David B. Layzell

Glutamine synthetase genes are regulated by ammonia provided externally or by symbiotic nitrogen fixation.

Glutamine synthetase is the key enzyme in the assimilation by plants of reduced nitrogen provided from either the soil or fixed symbiotically in association with Rhizobium. We have isolated a number of cDNA clones for soybean glutamine synthetase (GS) from a nodule‐cDNA library, using RNA from polysomes immunoprecipitated by GS antibodies. Transcripts corresponding to two clones differing in their 3′ non‐translated sequences were present in both root and nodule tissue; however, the concentration in the nodules was several times higher. The relative concentrations of these sequences in both tissues is about 9:1. Availability of ammonium ions [provided as NH4NO3 or (NH4)2SO4] enhanced the expression of both sequences in root tissue within 2 h, reaching a level similar to that in nodules by 8 h, while KNO3 had no effect during this period. When nitrogen fixation was prevented by replacing nitrogen with argon in the root environment or when the nodules were formed by a Fix‐ mutant of Bradyrhizobium japonicum, the amounts of GS mRNA did not increase over that in roots. These experiments, together with the time course of increase in GS mRNA transcripts, suggest that the genes encoding cytosolic GS are directly induced by the available ammonia.

Phloem Glutamine and the Regulation of O2 Diffusion in Legume Nodules

The aim of the present study was to test the hypothesis that the N content or the composition of the phloem sap that supplies nodulated roots may play a role in the feedback regulation of nitrogenase activity by increasing nodule resistance to O2 diffusion. Treating shoots of lupin (Lupinus albus cv Manitoba) or soybean (Glycine max L. Merr. cv Maple Arrow) with 100 [mu]L L-1 NH3 caused a 1.3-fold (lupin) and 2.6-fold (soybean) increase in the total N content of phloem sap without altering its C content. The increase in phloem N was due primarily to a 4.8-fold (lupin) and 10.5-fold (soybean) increase in the concentration of glutamine N. In addition, there was a decline in both the apparent nitrogenase activity and total nitrogenase activity that began within 4 h and reached about 54% of its initial activity within 6 h of the start of the NH3 treatment. However, the potential nitrogenase activity values in the treated plants were not significantly different from those of the control plants. These results provide evidence that changes in the N composition of the phloem sap, particularly the glutamine content, may increase nodule resistance to O2 diffusion and, thereby, down-regulate nodule metabolism and nitrogenase activity by controlling the supply of O2 to the bacteria-infected cells.

Model of gas exchange and diffusion in legume nodules : I. Calculation of gas exchange rates and the energy cost of N2 fixation.

A mathematical model is described which allows the estimation of rates of O2, CO2, N2, and H2 exchange from legume nodules under steady state conditions of N2 fixation. Calculated rates of gas exchange under defined conditions of nodule size, relative growth rate (RGR), specific total nitrogenase activity (TNA), nitrogenase electron allocation coefficient (EAC), uptake-hydrogenase activity (HUP) and nature of the N export product compared favorably with experimentally-obtained rates reported in the literature. Therefore the model was used to predict the effects of varying each of these nodule characteristics on the rates of gas exchange, and on the apparent respiratory cost (CO2/NH3) and sucrose cost (sucrose consumed/NH3) of N2 fixation.The model predicted that, all other characters being equal, ureide-producing nodules would consume 8% less sucrose per N fixed than asparagine-producing nodules, but would display an apparent respiratory cost which would be 5% higher than that in asparagine-producing nodules. In both ureide-producing and asparagine-producing nodules, the major factor affecting the apparent respiratory cost of N2 fixation was predicted to be EAC, followed by TNA, nodule RGR and nodule size. The relative importance of HUP in improving the apparent respiratory cost of N2 fixation was predicted to be largely dependent upon its potential role in the regulation of EAC.

Model of gas exchange and diffusion in legume nodules: II. Characterisation of the diffusion barrier and estimation of the concentrations of CO2, H2 and N2 in the infected cells

The rates of nodule O2, CO2, N2 and H2 exchange calculated in the previous modeling study (D.B. Layzell et al., 1987, Planta 173, 117–127) were combined with information on the diffusion characteristics of each gas, and the structural characteristics of soybean nodules, to produce a comprehensive mathematical model of nodule structure and function. The model assumed that an aqueous barrier to gas diffusion exists in the nodule cortex which may be regulated to maintain an O2 concentration of 10 nM in the centre of the infected cells of the central zone. The model was used to predict the concentration of N2, CO2 and H2 in the infected cells as the physical and physiological characteristics of the nodule were varied. The model predicted that (a) the diffusion barrier may be represented by plugs of water in the intercellular spaces of a layer of cells between the inner and outer cortex, the depth of which may be varied to vary the resistance of the barrier; (b) facilitated diffusion of O2 by oxyleghemoglobin is essential to the regulation of free O2 concentration in the infected cells; (c) the diffusion barrier is less effective in regulating CO2 flux than the fluxes of other gases with the result that the total gas pressure in the central zone is less than atmospheric pressure; (d) concentrations of N2 and HCO3-in the infected cells are saturating with respect to nitrogenase activity and phosphoenolpyruvate carboxylase activity respectively and (e) under atmospheric conditions the concentration of H2 in the infected cells is similar to, or greater than the Ki. (H2) for N2 fixation, which may account for values of nitrogenase electron allocation coefficient below 0.75.

H2 oxidation, O2 uptake and CO2 fixation in hydrogen treated soils

In many legume nodules, the H2 produced as a byproduct of N2 fixation diffuses out of the nodule and is consumed by the soil. To study the fate of this H2 in soil, a H2 treatment system was developed that provided a 300 cm3 sample of a soil:silica sand (2:1) mixture with a H2 exposure rate (147 nmol H2 cm−3hr−1) similar to that calculated exist in soils located within 1–4 cm of nodules (30–254 nmol H2 cm−3hr−1). After 3 weeks of H2 pretreatment, the treated soils had a Km and Vmax for H2 uptake (1028 ppm and 836 nmol cm−3 hr−1, respectively) much greater than that of control, air-treated soil (40.2 ppm and 4.35 nmol cm−3 hr−1, respectively). In the H2 treated soils, O2, CO2 and H2 exchange rates were measured simultaneously in the presence of various pH2. With increasing pH2, a 5-fold increase was observed in O2 uptake, and CO2 evolution declined such that net CO2 fixation was observed in treatments of 680 ppm H2 or more. At the H2 exposure rate used to pretreat the soil, 60% of the electrons from H2 were passed to O2, and 40% were used to support CO2 fixation. The effect of H2 on the energy and C metabolism of soil may account for the well-known effect of legumes in promoting soil C deposition.

Drought Stress, Permeability to O2 Diffusion, and the Respiratory Kinetics of Soybean Root Nodules

In legume nodules, treatments such as detopping or nitrate fertilization inhibit nodule metabolism and N2 fixation by decreasing the nodules permeability to O2 diffusion, thereby decreasing the infected cell O2 concentration (Oi) and increasing the degree to which nodule metabolism is limited by O2 availability. In the present study we used nodule oximetry to assess and compare the role of O2 limitation in soybean (Glycine max L. Merr) nodules inhibited by either drought or detopping. Compared to detopping, drought caused only minor decreases in Oi, and when the external O2 concentration was increased to raise Oi, the infected cell respiration rate in the drought-stressed plants was not stimulated as much as it was in the nodules of the detopped plants. Unlike those in detopped plants, nodules exposed to moderate drought stress displayed an O2-sufficient respiration rate that was significantly lower than that in control nodules. Despite possible side effects of oximetry in altering nodule metabolism, these results provided direct evidence that, compared to detopping, O2 limitation plays a minor role in the inhibition of nodule metabolism during drought stress and changes in nodule permeability are the effect, not the cause, of a drought-induced inhibition of nodule metabolism and the O2-suffiecient rate of respiration.

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.

A Re-Evaluation of the Role of the Infected Cell in the Control of O2 Diffusion in Legume Nodules

Two different simulation models were constructed to describe O2 diffusion into the bacteria-infected cells of legume nodules: one based on a central zone of uniform spherical cells and the other on a central zone of packed, uniform cubical cells with air spaces along the edges. The cubical model more closely approximated the geometry and gas diffusion characteristics of infected cells than did the spherical model. The models relied on set values for the innermost O2 concentration in the infected cell (1–20 nM) and predicted values for the free O2 and oxygenated leghemoglobin gradients toward the cell:space interface. The cubical model but not the spherical model predicted saturation of leghemoglobin (Lb) oxygenation at or within a few micrometers of the gas-filled intercellular space and predicted that the space concentration could be as high as 1.3% O2 when the fractional oxygenation of Lb and respiration rate within the infected cell were typical of that which has been measured in vivo. In the model, the higher the space O2 concentration, the greater the saturation of Lb by O2 and the greater the collapse of Lb-facilitated diffusion near the cell:space interface. This was predicted to result in a greater resistance to O2 diffusion from the space to the bacteroids, thereby providing an intrinsic, homeostatic mechanism for controlling the rate of O2 influx into infected cells. Changes in the physiological features of the simulated cubical infected cell, such as the proportion of the cell as cytosol, the surface area of the cell exposed to a space, the maximum rate of cellular respiration, or the concentration of Lb in the cytoplasm, significantly altered the extent to which the infected cell would be able to regulate its diffusive resistance. These results demonstrate the possibility of a Lb-based mechanism for controlling the O2 concentration within the infected cells. If such a mechanism exists in legume nodules, it would give the infected cell an ability to exercise fine control over its internal environment, a process that could complement a physical diffusion barrier that may exist in the inner cortex or elsewhere in the nodule and provide coarse control over O2 diffusion.

Pyrolysis of wood to biochar: increasing yield while maintaining microporosity.

The objective of this study was to determine if biochar yield could be increased by the deposition of volatile pyrolysis species within the bed during production, without negatively influencing the microporosity and adsorption properties. Aspen (Populus tremuloides) wood chips were loaded into three vertically stacked zones within a reactor and heated in nitrogen to temperatures between 420 and 650°C (i.e., pyrolyzed). The yield did increase from the zone at the reactor inlet to the subsequent zones as volatile species deposited and carbonized, and importantly, the carbonized deposits had a similar microporous structure and organic vapor uptake (1,1,1,2-tetrafluoroethane) to that of the primary biochar. Based on these results, bio-oil from previous runs at 600°C was recycled to the bed, which further increased the yield while maintaining the desirable adsorption properties of the biochar.

Production of bio-synthetic natural gas in Canada.

Large-scale production of renewable synthetic natural gas from biomass (bioSNG) in Canada was assessed for its ability to mitigate energy security and climate change risks. The land area within 100 km of Canadas network of natural gas pipelines was estimated to be capable of producing 67-210 Mt of dry lignocellulosic biomass per year with minimal adverse impacts on food and fiber production. Biomass gasification and subsequent methanation and upgrading were estimated to yield 16,000-61,000 Mm(3) of pipeline-quality gas (equivalent to 16-63% of Canadas current gas use). Life-cycle greenhouse gas emissions of bioSNG-based electricity were calculated to be only 8.2-10% of the emissions from coal-fired power. Although predicted production costs (