The Permeability of the Guard Cell Plasma Membrane and Tonoplast

Uptake experiments and efflux compartmental analysis of planthormones, osmotica and toxins using ‘isolated’ guardcells of Valerianella locusta and guard cell protoplasts (GCP)of Vicia faba were performed in order to study the permeabilityproperties of guard cell plasma membrane and tonoplast. Theplasma membrane of guard cells exhibits a higher permeabilitythan plasma membranes of mesophyll cells for most solutes investigated.The permeability coefficients (P_s calculated for the guard cellplasma membranes are also significantly higher than the P_s valuesfor the guard cell tonoplast. This applies also for protonatedABA. We suppose that the high permeability for ABAH could bepart of the target cell properties. A Collander analysis demonstratesa linear correlation between P_s, values and the ratio K_r/M_r^1,5for both plasma membrane (r = 0.87) and for the tonoplast (r=0.93). Because of deviations from the observed correlations,the permeation of some solutes (ABA, GA, IAA through the tonoplast;methylamine through the plasma membrane) seems to be facilitatedby an additional transport mechanism. The Collander analysisof the plasma membrane of GCP shows very similar results tothe analysis of the plasma membrane of ‘isolated’guard cells, indicating that isolation of protoplasts does notalter the permeability of the guard cell plasma membrane. Key words: Permeability coefficient, guard cells, plasma membrane, tonoplast     

Cl- Fluxes and Cl- Content of Dunaliella acidophila--An Alga with a Positive Membrane Potential

The Cl^– fluxes across the plasma membrane and the Cl^–content of the acid–resistant alga Dunaliella acidophila(optimal growthat pH 1.0, positive membrane potential) werestudied in the presence of 0.01–300 mM Cl^–. Up to40 mM Cl^– in the medium, theinternal Cl^– concentrationis higher than that predicted by the electrochemical equilibrium,whereas at higher external Cl^– concentrations internalCl^– levels are lower than expected for the electrochemicalequilibrium. Growth in the absence of Cl^– is significantlylower than in the standard growth medium (2.2 mM Cl^–)and this reduction cannot be overcome by the addition ofothermonovalent anions such as Br^– or NO^–₃ The latterimplies a specific Cl^– requirement in addition to therole of Cl^– as apermeant anion during ion translocations.Growth and photosynthesis tolerate an excess of Cl^– upto 300 mM (without stepwiseadaptation to increasing salinity).The uptake of Cl^– (measured by tracer techniques) exhibitsMichaelis–Menten kinetics (K_M = 0.75 mM Cl^–) andis stimulated by light and high H⁺ concentrations. Internalacidification by acetic acid causes an inhibition of Cl^–uptake. The uptake of Cl^– is inhibited by the monovalentanions Br^–, I^–, and NO^–₃ with K¹, values notvery much different from the K_M. value for Cl^–. The aniontransport inhibitors SITS and DIDS do not affect photosynthesis,but strongly suppressthe uptake of Cl^–. The Cl^–channel blockers A–9–C and NPPB cause inhibitionsof Cl^– uptake as well as of photosynthesis andthe ATPpool. FCCP strongly depresses the internal ATP–pool withouta marked effect on Cl^– uptake. Cl^– efflux was inhibitedbyDIDS and SITS, but stimulated or inhibited by FCCP, dependingon the external Cl^– concentration. Results are in agreementwiththe hypothesis that Cl^– uptake into D. acidophila is dueto catalysed diffusion and is primarily independent of the hydrolysisofATP. Cl^– efflux is assumed to be coupled to an activepump. Data suggest tight co–operativity between the systemsresponsiblefor Cl^– uptake and Cl^– efflux, with thecytoplasmic pH and the membrane potential being important mediators. Key words: Acid resistance, chloride carrier, chloride channels, Dunaliella acidophila, membrane potential, plasma membrane     

Dunaliella acidophila: an algae with a positive zeta potential at its optimal pH for growth*

Abstract. The zeta potential (which approximates the surface potential) of the acid resistant green alga Dunaliella acidophila (optimal growth at pH 1.0) and the salt resistant D. parva (grown at pH 7.6) were calculated from the electrophoretic mobility of cells as determined by means of free-flow electrophoresis. Dunaliella acidophila cells exhibit a positive zeta potential (+5 to +20mV) at acidic external pH values, whereas negative zeta potentials (-30 mV) were measured at neutral pH values. Negative zeta potentials of the same order of magnitude were also measured for D. parva cells (pH 7.6). Low concentrations of La³⁺ and A1³⁺ did not affect the positive zeta potential of D. acidophila at acidic pH values, whereas higher concentrations caused a shift to more positive potentials. However, at neutral pH these cations caused a significant decrease of the negative zeta potential. The impermeant polycation poly-L-lysine acted in a similar manner to A1³⁺ or La³⁺. The effect of Impermeant cations and anions on various physiological reactions also supports the existence of a positive zeta potential for D. acidophila and of a negative zeta potential for D. parva: polycations such as DEAE-dextran and poly-L-lysine strongly inhibitied photosynthesis and mobility of D. parva, but did not affect these reactions in D. acidophila. Comparable differential inhibitions were also observed for A1³⁺ and La³⁺. Impermeant anions such as Dextran-sulfate exhibited effects in the opposite direction: inhibition was stronger with D. acidophila and weaker with D. parva. However, the differential inhibition by impermeant anions was much less pronounced in comparison with impermeant cations due to the relatively high pK_a values of anionic solutes and consequently relatively high protonation at pH 1.0. The physiological consequences of an asymmetrically charged plasma membrane (excess of positive charges outside, excess of negative charges on the cytoplasmic side) of D. acidophila are discussed in regard to the extreme acid resistance of this alga and its resistance to cationic toxic solutes in industrial wastes.     

Salinity-dependent regulation of starch and glycerol metabolism in Dunaliella parva

Abstract. The glycerol and starch metabolism of synchronized Dunaliella parva cells as a function of the salinity of the medium has been investigated.
The higher the salinity of the medium the higher is the rate of glycerol synthesis and the endogenous glycerol concentration, whereas starch content and salinity of the medium are inversely related. Upon transfer to a hyperosmotic NaCl-medium cells respond by an immediate increase in glycerol synthesis and an inhibition of starch formation in the light. Under corresponding conditions in darkness, starch degradation is stimulated. In both light and darkness hyperosmotic shocks are followed by a rapid increase in the endogenous pool of inorganic phosphate (P_i). It is suggested that in the light the increase in the endogenous phosphate level inhibits the chloroplast ADPG-pyrophosphorylase (E.G.2.7.7.27), and thereby starch synthesis, and promotes starch phosphorolysis. Photosynthetically produced triosephosphates and triosephosphates derived from starch degradation are converted to glycerol. Also, in the dark the increase in the P_i-level stimulates phosphorolytic starch degradation and thereby synthesis of glycerol. Reasons for the salt stress induced increase in the endogenous P_i-level are discussed.     

Excretion of glycolate during synchronous culture of Ankistrodesmus braunii in the presence of disalicylidenepropanediamine or hydroxypyridinemethanesulfonate

The rate of excretion of glycolate by the unicellular greenalga Ankistrodesmus braunii changes during its life cycle. Itis high in the main growth phase during the light period witha maximum 6 hr after the start of illumination, and low duringthe period of cell division in the dark. The glycolate excretion is stimulated by DSPD and HPMS, whilethe total ¹⁴CO₂-fixation is inhibited by DSPD and enhanced byHPMS. Changes in the effects of DSPD and HPMS on glycolate excretionas well as on photosynthetic ¹⁴CO₂-fixation during the courseof the algal life cycle were followed using the technique ofsynchronous culture. How far the change of glycolate excretion is due to a changeof glycolate oxidase activity during the life cycle and to achange of C₂-supply from the carbon reduction cycle is discussed.The effect of DSPD on glycolate excretion suggests a participationof ferredoxin in the glycolate pathway. (Received August 10, 1968; )     

Flexible Coupling of Phosphate Uptake in Dunaliella acidophila at Extremely Low pH Values

The acidophilic alga Dunaliella acidophila exhibits optimalgrowth at pH 1. We have investigated the regulation of phosphateuptake by this alga using tracer techniques and by performingintracellular phosphate measurements under different growthconditions including phosphate limitation. In batch culturewith 2·2 mol m^–3 phosphate in the medium the uptakeof phosphate at micromolar phosphate concentrations followeda linear time dependence in the range of minutes and rates werein the range of 1 µmol phosphate mg^–1 chl h^–1,only. However, under discontinuous phosphate-limited growthconditions, tracer influx revealed a biphasic pattern at micromolarphosphate concentrations: An initial burst phase resulted ina 10⁴-fold internal phosphate accumulation and levelled offafter about 10 s. A double reciprocal plot of the initial influxrates obtained for phosphate-limited and unlimited algae exhibitedMichaelis-Menten kinetics. Phosphate limitation caused a significantactivation of the maximum velocity of uptake, yielding V_maxup to 1 mmol mg^–1 chl h^–1 as compared to valuesin the order of 50 µmol phosphate mg^–1 chl h^–1for the second phase (this magnitude is also representativefor non-limited batch cultures). Concomitantly the Michaelisconstant was altered from 4 mmol m^–3 to 0·7 mmolm^–3. The rapid uptake of phosphate was inhibited by arsenateand FCCP and was not stimulated by Na⁺. The pH dependence oftracer accumulation and measurements of the intracellular phosphatepool under different growth conditions indicate that at lowpH and low external phosphate concentrations the high protongradient present under these conditions is utilized for a H₃PO₄uptake or a H⁺/H₂PO^–4 cotransport. However, when the externalphosphate concentration was increased to levels sufficientlyhigh for transport to be driven by the positive membrane potential(10 mol m^–3 phosphate), the pH dependence of phosphateuptake was more complex, but could be explained by the uptakeof H₃PO₄ or a H⁺/H₂PO^–₄-cotransport at low pH and a differenttype H₂PO^–4-transport (with unknown type of ion coupling)at high pH-values. It is suggested that this flexible couplingof phosphate transport is of essential importance for the acidresistance of Dunaliella acidophila. Key words: Acid resistance, Dunaliella acidophila, phosphate cotransport, phosphate limitation, plasma membrane, sodium     

Effect of Ammonium on Dark-CO2 Fixation and on Cytosolic and Vacuolar pH Values in Eremosphaera viridis de Bary (Chlorococcales)

At constant external [CO₂], rates of dark-CO₂ fixation of theunicellular green alga Eremosphaera viridis were drasticallyincreased (up to 40-fold) by addition of ammonium (NH₃+ NH₄⁺)at external pH values (pH₀) between 6.0 and 8.0. The cytosolicpH was monitored under identical conditions by micro-pH-electrodemeasurements, and cytosolic and vacuolar pH by the ³¹P-NMR technique.Addition of ammonium (5.0 mol m^– pH₀ 7.0) caused a rapidand transient acidification of the cytosol during the first4 min. Thereafter, the cytosolic pH remained constant at itsoriginal value. A rather constant cytosolic pH was also confirmedby ³¹P-NMR measurements, which, in addition, indicated a slowalkalization of the vacuole (about 0.5 units within 30 min afteraddition of ammonium). Since the dramatic stimulation of dark-CO₂ fixation by ammoniumis not mediated by an alkalization of the cytosol, nor by directammonium effects on phosphoenolpyruvate carboxylase (PEPC, E.C.4.1.1.31 [EC] ), the role of vacuolar alkalization as a possible triggerfor the stimulation of PEP-carboxylase is discussed. Key words: Cytosolic pH, dark-CO₂ fixation, pH-regulation, vacuolar pH     

Tetraphenylphosphonium (TPP+) is not suitable for the assessment of electrical potentials in Chlorella emersonii

Abstract. For Chlorella emersonii , plausible membrane potentials between –80 and –120 mV were calculated from the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the cells and the medium. Furthermore, these calculated membrane potentials were influenced in a way expected from the literature, by different metabolic conditions induced by light or dark, anaerobiosis, glucose, and by inhibition or uncoupling of electron transport.
Nevertheless, the experiments presented here indicate that TPP⁺ is unsuitable as a probe for electrical potentials, at least in Chlorella emersonii. The reasons for this conclusion are as follows:

• 1. 

  Much of the incorporated TPP⁺-¹⁴C could not be exchanged against unlabelled TPP⁺.
• 2. 

  The uptake of TPP⁺-¹⁴C was very slow and exhibited complex rather than simple saturation kinetics.
• 3. 

  A large adsorption of TPP⁺-¹⁴C took place even after the cells were killed; the adsorption by living cells was only 20–60% higher than with killed cells. Furthermore, the adsorption by killed cells showed kinetics similar to living cells.

     

The Conductance of the Plasmalemma for CO2

Permeability coefficients (P_S values) for CO₂ of the plasmamembrane (PM) of the unicellular green algae Eremosphaera viridis,Dunaliella parva, and Dunaliella acidophila, and of mesophyllprotoplasts isolated from Valerianella locusta were determinedfrom ¹⁴CO₂ uptake experiments using the rapid separation ofcells by the silicone oil layer centrifugation technique. Theexperimental P_S values were compared with calculated numbersobtained by interpolation of Collander plots, which are basedon lipid solubility and molecular size, for D. parva cells,mesophyll protoplasts isolated from Spinacia oleracea, mesophyllcells and guard cells of Valerianella, and guard cell protoplastsisolated from Vicia faba. The conductivity of algal plasma membranes for CO₂ varies between0.1 and 9 ? 10^–6 m s^–1, whereas for the plasmalemmaof cells and protoplasts isolated from leaves of higher plantsvalues between 0.3 and 11 ? 10^–6 m s^–1 were measured.By assuming that these measurements are representative for plantsand algae in general, it is concluded that the CO₂ conductivityof algal PM is of the same order of magnitude as that of thehigher plant cell PM. P_s values of plasma membranes for CO₂are lower than those for SO₂, but are in the same order of magnitudeas those measured for H₂O. On the basis of these results itis concluded that theoretical values of about 3000 ? 10^–6m s^–1 believed to be representative for higher plant cells(Nobel, 1983) and which are frequently used for computer-basedmodels of photosynthesis, lack experimental confirmation andrepresent considerable overestimations. However, with severalsystems, including higher plant cells, the conductance of thePM for CO₂ was significantly higher in light than in darkness.This suggests that in light, additional mechanisms for CO₂ uptakesuch as facilitated diffusion or active uptake may operate inparallel with diffusional uptake. Key words: Conductivity, CO₂, permeability coefficient, photosynthesis, plasmalemma