Influence of salinity and abscisic acid on the O2 uptake by N2-fixing nodules of common bean

The effects of NaCl and ABA on the respiration of N₂-fixing nodules were analysed in common bean (Phaseolus vulgaris) inoculated with Rhizobium tropici the reference strain CIAT899. Shoot and nodule growth was more inhibited by NaCl than root growth. The O₂ uptake by nodulated roots at 21 kPa O₂ was significantly inhibited by salinity. Raising pO₂ stimulated nodule respiration more under NaCl treatment than for the control, although it did not compensate totally for the inhibitory effect of NaCl. Short NaCl application was less destructive than long term application. Also, the external application of ABA inhibited nodule respiration, and this inhibition was partly compensated by raising pO₂.     

Short-term effect of nitrate or water stress on nitrate reduction and malate fermentation pathways in yellow lupine (<Emphasis Type="Italic">Lupinus luteus</Emphasis>) nodules

Seven-week-old plants (symbiotic stage) of yellow lupine (Lupinus luteus L. cv. Ventus) were subjected for 8 days to 5 mM nitrate treatment or to drought stress to search for possible activation of bacteroidal nitrate and nitrite reductases. Both treatments affected activities of malate dehydrogenase and aspartate aminotransferase in nodule cytosol and therefore are presumed to impose O₂-limitation to nodule metabolism. However, no significant symptoms of senescence of nodules were found. Both nitrate treatment and drought stress increased rhizobial nitrate and nitrite reductase activities in contrast to noted decrease of corresponding activities in nodule cytoplasm. Differential regulation supports the hypothesis that bacteroidal enzymes can act in dissimilatory mode when nodule respiration is limited due to environmental stresses.     

Possible Involvement of Cytokinin in Nitrate-mediated Root Growth in Maize

Response of root system architecture to nutrient availability in soils is an essential way for plants to adapt to soil environments. Nitrate can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Nevertheless, less is known about the physiological mechanisms. In the present study, two maize (Zea mays L.) inbred lines (478 and Wu312) were used to study a possible role of cytokinin in nitrate-mediated root growth in nutrient solutions. Root elongation of 478 was more sensitive to high nitrate supply than that of Wu312. Medium high nitrate (5 mM) inhibited root elongation in 478, while, root elongation in Wu312 was only inhibited at high NO ₃ ⁻ supply (20 mM). Under high nitrate supply, the root elongation zone in 478 became swollen and the site of lateral root elongation was close towards the root tip. Both of the phenomena are typical of root growth induced by exogenous cytokinin treatments. Correspondingly, zeatin and zeatin nucleotide (Z + ZR) concentrations were increased at higher nitrate supply in 478, whereas they were constant in Wu312. Furthermore, exogenous cytokinin 6-benzylaminopurine (6-BA) completely reversed the stimulatory effect of low nitrate on root elongation. Therefore, it is supposed that the inhibitory effect of high concentration of nitrate on root elongation is, at least in part, mediated by increased cytokinin level in roots. High nitrate supply may have negative influences on root apex activity by affecting cytokinin metabolism so that root apical dominance is weakened and, therefore, root elongation is suppressed and lateral roots grow closer to the root apex. Nitrate suppressed lateral root elongation in Wu312 at concentration higher than 5 mM. In 478, however, this phenomenon was not significant even at 20 mM nitrate. Although exogenous 6-BA (20 nM) could suppress lateral root elongation as well, the inhibitory effect of high NO ₃ ⁻ concentration of nitrate on lateral root growth cannot be explained by changes in endogenous cytokinin alone.     

Effect of changes in shoot carbon-exchange rate on soybean root nodule activity

The effect of short- and long-term changes in shoot carbon-exchange rate (CER) on soybean (Glycine max [L.] Merr.) root nodule activity was assessed to determine whether increases in photosynthate production produce a direct enhancement of symbiotic N₂ fixation. Shoot CER, root + nodule respiration, and apparent N₂ fixation (acetylene reduction) were measured on intact soybean plants grown at 700 microeinsteins per meter per second, with constant root temperature and a 14/10-hour light/dark cycle. There was no diurnal variation of root + nodule respiration or apparent N₂ fixation in plants assayed weekly from 14 to 43 days after planting. However, if plants remained in darkness following their normal dark period, a significant decline in apparent N₂ fixation was measured within 4 hours, and decreasing CO₂ concentration from 320 to 90 microliters CO₂ per liter produced diurnal changes in root nodule activity. Increasing shoot CER by 87, 84, and 76% in 2-, 3-, and 4-week-old plants, respectively, by raising the CO₂ concentration around the shoot from 320 to 1,000 microliters CO₂ per liter, had no effect on root + nodule respiration or acetylene-reduction rates during the first 10 hours of the increased CER treatment. When the CO₂-enrichment treatment was extended in 3-week-old plants, the only measured parameter that differed significantly after 3 days was shoot CER. After 5 days of continuous CO₂ enrichment, root + nodule respiration and acetylene reduction increased, but such changes reflected an increase in root nodule mass rather than greater specific root nodule activity. The results show that on a 24-hour basis the process of symbiotic N₂ fixation in soybean plants grown under controlled environmental conditions functioned at maximum capacity and was not limited by shoot CER. Whether N₂-fixation capacity was limited by photosynthate movement to root nodules or by saturation of metabolic processes in root nodules is not known.     

Morphogenesis of lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen

Combined light and transmission electron microscopy were used to examine the effect of nitrate on the development of root nodules in lucerne (alfalfa, Medicago sativa L.) following induction by the nitrogen-fixing symbiont, Rhizobium meliloti. The timing of NO ₃ ^- addition was varied in order to study its effect on all of the recognized morphogenetic steps of nodule formation. Roots of plants inoculated in the presence of 18 mM NO ₃ ^- had straight root hairs which were devoid of adherent rhizobia and infection threads, and developed no nodules. However, nodules were formed on roots if 18 mM NO ₃ ^- was added 5 d after inoculation. At this time, the initiation of nodule primordia had already commenced in the root cortex. The histology and ultrastructure of young nodules which had developed for 5 d in the absence of NO ₃ ^- and another 5 d in the presence of 18 mM NO ₃ ^- resembled nodules developing under N-free conditions, except that in the infection threads within the infection zone of the nodule 1) some bacteria tended to loose their normal shape and gain more electron density, indicating premature degradation, and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO ₃ ^- levels in the medium, lysis and degeneration of the bacteria released from the infection threads were observed in the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other hand, the nodule meristems continued to proliferate even after 12 d of exposure of 18 mM NO ₃ ^- . This was the only morphogenetic step of root nodulation which was insensitive to levels of combined nitrogen that completely prevented infection if present at the time of inoculation. These data indicate that all of the recognized steps of root nodule morphogenesis in which the bacteria play a key role are sensitive to the inhibitory effect of combined nitrogen.     

Metabolite effectors for short-term nitrogen-dependent enhancement of phosphoenolpyruvate carboxylase activity and decrease of net sucrose synthesis in wheat leaves

It has been demonstrated previously that field pea (Pisum sativum L. cv. Express) grown in hydroponic culture on a complete nutrient solution with low NH₄⁺ concentrations (<0.5 mM) will produce a larger than normal proliferation of nodules. Peas grown in the absence of mineral N in hydroponic culture have been shown to rapidly autoregulate nodulation, forming a static nodule number by 14 to 21 days after planting. The present study further characterizes the effect of NH₄⁺ concentration in hydroponic culture on nodulation and nodule growth. Peas were grown continually for 4 weeks at NH₄⁺ concentrations that were autoregulatory (0.0 mM), stimulatory (0.2 mM) or inhibitory (1.0 mM), or peas were transferred between autoregulatory or NH₄⁺ inhibited and stimulatory solutions after 2 weeks. The peas nodulated as expected when grown under constant autoregulatory, stimulatory or inhibitory concentrations of NH₄⁺. When peas were transferred from the inhibitory (1.0 mM) to the stimulatory solution (0.2 mM) a massive proliferation of nodule primordia over the entire root system was observed within 3 days of the transfer. When they were transferred from the autoregulatory (0.0 mM) to the stimulatory (0.2 mM) solution a 10-day delay occurred before a proliferation in nodule primordia occurred at distal regions of the root system. These findings support our hypothesis that low concentrations (<1.0 mM) of NH₄⁺ in hydroponic culture cause a suppression of autoregulation in pea. In addition, the temporal and spatial differences in nodule proliferation between transfer treatments demonstrate at a whole plant level that autoregulation and NH₄⁺ inhibition suppress early nodule development via different mechanisms.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Respiration and oxygen transport in soybean nodules

Summary The respiration rate of individual soybean (Glycine max Merr.) nodules was measured as a function of pO₂ and temperature. At 23°, as the pO₂ was increased from 0.1 to 0.9 atm, there was a linear increase in respiration rate. At 13°, similar results were obtained, except that there was an abrupt saturation of respiration at approximately 0.5 atm pO₂. When measurements were made on the same nodule, the rate of increase in respiration with pO₂ was the same at 13° and 23°. Additional results were that 5% CO in the gas phase had no effect on respiration, except for a small decrease in the pO₂ at which respiration became saturated. Also, nodules still attached to the soybean root displayed the same respiratory behavior as detached nodules. A model for oxygen transport in the nodule is presented which explains these results quantitatively. The essence of the model is that the respiration rate of the central tissue of the nodule is almost entirely determined by the rate of oxygen diffusion to the respiratory enzymes. Evidence is given that the nodule cortex is the site of almost all of the resistance to oxygen diffusion within the nodule.     

Effect of gibberellins and the growth retardant CCC on the nodulation of soya

Summary The effect of exogenous applications of gibberellins (GAs) or the growth retardant -chloroethyltrimethylammonium chloride (CCC) on root nodule formation and activity (C₂H₂-reduction) in soya was studied. Daily foliar application of GA₃ (2.89×10^–6 M) delayed the formation of nodule initials and reduced the numbers mass nodule^–1 and specific activity of nodules by 43%, 31% and 47% respectively, without affecting plant growth. Similar effects on nodulation were produced by foliar application of GA₄ (3.01×10^–5 M) or GA₇ (3.03×10^–5 M), or by the addition of GA₃ (2.89×10^–6 M) to the rooting medium. GA effectiveness in reducing nodule numbers was decreased by delaying its application until after the initial infection process had occurred, but the nodules formed were smaller and less active than those of the untreated control plants. The GA effect on nodulation and nodule activity was not associated with alterations in root exudate or due to a direct inhibitory effect of the hormone on the nitrogenase system. When the endogenous root content of GA-like substances was reduced (86% decrease) by foliar application of CCC (6.30×10^–5 M), nodule numbers were increased by 56%, but nodule size and total nodule activity were similar to those of control plants. The GA and CCC treatments had no effect on rhizobial growth in liquid culture nor on root colonisation by rhizobia.The results suggest that the endogenous content of root GA may have a regulatory role in both the infection process and in subsequent nodule morphogenesis, thus controlling both the number and effectiveness of the root nodules formed.     

Induction of Root Nodule Senescence by Combined Nitrogen in Pisum sativum L

Root nodule senescence induced by nitrate and ammonium in Pisum sativum L. was defined by determining nitrogenase activity and leghemoglobin content with the acetylene reduction and pyridine hemochrome assays. Root systems supplied with 100 mm KNO(3) or 100 mm NH(4)Cl exhibited a decrease in nitrogenase activity followed by a decline in leghemoglobin content. Increasing the CO(2) concentration from 0.000320 atm to 0.00120 atm had no effect on the time course of root nodule senescence when 20 mm KNO(3) was supplied to the roots; in vitro nitrate reductase activity was detected in leaves and roots, but not bacteroids. Nitrate appeared in leaves, roots, and the nodule cytosol fraction but not bacteroids when 20 mm KNO(3) was supplied to roots. When nitrate entered through the shoots, however, no root nodule senescence was observed, and no nitrate was detected in root or nodule cytosol fractions although nitrate and nitrate reductase were found in leaves. The results suggest that nitrate does not induce root nodule senescence through competition between nitrate reductase and nitrogenase for products of photosynthesis.     

Symbiotic dinitrogen fixation as affected by short-term application of nitrate to nodulatedPisum sativum L.

Effect of nitrate on the nitrogenase (C₂H₂-reduction) activity, growth of nodule tissue accumulation of nitrate and nitrate reductase activity in 4-weeks-old nodulated peas (Pisum sativum l.) was investigated. A relatively slow decrease of the total nitrogenase activity (μmol C₂H₄ per root per h), as compared with plants cultivated without nitrate, was due to both retardation of further growth of the nodule tissue and to a decrease of their specific nitrogenase activity (μmol C₂H₄ per g_f.wt. per h). However, an absolute and pronounced decrease of both nitrogenase activities occurred only 4 or 7 d after the application of nitrate. The addition of nitrate led to its rapid accumulation in the nodule and leaf tissue with a simultaneous induction of the nitrate reductase activity. The nitrogenase activity was not completely inhibited even after a 7-d cultivation with 280 ppm NO₃ ^?-N in the nutrient medium and after accumulation of up to 180 ppm NO₃ ^?-N_f.wt. in the nodule tissue. The results obtained indicate that the “photosynthate deprivation” reflects competition between assimilation of nitrate and fixation of dinitrogen.     

Gas-exchange activity,carbohydrate status,and protein turnover in root nodule subpopulations of field pea (Pisum sativum L. cv. Century)

Root nodule ontogeny was followed in different parts of the root system of field peas (Pisum sativum L. cv. Century) to investigate the contribution to total nitrogen fixation by different nodule subpopulations. Seed-inoculated plants were grown to maturity in controlled-environment growth chambers. In a flow-through system nitrogenase activity (H₂-evolution in air) and nodulated-root respiration (net CO₂-evolution) were measured weekly or biweekly in different parts (top and mid) of the root system. Root nodule extracts were assayed for total soluble cytosolic protein, total heme, proteolytic capacity (at pH 7.0), soluble carbohydrates and starch. Total nitrogenase activity and nodule respiration were higher in the top zone, which was explained by differences in root and nodule mass. Nodule specific nitrogenase activity was similar in both zones, and gradually declined throughout the experiment. No differences were found between nodule subpopulations in the dry-matter specific concentrations of glucose, fructose, sucrose or starch. Neither did nodule concentrations of protein or leghemoglobin differ between the zones. Throughout reproductive growth, no decline was found in total or nodule specific nitrogenase activity, in any of the nodule subpopulations. Growth of the root nodules continued throughout the experiment, though growth of shoot and roots had ceased. The data gives no support for carbohydrate limitation in root nodules during pod-filling, since nodule respiration remained high, the concentration of soluble carbohydrates increased significantly, and the amount of starch was not reduced. We conclude that when this symbiosis is grown under controlled conditions, nitrogenase activity in nodules sub-sampled from the crown part of the root system is representative for the whole nodule population.     

Ontogenetic interactions between photosynthesis and symbiotic nitrogen fixation in pigeonpea

Total nodule nitrogenase activity (TNA, μmols ethylene plant^-1 h^-1) in pigeonpea (Cajanus cajari) increased with plant growth to reach maximum at flowering (75 days after sowing), decreasing thereafter until maturity (120 days after sowing). However, specific nodule nitrogenase activity (SNA, μmols ethylene g^-1 nodule fresh wt h^-1) reached its maximum earlier (45 days after sowing). The rate of photosynthesis and shoot and nodule respiration followed a similar pattern to TNA. However, higest rates of root respiration were observed at flowering and again immediately before final harvest. ¹⁴CO₂ feeding studies showed that assimilates produced in leaves before flowering were retained in the vegetative parts. Assimilates produced after flowering were exported to the reproductive structure at the expense of the nodules. It is suggested that the decreased availability of photosynthate to nodules decreased nitrogen fixation.     

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.     

Phosphorus deficiency-induced modifications in citrate catabolism and in cytosolic pH as related to citrate exudation in cluster roots of white lupin

A possible contribution of alterations in metabolic sequences involved in citrate catabolism, to intracellular accumulation and subsequent release of citrate was investigated in cluster roots of phosphorus (P)-deficient white lupin (Lupinus albus L.). Citrate accumulation during maturation of root clusters was associated with decreased levels of intracellular soluble P_i and ATP, and with reduced rates of respiration. Inhibitor studies with KCN and salicylhydroxamic acid (SHAM) suggest a reduced capacity of both the cytochrome pathway and of the alternative respiration with a concomitant decrease of immunochemically detectable protein levels of the alternative oxidase. Reduced respiration seems to be related to a general impairment of the respiratory system, rather than to limitation of respiratory substrates such as P_i and adenylates, as indicated by the absence of stimulatory effects of the uncoupler CCCP. The citrate/malate ratio in juvenile root clusters with high rates of respiration and low inherent levels of citrate accumulation was increased by short-term application (4–8 h) of azide and SHAM as respiration inhibitors. During maturation of root clusters, a shift from intracellular malic acid to citric acid accumulation was associated also with down-regulation of ATP citrate lyase (ACL), which catalyzes cleavage of citrate into acetyl-CoA and oxaloacetate with a putative function as anapleurotic source for the production of acetyl-CoA under P-deficient conditions. Inhibition of nitrate uptake and assimilation is a general response to P limitation in many plant species including white lupin. Reduced consumption of the amino acceptor 2-oxoglutaric acid as a product of citrate turnover may therefore contribute to increased citrate accumulation. Accordingly, artificial inhibition of nitrate reduction by localized application of tungstate significantly increased the citrate/malate ratio in juvenile root clusters. Lowering the cytosolic pH by external application of propionate stimulated citrate and malate exudation in non-cluster lateral roots and in developing root clusters. This effect was reverted by preincubation with phosphonate to buffer the cytosol. The results suggest that acidification of the cytosol may be an important factor, triggering the transient release of citrate and protons from mature root clusters in P-deficient white lupin.     

Alternative Path Mediated ATP Synthesis in Roots of Pisum sativum upon Nitrogen Supply

Changes in the efficiency of root respiration were examined on intact plants of Pisum sativum L. cv Rondo after addition of nitrate or ammonium to the culture solutions. Nitrate was absorbed immediately after addition and elicited a respiratory rise (O₂-uptake as well as CO₂-production) to 160% at most. This occurred both in roots of plants fixing N₂ and in those of non-nodulated plants pregrown for 1 or 2 weeks on a nitrogen-free culture solution. In older plants, used after 2 weeks of N-free growth, the full capacity of the cytochrome path was engaged in root respiration. This was demonstrated by the absence of an effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone in the presence of 25 millimolar salicylhydroxamate, an inhibitor of the alternative path. In these plants more than 90% of the nitrate-induced stimulation of root respiration was salicylhydroxamate-sensitive. In young plants, used after 1 week of N-free growth, the cytochrome path was not saturated. Its activity increased instantaneously at the expense of alternative path activity, which initially dropped to zero and subsequently increased to 160% of the control 7 hours after nitrate supply. The rate of photosynthesis rose to 120% of the control, but not before 1 hour after nitrate supply, suggesting that the stimulation of root respiration was not due to a higher rate of photosynthesis. Experiments with plants grown with a split-root system showed that respiration rate and alternative path activity only increased in the root halves exposed to nitrogen. Ammonium was equally effective as nitrate in stimulating root respiration. These results lead to the conclusion that alternative-path mediated root respiration contributes to synthesis of ATP during at least the first 24 hours following nitrogen supply.     

Low concentrations of nitrate and ammonium stimulate nodulation and N2 fixation while inhibiting specific nodulation (nodule DW g−1 root dry weight) and specific N2 fixation (N2 fixed g−1 root dry weight) in soybean

Nitrate N is a major inhibitor of the soybean/Bradyrhizobium symbiosis in legumes and although this inhibition has been studied for many years, as yet no consensus has been reached on the specific and quantitative interactions between nitrate and ammonium supply and N₂ fixation. The effect of nitrate and ammonium supply on plant growth, nodulation and N₂ fixation capacity during the full growth cycle was investigated in both greenhouse and growth chamber experiments with three soybean genotypes. The results show that a high concentration of mineral N (10 mM), either as nitrate or ammonium or ammonium nitrate significantly suppressed nodule number, nodule dry weight and total N₂ fixed per plant of nodulated soybeans. However, lower mineral N concentrations, either 1 mM or 3.75 mM significantly enhanced nodule number, nodule dry weight and total N₂ fixed per plant, while specific nodulation (nodule dry weight g^–1 root DW, SNOD) and specific N₂ fixation (total N₂ fixed g^–1 root DW, SNF) were significantly reduced, particularly at the early vegetative growth stage V4, compared to the treatment with N₂ fixation as the only N source, in both growth chamber and greenhouse experiments. Therefore, we suggest that SNOD or SNF might be better indicators to express the suppressing effect of mineral N addition on nodule performance and N₂ fixed. Our studies also showed that ammonium alone was the more efficient N source than either ammonium nitrate or nitrate for soybean, as it resulted in higher biomass accumulation, nodule dry weight, total N accumulation and total N₂ fixed by 23, 20, 18 and 44%, respectively, compared to NO₃ ^– as the N source.     

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.     

Elicitor-Inducible Caffeoyl-Coenzyme A 3-O-Methyltransferase from Petroselinum crispum Cell Suspensions : Purification, Partial Sequence, and Antigenicity

Physiological regulation of nodule gas permeability has a central role in the response of legumes to such diverse factors as drought, defoliation, and soil nitrate. A new method for quantifying nodule respiration and O₂ permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O₂ consumption) rate and internal O₂ concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N₂. The rate of increase of FOL under 100% O₂ was then used to calculate nodule O₂ permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O₂ for 15 minutes led to decreases in both permeability and O₂-saturated respiration (V_max), but the brief (<15 seconds) exposures to 100% O₂ required by the assay itself had little effect on either parameter. A gradual increase in external O₂ concentration from 20 to 40% resulted in a reversible decrease in permeability, but no change in V_max. The new method is likely to be useful for research on nodule physiology and might also be applicable to agronomic research and crop improvement programs.     

Effects of substrate nitrate concentration on symbiotic nodule formation in actinorhizal plants

The response ofAlnus glutinosa, Casuarina cunninghamiana, Elaeagnus angustifolia andMyrica cerifera to a range of substrate nitrogen levels in solution, in relation to plant growth, infection, nodulation and root fine structure was studied. Nine concentrations of potassium nitrate ranging from 0.05 to 3.0 mM, were tested on each of the species. Plants were inoculated withFrankia pure cultures after a two week exposure to one of the nine levels of added nitrate. After six more weeks with constant exposure to nitrate, plants were harvested and assayed. With the exception of Myrica, regression analyses of whole plant dry weights as a function of added nitrate were highly significant. There was a tendency for nodulated plants grown at intermediate levels of added nitrate to exhibit higher relative growth rates, probably due to the additive effect of substrate nitrogen and fixation of atmospheric nitrogen. The mean numbers of nodules per plant were, with the exception of Alnus, significantly higher at intermediate levels of added nitrate, as were mean nodule dry weights. A highly significant inverse relationship between nodule weight as a percentage of whole plant weight was found in Elaeagnus and Myrica. The observed response of Elaeagnus to added nitrate compared to other actinorhizal plants appears to demonstrate that root hair infected plants are much more sensitive to the inhibitory effects of added nitrate than plants infected by intercellular penetration. A sharp reduction in the presence of root hairs at high concentrations of nitrate was observed. This indicates that the inhibition of nodulation in some actinorhizal plant species results from nitrate induced root hair suppression.