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)     

Possible causes of the physiological decline in soybean nitrogen fixation in the presence of nitrate

Nodulated soybean plants (Glycine max (L.) Merr. cv. Clarke)were supplied with 10 mol m^-3 nitrate at the vegetative stage.This treatment caused a rapid decline in nitrogen fixation (acetylenereduction) activity and a consequent decline in ureides in thexylem sap. However, there was virtually no effect on the nitrogenasecomplex, according to Western blots against components 1 and2. The effect on nitrogen fixation was matched by a decreasein nitrogenase-linked respiration and increases in nodule oxygendiffusion resistance and the carbon cost of nitrogen fixation.The addition of nitrate had little effect on protein contentfrom either nodule plant or bacteroid fractions. Activitiesof nitrate reductase (NR) and nitrite reductase (NiR) from eitherthe plant fraction or the bacteroids were affected in differentways during 8 d of supply. Nodule plant NR and bacteroid NiR were not affected. However,nodule plant NiR increased 5-fold within 2 d of supplying Bacteroid NR only increased after6 d. These results could be interpreted in terms of a restrictednitrate access into the infected region of nodules. However,denitrification was detected within 2 d of nitrate supply insoybean nodules. The results are discussed in relation to possiblecauses of the nitrate-induced decline in nitrogenase activity. Key words: Glycine max, nitrate, nitrogen fixation, nodules     

Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate II. Accumulation and Properties of Nitrosylleghemoglobin in Nodules

The accumulation of nitrosylleghemoglobin (LbNO) in nodulesand the properties of LbNO in vitro were investigated in connectionwith the inhibition of nitrogen fixation in soybean nodulesby nitrate. The leghemoglobin extracted under argon gas from nodules ofplants supplied with nitrate consisted mainly of LbNO, as judgedfrom the spectrum which corresponded to that of LbNO formedin vitro by the reaction of leghemoglobin with nitrite in thepresence of dithionite or by the combination of ferrous leghemoglobin(Lb²⁺) with nitric oxide. Further, LbNO formed in vivo was easilydissociated by visible light, as was LbNO formed in vitro. Thus,authentic LbNO does actually accumulate in nodules. Most of the leghemoglobin was of the ferrous type in nodulesof plants supplied with nitrate. Some LbNO appeared to be derivedfrom LbO₂ which was deoxygenated by nitrite. The increase inlevels of LbNO in nodules paralleled the decrease in acetylenereducing activity. These results indicate that the decrease in nitrogenase activityin nodules of soybean plants supplemented with nitrate is causedby the decrease in levels of LbO₂ that carries oxygen into bacteroids,which results from the formation of LbNO (Received August 22, 1989; Accepted December 4, 1989)     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Nitrate metabolism in soybean root nodules

The nitrate metabolism in nodules induced by Bradyrhizobium japonicum strain PJ17 on roots of soybean [ Glycine max (L.) Merr. cv. Hodgson] has been characterized by the nitrate reductase (NR; EC 1.6.6.1 and EC 1.6.6.3) activity of both partners of the symbiosis. NR activities of bacteroids and nodular cytosol were comparable and significantly higher than those of the roots. Nitrate reduction led to nitrite accumulation in root nodules, which was maximum after pod filling. The nodule had the capacity to metabolize nitrite via nitrite reductase (NiR; EC 1.6.6.4), at least in the cytosolic fraction. This activity was partly inhibited by the low content of free O₂ in the nodule. Indeed, nitrite accumulation decreased in the presence of an increased external pressure of O₂.     

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)     

Effect of low nitrate supply to nodulated lucerne on time course of activites of enzymes involved in inorganic nitrogen metabolism

Plants of lucerne ( Medicago sativa L. cv. Aragón) inoculated with several strains of Rhizobium meliloti were supplied with a low level of nitrate (5 m M ). After 1 week, normalised nodule mass, obtained by dividing nodule weight by shoot weight, was decreased by one-fourth. This result closely paralleled the bacteroid protein content of nodules, whereas the cytosolic content remained constant. Nitrate reductase activity (NRA, EC 1.7.99.4) of bacteroids increased rapidly after nitrate supply, with actual rates being highly dependent on the Rhizobium strain. The expression of cytosolic NR (EC 1.6.6.1) also varied depending on the bacterial strain but was largely insensitive to nitrate feeding. Nitrite reductase activity (NiRA, EC 1.7.2.2) of either bacteroid or plant origin was independent of the R. meliloti strain. Activation occurred after 3 and 7 days, respectively, of nitrate feeding. Significant amounts of nitrite were obtained throughout the experimental period from buffered extracts of both bacteroids and cytosol of nodules. However, when these nodules were ground in the presence of inhibitors of enzyme activity, nitrite was only found in nodules containing strain 102-F-51 after 1 week of treatment. These results agree with the recent hypothesis that nitrite plays a role in a secondary stage of nodule damage by nitrate. We propose that NiRA rather than NRA can be used as an internal probe of nitrate access to the infected region of nodules.     

Effect of water stress on the reduction of nitrate and nitrite by soybean nodules

The effects of water stress on nitrate reductase and nitrite reductase activities in symbiotic nodules were examined in field-grown soybean plants (Glycine max L Merr. cv Clark). The in vitro assays of enzyme activity indicated that the nodule cytosol and bacteroids contained both nitrate reductase and nitrite reductase activities. The reduction of nitrate in bacteroids increased significantly as nodule water potential declined from −0.6 to −1.4 megapascals, and then decreased when −1.8 megapascals water potential was reached. On the contrary, the reduction of nitrate in nodule cytosol was inhibited as water stress progressed. Increases in water stress intensity also caused a significant inhibition in nitrite reductase activities of bacteroids and nodule cytosol within soybean nodules. The results show that nitrate reduction occurred both in the cytosol and bacteroids of water-stressed soybean nodules. The reduction of nitrate functioned at different physiological modes in these two fractions.     

Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.     

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 entry and nitrite formation in the infected region of soybean nodules

The entry of nitrate into the infected region of soybean nodules and the possibility of a subsequent nitrite accumulation was studied. Nitrate was observed to gain access to the infected region in the short-term and significant amounts could be measured within 2 d of nitrate supply. The availability of nitrate in the bacteroid-containing region did not cause free-nitrite accumulation for at least 8d. However, to avoid the artefactual production of nitrite during extraction it was necessary to disrupt nodules in the presence of zinc acetate and ethanol, to prevent bacteroid nitrate reductase activity. Nitrite rapidly accumulated if nodules were extracted without prior enzyme-inactivation, or if bacteroids were allowed access to nitrate, or, more significantly, if nodules were not extracted immediately following detachment. Nitrate accumulation in detached nodules was mediated by oxygen concentration within the nodule; in the presence of pure N2 gas, nitrite accumulation was three times greater than in air and, conversely, it was prevented by exposure to pure O2. Furthermore, nitrite produced in detached nodules under atmospheric conditions was scavenged by transferring these nodules into 100% oxygen. However, measurements of apparent functional leghaemoglobin, using a nodule oximeter, suggested that after 8 d nitrate exposure up to 83% of Lb activity was lost, possibly due to interactions with nitrite produced in the nodule interior leading to the formation of nitrosylleghaemoglobin.Key words: Glycine max, cortex, infected region, leghaemoglobin, nitrate, nitrite, nodules, soybean      

Main centers of nitrate and nitrite reduction in young and nodulated yellow lupine (<Emphasis Type="Italic">Lupinus luteus</Emphasis>)

Nitrate and nitrite reduction centers in non-nodulated and symbiotic yellow lupine were analyzed. In young seedlings, nitrate was exclusively accumulated in roots, which also was shown as the main nitrate reduction center. In contrast, leaves were shown to play a key role in nitrite reduction. A similar distribution of nitrate reductase (NR) and nitrite reductase was found in nodulated plants. However, in field conditions characterized by low nitrate content, a disproportionately high level of NR activity in nodules was also observed during all stages of symbiotic growth. This feature was confirmed in nitrate-fed hydroponic cultures. Nodule NR activity was one order of magnitude higher than in roots, in spite of the small stored nitrate pool found inside nodules. This suggests that nodule NR activity had been induced not by nitrate itself but indirectly. Since bacteroids were shown to be responsible for the vast majority of nodule NR activity, the plausible explanation of this effect seems to be a dissimilatory nature of rhizobial NR. Considering that environmental nitrate could cause hypoxia inside nodules, this is the proposed way of the observed nodule NR induction.     

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%).

     

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

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

Effect of Nitrogen on Nitrate Reductase Activity in the Nodules and Leaves of Summer Moong (Vigna radiata)

The relation of the in vivo nitrate reductase (NR) activityto growth period was studied in the nodules and the leaves ofthe summer moong (Vigna radiata). The maximum NR activity wasobserved 31 days after sowing (DAS) in the leaves and 28 DASin the case of the nodules. In a pot experiment, the effectof the various nitrogen concentrations, namely 0, 3, 6, 9 and12 mg kg^–1 was studied on NR activity at three growthstages. The maximum NR activity was observed at 6 mg kg^–1N during the pre-flowering stage (26 DAS). Though the noduleshave higher NR activity, its expression was limited by substrateavailability. The NR activity in the leaf could be used as anindex of NR activity in the nodules. Nitrate reductase, nitrogen, nitrate, moong, Vigna radiata     

A comparison of inhibition of French-bean and soybean nitrogen fixation by nitrate, 1% oxygen or direct assimilate deprivation

Inhibition by NO⁻₃ of acetylene reduction in bean ( Phaseolus vulgaris L. cv. Contender) and soybean ( Glycine max L. cv. Amsoy 71) was measured in parallel with nodule carbohydrate and nitrate metabolism. In bean the onset of inhibition of C₂H₂ reduction (6 h) coincided with decreased import of assimilates and a lowering of carbohydrate pools (sucrose, glucose and starch). Nitrate reductase (EC 1.6.6.1) activity was induced in all plant organs after 3 h but no nitrite was detected in the nodules. In soybean, nodule carbohydrate concentrations and import of assimilates into the nodules increased markedly between 6 to 24 h after supply of nitrate when the nitrogenase (EC 1.7.99.2) was progressively inhibited. High nitrate reductase activity was observed in the nodules and nitrites accumulated because of insufficient nitrite reductase activity. The nitrate-induced inhibition of nitrogenase was compared with the inhibition observed with low oxygen around the roots (1% O²) or with direct assimilate deprivation (girdling or decapitation). Soybean and bean appeared equally sensitive to these treatments as regards to acetylene reduction. The results are discussed in relation to the current hypotheses explaining nitrate-induced inhibition of dinitrogen fixation: assimilate deprivation or nitrite poisoning. Present data are in favour of the first for bean and of the second for soybean.     

Effects of Nitrate Level on Nitrogen Metabolism in Winged Bean and Soya Bean

The effects of high (15 mM) and low (0.75 mM) solution nitratelevels on nitrogen metabolism in three genotypes (IL 7A, IL13 and IL 21) of winged beans [Psophocarpus tetragonolobus (L.)DC.] and one genotype (Williams) of soya bean [Glycine max (L.)Merrill] were investigated. Plants were grown for 42 days ina greenhouse in solution culture prior to sampling. The 15 mM nitrate treatment resulted in greater growth of allplant parts except roots. Growth of soya beans was more responsiveto nitrate level than was growth of winged beans. The high nitratelevel inhibited nodulation in all plants. The IL 13 and IL 21winged bean genotypes had similar nitrogenase activity (acetylenereduction per plant) as the soya bean and IL 7A winged beangenotype had lower activity. However, the IL 13 winged beangenotype had higher nitrogenase activity (acetylene reductionper unit nodule mass) than the other three genotypes which allhad similar activity. The 15 mM solution nitrate level stimulatedleaf and root nitrate reductase (NR) activity for all plants.All winged bean genotypes had higher leaf NR activity and higherpercentage reduced- and nitrate-nitrogen contents of leavesand stems compared with soya beans. However, total protein (reducednitrogen) was greater in soya beans when sampled indicatingthat more nitrate had been metabolized by soya beans than bywinged beans during the 42-day growth period. Psophocarpus tetragonolobus (L.) DC., winged bean, Glycine max (L.) Merrill, Soya bean, nitrate reductase, nitrogen fixation, nitrogenase activity, nodulation     

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     

Effect of multiple factor source-sink manipulation on nitrogen and carbon assimilation by soybean

The objectives of this study were to determine the effect of light enhancement and hastened reproductive development on nitrogen and dry matter accumulation by field-grown soybean (Glycine max [L.] Merr.). The impacts of photosynthate supply and reproductive development on change in the season-long profiles of in vivo leaf nitrate reductase (NR) activity and root nodule acetylene reduction (AR) activity were evaluated.

Light enhancement resulted in significant increases in dry matter accumulation, root nodule fresh weight and AR activity. Seed yield was increased in both light enhanced treatments in 1978 and in one in 1979.

Hastened flowering and seed development was accomplished through photoperiod manipulation within a single genotype. Seasonal decline in leaf NR activity was most rapid in plants entering reproductive development early. An early increase in root nodule fresh weight and AR activity was also observed in response to this treatment and was followed similarly by early decline.

The addition of high levels of soil-applied nitrogen increased leaf NR activity and delayed late season decline in NR activity for both control and early reproductive plants. Nitrate supply was therefore implicated as limiting to leaf NR activity during the decline associated with flowering and early seed development. A limited additional increase in leaf NR activity was observed in response to light enhancement plus soil-applied nitrogen. As no significant increase in leaf NR activity was observed in response to light enhancement alone, leaf nitrate supply was further implicated as more limiting to leaf NR activity than was photosynthate supply during flowering and early seed development.

     

The Physiology and Nitrogen-fixing Capability of Aquatically and Terrestrially Grown Neptunia plena: The Importance of Nodule Oxygen Supply

The aquatic legume Neptunia plena (L.) Benth. was grown in non-aeratedwater culture or vermiculite. Growth, nodulation, nitrogen fixationand nodule physiology were investigated. Over an 80-d period,plants grew and fixed nitrogen and carbon equally well in bothrooting media, although distribution of growth between plantparts varied. Total nodule dry weights and volumes were similarbut vermiculite-grown plants had three times as many (smaller)nodules than those grown in water. Oxygen diffusion resistanceof nodules exposed to 21% oxygen and 10% acetylene did not differsignificantly. Both treatments showed similar declines in rootrespiration and acetylene reduction activity (approx. 10%) whenroot systems were exposed to stepped decreases and increasesin rhizosphere oxygen concentration. However, nitrogenase activityof aquatically grown plants was irreversibly inhibited by rapidexposure of nodules to ambient air, whereas vermiculite-grownplants were unaffected. Aeration of water-cultured N. plenareduced stem length (but not mass) and number of nodules perplant. The concentration of nitrogen fixation by 163%. PossibleO₂ transport pathways from the shoot atmosphere to roots andnodules are discussed. Aquatic legume, diffusion resistance, Neptunia plena, nitrogen fixation, oxygen, root nodules