Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

A comparative analysis of the effects of in-vivo and in-vitro abscisic-acid treatment on the surface electrical properties of barley chloroplast membranes

The effects of in-vivo and in-vitro abscisic acid (ABA) treatments on the surface charge density () of barley (Hordeum vulgare L.) thylakoids were compared using 9-aminoacridine fluorescence. The estimated surface charge density of isolated thylakoid membranes from control (non-treated) barley leaves was-0.065 C · m^-2. The net negative surface charge density decreased after application of various concentrations of ABA (10^-6, 10^-5 M) for 7 d in-vivo, the more pronounced effect being observed at 10^-5 M ABA. When ABA was added to the suspension of isolated thylakoids the opposite effect was observed. The average charge density increased in in-vitro-treated thylakoids at 10^-5 M ABA to -0.081 C · m^-2. The results are discussed in terms of a specific ABA-induced influence of the composition and-or stoicheometry of charged protein complexes within the thylakoid membranes.Abbreviations and Symbols ABA abscisic acid - 9AA 9-aminoacridine - C, C K⁺ and Mg²⁺ concentrations giving equal relative fluorescence - F 9AA-fluorescence intensity - F_max maximum 9AA fluorescence - surface charge density The authors are grateful to Professor L.P. Popova (Institute of Plant Physiology, Sofia, Bulgaria) for continuous support. This work was supported in part by the Bulgarian Ministry of Science and High Education under research contract No. 519.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

The ecology of Lake Nakuru (Kenya)

Summary Abiotic factors, standing crop and photosynthetic production were studied in the equatorial alkaline-saline closed-basin Lake Nakuru (cond. 10,000–160,000 S). Meteorological conditions and abiotic factors offer suppositions for a high primary productivity: mean solar radiation is 450–550 kerg·cm^-2·s^-1, with little seasonal variation, regular winds circulate the lake every day and nutrient concentrations are usually high (>100 g P–PO₄·l^-1). Oxygen concentrations near sediments were <1 gO₂·m^-3 for at least 6 h·d^-1 in 1972/73, resulting in a release of 45 mg P–PO₄·m^-2·d^-1. Attenuation coefficients vary from 3.6–16.5 according to algal densities and mean depth from 0–400 cm. Algal biomass was 200 g·m^-3 (d.w.) in 1972/73, due to a lasting Spirulina platensis bloom (98.5% of algal biomass). In 1974 algal biomass suddenly dropped to 50 g·m^-3 (d.w.). Spirulina and several consumer organisms almost vanished, but coccoid cyanobacteria, Anabaenopsis and diatoms increased. Several causes for this change in ecosystem structure are discussed. The use of the light/dark bottle method to measure photosynthetic production in eutrophic alkaline lakes is discussed and relevant experiments were done. Oxygen tensions of 2–35 gO₂·m^-3 do not influence primary production rates. Net photosynthetic rates (mgO₂·m^-3·h^-1; photosynthetic quotient=1.18) reached 12–17.7 in 1972/73 and 2–3 in 1974, but vertically integrated rates were only 1–1.4 in 1972/73 and 0.8 in 1974, and daily net photosynthetic rates (gO₂·m^-3·24 h^-1) 3.5 in 1972/73 and 1 in 1974. 50% of areal rates were produced within the 10 most productive cm of the depth profile. The disproportion between high algal standing crops and relatively low production rates is due to self-shading of the algae, reducing the euphotic zone to 35 cm in 1972/73 and 77 cm in 1974. Efficiency of light utilization is 0.4–2%, varying with time of day and phytoplankton density. In situ efficiencies show an inverse relationship to light intensities. Photosynthetic rates of L. Nakuru remain within the range of other African lakes (0.1–3 gO₂·m^-2·h^-1). The relation of O₂ produced/Chl a of the euphotic zone is 50% lower then in tropical African freshwater lakes and conforms to lakes of temperate regions.     

Does photorespiration protect the photosynthetic apparatus in french bean leaves from photoinhibition during drought stress?

Dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidation and zeaxanthin formation in chloroplasts, inhibited blue-light-stimulated stomatal opening in epidermal peels of Vicia faba L. in a concentration-dependent fashion. Complete inhibition was observed at 3 mM DTT. The DTT effect was specific for the stomatal response to blue light, and the red-light-stimulated opening, which depends on photosynthetic reactions in the guard cells, was unaffected. Preirradiation of stomata in epidermal peels with increasing photon fluence rates of red light, prior to an incubation in 10 mol·m^-2·s^-1 of blue light and 100 mol·m^-2·s^-1 red light, resulted in a DTT-sensitive, blue-light-stimulated opening that was proportional to the fluence rate of the red light pre-treatment. Guard cells in epidermal peels and guard-cell protoplasts irradiated with red light showed increases in their zeaxanthin content that depended on the fluence rate of red light, or on the incubation time. The increases in zeaxanthin concentration were inhibited by DTT. The obtained results indicate that zeaxanthin could function as a photoreceptor mediating the stomatal responses to blue light.Abbreviation DTT dithiothreitol This work was supported by grants from the National Science Foundation and the US Department of Energy to E.Z.     

Azide treatment enhances cell-to-cell diffusion in staminal hairs ofSetcreasea purpurea

Summary The effect of azide on the diffusion of fluorescent molecular probes was examined in staminal hairs ofSetcreasea purpurea. Staminal hairs were treated with azide before being microinjected with fluorescent molecular probes of different size, charge, and structure. The cell-to-cell movement of these fluorescent molecules was videotaped, analyzed, and coefficients of diffusion through plasmodesmata (D) and coefficients of diffusion across the tonoplast (k₁) were calculated and compared to those of untreated cells. The D was larger and the k₁ was smaller for many fluorescent probes in azide treated cells compared to normal, untreated cells. In addition, the cell-to-cell diffusion selectivity based on molecule structure, size and charge no longer existed in azide treated cells. An average D of 3.3×10^–8cm²/s and an average k₁ of 2.9×10^–7/m²/s was calculated for the molecular probes tested. New size limits for permeation were observed indicating that the plasmodesmata had become enlarged.Abbreviations CF carboxyfluorescein - D diffusion coefficient for molecular probes in intercellular pores - FITC-Ang fluorescein isothiocyanate-angiotensin II - k₁ coefficient of diffusive loss across the tonoplast     

Compartmentation of fluorescent tracers injected into the epidermal cells of Egeria densa leaves

We have compared the movement of a series of fluorescent tracers of increasing molecular weight injected into the cytoplasm in the epidermal cells of leaves of Egeria densa Planch. In general, the tracers showed major movement into three cellular compartments: first, to the cytoplasm of adjacent cells; secondly, from the cytoplasm, to the vacuole (irreversible); and thirdly, from the cytoplasm to the nucleus (reversible). No visible accumulation in chloroplasts or mitochondria, or loss across the plasmalemma was observed. No evidence for metabolic breakdown was found in extracts from injected leaves. The time course of accumulation of the dye in the three major compartments (cytoplasm, nucleus, vacuole) was monitored using fluorescence microscopy. The rate measurements and the quantified geometry of the cells were used to generate a model of compartmentation during intercellular transport. Permeability coefficients were calculated and related to the molecular sizes of the tracers. The coefficients for the tonoplast and nuclear envelope were independent of the molecular sizes of the tracers, and were in the range 2.4·10^–6–4.1· 10^–6 cm·s^–1 for the tonoplast, and 2.6·10^–5-9.4.10^–5 cm· s^–1 for the nuclear envelope. For intercellular movement, permeabilities were strongly dependent on molecular size, and ranged from 1.1·10^–4 cm·s^–1 for 6-carboxyfluorescein (376 daltons (Da)) to 9·10^–9 cm·s^–1 for fluorescein leucyldiglutamylleucine (874 Da). Thus, the differences in cell-to-cell movement of these tracers are based upon their differing ability to cross the intercellular walls, not upon differences in their intracellular compartmentation.Abbreviations 6COOHF 6-Carboxyfluorescein - Da daltons - DAPI 4,6-diamidino-2-phenylindole - F fluorescein-isothiocyanate isomer I - FGlu fluorescein glutamic acid - F(Glu)₂ fluorescein glutamyl-glutamic acid - F(Gly)₆ fluorescein hexaglycine - FLGGL fluorescein leucyl diglutamyl-leucine This work was supported by the Australian Research Grant Scheme. The assistance of Professor B.E.S. Gunning (Australian National University, Canberra, ACT) in providing facilities for making the photometer measurements is gratefully acknowledged.     

Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H⁺/e^– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m^–2) (strong light) the H⁺/e^– ratio was 3. At low intensity (0.9 W · m^–2) (weak light) the H⁺/e^– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H⁺/e^– ratio due to underestimation of the initial rate of H⁺ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.     

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179–1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 mol photons · m^–2 · s^–1 (VHL) than in cells acclimated at 10 mol photons · m^–2 · s^–1 (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566–568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357–366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).Abbreviations Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - DCPIP 2,6-dichlorophenol indophenol - -dm dodecyl--d-maltoside - HL high light of 150 mol photons · m^–2 · s^–1 - LGB lower green band - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - LL low light of 10 mol photons · m^–2 · s^–1 - ML medium light of 50 mol photons · m^–2 · s^–1 - MES 2-(N-morpholino) ethanesulfonic acid - P700 reaction center of PS I - PFD photon flux density - Trizma tris(hydroxymethyl)aminomethane - UGB upper green band - VHL very high light of 280 mol photons · m^–2 · s^–1     

Cotyledonal chloroplasts in the hypogeal seeds of clementine

Clementine (Citrus nobilisxCitrus aurantium amara pumila) is a chloroembryophyte with green quiescent embryos and hypogeal germination. The cotyledonal chloroplasts have been studied during germination in the dark and under two different irradiances 120 and 240 mol·m^-2·s^-1 throughout a period of three weeks. The plastids of the outer adaxial and inner regions develop differently. In the light, the former differentiate a photosynthetically active thylakoid system with an ultrastructural organization and a polypeptide composition resembling that of leaf chloroplasts. The inner chloroplasts maintain an organization reminiscent of chloroplasts of the quiescent embryo and never get beyond the photosynthesis/respiration compensation point; their differentiation pattern appears essentially the same under the two different irradiances. These observations and the germination in the dark indicate that the above differentiation is not strictly photodependent. The greening ability of the cotyledons provides, on occasion, an additional photosynthetic supply to this plant.Abbreviations Chl a-P chlorophyll a-protein-complex - CF1 coupling factor complex - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A new technique for continuous measurement of the heat of fermentation

Summary A special temperature control system has been developed and applied to continuous measuring of the heat evolved during a fermentation process. In this system, the fermentation broth was overcooled by a given constant cooling water flow. The excess heat removed from the fermentor was then made up by an immersion electrical heater. The action of the temperature controller was precisely monitored as it varied in response to the amount of heat produced by the microbial activities.The technique was used for determining the heat evolution byEscherichia coli grown on glucose. The ratio between quantities of total heat release and total oxygen consumption has been determined to be 0.556 MJ/mol O₂.The newly developed technique can be employed as an online sensor to monitor the microbial activities of either aerobic or anaerobic fermentation systems.Symbols C_c Heat capacity of cooling water (MJ/kg · °C) - C_p Heat capacity (MJ/kg · °C) - I Current of immersion heater (A) - K Constant in Equation (2) (h) - K Constant in Equation (13) (m³ · h · °C/MJ) - Q_c Flow rate of cooling water (m³/h) - Heat of agitation (MJ/m³ · h) - Heat dissipated by the bubbling gas (MJ/m³ · h) - Heat removal by the action of controller (MJ/m³ · h) - Heat of fermentation (MJ/m³ · h) - Heat loss to the surroundings (MJ/m³ · h) - Q_pass Constant average power dissipated by the immersion heater (MJ/m³ · h) - Fluctuating power dissipated by the immersion heater (MJ/m³ · h) - Power dissipated by the immersion heater (MJ/m³ · h) - T Temperature of fermentation broth (°C) - Constant average temperature of fermentation broth (°C) - Fluctuating temperature of fermentation broth (°C) - T_a Temperature of the ambient air (°C) - T_c Inlet temperature of cooling water (°C) - U₁A₁ Specific heat transfer coefficient for determination of heat loss to the surroundings (MJ/m³ · h · °C) - U₂A₂ Specific heat transfer coefficient for cooling surfaces (MJ/m³ · h · °C) - U₃A₃ Constant in Equation (16) (MJ/m³ · h · °C) - V Voltage of immersion heater (V) - V_L Liquid volume (m³) - OUR Oxygen uptake rate (mol O₂/m³ · h) Greek Letters H_fo The ratio between the total heat release and the total oxygen uptake (MJ/mol O₂) - _c Density of cooling water (kg/m³) - Time constant defined in Equation (6) (h) - _iM_iC_pi Heat capacity of system components (fermentation broth + fermentor jar + stainless steel) (MJ/m³ · °C)     

Green- and blue-light-mediated chloroplast migration in the centric diatomPleurosira laevis

Summary The existence of two photoreceptors regulating chloroplast orientation was found in the centric diatomPleurosira laevis. Chloroplasts migrate through the transvacuolar cytoplasmic strands according to the light conditions. Weak white light of less than 46 mol/m² · s (10 W/m²) induces chloroplast movement to the cortical cytoplasm, which is located next to the plasma membrane (dispersion), while intense white light of more than 92 mol/m² · s (20 W/m²) induces chloroplast movement towards the nucleus, which is situated in the center of the cell (assemblage). Chloroplast dispersion was maintained as long as the cells were irradiated with weak white light. Conversely, chloroplast assemblage under intense white light was transient and the chloroplasts were released from assemblage after 15 min. Action spectra determined with the Okazaki Large Spectrograph revealed that the weak white light receptor and the intense white light receptor are characterized by 540 nm and 450 nm optima, respectively.     

Effects of dissolved oxygen concentration on lipase production by Rhizopus delemar

Summary The influence of the concentration of oxygen on lipase production by the fungus Rhizopus delemar was studied in different fermenters. The effect of oxygen limitation ( 47 mol/l) on lipase production by R. delemar is large as could be demonstrated in pellet and filamentous cultures. A model is proposed to describe the extent of oxygen limitation in pellet cultures. Model estimates indicate that oxygen is the limiting substrate in shake flask cultures and that an optimal inoculum size for oxygen-dependent processes can occur.Low oxygen concentrations greatly negatively affect the metabolism of R. delemar, which could be shown by cultivation in continuous cultures in filamentous growth form (D_optimal=0.086 h^-1). Continuous cultivations of R. delemar at constant, low-oxygen concentrations are a useful tool to scale down fermentation processes in cases where a transient or local oxygen limitation occurs.Symbols and Abbreviations CO Oxygen concentration in the gas phase at time = 0 (kg·m^-3) - C_{O 2i} Oxygen concentration at the pellet liquid interface (kg·m^-3) - C_{O 2i} Oxygen concentration in the bulk (kg·m^-3) - D Dilution rate (h^-1) - ID_{O 2} Diffusion coefficient for oxygen (m²·s^-1) - dw Dry weight of biomass (kg) - f Conversion factor (rs _{O 2} to oxygen consumption rate per m³) (-) - k Radial growth rate (m·s^-1) - K Constant - kla Volumetric mass transfer coefficient (s^-1) - klA Oxygen transfer rate (m^-3·s^-1) - kl Mass transfer coefficient (m·s^-1) - K _{O 2} Affinity constant for oxygen (mol·m^-3) - K _w Cotton plug resistance (m^-3·s^-1) - M Henry coefficient (-) - NV Number of pellets per volume (m^-3) - R Radius (m) - RO Radius of oxygen-deficient core (m) - RQ Respiration quotient (mol CO₂/mol O₂) - rs _{O 2} Specific oxygen consumption rate per dry weight biomass (kg O₂·s^-1[kg dw]^-1) - rX Biomass production rate (kg·m^-3·s^-1) - SG Soytone glucose medium (for shake flask experiments) - SG ₄ Soytone glucose medium (for tower fermenter and continuous culture experiments) - V Volume of medium (m^-3) - X Biomass (dry weight) concentration (kg·m^-3) - XR _o Biomass concentration within RO for a given X (kg·m^-3) - Y _{O 2} Biomass yield calculated on oxygen (kg dw/kg O₂) - Thiele modulus - Efficiency factor =1-(RO/R)³ (-) - Growth rate (m^-1·s^-1·kg^1/3) - Dry weight per volume of pellet (kg·m^-3)     

Production studies on protozoa

Summary In the river Saale and in the terrestrial moss Mnium cuspidatum Leyss. in 1974/75 the annual production of Testacea and loricate ciliated protozoa were investigated.The production was estimated in the Saale-Aufwuchs on a -meso ... oligosaprobic (Kaulsdorf, Thuringia, GDR) and on a -mesosaprobic (Rothenstein, Thuringia, GDR) area of the river. The mosses were investigated in a forest near Jena.The production was estimated on slides and in special productionchambers; the time of exposure was 2 weeks. Investigations concerned annual production of individuals and biomass, the ratio of annual production/standing crop (P/B), numbers of generations per year (G) and mortality (M%/d). In the mosses, the rainfall modified the production and dislocation of the protozoa.The values for production are: Aufwuchs Saale (-meso... oligosaprobic): 24·10⁶ i/m²·a (=1,0 g/m²·a=79·10³ i/m²·d); P/B: 12.6. Aufwuchs Saale (-mesosaprobic): 3.2·10⁶ i/m²·a (=0.35 g/m²·a=81·10³ i/m²·d); P/B: 34.9; G: 22; M: 5%/d. Moss: 145·10⁶ i/m²·a (=0.11 g/m²·a=40.6·10³ i/m²·d); P/B: 8.1; G: 16.5; M: 3.0%/d.     

Plasmodesmatal frequency and radial translocation rates in ray cells of poplar (Populus x canadensis Moench ‘robusta’)

The minimum radial translocation rate of sugars has been determined from the starchaccumulation rate for the wood rays of Populus x canadensis Moench robusta, and related to ultrastructural peculiarities of the cell walls to be passed. The minimum radial flux or flow of sugars through the tangential walls, the pit fields, and per plasmodesma was 80.7 pmol · cm^-2 · s^-1, 400 to 800 pmol · cm^-2 · s^-1, and 1.0 to 1.7 · 10^-7 pmol · plasmodesma^-1 · s^-1, respectively. These values exclude a transmembrane flux mechanism and indicate that the radial translocation in this tissue must proceed via plasmodesmata. In the isolation cells of the ray center we found 39 plasmodesmata per m² of pit field, 8.0 per m² of tangential wall, and 1.98% of the wall occupied by plasmodesmata. Cells of the ray margins show plasmodesmata on only 1.16% of their tangential wall area and thus appear to be slightly inferior for radial translocation. As judged from both the observed plasmodesmatal frequencies and the translocation rates, the ray parenchyma cells are comparable to cells specialized in short-distance translocation.Abbreviations CCR contact-cell row - IC isolation cell - ICR isolation-cell row     

Development of aldosterone-stimulation of short-circuit current across larval frog skin

Summary The short-circuit current (SCC) across isolated skin from bullfrog larvae in developmental stage XXI was small and insensitive to amiloride. Overnight incubation of this tissue with 10^-6 M aldosterone stimulated the SCC from 1.35±0.55 to 14.55±4.12 A·cm^-2 with 11.18±4.46 A·cm^-2 being blocked by 100 M amiloride. Histologic examination of aldosterone-treated skins revealed a separation of the apical cell layer from the underlying epidermis that was not seen in untreated preparations. The onset of amiloride-sensitive Na⁺ transport thus coincided with the exposure of the apical surface of newly differentiated epithelial cells. Similar results were obtained with skin from stage XXI larvae whose rate of metamorphosis had been stimulated by 10 g·l^-1 thyroxine (T₄) but not with skin from T₄-treated larvae in stages XIX and XX. Fluctuation analysis of the amiloride-sensitive SCC of the above preparations failed to show a consistent Lorentzian component in the power-density spectrum. Fluctuation analysis was possible on skins from larvae whose development had been accelerated by 7–9 days treatment with 10 g·l^-1 triiodothyronine (T₃). Aldosterone treatment of these tissues resulted in a significant increase in Na⁺ channel density.Abbreviations ASCC component of the short-circuit current (A·cm^-2) that is blocked by amiloride - fc frequency (Hz) at which the magnitude of the Lorenzian component of the power spectra is reduced by half - i current (pA) through individual amiloride-sensitive Na⁺ channels - I _Na+ amiloride-sensitive short-circuit current (A·cm^-2) that remains after treatment with a given amiloride concentration - k ₀₁ the rate constant (s^-1·M^-1) for the association of amiloride with Na⁺ channels - k ₁₀ rate constant (s^-1) for the dissociation of amiloride from Na⁺ channels - K _b magnitude of the power spectrum (A²·s·cm^-2) at a frequency of 1 Hz - KSCC short-circuit (A·cm^-2) current with K⁺ as the primary mucosal cation - M density of amiloride-sensitive Na⁺ channels in the apical cell membrane - SCC short-circuit current (A·cm^-2) - S _(f) magnitude of the power spectra (A²·s·cm^-2) at a given frequency - S ₀ the magnitude of the plateau region of the Lorentzian component of the power spectra (A²·s·cm^-2) - T ₃ Triiodothyronine - T ₄ Thyroxine     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans has been proposed to oxidize acetate to CO₂ via an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway rather than via the citric acid cycle. We report here the presence of the enzyme activities required for the operation of the novel pathway. In cell extracts the following activities were found (values in brackets=specific activities and apparent K _m; 1 U·mg^-1=1 mol·min^-1·mg protein^-1 at 37°C): Acetate kinase (6.3 U·mg^-1; 2 mM acetate; 2.4 mM ATP); phosphate acetyltransferase (60 U·mg^-1, 0.4 mM acetylphosphate; 0.1 mM CoA); carbon monoxide dehydrogenase (29 U·mg^-1; 13% carbon monoxide; 1.3 mM methyl viologen); 5,10-methylenetetrahydrofolate reductase (3 U·mg^-1, 0.06 mM CH₃–FH₄); methylenetetrahydrofolate dehydrogenase (3.6 U·mg^-1, 0.9 mM NAD, 0.1 mM CH₂=FH₄); methenyltetrahydrofolate cyclohydrolase (0.3 U·mg^-1); formyltetrahydrofolate synthetase (3 U·mg^-1, 1.4 mM FH₄, 0.4 mM ATP, 13 mM formate); and formate dehydrogenase (10 U·mg^-1, 0.4 mM formate, 0.5 mM NAD). The specific activities are sufficient to account for the in vivo acetate oxidation rate of 0.26 U·mg^-1.Non-standard abbreviations FH₄ Tetrahydrofolate - CHO-FH₄ N¹⁰-formyltetrahydrofolate - CHFH₄ N⁵,N¹⁰-methenyltetrahydrofolate - CH₂=FH₄ N⁵,N¹⁰-methylenetetrahydrofolate - CH₃–FH₄ N⁵-methyltetrahydrofolate - MOPS morpholinopropane sulfonic acid - DTT d,l-1,4-dithiothreitol - TRIS tris-(hydroxymethyl)-aminomethane - Ap₅A p¹,P⁵-di(adenosine-5)pentaphosphate - MV methyl viologen     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Effect of light intensity on pigments and main acyl lipids during ‘natural’ chloroplast development in wheat seedlings

The content and composition of pigments and acyl lipids (monogalactosyl diacylglycerol, digalactosyl diacylglycerol and phosphatidyl glycerol) have been investigated in developing chloroplasts isolated from successive 2-cm sections along the leaves of wheat seedlings grown either under 100, 30 or 3 W·m^-2. In all examined stages of plastid development chlorophyll a/b and chlorophyll/carotenoid ratios were higher with increasing irradiance, whereas chlorophyll content expressed on fresh weight basis gradually decreased.Concentrations of monogalactosyl diacylglycerol, digalactosyl diacylglycerol and phosphatidyl glycerol decreased per chlorophyll unit with increasing plastid maturity. The higher was the light intensity applied during plant growth, the higher were galactolipid and phosphatidyl glycerol contents in developing chloroplasts. During plastid development the percentage of -linolenic acid markedly increased in total and individual acyl lipids. Under high light conditions, the accumulation of this fatty acid proceeded more rapidly. Significantly higher proportion of -linolenic acid was found in acyl lipid fraction of chloroplasts differentiating in high light grown plants, than in those from plants exposed to lower light intensities. The differences in the double bond index may indicate higher fluidity of thylakoid membranes in sun-type chloroplasts.Trans-3-hexadecenoic acid, virtually absent in the youngest plastids, was found in much higher concentration (per chlorophyll unit and as mol % of phosphatidyl glycerol fatty acids) in chloroplasts developing at high light conditions.Abbreviations MGDG monogalactosyl diacylglycerol - DGDG digalactosyl diacylglycerol - PG phosphatidyl glycerol - PC phosphatidyl choline - DBI double bond index - PS I photosystem I - PS II photosystem II - PSU photosynthetic unit - LHCP light harvesting chlorophyll-protein complex     

Regulation of the orientation movement of chloroplasts in epidermal cells of Vallisneria: cooperation of phytochrome with photosynthetic pigment under low-fluence-rate light

In epidermal cells of the leaves of the aquatic angiosperm Vallisneria gigantea Graebner, the chloroplasts accumulate in the outer periclinal layer of cytoplasm (P side) under light at low fluence rates. The nature of such intracellular orientation of chloroplasts was investigated in a semiquantitative manner. Time-lapse video microscopy revealed that, while irradiation with red light (650 nm, 0.41 W · m^–2) rapidly accelerated the migration of chloroplasts, not only from the anticlinal layers of cytoplasm (A sides) to the P side but also from the P side to the A sides, the increased rate of migration in both directions returned to the control rate upon subsequent irradiation with far-red light (746nm, 0.14W · m^–2). These effects of red and far-red light could be observed repeatedly, both in the presence and in the absence of inhibitors of photosynthesis, suggesting the involvement of phytochrome as the photoreceptor. After saturating irradiation with red light, the increased rate of migration of chloroplasts from the P side to the A sides declined more rapidly than the increased rate of migration in the opposite direction. This imbalance in the migration of chloroplasts between the two opposing directions resulted in the accumulation of chloroplasts on the P side. The more rapid decline in the rate of migration of chloroplasts from the P side to the A sides than in the opposite direction was not observed in the presence of an inhibitor of photosynthesis. It appears, therefore, that phytochrome and photosynthetic pigment cooperatively regulate the accumulation of chloroplasts on the P side through modulation of the nature of the movement of the chloroplasts.Abbreviations A side cytoplasmic layer that faces the anticlinal wall - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - P side cytoplasmic layer that faces the outer periclinal wall This work was supported in part by Grants-in-Aid from the Japanese Ministry of Education, Science and Culture to S.T. and R.N. The authors are indebted to the Osaka branch of Kashimura Inc. for their kind cooperation in preparing the GREEN software.