Cultivar Effects on Hydrogen Evolution by Rhizobium leguminosarum ' Received

The effect of host plant cultivar on H2 evolution by root nodules was examined in symbioses between Pisum sativum L. and selected strains of Rhizobium leguminosarum. Hydrogen evolution from root nodules containing Rhizobium represents the sum of H2 produced by the nitrogenase enzyme complex and H2 oxidized by any uptake hydrogenase present in those bacterial cells. Relative efficiency (RE) calculated as RE 1 (H2 evolved in air/C2H2 reduced) did not vary significantly among 'Feltham First,"Alaska,' and 'JI1205' peas inoculated with R. kguminosarum strain 300, which lacks uptake hydrogenase activity (Hup-). That observation suggests that the three host cultivars had no effect on H2 production by nitrogenase. However, RE of strain 128C53 was significantly (P c 0.05) greater in symbiosis with cultivar JI1205 than in root nodules of Feltham First. At a similar rate of C2H2 reduction on a whole-plant basis, nearly 24 times more H2 was evolved from the Feltham First/128C53 symbiosis than from the J11205/128C53 association. Root nodules from the Alaska/ 128C53 symbiosis had an intermediate RE over the entire study period, which extended from 21 to 36 days after planting. Direct assays of uptake hydrogenase by two methods showed significant (P c 0.05) host cultivar effects on H2 uptake capacity of both strain 128C53 and the genetically related strain 3960. The 'H2 incorporation assay showed that strains 128C53 and 3960 in symbiosis with Feltham First had about 10% of the uptake hydrogenase activity measured in root nodules of Alaska or JI1205. These data are the first demonstration of significant host plant effects on rhizobial uptake hydrogenase in a single plant species. Hydrogen evolution from leguminous root nodules represents the sum of H2 formation by nitrogenase and H2 oxidation by any uptake hydrogenase present within the Rhizobium cells. No plant enzyme system which metabolizes H2 has been identified in root nodule cells. Bulen and LeComte (5) first demonstrated an ATPdependent formation of H2 by a bacterial nitrogenase enzyme complex, and it is now known that, various biochemical factors can influence such H2 formation (19). Hydrogen uptake by leguminous root nodules was first reported in the pea system in 1941 (25). That work was confirmed by Dixon (11), who localized uptake hydrogenase activity in pea root nodule bacteroids (12) and found it to be similar to Azotobacter hydrogenase in all respects examined (13). ' Supported by a Fulbright Fellowship, a postgraduate fellowship from the Natural Sciences and Engineering Research Council of Canada, and the United States National Science Foundation Grant PCM 8217187. 2On leave from the Department of Microbiology, Estacion Experimental del Zaidin, C.S.I.C. Granada, Spain. 3Present address: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6. Many data suggest that at least 25% of the reductant used by nitrogenase is allocated to protons for H2 formation, while the remaining fraction of reductant is used to convert N2 to NH3 (6). The EAC4 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). In the presence of saturating levels of C2H2, all reductant is used to produce C2H4 and no H2 formation is detected. Schubert and Evans (28) defined the relative efficiency of N2 fixation as RE = 1 (H2 evolved in air/C2H2 reduced) and showed that RE varied greatly among different Rhizobium-legume symbioses. Such RE values depend on the EAC of nitrogenase and on the capacity of the rhizobial cells to recover H2 by a separate uptake hydrogenase. Thus, in the absence ofuptake hydrogenase activity (Hupphenotype), RE presumably is a measure of apparent EAC. Previous reports indicate that both plant and bacterial factors can affect H2 evolution from leguminous root nodules. Dixon (13) showed that R leguminosarum strain ONA 311 expressed strongly Hup+, slightly Hup+, or Hupphenotypes on Pisum sativum, Vicia bengalensis, and V. faba, respectively. Similar reversals in uptake hydrogenase phenotype have been observed for two R. japonicum strains associated with Vigna unguiculata and Glycine max (20). Both strains were Hup+ on three cowpea cultivars and Hupon three soybean cultivars. Host plant effects on apparent rhizobial EAC have been indicated by the observation that lengthening the normal dark period for P. sativum and Trifolium subterraneum increased RE of HupRhizobium symbionts (15). Host plant age and long-term environmental factors such as availability of combined nitrogen, irradiance, and CO2 level also can affect apparent EAC (16). These latter findings are consistent with a previous study which concluded that both EAC and uptake hydrogenase activity of a Hup+ rhizobial strain were affected by irradiance treatment of the host pea plant (3). Effects of bacterial genotype were evident from the report that only two of four R. leguminosarum strains tested were Hup+ in pea root nodules (13). More extensive, subsequent surveys reported that six of 15 R. leguminosarum strains tested on 'Austrian Winter' peas had uptake hydrogenase activity (27) and that only 14 of 79 effective R. leguminosarum isolates tested on 'Homesteader' peas had significant uptake hydrogenase activity (24). No workers studying R. leguminosarum have reported uptake hydrogenase activity which was sufficient to recover all H2 evolved from nitrogenase. One Rhizobium strain of particular interest, which deserves further study, is R. leguminosarum 128C53. This strain has been variously described in the literature as exhibiting Hup+ (3, 23, 24, 26) and Hup(14) phenotypes on P. sativum. Each of the groups studying strain 128C53 used a different pea cultivar as a host 4 Abbreviations: EAC, electron allocation coefficient of nitrogenase; RE, relative efficiency of nitrogenase; Hup+, hydrogen-uptake positive; Hup-, hydrogen-uptake minus. 1011 www.plantphysiol.org on July 19, 2017 Published by Downloaded from Copyright © 1983 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 72, 1983 plant, and it is impossible to evaluate the apparently conflicting reports. The genetic determinants responsible for uptake hydrogenase activity in 128C53 have been transferred by conjugation to other strains (4) and were part of a recombinant plasmid which was used to construct a Hup strain that was symbiotically superior to both parent strains (8). If the genetic determinants responsible for the Hup+ phenotype are influenced by the host plant genotype, as the one contradictory report (14) seems to imply, then it is important to verify and examine that fact. The immediate goal of our study was to test whether strain 128C53 expressed Hup+ and Hupphenotypes on different cultivars of P. sativum. A clear demonstration of such a phenomenon could form the basis for future physiological and genetic studies of host plant factors affecting H2 evolution by Rhizobium. Such studies are not feasible in systems where different species of legumes are required to elicit varying phenotypes in symbiotic rhizobial cells. MATERIALS AND METHODS Plants. Pisum sativum L. cv Alaska (Burpee Seed, Riverside, CA), Feltham First (Finney Lock, Witham, Essex), and JI1205 (courtesy of B. Snoad, John Innes Institute, Norwich, Norfolk) were grown in controlled environmental chambers under a 16/8 h light/dark cycle, 21/15°C, 50%o RH, and a photosynthetic photon flux density (400-700 nm) of 650 ,uE* m-2 s. Seeds were sterilized, germinated, and selected for uniformity before planting in sterile 750-ml Leonard jars containing vermiculite (10). All plants were provided with sterile N-free nutrient solution (10) and a single strain of Rhizobium in the Leonard jar. Bacteria. Rhizobium leguminosarum strains 128C53, 300, and 3960 were used. Strains 128C53 and 300 originally were isolated from the field and show Hup+ and Hupphenotypes, respectively, on Alaska pea (4). Strain 3960 was constructed by transferring determinants for nodulation, N2 fixation, and Hup+ from 128C53 through conjugation to 300 (8). Bacteria were maintained asymbiotically on LMB medium (22). Physiological Assays. Nitrogenase activties were measured on detached root systems excised at the cotyledonary node. Hydrogen evolution in air and C2H2-dependent C2H4 production (C2H2