Labelling of lupine nodule metabolites with 14CO2 assimilated from the leaves

On feeding ¹⁴CO₂ to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of ¹⁴C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids.     

Carbon dioxide injection method for enhancing hydrogenotrophic denitrification of secondary wastewater effluent in fixed bed reactor

This study presents an optimal injection method for using carbon dioxide as a carbon source for the hydrogenotrophic denitrification of secondary wastewater effluent in a laboratory-scale fixed bed reactor (FBR). The FBR was operated under three conditions: a continuous CO₂ supply, periodic CO₂ supply, and without a CO₂ supply. The continuous operation of the FBR without carbon dioxide injection resulted in an increase in pH to 10 and a noticeable level of nitrite accumulation. The continuous co-injection of carbon dioxide and hydrogen gas decreased the pH to a range of 6 ～ 8, but the denitrification efficiency decreased to 29%. The co-injection of carbon dioxide decreased the maximum dissolved hydrogen concentration and hydrogen mass transfer rate by 25 and 61%, respectively. Compared to the continuous injection method, a periodic injection of carbon dioxide increased the denitrification efficiency from 28.6 to 85% as the hydrogen flow rate and hydraulic retention time (HRT) increased. With the periodic injection of carbon dioxide, the nitrite accumulation appeared to be insignificant as the hydrogen flow rate increased.     

In vitro growth and shoot multiplication of Wrightia tomentosa Roem et Schult in a controlled carbon dioxide environment

In vitro growth and multiplication of shoots of a woody tree species Wrightia tomentosa in a controlled carbon dioxide environment was studied. The cultures were grown on BA supplemented MS medium with or without 3% sucrose. A range of CO₂ concentrations (0.0, 0.6, 10.0 and 40.0 g m^–3) was controlled in small chambers by using solutions of NaHCO₃, Na₂CO₃, KHCO₃ and K₂CO₃. To obtain a CO₂-free environment, a saturated solution of KOH was kept in the chambers. It was concluded that the growing shoot cultures required either sucrose in the medium as a carbon source or an ambient CO₂ environment. Complete absence of a carbon source caused severe browning of the shoots and death within 30 days. The cultures grew better with 10.0 g m^–3 carbon dioxide in the environment than with 3.0% sucrose in the medium. With both CO₂ and sucrose being available, the best response was obtained at 0.6 g m^–3 CO₂ in the chamber. At this concentration the rate of shoot multiplication was nearly double the standard rate obtained when exposed to the natural CO₂ level and sucrose-supplemented medium. Total fresh and dry weight, leaf number and area per cluster also showed the best response under this condition.     

Ribulose-1, 5-bisphosphate carboxylase from comfrey: Isolation and characterization

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified to electrophoretic homogeneity from comfrey, Symphytum spp. Sodium dodecyl sulfate polyacrylamide and polyacrylamide gel electrophoresis studies on the purified product showed no extraneous proteins. Comparisons of the electrophoretic mobilities of the subunits to those of standard proteins indicated a large subunit MW of 50 000 and a small subunit of 12 700, which for an octameric structure of each subunit indicates a native MW of 502 000. Specific activities of the comfrey enzyme ranged from 1.2 to nearly 2 μmol ¹⁴CO₂ fixed/min.mg of protein over several preparations and were maintained for months when stored from the sucrose gradient at ? 70°. The specific activities depended critically on the amounts of enzyme used in the assay even under saturating conditions of substrates and cofactors. The effective pH dependence for carboxylase catalysis peaked near 7.4, which apparently is the lowest elective optimum yet reported for this enzyme from any source. However, on a constant carbon dioxide basis the pH dependence profile was reversed with a maximum near pH 8.6 which was 0.4 units higher than the value for the spinach enzyme. The K_ms for carbon dioxide and ribulose-1,5-bisphosphate at pH 7.5 were 130 μM and 30 μM, respectively, which are comparable to the accepted values for the carboxylase from spinach at pH 7.2.     

Carbon Partitioning in a Flaveria linearis Mutant with Reduced Cytosolic Fructose Bisphosphatase

Oxygen sensitivity and partitioning of carbon was measured in a mutant line of Flaveria linearis that lacks most of the cytosolic fructose-1,6-bisphosphatase found in wild-type lines. Photosynthesis of leaves of the mutant line was nearly insensitive to O₂, as found before. The mutant plants partitioned 2.5 times less carbon into sucrose than the wild type in a pulse chase experiment, with the extra carbon going mainly to starch but also to amino acids. From 10 to 50 min postlabeling, radioactivity chased out of the amino acid fraction to starch in both lines. In the middle of the light period, starch grains were larger in the mutant than in the wild type and covered 30% of the chloroplast area as seen with an electron microscope. Starch grains were found in both mesophyll and bundle sheath chloroplasts in both lines in these C₃-C₄ intermediate plants. At the end of the dark period, the starch levels were considerably reduced from what they were in the middle of the light in both lines. The concentration of sucrose was higher in the mutant line despite the lack of cytosolic fructose-1,6-bisphosphatase. The amino acid fraction accounted for about 30% of all label following a 10-min chase period. In the mutant line, most of the label was in the glycine + serine fraction, with 10% in the alanine fraction. In wild-type leaves, 35% of the label in amino acids was in alanine. These results indicate that this mutant survives the reduced cytosolic fructose-1,6-bisphosphatase activity by partitioning more carbon to starch and less to sucrose during the day and remobilizing the excess starch at night. However, these results raise two other questions about this mutant. First, why is the sucrose concentration high in a plant that partitions less carbon to sucrose, and second, why is alanine heavily labeled in the wild-type plants but not in the mutant plants?     

Photojet de CO2 et réserves internes en gaz carbonique chez Zea mays

Summary When a leaf of maize (Zea mays) is illuminated following a long enough period of darkness (t10 min) under pure nitrogen, the oxygen evolution occurs only after a lag time from one to several minutes. During this delay, a burst of CO₂ occurs the maximum of which corresponds to the start of oxygen evolution. The source of carbon dioxide is thought to be a stable internal CO₂ reserve which has been previously formed in the leaf and is assumed to be malic acid. For as yet unknown reasons the carbon dioxide issued from the reserve by action of light at the beginning of illumination is not fixed and escapes into the atmosphere; this process could require the phytochrome system.     

Effect of controlled carbon dioxide on in vitro shoot multiplication in Feronia limonia (L.) Swingle

The effect of controlled carbon dioxide environment on in vitro shoot growth and multiplication in Feronia limonia (a tropical fruit plant, Family- Rutaceae) was studied. Carbon dioxide available in the ambient air of the growth room was insufficient for in vitro growth of the shoots alone. Also, the presence of sucrose only as the C-source in the medium (without CO₂), was found to be inadequate for sustainable growth and multiplication of shoots. The carbon dioxide enrichment promoted shoot multiplication and overall growth. The promotory effect of CO₂ was independent of the presence of sucrose in the medium. In the presence of both CO₂ and sucrose, an additive effect was observed producing maximum shoot growth. In the absence of sucrose a higher concentration of CO₂ (10.0)g m⁻³ was required to achieve photoautotrophic shoot multiplication comparable to ambient air controls. Highest leaf area per shoot cluster promoting shoot growth and multiplication was recorded under this treatment. Shoots growing on sucrose containing medium under controlled CO₂ environment of 0.6 g m⁻³ concentration evoked better response than ambient air controls (shoots growing on sucrose containing medium) in growth room. This treatment produced the overall best response. The present study highlighted the possibility of photoautotrophic multiplication which might prove useful for successful hardening and acclimatization in tissue culture plants.     

The potential of a sucrose ester coating material for improving the storage and shelf-life qualities of Cox's Orange Pippin apples

A sucrose-ester coating material was tested for its potential as a storage technique and as an extender of the shelf life of apple (cv. Cox's Orange Pippin). Apples treated with 1·25% sucrose ester formulation were stored in air at 3·5°C for up to 5 months. Sucrose ester treatment did not reduce detrimental changes in terms of fruit firmness, yellowing and weight loss but did increase core flush incidence. When applied after storage, the sucrose ester reduced yellowing and loss of firmness and markedly increased internal carbon dioxide levels during a 21 day simulated marketing period. Effects were enhanced with increasing sucrose ester concentrations between 1% and 4%. Sucrose ester did not markedly reduce weight loss in the fruit, did not cause accumulation of alcohol or induce any internal physiological disorders during the simulated marketing period. Treatment of fruit with an external atmosphere containing 8% carbon dioxide, a level similar to that found in fruit treated with 3% sucrose ester, did not have the same effects as 3% sucrose ester on firmness or ground colour changes, suggesting that the effects of the sucrose ester are not solely the result of the raised carbon dioxide level.     

Rates of Production of Individual Volatile Fatty Acids in the Rumen of Lactating Cows

The rumen fermentation rates in individual lactating cows were measured in four different experiments. The results disclosed that the amounts and proportions of volatile acids formed could vary widely. In one case, a marked difference in the proportions of the acids produced arose within the experiment and correlated with a difference in the proportion of methane formed.

The average rate of production per day was 10.5 moles butyric acid, 12.8 moles propionic acid, and 40 moles acetic acid. Manometric estimations of rate gave lower results than those obtained by the zero-time method, due to delay after sampling and to failure of the acids to liberate stoichiometric quantities of carbon dioxide.

For those experiments in which zero-time rates were estimated, the average specific absorption rates, i.e., the amount absorbed per hour per micromole of acid in the rumen, were 0.37 for butyric acid, 0.38 for propionic acid, and 0.26 for acetic acid.

The carbon dioxide, acids, and microbial cells produced in the rumen fermentation are estimated to account for about 90% of the carbon found in the milk and respiratory CO₂ of the cows. The carbon dioxide from the fermentation was about 27% of the carbon dioxide exhaled.

     

High productivity and photosynthetic flexibility in a CAM plant

Summary In the annual succulent Mesembryanthemum crystallinum growing in situ, the balance between C₃ and CAM carbon fixation shifted rapidly in response to changes in water availability. When water was plentiful, M. crystallinum fixed carbon dioxide by the C₃ pathway and grew at rates comparable to other C₃ species. Under drought conditions, M. crystallinum fixed carbon by the CAM pathway at an average rate which exceeded 1 nanomole of carbon dioxide per square centimeter of leaf surface per second, a very high rate for a CAM plant.     

Studies on the Ventilation in Submerged Fermentations

Quantitative studies on the dissolution and dissociation of carbon dioxide in a cultured system were made. The inosine fermentation and the glutamic acid fermentation were employed for this study. According to the results obtained in this experiment, the quantity of dissociated carbonic acid in cultured liquid was given by Henderson-Hasselbalch’s equation with experimental pK′. The method for the direct determination of bicarbonate ion concentration was also investigated. The Warburg direct method gave a satisfactory result for this purpose.

By using the modified Severinghaus CO₂ electrode, the relationship between partial pressure of carbon dioxide in effluent gas and that in culturing system was investigated. Partial pressure of carbon dioxide in gas phase was almost equivalent to the average value of dissolved carbon dioxide tension in liquid phase for a given short time of the fermentation. The term of r_e was introduced in order to study the dynamic characteristics of carbon dioxide evolution in submerged fermentors. The dynamic characteristics of respiration in submerged fermentation was also studied by using biological r_ab and r_e.     

Metabolism of 14C-Chlorobenzilate and 14C-Chloropropylate by Rhodotorula gracilis

Rhodotorula gracilis metabolizes Chlorobenzilate (ethyl 4,4'-dichlorobenzilate) and Chloropropylate (isopropyl 4,4'-dichlorobenzilate) to several metabolites in a basal medium supplemented by sucrose and by several intermediates of the citric acid cycle. Three identified metabolites resulting from the degradation of either acaricide, were 4,4'-dichlorobenzilic acid, 4,4'-dichlorobenzophenone, and carbon dioxide. Chlorobenzilate, i.e., ethyl ester of 4,4'-dichlorobenzilic acid, was more easily hydrolyzed than Chloropropylate, i.e., isopropyl ester of this acid, so that larger amounts of carbon dioxide and 4,4'-dichlorobenzophenone were obtained from Chlorobenzilate degradation. Regardless of acaricides used, longer incubation caused a higher accumulation of 4,4'-dichlorobenzophenone. The probable steps of the degradation pathway are: Chlorobenzilate (or Chloropropylate) --> 4,4'-dichlorobenzilic acid --> 4,4'-dichlorobenzophenone plus carbon dioxide. It appears that the decarboxylation of 4,4'-dichlorobenzilic acid to 4,4'-dichlorobenzophenone was hindered by alpha-ketoglutarate and enhanced by succinate.     

Diurnal changes in allocation of newly fixed carbon in exporting sugar beet leaves

Storage of newly fixed carbon as starch and sucrose follows a regular daily pattern in exporting sugar beet leaves under constant day length and level of illumination. Up to the final two hours of the light period, when starch storage declines, a nearly constant proportion of newly fixed carbon was allocated to carbohydrate storage, principally starch. Sucrose is stored only early in the light period, when there is little accumulation of starch. Pulse labeling with ¹⁴CO₂ revealed that considerable starch synthesis was taking place at this time. Starch made the previous day was not mobilized during this period but breakdown of newly synthesized starch may occur when carbon flow into sucrose synthesis increases early in the day. At the end of the day, starch storage declined from the constant level observed during most of the day, but no diversion of label into export of specific alternative compounds could be detected. Lowered storage of starch persisted when the 14-hour light period was lengthened. Changed allocation of recently fixed carbon to sucrose and starch at the beginning and end of the light period was not the result of outright inactivation of pathways but of regulation of carbon flow.     

Evidence for circadian regulation of starch and sucrose synthesis in sugar beet leaves

Starch accumulation and sucrose synthesis and export were measured in leaves of sugar beet (Beta vulgaris L.) during a period of prolonged irradiance in which illumination was extended beyond the usual 14-hour day period. During much of the 14-hour day period, approximately 50% of the newly fixed carbon was distributed to sucrose, about 40% to starch, and less than 10% to hexose. Beginning about 2 hours before the end of the usual light period, the portion of newly fixed carbon allocated to sucrose gradually increased, and correspondingly less carbon went to starch. By the time the transition ended, about 4 hours into the extension of the light period, nearly 90% of newly fixed carbon was incorporated into sucrose and little or none into starch. Most of the additional sucrose was exported. Gradual cessation of starch accumulation was not the result of a futile cycle of simultaneous starch synthesis and degradation. Neither was it the result of a decrease in the extractable activity of adenosine diphosphoglucose pyrophosphorylase or phosphoglucose isomerase, enzymes important in starch synthesis. Nor was there a notable change in control metabolites considered to be important in regulating starch synthesis. Starch accumulation appeared to decrease markedly because of an endogenous circadian shift in carbon allocation, which occurred in preparation for the usual night period and which diverted carbon from the chloroplast to the cytosol and sucrose synthesis.     

Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by ¹⁴CO₂ assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total ¹⁴CO₂ fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = −0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.     

Up-regulation of sucrose metabolizing enzymes in Oncidium goldiana grown under elevated carbon dioxide

Experiments were conducted in controlled growth chambers to evaluate how increase in CO₂ concentration affected sucrose metabolizing enzymes, especially sucrose phosphate synthase (SPS; EC 2.4.1.14) and sucrose synthase (SS; EC 2.4.1.13), as well as carbon metabolism and partitioning in a tropical epiphytic orchid species (Oncidium goldiana). Response of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) to elevated CO₂ was determined along with dry mass production, photosynthesis rate, chlorophyll content, total nitrogen and total soluble protein content. After 60 days of growth, there was a 80% and 150% increase in dry mass production in plants grown at 750 and 1 100 μl l^?1 CO₂, respectively, compared with those grown at ambient CO₂ (about 370 μl l^?1). A similar increase in photosynthesis rate was detected throughout the growth period when measured under growth CO₂ conditions. Concomitantly, there was a decline in leaf Rubisco activity in plants in elevated CO₂ after 10 days of growth. Over the growth period, leaf SPS and SS activities were up‐regulated by an average of 20% and 40% for plants grown at 750 and 1100 μl l^?1 CO₂, respectively. Leaf sucrose content and starch content were significantly higher throughout the growth period in plants grown at elevated CO₂ than those at ambient CO₂. The partitioning of photosynthetically fixed carbon between sucrose and starch appeared to be unaffected by the 750 μl l^?1 CO₂ treatment, but it was favored into starch under the 1 100 μl l^?1 CO₂ condition. The activities of SPS and SS in leaf extracts were closely associated with photosynthetic rates and with partitioning of carbon between starch and sucrose in leaves. The data are consistent with the hypothesis that the up‐regulation of leaf SPS and SS might be an acclimation response to optimize the utilization and export of organic carbon with the increased rate of inorganic‐carbon fixation in elevated CO₂ conditions.     

A Method for Calculating Sucrose Synthesis Rates throughout a Light Period in Sugar Beet Leaves

Sucrose synthesis rate in an exporting sugar beet (Beta vulgaris L.) leaf was calculated from simultaneous measurements of export and changes in leaf sucrose level. The amount of recently fixed carbon exported was determined from net carbon assimilated minus the tracer carbon accumulated in the leaf. The relative amount of ¹⁴C accumulated in the leaf supplied with ¹⁴CO₂ throughout an entire light period was recorded continuously with a Geiger-Mueller detector. To produce a continuous time course for tracer carbon accumulated in the leaf during the light period, the latter curve was superimposed on values for tracer carbon accumulated in leaves sampled at hourly intervals. Validity of the method requires that nearly all of the carbon that is exported be sucrose and that nearly all of the sucrose that is synthesized be either exported or accumulated as sucrose in the exporting leaves. These conditions appeared to be fulfilled in the situations where the method was applied. The method was used to study the effect of increasing atmospheric CO₂ concentration on the rate of sucrose synthesis. Further, the method can be used in conjunction with the gathering of other data such as gas exchange, metabolite levels, and enzyme activities in a set of leaves of a similar age on the same plant. This assemblage of data was found to be useful for understanding how rates of photosynthesis, sucrose synthesis, and translocation are regulated in relation to each other in an intact plant.     

Sucrose translocation and storage in the sugar beet

Several physiological processes were studied during sugar beet root development to determine the cellular events that are temporally correlated with sucrose storage. The prestorage stage was characterized by a marked increase in root fresh weight and a low sucrose to glucose ratio. Carbon derived from ¹⁴C-sucrose accumulation was partitioned into protein and structural carbohydrate fractions and their amino acid, organic acid, and hexose precursors. The immature root contained high soluble acid invertase activity (V_max 20 micromoles per hour per milligram protein; K_m 2 to 3 millimolar) which disappeared prior to sucrose storage. Sucrose storage was characterized by carbon derived from ¹⁴C-sucrose uptake being partitioned into the sucrose fraction with little evidence of further metabolism. The onset of storage was accompanied by the appearance of sucrose synthetase activity (V_max 12 micromoles per hour per milligram protein; K_m 7 millimolar). Neither sucrose phosphate synthetase nor alkaline invertase activities were detected during beet development. Intact sugar beet plants (containing a 100-gram beet) exported 70% of the translocate to the beet, greater than 90% of which was retained as sucrose with little subsequent conversions.     

Gas Exchange of Algae: III. Relation between the Concentration of Carbon Dioxide in the Nutrient Medium and the Oxygen Production of Chlorella pyrenoidosa

The oxygen production of a photosynthetic gas exchanger containing Chlorella pyrenoidosa (1% packed cell volume) was measured when various concentrations of carbon dioxide were present within the culture unit. The internal carbon dioxide concentrations were obtained by manipulating the entrance gas concentration and the flow rate. Carbon dioxide percentages were monitored by means of electrodes placed directly in the nutrient medium. The concentration of carbon dioxide in the nutrient medium which produced maximal photosynthesis was in the range of 1.5 to 2.5% by volume. Results were unaffected by either the level of carbon dioxide in the entrance gas or the rate of gas flow. Entrance gases containing 2% carbon dioxide flowing at 320 ml/min, 3% carbon dioxide at 135 ml/min, and 4% carbon dioxide at 55 ml/min yielded optimal carbon dioxide concentrations in the particular unit studied. By using carbon dioxide electrodes implanted directly in the gas exchanger to optimize the carbon dioxide concentration throughout the culture medium, it should be possible to design more efficient large-scale units.     

Clostridium magnum sp. nov., a non-autotrophic homoacetogenic bacterium

A new mesophilic, sporeforming, strictly anaerboic bacterium was isolated form enrichments with 2,3-butanediol as sole substrate and pasteurized freshwater sediment as inoculum. Cells were large, motile rods, and elliptical spores were formed subterminally or centrally. They stained Gram-negative, but no typical outer membrane layer could be observed by electron microscopy of ultrathin sections. 2,3-Butanediol, acetoin, fructose, glucose, sucrose, xylose, malate and citrate served as substrates and were completely converted to acetate with concomitant reduction of carbon dioxide. Growth on glucose (t _dmin=1.4 h) was faster than on butanediol (t _dmin=3.6 h). No growth occurred on hydrogen/carbon dioxide, on formate or on methanol. The guanine plus cytosine content of the DNA was 29.1%. The new isolate is described as a new species, Clostridium magnum sp. nov.