Significance of Hydrogen Evolution in the Carbon and Nitrogen Economy of Nodulated Cowpea

Rates of respiratory CO2 loss and nitrogenase activities of H2 uptakenegative mutant strains and H2 uptake-positive revertant strains of Rhizobium japonium have been investigated. Two-dimensional gel protein patterns of bacteroids formed by inoculation of soybeans (Glycine max L.) with these two strains show that they are closely related and revealed only one obvious difference between them On the basis of molecular weight standards, it was concluded that the missing protein spot in the H2 uptakenegative mutant strain could be caused by a failure of the mutant to synthesize hydrogenase. Nodules formed by the H2 uptake-negative mutant strain evolved respiratory CO2 at a rate of about 10% higher than that of nodules formed by the H2 uptake-positive revertant strain. During shortterm experiments employed, rates of both C2H2 reduction and 16N2 fixation varied considerably among replicate samples and no statistically significant differences between mutant and revertant strains were observed. It was observed that increasing the partial pressure of 02 over nodules significantly decreased the proportion of nitrogenase electrons allocated to H'. Rhizobium bacteroids in root nodules of infected legumes convert atmospheric N2 into ammonium that the plant uses for its growth. This reduction of N2 requires energy supplied primarily in the form of carbohydrates from the photosynthesis of the host plant, of which the availability to the nodules is considered as a major factor limiting symbiotic N2 fixation (16,21). The nitrogenase, which catalyzes this reaction, reduces protons simultaneously (3,19) and the portion of electrons diverted to proton reduction varies with the turnover rate of MoFe component of the enzyme (4). The nitrogenase reaction is the origin of the H2 evolution by nodules first shown on soybeans in 1957 (18). According to surveys done so far, most of temperate Rhizobium-legume associations evolve significant amounts of this gas, with no known benefit (30)(31)(32)33). H2 evolution, therefore, is considered as a loss of energy, the extent of which averages about one-third of the electron flow through the nitrogenase (11). Some associations do not evolve H2 and the proportion of these appear higher among tropical legumes (32). In soybeans, 25% of the strains found in nodules of plants collected from various parts of the United States evolved little or no H2 (23). This is due to a membrane-bound hydrogenase system carried by the bacteroids, which catalyzes the oxidation of H2 to H20 (5, 7, 28). According to its level of activity, the hydrogenase system recycles part or all ofthe H2 formed by nitrogenase (8,30). This oxyhydrogen reaction could benefit the N2 fixation process by recovering part of the ATP and electrons expended in the conversion of protons to H2, by functioning as a supplementary mechanism for the protection of nitrogenase from 02 damage, and by preventing any inhibitory effect of H2 in the immediate environment of nitrogenase (9).The saving of carbohydrates in Hup+3 bacteroids supplied wi...

Section: Resultssupporting

confidence: 82%

Respiratory and Nitrogenase Activities of Soybean Nodules Formed by Hydrogen Uptake Negative (Hup⁻) Mutant and Revertant Strains of Rhizobium japonicum Characterized by Protein Patterns

Drevon

Frazier²,

Russell³

et al. 1982

“…Although altered H2 evolution could reflect changes in overall nitrogenase activity or the relative activities of uptake hydrogenase in materials from different O2 atmospheres, it seems unlikely that differences in the "relative efficiency" of nodules (10) (Fig. 6) compared favorably with those determined previously for this and similar symbioses (14,19,20). The sharp increase in CO2 evolved per unit substrate reduced at 1 and 80% 02 suggests that at these extremes, nodule functioning was especially inefficient, with respiration supporting very much lower levels of nitrogenase activity.…”

Section: Nitrogenase Activitymentioning

confidence: 55%

Effect of pO₂ on Growth and Nodule Functioning of Symbiotic Cowpea (Vigna unguiculata L. Walp.)

Dakora

Atkins²

1990

Self Cite

Nodulated cowpea (Vigna unguiculata L. Walp. cv Vita 3:Bradyrhizobium CB 756) plants were cultured with their whole root system or crown root nodulation zone maintained for periods from 5 to 69 days after planting in atmospheres containing a range Of P02 (1-80%, v/v) while the rest of the plant grew in normal air. Growth (dry matter yield) and N2 fixation were largely unaffected by PO2 from 10 to 40%. Decrease in fixation at PO2 below 5% was due to lower nodulation and nodule mass and, at P02 above 60%, to a fall in specific N2-fixing activity of nodules. Root:shoot ratios were significantly lower at PO2 below 2.5%. The effect of PO2 on nitrogenase activity (acetylene reduction), both of whole nodulated root systems and crown root nodulation zones, varied with plant age but was generally lower at supraand subambient extremes of 02. H2 evolution showed a sharp optimum at 20% 02 but was at most 4% of total nitrogenase activity. The ratio of CO2 evolved to substrate (C2H2+H ) reduced by crown root nodulation zones was constant (6 moles CO2 per mole substrate reduced) from 2.5 to 60% 02 but at levels below 2.5 and above 80% 02 reached values between 20 and 30 moles CO2 per mole substrate reduced. Effects of long-term growth with nonambient P02 on adaptation and efficiency of functioning of nodules are discussed.with water status (9). Thus, mechanisms regulating 02 supply may underlie variation in field performance of N2 fixation in legumes. Few studies have examined directly the response of fixation to changes in the permeability of nodules, although decreasing O2 concentrations from ambient to 10% around nodulated soybean roots resulted in a 63% increase in the permeability of nodules to gas, while increasing the rhizosphere O2 concentration led to a decrease in permeability (26). These data suggest that soybean nodules are able to regulate the permeability of their tissue components to O2 in response to changing rhizosphere PO2 and, as a consequence, maintain N2 fixation at relatively constant rates.The present study reports the effect of 02 level on plant growth, nodule development, N2 fixation, and nitrogenase activities of intact nodules on cowpea plants cultured either with their whole nodulated root systems or their root crown nodulation zones alone maintained in a range of sub-and supra-ambient 02 throughout extended periods of growth. MATERIALS AND METHODS Plant MaterialLowered nitrogenase activity of legume root nodules under a variety of imposed experimental conditions have been interpreted as being due to a limitation of 02 supply to bacteroids (4,8,12,17,29). Recent evidence further suggests that nodules of some symbioses are able to protect nitrogenase from 02 damage by a rapid increase in resistance to diffusion of gases (21,29). This response appears to be mediated, in part, by operation of a biologically controlled "variable diffusion barrier" possibly located at, or close to, the inner cortex of the nodule (13,26,30,31).Although the mode of operation of this diffusion barrier remains unknow...

“…At both 22 and 30°C, the slopes were constant at 2.0 C02/H2 giving cost for the reduction of I N2 at 6.0 CO2 or 36 ATP (assuming 36 ATP/mol glucose respired). Similar costs for in vivo nitrogenase functioning in cowpea nodules have been derived using different techniques (20).…”

Section: Resultsmentioning

confidence: 97%

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

Rainbird¹,

Hitz²,

Hardy³

1984

Self Cite

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)