibition by 100 micrograms Pb(2+) per milliliter, to 100% inhibition by 10 micrograms Cd(2+) per milliliter and 100 micrograms per milliliter As, Cu(2+), and Zn(2+) ions. Zn(2+) showed statistically significant inhibition of activity at 0.1 micrograms per milliliter. Acid treatments were not inhibitory above pH 2, at which pH nitric acid inhibited acetylene reduction activity more than did sulfuric acid. The inhibition of acetylene reduction by these ions was Zn(2+) > Cd(2+) > Cu(2+) > AsO(3) (-) > Pb(2+) > F(-). The sensitivity of acetylene reduction to the ions was roughly equal to the sensitivity of photosynthesis, respiration, and chlorophyll content when Pb(2+) was applied, but was 1,000 times more sensitive to Zn(2+). The relationship of the data to field conditions and industrial pollution is discussed.

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Interactions between Nitrogen Fixation, Mycorrhizal Colonization, and Host-Plant Growth in the Phaseolus-Rhizobium-Glomus Symbiosis.

Bean (Phaseolus vulgaris L. cv. Dwarf) roots were inoculated with Rhizobium phaseoli and colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum Gerd. and Trappe or left uncolonized as controls. The symbiotic associations were grown in an inert substrate using 0, 25, 50, 100, or 200 milligrams hydroxyapatite (HAP) (Ca(10)[PO(4)](6)[OH](2)) per pot as a P amendment. Plant and nodule dry weights and nodule activity increased for both VAM and control plants with increasing P availability, but values for VAM plants were significantly lower in all parameters than for controls. Inhibition of growth and of N(2) fixation in VAM plants was greatest at the lowest and highest P regimes. It was smallest at 50 milligrams HAP, where available P at harvest (7 weeks after planting) was 5 micrograms P per gram substrate. At this level of P availability, the association apparently benefited from increased P uptake by the fungal endophyte. Percent P values for shoots, roots, and nodules did not differ significantly (p > 0.05) between VAM and control plants. The extent of colonization, fungal biomass, and the fungus/association dry weight ratio increased several fold as HAP was increased from 0 to 200 milligrams. It is concluded that intersymbiont competition for P and photosynthate was the primary cause for the inhibition of growth, nodulation, and nodule activity in VAM plants. Impaired N(2) fixation resulted in N stress which contributed to inhibition of host plant growth at all levels of P availability.

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A highly sensitive, flow through h(2) gas analyzer for use in nitrogen fixation studies.

Studies of H(2) evolution by N(2) fixing systems are frequently limited by an inability to accurately measure H(2) gas concentrations of less than about 10 microliters per liter. In this study, a H(2) gas analyzer is described which is able to accurately and reproducibly detect up to 100 times lower H(2) concentrations than most thermal conductivity gas chromatographs or other conventional instruments used for the measurement of H(2) gas. This high level of sensitivity (maximum of about 0.02 microliter per liter H(2) per millivolt output) and the ability to continuously monitor H(2) concentration directly in a flowing gas stream, makes this instrument well suited for use in an open gas exchange system.Since the sensor used in the instrument was also sensitive to other combustible gases, it was necessary to demonstrate that H(2) was the only combustible gas produced by the N(2) fixing system being studied. When an air stream was passed through a pot containing nodulated soybean (Glycine max L.) roots, gas chromatographic analysis of the effluent gas stream revealed that H(2) was the only combustible gas present. These results were supported by other studies in which no combustible gases were detected in the effluent gas stream from soybean roots nodulated with USDA 110, a Rhizobium strain which displays active uptake hydrogenase activity.

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Response to drought stress of nitrogen fixation (acetylene reduction) rates by field-grown soybeans.

The effects of drought stress on soybean nodule conductance and the maximum rate of acetylene reduction were studied with in situ experiments performed during two seasons and under differing field conditions. In both years drought resulted in decreased nodule conductances which could be detected as early as three days after water was withheld. The maximum rate of acetylene reduction was also decreased by drought and was highly correlated with nodule conductance (r = 0.95). Since nodule conductance is equal to the nodule surface area times the permeability, the relationship of these variables to both whole-plant and unit-nodule nitrogenase activity was explored. Drought stress resulted in a decrease in nodule gas permeability followed by decreases in nodule surface area when drought was prolonged. Under all conditions studied acetylene reduction on a unit-nodule surface area basis was highly correlated with nodule gas permeability (r = 0.92). A short-term oxygen enrichment study demonstrated nodule gas permeability may limit oxygen flux into both drought-stressed and well-watered nodules of these field-grown soybeans.

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Carbohydrate partitioning and the capacity of apparent nitrogen fixation of soybean plants grown outdoors.

Patterns of leaf carbohydrate partitioning and nodule activity in soybean plants grown under natural conditions and the irradiance level required to produce sufficient carbohydrate to obtain maximum rates of apparent N(2)-fixation (acetylene reduction) were measured. Soybean plants, grown outdoors, maintained constant levels of leaf soluble sugars while leaf starch pools varied diurnally. When root temperature was kept at 25 degrees C and shoot temperature was allowed to vary with ambient temperature, the plants maintained constant rates of apparent N(2)-fixation and root+nodule respiration. Results from a second experiment, in which the entire plant was kept at 25 degrees C, were similar to those of the first experiment. Shoot carbon exchange rate of plants from the second experiment was light saturated at photosynthetic photon flux densities between 400 and 600 micromoles per square meter per second. When plants were subjected to an extended 40-hour dark period to deplete carbohydrate reserves, apparent N(2)-fixation was unaffected during the first 10 hours of darkness, decreased rapidly between 10 and 16 hours, and plateaued at one-third the initial level thereafter. After the extended dark period, plants were exposed to photosynthetic photon flux density from 200 to 1000 micromoles per square meter per second for 10 hours. Photosynthetic photon flux densities of 200 micromoles per square meter per second and greater resulted in maximum leaf soluble sugar content and nodule activity. Leaf starch content increased with irradiance levels up to 600 micromoles per square meter per second with no further increase at higher irradiance levels. Results presented here indicate that maximum nodule activity occurs at irradiance levels that do not saturate the plant's photosynthetic apparatus. This response would allow for maximum N(2)-fixation to occur in a nodulated legume during periods of inclement weather.

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Flavone limitations to root nodulation and symbiotic nitrogen fixation in alfalfa.

Transcription of the nodABC genes in Rhizobium meliloti is required for root nodule formation in alfalfa (Medicago sativa L.) and occurs when specific compounds, such as the flavone luteolin, are supplied by the host plant. Results reported here indicate how luteolin in the root and rhizosphere can affect subsequent N(2) fixation and plant growth. Previous experiments with ;Hairy Peruvian 32' (HP32), an alfalfa population produced from ;Hairy Peruvian' (HP) by two generations of selection for increased N(2) fixation and growth, found that HP32 had more root nodules and fixed more N(2) than the parental HP population. In the present study, flavonoid extracts of HP32 seedling roots are shown to contain a 60% higher concentration of compounds that induce transcription of a nodABC-lacZ fusion in R. meliloti than comparable extracts of HP roots. Chromatographic data indicated that HP32 roots had a 77% higher concentration of luteolin than HP roots. Adding 10 micromolar luteolin to the rhizosphere of HP seedlings increased nodulation, N(2) fixation, total N, and total dry weight but had no effect on nitrate assimilation. These data show that normal levels of flavone nodulation signals in the rhizosphere of HP alfalfa can limit root nodulation, symbiotic N(2) fixation, and seedling growth and suggest that one mechanism for increasing N(2) fixation can be the genetic enhancement of specific biochemical signals which induce nodulation genes in Rhizobium.

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Nitrogen Fixation (C(2)H(2) Reduction) by Broad Bean (Vicia faba L.) Nodules and Bacteroids under Water-Restricted Conditions.

Water potentials of leaves and nodules of broad bean (Vicia faba L.) cultivated on a sandy mixture were linearly and highly (r(2) = 0.99) correlated throughout a water deprivation of plants. A decrease of 0.2 megapascal of the nodule water potential (Psi(nod)) induced an immediate 25% inhibition of the highest level of acetylene reduction of broad bean nodules attached to roots. This activity continued to be depressed when water stress increased, but the effect was less pronounced. Partial recovery of optimal C(2)H(2) reduction capacity of mildly water stressed nodules (Psi(nod) = -1.2 megapascals) was possible by increasing the external O(2) partial pressure up to 60 kilopascals. The dense packing of the cortical cells of nodules may be responsible for the limitation of O(2) diffusion to the central tissue. Bacteroids isolated from broad bean nodules exhibited higher N(2) fixation activity with glucose than with succinate as an energy-yielding substrate. Bacteroids from stressed nodules appeared more sensitive to O(2), and their optimal activity declined with increasing nodule water deprivation. This effect could be partly due to decreased bacteroid respiration capacity with water stress. Water stress was also responsible for a decrease of the cytosolic protein content of the nodule and more specifically of leghemoglobin. The alteration of the bacteroid environment appears to contribute to the decline in N(2) fixation under water restricted conditions.

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Bimetallic system for nitrogen fixation: ruthenium-assisted protonation of coordinated N2 on tungsten with H2

Treatment of the tungsten dinitrogen complex cis-[W(N2)2(PMe2Ph)4] (Me = methyl, Ph = phenyl) with an equilibrium mixture of [RuCl(dppp)2]X and trans-[RuCl(eta2-H2)(dppp)2]X [X = BF4, PF6, or OSO2CF3; dppp = 1,3-bis(diphenylphosphino)propane] under 1 atmosphere of dihydrogen at 55 degrees Celsius for 24 hours gave NH3 in moderate yield. The same reaction in the presence of acetone produced acetone azine in high yield. None of these reactions proceeded in the absence of dihydrogen.

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Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation.

Six mutants of Rhizobium leguminosarum 3855 lacking uptake hydrogenase activity (Hup phenotype) as a result of Tn5-mob mutagenesis of the hup-containing plasmid pRL6JI were tested for symbiotic performance on Pisum sativum L. and Vicia benghalensis L. Three pea cultivars and one vetch line, which induce four different levels of Hup activity in strain 3855, were grown to flowering under microbiologically controlled conditions in the absence of combined N. Direct Kjeldahl N measurements showed that in every case at least one Hup mutant fixed as much N(2) as the isogenic Hup strain. Measures of C(2)H(2) reduction, H(2) evolution, H(2) incorporation, and plant dry weight were consistent with the interpretation that the oxidation of H(2) produced by the nitrogenase enzyme complex was not necessarily associated with increased N(2) fixation in these symbiotic associations. Tests with a smaller subset of the Hup strains under four different root environments ranging from pH 5.0 to 8.2 likewise showed no significant advantage for the isogenic Hup strain. It was concluded that the improvements in symbiotic N(2) fixation produced by pRL6JI are associated with some trait other than the Hup phenotype.

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Associative Nitrogen Fixation by Klebsiella spp.: Adhesion Sites and Inoculation Effects on Grass Roots.

Adhesion sites on grass roots for Klebsiella strains carrying type 3 or type 1 fimbriae or both were determined. Adhesion of the strains to the roots of Poa pratensis and Festuca rubra was highly localized; the bacteria adhered strongly to root hairs and with a markedly lower efficiency to the surface of the zone of elongation and to the root cap mucilage. No adhesion to the epidermal cells between root hairs was observed. The adhesion sites were identical for the type 3- and 1-fimbriated bacteria and for P. pratensis, F. rubra, and Trifolium pratense. Inoculation of P. pratensis seedlings with Klebsiella pneumoniae strain As resulted in morphological changes in plant roots. The roots of infected plants were heavily covered with root hairs, which often were deformed and branched.

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Nodulation, Nitrogen Fixation, and Hydrogen Oxidation by Pigeon Pea Bradyrhizobium spp. in Symbiotic Association with Pigeon Pea, Cowpea, and Soybean.

The pigeon pea strains of Bradyrhizobium CC-1, CC-8, UASGR(S), and F4 were evaluated for nodulation, effectiveness for N(2) fixation, and H(2) oxidation with homologous and nonhomologous host plants. Strain CC-1 nodulated Macroptilium atropurpureum, Vigna unguiculata, Glycine max, and G. soja but did not nodulate Pisum sativum, Phaseolus vulgaris, Trigonella foenum-graecum, and Trifolium repens. Strain F4 nodulated G. max cv. Peking and PI 434937 (Malayan), but the symbioses formed were poor. Similarly, G. max cv. Peking, cv. Bragg, PI 434937, PR 13-28-2-8-7, and HM-1 were nodulated by strain CC-1, and symbioses were also poor. G. max cv. Williams and cv. Clark were not nodulated. H(2) uptake activity was expressed with pigeon pea and cowpea, but not with soybean. G. max cv. Bragg grown in Bangalore, India, in local soil not previously exposed to Bradyrhizobium japonicum formed nodules with indigenous Bradyrhizobium spp. Six randomly chosen isolates, each originating from a different nodule, formed effective symbioses with pigeon pea host ICPL-407, nodulated PR 13-28-2-8-7 soybean forming moderately effective symbioses, and did not nodulate Williams soybean. These results indicate the six isolates to be pigeon pea strains although they originated from soybean nodules. Host-determined nodulation of soybean by pigeon pea Bradyrhizobium spp. may depend upon the ancestral backgrounds of the cultivars. The poor symbioses formed by the pigeon pea strains with soybean indicate that this crop should be inoculated with B. japonicum for its cultivation in soils containing only pigeon pea Bradyrhizobium spp.

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