Nitrogenase Activity In Response Research Articles

Potential of Phototrophic Purple Nonsulfur Bacteria to Fix Nitrogen in Rice Fields.

Biological nitrogen fixation catalyzed by Mo-nitrogenase of symbiotic diazotrophs has attracted interest because its potential to supply plant-available nitrogen offers an alternative way of using chemical fertilizers for sustainable agriculture. Phototrophic purple nonsulfur bacteria (PNSB) diazotrophically grow under light anaerobic conditions and can be isolated from photic and microaerobic zones of rice fields. Therefore, PNSB as asymbiotic diazotrophs contribute to nitrogen fixation in rice fields. An attempt to measure nitrogen in the oxidized surface layer of paddy soil estimates that approximately 6–8 kg N/ha/year might be accumulated by phototrophic microorganisms. Species of PNSB possess one of or both alternative nitrogenases, V-nitrogenase and Fe-nitrogenase, which are found in asymbiotic diazotrophs, in addition to Mo-nitrogenase. The regulatory networks control nitrogenase activity in response to ammonium, molecular oxygen, and light irradiation. Laboratory and field studies have revealed effectiveness of PNSB inoculation to rice cultures on increases of nitrogen gain, plant growth, and/or grain yield. In this review, properties of the nitrogenase isozymes and regulation of nitrogenase activities in PNSB are described, and research challenges and potential of PNSB inoculation to rice cultures are discussed from a viewpoint of their applications as nitrogen biofertilizer.

NLP1 reciprocally regulates nitrate inhibition of nodulation through SUNN-CRA2 signaling in Medicago truncatula

Most legume plants can associate with diazotrophic soil bacteria called rhizobia, resulting in new root organs called nodules that enable N2 fixation. Nodulation is an energy-consuming process, and nodule number is tightly regulated by independent systemic signaling pathways controlled by CLE/SUNN and CEP/CRA2. Moreover, nitrate inhibits legume nodulation via local and systemic regulatory pathways. In Medicago truncatula, NLP1 plays important roles in nitrate-induced inhibition of nodulation, but the relationship between systemic and local pathways in mediating nodulation inhibition by nitrate is poorly understood. In this study, we found that nitrate induces CLE35 expression in an NLP1-dependent manner and that NLP1 binds directly to the CLE35 promoter to activate its expression. Grafting experiments revealed that the systemic control of nodule number involves negative regulation by SUNN and positive regulation by CRA2 in the shoot, and that NLP1's control of the inhibition of rhizobial infection, nodule development, and nitrogenase activity in response to nitrate is determined by the root. Unexpectedly, grafting experiments showed that loss of CRA2 in the root increases nodule number at inhibitory nitrate levels, probably because of CEP1/2 upregulation in the cra2 mutants, suggesting that CRA2 exerts active negative feedback regulation in the root.

Dry-hot stress significantly reduced the nitrogenase activity of epiphytic cyanolichen

Nitrogen (N) fixed by epiphytic cyanolichens (i.e. lichens that contain cyanobacterial symbionts) is thought to be the most important resource of this nutrient in some natural forest ecosystems. Although a great deal of work has been carried out to evaluate the biomass of this group as well as its contribution to ecosystem N budgets, empirical studies are needed to confirm the N input responses by cyanolichens under climate change conditions (dry-hot stress) as well as to determine the factors that control this process. We simulated climate change conditions by transplanting Lobaria retigera, a common cyanolichen in the area, to lower elevations, and measured nitrogenase activity in response to warmer and drier conditions. In addition, we conducted a series of laboratory and greenhouse experiments to determine the dominant factors influencing nitrogenase activity in this species. The results of this study show that mean annual nitrogenase activity at the higher site was 1.5 and 2.4 times that at the simulated warmer and drier (middle and lower) sites, respectively. Combining laboratory experimental conclusions, we show that thallus water content is a key factor determining the nitrogenase activity of L. retigera in early transplantation while insufficient carbon storage resulting from a combination of warming and desiccation was likely responsible for reducing nitrogenase activity in later months of the transplant experiment. The results of this study imply that the negative impact of climate change (dry-hot stress) on ecosystems not only impacts the distribution and growth of species, but also nutrient circles and budgets.

Role of nitrogen fixation in the autecology of Polaromonas naphthalenivorans in contaminated sediments

Polaromonas naphthalenivorans strain CJ2 is a Gram-negative betaproteobacterium that was identified, using stable isotope probing in 2003, as a dominant in situ degrader of naphthalene in coal tar-contaminated sediments. The sequenced genome of strain CJ2 revealed several genes conferring nitrogen fixation within a 65.6 kb region of strain CJ2's chromosome that is absent in the genome of its closest sequenced relative Polaromonas sp. strain JS666. Laboratory growth and nitrogenase assays verified that these genes are functional, providing an alternative source of nitrogen in N-free media when using naphthalene or pyruvate as carbon sources. Knowing this, we investigated if nitrogen-fixation activity could be detected in microcosms containing sediments from the field site where strain CJ2 was isolated. Inducing nitrogen limitation with the addition of glucose or naphthalene stimulated nitrogenase activity in amended sediments, as detected using the acetylene reduction assay. With the use of fluorescence microscopy, we screened the microcosm sediments for the presence of active strain CJ2 cells using a dual-labelling approach. When we examined the carbon-amended microcosm sediments stained with both a strain CJ2-specific fluorescent in situ hybridization probe and a polyclonal fluorescently tagged antibody, we were able to detect dual-labelled active cells. In contrast, in sediments that received no carbon addition (showing no nitrogenase activity), no dual-labelled cells were detected. Furthermore, the naphthalene amendment enhanced the proportion of active strain CJ2 cells in the sediment relative to a glucose amendment. Field experiments performed in sediments where strain CJ2 was isolated showed nitrogenase activity in response to dosing with naphthalene. Dual-label fluorescence staining of these sediments showed a fivefold increase in active strain CJ2 in the sediments dosed with naphthalene over those dosed with deionized water. These experiments show that nitrogen fixation may play an important role in naphthalene biodegradation by strain CJ2 and contribute to its ecological success.

Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness

Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to P(II) protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

Effect of Drought Stress on Traits Related to N2 Fixation in Eleven Peanut (Arachis hypogaea L.) Genotypes Differing in Degrees of Resistance to Drought

The aim of this study was to determine the effects of drought stress on nitrogenase activity, nodule number and nodule dry weight of eleven peanut (Arachis hypogaea L.) genotypes with different degrees of drought resistance. The relative values of these nitrogen fixation traits were evaluated under well watered and water-stressed conditions. Eleven peanut genotypes were tested in a split-plot design with four replications under field conditions in 2003/2004 and 2004/2005 in Northeast Thailand. Main-plot treatments were three water regimes [Field Capacity (FC), 2/3 Available soil Water (AW) and 1/3 AW] and sub-plot treatments were 11 peanut lines. Data were collected on nodule number, nodule dry weight and nitrogenase activity (acetylene reduction assay) at 30, 60 and 90 Days After Emergence (DAE). Severe drought stress reduced nitrogenase activity, nodule number and nodule dry weight about two times greater than did mild drought stress, causing uniform performance of peanut genotypes for nitrogenase activity under severe drought conditions. However, differences among peanut genotypes in nodule traits were found at all water levels. Tifton-8 and KK 60-3 in general performed better than the drought resistant lines from ICRISAT for all traits. Nodule dry weight was closely related with nitrogenase activity under drought conditions. High nitrogenase activity under mild drought conditions was related to high nitrogenase activity under well watered conditions (potential) and to a low rate of reduction in nitrogenase activity in response to stress. The contribution of the potential was lower under more severe drought conditions. Selection for high nitrogenase activity as a surrogate trait to improve nitrogen fixation under drought conditions should be more effective under mild than severe stress.

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

The AmtB protein transports uncharged NH(3) into the cell, but it also interacts with the nitrogen regulatory protein P(II), which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P(II) homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB(1) and amtB(2), have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB(1) are defective in their response to both addition and darkness, while mutants lacking AmtB(2) show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB(1) is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB(1)-P(II) regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P(II) is proposed. Other results demonstrate a degree of specificity in interaction of AmtB(1) with the different P(II) homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P(II) proteins regulate processes involved in nitrogen acquisition and utilization.

AmtB is necessary for NH(4)(+)-induced nitrogenase switch-off and ADP-ribosylation in Rhodobacter capsulatus.

Rhodobacter capsulatus possesses two genes potentially coding for ammonia transporters, amtB and amtY. In order to better understand their role in the physiology of this bacterium and their possible significance in nitrogen fixation, we created single-knockout mutants. Strains mutated in either amtB or amtY did not show a growth defect under any condition tested and were still capable of taking up ammonia at nearly wild-type rates, but an amtB mutant was no longer capable of transporting methylamine. The amtB strain but not the amtY strain was also totally defective in carrying out ADP-ribosylation of Fe-protein or the switch-off of in vivo nitrogenase activity in response to NH(4)(+) addition. ADP-ribosylation in response to darkness was unaffected in amtB and amtBY strains, and glutamine synthetase activity was normally regulated in these strains in response to ammonium addition, suggesting that one role of AmtB is to function as an ammonia sensor for the processes that regulate nitrogenase activity.

Response of the endophytic diazotroph Gluconacetobacter diazotrophicus on solid media to changes in atmospheric partial O(2) pressure.

Gluconacetobacter diazotrophicus is an N(2)-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O(2) pressures (pO(2)) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO(2) (5 to 60 kPa). Nitrogenase activity was measured by H(2) evolution in N(2)-O(2) and in Ar-O(2), and respiration rate was measured by CO(2) evolution in N(2)-O(2). To validate the use of H(2) production as an assay for nitrogenase activity, a non-N(2)-fixing (Nif(-)) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup(+)) activity (0.016 +/- 0.009 micromol of H(2) 10(10) cells(-1) h(-1)) when incubated in an atmosphere enriched in H(2). However, Hup(+) activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO(2) tested. However, when the assay atmospheric pO(2) was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO(2) for nitrogenase activity was 0 to 20 kPa above the pO(2) at which the bacteria had been grown. As atmospheric pO(2) was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO(2) was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO(2) from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O(2), 80% of nitrogenase activity was recovered within 10 min, indicating a "switch-off/switch-on" O(2) protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N(2) at a wide range of atmospheric pO(2) and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO(2).

Role of a ferredoxin gene cotranscribed with the nifHDK operon in N(2) fixation and nitrogenase "switch-off" of Azoarcus sp. strain BH72.

The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a sigma(54)-type promoter. Shorter transcripts sequentially missing genes of the 3' part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% +/- 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxN deletion also had an effect on the rapid "switch-off" of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.

Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum.

Homologs of ntrB and ntrC genes from Rhodospirillum rubrum were cloned and sequenced. A mutant lacking ntrBC was constructed, and this mutant has normal nitrogenase activity under nif-derepressing conditions, indicating that ntrBC are not necessary for the expression of the nif genes in R. rubrum. However, the post-translational regulation of nitrogenase activity by ADP-ribosylation in response to NH4+ was partially abolished in this mutant. More surprisingly, the regulation of nitrogenase activity in response to darkness was also affected, suggesting a physiological link between the ntr system and energy signal transduction in R. rubrum. The expression of glutamine synthetase, as well as its posttranslational regulation, was also altered in this ntrBC mutant.

Amino acid transport systems required for diazotrophic growth in the cyanobacterium Anabaena sp. strain PCC 7120.

Uptake of 16 amino acids by the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was characterized with regard to kinetic parameters of transport, intracellular accumulation of the transported amino acids, and sensitivity of the transport process to energy metabolism inhibitors. Mutants resistant to certain toxic analogs of some amino acids were isolated that were impaired in amino acid transport. Results obtained in this study, together with those reported previously (A. Herrero and E. Flores, J. Biol. Chem. 265:3931-3935, 1990), suggest that there are at least five amino acid transport systems in strain PCC 7120: one high-affinity, active system for basic amino acids; one low-affinity, passive system for basic amino acids; two high-affinity, active systems with overlapping, but not identical, specificities for neutral amino acids; and one putative system for acidic amino acids. Some of the amino acid transport mutants were impaired in diazotrophic growth. These mutants were unable to develop a normal percentage of heterocysts and normal nitrogenase activity in response to nitrogen stepdown. Putative roles for the amino acid transport systems in uptake of extracellular amino acids, recapture of amino acids that have leaked from the cells, and intercellular transfer of amino acids in the filaments of Anabaena sp. strain PCC 7120 are discussed.

Nodule gas exchange and water potential response to rapid imposition of water deficit

ABSTRACTThe permeability (P) of the gaseous diffusion barrier in the nodules of soybean [Glycine max (L.) Merr.] decreases when water deficits are extended over a 7 to 10 d period. The mechanism controlling P changes is unclear, but may result from the release of water to intercellular pathways, and an associated change in the nodule water potential. The purpose of these experiments was to impose water deficit treatments rapidly in order to determine the early sequence of the responses of nodule water potential and nodule gas exchange without the complications that arise from long‐term water deficit treatments. A vertical, split‐root system was used to separate nodule drying effects from plant water deficits by replacing humidified air that was passed over upper root nodules in well‐watered plants with dry air, or by replacing the nutrient solution that surrounded lower roots with ‐1.0 MPa polyethylene glycol (PEG) solution, or by a combination of the dry air and PEG treatments. The PEG treatment caused large decreases in both the components of nodule water potential and nodule relative water content, but there was no indication that these factors had immediate, direct effects on either nitrogenase activity or P. After 7 h of the PEG treatment a significant decrease in nitrogenase activity was found but no decrease in P was detected. These results indicate that changes in nitrogenase activity in response to water deficits precede decreases in P. Exposure of nodules to dry air in well‐watered plants had no significant effect on either nitrogenase activity or P during the 7 h treatment.

The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase

Synthetic oligonucleotides, which were designed according to amino acid sequences conserved between Rhodospirillum rubrum and Azospirillum brasilense DraT and DraG, respectively, were used to identify the corresponding genes of Rhodobacter capsulatus. Sequence analysis of a 1904 bp DNA fragment proved the existence of R. capsulatus draT and draG. These two genes were separated by 11 bp only, suggesting that R. capsulatus draT and draG were part of one transcriptional unit. In contrast to R. rubrum, A. brasilense and Azospirillum lipoferum, the R. capsulatus draTG genes were not located upstream of the structural genes of nitrogenase nifHDK but close to the dctP gene at a distance of about 1000 kb from the nifHDK genes. Deletion mutations in the draTG gene region were constructed and introduced into R. capsulatus wild-type and a nifHDK deletion strain. The resulting mutant strains were examined for post-translational regulation of the molybdenum and the alternative nitrogenase in response to ammonia and darkness. Under 'switch-off' conditions the modified (ADP-ribosylated) and the non-modified forms of component II of both the molybdenum and the alternative nitrogenase were detected in a draTG wild-type background by immunoblot analysis, whereas only the non-modified forms were present in the draTG deletion strains. Nitrogenase activity in these strains was followed by the acetylene reduction assay. In contrast to the wild-type, draTG mutants were not affected in nitrogenase activity in response to ammonia or darkness. These results demonstrated that the draTG genes are required for post-translational regulation of both the molybdenum and the heterometal-free nitrogenase in R. capsulatus.

The use of biofertilizer of Calothrix sp. M103, enhanced by addition of iron and siderophore production

Calothrix sp. M103, enhanced by addition of iron and siderophore production was used as a biofertilizer. The strain was isolated from a colony on the surface root of a rice plant from a rice field in Bangladesh. It showed a marked and immediate increase in nitrogenase activity in response to the addition of iron and also a normal activity (compared with the control) under siderophore production. The addition of iron to iron-limited cultures resulted in a rapid increase in algal iron (11-fold) followed by a marked increase (9-fold) in nitrogenase activity, reaching about 11.8 kg N/ha/crop. This was more than twice the amount of nitrogen usually fixed by the alga during normal growth (controlled conditions), suggesting that pulsing algal inocula with iron would increase the efficiency of algae as a biofertilizer. Indeed, the use of an iron-pulsed inoculum of this strain as a biofertilizer resulted in an increase in the yield of sweetcorn by ca 143% over that produced by the control (no algal inoculation) and to ca a 73% increase over that produced by the normal inoculum (unpulsed with iron). The ability of this strain to produce siderophores has also led to both normal (compared with the control) nitrogenase activity as well as normal growth, which shows a marked contrast to other circumstances where the non-siderophore producing strains showed no growth at all. The significance of these observations on the enhancement of biofertilizer technology is discussed.

Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.

Ca2+ Requirement for Aerobic Nitrogen Fixation by Heterocystous Blue-Green Algae

The requirement of Ca(2+) for growth and nitrogen fixation has been investigated in two strains of heterocystous blue-green algae (Anabaena sp. and Anabaena ATCC 33047). With combined nitrogen (nitrate or ammonium) or with N(2) under microaerobic conditions, Ca(2+) was not required for growth, at least in concentrations greater than traces. In contrast, Ca(2+) was required as a macronutrient for growth and nitrogen fixation with air as the nitrogen source. Addition of Ca(2+) to an aerobic culture without Ca(2+) promoted, after a lag of several hours, development of nitrogenase activity and cell growth. Provision of air to a microaerobic culture in the absence of Ca(2+) promoted a drastic drop in nitrogenase activity, which rapidly recovered its initial level upon restoration of microaerobic conditions. Development of nitrogenase activity in response to either Ca(2+) or low oxygen tension was dependent on de novo protein synthesis. The role of Ca(2+) seems to be related to protection of nitrogenase from inactivation, by conferring heterocysts resistance to oxygen.

Effects of water stress on nitrogenase activity in Abuts incana

Tolerance to water stress was studied in plants of grey alder, Alnus incana (L.) Moench, grown in a climate chamber in pots of sand supplied with a nitrogen‐free nutrient solution. The plants were subjected to a single drying and recovery cycle, during which acetylene reduction, transpiration and stomatal resistance were measured. At different stress levels the plants were placed in a closed system to equilibrate the water potential in the plant‐soil system. The water potential of the plants was determined, after which they were watered and their recovery studied. Nitrogenase activity showed low tolerance to water deficit. At moderate stress (−0.6 to −0.8 MPa) acetylene reduction was reduced by half, and at more severe stress, (< −1 MPa) activity was near zero. There was a rapid decrease in nitrogenase activity coincident with stomatal closure, which indicates a continuous need for photoassimilates for nitrogenase activity. Nodules or nitrogenase activity seemed to be weak sinks for assimilates compared with root pressure bleeding. Measurements of nitrogenase activity in root nodule homogenates supplied with ATP and reductant suggested a loss of active nitrogenase in the nodules in response to water stress. The recovery from moderate stress or long dark treatment took several days, and recovery from severe stress took still longer. Shortage of assimilates and disturbances in oxygen and nitrogen balances in the nodules are discussed as reasons for the reduced nitrogenase activity in response to water stress.

Nitrogenase Activity in Response to Darkening and Defoliation ofAlnus incana

Huss-Danell, K. and Sellstedt, A. 1985. Nitrogenase activity in response to darkening and defoliation of Alnus incana.—l. exp. Bot. 36: 1352-1358. In the Alnus-Frankia symbiosis the nitrogen-fixing root nodules are one of the sinks for carbon compounds newly formed in photosynthesis and exported from the leaves (source). The source-sink ratio of cloned plants of Alnus incana was reduced by darkening or by total or partial defoliation and the resulting nitrogenase activity (C2H2-reduction) was measured. Nitrogenase activity had nearly ceased 5 h after total defoliation but not until ca. 5 d after total darkening. Most of the activity was lost during the initial hours and days, respectively. When leaf area was reduced approximately by half nitrogenase activity decreased slightly less than by half. Removal of upper leaves seemed less harmful

Nitrogenase activity in response to restricted shoot growth in Alnus incana

In the Alnus–Frankia symbiosis the nitrogen-fixing root nodules are one of the sinks for carbon compounds newly formed in photosynthesis and exported from the leaves (source). The competition for assimilates between shoot tips and root nodules was studied by reducing shoot growth. Cloned plants of Alnus incana (L.) Moench were grown without combined nitrogen in a growth chamber. Shoot growth was inhibited by excision of all buds or by induced dormancy. The experiments showed an increased root biomass in the treated plants, indicating a changed source–sink balance. The treatments never caused an increased nitrogenase activity (C2H2-dependent C2H4 production); rather it was decreased. The nitrogenase activity was always correlated with leaf areas. It was also correlated with growth, which is shown by the similar nitrogen percentage in intact and treated plants.