Carbon Dioxide Fixation by Lupin Root Nodules

The magnitude and role of dark CO2 fixation were examined in nodules of intact soybean plants (Harosoy 63 x Rhizobium japonicum strain USDA 16). The estimated rate of nodule dark CO2 fixation, based on a 2 minute pulse-feed with '4CO2 under saturating conditions, was 102 micromoles per gram dry weight per hour. This was equivalent to 14% of net nodule respiration. Only 18% of this CO2 fixation was estimated to be required for organic and amino acid synthesis for growth and export processes. The major portion (75-92%) of fixed label was released as CO2 within 60 minutes. The labeling pattern during pulse-chase experiments was consistent with CO2 fixation by phosphoenolpyruvate carboxylase. During the chase, the greatest loss of label occurred in organic acids. Exposure of nodulated roots to Ar:02 (80:20) did not affect dark CO2 fixation, while exposure to 02:CO2 (95:5) resulted in 54% inhibition. From these results, it was concluded that at least 66% of dark CO2 fixation in soybean may be involved with the production of organic acids, which when oxidized would be capable of providing at least 48% of the requirement for ATP equivalents to support nitrogenase activity.Dark CO2 fixation is widespread in the root nodules oflegumes and has been estimated to recycle 9 to 30% of nodule respiratory carbon in soybean (5). It has been suggested that reassimilation of respired CO2 may increase the apparent energy use efficiency of legume symbioses (13), and that selection for increased dark CO2 fixation may be a feasible means of increasing legume productivity (8).Several CO2 fixing enzymes are known to occur in nodules, including carbamoyl phosphate synthetase (16) and phosphoribosylaminoimidazole carboxylase (2). Ribulose bisphosphate carboxylase has been demonstrated to occur in free-living Rhizobium japonicum but not in bacteroids (25). However, the primary CO2 fixing enzyme in legume nodules is PEP2 carboxylase (4,5,15,27 Harosoy 63) were inoculated with liquid cultures of USDA 16, a Rhizobium japonicum strain which lacks measurable uptake hydrogenase activity, and grown in a greenhouse with supplemental metal arc lighting (14-18 h day, 25-31C). Plants were cultured in silica sand and watered with N-free nutrient solution.Nodule N increment was determined as described previously (29) Measurement of CO2 Fixation. In the pulse-chase experiments, root systems with shoot attached were sealed into a 50 ml sidearm Erlenmeyer flask or 250 ml Mason jar equipped with fittings to permit gas flow. Humidified air containing 5% CO2 was passed over the root systems at 200 to 250 ml min-' for at least 5 min; then the gas lines were closed and "'CO2 was injected to give a specific activity of approximately 3 x 103 to 6 x 104 MBq L-'. After a 2 min pulse-feed the gas lines were opened and the container flushed with 5% CO2 for a chase period of0 to 6Q min; then the plants were removed and frozen rapidly in liquid N2.The specific radioactivity of the gas phase during the feed period was determined by removing 0.1 ml gas sampl...

Section: Methodssupporting

confidence: 81%

The Role of Dark Carbon Dioxide Fixation in Root Nodules of Soybean

King¹,

Layzell²,

Canvin³

1986

“…The cost in terms of ATP could vary depending on the P/2e ratio of the oxidative phosphorylation being different from 3. Support for CO2 fixation varying with total nitrogenase activity can be gained from the loose correlation between these processes in other legumes and cultivars of soybean (3,4). However, under an environment ofAr.02, similar to the measurement conditions in this study, the relationship between CO2 fixation and nitrogenase function could not be shown in lupin (10), and these authors suggested that nodule CO2 fixation may be primarily a function of ammonia assimilation (10). If CO2 fixation in fact varied with total e flow, then the cost estimated by the slope would be a minimal value and can be regarded throughout the discussion as a minimum of 2 C02/e pair since root respiration declined only slightly as nodule respiration decreased.…”

Section: Resultsmentioning

confidence: 40%

Experimental Determination of the Respiration Associated with Soybean/Rhizobium Nitrogenase Function, Nodule Maintenance, and Total Nodule Nitrogen Fixation

Rainbird¹,

Hitz²,

Hardy³

1984

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.Rates of CO2 evolution and H2 evolution from intact, nodulated root systems under Ar:02 atmospheres decreased in parallel when plants were maintained in an extended dark period. While H2 evolution approached zero after 36 hours of darkness at 22°C, CO2 evolution rate remained at 380 of the rate measured in light. Of the remaining CO2 evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO2 (per gram dry weight nodule) per hour at 22°C and 30°C, respectively.The cost of N2 fixation in terms of CO2 evolved per electron pair utilized by nitrogenase was estimated from the slope of H2 evolution rate versus CO2 evolution rate. The cost was 2 moles CO2 evolved per mole H2 evolved and was independent of temperature.In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H2 evolution.The ability of legume/Rhizobium symbioses to reduce dinitrogen has led to a large effort to understand processes regulating and affecting the efficiency of this process (12,15,18,24). Symbiotic N2 fixation has been shown to be sensitive to photosynthate supply (7) (14), a strong correlation between e flow through nitrogenase and respiration has been shown. This relationship led Mahon (13) to propose that the total respiration of an intact nodulated root system could be partitioned according to the following equation:where R is total respiration, W is nodule dry weight, t is time, N2-ase is nitrogenase activity, and RM, RG, and RFJx are maintenance, growth, and fixation coefficients, respectively. Based on this equation, nodule respiration becomes linearly related to nitrogenase activity if the growth and maintenance components remain constant. In mature nodule systems, assuming no growth, the slope of this relationship could be considered as the CO2 respired/e pair reduced and the intercept would be associated with nodule maintenance.Exploiting this relationship between nodule respiration and nitrogenase activity, the cost of total nodule function, as well as the individual cost of nitrogenase operation and nodule maintenance, were experimentally determined. In this report, H2 and CO2 evolution under varied plant light regimes was measured, while in another study (8)

“…13 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.…”

mentioning

confidence: 99%

Alfalfa Root Nodule Carbon Dioxide Fixation

Vance¹,

Stade²,

Maxwell³

1983

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 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, as...