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.

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.

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Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe"'EDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (3040 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts.Ferrocyanide has no effect. In the state of ferricyanide reduction, H' (1H/2 electrons) and K' ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.

The electrical properties of the tonoplast from a large variety of plant materials such as mesophyll cells, storage cells, tumor cells, suspension cultured cells, guard cells, coleoptile cells, and liverwort cells have been investigated using the patch‐clamp technique. Whole‐vacuole recordings were employed to study the dynamics of an ATP‐dependent proton pump by directly measuring the electrogenic currents. The addition of Mg‐ATP induced an inwardly directed current which depolarized the tonoplast (the vacuole becoming positive inside). Furthermore, voltage‐dependent passive ion fluxes were analyzed using whole vacuoles and isolated membrane patches. Whole‐vacuolar currents and single‐channel currents were induced at hyperpolarizing potentials, whereas currents decreased at positive trans‐tonoplast potentials. The electrical properties of the tonoplast of vacuoles from various plant tissues were similar and it was concluded that ion fluxes across the tonoplast follow the same general mechanisms.

The utiiLzation of HC03-as carbon source for photosynthesis by aquatic angiosperms results in the production of 1 mole OH-for each mole CO2 assimilated. The OH-ions are subsequently released to the medium. In several Potamogeton and Elodea species, the site of the HC03-influx and OH-efflux are spatially separated. Described here are light-and darkinduced pH changes at the lower and upper epidermis of the leaves of Potamogeton lucens, Elodea densa, and Elodea canadensis.In the light, two phases could be discerned. During the first phase, the pH increased at both sides of the leaves. geton and Elodea are accompanied by cation transport from the lower to the upper side of the leaf and by the formation of an electrical PD across the leaf, making the upper side negative with respect to the lower side (5, 6, 7). Comparable PD were also observed in Chara corallina cells. In these latter cells, electric currents are generated between the alkaline and acid regions and may result in electrical PD of 7 mv between the centers of the bands (4, 18, 28). The highest value we observed so far with a Potamogeton lucens leaf was 40 mv, upper side negative.We analyzed the time course of the light induced pH changes at both sides and the trans-leaf PD of leaves of Potamogeton and Elodea with miniature pH and reference electrodes positioned against the leaf surface.For comparison, we studied the pH changes at the leaf surface of Vallisneria spiralis, a HCO3 -fixing aquatic species with no separation of the HCO3 and OH-transport sites, and of Ludwigia natans, a waterplant that used CO2 only as a C source for photosynthesis. MATERIALS AND METHODSP. lucens and Elodea canadensis were grown in concrete tanks outside the building, whereas Elodea densa, V. spiralis and L. natans were grown inside under artificial light as described earlier (21). All plants were cultivated in H20 at pH 7.8 to 9.5.The pH near the surface on both sides of the leaves was measured simultaneously with two miniature pH electrodes (Microelectrodes Ml 440) with the sensing bulb touching gently on the leaf surface. Two reference electrodes (Microelectrodes Ml 401) filled with 3 M KCI were also positioned with their tips very near the leaf surface on both sides. These reference electrodes were also used to measure the electrical PD across the leaf (Fig. 1). The pH-sensitive part of the glass electrode is a hemisphere of radius 0.75 mm and the wall is not of uniform thickness. The end which touches the leaf surface is much thinner and, thus, is the most sensitive part. To enlarge the contact between the sensing bulb and the leaf surface, we folded the leaf slightly around the bulbs (Fig. 1B)