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     

Nitrate Effect on Nitrogen Fixation (Acetylene Reduction): ACTIVITIES OF LEGUME ROOT NODULES INDUCED BY RHIZOBIA WITH VARIED NITRATE REDUCTASE ACTIVITIES

The effect of nitrate on symbiotic nitrogen fixation by root nodules of cowpea (Vigna unguiculata L., Walp., cv. California Blackeye) and lupine (Lupinus augustifolius L., cv. Frost) plants inoculated with nitrate reductase-expressing and nitrate reductase-nonexpressing Rhizobium strains were examined. Nitrate reductase of Rhizobium bacteroids in the nodules of cowpea and lupine reduced nitrate to nitrite. Both cowpea and lupine nodules accumulated nitrite when grown in the presence of 15 millimolar nitrate and induced by Rhizobium strains which express nitrate reductase activity (Rhizobium sp. 32H1 and 127E15). The nitrogen fixation (acetylene reduction) activities of cowpea and lupine nodules were inhibited by nitrate whether the nodules were induced by Rhizobium strains that express (Rhizobium sp. 32H1 and 127E15) or do not express (Rhizobium sp. 127E14 and R. lupini ATCC 10318) nitrate reductase activity. These findings indicate that nitrite, the product of bacteroid nitrate reductase, may not play a role in the inhibitory effect of nitrate on nitrogen fixation activities of legume root nodules. However, the degree of inhibition on the fixation activity by nitrate varied in different legume-Rhizobium combinations.     

Alfalfa root nodule carbon dioxide fixation : I. Association with nitrogen fixation and incorporation into amino acids

In vivo CO₂ fixation activity and in vitro phosphoenolpyruvate carboxylase activity were demonstrated in effective and ineffective nodules of alfalfa (Medicago sativa L.) and in the nodules of four other legume species. Phosphoenolpyruvate carboxylase activity was greatly reduced in nodules from both host and bacterially conditioned ineffective alfalfa nodules as compared to effective alfalfa nodules.

Forage harvest and nitrate application reduced both in vivo and in vitro CO₂ fixation activity. By day 11, forage harvest resulted in a 42% decline in in vitro nodule phosphoenolpyruvate carboxylase activity while treatment with either 40 or 80 kilograms nitrogen per hectare reduced activity by 65%. In vitro specific activity of phosphoenolpyruvate carboxylase and glutamate synthase were positively correlated with each other and both were positively correlated with acetylene reduction activity.

The distribution of radioactivity in the nodules of control plants (unharvested, 0 kilograms nitrogen per hectare) averaged 73% into the organic acid and 27% into the amino acid fraction. In nodules from harvested plants treated with nitrate, near equal distribution of radioactivity was observed in the organic acid (52%) and amino acid (48%) fractions by day 8. Recovery to control distribution occurred only in those nodules whose in vitro phosphoenolpyruvate carboxylase activity recovered.

The results demonstrate that CO₂ fixation is correlated with nitrogen fixation in alfalfa nodules. The maximum rate of CO₂ fixation for attached and detached alfalfa nodules at low CO₂ concentrations (0.13-0.38% CO₂) were 18.3 and 4.9 nanomoles per hour per milligram dry weight, respectively. Nodule CO₂ fixation was estimated to provide 25% of the carbon required for assimilation of symbiotically fixed nitrogen in alfalfa.

     

Nitrate and Carbohydrate Effects on Nodulation and Nitrogen Fixation (Acetylene Reduction) Activity of Lentil (Lens esculenta Moench)

Lentils (Lens esculenta Moench, cv. Tekoas) grown in a nutrient solution containing 15 millimolar nitrate had 84% fewer nodules than lentils grown in nitrate-free nutrient solution. Nodules from the nitrate-grown plants weighed 71% less than nodules from the nitrate-free plants. Nitrate-grown plants also fixed much less nitrogen (measured by acetylene reduction) than the nitrate-free plants. When lentils were grown in a solution containing 15 millimolar nitrate and 75 millimolar fructose, glucose, or sucrose, however, the nitrogen fixation activity of their nodules was similar to that of nodules from nitrate-free plants. Leaves of lentils grown in the nitrate-sugar solutions had only about 7% as much nitrate reductase activity and accumulated only 10% as much nitrate as leaves from lentils grown in the nitrate solution alone. Roots of lentils grown in the nitrate-sugar solutions had similar nitrate reductase activity but accumulated only 17 to 25% as much nitrate as roots from lentils grown in the nitrate solution. The results indicate that the added sugars alleviated the inhibitory effects of nitrate on symbiotic nitrogen fixation not only by increasing the carbohydrate supply so lentils could support both nitrogen fixation and nitrate reduction but also by inhibiting the accumulation of nitrate and, hence, lowering nitrate reductase activity in the leaves.     

psi, a plasmid-linked Rhizobium phaseoli gene that inhibits exopolysaccharide production and which is required for symbiotic nitrogen fixation

Summary A strain of R. phaseoli cured of its symbiotic plasmid, pRP2JI, retained the ability to make exopolysaccharide (EPS). However, a region of pRP2JI, when cloned at an increased copy number in wide host-range vectors and transferred to this and other strains of Rhizobium, inhibited EPS synthesis. The gene responsible was termed psi (polysaccharide inhibition) and was located in a region of the symbiotic plasmid close to nodulation and nitrogen fixation genes. psi is important in the symbiosis since a wild-type strain containing psi cloned on a multicopy plasmid failed to form Phaseolus nodules, and mutant strains containing psi::Tn5 mutations failed to fix nitrogen in Phaseolus nodules. It is proposed that the function of psi may be to repress in the bacteriod the expression of genes such as those for EPS synthesis which are normally expressed in free-living culture.     

Physiological and biochemical studies on senescing tap root nodules of soybeans.

Senescence of soybean (Glycine max L. Merr.) tap root nodules was investigated by comparing changes in various physiological and biochemical activities with changes in capacity to fix nitrogen. Field-grown Beeson and Calland varieties of soybeans of various ages were sources of tap root nodules. With both varieties, the number of tap root nodules per plant remained constant between 56 and 86 days after planting but fresh weight, dry weight, and mass of tap root nodules increased duing this period. Nitrogen (C2H2)fixation by attached tap root nodules was maximum on a fresh weight, dry weight, or nitrogen basis about 56 days after planting for either variety. Metabolic activities of bacteroids as measured by carbon dioxide evolution from glucose and succinate did not appear to vary among nodules of different ages. There was also no indication of mobilization or deposition or deposition of iron, molybdenum, calcium, zinc, and nitrate in aging tap root nodules. Nitrate levels in the aerial portion of the plants decreased significantly after the initial decline in acetylene reduction. Nicotinamide deamidase activity in the cytosol and in extracts of bacteroids did not change significantly as tap root nodules aged. However, significant and consistent changes were observed in initial pH values of nodule breis and the initial decline occurred before (Calland) or concurrently (Beeson) with the initial decline of nitrogen fixation.     

Actual and potential nitrogen fixation ofAlbizia rhizobium symbioses as affected by nitrate

Actual nitrogen fixation of root nodules of differentAlbizia-rhizobium symbioses, was compared with the potential nitrogen fixation of isolated bacteroids. The potential nitrogen fixation exceeded actual nitrogen fixation in all symbionts. After addition of nitrate the actual nitrogen fixation decreased more than did potential nitrogen fixation in effective symbiosis, whereas in a less effective symbiosis, the actual and potential nitrogen fixation increased as a result of better photosynthate supply to the roots and nodules. As confirmed by correlation analysis, the nitrogen fixation and photosynthetic yield of suboptimum symbioses were relatively enhanced by dressing with inorganic nitrogen fertilizer.     

Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate

The effect of nitrate on N₂ fixation and the assimilation of fixed N₂ in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, ¹⁵N₂ fixation and relative abundance of ureides ([ureides/(ureides + nitrate + α-amino nitrogen)] × 100) in xylem exudate, gave similar results for the effect of nitrate on N₂ fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N₂ fixation precedes the loss of the potential of bacteroids to fix N₂. This in turn precedes the loss of the potential of nodules to assimilate fixed N₂.     

MtMOT1.2 is responsible for molybdate supply to Medicago truncatula nodules

Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron‐molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss‐of‐function mot1.2‐1 mutant showed reduced growth compared with wild‐type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum‐dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen‐fixing nodules, since genetic complementation with a wild‐type MtMOT1.2 gene or molybdate‐fortification of the nutrient solution, both restored wild‐type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.     

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     

Influence of combined nitrogen level andEucalyptus competition on dinitrogen fixation in nodulatedCasuarina

Summary Experiments were conducted to determine the effect on biological dinitrogen fixation byCasuarina of available nitrogen (N) in the substrate and competition by interplantedEucalyptus. In these experiments, combined N was applied to the plants after nodules were developed and functioning. Both environmental factors, nitrate and competition, were observed to influence biological dinitrogen fixation byCasuarina, but not yield (total dry weight). In one experiment, the proportion of nitrogen derived byCasuarina from atmospheric fixation (pNdfa) was observed to be inhibited by potassium nitrate in a linear fashion. However, substrate N did not significantly affect the weight of root nodules. Thus nodule dry weight was not highly correlated with the proportion of nitrogen fixed. In a second experiment, the presence of a non-fixing interplanted species,Eucalyptus, increased dinitrogen fixation inCasuarina.Casuarina interplanted withEucalyptus obtained a greater proportion of its nitrogen (94.75%) from fixation than didCasuarina grown alone (86.68%) suggesting that competition for substrate N influences the proportion of nitrogen fixed by this actinorhizal plant.Dedicated to the memory of Professor John G. Torrey     

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)     

Purification and Properties of Glutamine Synthetase in Leaves and Roots of Pinus banksiana Lamb

A method is described for the purification of glutamine synthetase (GS; EC. 6.3.1.2) from the leaves and roots of Pinus banksiana Lamb., a conifer which utilizes ammonium as its primary nitrogen source. The enzyme was purified to apparent homogeneity by a procedure involving salt fractionation as well as ion-exchange, size exclusion, and affinity chromatography. Since the final preparation produced two bands on SDS polyacryamide gels but only one band on a nondenaturating gel, it is concluded that the two subunits (44 and 40 kilodaltons, respectively) are part of a single enzymatic protein which shows GS activity. The pH optimum for leaf GS ranged between 6.2 and 6.5, one pH unit lower than the values reported for higher plants which utilize primarily nitrate nitrogen. Magnesium requirements for GS in P. banksiana were different for leaves and roots, showing V_max/2 values of 2.5 and 8 millimolar, respectively at 5 millimolar ATP. Furthermore, K_m values for ammonium were higher for the enzyme in leaves (33.1 micromolar) than in roots (19.2 micromolar). K_m values for ATP and for glutamate, on the other hand, were similar for the two tissues. A polyclonal antibody was produced against the purified leaf GS. Western blots of leaf homogenates produced two bands, the lighter one being more abundant. The same pattern was found when immunodetection was performed using an anti GS IgG produced against purified GS from Phaseolus nodules thus indicating common antigenic determinants. At least 30% of total GS was recovered in a plastid-fraction of dark-grown calli produced from the basal part of P. banksiana hypocotyls.     

Changes in growth and oxidative response of leaves and nodules in <Emphasis Type="Italic">Medicago ciliaris</Emphasis> during salt stress recovery

In the present study, we exposed Medicago ciliaris isolated from a saline sebhka and growing symbiotically with Sinorhizobium medicae to 100 mM of NaCl and followed this stress by a recovery period (complete NaCl withdrawal from the root medium for two or one weeks). This experiment was conducted in order i) to check whether a reduction in growth and nitrogen fixation could be reversed by alleviating salt stress and ii) to determine specific changes related to salt-induced growth and nitrogen fixation decline. Salt stress reduced growth of all organs (particularly nodules), leaf area, photosynthetic activity and nitrogen fixation. The depressive effect of salt was not linked to osmotic stress. The removal of saline conditions restored growth of all organs after 2 weeks of recovery, with aerial organs recovering quickly after 1 week. Both photosynthetic and nitrogen fixation recovered only after a 2 week period of recovery. Salt stress in these saline-tolerant plants was accompanied by oxidative damage (electrolyte leakage), which was more accentuated in nodules in comparison with leaves. The observed recovery in growth, nitrogen fixation and photosynthesis was mainly linked to a preferential preservation of shoots growth owing to the maintenance of SOD activity (namely the isoforms A₂ and A₃); such maintenance would allow higher photosynthetic activity permitting an adequate supply of nodules with photoassimilates and thus facilitating stress withstand.     

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.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

Development of the nitrogen fixing apparatus in the legumes,Centrosema pubescens Benth., and Vigna unguiculata L. Walp.

The sequence of events leading up to the establishment of symbiotic nitrogen-fixation were studied in two tropical legumes, Centrosema pubescens Benth, and Vigna unguiculata L. Walp. Parameters measured included fresh and dry weights, chlorophyll and leghaemoglobin contents, as well as the activities of NADH-nitrate reductase (EC 1.6.6.1), and nitrogenase (nitric-oxide reductase-EC 1.7.99.2) in plants that were inoculated with suitable rhizobia or which were watered with potassium nitrate. Dry weight and photosynthetic activity of both species followed the sigmoidal pattern which is characteristic of most plants. Growth was little different in either a qualitative or quantitative sense whether nitrogen was supplied as nitrate or through dinitrogen fixation. Although the biochemical sequence of events was dependent on the limiting sensitivities of the individual assays used, the data suggest that nitrate reductase is the first measurable enzymatic activity in the nodules (and roots), followed by acetylene reduction and leghaemoglobin in that order. It is possible therefore, that low levels of symbiotic nitrogen fixation occur in the nodules in the absence of leghaemoglobin. Nitrate reductase activity in C. pubescens nodules was negatively exponentially correlated with nitrogenase activity of the same nodules, suggesting a changing metabolism in old nodules. These data are discussed in terms of environmental and physical factors known to control nitrogen fixation.     

Nitrogen fixation byAlnus viridis (Chaix) DC.

Summary InAlnus viridis nodule growth relative to plant growth was inversely related to the quantity of nitrate added to nutrient solutions. Nodulated plants showed maximum growth when grown independently of supplied nitrogen and made better growth in its absence than unnodulated plants at any level of added nitrogen. Low levels of nitrate caused a depression of growth of nodulated plants, apparently by suppressing both nitrogen fixation and nodule growth. Nodules in nitrogen-free sand culture fixed atmospheric nitrogen at a rate of 6.6 mg/day/g nodule. Phosphorus deficiency was induced by low levels of phosphate and resulted in small plants with dark-green foliage. Root and nodule growth as a percentage of total plant growth and the percentage of total accumulated plant nitrogen below ground were greater at a root temperature of 11°C than 21°C. Thus at low root temperature processes other than nitrogen fixation were limiting to plant growth. Excised nodules were exposed to an N ₂ ¹⁵ -enriched atmosphere. A positive correlation between rate of nitrogen fixation and temperature was obtained, with optimum fixation occurring at about 20°C. It was shown that in spite of decreasing mean temperatures with increase in altitude, rate of nitrogen fixation by nodules of plants growing in the field increased with increase in altitude. This latter trend was deduced to be a reflection of the extent to which the field sites were nitrogen deficient in relation to climatically possible growth.     

Gabaculine Inhibition of Chlorophyll Biosynthesis and Nodulation in Phaseolus lunatus L

Gabaculine (3-amino-2,3-dihydrobenzoic acid) was an inhibitor of in vivo chlorophyll biosynthesis in lima bean (Phaseolus lunatus L. cv Henderson). When applied to roots of 9-day-old plants, 10 micromolar gabaculine was sufficient to terminate biosynthesis of new chlorophyll. The trifoliolate leaves which emerged after gabaculine treatment were yellow. Gabaculine-treated plants had slightly lower dry weights; yet, overall plant size showed very little change. Chlorophyll fluorescence induction kinetics and CO₂ exchange measurements were used to monitor both immediate and long-term effects of gabaculine on photosynthesis. A lowered rate of the decline from the maximum level of fluorescence was observed after 10 hours for nitrate-supplemented plants, and all treated plants showed a slightly increased level of original fluorescence after 6 days. No change was observed in the rate of photosynthesis by unifoliolate leaves. The trifoliolate leaves, though not able to photosynthesize, were able to continue respiration. This suggested that heme biosynthesis for mitochondrial cytochromes was not abolished. In untreated lima bean, root nodules were induced by Rhizobium sp. 127E15. Following gabaculine treatment, root nodules formed, but were largely ineffective in nitrogen fixation. Nodule dry weight, nitrogen fixation activity, and leghemoglobin content were decreased by gabaculine.