Analysis of oxygen evolution during photosynthetic induction and in multiple-turnover flashes in sunflower leaves

Exchange of CO₂ and O₂ and chlorophyll fluorescence were measured in the presence of 360 1 · 1^–1 CO₂ in nitrogen in Helianthus annuss L. leaves which had been preconditioned in the dark or at a photon flux density (PFD) of 24 mol · m^–2 · s^–1 either in 21 or 0% O₂. An initial light-dependent O₂ outburst of 6 mol · m^–2 was measured after aerobic dark incubation. It was attributed to the reduction of electron carriers, predominantly plastoquinone. The maximum initial rate of O₂ evolution at PFD 8000 mol · m^–2 · s^–1 was 170 mol · m^–2 · s^–2 or about four times the steady CO₂-and light-saturated rate of photosynthesis. Fluorescence measurements showed that the rate was still acceptor-limited. Fast O₂ evolution ceased after electron carriers were reduced in the dark-adapted leaf, but continued for a short time at the lower rate of 62 mol · m^–2 · s^–1 in the light-adapted leaf. The data are interpreted to show that enzymes involved in 3-phosphoglycerate reduction are dark-inhibited, but were fully active in low light. In a dark-adapted leaf, respiratory CO₂ evolution continued under nitrogen; it was partially inhibited by illumination. Prolonged exposure of a leaf to anaerobic conditions caused reducing equivalents to accumulate. This was shown by a slowly increasing chlorophyll fluorescence yield which indicated the reduction of the PSII acceptor Q_A in the dark. When the leaf was illuminated, no O₂ evolution was detected from short light pulses, although transient O₂ production was appreciable during longer light pulses. This indicates that an electron donor (pool size about 2–3 e/PSII reaction center) became reduced in the dark and the first photons were used to oxidise this donor instead of water.Abbreviations Chl chlorophyll - CRC carbon reduction cycle - GAPDH NADP-glyceraldehyde-phosphate dehydrogenase - PFD photon flux density - PGA 3-phosphoglycerate - RuBP ribulose bisphosphate - TCA tricarboxylic acid cycle To whom correspondence should be addressedThis work received support by the Estonian Academy of Sciences, the Gottfried-Wilhelm-Leibniz Program of the Deutsche For-schungsgemeinschaft and the Sonderforschungsbereich 251 of the University of Würzburg.     

The effect of carbon dioxide on the growth kinetics of fructose-limited chemostat cultures of Acetobacterium woodii DSM 1030

The growth of the anaerobic acetogenic bacterium Acetobacterium woodii DSM 1030 was investigated in fructose-limited chemostat cultures. A defined medium was developed which contained fructose, mineral salts, cysteine · HCl and Ca pantothenate (1 mg · 1^–1) supplied in a vitamin supplement. Growth at high dilution rates was dependent on the presence of CO₂ in the gas phase. The ^max was found to be 0.16 h^–1 and the fructose maintenance requirement was 0.1 to 0.13 mmol fructose · (g dry wt)^–1 · h^–1. A growth yield of 61 g dry wt · (mol fructose)^–1, corrected for the cell maintenance requirement and for incorporation of fructose carbon into cell biomass, was determined from the fructose consumption. A corresponding growth yield of 69 g dry wt · (mol fructose)^–1 was calculated from the acetate production assuming that fructose fermentation was homoacetogenic. A Y_ATP of 12.2 to 13.8 g dry wt · (mol ATP)^–1 was calculated from these growth yields using a value of 5 mol ATP · (mol fructose)^–1 as an estimate of the amount of ATP synthesised from fructose fermentation. The addition of yeast extract (0.5 g · 1^–1) to the medium did not influence the ^max or cell yield. After prolonged growth under fructose-limited conditions the requirement of the culture for CO₂ in the gas phase was reduced.Abbreviations YE yeast extract - IC inorganic carbon - D fermenter dilution rate : h^–1 - M_X maintenance requirement for X: mmol X · (g dry wt)^–1 · h^–1 - X may be fructose (Fruct), fructose consumed in energy metabolism (Fruct [E]), acetate (Ac) - ATP CO₂, NH _inf4 ^sup+ or Pi - qX specific rate of utilisation or consumption of X: mmol X · (g dry wt)^–1 · h^–1 - V fermenter volume: litre - rC · Cell, fermenter cell carbon production: mmol C · h^–1 - Y_X yield of cells on X: g dry wt · (mol X)^–1 - Y _infx ^supmax the yield corrected for cell maintenance: g dry wt · (mol X)^–1 - S_ATP stoichiometry of ATP synthesis from fructose: mol ATP · (mol frucose)^–1 - x cell concentration: g dry wt · 1^–1 - specific growth rate : h^–1 - ^max maximum specific growth rate: h^–1     

Phosphatase activity ofPenicillium citrinum submerged batch cultures and its relationship to fungal activity

Acid and alkaline phosphatase activities were evaluated using batch fermenter cultues ofPenicillium citrinum, an organism used in studies of fungal functioning in soil. Fungal activity was assessed by monitoring rates of O₂ utilization, glucose utilization, dry weight changes over time, and lengths of FDA-stained hyphae. At low growth rates (7 g dry wt increases·h^–1·ml^–1) and low culture activity, phosphatase activity at both pH 8.5 and 5.5 tended to decrease with culture age, with the exception that phosphatase activity at pH 8.5 peaked during early stationary phase. At higher growth rates (25 g dry wt increase·h^–1·ml^–1) and high culture activity, phosphatase activity tended to remain constant throughout the course of the experiment. The relationship between phosphatase activity and other measures of fungal activity was consistent only at low growth rates for acid phosphatase. These results suggest that phosphatase measurements will be of limited utility in assessing activity, except at low growth rates.     

Transient state characteristics of adaptation to changes in light conditions for the cyanobacterium Oscillatoria agardhii

Transitions in growth irradiance level from 92 to 7 Em^-2 s^-1 and vice versa caused changes in the pigment contents and photosynthesis of Oscillatoria agardhii. The changes in chlorophyll a and C-phycocyanin contents during the transition from high to low irradiance (HL) were reflected in photosynthetic parameters. In the LH transition light utilization efficiencies of the cells changed faster than pigment contents. This appeared to be related to the lowering of light utilization efficiencies of photosynthesis. As a possible explanation it was hypothesized that excess photosynthate production led to feed back inhibition of photosynthesis. Time-scales of changes in the maximal rate of O₂ evolution were discussed as changes in the number of reaction centers of photosystem II in relation to photosynthetic electron transport. Parameters that were subject to change during irradiance transitions obeyed first order kinetics, but hysteresis occurred when comparing HL with LH transients. Interpretation of first order kinetic analysis was discussed in terms of adaptive response vs changes in growth rate.Non-standard abbreviations Chla chlorophyll a - CPC C-phycocyanin - PS II photosystem II - PS I photosystem I - RC II reaction center of photosystem II - P photosynthetic O₂-evolution - I irradiance, Em^-2 s^-1 - light utilization efficiency of cells, mmol O₂·mg dry wt^-1·h^-1/Em^-2 s^-1 - light utilization efficiency of photosynthetic apparatus, mol O₂·mol Chla ^-1·h^-1/Em^-2 s^-1 - P_max maximal rate of O₂ evolution by cells, mol O₂·mg dry wt^-1·h^-1 - P_max maximal rate of O₂ evolution by photosynthetic apparatus, mol O₂·mol·Chla ^-1·h^-1 - LL low light, E m^-2 s^-1 - HL high light, E m^-2 s^-1 - LH low to high light transition - HL high to low light transition - k specific rate of adaptation, h^-1 - specific growth rate, h^-1 - Q pool size of cell constituent, mol·mg dry wt^-1 - q net synthesis rate of cell constituent, mol·mg dry wt^-1·h^-1     

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     

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     

Nitrate induction in spruce: an approach using compartmental analysis

Using ¹³NO ₃ ^– -efflux analysis, the induction of nitrate uptake by externally supplied nitrate was monitored in roots of intact Picea glauca (Moench) Voss. seedlings over a 5-d period. In agreement with our earlier studies, efflux analysis revealed three compartments, which have been identified as surface adsorption, apparent free space, and cytoplasm. While induction of nitrate uptake was pronounced, NO ₃ ^– fluxes in induced plants were decidedly lower and the induction response was slower than in other species. Influx rose from 0.1 mol·g^–1·h^–1 (measured at 100 M [NO ₃ ^– _o) in uninduced plants to a maximum of 0.5 mol·g^–1h^–1 after 3 d of exposure to 100 M [NO ₃ ^– _o and declined to 0.3–0.4 mol·g^–1h^–1 at the end of the 5-d period. Efflux remained relatively constant around 0.02-0.04 mol·g^–1h^–1, but its percentage with respect to influx declined from initially high values (around 30%) to steady-state values of 4–7%. Cytoplasmic [NO ₃ ^– ] ranged from the low micromolar in uninduced plants to a maximum of 2 mM in plants fully induced at 100 M [NO ₃ ^– ]_o. In-vivo root nitrate reductase activity (NRA) was measured over the same time period, and was found to follow a similar pattern of induction as influx. The maximum response in NRA slightly preceded that of influx. It increased from 25 nmol·g^–1·h^–1 without prior exposure to NO ₃ ^– to peak values around 150 nmol· g^–1h^–1 after 2 d of exposure to 100 M [NO ₃ ^– ]_o. Subsequently, NRA declined by about 50%. The dynamics of flux partitioning to reduction, to the vacuole, the xylem, and to efflux during the induction process are discussed.The research was supported by an Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. J. Vidmar for essential assistance in experiments.     

Proton translocation in corn coleoptiles: ATPase or redox chain?

The O₂ dependence of net H⁺ efflux of maize coleoptiles has been investigated. Below 100 M O₂, H⁺ efflux in young (1 cm long) coleoptiles is markedly decreased while old (7 cm long) coleoptiles show a decline only at 10 M O₂. Old coleoptiles show the same decrease in net H⁺ efflux as young ones if treated with fusicoccin. The ratio of alteration of CO₂ production to the change in net proton efflux is about 1:1 at 40–80 M O₂ but not at 10 M O₂. An influx can be observed at 10 M O₂ in young as well as in old coleoptiles if the H⁺ concentration is held at values below pH 6.5. Lower O₂ concentrations lead to an increase of net H⁺ efflux, which might be caused by leaching of organic acids resulting from anaerobic processes, but CO₂ production is not significantly changed at these values. It is proposed that more than one system is responsible for proton translocation across the plasmalemma. One of the systems has a high sensitivity to reduced O₂ concentration which is within the same range as the high K_m of the alternative path.Abbreviation FC fusicoccin     

Water chemistry changes in the gill micro-environment of rainbow trout: experimental observations and theory

Summary Soft water of low buffer capacity was drawn from near the branchial surface of rainbow trout (Salmo gairdneri) at 15°C, using opercular catheters, to determine pH changes in water passing over the gills. Latex masks allowed measurement of ventilation volume, and concentrations of carbon dioxide, oxygen, ammonia, and titratable base in expired water were compared to concentrations in inspired water. Water passing over the gills was more basic than inspired water if the inspired water was pH 4–6 (maximum increase: +0.7 pH units near pH 5). Expired water was more acidic than inspired water if the inspired water was pH 6–10 (maximum decrease: –1.7 pH units near pH 9). Ventilation volume (0.37 l·kg^–1·min^–1) and oxygen consumption (1.7 mmol·kg^–1·h^–1) were constant in the pH range 4.6–10.1, but both increased by 1.6–2.4× near pH 4. Carbon dioxide transfer near the gills was about 100 M, ammonia transfer about 15 M, and titratable base added at the gills was about 30 M. A theoretical model using CO₂, titratable base, and ammonia added at the gills, the titration characteristics of the defined soft water medium, and aquatic equilibria for CO₂ and ammonia, adequately explained the experimentally observed changes in pH near trout gills. Our observations and predictive model indicate that any gill contaminant whose toxicity varies with pH may be more or less toxic at the gills than predicted from bulk water chemistry alone.Abbreviations pH _ex expired pH - pH _in inspired pH     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Temperature insensitive O2 in blood of the tree frogChiromantis petersi

Summary Respiratory gas exchange and blood respiratory properties have been studied in the East-African tree frogChiromantis petersi. This frog is unusually xerophilous, occupies dry habitats and prefers body temperatures near 40°C and direct solar exposure. Total O₂ uptake was low at 81 l O₂·g^–1·h^–1±19.0 (SD) at 25°C increasing to 253.5 l O₂·g^–1·h^–1±94.8 (SD) at 40°C giving aQ ₁₀ value of 2.1. Skin O₂ uptake at 25°C was 38.5% of total. The gas exchange ratio was 0.71 for whole body gas exchange, 0.61 for the lungs and 1.02 for the skin at 25°C.Blood O₂ affinity was low with aP ₅₀ of 47.5 mmHg at 25°C and pH 7.65. Then _H-value at 25°C increased from 2.7 aroundP ₅₀ to 5.0 at O₂ saturations exceeding 70–80%. Surprisingly, blood O₂ affinity was nearly insensitive to temperature expressed by a H value of ±1.0 kcal·mole between 25 and 40°C.The adaptive significance of the low O₂ affinity, the increase ofn _H with O₂ saturation and the temperature insensitive O₂-Hb binding is discussed in relation to the high and fluctuating body temperatures ofChiromantis.     

Photosynthesis in enzymatically isolated leaf cells from the CAM plant Sedum telephium L.

A technique has been developed for the enzymatic isolation of leaf cells from the Crassulacean acid-metabolism plant Sedum telephium. The cells exhibited high activity in both ¹⁴CO₂ incorporation (30–70 mol CO₂ mg^-1 chlorophyll h^-1) and O₂ evolution in the presence of bicarbonate (60–110 mol O₂ mg^-1 chlorophyll h^-1). Half-maximum saturation of ¹⁴CO₂ incorporation occurred at a bicarbonate concentration of ca. 2 mM (20 M CO₂) at pH 8.4 and 30°C. Two types of light-dependent O₂ evolution are reported: O₂ evolution in the absence of exogenously supplied bicarbonate (endogenous O₂ evolution), and bicarbonate-stimulated O₂ evolution. Oxygen evolution in the presence of approximately ambient concentrations of CO₂ appeared to be a combination of the endogenous O₂ evolution and O₂ evolution from fixation of the exogenously supplied CO₂.Abbreviations CAM Crassulacean acid metabolism - cirlo chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - RuDP ribulose-1,5-diphosphate     

n-3 PUFA productivity in chemostat cultures of microalgae

Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid productivities from chemostat cultures of an isolate of Isochrysis galbana have been studied. The productivities reached in the interval of dilution rates between 0.0295 h^–1 and 0.0355 h^–1 were 1.5mg·1^–1·h^–1 for lipids, 300 g·1^–1·h^–1 for EPA and 130g1·1^–1·h^–1 for DHA. Furthermore, light attenuation by mutual shading, and agitation speed influences on growth and fatty acid composition were analysed. A model relating steady-state dilution rates to internal average light intensity has been proposed, the parameter values of which obtained by non-linear regression were: maximum specific growth rate (_max)=0.0426 h^–1; the affinity of cells to light (I_k) = 10.92 W·m^–2; the exponent (n) = 5.13; regression coefficient (r ²)=0.9999. Correspondence to: E. Molina Grima     

Nitrogen fixation byRubus ellipticus J. E. Smith

Summary Nitrogenase activity as assayed by acetylene reduction was observed in detachedRubus ellipticus J. E. Smith root nodules collected in the field and tested under ambient conditions. The nitrogenase activity was 8.4 moles C₂H₄.gfr. wt nodule^–1.h^–1 or 24.0 moles C₂H₄.g dry wt nodule^–1.h^–1 being at a rate comparable with that measured in some other non-legumes assayed in Java at the same time under similar conditions. Nodule morphology bore little resemblance to the root nodules of other non-leguminous plants and nodule structure was different from the other rosaceous examples.The endophyte inhabiting the root nodules was actinomycetal.     

CO2 gas exchange of the understory plantAsarum europaeum L. in November

The CO₂ gas exchange rates of the Central European perennial understory plantAsarum europaeum L. were measured in late autumn (October 30 to November 30) in its natural habitat day and night.During these measurements the temperature ranged from 0 to 15°C and the absolute air humidity from 3 to 10 mg H₂O·1^–1. Temperature and absolute air humidity over these ranges did not affect CO₂ net assimilation which was determined almost entirely by quantum flux density.CO₂ net assimilation was light saturated at about 100 M·m^–2·s^–1 quantum flux density. The uptake rate at this point was 4.3 mg·dm^–2·h^–1. The compensation point occurred at approximately 1 M·m^–2·s^–1.     

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl^– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^–1), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol·l^–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l^–1 as NH₄Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO ₃ ^– buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol·l^–1). Neither bafilomycin A₁ (3 mol·l^–1) nor Cl removal altered the intracellular pH recovery rate. The K _m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol·l^–1, and the rate constant at V _max was 0.008·s^–1 (equivalent to 5.60 mmol H⁺·l^–1 cell water·min^–1), which was achieved at external Na⁺ levels from 25 to 140 mmol·l^–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO ₃ ^– -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺-ATPase.Abbreviations BCECF 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein - BCECF/AM 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester - Cholin-Cl choline chloride - DMSO dimethyl sulfoxide - EDTA ethylene diamine tetra-acetic acid - FBS foetal bovine serum - H ⁺ -ATPase Proton-dependent adenosine triphosphatase - HEPES N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid] - pH _i intracellular pH - pH _e extracellular pH - PBS phosphate-buffered saline - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to decrease the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO₂ at 20°C at 100, 300 and 750 mol·m^–2·s^–1 irradiance, and at 28°C at 100, 300 and 1000 mol·m^–2·s^–1 irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (C _infRubisco ^supA ) was very low (0.01–0.03) at low growth irradiance, and still fairly low (0.24–0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that C _infRubisco ^supA increases as incident irradiance rises, (iii) When low-light (100 mol·m^–2·s^–1)-grown plants are exposed to high (750–1000 mol·m^–2·s^–1) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750–1000 mol·m^–2·s^–1) are suddenly exposed to high and saturating irradiance (1500–2000 mol·m^–2·s^–1), C _infRubisco ^supA remained relatively low (0.23–0.33), showing that in saturating light Rubisco only exerts partial control over the light-saturated rate of photosynthesis in sun leaves; apparently additional factors are co-limiting photosynthetic performance, (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the light reactions and Rubisco; this was shown by the low value of C _infRubisco ^supA (see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions, (v) Antisense plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.Abbreviations A rate of photosynthesis - C _infRubisco ^supA flux control coefficient of Rubisco for photosynthesis - c_i internal CO₂ concentration - qE energy-dependent quenching of chlorophyll fluorescense - qQ photochemical quenching of chlorophyll fluorescence - NADP-MDH NADP-dependent malate dehydrogenase - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose-1,5-bisphosphate This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137).