C(4) Acid Metabolism and Dark CO(2) Fixation in a Submersed Aquatic Macrophyte (Hydrilla verticillata)

The CO₂ compensation point of the submersed aquatic macrophyte Hydrilla verticillata varied from high (above 50 microliters per liter) to low (10 to 25 microliters per liter) values, depending on the growth conditions. Plants from the lake in winter or after incubation in an 11 C/9-hour photoperiod had high values, whereas summer plants or those incubated in a 27 C/14-hour photoperiod had low values. The plants with low CO₂ compensation points exhibited dark ¹⁴CO₂ fixation rates that were up to 30% of the light fixation rates. This fixation reduced respiratory CO₂ loss, but did not result in a net uptake of CO₂ at night. The low compensation point plants also showed diurnal fluctuations in titratable acid, such as occur in Crassulacean acid metabolism plants. However, dark fixation and diurnal acid fluctuations were negligible in Hydrilla plants with high CO₂ compensation points.     

Glutamate Oxaloacetate Transaminase in Pea Root Nodules : Participation in a Malate/Aspartate Shuttle between Plant and Bacteroid

Glutamate oxaloacetate transaminase (l-glutamate: oxaloacetate aminotransferase, EC 2.6.1.1 [GOT]), a key enzyme in the flow of carbon between the organic acid and amino acid pools in pea (Pisum sativum L.) root nodules, was studied. By ion exchange chromatography, the presence of two forms of GOT in the cytoplasm of pea root nodule cells was established. The major root nodule form was present in only a small quantity in the cytoplasm of root cells. Fractionation of root nodule cell extracts demonstrated that the increase in the GOT activity during nodule development was due to the increase of the activity in the cytoplasm of the plant cells, and not to an increase in activity in the plastids or in the mitochondria. The kinetic properties of the different cytoplasmic forms of GOT were studied. Some of the K_m values differed, but calculations indicated that not the kinetic properties but a high concentration of the major root nodule form caused the observed increase in GOT activity in the pea root nodules. It was found that the reactions of the malate/aspartate shuttle are catalyzed by intact bacteroids, and that these reactions can support nitrogen fixation. It is proposed that the main function of the nodule-stimulated cytoplasmic form of GOT is participation in this shuttle.     

Ammonia (C-Methylamine) Transport across the Bacteroid and Peribacteroid Membranes of Soybean Root Nodules

[¹⁴C]Methylamine (MA; an analog of ammonia) was used to investigate ammonia transport across the bacteroid and peribacteroid membranes (PBM) from soybean (Glycine max) root nodules. Free-living Bradyrhizobium japonicum USDA110 grown under nitrogen-limited conditions showed rapid MA uptake with saturation kinetics at neutral pH, indicative of a carrier. Exchange of accumulated MA for added ammonia occurred, showing that the carrier recognized both NH₄⁺ and CH₃NH₃⁺. MA uptake by isolated bacteroids, on the other hand, was very slow at low concentrations of MA and increased linearly with increasing MA concentration up to 1 millimolar. Ammonia did not inhibit MA by isolated bacteroids and did not cause efflux of accumulated MA. PBM-enclosed bacteroids (peribacteroid units [PBUs]) were qualitatively similar to free bacteroids with respect to MA transport. The rates of uptake and efflux of MA by PBUs were linearly dependent on the imposed concentration gradient and unaffected by NH₄Cl. MA uptake by PBUs increased exponentially with increasing pH, confirming that the rate increased linearly with increasing CH₃NH₂ concentration. The results are consistent with other evidence that transfer of ammonia from the nitrogen-fixing bacteroid to the host cytosol in soybean root nodules occurs solely by simple diffusion of NH₃ across both the bacteroid and peribacteroid membranes.     

Effect of Salt Stress on Carbon Metabolism and Bacteroid Respiration in Root Nodules of Common Bean (Phaseolus vulgaris L.)

Abstract: In the present work, we examined the effect of salinity on growth, N fixation and carbon metabolism in the nodule cytosol and bacteroids of Phaseolus vulgaris, and measured the O₂ consumption by bacteroids incubated with or without the addition of exogenous respiratory substrates. The aim was to ascertain whether the compounds that accumulate under salt stress can increase bacteroid respiration and whether this capacity changes in response to salinity in root nodules of Phaseolus vulgaris. The plants were grown in a controlled environment chamber, and 50, 100 mM or no NaCl (control) was added to the nutrient solution. Two harvests were made, at the vegetative growth period and at the beginning of the reproductive period. The enzyme activities in the nodule cytosol were reduced by the salt treatments, while in the bacteroid cytosol the enzyme activities increased at high salt concentrations at the first harvest and for ADH in all treatments. The data presented here confirm that succinate and malate are the preferred substrates for bacteroid respiration in common bean, but these bacteroids may also utilize glucose, either in control or under saline conditions. The addition of proline or lactate to the incubation medium significantly raised oxygen consumption in the bacteroids isolated from plants treated with salt.     

Essentiality of Boron for Symbiotic Dinitrogen Fixation in Pea (Pisum sativum) Rhizobium Nodules

The effect of boron deficiency on symbiotic nitrogen fixation in pea (Pisum sativum) was examined. The absence of boron in the culture medium resulted in a decrease of the number of nodules and an alteration of nodule development leading to an inhibition of nitrogenase activity. Examination of boron-deficient nodules showed dramatic changes in cell walls and in both peribacteroid and infection thread membranes, suggesting a role for boron in the stability of these structures. These results indicate that boron is a requirement for normal nodule development and functionality.     

Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation

Detached roots and nodules of the N₂-fixing species, Albus glutinosa (European black alder), actively assimilate CO₂. The maximum rates of dark CO₂ fixation observed for detached nodules and roots were 15 and 3 micromoles CO₂ fixed per gram dry weight per hour, respectively. The net incorporation of CO₂ in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO₂ fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO₂ fixation and N₂ fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total ¹⁴C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N₂-fixing nodules and roots of alder.     

Dark CO(2) Fixation and its Role in the Growth of Plant Tissue

Experiments were designed to determine the significance of dark CO₂ fixation in excised maize roots, carrot slices and excised tomato roots grown in tissue culture. Bicarbonate-¹⁴C was used to determine the pathway and amounts of CO₂ fixation, while leucine-¹⁴C was used to estimate protein synthesis in tissues aerated with various levels of CO₂.

Organic acids were labeled from bicarbonate-¹⁴C, with malate being the major labeled acid. Only glutamate and aspartate were labeled in the amino acid fraction and these 2 amino acids comprised over 90% of the ¹⁴C label in the ethanol-water insoluble residue.

Studies with leucine-¹⁴C as an indicator of protein synthesis in carrot slices and tomato roots showed that those tissues aerated with air incorporated 33% more leucine-¹⁴C into protein than those aerated with CO₂-free air. Growth of excised tomato roots aerated with air was 50% more than growth of tissue aerated with CO₂-free air. These studies are consistent with the suggestion that dark fixation of CO₂ is involved in the growth of plant tissues.

     

Initial Organic Products of Fixation of [N]Dinitrogen by Root Nodules of Soybean (Glycine max)

When detached soybean Glycine max (L.) Merr. cv. Hark, nodules assimilate [¹³N]N₂, the initial organic product of fixation is glutamine; glutamate becomes more highly radioactive than glutamine within 1 minute; ¹³N in alanine becoms detectable at 1 minute of fixation and increases rapidly between 1 and 2 minutes. After 15 minutes of fixation, the major ¹³N-labeled organic products in both detached and attached nodules are glutamate and alanine, plus, in the case of attached nodules, an unidentified substance, whereas [¹³N]glutamine comprises only a small fraction of organic ¹³N, and very little ¹³N is detected in asparagine. The fixation of [¹³N]N₂ into organic products was inhibited more than 99% by C₂H₂ (10%, v/v). The results support the idea that the glutamine synthetase-glutamate synthase pathway is the primary route for assimilation of fixed nitrogen in soybean nodules.     

Enhanced Dark CO(2) Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

The products of short time photosynthesis and of enhanced dark ¹⁴CO₂ fixation (illumination in helium prior to addition of ¹⁴CO₂ in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the ¹⁴C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C₆ chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of ¹⁴CO₂ uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO₂ fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase.

It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

Long-Term Anaerobic Metabolism in Root Tissue (Metabolic Products of Pyruvate Metabolism)

The onset of anaerobiosis in barley root tissue (Hordeum vulgare L. cv Himalaya) results in the following metabolic responses. There are rapid increases in the levels of pyruvate, lactate, and ethanol. Malate and succinate concentrations increase over the first 12 h, after which they return to the levels found in oxygenated root tissue. Alanine concentration increases over the first 12 h, and this is matched by a corresponding decrease in aspartate. The initial stoichiometric decline in aspartate and increase in alanine suggests that the amino group of aspartate is conserved by transaminating pyruvate to alanine. Aspartate catabolism also probably provides the initial source of carbon for reduction to succinate under anoxic conditions. Under long-term anaerobiosis (>24 h), there is no further accumulation of any of the fermentative end products other than ethanol, which also represents the major metabolic end product during long-term anaerobiosis. Although a number of the enzymes involved in fermentative respiration have been found to be induced under anaerobic conditions, neither aspartate amino-transferase nor malate dehydrogenase is induced in barley root tissue. The observations suggest that the long-term adaptations to hypoxic conditions may be quite different than the more well-characterized short-term adaptations.     

Diffusional Contribution to Carbon Isotope Fractionation during Dark CO(2) Fixation in CAM Plants

A mathematical model is developed which can be used to predict in vivo carbon isotope fractionations associated with carbon fixation in plants in terms of diffusion, CO₂ hydration, and carboxylation components. This model also permits calculation of internal CO₂ concentration for comparison with results of gas-exchange experiments. The isotope fractionations associated with carbon fixation in Kalanchoë daigremontiana and Bryophyllum tubiflorum have been measured by isolation of malic acid following dark fixation and enzymic determination of the isotopic composition of carbon-4 of this material. Corrections are made for residual malic acid, fumarase activity, and respiration. Comparison of these data with calculations from the model indicates that the rate of carbon fixation is limited principally by diffusion, rather than by carboxylation. Processes subsequent to the initial carboxylation also contribute to the over-all isotopic composition of the plant.     

豌豆根瘤中细菌的扫描电镜观察

用扫描电镜观察了豌豆根瘤的侵染细胞.结果表明,在这些细胞中有大量的细菌,它们主要是杆状细菌,其次是球形、Y形和T形细菌,其它形状的细菌很少.除了细菌形状不同外,还有一些细菌比较特殊,如有的细菌较长,菌体出现部分收缩并形成一个或一个以上的收缩环,其形状类似一条莲根;有的细菌很大,它的体积是普通细菌的2倍或2倍以上;有的细菌粗细不均匀,端部膨大,呈棒槌状.侵染细胞中有许多小泡,它们大小不同,呈球形.它们存于细菌之间,其中一些小泡还位于细菌的表面上,而且附近细菌的表面有时还有各种隆起.     

Effect of Fusicoccin on Dark CO(2) Fixation by Vicia faba Guard Cell Protoplasts

When Vicia faba guard cell protoplasts were treated with fusicoccin, dark ¹⁴CO₂ fixation rates increased by as much as 8-fold. Rate increase was saturated with less than 1 micromolar fusicoccin. Even after 6 minutes of dark ¹⁴CO₂ fixation, more than 95% of the incorporated radioactivity was in stable products derived from carboxylation of phosphoenolpyruvate (about 50% and 30% in malate and aspartate, respectively). The relative distribution of ¹⁴C among products and in the C-4 position of malate (initially more than 90% of [¹⁴C]malate) was independent of fusicoccin concentration. After incubation in the dark, malate content was higher in protoplasts treated with fusicoccin. A positive correlation was observed between the amounts of ¹⁴CO₂ fixed and malate content.

It was concluded that (a) fusicoccin causes an increase in the rate of dark ¹⁴CO₂ fixation without alteration of the relative fluxes through pathways by which it is metabolized, (b) fusicoccin causes an increase in malate synthesis, and (c) dark ¹⁴CO₂ fixation and malate synthesis are mediated by phosphoenolpyruvate carboxylase.

     

Dark CO(2) Fixation in Gladiolus Cormels and Its Regulation during the Break of Dormancy

The increase in dark CO₂ fixation during cold storage of Gladiolus x gandavensis van Houtte-type grandiflorus cormels is used to monitor changes in their state of dormancy. Dark fixation is also promoted by benzyladenine, which breaks cormel dormancy, and is inhibited by abscisic acid and gibberellin A₃, which inhibit cormel germination. The rate of dark fixation by nondormant cormels is five times higher than that in dormant ones. Dark fixation is not due to microorganisms. It is temperature-dependent and can be measured stoichiometrically in vivo. The apex and base of the cormels accumulate more label than the central part. Dark fixation of both dormant and nondormant cormels is also promoted by imbibition in water. The fate of the labeled assimilates was followed by ion exchange chromatography.