Truncated hemoglobins in actinorhizal nodules of Datisca glomerata.

Three types of hemoglobins exist in higher plants, symbiotic, non-symbiotic, and truncated hemoglobins. Symbiotic (class II) hemoglobins play a role in oxygen supply to intracellular nitrogen-fixing symbionts in legume root nodules, and in one case ( Parasponia Sp.), a non-symbiotic (class I) hemoglobin has been recruited for this function. Here we report the induction of a host gene, dgtrHB1, encoding a truncated hemoglobin in Frankia-induced nodules of the actinorhizal plant Datisca glomerata. Induction takes place specifically in cells infected by the microsymbiont, prior to the onset of bacterial nitrogen fixation. A bacterial gene (Frankia trHBO) encoding a truncated hemoglobin with O (2)-binding kinetics suitable for the facilitation of O (2) diffusion ( ) is also expressed in symbiosis. Nodule oximetry confirms the presence of a molecule that binds oxygen reversibly in D. glomerata nodules, but indicates a low overall hemoglobin concentration suggesting a local function. Frankia trHbO is likely to be responsible for this activity. The function of the D. glomerata truncated hemoglobin is unknown; a possible role in nitric oxide detoxification is suggested.

The Resistance of the Diffusion Barrier in Nodules of Myrica gale L. Changes in Response to Temperature but Not to Partial Pressure of O2.

Rates of C2H2 reduction and CO2 evolution by nodules were measured in a flowthrough system using intact plants of Myrica gale L. Both activities increased linearly with increasing partial pressure of O2 (pO2) up to 18 kPa. The linear relationship between CO2 evolution and pO2 at pO2 values between 6 and 18 kPa suggests that the diffusion barrier has a constant resistance. The lack of a variable resistance was further supported by sustained increases and decreases in nodule activities in response to changes in pO2 in the range of 6 to 20 kPa O2. When pO2 was increased above 20 kPa, C2H2 reduction and CO2 evolution continually declined with time. These results confirm that the diffusion barrier in nodules of M. gale is not variable in response to changes in pO2. The effect of temperature was examined at 8 and 20 kPa O2. Rates of C2H2 reduction and CO2 evolution increased with increasing temperature from 10 to 30[deg]C at both pO2 values. These results indicate that the diffusion resistance of the barrier changes as temperature changes, with the resistance decreasing as temperature increases.

Purification of Hemoglobin from the Actinorhizal Root Nodules of Myrica gale L.

Hemoglobins are generally absent or present in low concentrations in the nodules of actinorhizal plants. An exception is Casuarina, where a hemoglobin occurs at relatively high concentration. However, this plant is unique in that Frankia, the microsymbiont, lacks the vesicles that are normally the site of nitrogen fixation. The present paper shows that a hemoglobin also occurs at high concentrations in Myrica gale L., an actinorhizal plant in which Frankia does form vesicles. Hemoglobin was extracted from root nodules under anaerobic conditions using a buffer containing CO, detergent, and a reducing agent. Carboxyhemoglobin was purified using gel filtration followed by aerobic ion-exchange chromatography. The optical absorption spectra of the oxy-, deoxy-, and carboxyhemoglobins were similar to those of other hemoglobins. The molecular mass of the native hemoglobin estimated by gel filtration was 38,500 D. The molecular mass of the subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 16,200 D, consistent with the mass of other hemoglobin subunits. Thus, the native hemoglobin is probably a dimer.

Factors Affecting the Acetylene to 15N2 Conversion Ratio in Root Nodules of Myrica gale L.

When nodules of actinorhizal plants are exposed to acetylene, there is often an initial peak rate of acetylene reduction followed by a decline and a partial recovery. Treatment of hydroponically grown Myrica gale L. with water deficiency or dark stress increased the magnitude of the acetylene-induced decline and decreased the extent of the recovery. When N2 fixation was measured with 15N2 in unstressed plants, the ratio of acetylene reduction (peak) to N2 fixation prior to acetylene exposure was 3.73 [plus or minus] 0.14 (mean [plus or minus] SE). This value does not differ significantly (P < 0.05) from the theoretical minimum value of 4.0. In water-stressed plants the conversion ratio for the peak rate was greater (4.32 [plus or minus] 0.10) and in dark-stressed plants it was lower (2.54 [plus or minus] 0.33) than 4.0. The conversion ratio for the recovered rate of acetylene reduction was much lower than 4.0 in all cases, with mean values ranging from 1.16 to 2.60. We conclude that the peak rate of acetylene reduction provides the most reliable estimate of N2 fixation. The recovered rate of acetylene reduction consistently underestimates N2 fixation, sometimes severely, and thus measurements of acetylene reduction made in closed systems also underestimate N2 fixation to varying degrees.

Factors Affecting the Acetylene-Induced Decline during Nitrogenase Assays in Root Nodules of Myrica gale L.

Our goal was to determine why the rate of acetylene reduction by nodules of actinorhizal plants declines after an initial peak value. The decline was eliminated by pretreatment with argon, indicating that the decline is initiated by cessation of ammonia synthesis. When O(2) concentration was decreased during the decline, the rate of acetylene reduction increased. This shows that during the decline there is either O(2) toxicity or competition between respiration and nitrogenase for reductant. The decline was not eliminated when uptake hydrogenase was inactivated by pretreatment with acetylene, showing that cessation of H(2) oxidation is not the primary cause of the decline. The effects of a variety of other treatments on the decline were also studied. Overall, we conclude that the cessation of ammonia formation is the primary cause of the acetylene-induced decline. We hypothesize that the supply of reductant for nitrogenase depends on amino acids that are depleted following cessation of ammonia formation. We also conclude that the initial peak rate of acetylene reduction provides the best measure of nitrogenase activity.

Time course of acetylene reduction in nodules of five actinorhizal genera.

The rate of acetylene reduction was measured as a function of time after addition of 10% acetylene in Alnus, Casuarina, Ceanothus, Datisca, and Myrica. The maximum rate occurred after 45 to 60 seconds and was maintained for an additional 0.5 to 4 minutes before a decline in rate to 30 to 90% of the maximum. The rate then recovered to a value of 63 to 98% of the maximum. Removal of the shoot and lower roots did not affect nodule activity.

Oxygen protection of nitrogenase in Frankia sp. HFPArI3.

O2 protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O2 uptake and acetylene reduction rates in response to substrate concentration. O2 of NH4+-grown cells showed an apparent KmO2 of approximately 1 microM O2. In N2-fixing cultures a second Km O2 of about 215 microM O2 was observed. Thus, respiration remained unsaturated by O2 at air-saturation levels. In vivo, the apparent Km for acetylene was more than 10-fold greater than reported in vitro values. These data were interpreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.

Hemoglobin in a nonleguminous plant, parasponia: possible genetic origin and function in nitrogen fixation.

A dimeric hemoglobin was purified from nitrogen-fixing root nodules formed by association of Rhizobium with a nonleguminous plant, Parasponia. The oxygen dissociation rate constant is probably sufficiently high to allow Parasponia hemoglobin to function in a fashion similar to that of leghemoglobin, by oxygen buffering and transport during symbiotic nitrogen fixation. The identification of hemoglobin in a nonlegume raises important questions about the evolution of plant hemoglobin genes.

Simultaneous measurement of acetylene reduction and respiratory gas exchange of attached root nodules.

A method was developed for the simultaneous measurement of acetylene reduction, carbon dioxide evolution and oxygen uptake by individual root nodules of intact nitrogen-fixing plants (Alnus rubra Bong.). The nodules were enclosed in a temperature-controlled leak-tight cuvette. Assay gas mixtures were passed through the cuvette at a constant, known flow rate and gas exchange was measured by the difference between inlet and outlet gas compositions. Gas concentrations were assayed by a combination of an automated gas chromatograph and a programmable electronic integrator. Carbon dioxide and ethylene evolution were determined with a coefficient of variation which was less than 2%, whereas the coefficient of variation for oxygen uptake measurements was less than 5%. Nodules subjected to repeated removal from and reinsertion into the cuvette and to long exposures of 10% v/v acetylene showed no irreversible decline in respiration or acetylene reduction. This system offers long-term stability and freedom from disturbance artifacts plus the ability to monitor continuously, rapidly and specifically the changes in root nodule activity caused by environmental perturbation.

Diffusion Limitation of Oxygen Uptake and Nitrogenase Activity in the Root Nodules of Parasponia rigida Merr. and Perry.

Parasponia is the first non-legume genus proven to form nitrogen-fixing root nodules induced by rhizobia. Infiltration with India ink demonstrated that intercellular air spaces are lacking in the inner layers of the nodule cortex. Oxygen must diffuse through these layers to reach the cells containing the rhizobia, and it was calculated that most of the gradient in O(2) partial pressure between the atmosphere and rhizobia occurs at the inner cortex. This was confirmed by O(2) microelectrode measurements which showed that the O(2) partial pressure was much lower in the zone of infected cells than in the cortex. Measurements of nitrogenase activity and O(2) uptake as a function of temperature and partial pressure of O(2) were consistent with diffusion limitation of O(2) uptake by the inner cortex. In spite of the presumed absence of leghemoglobin in nodules of Parasponia rigida Merr. and Perry, energy usage for nitrogen fixation was similar to that in legume nodules. The results demonstrate that O(2) regulation in legume and Parasponia nodules is very similar and differs from O(2) regulation in actionorhizal nodules.

Dinitrogen Fixation by Cultures of Frankia sp CpIl Demonstrated by N(2) Incorporation.

The filamentous bacterium Frankia of the Actinomycetales, isolated from the nitrogen-fixing root nodules of certain woody plants, has shown nitrogenase activity in culture, using the acetylene reduction method. In the present work, nitrogenase activity in pure cultures of Frankia sp. CpIl is confirmed using mass spectrometric measurements of (15)N(2) incorporation. After addition of carrier NH(4) (+) to digested cultures, those exposed to (15)N(2) (25 atom%) had a (15)N content of 3.16 atom% compared to 0.354 atom% (15)N in the controls.

Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in Frankia sp. CpI1.

Vesicle formation and acetylene reduction (nitrogenase activity) were observed when washed hyphae from cultures of Frankia sp. CpI1 were transferred to a nitrogen-free medium containing ethylenediaminetetraacetic acid and succinate. Succinate could be replaced by malate or fumarate, but not other carbon sources. Maximum acetylene reduction and vesicle numbers were observed at a pH of 6.0-6.5, at 25-30 degrees Centigrade, and at atmos pheric Po2 or somewhat less (5-20 kPa). Addition of 1 mM NH4Cl almost completely inhibited vesicle formation and acetylene-reducing activity, but did not immediately inhibit such reducing activity by cultures with preexisting vesicles. Acetylene-reducing activity was never observed in the absence of vesicle formation.

Energy requirement for nitrogen fixation in actinorhizal and legume root nodules.

The ratio of respiration to nitrogenase activity was measured in five species of actinorhizal root nodules and eight species of legume nodules. The two types of nodules could not be distinguished on the basis of this ratio; this evidence thus indicates that the energy cost of nitrogen fixation is similar for both.

Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum.

Sorghum and corn breeding lines were grown in soil in field and greenhouse experiments with and without an inoculum of N2-fixing in Spirillum strains from Brazil. Estimated rates of N2 fixation associated with field-grown corn and sorghum plants were less than 4 g of N2/ha per day. The mean estimated N2-fixation rates determined on segments of roots from corn inoculated with Spirillum and grown in the greenhouse at 24 to 27 degrees C were 15 g of N2/ha per day (16 inbreds), 25 g of N2/ha per day (six hybrids), and 165 g of N2/ha per day for one hybird which was heavily inoculated. The corresponding mean rates determined from measurements of in situ cultures of the same series of corn plants (i.e., 16 inbreds, six hybrids, and one heavily inoculated hybrid) were 0.4, 2.3, and 1.1 g of N2/ha per day, respectively. Lower rates of C2H2 reduction were associated with control corn cultures which had been treated with autoclaved Spirillum than with cultures inoculated with live Spirillum. No C2H2 reduction was detected in plant cultures treated with ammonium nitrate. Numbers of nitrogen-fixing bacteria on excised roots of corn plants increased an average of about 30-fold during an overnight preincubation period, and as a result acetylene reduction assays of root samples after preincubation failed to serve as a valid basis for estimating N2 fixation by corn in pot cultures. Plants grown without added nitrogen either with or without inoculum exhibited severe symptoms of nitrogen deficiency and in most cases produced significantly less dry weight than those supplied with fixed nitrogen. Although substantial rates of C2H2 reduction by excised corn roots were observed after preincubation under limited oxygen, the yield and nitrogen content of inoculated plants and the C2H2-reduction rates by inoculated pot cultures of corn, in situ, provided no evidence of appreciable N2 fixation.

Measurement of oxygen partial pressure within soybean nodules by oxygen microelectrodes.

The internal pO2 of soybean (Glycine max Merr.) nodules was measured with oxygen microelectrodes. For nodules in air at 23°, the pO2 decreased sharply across the nodule cortex, and was too low to measure in the central tissue. At 1° in 1.0 atm O2, the pO2 in the central tissue was measurable, and was approximately uniform from the center to the edge of the central tissue. This uniformity was probably due to the intercellular air spaces of the central tissue, since infiltrating the spaces with water substantially decreased the pO2 in the central tissue. The results strongly suggest that most of the resistance to O2 diffusion into the nodule occurs within the cortex.

Respiration and oxygen transport in soybean nodules.

The respiration rate of individual soybean (Glycine max Merr.) nodules was measured as a function of pO2 and temperature. At 23°, as the pO2 was increased from 0.1 to 0.9 atm, there was a linear increase in respiration rate. At 13°, similar results were obtained, except that there was an abrupt saturation of respiration at approximately 0.5 atm pO2. When measurements were made on the same nodule, the rate of increase in respiration with pO2 was the same at 13° and 23°. Additional results were that 5% CO in the gas phase had no effect on respiration, except for a small decrease in the pO2 at which respiration became saturated. Also, nodules still attached to the soybean root displayed the same respiratory behavior as detached nodules. A model for oxygen transport in the nodule is presented which explains these results quantitatively. The essence of the model is that the respiration rate of the central tissue of the nodule is almost entirely determined by the rate of oxygen diffusion to the respiratory enzymes. Evidence is given that the nodule cortex is the site of almost all of the resistance to oxygen diffusion within the nodule.