Regulation of 02 Concentration in Soybean Nodules Observed by in Situ Spectroscopic Measurement of Leghemoglobin Oxygenation

A flber 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 P02 of 20 kilopascals, leghemoglobin was 36.6 ± 5.4% oxygenated, a value estimated to represent an infected cell 02 concentration of 21.5 nanomolar. Increasing the external P02 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 02, the rates of H2 and CO2 evolution were similar to those at 20 kilopascals. Since respiration had not increased, the results support the proposal that nodules adapt to increased external P02 by regulating their resistance to O2 diffusion. N2 fixation by Rhizobium bacteria in symbiosis with legumes requires a large supply of reductant and ATP from aerobic respiration, yet nitrogenase, the N2-fixing enzyme, is irreversibly inhibited by even low levels of 02 (13). Therefore, it has been proposed that the legume root nodule must be capable of regulating its 0.2 (14). Tjepkema and Yocum (18) provided evidence that a barrier to 02 diffusion is present in the nodule cortex surrounding the infected cells. It has been suggested (16-18) that this barrier 1 Supported by grants from the Natural Sciences and Engineering Research Council of Canada (D. B. L., D. T. C.), the Department of National Defence (R. H. P.), and the Queen's University Advisory Research Council (B. J. K.). B. J. K. and K. B. W. acknowledge support from a Natural Sciences and Engineering Research Council of Canada Post-Graduate Scholarship and a Commonwealth Scholarship, respectively. 2 Abbreviations: Oi, concentration (nM) of free 02 in the infected cells of the nodule; Oe, 02 partial pressure (kPa) outside the nodule; Lb, ferrous leghemoglobin; Y, fractional oxygenation of Lb as percent; A,md, nodule absorbance (642 nm) at Qe = 0 kPa (fully deoxygenated Lb); Aoxy nodule absorbance (642 nm) at °e = 100 kPa (fully oxygenated Lb); A,, nodule absorbance at time t; k, rate constant for dissociation of 02 from Lb; k2, rate constant for binding of 02 with Lb. consists of a continuous layer of cells with water-filled intercellular spaces. Gas exchange experiments have indicated that the barrier is physiologically regulated (7, 14, 21, 23), presumably through changes in thickness of the water-filled layer (8, 15). Witty et al. (24), using °2 microelectrodes, provided direct evidence that Oi is regulated in response to changes in °e; however, this technique was invasive and was not sufficiently sensitive to provide an estimate of O0 under ambient conditions. An aim of the present study was to provide evidence for regulation of O0 using a noninvasive technique of much higher sensitivity. Upon increasing 0e, an immediate, temporary decline in nitrogenase activity is observed (7, 21), which cannot be accounted for by destruction and de novo synthesis of nitrogenase (7). Either of two mechanisms could account for this decline: (a) an immediate, overcompensating increase in the diffusion resistance may occur, causing a rapid decrease in Oi and limiting respiration to support nitrogenase activity; or (b) a more gradual increase in the diffusion resistance may occur, allowing O0 to undergo a transient increase, which in turn causes reversible inhibition of nitrogenase. A further aim of this study was to investigate these two hypotheses by direct measurement of O0. The infected cells of the nodule contain Lb, a myoglobin-like protein that binds reversibly with 02 with high affinity. Lb serves as a carrier of bound 02 to the bacterial electron transport chain while maintaining a low O0 (3, 22). Spectrophotometric measurement of oxygenation-dependent changes in the optical absorbance of Lb is a highly sensitive, noninvasive means for monitoring O0. However, it is difficult to obtain meaningful absorbance spectra from intact soybean nodules due to their thickness (about 5 mm), which results in high light absorbance and scattering, and their spherical shape and heterogeneous structure, which result in preferential transmission of light through the non-Lbcontaining cortex (4, 9). Therefore, in most studies Lb oxygenation has been measured in nodule slices (2, 4). Although Klucas et al. (9) measured Lb oxygenation in intact, attached nodules of sweet clover, they did not study the regulation of Oi under changing environmental conditions. In the present study, an improved spectroscopic technique is described, which was used to measure Oi in intact, attached soybean nodules that were flattened during development by growth in narrow glass-walled cuvettes. This technique was used to obtain evidence for regulation of Oi with increases in °e and to determine the speed with which this regulation occurs. 296 www.plantphysiol.org on July 22, 2017 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. REGULATION OF Or CONCENTRATION IN SOYBEAN NODULES MATERIALS AND METHODS Plant Material and Growth and Experimental Conditions. Seeds of soybean (Glycine max L. Merr. cv Harosoy 63) were inoculated with Bradyrhizobium japonicum strain USDA 16 and grown as described previously (7) in a growth chamber with 16 h photoperiod, 25°C day, and 20°C night. At age 10 d, seedlings were placed with the root systems enclosed in cuvettes as shown in Figure 1. The cuvettes were then placed in 10 cm diameter plastic pots, covered with moist vermiculite, and returned to the growth cabinet. Plants were used for experiments at age 30 to 32 d, and were kept in a greenhouse at the site of the spectrophotometer (Royal Military College of Canada) on the day of the experiments. A total period of 5 to 10 min elapsed between removal of a plant from the greenhouse and beginning of the measurements. The assays were carried out at room temperature (approximately 22°C). The shoots were maintained in darkness during the experiments because of the need to prevent stray light from reaching the spectrophotometer. However, this is unlikely to have affected nitrogenase activity since soybeans do not show a pronounced diurnal variation in nitrogenase activity (20), and since the rates of H2 and CO, evolution were stable. Gas Exchange and Spectrophotometric Measurements. Each plant chosen had a relatively homogeneous population of nodules that were flattened against the glass walls of the cuvette. For an experiment, a cuvette (Fig. 1) was removed from its growth pot, sealed with silicon rubber (Dow Corning 3110 RTV cured with RTV 4 catalyst), and equipped with 18.5 gauge hypodermic syringe needles for connection to an open circuit gas exchange system to monitor H2 and CO, evolution, as described previously (7). For spectrophotometric measurements, quartz fiber optic probes were placed on either side of a typical nodule. The illuminating probe (a fiber bundle of 2 mm diameter) was directed to a xenon arc light source equipped with a lens for focusing and a band pass filter to reduce illumination in the red region. The collecting probe (a single fiber of 1.5 mm diameter) was directed to a Tractor Northern TN1710 spectrophotometer. The complete spectrophotometric system is described elsewhere (12). The lag time between the spectrophotometric and gas exchange measurements was approximately 1 min. Experimental Procedure and Calculations. During each experiment the absorbance spectrum of a selected nodule was monitored over the range of 500 to 650 nm. Each complete scan lasted 40 to 80 ms, and at selected intervals of 30 s or more, 200 sequential scans were averaged to obtain a mean spectrum which covered 8 to 16 s. Meanwhile, the nodule cuvette was flushed with 20 kPa 02 until a stable spectrum and steady state conditions of CO2 and H2 evolution were attained (generally within 10 min). Then °e was increased to 25 kPa and the absorbance spectrum was monitored until new, steady state rates of gas exchange were attained (after 30-45 min). Finally, the nodules were exposed for 5 to 10 min to 0 and then to 100 kPa 02 to obtain the spectra for the fully deoxygenated and fully oxygenated forms of Lb, respectively. Figure 2 illustrates typical, steady state absorbance spectra obtained sequentially for a single nodule under 0, 20, and 100 kPa O. Fractional oxygenation of Lb (Y), or percent of Lb bound to O, was calculated as by Appleby (1): Ared x 100 Ared A(x%,