Alfalfa Root Nodule Carbon Dioxide Fixation

Plant Physiol. (1983) 72, 469-473 0032-0889/83/72/0473/05/$00.50/0 Alfalfa Root Nodule Carbon Dioxide Fixation1 2 I. ASSOCIATION WITH NITROGEN FIXATION AND INCORPORATION INTO AMINO ACIDS Received for publication December 27, 1982 and in revised form February 22, 1983 CARROLL P. VANCE, SUSAN STADE, AND CARL A. MAXWELL United States Department of Agriculture, Science and Education Administration, Agricultural Research Service, and the Department of Agronomy and Plant Genetics, The University of Minnesota, St. Paul, Minnesota 55108 ABSTRACT In vivo CO2 fixation activity and in vitro phosphoenolpyruvate carboxylase activty were demonstrated in effective and ineffective nodules of alfalfa (Medicago sativa L.) and in the nodules offour other legume species. Phosphoenolpyruvate carboxylase activity was greatly reduced in nodules from both host and bacterially conditioned ineffective alfalfa nodules as compared to effective alfalfa nodules. Forage harvest and nitrate application reduced both in vivo and in vitro CO2 fixation activty. By day 11, forage harvest resulted in a 42% decline in in vitro nodule phosphoenolpyruvate carboxylase activity while treatment with either 40 or 80 kilograms nitrogen per hectare reduced activity by 65%. In vitro specific activity of phosphoenolpyruvate carboxylase and glutamate synthase were positively correlated with each other and both were positively correlated with acetylene reduction activity. Tlhe distribution of radioactivity in the nodules of control plants (unharvested, 0 kilgrams nitrogen per hectare) averaged 73% into the organic acid and 27% into the amino acid fraction. In nodules from harvested plants treated with nitrate, near equal distribution of radioactivity was observed in the organic acid (52%) and amino acid (48%) fractions by day 8. Recovery to control distribution occurred oniy in those nodules whose in vitro phosphoenolpyruvate carboxylase activity recovered. The results demonstrate that CO2 fixation is correlated with nitrogen fixation in alfalfa nodules. The maximum rate of CO2 fixation for attached and detached alfalfa nodules at low CO2 concentrations (0.13-0.38% C02) were 18.3 and 4.9 nanomoles per hour per milligram dry weight, respectively. Nodule CO2 fixation was estimated to provide 25% of the carbon required for assimilation of symbiotically fixed nitrogen in alfalfa. Current estimates with annual legumes suggest that 30%o of the carbon gained through photosynthesis in the shoot is used for nodule function and maintenance and that approximately 60%o of the carbon partitioned to the nodules is lost as CO2 through respiration (14, 16). This results in a loss of 18% of the total photosynthate through the nodule to the atmosphere. Recent studies of annual legumes suggest that nonphotosynthetic CO2 fixation via nodule PEP3 carboxylase (EC 4.1.1.3 1) acts as a mechanism for recovery ofsome ofthis respired C02, thus increasing nodule efficiency and providing an added source of carbon for assimilation of fixed N. Studies with lupine and soybean suggest different relationships between N2 fixation and PEP carboxylase. In lupine, manipulations that reduced nitrogen fixation activity caused a concomitant decrease in PEP carboxylase activity (12). Coker and Schubert (7) found that CO2 fixation activity declined in advance of the decrease in N2 fixation activity in soybean nodules. Several investigations on the role of PEP carboxylase in nodule metabolism have involved exposure of either excised or attached nodules to 14CO2. Lawrie and Wheeler (13) demonstrated that high levels ofradioactivity were initially associated with glutamate and aspartate and later with asparagine in excised nodules of Vicia faba after exposure to 14CO2. In excised lupine nodules, aspartate was the only amino acid labeled within the first 10 min of exposure to [3,4-'4CJglucose (12). Coker and Schubert (6) showed that when intact nodulated roots of soybean were exposed to 14CO2, label was initially incorporated into organic acids with subsequent incorporation into aspartate and glutamate. The first detectable labeled product in Pisum sativum nodules exposed to 14CO2 was malate with subsequent rapid conversion to amino acids (10). Cookson et aL (8) showed that amino.acids accounted for approximately 25% of the carbon fixed from 4CO2 in excised nodules of Phaseolus vulgaris and that tricarboxylic acid cycle intermediates accounted for 60 to 70%o of the radioactivity recovered in bleeding xylem sap, while the remainder was found in aspartate, arginine, lysine, and allantoin. Comparable investigations on the role of nodule PEP carboxylase in perennial legumes are lacking. In addition, there is a large disparity in the estimated carbon input from PEP carboxylase to nodule N assimilation in annual legumes (5, 7, 26), indicating that the contribution of this system to N2 fixation and N assimilation is poorly understood. The contribution of PEP carboxylase to nodule N and C economy of perennial forage legumes has not been investigated. Earlier studies on alfalfa nodule metabolism in our laboratory have detailed the changes in nodule structure, the activity of nitrogenase, and the major N-assimilating enzymes following forage harvest and N application (11, 22). The objectives of this study were to identify the major products of CO2 fixation in alfalfa nodules before and after forage harvest and N application, and to investigate the relationship between PEP carboxylase activity and 'Contribution No. 13,088 from the Minnesota Agricultural Experiment N2 fixation. Station. This research was supported in part by the United States Department of Agriculture, Science and Educational Administration, under 3Abbreviations: PEP, phosphoenolpyruvate; GOGAT, NADH-dependGrant 82-CRCR-1-1 124 from the Competitive Research Grants Office. 2 Mention of a trademark or proprietary product does not constitute a ent glutamate synthase; 2,4-DNPH, 2,4-dinitrophenylhydrazine; GS, NH3guarantee or warranty of the product by either the United States Depart- dependent glutamine synthetase; OA, organic acid; AA, amino acid; MDH, ment of Agriculture or approval to the exclusion of other products that malic dehydrogenase; LSC, liquid scintillation counting; AS, asparagine synthetase; AAT, aspartate aminotransferase. may also be suitable. 469 470 VANCE ET AL. MATERIALS AND METHODS Plant Material. Adzuki bean ( Vigna angularis WILLD. Ohioi and Ohashi), navy bean (Phaseolus vulgaris L.), and soybean (Glycine max L.) nodules were obtained from field-grown plants in the RI stage. Alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) nodules were obtained from vegetative plants grown under conditions previously described by Vance et al. (21). Ineffective nodules regulated by host plant factors were produced on alfalfa clones MnPL-480(In), MnNC-3226(In), MnNC-381 1(In), MnSa(In), and MnAg(In) (17). Characterization and conditions required for the growth and maintenance of these clones is as described for MnPL-480(In) by Viands et al. (24). Bacterially induced ineffective nodules and effective nodules were produced on alfalfa plants (cv Saranac) grown in enclosed culture tubes on agar (23). Seedlings in culture tubes were inoculated either with Rhizobium meliloti strain 102F51 (effective) or with one of the following ineffective strains: 1029, 1054, 1064, or 1058 (donated by Dr. S. Long, Stanford University, Palo Alto, CA). Nodules were collected and PEP carboxylase activity was assayed 6 to 8 weeks after inoculation. Alfalfa plants (cv Saranac) used in NO3- and forage harvest (75% shoot removal) experiments were grown in a sand bench as described by Groat and Vance (1 1). Plants were sampled at 0, 1, 8, 11, 15, and 22 d after treatment. Nodules in all experiments reported in this paper were picked manually, placed in beakers on ice, and used within 30 min for enzyme extraction or for determination of in vivo CO2 fixation activity. Preparation of Nodule Cell-Free Extracts and Enzyme Assays. Excised nodules of all plant types (100-200 mg) were extracted in a Mes-NaOH buffer system (11) for stabilization of the plant cytosol enzyme activities. The homogenates were centrifuged at 18,1OOg for 20 min and the clear supernatant fraction was placed on ice in capped vials until assayed for plant enzyme activities and soluble protein. Conditions for the assay of nodule NADH-GOGAT and GS activities were described previously (11). Rates of PEP carboxylase activity were measured spectrophotometrically in a coupled assay system involving MDH as previously described for the soybean nodule enzyme (18). PEP carboxylase activity was assayed at 250C in 100 mm Bicine, pH 8.5, containing 2 mm PEP, 5 mm MgCl2, 10 mM NaHCO3, and 1.6 mm NADH. Exogenous addition of MDH activity did not increase the in vitro PEP carboxylase activity in any of the nodule extracts assayed. Protein Determination. Soluble protein was measured in nodule extracts by the method of Lowry et al. (15) following precipitation of the protein with 7% TCA. A standard curve was constructed using BSA (12.5-87.5 pg protein). Acetylen Reduction Activity. The acetylene reduction assay described by Vance et al. (21) was used to estimate rates of N2(C2H2) fixation by intact alfalfa nodules on excised root systems. Product Identification. Oxaloacetate was identified as the initial radioactive product of the "'CO2 fixation reaction in alfalfa root nodules by 2,4-DNPH derivatization and TLC with authentic standards. The 2,4-DNPH derivative was prepared according to Bachelard (2) and separated by TLC on silica gel plates (Brinkmann Instruments Inc., Des Plaines, IL)2 in a petroleum ether:ethyl acetate:acetic acid (13:7:2) solvent system for 90 mi (19). In ViVo CO2 Fixation Assay. The in vivo CO2 fixation assay was modified from Christeller et aL (5). Alfalfa root nodules (100 mg fresh weight) were placed on moist filter paper at the bottom of a sealed 10-ml reaction flask (Kontes, Vineland, NJ). The assay was initiated by injection of 4 M lactic acid into center wells contaning 8 ,uCi of aqueous NaH 4CO3 (42 to 52 mCi mmol1, ICN, Irvine, CA) suspended above the nodules. After incubation for 30 min at 230C, the reaction was terminated by injection of 1.5 ml of hot Plant Physiol. Vol. 72, 1983 50%o ethanol (70°C) onto the nodules and the flasks were opened to release any unreacted "CO2. The nodule samples were homogenized in 50% ethanol, followed by extraction in a 45°C water bath for 20 min and centrifugation at 18,100g for 15 min. An aliquot of the supernatant was treated with HCl and the acidstable radioactivity was determined by LSC. Separation of Labeled Nodule Extracts. "4C-labeled alfalfa root nodule extracts were separated into AA, OA, and neutral fractions using Dowex ion exchange resins prepared according to Atkins and Canvin (1). Samples were successively passed through Dowex 5OW, and Dowex 1 columns followed by 25 ml of 50%o ethanol. This eluant was collected as the neutral fraction. The AA fraction was eluted from the Dowex 50W column with 25 ml of 2 M NH40H while the OA fraction was eluted from the Dowex 1 column with 25 ml of 6 M formic acid. Fractions were lyophilized to dryness and resuspended in 600 ,ul of 50%o ethanol (OA and neutral fractions) or 20% ethanol (AA fraction). The total radioactivity incorporated into each fraction was determined by LSC. RESULTS Legume in vitro nodule PEP carboxylase, GOGAT, and GS specific activities are shown in Table I. Alfalfa nodule PEP carboxylase specific activity averaged 644 ± 60 nrmol min-' mg-' protein, the highest of any of the nodules investigated, while birdsfoot trefoil nodules, with an average PEP carboxylase specific activity of 296 ± 47 nmol min-' mg-' protein, had the lowest specific activity. Adzuki bean, soybean, and navy bean nodules had levels of PEP carboxylase activity intermediate to those of alfalfa and birdsfoot trefoil. Nodule GOGAT specific activities Table I. Phosphoenolpyruvate Carboxylase (PEPC), Glutamine Synthetase, and Glutamate Synthase Activity in Several Legumes Each value is the mean ± SE of three replications. Legume Nodule Protein mgprotein g-' fresh wt 14.8 ± 0.5 14.1 ± 0.3 11.1± 1.4 11.5 ±0.8 20.1 ± 1.4 Soybean Adzukibean Navy bean Birdsfoottrefoil Alfalfa PEPC GS GOGAT nmol min'1 mg' protein 488 ± 26 415 + 8 494 ± 54 296 ±47 644 ± 60 254 + 2 198 ± 8 284 ± 13 112 ± 17 140 ± 20 23 + 4 14 1 22 3 18 5 53 3 Table II. Comparisons of PEP Carboxylase (PEPC) in Nodules Induced by Various Strains of R meliloti and in Ineffective Alfalfa Genotypes Nodule Source PEPCb Nitrogenasea nmol min-' mg-' protein R. meliloti strain 1029 1054 1064 1058 102F51 Alfalfa genotype MnSa(In) MnAg(In) MnPL-480(In) MnNC-3226(In) MnNC-381 Saranac I(In) + 74 ± 10 31 ± 4 24 ± 3 87 ± 4 309 ± 56 68 ± 20 119 ± 30 19± 4 34 ± 7 83 ± 10 636 ± 50 a Nitrogenase is based on acetylene reduction activity after 120 min incubation period. + = activity; - = no activity. b Each value is the mean + SE of three replications. ROOT NODULE CARBON DIOXIDE FIXATION 471 min-' mg-' protein. Nodule CO2 fixation was associated with effective N2 fixation. Forage harvest and N03 reduced in vitro PEP carboxylase activity of alfalfa nodules (Fig. 1). PEP carboxylase activity in harvested plants not treated with NO3- declined to 58% of the control (unharvested, 0 kg N ha-') by 11 d after cutting and then recovered to 85% of the control value by day 22. Treatment with either 40 or 80 kg N ha-' resulted in a steady decline in PEP carboxylase specific activity in the nodules of unharvested and harvested plants. By day 15, PEP carboxylase specific activities in nodules from NO03-treated plants (harvested and unharvested) averaged 29% of the day 0 value. PEP carboxylase activity in nodules of harvested plants treated with 40 kg N ha-' began to show a slight recovery trend between days 15 and 22 although the differences between harvested and unharvested values were not statistically significant. Nodule soluble protein concentrations substantiated this recovery (data not shown). No recovery of PEP carboxylase activity was evident in nodules from plants treated with 80 kg N ha-'. Patterns of in vivo nodule CO2 fixation activity were similar to in vitro nodule PEP carboxylase activity for all treatments. Similar to previous studies (1 1), in vitro nodule GOGAT activity changed in response to harvest and applied N03 . Nodule GOGAT specific activity decreased sharply as a result of harvest in C B _DA not treated with NO3 between days 1 and 8 and then plants .r600 showed a steady increase, with activity recovering to control levels by day 22 (Fig. 2). Nodule GOGAT specific activity of plants treated with either 40 or 80 kg N ha-' in both the harvested and unharvested treatments displayed a steady decline through day 15 T / h sa00 and subsequently began to recover between days 15 and 22. I Recovery of GOGAT activity was greater in nodules treated with .300 40 kg N ha-' than in those nodules treated with 80 kg N ha-'. oE Statistical analysis of the data revealed that in vitro nodule PEP carboxylase specific activity and GOGAT specific activity were significantly correlated (P = 0.01) when analyzed by harvest treatment (data for each harvest treatment combined over all N03- levels and all days of the experiment) and by NO3 levels (data for each N03- level combined over all harvest treatments and all days of the experiment). Correlation coefficients for in vitro PEP carboxylase specific activity and GOGAT specific activity of 0.82 and 0.81 were obtained for unharvested and harvested B A treatments, respectively. Analysis of the data by N03 levels gave oE correlation coefficients of 0.69, 0.71, and 0.86 for the 0, 40, and 80 (C). Shoots were harvested - - -) and allowed to regrow for 22 d while kg N ha-' treatment levels, respectively. Both nodule in vitro PEP shoots of control plants ( ) were not harvested. Each point is the mean carboxylase specific activity and GOGAT specific activity were of three replicates ± SE. highly correlated (P = 0.01) with acetylene reduction activity on a per plant basis when the data were analyzed by harvest treatment. Correlation coefficients of 0.63 and 0.62 were obtained for 0 nodule GOGAT specific activity and acetylene reduction activity while correlation coefficients of 0.59 and 0.58 were obtained for in vitro nodule PEP carboxylase specific activity and acetylene W Pttem of invitro VLIPEP FIG.1. carboxylm speifi activity,foowing reduction activity in unharvested and harvested treatments, respectively. Highly significant correlations (P = 0.01) between nodule GOGAT specific activity and acetylene reduction activity (0.78) and between in vitro PEP carboxylase specific activity and acetylene reduction activity (0.66) occurred at the 80 kg N ha-' treatment level but not at the 0 or 40 kg N ha-' treatment levels. There was no significant harvest by N03 level interaction. Both forage harvest and applied N03- caused a shift in distribution of fixed "4CO2 into the OA and AA fractions in alfalfa 22 15 22 1 8 11 15 8 11 15 22 nodules (Table III). The distribution of radioactivity in nodules of 1 DAYS At AFTER AND/OR 1MN At-rUjA APPI CIATION unharvested plants not treated with NOS- was relatively constant I IVIM ILKlHAREI AVcLZ NGAM/r U^ATZ and averaged 73% incorporated into the OA fraction while 27% 40 of 0 and 2. of FIG. Effects harvesting and/or application (A), (B), 80 kg N03--N ha-' (C) on alfalfa nodule NADH-GOGAT specific was incorporated into the AA fraction. By day 8, forage harvest activity. Shoots were harvested (- -) and allowed to regrow for 22 d of plants not treated with N03- resulted in 62% of the total while shoots of control plants (-) were not harvested. Each point is the radioactivity in the OA fraction and 38% in the AA fraction, with recovery to control (unharvested, 0 kg N ha-' values given above) mean of three replicates i SE. were positively correlated to PEP carboxylase, with alfalfa nodules displaying the highest activity and adzuki bean nodules the lowest specific activity. Nodule GS activity of bean species was substantially higher than that of either alfalfa or birdsfoot trefoil. In vivo CO2 fixation assays were also performed on excised nodules of all of these legumes and the ranking of activity was similar to that obtained with the in vitro assay. To evaluate how alfalfa nodule PEP carboxylase activity was related to N2 fixation capability, in vitro PEP carboxylase activity was measured in both bacterial and host plant conditioned-ineffective nodules. In all cases, whether ineffectiveness was the result of bacterial or host genetic factors, the in vitro PEP carboxylase activity of ineffective nodules was substantially lower than that of effective nodules (Table II). The mean PEP carboxylase specific activity of Rhizobium-induced ineffective nodules on plants grown in tube culture (23) was 54 nmol min-' mg-' protein whereas that of comparably grown effective nodules was 309 nmol min-' mg-' protein. The unusually low activity obtained for effective nodule PEP carboxylase of plants in the Rhizobium-induced experiments was probably the result of the plants growing in the tube culture environment. Similarly, the mean PEP carboxylase specific activity of host plant-induced ineffective nodules was 64 nmol min-' mg-1 protein whereas that for effective nodules was 636 nmol o .E - Plant Physiol. Vol. 72, 1983 VANCE ET AL. 472 Table III. Distribution of Total Radioactivity into Alfalfa Nodule Organic Acid and Amino Acid Fractions Days after Harvest and/or N Applicationa Sample OA 11 8 1 AA OA AA AA OA dpm x 10-3 22 15 OA AA OA AA 105 (74) 65 (74) 37 (26) 23 (26) 81 (66) 69 (66) 42 (34) 35 (34) 27 (52) 19 (53) 25 (48) 17 (47) 27 (67) 34 (67) 13 (33) 17 (33) 24 (58) 22 (52) 18 (42) 20 (48) 23 (59) 24 (52) 16 (41) 22 (48) 0 kg N ha-' 21 (21) 78 (79) 51 (72)b 21 (17) 20 (28)b 103 (83) Unharvested 81 (76) 25 (24) 35 (62) 22 (38) Harvested 71 (64) 40 (36) 40 kg N ha-' 65 (73) 24 (27) 46 (73) 17 (27) Unharvested 45 (76) 14 (24) 79 (81) 19 (19) Harvested 21 (57) 16 (43) 22 (63) 13 (37) 80 kg N ha-' 72 (79) 19 (21) 37 (74) 27 (73) Unharvested 13 (26) 10 (27) 44 (65) 24 (35) 17 (49) Harvested 18 (51) 18 (46) 21 (54) a Each value is the mean of at least two replications. Numbers in parentheses, percentage of total. b A portion of this sample was lost during preparation. distribution by day 15. The distribution of total radioactivity incorporated into the OA and AA fractions in nodules from unharvested plants treated with 40 kg N ha-' remained unchanged until day 15 when 52 and 48% was found in the OA and AA fractions, respectively. By day 22, distribution of radioactivity in nodules of unharvested, 40 kg N ha-' plants approximated the control. This recovery between days 15 and 22 corresponds with the recovery trend observed in in vitro PEP carboxylase activity (Fig. 1). A similar pattern was observed in unharvested plants treated with 80 kg N ha-', but there was no recovery by day 22. Plants that were both harvested and treated with N showed the most rapid and pronounced shift in distribution. Nodules from harvested plants treated with 40 kg N ha-' showed a decrease in per cent radioactivity in the OA fraction with a concomitant increase in per cent radioactivity in the AA fraction by day 8 after treatment. This pattern was retained through day 15 and again approached control values by day 22. In comparison, nodules from harvested plants treated with 80 kg N ha-' showed an immediate and persistent response to treatment. The distribution of total radioactivity on day 1 was 65% into the OA fraction and 35% into the AA fraction. By day 8 and through the end of the experiment, nearly equal distribution of radioactivity was observed in the OA (52%) and AA (48%) fractions. increased ureide transport in the xylem sap. The transport form of fixed N in legume species may determine the nature of the relationship between nodule CO2 fixation activity and N2 fixation activity. Nodule CO2 fixation and N2 fixation appear to be positively correlated in species that transport N primarily as amides (lupine, alfalfa) (5). No correlation was observed between nodule CO2 fixation and N2 fixation in two species that transport fixed N as ureides (soybean, broad bean) (7, 26). The initial carbon skeleton of the amide asparagine can be derived directly from oxaloacetate, the initial product of PEP carboxylase. The pathway for incorporation of fixed C from nodule CO2 fixation into ureides is less clear (4, 8). Coker and Schubert (7) reported incorporation of 14CO2 into soybean nodules decreased with the onset of N2 fixation but they did not demonstrate a role for CO2 fixation in ureide biosynthesis. Cookson et al. (8), however, suggested that the "C label in dwarf French bean ureides originated from 14C incorporation into oxaloacetate via PEP carboxylase. In any event, additional research is needed to verify the extent and pathway of 14C incorporation from 14CO2 into ureides. Our observations that PEP carboxylase activity is highly correlated with GOGAT activity and that PEP carboxylase and GOGAT activities are higher in alfalfa than in bean species support the interpretation that alfalfa nodule CO2 fixation and N2 fixation are directly linked through oxaloacetate. Boland et al. (3) and Groat and Vance (11) have previously noted that nodule GODISCUSSION GAT/GS ratios are consistently lower in ureide-transporting legAlfalfa root nodules actively fix CO2 via the enzyme PEP umes as compared to amide-transporting legumes. We again carboxylase and root nodule CO2 fixation is closely associated confirm that observation here. Inasmuch as GOGAT tends to be with nodule effectiveness and N2 fixation capacity. Specific activ- higher in amide transporters and since glutamine amide N may ity for in vitro PEP carboxylase of effective alfalfa nodules was be directly utilized for purine biosynthesis (4), nodule CO2 fixation significantly greater than that of ineffective alfalfa nodules, re- is probably more directly related to N2 fixation and assimilation gardless of whether ineffectiveness was the result of bacterial or of in amide-transporting legumes than in ureide-transporting leghost genetic factors. Treatments that reduced acetylene reduction umes. Forage harvest and applied NO3- reduced in vitro PEP carbox(i.e. applied N and forage harvesting) reduced in vitro PEP carboxylase activity, and recovery of acetylene reduction was accom- ylase activity (and in vivo CO2 fixation activity) and shifted the panied by increased PEP carboxylase activity. Identical results distribution of radioactivity incorporated into the OA and AA were obtained for in vivo CO2 fixation (data not shown). Highly fractions of nodules exposed to 1 CO2. Although less CO2 was significant positive correlations were observed between alfalfa fixed and the distribution of radioactivity shifted as a result of nodule in vitro PEP carboxylase specific activity, GOGAT specific treatments, the total counts incorporated into the AA fraction activity, and acetylene reduction activity. Christeller et al. (5) remained relatively constant as the counts in the OA fraction reported significant positive correlations between both in vitro and decreased substantially (Table III). This suggests there is a bufferin vivo PEP carboxylase activity and acetylene reduction activity ing effect associated with the amino acid fraction that may reflect during the development of lupine nodules. In contrast, Coker and maintenance of a steady state pool of amino acids in the nodule. Schubert (7) reported CO2 fixation was high as soybean nodules This observation and the presence of PEP carboxylase in ineffecstarted to fix N2 but then declined prior to attainment ofmaximum tive nodules suggests that CO2 fixation may be involved in several N2 fixation rates. The decline in soybean nodule CO2 fixation pathways of metabolism throughout the ontogeny of the nodule rates corresponded to a time of decreased amide transport and and the fate of the products of CO2 fixation change as plant ROOT NODULE CARBON DIOXIDE FIXATION 473 conditions change (7, 9). Malate appeared to be the primary metabolism of alfalfa root nodules and that a substantial portion compound in the OA fraction while the neutral fraction consist- of the carbon fixed by this enzyme in alfalfa nodules is utilized ently contained less than 1% of the total radioactivity incorporated for the assimilation of symbiotically fixed N. into the nodules. LITERATURE CITED Although total radioactivity in the nodule AA fraction remained relatively constant with treatments, applied N may induce changes 1. ATKINS CA, DT CANVIN 1971 Photosynthesis and CO2 evolution by leaf discs: in the distribution of that radioactivity among specific amino gas exchange, extraction, and ion-exchange fractionation of "4C-labeled photosynthetic products. Can J Bot 49: 1225-1234 acids. Addition of N03 appeared to reduce the amount of HS 1965 Glucose metabolism and a-keto acids in rat brain and liver radioactivity incorporated into asparagine and alanine while the 2. BACHELARD in vivo. Nature 205: 903-904 radioactivity incorporated into aspartate increased (data not 3. BoLAND MJ, AM FORDYCE, RM GREENWOOD 1978 Enzymes of nitrogen metabshown). This indicates that applied NO3 alters nodule N assimolism in legume nodules: a comparative study. Aust J Plant Physiol 5: 553-559 ilation. Our observations of GOGAT activity support this inter- 4. BoLAND MJ, KR SCHUBERT 1982 Purine biosynthesis and catabolism in soybean root nodules: incorporation of 14C from "CO2 into xanthine. Arch Biochem pretation. Previous studies have shown GS is less affected than Biophys 213: 486491 GOGAT by forage harvest and applied N03 . These observations 5. CHRISTELLER JT, WA LAING, WD SUrrON 1977 Carbon dioxide fixation by imply a more crucial role for AS and AAT in nodule metabolism lupin root nodules. I. Characterization, association with phosphoenolpyruvate carboxylase, and correlation with nitrogen fixation during nodule development. than previously noted. Both AS and AAT have been detected in Plant Physiol 60: 47-50 is not well of their legume nodules; however, regulation activity 6. COKER III GT, KR SCHUBERT 1979 The role of dark CO2 fixation in amino acid understood (20). Because a major portion of the fixed N is biosynthesis in soybean root nodules. Plant Physiol 63: S-621 transported from alfalfa nodules as asparagine, it seems apparent 7. COKER III GT, KR SCHUBERT 1981 Carbon dioxide fLxation in soybean roots and nodules. I. Characterization and comparison with N2 fixation and comwe need to evaluate critically the regulation of AAT and AS with of xylem exudate during early nodule development. Plant Physiol 67: position N. of fixed respect to constraints associated with the transport 691-696 Although the in vivo technique was used in the forage harvest 8. COOKSON C, H HUGHES, J COOMBS 1980 Effects of combined nitrogen on and N03 application experiment to measure both quantitative anapleurotic carbon assimilation and bleeding sap composition in Phaseolus vulgaris L. Planta 148: 338-345 differences in the CO2 fixation activity between treatments and 9. DAVIES DD 1979 The central role of phosphoenolpyruvate in plant metabolism. qualitative changes in the labeling patterns resulting from the Annu Rev Plant Physiol 30: 131-158 treatments, we believe that this assay can also be used to give a 10. DEVRuEs GE, P IN'T VELD, JW KIJNE 1980 Production of organic acids in Pisum sativum root nodules as a result of oxygen stress. Plant Sci Lett 20: 115-123 reliable estimate of the true CO2 fixation rate in alfalfa nodules. 11. GROAT RG, CP VANCE 1981 Root nodule enzymes of ammonia assimilation in measured alfalfa nodule that We have found CO2 fixation, using alfalfa (Medicago sativa L.). Developmental pattern and response to applied excised nodules, is linear for at least 20 min. Maximum CO2 nitrogen. Plant Physiol 67: 1198-1203 fixation rates obtained in 20-min assays for excised alfalfa nodules 12. LAING WA, JT CHRISTELLER, WD SUTrON 1979 Carbon dioxide fixation by lupin root nodules. II. Studies with "C-labeled glucose, the pathway of glucose were 4.4 ± 0.6 and 32.2 ± 2.1 nmol CO2 mg-' nodule dry weight metabolism and the effect of some treatments that inhibit nitrogen fixation. h-' for 0.38% (subsaturating) and 4.9 to 8.4% (saturating) CO2 63: 450-454 Plant concentrations, respectively. The CO2 fixation rate observed for 13. LAWRIE Physiol AC, CT WHEELER 1975 Nitrogen fixation in the root nodules of Vicia to the assimilation ofcarbon. II. The dark fixation ofcarbon L. in relation nodule alfalfa nodules on roots was 18.3 ± 0.3 nmol CO2 mg-' faba dioxide. New Phytol 74: 437-445 dry weight h'1 at a CO2 concentration of 0.13% while the acetylene 14. LAYzELL DB, RM RAINBIRD, CA ATKINS, JS PATE 1979 Economy of photosynreduction rate was 178 nmol ethylene produced mg-' nodule dry thate use in nitrogen fLxing legume nodules. Plant Physiol 64: 888-891 weight h-1. If it is assumed that three acetylene molecules are 15. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 reduced for each dinitrogen fixed and that all of the fixed carbon FR, RJ SUMMERFIELD, P HADLEY, EH ROBERTS, S RAWSTHORNE 1981 and nitrogen is used in the synthesis of asparagine, the major N 16. MINCHIN Carbon and nitrogen nutrition of nodulated roots of grain legumes. Plant Cell transport compound in alfalfa, then CO2 fixation by nodule PEP Environ 4: 5-26 carboxylase is calculated to provide at least 25% of the carbon 17. PErERSON MA, DK BARNES 1981 Inheritance of ineffective nodulation and nonnodulation traits in alfalfa. Crop Sci 21: 611-616 required for the assimilation and transport of symbiotically fixed 18. PETERSON JB, HJ EVANS 1979 Phosphoenolpyruvate carboxylase from soybean N in alfalfa. nodule cytosol. Evidence for isoenzymes and kinetics of the most active Incorporation of acid stable radioactivity into nodules exposed component. Biochim Biophys Acta 567: 445-452 to 14Co2, as an accurate estimate of CO2 fixation, should be viewed 19. PLATT SG, L RAND 1979 Thin-layer chromatographic separation of "C-labeled 2: 239-253 Liquid Chromatogr with caution (5, 7, 9, 25). Therefore, our standard in vivo assay 20. metabolites fromFRphotosynthate. JSUMMERFIELD, C COOKSON, J COOMBS 1980 MINCHIN, RJ S, RAwsTHORNE 30 for conditions ambient under CO2 technique was performed Carbon and nitrogen metabolism in legume root nodules. Phytochemistry 19: min to allow maximum label incorporation into the nodules and 341-355 to be ensured of reaching steady state conditions in the nodules. 21. VANCE CP, GH HEICHEL, DK BARNES, JW BRYAN, LEB JOHNSON 1979 Nitrogen fixation, nodule development and vegetative regrowth of alfalfa (Medicago The final CO2 concentration of the in vivo assay ranged from 0.3 sativa L.) following harvest. Plant Physiol 64: 1-8 to 0.6% due to nodule respiration during the assay period. 22. VANCE CP, LEB JOHNSON, AM HALVERSON, GH HEICHEL, DK BARNES 1980 Estimates of the carbon input from PEP carboxylase to N Histological and ultrastructural observations of Medicago sativa root nodule senescence after foliage removal. Can J Bot 58: 295-309 assimilation in legumes vary widely (5, 7, 26). These variations VANCE 23. CP, LEB JOHNSON, G HARDARSON 1980 Histological comparisons of of the of calculation methods the to in due may be part differing and Rhizobium induced ineffective nodules in alfalfa. Physiol Plant plant estimates. However, it also seems feasible that these estimates vary Pathol 17: 167-173 greatly because the contribution of PEP carboxylase to nodule N 24. VLANDS DR, CP VANCE, GH HEICHEL, DK BARNES 1979 An ineffective nitrogen fLxation trait in alfalfa. Crop Sci 19: 905-908 and C economy likely varies with: (a) ontogeny, (b) environment, 25. WALKza DA 1962 Pyruvate carboxylation and plant metabolism. Biol Rev 37: (c) genotype, (d) species, and (e) primary transport product. 215-256 These studies show that nonphotosynthetic CO2 fixation cata- 26. WHELER CT 1978 Carbon dioxide fixation in the legume root nodule. Ann Appl Biol 88: 481-484 lyzed via the enzyme PEP carboxylase plays a major role in the