Proc. Natl. Acad. Sci. USA Vol. 83, pp. 3811-3815, June 1986 Botany Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules (nitrogen fixation/oxygen toxicity/antioxidant) DAVID A. DALTON*, STERLING A. RUSSELL*, F. J. HANUS*, GARY A. PASCOEt, AND HAROLD J. EVANS* *Laboratory for Nitrogen Fixation Research and tEnvironmental Health Sciences Center, Oregon State University, Corvallis, OR 97331 Contributed by Harold J. Evans, January 27, 1986 ABSTRACT The critical problem of oxygen toxicity for H202 2GSH NADP' nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides. An enzymatic mechanism is a b described for removal of peroxides in root nodules of soybean (Glycine max). The system utilizes ascorbate as an antioxidant 2H20 GSSG NADPH + H+ and glutathione as a reductant to regenerate ascorbate. The enzymes involved are ascorbate peroxidase (ascorbate:hydro- gen-peroxide oxidoreductase, EC 1.11.1.7), dehydroascorbate H202 ASC GSSG NADPH + H+ reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-gluta- c d thione oxidoreductase, EC 1.6.4.2). The reactions are essen- 2H20 DHA 2GSH NADP+ tially the same as those involving scavenging of H202 in chloroplasts. Glutathione peroxidase (glutathione:hydrogen- FIG. 1. Oxidation-reduction pathways involved in scavenging of peroxide oxidoreductase, EC 1.11.1.9) was not detected. Dur- H202. The upper scheme occurs in mammalian cells. The lower ing the course of early nodule development, ascorbate perox- scheme occurs in chloroplasts, cyanobacteria, and soybean nodules. idase and dehydroascorbate reductase activities and total Enzymes: a, glutathione peroxidase; b, glutathione reductase; c, glutathione contents ofnodule extracts increased strikingly and ascorbate peroxidase; d, dehydroascorbate reductase. ASC, ascor- were positively correlated with acetylene reduction rates and bate; DHA, dehydroascorbate. nodule hemoglobin contents. The evidence indicates an impor- tant role of glutathione, ascorbate, ascorbate peroxidase, reaction, essentially the same as that which occurs in the dehydroascorbate reductase, and glutathione reductase as mammalian system described above, that involves gluta- components of a peroxide-scavenging mechanism in soybean thione reductase and NADPH. The enzymes involved are root nodules. ascorbate peroxidase (ascorbate:hydrogen-peroxide oxido- reductase, EC 1.11.1.7), dehydroascorbate reductase (gluta- All aerobic organisms are subject to oxygen toxicity that thione:dehydroascorbate oxidoreductase, EC 1.8.5.1, also results from the formation of reactive intermediates such as called glutathione dehydrogenase), and glutathione reduc- hydrogen peroxide (H202), superoxide radical (02w), and tase. The initial product of the ascorbate peroxidation reac- hydroxyl radical (OH7). One of the main defensive mecha- tion is actually monodehydroascorbate, which spontaneous- nisms against these reactive intermediates is superoxide ly forms dehydroascorbate. Also present in spinach chloro- dismutase (superoxide:superoxide oxidoreductase, EC plasts is an enzyme that catalyzes the direct conversion of 1.15.1.1), an enzyme that is ubiquitous in aerobic organisms monodehydroascorbate back to ascorbate (5). At least some including both eukaryotes and prokaryotes (1, 2). Superoxide of these enzymes are also present in cyanobacteria (6, 7). dismutase catalyzes the conversion of 027 to H202. The Nitrogen-fixing organisms are especially vulnerable to 02 H202 may then be decomposed in the presence of catalase, toxicity because of the extreme sensitivity ofthe nitrogenase which is widespread in plants and animals (1). In addition, proteins to 02 damage. Consequently, nitrogen-fixing orga- many higher animals contain the selenoenzyme glutathione nisms have evolved various strategies to protect nitrogenase peroxidase (glutathione:hydrogen-peroxide oxidoreductase, from 02 damage. These adaptations include respiratory EC 1.11.1.9) which catalyzes the peroxidation of reduced protection, physical protection (slime production, thick cell glutathione (GSH), forming the oxidized disulfide form of walls, cortical diffusion barriers, etc.), an 02-buffering sys- glutathione (GSSG) as a product (3). The supply of GSH is tem such as leghemoglobin, and temporal or spatial separa- in tion of photosynthesis and nitrogen fixation [as in many regenerated an NADPH-dependent reaction catalyzed by cyanobacteria (8)]. glutathione reductase (NADPH:oxidized-glutathione oxido- Part of the 02 sensitivity of these organisms may be reductase, EC 1.6.4.2.; Fig. 1). In mammals, this system is explained by the production ofreactive oxygen intermediates critical in the protection of hemoglobin and some other by reactions associated with nitrogen fixation. For example, proteins from peroxide damage. reactions involving ferredoxin, hemoglobin, and hydrogen- Another type of peroxidase system (Fig. 1) has been ase can generate 027(2, 9). H202 is produced by the reaction identified in chloroplasts (4). The initial peroxidase reaction catalyzed by superoxide dismutase, which is found in both of this system utilizes ascorbate as an antioxidant and free-living Rhizobium and bacteroids (10, 11). Some legume produces dehydroascorbate. Ascorbate is then regenerated nodules also produce H202 via the urate oxidase reaction, in a GSH-dependent reaction catalyzed by dehydroascorbate which converts urate to allantoin (12). This is a key step in the reductase. Finally, the GSSG is reduced back to GSH in a production of ureides, which are the major form in which nitrogen is transported out of nodules in legumes such as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: GSH, reduced glutathione; GSSG, oxidized in accordance with 18 U.S.C. §1734 solely to indicate this fact. glutathione. 3811 Downloaded by guest on September 23, 2021 3812 Botany: Dalton et al. Proc. Natl. Acad. Sci. USA 83 (1986) soybean, snap bean, and cowpea. Although catalase may be min. This problem was found to be due to the inclusion of0.1 present in nodules (13), this enzyme alone may not be mM EDTA in the reaction buffers. When buffers without sufficient to eliminate all the H202, because catalase has a EDTA were used, the lag period was reduced to about 20 sec. high Km for H202 and tends to be restricted largely to Rates were then determined during the linear phase of the peroxisomes (3). Further, catalase also seems poorly suited reaction from 20 to 60 sec. The persistence of this 20-sec lag for disposal of H202 in nitrogen-fixing organisms because the period was probably due to the small amount of EDTA (3.8 catalase reaction produces free 02. nmol in 1.5-ml final volume) introduced into the reaction In this report, we present evidence for the presence of a mixture along with the crude extract. H202-scavenging system in soybean nodules which is similar Dehydroascorbate reductase was measured by the rate of to that identified in chloroplasts. increase in absorbance at 265 nm due to ascorbate formation (19). The assay mixture contained 50 mM KH2PO4/K2HPO4 MATERIALS AND METHODS at pH 7.0, 0.2 mM dehydroascorbate (freshly prepared in N2-saturated buffer), 0.1 mM EDTA, 2.5 mM GSH, and 37.5 Growth of Plants. Soybean seeds [Glycine max (L.) Merr. 1.l of extract in a final volume of 1.5 ml. Correction was made cv. Williams] were planted in 20-cm plastic pots containing for the nonenzymatic reduction rate. The GSH-independent perlite and were inoculated with Bradyrhizobium japonicum rate was negligible. The absorbance of GSSG was corrected USDA 122 DES (14). Plants were grown in a greenhouse with for by multiplying by a factor of 0.98 (19). Glutathione a 16-hr photoperiod and a light intensity of 190 microeinsteins reductase was assayed by measuring the decrease in absorb- per m2 per sec. The day temperature was 270C and the night ance at 340 nm due to NADPH oxidization (20). Reaction temperature was 21'C. Plants were provided daily with a mixtures consisted of0.25 mM GSSG, 0.125 mM NADPH, 50 nitrogen-free nutrient solution consisting of 1.58 mM K2SO4, mM Tricine (pH 7.8), 0.5 mM EDTA, and 50 jul of extract in 2mM MgSO4, 0.57mM KH2PO4, 0.86mM K2HPO4, 12.0mM a final volume of 2.0 ml. Rates were corrected for GSSG-in- CaSO4, 1.0 mM CaCl2, 3.7 ,M H3BO3, 0.72 ,M MnSO4, 0.76 dependent NADPH oxidation. The nonenzymatic rate was ,uM ZnSO4, 0.31 ,M CuSO4, 0.10 ,uM Na2MoO4, 0.17 AM negligible. CoCl2, 0.17 ,uM NiCl2, and 40.4 ,M sodium ferric All reported values for enzyme activities are the means of ethylenediaminedi(o-hydroxyphenyl)acetate. Excess nutri- determinations on three separate extracts for each sampling ent solution drained through holes in the bottom of each pot. date. Each extract was prepared from the nodules of three Pots were flushed with water twice each week to prevent pots of plants. accumulation of minerals. After germination, the plants were Other Assays. GSH and GSSG contents of cytosol extracts thinned to five per pot. Samples of nodules were collected at were determined by HPLC analysis of the 2,4-dinitrophenyl each sampling date from each of three replicate groups of derivatives of 3-carboxymethyl-GSH and -GSSG (21). plants. Each replicate group consisted of three pots of plants. Ascorbate content of nodule extracts was determined by Nodules were collected only from the upper 5 cm of the main HPLC analysis using a Beckman Gradient LC model 334 with tap root. guard and analytical columns of 5-,um RP-18 (Brownlee Extraction Procedures. Nodules were harvested at various Laboratories, Santa Clara, CA; ref. 22). Separate extractions ages as indicated in Figs. 2-5 and ground at 4°C by mortar and were made for glutathione and ascorbate at each sampling pestle with polyvinylpolypyrrolidone (25% by weight) and 4 date from nodule samples from each ofthree groups of plants. volumes ofbuffer consisting of 50mM KH2PO4/K2HPO4 and Triplicate analyses were performed on each extract.