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Effects of elevated carbon dioxide concentration on growth and nitrogen fixation in Alnus glutinosa in a long-term field experiment.

Nitrogen-fixing plant species may respond more positively to elevated atmospheric carbon dioxide concentrations ([CO2]) than other species because of their ability to maintain a high internal nutrient supply. A key factor in the growth response of trees to elevated [CO2] is the availability of nitrogen, although how elevated [CO2] influences the rate of N2-fixation of nodulated trees growing under field conditions is unclear. To elucidate this relationship, we measured total biomass, relative growth rate, net assimilation rate (NAR), leaf area and net photosynthetic rate of N2-fixing Alnus glutinosa (L.) Gaertn. (common alder) trees grown for 3 years in open-top chambers in the presence of either ambient or elevated atmospheric [CO2] and two soil N regimes: full nutrient solution or no fertilizer. Nitrogen fixation by Frankia spp. in the root nodules of unfertilized trees was assessed by the acetylene reduction method. We hypothesized that unfertilized trees would show similar positive growth and physiological responses to elevated [CO2] as the fertilized trees. Growth in elevated [CO2] stimulated (relative) net photosynthesis and (absolute) total biomass accumulation. Relative total biomass increased, and leaf nitrogen remained stable, only during the first year of the experiment. Toward the end of the experiment, signs of photosynthetic acclimation occurred, i.e., down-regulation of the photosynthetic apparatus. Relative growth rate was not significantly affected by elevated [CO2] because although NAR was increased, the effect on relative growth rate was negated by a reduction in leaf area ratio. Neither leaf area nor leaf P concentration was affected by growth in elevated [CO2]. Nodule mass increased on roots of unfertilized trees exposed to elevated [CO2] compared with fertilized trees exposed to ambient [CO2]. There was also a biologically significant, although not statistically significant, stimulation of nitrogenase activity in nodules exposed to elevated [CO2]. Root nodules of trees exposed to elevated [CO2] were smaller and more evenly spaced than root nodules of trees exposed to ambient [CO2]. The lack of an interaction between nutrient and [CO2] effects on growth, biomass and photosynthesis indicates that the unfertilized trees maintained similar CO2-induced growth and photosynthetic enhancements as the fertilized trees. This implies that alder trees growing in natural conditions, which are often limited by soil N availability, should nevertheless benefit from increasing atmospheric [CO2].

Alnus↗

Effects of calcium deficiency on symbiotic nitrogen fixation.

The mechanism of the effect of mild calcium deficiency on nitrogen metabolism of the symbiotic plant was studied from the distribution of calcium and of nitrogen and carbohydrate fractions in plant organs.Nitrogen concentrations of all plant organs decreased with calcium deficiency. Addition of either a nitrogen or a calcium salt increased nitrogen concentrations. For roots as well as whole plants the effects of one salt were largely replaced by the other. These effects establish that calcium deficiency decreased the supply of fixed nitrogen from nodules to other organs. As weight of nodules was independent of calcium it follows that nodular efficiency was impaired.Since nitrogen concentrations of nodules decreased rather than increased with calcium deficiency it is suggested that calcium deficiency interferes with the rate of reduction of nitrogen in the nodule rather than with the export of reduced nitrogen. Distribution of alpha-amino, amide, and ammonium nitrogen are consistent with this suggestion.Nitrogen fixation was not limited by translocation of carbohydrates to nodules as calcium deficiency had little effect on the concentration of soluble carbohydrates and actually increased the concentration of starch in nodules.Calcium deficiency depressed the calcium content of nodules so that nitrogen fixation may have been impaired by inadequate calcium for nodular structure or metabolism.

Journal Article↗

Physiological and 15N-NMR analysis of molecular nitrogen fixation by Methanococcus thermolithotrophicus, Methanobacterium bryantii and Methanospirillum hungatei.

Two mesophilic methanogenic bacteria, Methanobacterium bryantii strain MOH and Methanospirillum hungatei strain GP1 were demonstrated, using several different experimental approaches, to fix dinitrogen. Evidence includes (1) growth with N2 as the sole nitrogen source; (2) incorporation of 15N2 into cellular material (both soluble amino acid pools and insoluble cell protein and other macromolecules) detected by 15N-NMR spectroscopy; (3) acetylene reduction to ethylene by the cells, and inhibition of this reaction by bromoethanesulfonic acid (BES), a methanogen inhibitor. High-resolution 15N-NMR analysis of ethanol extracts of these organisms and cross-polarization magic-angle sample spinning analysis of the solid debris from these extracts are compared to labeled material from Methanococcus thermolithotrophicus, a methanogen previously determined to fix dinitrogen.

Clostridium↗

Nitrogen fixation and vegetative regrowth of alfalfa and birdsfoot trefoil after successive harvests or floral debudding.

Nitrogenase-dependent acetylene reduction, leaf, herbage, and root growth, and total nonstructural carbohydrate accumulation of alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) were compared to learn how nitrogen fixation capacity and vegetative growth respond to partial (75-85%) or total shoot and leaf removal, and floral debudding. Treatments were imposed on greenhouse-grown plants during two successive harvest cycles.Both species displayed an initial decline in total nitrogenase activity within 2 days of harvest and a subsequent recovery of activity after 10 to 21 days. Rate of recovery varied with the amount of leaf area removed. Periodic flower removal did not significantly alter total nitrogenase activity of either species compared with the unharvested controls. In the first harvest cycle, partial leaf area removal did not affect nitrogenase activity of alfalfa, but activity of trefoil was reduced 56%. In the second harvest cycle, partial leaf area removal reduced total nitrogenase activity of alfalfa 46% and that of trefoil 69%. Complete leaf and shoot removal reduced total nitrogenase activity of alfalfa 78% after the first harvest and 86% after the second harvest.Recovery of nitrogenase activity after harvest paralleled leaf area expansion in both species. After the initial decline following the first partial harvest, total nitrogenase activity and leaf area of alfalfa increased 170 and 500%, respectively. After the initial decline following the second partial harvest, nitrogenase activity and leaf area of alfalfa increased 280 and 800%, respectively. Partly harvested trefoil and completely harvested alfalfa showed similar response patterns. Release of bud dormancy and leaf area expansion after flowering of nonharvested alfalfa apparently caused an increase in nitrogenase activity, but patterns of acetylene reduction and leaf area were not otherwise closely correlated in controls of either species. Decline and accumulation of total nonstructural carbohydrates in both species varied with defoliation treatment. Patterns of nonstructural carbohydrates in root tissue were not closely related to the changes in total nitrogenase activity caused by shoot removal.

Journal Article↗

Diversity of Plasmid Profiles and Conservation of Symbiotic Nitrogen Fixation Genes in Newly Isolated Rhizobium Strains Nodulating Sulla (Hedysarum coronarium L.).

Forty-five Rhizobium strains nodulating sulla (Hedysarum coronarium L.), isolated from plants grown in different sites in Menorca Island and southern Spain, were examined for plasmid content and the location and organization of nif (nitrogen fixation) and nod (nodulation) sequences. A great diversity in both number and size of the plasmids was observed in this native population of strains, which could be distributed among 19 different groups according to their plasmid profiles. No correlation was found between plasmid profile and geographical origin of the strains. In each strain a single plasmid ranging from 187 to 349 megadaltons hybridized to Rhizobium meliloti nifHD and nodD DNA, and in three strains the spontaneous loss of this plasmid resulted in the loss of the nodulation capacity. In addition to the symbiotic plasmid, 18 different cryptic plasmids were identified. A characteristic cryptic plasmid of >1,000 megadaltons was present in all strains. Total DNA hybridization experiments, with nifHD and portions of nodC and nodD genes (coding for common nodulation functions) from R. meliloti as probes, demonstrated that both the sequence and organization of nif and common nod genes were highly conserved within rhizobia nodulating sulla. Evidence for reiteration of nodD sequences and for linkage of nodC to at least one copy of nodD was obtained for all the strains examined. From these results we conclude that Rhizobium strains nodulating sulla are a homogeneous group of symbiotic bacteria that are closely related to the classical fast-growing group of rhizobia.

Journal Article↗

Quantifying functional gene populations: comparing gene abundance and corresponding enzymatic activity using denitrification and nitrogen fixation in pulp and paper mill effluent treatment systems.

The relationship between the abundance of three functional genes and their corresponding biochemical reaction rates was investigated in several activated sludge and mill effluent microbial communities. Gene probes were prepared for two key denitrification genes (nirS and nirK) and for one nitrogen-fixation gene (nifH) and were validated using a variety of strains of known nir and nif genotype. ATP-based measures of viable cell numbers were used to provide total population sizes. In certain microbial communities (activated sludge enrichment cultures and multiple samples taken from the same mill primary clarifier), a strong correlation was observed between gene abundance and biochemical activity rates. However, when comparing several different nonenriched activated sludge bioreactors and separate primary clarifier microbial communities, the ratio of specific gene abundance to biochemical activity rates varied widely. These results suggest that in cases where a microbial community is not fully induced for a given biochemical activity or when very different communities are compared, quantitative gene probing can give a better measure of a community's potential to carry out the encoded function than can the relevant biochemical assay. However, the gene quantitation method employed here probably underestimated the true number of probed genes present in the microbial communities due to nirS and nifH genes in the communities having reduced DNA sequence similarity with the probes used.

Adenosine Triphosphate↗