Effect of the Host Legume on Acetylene Reduction and Hydrogen

The relative efficlency (RE) ofN2 fxation (RE -1 H2 evolved In airl /[acetylene reducedi) was Investigated In a Rhizobiwm strain lackng uptake hy se actMity (Hup-). Vadation in RE of such strains presmably reflects changes In the electron allocation coefficient of nitrogenase. By artificially exteming the normal dark period of 24day-old Pism sativwn L cv 'Alaska' inoculated with the HupR. kgwninsawn strain 3740, reproducible changes in RE were obtained. The RE showed no change during a normal S-hour n4iht, but a signficant increase in RE was measured after 20 hours In the dark. Upon retrning to the normal 550 microEinsteins per square meter per second light treatment, RE declined to previous levels within 2 hours. If, after the 20-hour dark treatment, plants were returned to 90 or 190 microEinsteins per square meter per second or maintaied In the dark, RE did not decline signifantly. The RE varied inversely with changes in soluble sugar content of root nodules. A silar pattern of changes In RE during an extended dark period and subsequent light treatment was measured In 28-day-old Alaska peas and In the HupR. trifodii strain 162X99 In symbiosis with Trufoliwm subtenanewm L. cv 'Woogenellup.' These results suggest that Rhizobiwm cells may produce short-term alterations in the electron allocation coefficient of nitrogenase in response to physiological changes. The observed changes in the bacterial RE favored N2 reduction over proton reduction when soluble sugars provided by the host plant declined. Symbiotic N2 fixation in legumes is a function of the Rhizobium nitrogenase enzyme complex. Nitrogenase normally catalyzes a concurrent reduction of N2 to NH3 and protons to H2, but under a 90% air, 10% acetylene atmosphere, essentially all electrons are transferred to acetylene (6). EAC3 is a convenient expression that reflects the partitioning of reductant among protons and alternative substrates such as N2 or C2H2 (6): EAC = (exogenous substrate reduced)/(H30' reduced + exogenous substrate reduced). The view that N2 is the primary substrate of nitrogenase led Schubert and Evans (19) to defme RE as a parameter for evaluating the significance of H2 production by nitrogenase: RE = 1 (H2 evolved in air)/(C2H2 reduced). The RE in vivo depends on the EAC of nitrogenase and the capacity of the Rhizobium to recover H2 by a separate uptake hydrogenase (19). Thus, in the absence of 'Supported by a postgraduate fellowship from the Natural Sciences and Engineering Research Council of Canada and by United States National Science Foundation Grant No. 78-01146. 2Prewnt address: Department ofBiology, Queen's University, Kingston, Ontario K7L 3N6 Canada. 3Abbreviations: EAC, electron allocation coefficient of nitrogenase; RE, relative efficiency of N2 fLxation; HE, hydrogen evolution in air, AR, acetylene reduction; CER, carbon exchange rate. uptake hydrogenase activity (Hupphenotype), RE presumably is a measure of apparent EAC. The potential agronomic advantages postulated for Rhizobium phenotypes with uptake hydrogenase activity (19) led to studies of various factors that influence RE. Bethlenfalvay and Phillips (2, 3) showed that RE was altered by plant age and irradiance in Pisum sativum L. cv 'Alaska' with the Hup+ R leguminosarum strain 128C53. Using 3H2 incorporation as an assay for uptake hydrogenase, they suggested that changes in RE ofthat host-strain combination resulted from variations in both uptake hydrogenase activity and EAC (4). In other studies of a symbiosis between the Hup+ Rhizobium strain 176A28 and Vigna unguiculata (L.) Walp., where essentially no H2 was measured outside the nodule, plant age had no effect on RE (18). Results with purified nitrogenase showed that the availability of MgATP and other factors influenced EAC (6, 7, 11). If such changes occur in vivo, they could have physiological significance. The present study was conducted to determine whether the RE, and thus presumably the apparent EAC, changed in the HupR leguminosarum strain 3740 (5) on Alaska peas during an extension of the normal dark period. The hypothesis underlying this experiment was that an extended dark treatment could affect carbohydrate supply within the root nodule and possibly alter energy available to the bacterial nitrogenase complex. MATERIALS AND METHODS Biological Materials. Alaska peas (Pisum sativum L.) were grown aseptically in vermiculite with N-free nutrient solution as described by DeJong and Phillips (8), except for the following modifications. Leonard jars were constructed from 1.5-liter wine bottles, and two plants were maintained in each jar. The normal conditions in environmental cabinets were a 16/8 h light/dark, 19/14°C day/night with a 550 ,uE m-2 s-1 (400-700 nm) photosynthetic photon flux density measured 25 cm above the jar surface with the LI-185B quantum sensor (Licor, Inc.). 'Woogenellup' subclover (Trifolium subterraneum L.) was grown under identical conditions with nine seeds in each Leonard jar. When the normal dark period was extended, the customary night temperature was maintained. Upon reillumination, the normal day temperature was established under various irradiance levels. Peas were inoculated with Rhizobium leguminosarum strain 3740; subclover was inoculated with R trifolii strain 162X99 (supplied originally by J. Burton, The Nitragin Co., Milwaukee, WI). Both strains 3740 and 162X99 show a Hupphenotype in the 3H2 incorporation assay (5; S. S. Jue and D. A. Phillips, unpublished data). Bacterial stocks were maintained on agar, usingTY medium (1) and LMB medium (14) for strains 3740 and 162X99, respectively. Physiological Analyses. Detached root systems were analyzed for HE, AR, and H2 evolution in 80% argon, 20% 02. Assays were conducted in 135 ml bottles sealed with rubber septa. Ethylene and H2 were quantified by GC using flame ionization and therm156 www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 1983 American Society of Plant Biologists. All rights reserved. HOST EFFECTS ON RHIZOBIUM NITROGENASE istor detectors, respectively, with columns and conditions described previously (2). The linear nature of measured rates in the excised root systems were verified under all conditions, and the RE was calculated as RE = 1 (HE/AR) (19). HE and AR (or HE and H2 evolution in argon) were measured successively on the same root systems and assay containers were flushed thoroughly with air following the HE determinations. All measurements were completed within 30 min after the roots were excised from the plant. Shoot CER was determined in Plexiglas chambers with an open system gas analysis apparatus (8). Assay chambers were located within the environmental cabinets used to grow the plants. Temperature and light in the assay chamber were maintained at levels identical to those under which the plants were grown. Gas exchange measurements of shoots and roots were obtained separately by covering the vermiculite surface with a Plexiglas lid containing holes for shoots and air lines. Respiration of detached root nodules was measured at 250C with a small portion of root attached. Nodules were placed in 24ml vials, which were sealed with rubber septa and flushed with 312 Abar CO2 in air. The CO2 concentration of samples collected 1 and 4 min after sealing the vials was measured by IRGA analysis. Compositional Analyses. Root nodules were frozen with liquid N2, lyophilized, weighed, and ground. Sugars were determined by an anthrone method (21) separately on materials extracted by hot 80%1o ethanol and those released from the residue by amyloglucosidase. Soluble sugars and starch, respectively, were expressed as glucose equivalents. Free amino acids were measured in the ethanol extracts with a modified ninhydrin technique (17). Leucine in 0.1 M, pH 5.0 citrate buffer was used as the standard. Statistical Analyses. The RE values were constrained between 0 and 1 and did not always show a normal distribution. RE data therefore were transformed according to the equation m = In (RE/1-RE), and the parameter m was used for all statistical analyses. Only untransformed values, however, are reported. Treatment effects within a single harvest were assessed by oneway analysis of variance. If the F test was significant at P c 0.01, pairwise comparisons among treatments at each assay time were carried out using the Tukey-Kramer method for unbalanced conditions (12). Treatment means followed by the same letter were not significantly different at P c 0.01.