John D. Tjepkema

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.

Nitrogen fixation and respiration by root nodules of Alnus rubra Bong.: effects of temperature and oxygen concentration

SummaryUsing a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO2=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.

Total and CO-reactive heme content of actinorhizal nodules and the roots of some non-nodulated plants

The concentration of total and CO-reactive heme was measured in actinorhizal nodules from six different genera. This gave the upper limit to hemoglobin concentration in these nodules. Quantitative extraction of CO-reactive heme was achieved under anaerobic conditions in a buffer equilibrated with CO and containing Triton X-100. The concentration of CO-reactive heme in nodules of Casuarina and Myrica was approximately half of that found in legume nodules, whereas in Comptonia, Alnus and Ceanothus the concentrations of heme were about 10 times lower than in legume nodules. There was no detectable CO-reactive heme in Datisca nodules, but low concentrations were detected in roots of all non-nodulating plants examined, including Zea mays. Difference spectra of CO treated minus dithionite-reduced extracts displayed similar wavelengths of maximal and minimal light absorption for all extracts, and were consistent with those of a hemoglobin. The concentration of CO-reactive heme was not correlated to the degree to which CO inhibited nitrogenase activity nor was it affected by reducing the oxygen concentration in the rooting zone. However, there was a positive correlation between heme concentration and suberization or lignification of the walls of infected host cells. These observations demonstrate that, unlike legume nodules, high concentrations of heme or hemoglobin are not needed for active nitrogen fixation in most actinorhizal nodules. Nonetheless, a significant amount of CO-reactive heme is found in the nodules of Alnus, Comptonia, and Ceanothus, and in the roots of Zea mays. The identity and function of this heme is unknown.

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 Km O2 of approximately 1μM O2. In N2-fixing cultures a second Km O2 of about 215 μM 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 inter oreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.

Hemoglobin in Frankia, a Nitrogen-Fixing Actinomycete

ABSTRACT Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s−1, and the oxygen association rate constant was 206 μM−1 s−1.

Natural abundance of 15N in actinorhizal plants and nodules

Substantial enrichment of some plant parts in 15N relative to the rest of the plant is unusual, but is found in the nitrogen-fixing nodules of many legumes. A range of actinorhizal plants was surveyed to determine whether the nodules of any of them are also substantially enriched in 15N. The nonlegume Parasponia, nodulated by a rhizobium, was also included. Four of the actinorhizal genera and Parasponia were grown in N-free culture, and three actinorhizal genera were collected from the field. Nodules of Parasponia, Casuarina and Alnus were15N enriched relative to other plant parts, but only Parasponia approached the degree of enrichment found in some legume nodules. The nodules of Datisca, Myrica, Elaeagnus, Shepherdia, and Coriaria were depleted in 15N. Thus many actinorhizal nodules are depleted in 15N compared to other plant parts and enrichment is modest when it does occur. Whole plant 15N content (δ15N) in four actinorhizal plants and Parasponia showed a relatively narrow range of −1.41 to −1.90. Hence regardless of the degree of nodule enrichment or depletion, whole plant 15N content appears to vary little in plants grown in N-free culture.

The wall of the infected cell may be the major diffusion barrier in nodules of Myrica galE L

Abstract Nitrogenase activity (C 2 H 2 reduction) by single- or two-lobed nodules of Myrica gale L. with long nodule roots was measured at 6 and 8 kPa O 2 in a flow-through system after various treatments. Neither excision of nodule roots nor covering the cut surface with silicone grease after nodule root removal had a significant effect on C 2 H 2 reduction, while covering the entire nodule with grease eliminated C 2 H 2 reduction. Likewise, covering the nodule lobe while leaving nodule roots uncovered had little effect on C 2 H 2 reduction. These results suggest that the diffusion resistance to gases is low at the surface of the nodule lobe and the nodule roots. Nodule slices ( ca 100 μm thick) retained high C 2 H 2 reduction rates which were O 2 -limited up to 18 kPa, indicating that the diffusion barrier is within the slices. The O 2 gradient from the gas phase to the infected cells in the slices was calculated to be ca 0.65 kPa out of a total gradient of 20 kPa. The addition of 2 kPa CO had little effect on nodule respiration (CO 2 evolution), suggesting that the diffusion barrier is outside the cytoplasm of the infected cells. Thus the major diffusion barrier may be in the wall of the infected cells.

Gas diffusion pathway in nodules ofCasuarina cunninghamiana

The gas diffusion pathway in nodules was traced by vacuum infiltration with India ink or aniline blue and by electron microscopy. India ink infiltration was observed in the outermost and the innermost cortex in sliced nodules, but not in intact nodules. With aniline blue infiltration, it was observed that intercellular air spaces in the outermost and the innermost cortex were connected to those in nodule roots. No air spaces were in contact with walls of infected cells, although intercellular air spaces existed in some groups of uninfected cells within the infected zone. Infiltration with either India ink or aniline blue could not be observed in the infected zone in essentially all cases. Thus it is suggested that the discontinuity of the intercellular air spaces represents a major resistance to O2 diffusion in nodules ofCasuarina cunninghamiana.

Respiration and nitrogenase activity in nodules ofCasuarina cunninghamiana and cultures ofFrankia sp. HFP020203: Effects of temperature and partial pressure of O2

The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant.Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction.Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO2 (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O2 (vesicles absent) or 20 kPa O2 (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO2 at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO2, but the Q10 was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O2-saturated at low pO2 values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.

Oxygen, Hemoglobins, and Energy Usage in Actinorhizal Nodules

The nitrogen-fixing symbioses between higher plants and Rhizobium, Frankia, and cyanobacteria differ in a number of important ways. Identification of the nature and range of these differences may help in understanding how these systems function and the possible means by which new symbioses may be created by genetic engineering. My own recent studies have emphasized comparisons between actinorhizal plants where Frankia is the nitrogen-fixing symbiont, and legumes and Parasponia where Rhizobium is the symbiont