Decline In Nitrogenase Activity Research Articles

Short-Term Molecular Acclimation Processes of Legume Nodules to Increased External Oxygen Concentration

Nitrogenase is an oxygen labile enzyme. Microaerobic conditions within the infected zone of nodules are maintained primarily by an oxygen diffusion barrier (ODB) located in the nodule cortex. Flexibility of the ODB is important for the acclimation processes of nodules in response to changes in external oxygen concentration. The hypothesis of the present study was that there are additional molecular mechanisms involved. Nodule activity of Medicago truncatula plants were continuously monitored during a change from 21 to 25 or 30% oxygen around root nodules by measuring nodule H2 evolution. Within about 2 min of the increase in oxygen concentration, a steep decline in nitrogenase activity occurred. A quick recovery commenced about 8 min later. A qPCR-based analysis of the expression of genes for nitrogenase components showed a tendency toward upregulation during the recovery. The recovery resulted in a new constant activity after about 30 min, corresponding to approximately 90% of the pre-treatment level. An RNAseq-based comparative transcriptome profiling of nodules at that point in time revealed that genes for nodule-specific cysteine-rich (NCR) peptides, defensins, leghaemoglobin and chalcone and stilbene synthase were significantly upregulated when considered as a gene family. A gene for a nicotianamine synthase-like protein (Medtr1g084050) showed a strong increase in count number. The gene appears to be of importance for nodule functioning, as evidenced by its consistently high expression in nodules and a strong reaction to various environmental cues that influence nodule activity. A Tnt1-mutant that carries an insert in the coding sequence (cds) of that gene showed reduced nitrogen fixation and less efficient acclimation to an increased external oxygen concentration. It was concluded that sudden increases in oxygen concentration around nodules destroy nitrogenase, which is quickly counteracted by an increased neoformation of the enzyme. This reaction might be induced by increased formation of NCR peptides and necessitates an efficient iron supply to the bacteroid, which is probably mediated by nicotianamine.The paper is dedicated to the 85th birthday of Prof. Dr. Günther Schilling, University of Halle/Wittenberg, Germany, https://de.wikipedia.org/wiki/Günther_Schilling

Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain display nitrogen-fixing activity with enhanced tolerance to salinity stress

Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules. We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate-glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxin-containing nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed.

Biological dinitrogen fixation by selected soil cyanobacteria as affected by strain origin, morphotype, and light conditions

The potential for N(2) fixation by heterocystous cyanobacteria isolated from soils of different geographical areas was determined as nitrogenase activity (NA) using the acetylene reduction assay. Morphology of cyanobacteria had the largest influence on NA determined under light conditions. NA was generally higher in species lacking thick slime sheaths. The highest value (1446 nmol/h C(2)H(4) per g fresh biomass) was found in the strain of branched cyanobacterium Hassalia (A Has1) from the polar region. A quadratic relationship between NA and biomass was detected in the Tolypothrix group under light conditions. The decline of NA in dark relative to light conditions ranged from 37 to 100 % and differed among strains from distinct geographical areas. Unlike the NA of temperate and tropical strains, whose decline in dark relative to light was 24 and 17 %, respectively, the NA of polar strains declined to 1 % in the dark. This difference was explained by adaptation to different light conditions in temperate, tropical, and polar habitats. NA was not related to the frequency of heterocysts in strains of the colony-forming cyanobacterium Nostoc. Colony morphology and life cycle are therefore more important for NA then heterocyst frequency. NA values probably reflect the environmental conditions where the cyanobacterium was isolated and the physiological and morphological state of the strain.

The argon-induced decline in nitrogenase activity commences before the beginning of a decline in nodule oxygen uptake

Replacement of N(2) by argon in the air around nodules directs nitrogenase electron flow in its total onto H(+) resulting in increased nodule H(2) evolution (total nitrogenase activity (TNA)). However, argon application induces a so-called argon-induced decline in nitrogenase activity (Ar-ID) connected with decreased nodule oxygen permeability. Consequently, TNA measurements tend to underestimate total nitrogenase activity. It is unclear whether the decline in oxygen diffusion into nodules induces the Ar-ID, or whether a decline in nitrogenase activity is followed by lower nodule O(2) uptake. The objective of the present work was to examine the time sequence of the decline in nodule H(2) evolution and O(2) uptake after argon application. In addition, the reliability of TNA values, taken as quickly as possible after the switch to Ar/O(2), was tested through comparative measurement of (15)N(2) uptake of the same plants. Short-term TNA measurements in an optimized gas exchange measurement system yielded reliable results, verified by parallel determination of (15)N(2) uptake. A five min application of Ar/O(2) was without effect on the subsequent H(2) evolution in ambient air. A parallel experiment on control plants revealed that a decrease in nodule oxygen uptake began several minutes after the onset of the decline in H(2) evolution. We conclude that the primary effect of the replacement of N(2) by argon differs from oxygen diffusion control. A gas exchange system allowing an immediate taking of TNA yields reliable results and does not disturb nodule activity. Gas exchange measurements provide a powerful tool for studying nodule physiology and should be combined with material from molecular studies.

Overexpression of flavodoxin in bacteroids induces changes in antioxidant metabolism leading to delayed senescence and starch accumulation in alfalfa root nodules.

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.

The effect of potassium deficiency on growth and N2‐fixation in Trifolium repens

The effects of potassium (K) deficiency on growth, N2‐fixation and photosynthesis in white clover (Trifolium repens L.) were investigated using natural occurring gas fluxes on the nodules in real time of plants under three contrasting relative addition rates of K causing mild K deficiency, or following abrupt withdrawal of the K supply causing strong K deficiency of less than 0.65% in dry matter. A steady‐state below‐optimum K supply rate led to an increase in CO2‐fixation per unit leaf surface area as well as per plant leaf surface. However, nitrogenase activity per unit root weight and per unit nodule weight was maintained, as was the efficiency with which electrons were allocated to the reduction of N2 in the nodules. Abrupt K removals stimulated nodule growth strongly without delay, but as K concentrations decreased in the plant tissue a significant decline in nitrogenase activity per unit root weight as well as per unit nodule mass occurred. Further, the rate of photosynthesis per unit leaf area was unaffected, while the CO2 acquisition for the plant as a whole increased due to an expansion of total leaf area whereas the leaf area per unit leaf weight was unaffected. The ratio between CO2‐fixation and N2‐fixation increased, although not statistically significant, under short‐term K deprivation as well as under long‐term low K supply indicating a downregulation of nodule activity following morphological and growth adjustments. This downregulation took place despite a partly substitution of the K by Na. It is concluded that N2‐fixation does not limit the growth of K‐deprived clover plants. K deprivation induces changes in the relative growth of roots, nodules, and shoots rather than changes in N and/or carbon uptake rates per unit mass or area of these organs.

Nitrogenase activity and root nodule metabolism in response to O2 and short‐term N2 deprivation in dark‐treated Frankia‐Alnus incana plants

Inhibition of nitrogenase (EC 1.18.6.1) activity by O2 has been suggested to be an early response to disturbance in carbon supply to root nodules in the Frankia‐Alnus incana symbiosis. Intact nodulated root systems of plants kept in prolonged darkness of 22 h were used to test responses to O2 and short‐term N2 deprivation (1 h in Ar:O2). By using a Frankia lacking uptake hydrogenase it was possible to follow nitrogenase activity over time as H2 evolution in a gas exchange system. Respiration was simultaneously recorded as CO2 evolution. Dark‐treated plants had lower initial nitrogenase activity in N2:O2 (68% of controls), which declined further during a 1‐h period in the assay system in N2:O2 at 21 and 17% O2, but not at 13% O2. When dark‐treated plants were deprived of N2 at 21 and 17% O2 nitrogenase activity declined rapidly to 61 and 74%, respectively, after 20 min, compared with control plants continuously kept in their normal light regime. In contrast, there was no decline in dark‐treated plants at 13% O2, and only a smaller and temporary decline in control plants at 21% O2. When dark‐treated plants were kept at 21% O2 during 45 min prior to N2 deprivation at 17% O2 the decline was abolished. This supports the idea that the decline in nitrogenase activity observed in N2:O2 at 21% O2 and during N2 deprivation was caused by O2, which affected a sensitive nodule fraction. Nodule contents of the amino acids Gln and Cit decreased during N2 deprivation, suggesting decreased assimilation of NH4+. Contents of ATP and ADP in nodules were not affected by short‐term N2 deprivation. ATP/ADP ratios were about 5 indicating a highly aerobic metabolism in the root nodule. We conclude that nitrogenase activity of Alnus plants exposed to prolonged darkness becomes more sensitive to inactivation by O2. It seemed that dark‐treated plants could not adjust their nodule metabolism at higher perceived pO2 and during cessation of NH4+ production.

The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants.

The effects of P deficiency on growth, N(2)-fixation and photosynthesis in white clover (Trifolium repens L.) plants were investigated using three contrasting relative addition rates of P, or following abrupt withdrawal of the P supply. Responses to a constant below-optimum P supply rate consisted of a decline in N(2)-fixation per unit root weight and a small reduction in the efficiency with which electrons were allocated to the reduction of N(2) in nodules. Abrupt removal of P arrested nodule growth and caused a substantial decline in nitrogenase activity per unit root weight, but not per unit nodule mass. Similarly, the rate of photosynthesis per unit leaf area was unaffected by abrupt P removal, whereas CO(2) acquisition for the plant as a whole decreased due to a decline in total leaf area, leaf area per unit leaf weight and utilization of incoming radiation. These changes followed the decline in tissue P concentrations. The ratio between CO(2)-fixation and N(2)-fixation was maintained under short-term P deprivation but increased under long-term low P supply, indicating a regulatory inhibition of nodule activity following morphological and growth adjustments. It is concluded that N(2)-fixation did not limit the growth of clover plants experiencing P deficiency. A low P status induced changes in the relative growth of roots, nodules and shoots rather than changes in N and/or C uptake rates per unit mass or area of these organs.

Effect of NaCl on nitrogen fixation of unadapted and NaCl-adapted Azollapinnata–Anabaenaazollae

Exposure of Azolla pinnata–Anabaena azollae to NaCl induced a sharp decline in nitrogenase activity. A lethal dose of NaCl (40 mM) for the growth of A. pinnata–A. azollae complex could inhibit the nitrogenase activity by 39%, revealing for higher sensitivity of general growth to NaCl than the nitrogenase activity. Salinity caused a distinct shift in the profile of heterocyst differentiation in cavities of individual leaves with an increase in heterocyst frequency towards the apex, as well as increased the heterocyst frequency. An observed 16% heterocyst frequency in unadapted complex under non-saline condition rose to 18.7% after exposing it to 60 mM NaCl in comparison to 17.4% in adapted complex at identical NaCl concentration. The heterocyst frequency and its pattern in different leaf lobes of unadapted complex exposed to 60 mM NaCl was significantly different, whilst the difference was non-significant between unadapted complex under non-saline condition and the adapted complex at 60 mM NaCl. The increase in heterocyst frequency did not match with the nitrogenase activity. Dinitrogen fixation ability was maximum in mature median leaf cavities and minimum in apex, while NaCl-induced reduction in the nitrogenase activity was most marked in the cyanobionts occupying mature median leaf cavities.

Stress-induced legume root nodule senescence. Physiological, biochemical, and structural alterations.

Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O(2) diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O(2) limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2-4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by gamma-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress.

Glycolytic flux is adjusted to nitrogenase activity in nodules of detopped and argon-treated alfalfa plants

To investigate the short-term (30-240 min) interactions among nitrogenase activity, NH4+ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O2 (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O2-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O2-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.

The feedback mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism

A feedback mechanism which involves sensing of change in phloem N concentration has been proposed to control nodulation and dinitrogen fixation in the presence of external combined N. Whether this control is in response to a change in total N or in some specific signal compound(s) is not known. In the present study we reevaluated the hypothesis that control of nodulation and N2 fixation involves sensing of change in tissue N composition and attempted to identify potential signal molecule(s) involved. Two soybean (Glycine max [L.] Merr.) genotypes (Williams 82 and NOD1‐3) differing in nodule number and tolerance to nitrate were germinated in sand trays. Seven‐day‐old seedlings were inoculated with a solution of Bradyrhizobium japonicum and grown for 28 days in growth chambers, using a hydroponic system with limited N supply to promote nodulation. Half of 28‐day‐old plants were treated with 15 mM NO3−, then control and treated plants were sampled at the onset of nitrogenase inhibition (24 h following NO3−, treatment) for evaluation of nitrogenase activity and tissue concentration of total N and of each individual free amino acid. Phenylisothiocyanate‐(PITC) amino acid derivatives were separated and quantified using HPLC. The decline in nitrogenase activity following the short‐term nitrate treatment was associated with a dramatic asparagine concentration increase in the shoot and an increase in nodule aspartate and glutamate in both genotypes. Asparagine concentration in the shoot increased 35 times from a barely detectable level of 95 to 3 327 nmol g−1 fresh weight in Williams 82, and more than tripled from 509 to 1 753 nmol g−1 fresh weight in NOD1‐3. Increase in levels of free Asn and in total free amino acids in the shoot following the short‐term nitrate treatment was more pronounced in Williams 82 than in its partially nitrate‐tolerant mutant NOD1‐3. These results indicate that the feedback control of nodule activity may involve sensing changes in shoot asparagine levels and/or products of its metabolism (aspartate and glutamate) in the nodule. These results also indicate that partial‐nitrate tolerance of nodulation in the hypernodulated NOD1‐3 mutant is associated with a lesser change in tissue N following nitrate treatment.

Phosphorus deficiency increases the argon-induced decline of nodule nitrogenase activity in soybean and alfalfa

Open-flow assays of H2 evolution in Ar:O2 (80:20, v/v) by nodulated roots were performed in situ with soybean [Glycine max (L.) Merr.] and alfalfa [Medicago sativa L.) grown in sand with orthophosphate (Pi) nutrition either limiting (low-P) or non-limiting (control) for plant growth. Nodule growth was more limited than shoot growth by P deficiency. Phosphorus concentration was less affected in nodules than in other parts of the low-P plants. During assays, nitrogenase activity declined a few minutes after exposure of the nodulated roots to Ar. The magnitude of this argon-induced decline (Ar-ID) was less in alfalfa than in soybean. In both symbioses the magnitude of the Ar-ID was larger in low-P than control plants. Moreover, the minimum H2 evolution after the Ar-ID, was reached earlier in low-P plants. The Ar-ID was partly reversed by raising the external partial pressure of O2 in the rhizosphere. The magnitude of the Ar-ID in soybean was correlated negatively to nodule and shoot mass per plant, individual nodule mass, H2 evolution in air prior to the assay, and nodule N and P concentrations. Possible reasons, including nodule size and nodule O2 permeability, for the increase in Ar-ID in P-deficient plants are discussed and an interpretation of the P effect on nodule respiration and energetic metabolism is proposed.

C2H2 and Ar induce rapid declines in nitrogenase activity and CO2 evolution in nodules of Datisca glomerata

Preliminary studies have indicated that after addition of C2H2 there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata. The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C2H4 and CO2 evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C2H2. The peak rate of C2H4 evolution occurred at 30 s after C2H2 exposure, while the rate of CO2 evolution started to decline at 60 s after exposure to C2H2. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO2 evolution. Further, pretreatment with Ar eliminated most of the C2H2‐induced decline in nitrogenase activity and CO2 evolution. These C2H2‐ and Ar‐induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.

The acetylene-induced decline in nitrogenase activity in root nodules of Elaeagnus angustifolia

The rate of C2H2 reduction by nodulated seedlings of Elaeagnus angustifolia (Russian olive) was followed as a function of time. Our goals were to: 1) determine whether there is an C2H2-induced decline in nitrogenase activity; and 2) investigate the mechanism of any decline. We found a peak rate of C2H2 reduction at 1.5 min after the introduction of C2H2 that was followed by a rapid decline in activity to 56% of the peak value. After the decline there was a partial recovery to 67% of the peak value at 60 min. When the pO2 was decreased during the decline there was no significant effect (p≤0.05) on nitrogenase activity. When the C2H2 reduction assay was preceded by an incubation in a gas mixture (20 kPa O2) with Ar substituted for N2, there was little decline in nitrogenase activity as a function of time, but the rate of C2H2 reduction per gram nodule was reduced by approximately 50%. From these results we conclude that t Elaeagnus angustifolia exhibits a pronounced C2H2-induced decline and consequently the initial peak rate C2H2 reduction must be determined to obtain a valid measure of nitrogenase activity. We further suggest that cessation of NH3 formation initiates the decline and that the decline is not caused by a change in nodule permeability to gases.

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 (acetylene reduction) activity and a consequent decline in ureides in the xylem sap. However, there was virtually no effect on the nitrogenase complex, according to Western blots against components 1 and 2. The effect on nitrogen fixation was matched by a decrease in nitrogenase-linked respiration and increases in nodule oxygen diffusion resistance and the carbon cost of nitrogen fixation. The addition of nitrate had little effect on protein content from either nodule plant or bacteroid fractions. Activities of nitrate reductase (NR) and nitrite reductase (NiR) from either the plant fraction or the bacteroids were affected in different ways during 8 d of NO3- supply. Nodule plant NR and bacteroid NiR were not affected. However, nodule plant NiR increased 5-fold within 2 d of supplying NO3- Bacteroid NR only increased after 6 d. These results could be interpreted in terms of a restricted nitrate access into the infected region of nodules. However, denitrification was detected within 2 d of nitrate supply in soybean nodules. The results are discussed in relation to possible causes of the nitrate-induced decline in nitrogenase activity.

The O 2-lnduced transient response of nitrogenase activity in nodules of Myrica gale L.

Summary There is a transient decline of nitrogenase activity in nodules of Myrica gale L. when the partial pressure of O 2 (pO 2 ) in the rhizosphere is increased. To help determine the cause of this decline, nodule respiration (CO 2 evolution) was measured. When pO 2 was increased, there was a transient decline in nitrogenase activity but not in nodule respiration, which increased over a period of about 6 min to a new steady-state rate. Thus the decline in nitrogenase activity was not caused by a decline in respiration. When nodules were exposed to 9 kPa O 2 and then returned to 20 kPa, there was full recovery of nitrogenase activity if the nodules remained at 9 kPa for only 3 min. However, if the nodules remained at 9 kPa for 30 min, about 30% of nitrogenase activity and 6% of respiration were lost upon return to 20 kPa O 2 . These observations suggest that the rate of respiration adjusts over a period of minutes when pO 2 is changed. Thus when external pO 2 is increased, there may be a temporary rise in internal O 2 concentration until respiration increases to a higher rate. This temporary rise in pO 2 may cause a transient inhibition of nitrogenase activity. There was no evidence for changes in nodule resistance to gas diffusion during or following changes in pO 2 .

Phosphorus deficiency increases the acetylene-induced decline in nitrogenase activity in soybean (Glycine max (L.) Merr.)

Open-flow assays of acetylene reduction activity {ARA)and CO2 production in nodulated roots were performed in situ with soybean {Glycine max (L.) Merr.) cv. Kingsoy grown hydroponically with orthophosphate (Pi) nutrition either limiting (low-P) or non-limiting (control) for plant growth. Nodule growth was more limited than shoot growth by P deficiency. During ARA assays, nitrogenase activity declined a few minutes after exposure of the nodulated roots to C2H2, and this acetyleneinduced decline (C2H2-ID) was twice as intense at low-P. Moreover, the minimum ARA after the C2H2-ID was reached about 10 min earlier at low-P. The intensity of the C2H2-ID was correlated negatively with nodule mass per plant and positively with the ratio of shoot/nodule mass. After initial exposure to C2H2, the nodulated-root CO2 production was transiently stimulated and, moreover, this increase was 2-fold higher at low-P. Then, the nodulated-root CO2 production decreased with nodule C2H4 production. During the C2H2-ID, the nodule nitrogenase-linked respiration, which was computed as the variable component of the linear regression between CO2 and C2H4 production, was 2-fold higher at low-P. Furthermore, the microscopic observation of nodule sections revealed that starch deposits were decreased at low-P. However, nitrogenase activity, i.e. ARA before the C2H2-ID, was not affected by P deficiency. It is argued that P deficiency increased the C2H2-ID because it increased nodule permeability to O2 diffusion.

Interaction of boron and calcium in the cyanobacteria Anabaena and Synechococcus

Although some studies have reported an interaction between boron (B) and calcium (Ca2+) in higher plants, there is little evidence for a similar relationship in cyanobacteria. The present study was designed to determine the effect of a supplement of boron to Ca2+‐deficient cultures of Anabaena PCC 7119 and Synechococcus PCC 7942. Grown under Ca2+ deprivation, Anabaena had a slow growth rate and a low photosynthetic pigment content that was related to an inhibition of photosynthesis. Ca2+‐deficient cells showed a lack of cohesiveness of the heterocyst envelope layers, which was consistent with a rapid decline in nitrogenase activity. A supplement of B led to partial recovery from the effects caused by lack of Ca2+. Similarly, low Ca2+ had inhibitory effects on growth and metabolism of Synechococcus cultures. In this case, the effect of a B supplement depended on the concentration of Ca2+ in the growth medium. When Ca2+ was present at normal concentration. B was not required, at least no more than trace amounts. However, when the Ca2+ concentration decreased, B was required at increasing levels. An effect of boron on uptake and/or on the binding of Ca2+ in cyanobacteria is proposed.

The Role of Oxygen in the Regulation of Nitrogenase Activity in Drought-Stressed Soybean Nodules.

The aim of this study was to investigate the mechanism of nitrogenase inhibition in drought-stressed soybean (Glycine max L.) nodules to determine whether this stress was similar to other inhibitory treatments (e.g. detopping) known to cause an O2 limitation of nodule metabolism. Nodulated soybean plants were either detopped or subjected to mild, moderate, or severe drought stress by growth in different media and by withholding water for different periods. All treatments caused a decline in nitrogenase activity, and in the drought-stressed nodules, the decline was correlated with more negative nodule water potentials. Increases in rhizosphere O2 concentration stimulated nitrogenase activity much more in detopped plants than in drought-stressed plants, reflecting a greater degree of O2 limitation with the detopped treatment than with the drought-stressed treatment. These results indicated that drought stress differs from many other inhibitory treatments, such as detopping, in that its primary cause is not a decrease in nodule permeability and a greater O2 limitation of nodule metabolism. Rather, drought stress seems to cause a decrease in the maximum O2-sufficient rate of nodule respiration or nitrogenase activity, and the changes in nodule permeability reported to occur in drought-stressed nodules may be a response to elevated O2 concentrations in the infected cell that may occur as nodule respiration declines.