The utilization of inorganic carbon species by the marine microalga Phaeocystis globosa (Prymnesiophyceae) and several other algal species from different taxa, was investigated by determining the time course of 14 C incorporation in isotopic disequilibrium experiments. From these kinetic data, conclusions can be drawn about the carbon species, CO 2 or HCO 3 Ϫ , that is being utilized. By comparing the uptake kinetics in the absence and presence of acetazolamide (AZ) or dextran-bound sulfonamide, inhibitors of external carbonic anhydrase (CA), it was determined that P. globosa, Dunaliella tertiolecta, and some strains of Emiliania huxleyi do use HCO 3 Ϫ by extracellular, CA-catalyzed conversion to CO 2 , which then diffuses across the membrane. Nannochloropsis, Thalassiosira pseudonanna, and often Synechococcus use HCO 3 Ϫ without extracellular conversion. Thalasiosira punctigera, some strains of E. huxleyi, and Rhodomonas sp. use exclusively free CO 2 . The presence of extracellular CA activity in Phaeocystis is not constitutive but is induced under low inorganic carbon conditions. Thus, marine microalgae show variability in carbon acquisition strategy for one single species, depending on external conditions, and in carbon acquisition strategy between species. Determining AZ-induced changes in carbon uptake kinetics provides a sensitive test for the presence of extracellular CA activity. With the potentiometric method, no CA activity could be measured, whereas with the isotopic disequilibrium technique, significant CA activity could be detected.For photosynthesizing aquatic macro-and microphytes, the chemical composition of the unstirred layer surrounding the cells differs from that of the bulk of the medium. Uptake of CO 2 or any other nutrient by the cell depletes the immediate environment and creates a concentration gradient. In a steady state, the uptake of CO 2 is balanced by diffusion from the bulk medium into the unstirred layer and by production of CO 2 from HCO 3 Ϫ and CO 3 2Ϫ in the unstirred layer itself (Wolf-Gladrow and Riebesell 1997). Because of the slow rate of formation from HCO 3 Ϫ and diffusional limitation through the unstirred layer around the cells, the availability of CO 2 may limit photosynthesis and growth of marine algal species (Riebesell et al. 1993;Chen and Durbin 1994). Strategies used by aquatic species to overcome these limitations include the active uptake of one of the carbon species across one of the membranes, either the plasmamembrane or the chloroplast envelope that separate the external medium from the site of fixation. This mechanism is commonly referred to as carbon concentrating mechanism (CCM). Another strategy is the carbonic anhydrase-catalyzed extracellular conversion of HCO 3 Ϫ to CO 2 , followed by diffusion of CO 2 across the membrane. These mechanisms are often only induced by conditions where CO 2 is 1 Corresponding author (j.t.m.elzinga@biol.rug.nl). AcknowledgmentsThis work is a contribution to the European Union ELOISE Programme (ELOISE No. 108) in t...

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Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K+ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (Em) was not sensitive to the external K+ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K(+)-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235-246). The activation and inactivation times and the activation potential, all depended on the reversal potential (Erev) of the s-ORC conductance. The s-ORC was blocked by Ba2+ (K1/2 = 0.3-1.3 mM) and verapamil (K1/2 = 15-20 microM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba2+. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K+ concentration. The slow activation of the IRC (t1/2 approximately 0.5 s) and its negative activation potential (Vthreshold = -155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701-2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes.

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Photosynthetic utilization of HCO, in leaves of Pot(tmogeton and Elodea occurs at the lower leaf side, with subsequent OH~ release at the upper side. It is accompanied by transport of cations, in the present experiment K^, across the leaf. The resulting pH and K"^ concentration changes near the leaf surface were recorded with miniature electrodes. From the pH and K^ concentration the concentrations of the different inorganic carbon species were calculated and compared with photosynthetic O2 production, HCO3' utilization is accompanied by a drastic increase in the free CO2 concentration near the lower epidermis. Experiments with CO2-and HCO3"-free solutions showed an oscillating acidification near the lower epidermis and alkalinization near the upper epidermis. It is concluded that the acidification results from the activity of light-dependent H"^ pumps. The finding that an increase in pH at the upper side always coincided with a decrease at the lower in these experiments shows that the H ^ pumps and the OH " extruding mechanism are coupled although occurring in different cell layers. Previously we have suggested that the first step in the process of photosynthetic HCO.^ utilization is external conversion of HCO3" by acidification caused by light-dependent H^ pumps. The present results strongly support this hypothesis. Two possible pathways for the accompanying Kt ransport are discussed. The model presented here explains the known inhibiting effects of buffers and high pH on photosynthetic HCO^ utilization.