Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate III. Kinetics of the Formation of Nitrosylleghemoglobin and of the Inhibition of Formation of Oxyleghemoglobin

In order to elucidate the mechanism of inhibition, by nitrite,of the formation of oxyleghemoglobin (LbO₂ and the mechanismof generation of nitrosylleghemoglobin (LbNO), kinetic analysesof results of measurements of oxygen uptake and spectrophotometricassays of leghemoglobin were performed. The decrease, by nitrite, in the oxygen-binding capacity ofleghemoglobin was caused by the increase in levels of LbNO formedfrom ferrous leghemoglobin. In this case, the oxygen-bindingsite of leghemoglobin was competitively occupied by nitric oxideproduced from nitrite. The kinetic constants for the generation of LbNO from leghemoglobinand nitrite were 5.7 ? 10 for the association rate constant,4.4 ? 10^–5 for the dissociation rate constant, and 1.3? 10⁶ for the equilibrium constant. From calculations basedon the equilibrium constant, it appears that LbO₂ is presentin small amounts in nodules of plants supplied with nitrate.Furthermore, the dissociation rate constant for LbNO was muchsmaller than that for LbO₂ or carboxyleghemoglobin (LbCO). Thisdifference indicates that, once formed, LbNO is harder to dissociatethan LbO₂ or LbCO. Thus, the accumulation of LbNO in the nodule cytosol, as a resultof the supply of nitrate, would inhibit nitrogenase activitythrough a decrease in the diffusion of oxygen that results froma lack of LbO₂. (Received December 18, 1989; Accepted April 13, 1990)     

Formation of Nitrosylleghemoglobin in Nodules of Nitrate-Treated Cowpea and Pea Plants

The formation of nitrosylleghemoglobin (LbNO) was examined incowpea and pea nodules in relation to the inhibition of nitrogenfixation by nitrate. Leghemoglobin was of the ferrous type andwas mainly converted to LbNO in cowpea nodules when the acetylene-reducingactivity decreased to 45% of control values as a result of thesupply of nitrate. In nodules of nitrate-treated pea plants,leghemoglobin was also of the ferrous type and LbNO was a minorcomponent of leghemoglobin. The levels of LbNO isolated fromnodules corresponded to the levels of LbNO calculated from equilibriumconstants for LbNO and the concentration of nitrite in nodules.The dissociation rate constants for LbNO from both cowpea andpea were much smaller than those for LbO₂ or LbCO, as is alsothe case in soybean. These results indicate that the inhibition of the functionsof leghemoglobin, due to the accumulation of LbNO, induces adecrease in nitrogen fixation in cowpea nodules, and that theinhibition of nitrogen fixation in pea nodules is not relatedto the formation of LbNO. (Received July 2, 1990; Accepted October 9, 1990)     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate I. Nitrite Accumulation and Formation of Nitrosylleghemoglobin in Nodules

The accumulation of nitrite in nodules was investigated to elucidatethe mechanism of inhibition of nitrogen fixation in nodulesof soybean (Glycine max. [L.] Merr.) plants supplied with nitrate.Acetylene-reducing activity (ARA) in nodules fell within 24h as a result of the supply of exogenous nitrate, accompaniedby an increase in the accumulation of nitrite in the cytosolbut not in the bacteroids of nodules. Nitrate reductase (NR)activity in the nodule cytosol remained high, irrespective ofthe supply of nitrate. Nitrosylleghemoglobin (LbNO) was detectedspectrophotometrically in the extract from nodules in whichnitrogen fixation was inhibited by nitrate. In experiments invitro, it was found that LbNO was easily formed from leghemoglobinin the presence of nitrite and dithionite. Thus, it is suggested that nitrogen fixation was inhibited primarilyby a decrease in the function of leghemoglobin, attributableto the formation of LbNO, which was caused by the accumulationof nitrite generated from nitrate by NR in the nodule cytosol. (Received August 22, 1989; Accepted January 24, 1990)     

Nicotinate, nicotinamide, and the reactivity of leghemoglobin in soybean root nodules

Nicotinate has been postulated to interfere with the binding of O₂ to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O₂. Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O₂ in soybean root nodules.     

Oxidation and reduction of leghemoglobin in root nodules of leguminous plants

Reactions involving changes that affect the function of leghemoglobin (Lb) are reviewed. The chemical nature of Lb and conditions inside nodules, such as slightly acid pH and the presence of metal ions, chelators, and toxic metabolites (nitrite, superoxide radical, peroxides), are conducive for oxidation of ferrous Lb (Lb²⁺) or its oxygenated form (LbO₂) to nonfunctional ferric Lb (Lb³⁺) and ferryl Lb. Because Lb³⁺ is nearly nonexistent in nodules and undergoes observable reduction in vivo, mechanisms must operate in nodules to maintain Lb in the Lb²⁺ state. Redox reactions of Lb are mediated, for the most part, by activated oxygen species: (a) oxidation of LbO₂ to Lb³⁺ involves superoxide; (b) excess peroxide oxidizes LbO₂ and Lb³⁺ to ferryl Lb and may cause breakdown of heme, release of iron, and generation of hydroxyl radicals (protein radicals may be formed in this process); (c) enzymatic reduction of Lb³⁺ requires active flavin and thiol groups and involves formation of peroxide; and (d) direct reduction of Lb³⁺ by NADH is mediated by superoxide and peroxide. Transition metal ions and certain small molecules of nodules such as flavins may act as intermediate electron carriers between NADH and Lb³⁺, increasing the rate of reaction, which then proceeds via superoxide or flavin radicals, respectively.     

Assimilate Partitioning in Red and White Clover Either Dependent on N2 Fixation in Root Nodules or Utilizing Nitrate Nitrogen

During vegetative growth in controlled environments, the patternof distribution of ¹⁴C-labelled assimilates to shoot and root,and to the meristems of the shoot, was measured in red and whiteclover plants either wholly dependent on N₂ fixation in rootnodules or receiving abundant nitrate nitrogen but lacking nodules. In experiments where single leaves on the primary shoot wereexposed to ¹⁴CO₂, nodulated plants of both clovers generallyexported more of their labelled assimilates to root (+nodules),than equivalent plants utilizing nitrate nitrogen, and thiswas offset by reduced export to branches (red clover) or stolons(white clover). The intensity of these effects varied with experiment.The export of labelled assimilate to growing leaves at the terminalmeristem of the donor shoot was not influenced by source ofnitrogen. Internode elongation in the donor shoot utilized nolabelled assimilate. Whole plants of white clover exposed to ¹⁴CO₂ on seven occasionsover 32 days exhibited the same effect on export to root (+nodules),which increased slightly in intensity with increasing plantage. Nodulated plants had larger root: shoot ratios than theirequivalents utilizing nitrate nitrogen. Trifolium repens, Trifolium pratense, red clover, white clover, nitrogen fixation, nitrate utilization, assimilate partitioning     

Nitrate inhibition of legume nodule growth and activity : I. Long term studies with a continuous supply of nitrate

The synthesis and accumulation of nitrite has been suggested as a causative factor in the inhibition of legume nodules supplied with nitrate. Plants were grown in sand culture with a moderate level of nitrate (2.1 to 6.4 millimolar) supplied continuously from seed germination to 30 to 50 days after planting. In a comparison of nitrate treatments, a highly significant negative correlation between nitrite concentration in soybean (Glycine max [L.] Merr.) nodules and nodule fresh weight per shoot dry weight was found even when bacteroids lacked nitrate reductase (NR). However, in a comparison of two Rhizobium japonicum strains, there was only 12% as much nitrite in nodules formed by NR⁻R. japonicum as in nodules formed by NR⁺R. japonicum, and growth and acetylene reduction activity of both types of nodules was about equally inhibited. In a comparison of eight other NR⁺ and NR⁻R. japonicum strains, and a comparison of G. max, Phaseolus vulgaris, and Pisum sativum, the concentration of nitrite in nodules was unrelated to nodule weight per plant or to specific acetylene reduction activity. The very small concentration of nitrite found in P. vulgaris nodules (0.05 micrograms NO₂⁻-N per gram fresh weight) was probably below that required for the inhibition of nitrogenase based on published in vitro experiments, and yet the specific acetylene reduction activity was inhibited 83% by nitrate. The overall results do not support the idea that nitrite plays a role in the inhibition of nodule growth and nitrogenase activity by nitrate.     

Nitrate Inhibition of Legume Nodule Growth and Activity : II. Short Term Studies with High Nitrate Supply

Soybean plants (Glycine max [L.] Merr) were grown in sand culture with 2 millimolar nitrate for 37 days and then supplied with 15 millimolar nitrate for 7 days. Control plants received 2 millimolar nitrate and 13 millimolar chloride and, after the 7-day treatment period, all plants were supplied with nil nitrate. The temporary treatment with high nitrate inhibited nitrogenase (acetylene reduction) activity by 80% whether or not Rhizobium japonicum bacteroids had nitrate reductase (NR) activity. The pattern of nitrite accumulation in nodules formed by NR⁺ rhizobia was inversely related to the decrease and recovery of nitrogenase activity. However, nitrite concentration in nodules formed by NR⁻ rhizobia appeared to be too low to explain the inhibition of nitrogenase. Carbohydrate composition was similar in control nodules and nodules receiving 15 millimolar nitrate suggesting that the inhibition of nitrogenase by nitrate was not related to the availability of carbohydrate.

Nodules on plants treated with 15 millimolar nitrate contained higher concentrations of amino N and, especially, ureide N than control nodules and, after withdrawal of nitrate, reduced N content of treated and control nodules returned to similar levels. The accumulation of N₂ fixation products in nodules in response to high nitrate treatment was observed with three R. japonicum strains, two NR⁺ and one NR⁻. The high nitrate treatment did not affect the allantoate/allantoin ratio or the proportion of amino N or ureide N in bacteroids (4%) and cytosol (96%).

     

Transport of Nitrate and Calcium into Legume Root Nodules

Nitrate transport into nodulated plants of soybean (Glycinemax), cowpea (Vigna unguiculata) and faba bean (Vicia faba)was investigated. Nitrate entering the root system of soybeandid not pass out of the vascular system into nodular tissuesin detectable quantities. On the other hand, nitrate could passfrom soil through the outer surface of nodules but did not penetratethe infected tissue. Similarly, nitrate was restricted to corticaltissues of cowpea and faba bean. Thus, nitrate cannot inhibitnitrogen fixation as a result of reduction to nitrite by nitratereductase within the bacteroid zone. These results are, however,consistent with an effect of nitrate on an oxygen diffusionresistance located in the nodule cortex. Unlike nitrate, measurable quantities of ⁴⁵calcium were transportedvia the xylem into infected and cortical tissues of soybeannodules: it also passed from the soil into the free space ofthe nodule cortex. Key words: Nitrate, legume nodules, calcium     

Inhibition of Nodule Development in Soybean by Nitrate or Reduced Nitrogen

Imsande, J. 1986. Inhibition of nodule development in soybeanby nitrate or reduced nitrogen.—J. exp. Bot. 37: 348–355. Nodulation of hydroponically grown soybean plants [Glycine max(L.) Merr.] is inhibited by continuous growth in the presenceof 4· mol m^–3 KNO₃ The presence of 4·0 molm^–3 ‘starter nitrate’ for 3-6 d during noduledevelopment, however, subsequently stimulates nodule dry weightaccumulation and nitrogenase activity. These stimulations occureven though 4· mol m^–3 nitrate temporarily delaysnodule development, i.e. the late steps of nodule developmentare reversibly inhibited by a short-term exposure to 4·0mol m^–3 nitrate. On the other hand, treatment with 4·0mol m^–3 nitrate in excess of 14 d significantly reducesnodule dry weight Thus, extended growth in the presence of 4·0mol m^–3 KNO₃ seems to block both early and late stepsof nodule development. Nodulation of hydroponically grown soybeansis also inhibited by continuous growth in the presence of 2·0mol m^–3 (NH₄)₂SO₄ This inhibition is not caused by acidityof the growth medium. On the other hand, nodule development6 d after inoculation with Rhizoblum japonicum is not delayedby a 7-d exposure to 2·0 mol m^–3 (NH₄)₂SO₄ butis partially inhibited by a prolonged exposure to (NH₄)₂SO₄Because repression of nodulation by 4·0 mol m^–3KNO₃ is more severe than that by 2·0 mol m^–3 (NH₄)₂SO₄and because ammonium taken up by the soybean plant is not activelyoxidized to nitrate, it is suggested that there are at leasttwo mechanisms by which nitrate utilization represses noduleformation in soybean. Key words: Glycine max, nitrogen, nitrogen fixation, nodulation     

Relationships Between Nitrate and Oxygen Supply in Symbiotic Nitrogen Fixation by White Clover

Exposure of mature, nodulated plants of white clover (Trifoliumrepens) cv. Blanca to 330 mg dm^–3 NO₃-N for 8 d causednitrogenase activity per plant to decrease by 80%. Total nodulatedroot respiration was not significantly affected but analysisof its components showed an 81% decrease in nitrogenase-linkedrespiration and a 340% increase in growth and maintenance respiration.Carbon costs of nitrogenase activity (mol CO₂ respired per molC₂H₄ produced) increased by 45% over the exposure period. Sucrosecontent of the nodules decreased, but the pattern of decreasedid not correlate with that of nitrogenase activity. The oxygendiffusion resistance of the nodules was increased by a factorof five. Characterization of this resistance increase suggestsan abnormal modification of the diffusion barrier and it isconcluded that alteration in the oxygen supply to the bacteroidsis involved in the effect of nitrate on nitrogenase activity. Key words: Nitrogenase activity, nitrate, oxygen     

Effects of univalent cations on the formation of nitrate reductase and nitrite reductase in rice seedlings

An investigation was made to determine the effects of univalentcations as activators on the formation of nitrate reductaseand nitrite reductase in rice seedlings. K⁺ functioned moreeffectively as a univalent cation activator than did other univalentcations examined. Substitution of Rb⁺ for K⁺ resulted in stimulationof nitrate reductase formation at about half the rate obtainedwith K⁺. There was no effect on nitrite reductase formation.Na⁺ could be partially substituted for K⁺ in the formation ofboth enzymes. NH₄⁺ slightly inhibited formation of the enzymes.In the absence of univalent cations, enzyme formation proceededat a slower rate during the initial 15-hr period, but thereafterproceeded at a higher rate. This delayed formation was not observedin the presence of K⁺. Results from inhibitor experiments suggestthat K⁺ stimulates the formation of nitrate reductase and nitritereductase. In conclusion, when nitrate nitrogen is supplied to rice plantsutilization of the nitrogen may be accelerated by increasedformation of enzymes involved in nitrate assimilation in thepresence of K⁺. (Received February 21, 1969; )     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 1. Development and Application of 13CO2 Assimilation System at a Constant 13C Abundance

A long-term, steady-state ¹³CO₂ assimilation system at a constantCO₂ concentration with a constant ¹³C abundance was designedand applied to quantitative investigations on the allocationof photoassimilated carbon in nodulated soya bean (Glycine maxL.) plants. The CO₂ concentration in the assimilation chamberand its ¹³C abundance were maintained constant with relativevariances of less than ±0.5 per cent during an 8-h assimilationperiod. At the termination of 8-h ¹³CO₂ assimilation by plantsat early flowering stage, the currently assimilated carbon relativeto total tissue carbon (measured by the degree of isotopic saturation)were for young leaves (including flower buds), 13.9 per cent;mature leaves, 15.7 per cent; stems+petioles, 5.9 per cent;roots, 5.4 per cent and nodules, 6.9 per cent, 48 h after theend of the ¹³CO₂ assimilation period, they were 12.3, 7.5, 7.4,6.8 and 6.1 per cent, respectively. The treatment with a highconcentration of nitrate in the nutrient media significantlydecreased the allocation of ¹³C into nodules. Experiments on¹³CO₂ assimilation by plants at the pod-filling stage were alsoconducted. Labelling by ¹³C was weaker than at the early floweringstage, but an intense accumulation of ¹³C into reproductiveorgans was observed. Glycine max L., nodulated soya bean plants, ¹³CO₂ assimilation, carbon dynamics     

Promotion of Assimilation of Ammonium Ions by Simultaneous Application of Nitrate and Ammonium Ions in Radish Plants

When radish plants were grown in nutrient solutions that containedammonium ions (NH₄⁺) as the sole source of nitrogen, they grewpoorly and accumulated high levels of NH₄⁺ in their leaves.However, radish plants cultured in 5 mM NH₄⁺ plus 1 mM NO₃^–(a ratio of 5 : 1 in forms of nitrogen; referred to as 5:lmix-N)grew well and accumulated very low levels of NH₄⁺ in their leaves.After radish plants were cultured in solutions that containedNO₃^–, or NH₄⁺, or 5: lmix-N for a week, they were thensupplied with the same nitrogen source labeled with ¹⁵N forone day. The uptake of ¹⁵N from labeled NH₄⁺ into total nitrogenwas the highest in plants supplied with 5:1mix-N. These plantsconverted far fewer labeled NH₄⁺ into free NH₄⁺ than did NH₄⁺-fedplants, but converted many more labeled NH₄⁺ into the insolublefraction than did NH₄⁺- or NO₃^–-fed plants. The presence of a small amount of nitrate was shown to stimulatethe assimilation of ammonium ions and the synthesis of proteins. (Received October 26, 1988; Accepted January 24, 1989)     

Nitrogen Partitioning During Early Development of Supernodulating Soybean (Glycine max [L.] Merrill) Mutants and their Wild-Type Parent

The relative importance of fixed N², cotyledonary N, and nitratefor growth of seedlings of soybean cv. Bragg and two of itsnitrate tolerant supernodulating (nts) mutants (intermediatents 1116 and extreme nts1007) was investigated during symbioticdevelopment in the presence of nitrate (3.0 mol m^–3) using¹⁵N techniques. Newly-fixed N₂ and nitrate were both major sourcesof N for nodule development and nitrate principally supportedearly shoot and root growth in Bragg. In the nts mutants, however,all plant parts and nodules in particular, relied more on Nstored in the cotyledons. This resulted in later nodule maturityand a period of prolonged N-starvation for the seedlings ofthe extreme supernodulator, and could be responsible for theirsubsequently lowered biomass accumulation compared to the parentcultivar. Key words: Nodules, N partitioning, supernodulating soybeans     

Hydrogen Effect on Nitrogenase (C2H2 Reduction) Response to Oxygen in Bradyrhizobium japonicum-Phaseoleae Symbioses

The influence of hydrogenase in Bradyrizobum-Phaseoleae symbioseswas studied ex-planta and in-planra in soybean (Glycine max)and cowpea (Vigna unguiculata). The hydrogenase was activatedby the addition of hydrogen in the incubation gas phase whichmodified the response of nitrogenase activity of Hup⁺ (hydrogenuptake positive) symbiosis to the external oxygen partial pressure.For bacteroids the hydrogenase expression increased nitrogenaseactivity at supraoptimal pO₂, acting possibly as a respiratoryprotection of nitrogenase. However, at suboptimal pO₂, nitrogenaseactivity of Hup⁺ bacteroids decreased with hydrogen, a phenomenonattributed to the lower efficiency of ATP synthesis from hydrogenthan from carbon substrates oxidation. For undisturbed nodules,the hydrogenase expression in soybean increased the optimalpO₂ for ARA (COP), from 35.3 to 40.3 kPa O₂, and the ARA atsupraoptimal pO₂; at suboptimal PO₂ there was a negative effectof hydrogenase on ARA, although this inhibition was less thanon bacteroids and was not detected if plants were grown at 15°C rather than 20 °C root temperature. No H₂ effectwas detected on cowpea nodules. The results on soybean nodulesare consistent with the concept that symbiotic nitrogen fixationis oxygen-limited and that hydrogenase activity has no beneficialeffect on nitrogen fixation in O₂ limitation. Key words: Glycine max, hydrogenase, nitrogenase, nitrogen fixation, nodules, Vigna unguiculata     

Ethylene, Nitrate and Nitrite Interactions in the Promotion of Dark Germination of Common Purslane Seeds

Ethylene (10 µ1^–1) caused about one-third of highlydark-dormant seeds of common purslane (Portulaca oleracea L.)to germinate in the dark. Attempts were made to increase germinationin the dark with nitrate and ethylene combinations. When applieddirectly to the seeds, KNO₃ did not stimulate germination andKNO₃ plus ethylene did not increase germination above that ofethylene alone. Pre-incubation of seeds in KNO₃ for 4 to 7 dbefore the ethylene applications significantly increased germination.The effects of the KNO₃ pre-incubation were additive at eachof four ethylene concentrations (0.1–100 µ11^–1).Potassium nitrate was effective only when ethylene followedthe KNO₃ pre-incubation period. Potassium nitrite stimulatedabout 25 per cent of the seeds to germinate without a pre-incubationperiod and without ethylene. Also, ethylene plus KNO₂ enhancedgermination above that achieved by either stimulus alone. Silvernitrate did not block the ethylene promotion of germination,but reversed the typical ethylene inhibition of seedling growthfollowing germination. The results support the views that nitrateexerted its effect via conversion to nitrite within the seedand that the rate of nitrate conversion may be a limiting factorin the dark germination of common purslane seeds. Ethylene mayfacilitate nitrite activity by increasing seed sensitivity tothe stimulus. Common purslane, Portulaca oleracea L., ethylene, nitrate, nitrite, germination, dormancy     

Serinol (2-Amino-1,3-propanediol) and 3-Amino-1,2-propanediol in Soybean Nodules

The compound X, which had previously been found to be accumulatedin the soybean nodules formed by infection with wild-type H₂-uptakenegative Bradyrhizobium japonicum strains, was identified asserinol (2-amino-1,3-propanediol) by means of elementary analysis,infrared spectrometry, ¹H-nuclear magnetic resonance, ¹³C-nuclearmagnetic resonance, high-performance liquid chromatography andgas chromatography/mass spectrometry. During the process ofpurification of compound X, it was also elucidated that 3-amino-1,2-propanediolwas present in the soybean nodules as a minor component. (Received January 6, 1986; Accepted June 16, 1986)     

Root Nodules of Phaseolus: Efficient Scavengers of Molybdenum for N2-fixation

Molybdenum is thought to be of intermediate mobility in thephloem and this may limit N₂-fixation by restricting the supplyof molybdenum to the nodules of legumes. When no molybdenumwas supplied to Phaseolus vulgaris nodule Mo content increasedat the expense of shoots and roots even when seed molybdenumcontent was large. Nodules sampled from plants receiving molybdenumin the feeding solution had a concentration of 21–78 µgMo g^-1. In the absence of molybdenum and with deficient seedcontent (<0.5 µg Mo seed^-1) nodule concentrations rangedfrom 1.9 to 3.5 fig Mo g^-1 in a small seeded genotype and 8.7±0.48µg Mo g^-1 in a large seeded genotype. N₂-fixation in theseplants was not impaired except in one instance where noduleconcentration was 1.9 µg Mo g^-1. Evidence that molybdenumis effectively translocated from leaves to roots and noduleswas obtained using foliar treatments. All of the 3.3 µgMo applied to a leaf was recovered in the plant after 10 d.Mo content of the nodules increased by 81%, whilst Mo contentof shoots increased by 56%. Root Mo content was eight timesgreater than that in plants not receiving a foliar treatmentof molybdenum. We conclude that when molybdenum was scarce inthe plant it was mobile and was translocated from roots andshoots to the nodules. As a result, nodule concentrations andcontents of molybdenum were frequently maintained at amountssufficient for N₂-fixation even when the plant was entirelydependent on a small seed reserve of molybdenum.