Active Transport of CO(2) by the Cyanobacterium Synechococcus UTEX 625 : Measurement by Mass Spectrometry

Mass spectrometry has been used to confirm the presence of an active transport system for CO₂ in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO₃ in the absence of Na⁺ (to prevent HCO₃⁻ transport). Upon illumination the cells rapidly removed almost all the free CO₂ from the medium. Addition of carbonic anhydrase revealed that the CO₂ depletion resulted from a selective uptake of CO₂, rather than a total uptake of all inorganic carbon species. CO₂ transport stopped rapidly (<3 seconds) when the light was turned off. Iodoacetamide (3.3 millimolar) completely inhibited CO₂ fixation but had little effect on CO₂ transport. In iodoacetamide poisoned cells, transport of CO₂ occurred against a concentration gradient of about 18,000 to 1. Transport of CO₂ was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO₂ transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na⁺ (<100 microequivalents per liter), but not of K⁺, stimulated CO₂ transport as much as 2.4-fold. Unlike Na⁺-dependent HCO₃⁻ transport, the transport of CO₂ was not inhibited by high concentrations (30 milliequivalents per liter) of Li⁺. During illumination, the CO₂ concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO₂ transport was continuously occurring at a rapid rate, since the continuing dehydration of HCO₃⁻ to CO₂ would rapidly raise the CO₂ concentration to its equilibrium value if transport ceased. Measurement of the rate of dissolved inorganic carbon accumulation under these conditions indicated that at least part of the continuing CO₂ transport was balanced by HCO₃⁻ efflux.     

Measurement of CO(2) and H(2)O Vapor Exchange in Spinach Leaf Discs : Effects of Orthophosphate

A leaf chamber has been designed which allows the measurement of both CO₂ and water vapor exchange in Spinacia oleracea leaf discs. The center of the disc lies within a cylindrical gas chamber and its margins are enclosed within a cavity through which water or various metabolites can be pumped. In saturating light and normal atmospheres, the leaf discs have a relatively low resistance to H₂O vapor transfer (r_w = 1.87 seconds per centimeter) and can support high rates of photosynthesis for several hours. The abaxial surface of a disc had a higher resistance to water vapor transfer (r_w = 3.22 seconds per centimeter) than the adaxial (r_w = 2.45 seconds per centimeter) despite having a higher stomatal frequency (abaxial, 105/square millimeter; adaxial, 58/square millimeter). In 2% O₂, the discs required an internal concentration of CO₂ of 115 microliters per liter to support one-half of the maximal velocity of apparent photosynthesis (average value, 66 milligrams CO₂ per square decimeter per hour). In 20% O₂, the comparable values are 156 microliters per liter and 56 milligrams CO₂ per square decimeter per hour. In air, apparent photosynthesis saturated at intensities (750 microeinsteins per square meter per second) well below that of daylight but, when the internal CO₂ was raised to 700 to 900 microliters per liter, photosynthesis was not saturated even at daylight intensities (2025 microeinsteins per square meter per second). The distribution of Prussian blue crystals, formed after ferrocyanide feeding, showed that water entered the disc via the vasculature. When 25-minute pulses of orthophosphate were provided in the feeding solution, there were concentration-dependent increases in both r_w and r_m leading to inhibition of photosynthesis. The orthophosphate-dependent inhibitions were reversible.     

Control of CO(2) Fixation by Cell-free Extracts of Chlamydomonas reinhardtii

Contrary to earlier reports, CO₂ fixation by extracts of Chlamydomonas is inhibited by glutamate and aspartate. These amino acids and some organic acids are shown to be inhibitors of phosphoenolpyruvate carboxylase. Inorganic phosphate is shown to activate CO₂ fixation, but there is a time lag before inorganic phosphate exerts its full activating effect.     

Control of smoulder (Botrytis narcissicold) in narcissus with fungicides

Smoulder, caused by Botrytis narcissicola, is the most widespread foliar disease of narcissus (Narcissus cultivars) in the UK and causes significant losses in bulb and flower yield. A range of current and novel fungicide treatments was examined to determine if control of the disease could be improved and to measure the effect of treatments on bulb yield. In assays on pot-grown plants, carbendazim+flusilazole, epoxiconazole, iprodione + thiophanate-methyl, tebuconazole and vinclozolin reduced lesion size by more than 90%, compared with untreated plants, when applied 1 day before inoculation with the fungus; vinclozolin and tebuconazole were also effective when applied 2 days after inoculation. In field experiments in Cambridgeshire and Lincolnshire, significant reductions were observed in secondary smoulder symptoms (leaf lesions and stem rot) following fungicide sprays. Spray programmes of the novel fungicides azoxystrobin, cyprodinil, kresoxim-methyl, tebuconazole and pyrimethanil were as effective, or more so, than the standard treatments (chlorothalonil, vinclozolin) used by growers. Mixtures of carbendazim + tebuconazole and azoxystrobin + tebuconazole also gave effective control of smoulder. Programmes of four to six sprays, using two or three fungicides with different modes of action, applied alternately, reduced smoulder by 35–69% and increased bulb yields by 7–59%. Although treatment around and after flowering resulted in the greatest control of secondary smoulder, treatment before flowering was required for the highest bulb yields. The choice of fungicides for use in spray programmes to provide effective and reliable control of smoulder, and the possibility of control using fewer sprays, are discussed.     

Assimilatory Power (Postillumination CO(2) Uptake) in Leaves: Measurement, Environmental Dependencies, and Kinetic Properties

Assimilatory power was measured in ten C₃ species by means of a rapid-response gas exchange device as the total amount of CO₂ fixed in N₂-CO₂ atmosphere after switching the light off. Different steady-state levels of the assimilatory power were obtained by varying light intensity and O₂ and CO₂ concentrations during the preexposition periods in the leaf chamber.

Within the limits of the linear part of the CO₂ curve of photosynthesis in N₂, the assimilatory power is constant, being sufficient for the assimilation of about 20 nanomoles CO₂ per square centimeter leaf. The pool starts to decrease with the onset of the CO₂ saturation of photosynthesis. Increase in O₂ concentration from 0 to 100% at 350 microliters CO₂ per liter produces a considerable decrease in the assimilatory power.

The mesophyll conductance (M) was found to be proportional to the assimilatory power (A): M = mA. The most frequently occurring values of the proportionality constant (m) (called the specific efficiency of carboxylation) were concentrated between 0.03 and 0.04 centimeter per second per nanomole A per square centimeter but the measured extreme values were 0.01 and 0.06 centimeter per second per nanomole A per square centimeter. The specific rate of carboxylation (the rate per unit A) showed a hyperbolic dependence on CO₂ conentration with the most frequent values of K_m (CO₂) ranging from 25 to 35 micromolar in the liquid phase of mesophyll cells (extremes 23 and 100 micromolar).

It is concluded that the CO_2⁻ and light-saturated rate of photosynthesis is limited by the reactions of the formation of the assimilatory power and not by ribulose-1,5-bisphosphate carboxylase. O₂ is a competitive consumer of the assimilatory power, and the inhibitory effect of O₂ on photosynthesis is caused mainly by a decrease in the pool of the assimilatory power at high O₂ concentrations. In intact leaves, the kinetic properties of ribulose-1,5-bisphosphate carboxylase seem to be variable.

     

A Numerical Approach to Measurement of CO(2) Exchange Transients by Infrared Gas Analysis

Open flow-through systems coupled to infrared gas analyzers have been frequently employed in the study of CO₂ exchange transients such as the postillumination burst observed in leaves of C₃ plants. A major limitation associated with use of such systems is their non-steady state response to rapid changes in leaf CO₂ exchange rate. A previous publication outlined a numerical approach to model the analyzer response as a function of CO₂ exchange rate and thus permit estimation of the postillumination burst (Peterson 1983 Plant Physiol 73: 978-982). The model is critically analyzed within the framework of the physics of solute dispersion as previously described for linear flow systems. Thus, the numerical simulation is validated on the basis of physical principle. Additional improvements to the previous model are described which enhance the accuracy and efficiency of use of this technique for estimation of photorespiration.     

Control of Botrytis cinerea and Botrytis squamosa in overwintered salad onions by fungicide sprays

In 1974/75, 13 sprays of 0·2% a.i. thiram applied at 14 day intervals to overwintered salad onions reduced the incidence of Botrytis cinerea and significantly increased onion yields. In 1977/78 both B. cinerea and B. squamosa occurred, and 12 iprodione sprays at 0·1% a.i. applied at 14-day intervals or 6 sprays at 0·2% a.i. applied at 28-day intervals gave good control of B. cinerea and B. squamosa and significantly increased onion yields. Benomyl (0·1% a.i. at 14-day intervals, or 0·2% a.i. at 28-day intervals) failed to control either pathogen because of the development of carbendazim-insensitive strains of the fungi. Effective control of both pathogens and increased yields were obtained with an application of 0·4% a.i. thiram in October and November followed by an application of 0·2% a.i. iprodione in December and January.     

Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts

A system has been developed for the study of photosynthetic CO₂ fixation by isolated spinach chloroplasts at air levels of CO₂. Rates of CO₂ fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO₂ fixation, suggesting that the decline in CO₂ fixation may have been caused by deactivation of the enzyme.     

Determination of biogenic and fossil CO(2) emitted by waste incineration based on (14)CO(2) and mass balances

A field application of the radiocarbon ((14)C) method was developed to determine the ratio of biogenic vs. fossil CO(2) emissions from waste-to-energy plants (WTE). This methodology can be used to assign the Kyoto relevant share of fossil CO(2) emissions, which is highly relevant for emission budgets and emission trading. Furthermore, heat and electricity produced by waste incinerators might be labelled depending on the fossil or biogenic nature of the primary energy source. The method development includes representative on-site CO(2) absorption and subsequent release in the laboratory. Furthermore, a reference value for the (14)C content of pure biogenic waste (f(M,bio)) was determined as 1.130+/-0.038. Gas samples for (14)CO(2) analysis were taken at three WTEs during one month each. Results were compared to an alternative approach based on mass and energy balances. Both methods were in excellent agreement and indicated a fraction of biogenic CO(2) slightly above 50%.     

Vibrational spectra and structure of M(CO2) and M2(CO2) molecules

Atomic Na, K and Cs were codeposited with CO₂ in excess of matrix gas at the temperature of 12 K. The IR spectra revealed the presence of ionic aggregates corresponding to the molecules M(CO)₂ and M₂(CO₂) (M=Na, K, Cs). Both molecular species have C_2v symmetry; M(CO₂) species have a planar ring structure while M₂(CO₂) have a W-shape structure. M₂(CO₂) molecules with C_s symmetry were also identified. The geometrical parameters of all the molecules were determined by ¹²C/¹³C and ¹⁶O/¹⁸O isotopic shifts. Raman spectra were also recorded and the results are reported in this study. The effect of photolysis on the structure of these molecules was examined. It was determined that photolysis promotes the formation of Na(CO₂) and transforms the M₂(CO₂) molecules with C_2v symmetry into C_s symmetry isomers.     

Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

Ethylene Contamination of CO(2) Cylinders: Effects on Plant Growth in CO(2) Enrichment Studies

The mechanical extensibilities of stage IVb Phycomyces were measured before and after a humidified wind stimulus. We find that when the humidity of the wind is greater than that of the ambient air, there is an increase in the mechanical extensibility of the cell wall. We also find that a step decrease in wind humidity results in a decrease in the mechanical extensibility of the cell wall.     

Two-breath CO(2) test detects altered dynamic cerebrovascular autoregulation and CO(2) responsiveness with changes in arterial P(CO(2))

The new two-breath CO(2) method was employed to test the hypotheses that small alterations in arterial P(CO(2)) had an impact on the magnitude and dynamic response time of the CO(2) effect on cerebrovascular resistance (CVRi) and the dynamic autoregulatory response to fluctuations in arterial pressure. During a 10-min protocol, eight subjects inspired two breaths from a bag with elevated P(CO(2)), four different times, while end-tidal P(CO(2)) was maintained at three levels: hypocapnia (LoCO(2), 8 mmHg below resting values), normocapnia, and hypercapnia (HiCO(2), 8 mmHg above resting values). Continuous measurements were made of mean blood pressure corrected to the level of the middle cerebral artery (BP(MCA)), P(CO(2)) (estimated from expired CO(2)), and mean flow velocity (MFV, of the middle cerebral artery by Doppler ultrasound), with CVRi = BP(MCA)/MFV. Data were processed by a system identification technique (autoregressive moving average analysis) with gain and dynamic response time of adaptation estimated from the theoretical step responses. Consistent with our hypotheses, the magnitude of the P(CO(2))-CVRi response was reduced from LoCO(2) to HiCO(2) [from -0.04 (SD 0.02) to -0.01 (SD 0.01) (mmHg x cm(-1) x s) x mmHg Pco(2)(-1)] and the time to reach 95% of the step plateau increased from 12.0 +/- 4.9 to 20.5 +/- 10.6 s. Dynamic autoregulation was impaired with elevated P(CO(2)), as indicated by a reduction in gain from LoCO(2) to HiCO(2) [from 0.021 +/- 0.012 to 0.007 +/- 0.004 (mmHg x cm(-1) x s) x mmHg BP(MCA)(-1)], and time to reach 95% increased from 3.7 +/- 2.8 to 20.0 +/- 9.6 s. The two-breath technique detected dependence of the cerebrovascular CO(2) response on P(CO(2)) and changes in dynamic autoregulation with only small deviations in estimated arterial P(CO(2)).     

Measurement of CO(2) Assimilation in Soils: an Experiment for the Biological Exploration of Mars

A method is described for the measurement of ¹⁴CO₂ assimilation by microorganisms in soils. A determination involves exposing soil to ¹⁴CO₂, pyrolyzing the exposed soil, trapping the organic pyrolysis products on a column of firebrick coated with CuO, combusting the trapped organics by heating, and measuring the radioactivity in the CO₂ produced in the combustion. The detection of significant levels of ¹⁴C in the trapped organic fraction appears to be an unambiguous indication of biological activity. The ¹⁴CO₂ which is adsorbed or exchanged into soils by nonbiological processes does not interfere. The method easily detects the ¹⁴CO₂ fixed by 10² to 10³ algae after light exposure for 3 to 24 hr. Assimilation of ¹⁴C is also demonstrable in dark-exposed soils containing 10⁵ to 10⁶ heterotrophic bacteria. Possible applications of the method in the biological exploration of Mars are discussed.     

Biotransformation of (-)-a-pinene by Botrytis cinerea

(-)-alpha-Pinene (1), a major constituent of many aromatic plants was biotransformed by the plant pathogenic fungus, Botrytis cinerea to afford three new metabolites, characterized as 3beta-hydroxy-(-)-beta-pinene (10%) (3), 9-hydroxy-(-)-a-pinene (12%) (4), 4beta-hydroxy-(-)-alpha-pinene-6-one (16%) (5) by physical and spectroscopic methods. A known metabolite verbenone (2) was also obtained.     

Quantum Yields for CO(2) Uptake in C(3) and C(4) Plants: Dependence on Temperature, CO(2), and O(2) Concentration

The quantum yields of C₃ and C₄ plants from a number of genera and families as well as from ecologically diverse habitats were measured in normal air of 21% O₂ and in 2% O₂. At 30 C, the quantum yields of C₃ plants averaged 0.0524 ± 0.0014 mol CO₂/absorbed einstein and 0.0733 ± 0.0008 mol CO₂/absorbed einstein under 21 and 2% O₂. At 30 C, the quantum yields of C₄ plants averaged 0.0534 ± 0.0009 mol CO₂/absorbed einstein and 0.0538 ± 0.0011 mol CO₂/absorbed einstein under 21 and 2% O₂. At 21% O₂, the quantum yield of a C₃ plant is shown to be strongly dependent on both the intercellular CO₂ concentration and leaf temperature. The quantum yield of a C₄ plant, which is independent of the intercellular CO₂ concentration, is shown to be independent of leaf temperature over the ranges measured. The changes in the quantum yields of C₃ plants are due to changes in the O₂ inhibition. The evolutionary significance of the CO₂ dependence of the quantum yield in C₃ plants and the ecological significance of the temperature effects on the quantum yields of C₃ and C₄ plants are discussed.