Biomedical subjects
Publications and source records attributed to D B Layzell.
Elevated CO(2), in the dark, is sometimes reported to inhibit leaf respiration, with respiration usually measured as CO(2) efflux. Oxygen uptake may be a better gauge of respiration because non-respiratory processes can affect dark CO(2) efflux in elevated CO(2). Two methods of quantifying O(2) uptake indicated that leaf respiration was unaffected by coincident CO(2) level in the dark.
Simultaneous measurements of CO(2) (CER) and O(2) (OER) exchange in roots and shoots of vegetative white lupin (Lupinus albus) were used to calculate the flow of reducing power to the synthesis of biomass that was more reduced per unit of carbon than carbohydrate. On a whole-plant basis, the diverted reductant utilization rate (DRUR which is: 4 x [CER + OER]) of shoot tissue was consistently higher than that of roots, and values obtained in the light were greater than those in the dark. An analysis of the biomass being synthesized over a 24-h period provided an estimate of whole-plant DRUR (3.5 mmol e(-) plant(-1) d(-1)), which was similar to that measured by gas exchange (3.2 mmol e(-) plant(-1) d(-1)). Given that nitrate reduction to ammonia makes up about 74% of whole-plant DRUR, root nitrate reduction in white lupin was estimated to account for less than 43% of whole-plant nitrate reduction. The approach developed here should offer a powerful tool for the noninvasive study of metabolic regulation in intact plants or plant organs.
Regulation of the intracellular concentration of substrates is essential for the maintenance of a stable cellular environment. Diffusion and reaction processes supply and consume substrates within cells and determine their steady-state concentrations. To realistically represent these processes by computer simulation they must be modeled in three dimensions. Yet three-dimensional models are inherently computing intensive. This study describes a method, which substantially simplifies the modeling of diffusion into a polyhedral body (a cube), that was used as a model representation of a cell. The method is applied to a case study of oxygen diffusion into nitrogen-fixing, rhizobia-infected cells in legume nodules. The method involved generating a one-dimensional representation of the three-dimensional problem to provide a "surface area profile" of three-dimensional diffusion. The one-dimensional models were significantly easier to program, several orders of magnitude faster to solve and in this study were validated by assessing their results against those of comparable three-dimensional models of diffusion into the same body. The results show the one-dimensional method to be a close approximation of a three-dimensional source-sink problem with systematic differences below 10% for fractional oxygenation of leghemoglobin, cell respiration and nitrogenase activity. Larger differences between models (up to 45%) in the predicted average and innermost O(2)concentrations had no effects on the physiological conclusions of the study, but were attributed to the poorer resolution of the three- than the one-dimensional model, and to an inherent simplification in the derivation of the one-dimensional surface area profiles. The one-dimensional modeling approach was found to be a simple, yet powerful tool for the study of diffusion and reaction in biological systems.
Control of the permeability to oxygen is critical for the function of symbiotic nitrogen fixation in legume nodules. The inner cortex (IC) seems to be a primary site for this regulation. In alfalfa (Medicago sativa) nodules, expression of the Msca1 gene encoding a carbonic anhydrase (CA) was previously found to be restricted to the IC. We have now raised antibodies against recombinant Msca1 protein and used them, together with antibodies raised against potato leaf CA, to demonstrate the presence of two forms of CA in mature nodules. Each antibody recognizes a different CA isoform in nodule tissues. Immunolocalization revealed that leaf-related CAs were localized primarily in the nitrogen-fixing zone, whereas the Msca1 protein was restricted exclusively to the IC region, in indeterminate and determinate nodules. In alfalfa nodules grown at various O(2) concentrations, an inverse correlation was observed between the external oxygen pressure and Msca1 protein content in the IC, the site of the putative diffusion barrier. Thus Msca1 is a molecular target of physiological processes occurring in the IC cells involved in gas exchange in the nodule.
An instrument for measuring low rates of biological O2 exchange using an open-flow gas analysis system is described. A novel differential O2 sensor that is capable of measuring as little as 0.4 Pa O2 against a back-ground of ambient air (20,900 Pa O2), yet has a dynamic range of +/- 2000 Pa O2 (i.e., +/- ca. 2% O2) is described. Baseline drift was typically less than 0.025 Pa min-1. The differential O2 sensor was incorporated into a respiratory quotient/photosynthetic quotient analyzer that contained other environmental sensors for atmospheric pressure, absolute O2 and CO2 concentration, temperature of the differential O2 sensor block, and differential pressure between reference and sample streams. Protocols for how these sensors can be used to calibrate the differential O2 sensor and to improve its stability with time are described. Together, the differential O2 sensor, the environmental sensors, and the simple calibration techniques allow for simultaneous, noninvasive, and accurate measurements of O2 and CO2 exchange in tissues with metabolic rates as low as about 0.1 mumol O2 or CO2 h-1. Example data are provided in which O2 differentials of 3 to 41 Pa O2 were measured in an open-flow system.
Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H(2) evolution in Ar:O(2) (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO(2) production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O(2) permeability decreased 50% within 5 hours in each species. A similar decrease in the O(2)-saturated respiration rate (V(max)) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V(max) in each species. V(max) may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O(2) inactivation of nitrogenase.
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 O(2) permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O(2) consumption) rate and internal O(2) concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N(2). The rate of increase of FOL under 100% O(2) was then used to calculate nodule O(2) permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O(2) for 15 minutes led to decreases in both permeability and O(2)-saturated respiration (V(max)), but the brief (<15 seconds) exposures to 100% O(2) required by the assay itself had little effect on either parameter. A gradual increase in external O(2) 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.
When intact nodulated roots of soybean (Glycine max L. Merr. nodulated with Bradyrhizobium japonicum strain USDA 16) were exposed to an atmosphere lacking N(2) gas (Ar:O(2) 80:20), total nitrogenase activity (measured as H(2) evolution) and respiration (CO(2) evolution) declined with time of exposure. In Ar-inhibited nodules, when the O(2) concentration in the rhizosphere was increased in a linear ;ramp' of 2.7% per minute, 93% of the original H(2) evolution and 99% of the CO(2) evolution could be recovered. The internal nodule O(2) concentration (estimated from leghemoglobin oxygenation) declined to 56% of its initial value after 60 minutes of Ar:O(2) exposure and could be partially recovered by the linear increases in O(2) concentration. Nodule gas permeability, as estimated from the lag in ethylene production following exposure of nodules to acetylene, decreased to 26% of its initial value during the Ar-induced decline. Collectively, the results provide direct evidence that the Ar-induced decline results from decreased nodule gas permeability and indicate that the decline in permeability, rather than being immediate, occurs gradually over the period of Ar:O(2) exposure.
A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell O(2) concentration (O(i)) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free O(2) in the infected cells of the nodule (O(i)). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO(3) (-) treatment, stem-girdling, continuous darkness, or nodule disturbance is caused by a reduction in O(i) and limitation of respiration in support of nitrogenase activity. A plot of nitrogenase activity (measured as peak H(2) evolution in Ar:O(2)) versus O(i) for the various treatments was consistent with the concept that O(i) limits in vivo nitrogenase activity in legume nodules under adverse conditions. The potential for using O(i) to estimate nitrogenase activity in laboratory and field-grown legumes is discussed.
Exposure of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mutants of Arabidopsis thaliana to photorespiratory conditions resulted in the accumulation of NH(4) (+) and the inhibition of photosynthesis. However, upon transfer from 2% O(2), 350 microliters per liter CO(2), to 21% O(2), 350 microliters per liter CO(2), net photosynthesis declined at a slower rate in methionine sulfoximine treated leaf discs relative to controls. The recovery of photosynthesis was also more rapid in MSO-treated leaf discs although ammonia levels were more than threefold higher. Photosynthesis in leaf discs treated with azaserine was inhibited more than controls when transferred to 21% O(2) and recovered less than controls when returned to 2% O(2) although NH(4) (+) levels were not significantly different. The results obtained are consistent with the view that the rapid inhibition of photosynthesis in the glutamate synthase mutants in photorespiratory conditions is not due to the accumulation of NH(4) (+) but rather to the depletion of amino donors for glyoxylate and the consequent effects of glyoxylate on the lack of return of carbon to the chloroplast.
The objectives of this study were to determine whether attached nodules of soybean (Glycine max L. Merr.) could adjust to gradual increases in rhizosphere pO(2) without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by pO(2) under ambient conditions. A computer-controlled gas blending apparatus was used to produce linear increases (ramps) in pO(2) around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H(2) production in N(2):O(2) and Ar:O(2)) and respiration (CO(2) evolution) were monitored continuously as pO(2) was ramped from 20 to 30 kilopascals over periods of 0, 5, 10, 15, and 30 minutes. The 0, 5, and 10 minute ramps caused inhibitions of nitrogenase and respiration rates followed by recoveries of these rates to their initial values within 30 minutes. Distinct oscillations in nitrogenase activity and respiration were observed during the recovery period, and the possible basis for these oscillations is discussed. The 15 and 30 minute ramps did not inhibit nitrogenase activity, suggesting that such inhibition is not a factor in the regulation of nodule diffusion resistance. During the 30 minute ramp, a stimulation of nitrogenase activity was observed, indicating that an O(2)-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions.
Ammonia production and assimilation(1) were examined in photorespiratory mutants of Arabidopsis thaliana L. lacking ferredoxin-dependent glutamate synthase (Fd-GluS) activity. Although photosynthesis was rapidly inhibited in these mutants in normal air, NH(4) (+) continued to accumulate. The accumulation of NH(4) (+) was also seen after an initial lag of 30 minutes in 2% O(2), 350 microliters per liter of CO(2) and after 90 minutes in 2% O(2), 900 microliters per liter of CO(2). The accumulation of NH(4) (+) in normal air and low O(2) was also associated with an increase in the total pool of amino acid-N and glutamine, and a decrease in the pools of glutamate, aspartate, alanine, and serine. Upon return to dark conditions, or to 21% O(2), 1% CO(2) in the light, the NH(4) (+) which had accumulated in the leaves was reassimilated into amino acids. The addition of methionine sulfoximine (MSO) resulted in higher accumulations of NH(4) (+) in glutamate synthase mutants and prevented the reassimilation of NH(4) (+) upon return to the dark. The addition of MSO also resulted in the accumulation of NH(4) (+) in glutamate synthase mutants in the light and in 21% O(2), 1% CO(2). These results indicate that glutamine synthetase is essential for the reassimilation of photorespiratory NH(4) (+) and for primary N assimilation in the leaves and strongly suggest that glutamate dehydrogenase plays only a minimal role in the assimilation of ammonia. Levels of NADH-dependent glutamate synthase (NADH-GluS) appear to be sufficient to account for the assimilation of NH(4) (+) by a GS/NADH-GluS cycle.
A fiber optic spectrophotometric system was used to monitor the in vivo oxygenation of leghemoglobin in intact, attached soybean root nodules (Glycine max L. Merr. x USDA 16 Bradyrhizobium japonicum) which were flattened during development by growth in narrow, glass-walled cuvettes. When equilibrated at an external pO(2) of 20 kilopascals, leghemoglobin was 36.6 +/- 5.4% oxygenated, a value estimated to represent an infected cell O(2) concentration of 21.5 nanomolar. Increasing the external pO(2) from 20 to 25 kilopascals caused a rapid increase in leghemoglobin oxygenation, followed by a recovery to the initial level, all within 7.5 minutes. At 25 kilopascals O(2), the rates of H(2) and CO(2) evolution were similar to those at 20 kilopascals. Since respiration had not increased, the results support the proposal that nodules adapt to increased external pO(2) by regulating their resistance to O(2) diffusion.
Increased concentrations of nitrate in a nutrient solution (2, 5, and 10 millimolar KNO(3)) were correlated with increased shoot:root ratios of non-nodulated soybeans (Glycine max [L.] Merr.) grown in sand culture. While altering the pattern of C and N partitioning, the N treatments did not affect whole plant photosynthesis over the study period. To determine the mechanism responsible for the observed changes in assimilate partitioning, detailed C and N budgets were worked out with plants from each N treatment over three consecutive 4-day periods of midvegetative growth. The information for the C and N budgets from the 2 and 10 millimolar NO(3) (-) treatments was combined with data on the composition of xylem and phloem exudates to construct a series of models of C and N transport and partitioning. These models were used to outine a ;chain-reaction' of cause-and-effect relationships that may account for the observed changes in assimilate partitioning in these plants. The proposed mechanism identifies two features which may be important in regulating the partitioning of N and other nutrients within the whole plant. (a) The concentration of N in the phloem is highly correlated with the N concentration in the xylem. (b) The amount of N which cycles through the root-from phloem imported from the shoot to xylem exported by the root-is regulated by the root's requirement for N: only that N in excess of the root's N requirements is returned to the shoot in the xylem. Therefore, roots seem to have the highest priority for N in times of N stress.
An open gas exchange system was used to monitor the nonsteady state and steady state changes in nitrogenase activity (H(2) evolution in N(2):O(2) and Ar:O(2)) and respiration (CO(2) evolution) in attached, excised, and sliced nodules of soybean (Glycine max L. Merr.) exposed to external pO(2) of 5 to 100%. In attached nodules, increases in external pO(2) in steps of 10 or 20% resulted in sharp declines in the rates of H(2) and CO(2) evolution. Recovery of these rates to values equal to or greater than their initial rates occurred within 10 to 60 minutes of exposure to the higher pO(2). Recovery was more rapid at higher initial pO(2) and in Ar:O(2) compared to N(2):O(2). Sequential 10% increments in pO(2) to 100% O(2) resulted in rates of H(2) evolution which were 1.4 to 1.7 times the steady state rate at 20% O(2) in Ar. This was attributed to a relief at high pO(2) from the 40% decline in nitrogenase activity that was induced by Ar at a pO(2) of 20%. Changes in nodule respiration rate could not account for the nodules' ability to adjust to high external pO(2), supporting the hypothesis that soybean nodules have a variable barrier to O(2) diffusion which responds slowly (within minutes) to changes in pO(2). Nodule excision and slicing resulted in 45 and 78% declines, respectively, in total specific nitrogenase activity at 20% O(2). In contrast with the result obtained with intact nodules, subsequent 10% increases in pO(2) in Ar:O(2) did not result in transient declines in H(2) evolution rates, but in the rapid attainment of new steady state rates. Also, distinct optima in nitrogenase activity were observed at about 60% O(2). These results were consistent with an increase in the diffusive resistance of the nodule cortex following nodule excision or nodule slicing. This work also shows the importance of using intact plants and continuous measurements of gas exchange in studies of O(2) diffusion and nitrogenase activity in legume nodules.
When arrival of shoot supplied carbohydrate to the nodulated root system of soybean was interrupted by stem girdling, stem chilling, or leaf removal, nodule carbohydrate pools were utilized, and a marked decline in the rates of CO(2) and H(2) evolution was observed within approximately 30 minutes of treatment. Nodule excision studies demonstrated that the decline in nodulated root respiration was associated with nodule rather than root metabolism, since within 3.5 hours of treatment, nodules respired at less than 10% of the initial rates. Apparently, a continuous supply of carbohydrate from the shoot is required to support nodule, but not root, function. Depletion of nodular carbohydrate pools was sufficient to account for the (diminishing) nodule respiration of girdled plants. Of starch and soluble sugar pools within the whole plant, only leaf starch exhibited a diurnal variation which was sufficient to account for the respiratory carbon loss of nodules over an 8 hour night. Under 16 hour nights, or in continuous dark, first the leaf starch pools were depleted, and then nodule starch reserves declined concomitant with a decrease in the rates of CO(2) and H(2) evolution from the nodules. Nodule soluble sugar levels were maintained in dark treated plants but declined in girdled plants. The depletion of starch in root nodules is an indicator of carbohydrate limitation of nodule function.
Low root temperature effects on vegetative growth of soybean (Harosoy 63 x Rhizobium japonicum USDA 16) were examined in 35 day old plants exposed to temperatures of 15 degrees C (shoots at 25 degrees C) for an 11 day period. Duing this period various aspects of C and N assimilation and partitioning were monitored including shoot night and nodulated root respiration, C and N partitioning to six plant parts, C(2)H(2) reduction, H(2) evolution, leaf area, transpiration, net photosynthesis, and N(2) fixation. The low temperature treatment resulted in a decrease in the net rate of N(2) fixation but nitrogenase relative efficiency increased. In response, the plant retained N in the tissues of the nodulated root and decreased N partitioning to young shoot tissues, thereby inducing the remobilization of N from older leaves, and reducing leaf area development. The leaf area specific rate of net photosynthesis was not affected over the study period; however, shoot and nodulated root respiration declined. Consequently, C accumulated in mature leaves and stems, partly in the form of increased starch reserves. Three possibilities were considered for increasing low temperature tolerance in nodulated soybeans: (a) decrease in temperature optima for nitrogenase, (b) increased development of nodules and N(2) fixation capacity at low temperature, and (c) alterations in the pattern of C and N partitioning in response to low temperature conditions.
The magnitude and role of dark CO(2) fixation were examined in nodules of intact soybean plants (Harosoy 63 x Rhizobium japonicum strain USDA 16). The estimated rate of nodule dark CO(2) fixation, based on a 2 minute pulse-feed with (14)CO(2) under saturating conditions, was 102 micromoles per gram dry weight per hour. This was equivalent to 14% of net nodule respiration. Only 18% of this CO(2) fixation was estimated to be required for organic and amino acid synthesis for growth and export processes. The major portion (75-92%) of fixed label was released as CO(2) within 60 minutes. The labeling pattern during pulse-chase experiments was consistent with CO(2) fixation by phosphoenolpyruvate carboxylase. During the chase, the greatest loss of label occurred in organic acids. Exposure of nodulated roots to Ar:O(2) (80:20) did not affect dark CO(2) fixation, while exposure to O(2):CO(2) (95:5) resulted in 54% inhibition. From these results, it was concluded that at least 66% of dark CO(2) fixation in soybean may be involved with the production of organic acids, which when oxidized would be capable of providing at least 48% of the requirement for ATP equivalents to support nitrogenase activity.