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Potassium transport in Chlamydomonas reinhardtii Malhotra, Bhupinder K. 1994

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Potassium transport in Chiamydomonas reinhardtiibyBHUPINDER K. MALHOTRAM.Sc. Punjab University, Chandigarh, India 1987A THESIS SUBMH1ED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYINTHE FACULTY OF GRADUATE STUDIES(Department of Botany)We accept this thesis asconforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1994© Bhupinder K. MaihotraIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of j\J ‘.1.The University of British ColumbiaVancouver, CanadaDate ‘ 1q9s.DE-6 (2/88)nABSTRACTSeveral lines of evidence have indicated that K uptake is mediated by both proteincarriers and channels according to external [K]. Evidence derived from biophysical,biochemical and molecular studies of K uptake in higher piants, fungi and bacteria indicatethat K uptake is mediated by proteinaceous carriers (active transport) and channels (passive)depending upon the external concentration of K. Only limited work has been undertaken usingmicroalgae.In Chiamydomonas reinhardtii, K influx appears to be mediated by two discretetransport systems, a high affinity transport system (HATS) at low external [Kfl and a lowaffinity transport system (LATS) at high external [K]. These two transport systems werefurther characterized by employing various metabolic inhibitors, a K channel blocker and asulfhydryl reagent. Effects of light and dark on K transport were also studied. To calculateAJIK across the plasma membrane, electrical potential difference (AN!) was measured usingTPP, and cytoplasmic [K] was obtained from compartmental analysis. According to thisvalue of AIK, K enters the cell against its electrochemical potential gradient at low [KJ0(<0.2 mM) and is probably moving down the electrochemical gradient at high [K]0 (>1 mM).In isolated chloroplasts, [K] was found to be 165±21 mM. This value was close tothat estimated by compartmental analysis (223±31.4 mM). Compartmental analysis of cellsusing 86Rb-’- and 42K as the tracers also showed the presence of three cellular compartmentsand their half-lives. Three compartments detected in Chiamydomonas probably correspond tothe cell wall, cytoplasm and chioroplast, respectively. Comparisons of compartmental analysis111of wild type and trkl cells showed that compartment ifi (putatively identified as thechioroplast) may serve as a reservoir for K.It is a common observation that plants show increased ion uptake when deprived ofnutrients. Chiamydomonas showed increased uptake of K(42K or 86Rb) when K supplydecreased in the external medium. Inhibitors of protein synthesis were used to study the role ofde novo protein synthesis in the development of increased K influx associated with Kdeprivation. There was no significant increase in K uptake before 3 h of K deprivation. Kinflux increased significantly after 3 h and peaked at about 18 h of K deprivation. In parallelexperiments, expression of membrane polypeptides was studied. Some polypeptides inresponse to K deprivation were expressed in the microsomal fraction. When cells weredeprived of K for 4 h, a 22 kD polypeptide showed increased expression. Anotherpolypeptide of Mr 51 kD was synthesised as a response to long term K deprivation. Whencomparisons were made between microsomal fractions from WT and trkl cells, a 21 kDpolypeptide was found to be absent from trkl microsomes.ivTABLE OF CONTENTSABSTRACT iiLIST OF TABLES xiiLIST OF FIGURES xivABBREVIATIONS xviiACKNOWLEDGEMENTS xviiiDEDICATION xix1. GENERAL INTRODUCTION1.1 Role of Kin Plant 11.1.1 Role of K in protein synthesis1.1.2 Role of K as an enzyme activator1.1.3 Role of K in osmoregulation and in charge balance1.1.4 Role of K in photosynthesis1.2 Energetics of ion transport 51.3 Energy coupling to K transport 91.3.1 K/H exchange1.3.2 K/H symport1.3.3 K -ATPase1.3.4 K channels1.4 Kinetics of K fluxes in plant cells 141.5 Regulation of K transport 17V1.6 Objectives.20II. GENERAL MATERIAL AND METHODS2.1 Strain, growth conditions and synchronization 232.2 Growth media and modifications for different [K] 252.3 Methods to monitor cell growth 261. Coulter counter2. Hemocytometer3. Fluorescence4. Chlorophyll content2.4 Determination of K content of the cells 281. Centrifugation through silicone oil2. Filter paper method3. Low speed centrifugation4. Compartmental analysis5. Time course studies2.5 Depletion of K from the growth media 332.6 Potassium deprivation 332.7 Determination of cell diameters and volumes 341. Coulter counter2. Light microscopy3. Kontron Imaging AnalysisviIll. GROWTH3.1 Introduction 363.2 Methods 383.3 Results 393.3.1. Growth of cells in media containing 0 to 10 mM Ka) Growth of cells in 0, 10, 25 and 50 jiM Kb) Growth of cells in 75 p.M to 10 K3.3.2. Growth of cells in the light and dark at 10 mM K3.3.3. Growth of cells in K -replete and K -deplete media3.4 Discussion 45IV. CHARACFERIZATION OF K TRANSPORT SYSTEMS4.1 Introduction 484.2 Methods 504.2.1. Strain and culture conditions4.2.2. Influx determinationsa). General methodsb). Effects of pH4.2.3. Inhibitor treatment and influx determination4.2.4. Lightldark effects4.3 Results 54VII4.3.1. K influxa). High affinity transport system (HATS)b). Low affinity transport system (LATS)4.3.2. Metabolic dependence of the transport systems4.3.3. Effects of external pH on K(86Rbj influx4.3.4. Influx in cells grown in the dark4.4 Discussion 64V. MEMBRANE POTENTIAL DETERMINATIONS5.1 Introduction 695.2 Methods 715.2.1. Chemicals5.2.2. Accumulation of TPP5.2.3. Calculation of electrical potential difference across the plasmamembrane from TPP accumulation5.2.4. Effects of external [KJ on membrane potential5.2.5. Effect of DNP on membrane potential5.2.6. Effects of external pH on membrane potential5.2.7. Effects of oligomycin and light/dark treatments on TPP accumulationa). Membrane potentialsb). Effect of oligomycin on respirationc). Effect of darkness on oxygen liberation by photosynthesisvu’5.3 Results 785.3.1. Time course of tetraphenyiphosphonium (TPP) distribution5.3.2. Effects of external [K] on membrane potential5.3.3. Response of membrane potential to external pH5.3.4. Metabolic dependence of Mi5.4 Discussion 85VI. COMPARTMENTAL ANALYSIS6.1 Introduction 896.2 Methods 926.2.1. Efflux using K(86Rbj in wild type and trkl cells6.2.2. Efflux using K(42K) in wild type cells6.2.3. Efflux using K(42K) in wild type and trkl cells6.2.4. Efflux in the dark in wild type cells using K(86Rb)6.2.5. Subcellular distribution of K in wild type and mutant cells6.2.6. Chloroplast Isolation6.2.7. Potassium concentrations of intact chioroplasts6.3 Results 1026.3.1. Efflux using K(86Rbj in wild type cells6.3.2. Efflux using K(86Rb) in trkl cells6.3.3. Efflux using K(42Kj in wild type and trkl cellsix6.3.4. Subcellular distribution and fluxes of K in wild type and mutantcells6.3.5. Efflux in the dark in wild type cells using K(86Rb-’-)6.3.6. Intactness and yield of isolated chioroplasts6.3.7. Potassium concentration of isolated chioroplasts6.3.8. Electrochemical potential difference across the plasma membrane6.4 Discussion 116VII. REGULATION7.1 Introduction 1237.2 Methods 1257.2.1. Effects of varying K concentrations in the growth media on Kuptake and cell K concentrationsa). Time course of K deprivationb). Influx determination7.2.2. Potassium influx and K content of wild type and trkl cells7.2.3. Effects of protein synthesis inhibitors on K influx7.3 Results 1277.3.1. Effects of varying K concentrations in the growth media on Kuptake and cell K concentrations7.3.2. Potassium influx and K content of wild type and trkl cells7.3.3. Effects of protein synthesis inhibitors on K influxx7.4 Discussion.133Vifi. BIOCHEMICAL ASPECTS OF K TRANSPORT IN CHLAMYDOMONAS8.1 Introduction 1358.2 Materials and Methods 1388.2.1. Culture and growth conditions8.2.2. Cell harvesting8.2.3.35S-methionine labeling8.2.4. 14C-arginine labeling1). Isolation of membrane fractions2). Sucrose step gradients3). Aqueous polymer two phase partitioning8.2.5. Protein determinations8.2.6. Electrophoresis and autoradiography8.2.7. Gel casting and buffer preparation8.2.8. Sample preparation, electrophoresis and autoradiography8.3 Results 1518.3.1.35S-methionine labeling8.3.2.‘4C-arginine labeling8.3.3. Recovery of plasma membranes by various techniques8.3.4. Comparison of Coomassie blue and silver stained polypeptides ofmicrosomal and plasma membranes of wild type and trkl cellsxi8.3.5. Time course of14C-arginine labeling of membrane polypeptides inrelation to K deprivation1). Polypeptides of interest2). Polypeptides of interest are specific in response to K deprivation8.4 Discussion 160IX. SUMMARY 164X. REFERENCES 168xiiLIST OF TABLESTable pg1. Composition of the standard TAPM medium 242. Concentrations and pretreatment times for various inhibitors employed forK(86Rb) influx experiments 523. K and r2 values for linear regressions for the starved and unstarvedcells. K( 86Rb or 42K) influx was determined from [K-’-]0 in the rangefrom 0 to 0.75 mM 574. Effects of various inhibitors onK(86Rb) influx from [K]0 at 0.005 and0.1 mM (HATS) and 4.0 and 10.0 mM (LATS) 575. Effect of oligomycin on respiration 826. Effect of darkness and oligomycin on oxygen liberation by photosynthesis 827. Preparation of 20%, 45% and 65% gradient mixture for chioroplast isolation 988. Solutions for the separation of Chiamydomonas chloroplasts 1. 5 x GR Mix 989. Compartmental analysis of K(86Rb) efflux at 10 mM [K]0 inChiamydomonas reinhardtii to obtain half lives of exchange (t05) of the threephases (I, II and III) 10510. Compartmental analysis of K(42K’-) efflux at 10 mM [K-’-]0 in wild type(WT) and mutant (trkl) strains of Chlamy&nnonas reinhardtii to show halflives of exchange (t05) of the three phases (I, II and III) 10511. Unidirectional K’- fluxes (0), net flux (J and Jh1). K concentrations ([Kj)and K’- contents (Q and Q) calculated from compartmental analysis at 10 mM[K]0 in wild type and trkl cells of Chiamydomonas 10712. Cytoplasmic K-’- concentrations calculated from compartmental analysis performedat 10 mM K’- 10713. Compartmental analysis of K-’-(86Rb) efflux at 10 mM [K’-]0 in Chiamydomonasxmreinhardtii in light and in dark to show half lives of exchange (t05) of thethree phases 10914. Unidirectional K fluxes (0), net flux (J00 and hl). K concentrations ([K])and K-’- contents (Q and Q) calculated from compartmental analysis inwild type cells of Chiamydomonas in the light and the dark 10915. Electrochemical potential differences for K between cytoplasm and externalmedia at different values of external K concentration 11516. Effect of different concentrations of NH4 on 35S accumulation 14017. Volumes of various stock solutions used to prepare the gradient gel mixtures 148xivLIST OF FIGURESFig pgFig. 1. Some of the possible mechanisms of K transport in various organisms 8Fig. 2. Cell growth shown by cell number in culture containing variousconcentrations (0 to 10 mM) of K-’- 40Fig. 3. Cell number and chlorophyll contentof cells grown in either light ordark 42Fig. 4. Cell number and K-’- content of cells plotted to show the change in Kcontent of cells during K’- deprivation or when cells were grown at 10 mMexternal [K] for 48 h 44Fig. 5. 42K influx from [Kj0 in the range 0-30 mM in Chiamydomonasreinhardtii cells 55Fig. 6. 42K influx from [K]0 in the range 0-0.75 mM showing the saturablehigh affinity system 56Fig. 7. 86Rb influx from [K]0 in the range 0-0.75 mM 56Fig. 8. 42K influx showing the linear system (LATS) obtained by subtracting theVmax of the HATS from observed fluxes in the range 1.5-30 mM[K-’-]0 59Fig. 9. 86Rb influx showing the linear system (LATS) obtained by subtracting theVmax of the HATS from observed fluxes in the range 1.5-30 mM [K-’-]0 59Fig. 10. 86Rb-’- influx from [Rb]0 in the range 0-40 mM in cells grown in K repleteand K deficient media 60Fig. 11. Effect of external pH in the range 3.5 to 9.0 on K(86Rb) influx 62Fig. 12. K(86Rb) influx in cells grown in light and dark 63Fig. 13. Time course for TPP accumulation over a period of 5 h 79Fig. 14. Effects of external [K-’-] in the range from 0 to 200 mM on the membranexvpotential .80Fig. 15. Effect of external pH in the range 3.5 to 9.0 on membrane potential 84Fig. 16. Membrane potential difference in the presence and absence of oligomycin,measured in light and dark 84Fig. 17. Filtering device used for compartmental analysis of Chiamydomonas cells ...93Fig. 18. Symbols used for various K fluxes across the cellular compartments andtheir [K] 94Fig. 19. Linear regression on semi-log plot of 86Rb elution data from wild typeChiamydomonas cells grown at 10 mM K1 103Fig. 20. Linear regression on semi-log plot of 86Rb elution data from trklChiamydomonas cells grown at 10 mM K 104Fig. 21. Isolated chioroplasts of Chiamydomonas reinhardtii under fluorescence lightmicroscope 110Fig. 22. Electrochemical potential differences for K (1.tK) for cells grown at 0.1mM K, across the plasma membrane 112Fig. 23. Electrochemical potential differences, LL (k.J mo11)for K across the plasmamembrane and chioroplast envelope at 0.1 and 10 mM [K10 in wild typeand trkl cells of Chiamydomonas 113Fig. 24. A model proposed to explain the mechanisms of K transport across theplasma membrane and chioroplast envelope at 0.1 mM and 10 mM [K]0 inwild type and trkl cells 120Fig. 25. K content and K influx from 0.1 mM [K]0 in cells deprived of K for differentdurations 128Fig. 26. K influx from 0.1 mM [KJ0 in in wild type and trkl cells deprived ofK for different durations 130Fig. 27. K content in wild type and trkl cells deprived of K for differentxvidurations .130Fig. 28. Effect of inhibitors of protein synthesis cycloheximide and anisomycin ondevelopment of increased K(86Rb) influx during K deprivation 131Fig. 29. Silver-stained polypeptides of microsomal proteins obtained from wild typeChiamydomonas cells and trkl cells 152Fig. 30. Changes in the expression of microsomal polypeptides in wild type cellswhen grown at 10 mM K, deprived of K for 4 h and 20 h 155Fig. 31. Microsomal polypeptides in wild type Chlainydomonas cells when grown at10 mM K, deprived of K for 8 h, 12 h, 20 h and 28 h 158Fig. 32. Microsomal polypeptides in wild type cells grown at 10 mM K throughout.Cells were harvested in their early light phases, late light phases and middark phases 159xviiLIST OF ABBREVIATIONSANISO: anisomycinCHX: cycloheximideDTr: DithiothreitolEDTA: disodium ethylenediamine tetra acetic acidHATS: High Affmity Transport SystemHEPES: (N-[2-Hydroxyethyl] Piperazine-N’-[2 ethanesulfonic acid])MES: 2-(N-Morpholini) ethane sulfonic acidPEG: Polyethylene glycolPMSF: PhenylmethylsulfonylfiouridePVP: Polyvinyl pyrrolidinemembrane potential grdientelectochemical potential gradientApH: pH gradientOK: TAPM medium with no K added10K: TAPM medium with 10 mM K addedTAPM: Tris acetate phosphate modified mediumWT: Wild type strain of Chiamydomonas (CC 125 mtjtrkl: K transport defective mutantLATS: Low Affmity Transport SystemKCN: potassium cyanideCCCP: carbonyl cyanide m - chiorophenythydrazonepCMBS: p-chloromercuribenzene sulfonic acidTEA: tetraethylammonium chlorideSDS: sodium dodecyl sulfateSDS-PAGE: SDS-Polyacrylamide gel electrophoresisxixACKNOWLEDGEMENTSI extend my sincere thanks to my research supervisor Dr. A.D.M. Glass, forhis guidance, supervision, encouragement and patience throughout this project. The time hespent on editing this thesis is gratefully appreciated. I thank my committee members Drs. P.J.Harrison, G.H.N. Towers and E.L. Camm for their useful suggestions, inspiration, the timespent at the committee meetings and for editing this thesis. Dr. P.J. Harrison is especiallyacknowledged for his guidance and useful comments during this research project. Specialthanks to my colleagues, especially Dr. M.Y. Siddiqi and family and J. Mehroke for theassistance and moral support given throughout this research project. My thanks are alsodirected to Dr. L.D. Polley for sending the mutants defective in K transport. I am alsothankful to Drs. B.R. Green, T. Crawford, A.J.F. Griffiths and I. Taylor for allowing me touse the faculties available in their laboratories. The warmest thanks are also extended to all myfriends who are in our lab and in the Botany department and the staff in the Botany workshop,Botany office, EM facility and Botany stores. Dr. C. French and G.H.N. Towers aregratefully acknowledged for their support given for work at Agriculture Canada.Invaluable moral support given by my husband, Bob, during the greater part ofthis study is warmly appreciated. Love and happiness given by my parents, sisters andbrothers is greatly appreciated. I also extend my thanks to my mother-in-law for the supportgiven during the later part of this project.Financial assistance through teaching assistantships and for the research grantsfrom The Potash institute and NSERC to Dr. A.D.M. Glass are gratefully acknowledged.xixTo my family1I. GENERAL INTRODUCTIONAll plants require at least 16 chemical elements to maintain their metabolism andgrowth. The flow of molecules and ions between a cell and its environment is preciselyregulated by specific transport systems. These transport systems maintain the internalconcentrations of many of the elements at a constant level despite wide variations inenvironmental and physiological factors. The required elements serve diverse roles asstructural components of the organic constituents of plants (C, H, 0, N, S and P), enzymeactivators (K, Ca, Mg and Mn), and redox reagents (Fe, Cu and Mo) as well as functions thatare, at present, still poorly understood, e.g., B, Na and Cl (see Glass, 1990).1.1 Role of K in PlantsThe involvement of K-’- in a number of plant functions has been the basis for thestudy of K-’- transport as a model system for ion transport in plants (Kochian and Lucas, 1988).These functions of K-’- in plants have been extensively reviewed (see Mengel and Kirkby,1982; Marschner, 1986)1.2.1 Role of K in protein synthesisIt is now well established that K is required for protein synthesis in plants. It hasbeen suggested that the requirement for high levels of K may be due to its involvement in theprocess of transfer of amino acids from tRNA to ribosomes (Lubin and Ennis, 1965; Evans2and Wildes, 1971; Wyn Jones et aL, 1983). In vitro protein synthesis requires 100-150 mMK and a high K: Na ratio, and any large deviations from these conditions may affect boththe quantity and type of protein produced (Leigh and Wyn Jones, 1984). For example, it hasbeen shown in cell-free systems that ribosomes from wheat germ show their optimal activity ataround 130 mM K (Wyn Jones et al., 1979).The role of K in protein synthesis is indicated by the accumulation of solublenitrogen compounds in K -deficient plants, but can also be shown directly by the effect of Kdeprivation on the incorporation of 15N -labeled inorganic nitrogen into the protein fraction.1.2.2 Role of K+ as an enzyme activatorIt has been shown that more than 50 enzymes either completely depend on, or arestimulated by K (Suelter, 1970). Potassium activates enzymes by inducing conformationalchanges in the enzyme protein. Some of the most important enzymes which are activated byK are those which participate in glycolysis: pyruvate kinase (Wildes and Pitman, 1975;Besford, 1978), starch synthesis: starch synthase (Hawker et al., 1979), RUBP carboxylaseand membrane-bound ATPases (see Marschner, 1986).1.2.3 Role of K+ in osmoregulation and in charge balanceThe maintenance of osmotic pressure is an important prerequisite for the growthand survival of plant cells. The ability to adjust their internal osmotic pressure, and thus turgorpressure, in response to salt and water stress has been observed in both higher plants and algal3cells. At the level of individual cells or in certain tissues, the same mechanism is responsiblefor cell extension and various kinds of plant movements. Potassium, as the most prominentinorganic solute, plays a key role in these processes.In nature, there is generally a high gradient of K concentration between the cell andthe external medium. Most higher plants generate turgor by means of the K salts of organicacids such as malic acid. When K is deficient, plants compensate for this deficiency byincreasing the accumulation of other solutes such as Mg2 and Ca2 (Kirkby and Mengel,1976) or organic solutes (Pitman et al., 1971). In barley, reducing sugars may replace theturgor function of K in low-salt (K -deficient) roots (Pitman et al., 1971).Several plant movements, such as turgor-mediated guard cell opening and closure,nyctinastic and seismonastic movements have been shown to be caused by turgor changesinvolving K-’- movement across cellular membranes. Increased turgor of guard cells duringstomatal opening results from increased absorption of K into these cells, through inwardlydirected K channels (Schroeder et al., 1987). During stomatal closure, K is lost from thecells. The accumulation of K’- in the guard cells has to be balanced by a counteranion, mainlymalate or Cl-, depending on the plant species and availability of Cl- (Van Kirk and Raschke,1978). The normal diurnal opening and closing of guard cells and uptake/release of K’- isthought to be linked to plasma membrane H pump. The plant growth regulator, abscisic acid,is known to cause stomatal closure (Mittelheuser and Van Steveninck, 1971) by increasedefflux of K-’- from the guard cells, which is probably caused through the inhibitory action ofABA on the proton pump (MacRobbie, 1981).4In Albizzia (Satter et aL, 1974), nyctinastic (circadian) movements of leaves havebeen observed. These movements are caused by K -mediated turgor changes in specializedtissues, the pulvini or motor organs. Like the stomatal movements, nyctinastic movementsoccur as a result of K fluxes driven by the H pump in the pulvini (Iglesias and Satter, 1983).A similar mechanism is responsible for the seismonastic movements of the leaves of Mimosapudica. The turgor-regulated response is caused by a redistribution of K within the pulvini(Allen, 1969). Relocation and/or change in Ca2 binding has been suggested to affectmembrane permeability to K in the pulvini (Campbell and Thompson, 1977).Potassium is an important cation for counterbalancing anions in the cytoplasm,xylem and phloem. The accumulation of organic acids is often the result of K transportwithout a counteranion into the cytoplasm (e.g., guard cells).1.2.4 Role of K in PhotosynthesisThe role of K+ in CO2 fixation has been demonstrated with isolated chioroplasts ofspinach. When the external K concentration was increased to 50-100 mM, CO2 fixation wasfound to be stimulated (Pfluger and Cassier, 1977; Kaiser et al., 1980). Robinson andDownton (1984) reported that K was the major monovalent cation in chioroplasts (160-200mM) and suggested a role of K in photosynthesis (Robinson, 1986). An increase in the leafK content is accompanied by increased rates of photosynthesis, photorespiration and RUEPcarboxylase activity, but a decrease in dark respiration (Peoples and Koch, 1979). Increasedrates of respiration are common in K-deficient plants (e. g. spinach; Bottril et al., 1970).51.1 Energetics of Ion TransportInternal concentrations of nutrients are kept constant due to the presence oftransport systems in cellular membranes. These transport systems can accumulate nutrientsagainst strong electrochemical gradients. Nutrients first move passively into the cell wallmatrix and then are taken up into the cell. When considering the movement of ions across theplasma membrane, the concentration gradients of ions as well as the electrical potentialdifferences across the membrane must be taken into account because of the charges associatedwith them. Movement of nutrients into the cell across the plasma membrane can occur by threemechanisms:1. Diffusion: is the movement of solutes through the lipids of the lipid bilayer, along theirelectrochemical potential gradients.2. Facilitated diffusion: is the movement of solutes through proteinaceous pores embedded inthe lipid bilayer, down their electrochemical potential gradients.3. Active transport: is the movement of solutes through protein carriers in the plasmamembrane, against their electrochemical potential gradients.Although active transport processes occur at the expense of metabolic energy, they have severalimportant roles. These include:1) regulating internal pH (Raven and Smith, 1979),2) energy storage and transduction (Mitchell, 1966),3) maintaining electrical and chemical potential gradients (Poole, 1978)64) scavenging nutrients from low external concentrations (Skulachev, 1977)5) maintaining internal ionic composition within a narrow range (Cram, 1976).It is now generally accepted that the ultimate source of energy responsible for iontransport is ATP (Hodges, 1976; Petraglia and Poole, 1980). Higinbotham (1973), and Poole(1978) have suggested that a significant portion of the resting membrane electrical potentialacross the plasma membrane is produced by an ATP-driven electrogenic H pump, and that theenergy conserved by this pump is used for the active transport of other ions. Theseelectrogenic proton pumps are also important for the maintenance of cytoplasmic pH in therange of 7.0 to 7.5 by extruding protons to the cell wall (Raven and Smith, 1979).Active transport systems can be of two kinds, namely primary active transportsystems and secondary active transport systems.Primary Active Transport Systems:In primary active transport systems, the energy required to move the solute againstits electrochemical potential difference is supplied directly by a chemical reaction mediated bythe transport complex. Up to now, three types of primary active transport systems have beenidentified in living organisms:1. ATPases and Pyrophosphatases: these are membrane-bound enzymes which couple thetransport of solutes to the hydrolysis of ATP (Pedersen and Carafoli, 1987) or pyrophosphates(Rea and Poole, 1985), respectively.2. Redox pumps: in these systems, oxidation-reduction (redox) reactions taking place on eitherside of a membrane result in the transport of protons across the membranes,73. Light driven pumps of certain halophiic bacteria: bacteriorhodopsin is the light-drivenprotein pump of Halobacterium haiobium which generates an electrochemical gradient acrossthe bacterial membrane.Among ATPases, the most extensively studied and characterized systems are:1. The Na/K ATPases of animal cells (Glynn, 1984),2. The Ca2-’- -ATPases of the sarcoplasmic reticulum and plasma membranes of animal cells(Guidotti, 1976; Michalak, 1984),3. H-’- -translocating ATPases in fungi and higher plants (Bowman, 1980; Bowman et al.,1986; Leonard and Hodges, 1973; Serrano, 1989).Primary active transporters which transport ions of the same charge in the oppositedirection with a 1:1 stoichiometry are known as “electrically silent” or “neutral” pumps sincethere is no net charge separation e.g. K/H ATPase from vertebrate gastric mucosa (Sachs etal., 1978). Transporters which transport ions in opposite directions with unequalstoichiometries generate an electric field across the cell membrane (Spanswick, 1981; Sze,1985) and thus are electrogenic in nature (Poole, 1978; Spanswick, 1981). These pumps arecapable of maintaining an electrical potential gradient (negative inside) of -60 to -300 mV(Poole, 1978).Secondary Active Transport Systems:The activity of the plasma membrane H -ATPase results in the generation of a pH8Fig. 1 Some of the possible mechanisms of K transport in various organisms.a = H ATPase; b = K/W symport; c = K/W exchange;d = K channel; e = K ATPase.HK(b)9gradient and an electrical gradient which may form a large inwardly directed electrochemicaldifference for H. This proton motive force is a source of free energy which can be used totransport other ions into the cell provided there is a coupling mechanism. An ion such as H orNa can symport with another ion when it enters the cell down its electrochemical gradient.For example, a K/H symport has been suggested for K transport in Neurospora(Rodriguez-Navarro et al., 1986); sucrose/H symport for sucrose transport (Humphreys,1988); glucoselW symport in Chiorella vulgaris (Komor and Tanner, 1976); Cl-/EP symportin Chara corallina (Sanders, 1980; Beilby and Walker, 1981) ; Cl/H and NO3iH symportsin Lemna gibba (Novacky and Luttge, 1981), and a SO4/H symport in Leinna gibba (Lassand Ulrich-Eberius, 1984).Antiport systems (Na/H) have been suggested in sea urchin eggs (Alberts et al.,1983), Neurospora (Slayman, 1974) and gastric mucosa of animal cells (K/H; West, 1983).1.3 Energy Coupling for K TransportIt has long been accepted that K influx into plant cells from low external [Kfl mustbe coupled to a source of energy. Several models have been proposed to explain the mode ofcoupling between K fluxes and H efflux, but the controversy has not been resolved yet.Figure 1 depicts some of the possible mechanisms of K transport in various organisms.1) K/H exchangeSeveral workers suggested that K uptake across the plasma membrane occursthrough direct coupling with the H ATPase (Cheeseman and Hanson, 1979; Lin and Hanson,101976), but Poole (1974) was probably the first to suggest that the plasma membrane H pumpof plant cells was actually a KIH exchange ATPase. Working with red beet root slices, heobserved that as the external pH was increased from 5.5 to 8.0, there were parallel increasesof H efflux and K uptake and a hyperpolarization of the membrane potential.The correlation between plasma membrane-associated, K-stimulated ATPaseactivity and K influx, and the similarity between the sequence for monovalent cationstimulation of ATPase activity (K >NH4Rb > Cs > Li) and specificity of monovalentcation uptake into roots (Sze and Hodges, 1977), was used as evidence that this ATPase isinvolved in K uptake operating as a KfH exchange system.Plasma membrane ATPase forms a covalently bound, phosphorylated intermediateduring the course of ATP hydrolysis (Briskin and Leonard, 1982b; Vara and Serrano, 1983;Briskin and Poole, 1983). Briskin and Leonard (1982b) showed that K stimulated thebreakdown of this phosphorylated intermediate in corn roots and concluded that thisstimulation is evidence for a K -transport role of H ATPase.In Chiorella, coupling between K and H fluxes was suggested by Schaedle andJacobson (1965), Shieh and Barber (1971). Tromballa (1983) found that net K uptake inChiorella was stimulated by permeant acids, low uncoupler concentrations and glucose. Allthese agents acidified the cell interior by 0.2-0.4 pH units. These effects were explained as theresult of stimulation of a K/H exchange which reduces intracellular acidification and thus,acts as a pHstat (see Tromballa, 1983).Although a K/H exchange has been suggested, other workers argue that this is11not the case. For example, Vara and Serrano (1983) found no effect of K on thephosphorylation and dephosphorylation of the plasma membrane ATPase from oat roots.Thus, they concluded that the ATPase was not a K -dependent enzyme involved in Ktransport. Similarly, Spanswick and Anton (1988) suggested that stimulation of thebreakdown of the phosphorylated intermediate by cytoplasmic K appears to be sufficient toexplain the K -stimulation of ATPase activity, without invoking a K transport role for theATPase. Such a K/H exchange is also unlikely because the ratio of H efflux to K influxhas been found to be considerably lower than 1 (Glass and Siddiqi, 1982; Kochian et aL,1989).2). KJH SymportStrong evidence for the existence of a K/H cotransport system in fungi has beenrecently provided by work on Neurospora (Rodriguez-Navano et al., 1986; Blatt andSlayman, 1987). Measurements of internal and external K and membrane potential,suggested that this uptake system may mediate active K uptake, and the system appears to becoupled to the very active plasma membrane H -ATPase of Neurospora. Current-voltageanalysis conducted by these workers indicated that the K associated inward current was twicethat of the net K influx. Thus, one additional positive charge enters with every K. Thesecond charge coming in with K was suggested to be a H, which indicates that the highaffinity K transport system may operate as a K/W symport. In corn roots, K uptake washighly electrogenic as would be expected for a K/H symport system (Newman et at, 1987).12Smith and Walker (1989) and Walker and Sanders (1991) have described aNa -coupled symport system in Chara australis and Nitella translucens. Chara australisdemonstrated a strict Na requirement for the onset of an inward electrical current when Kwas supplied to cells starved of K. These authors have also suggested that Na -coupledsolute transport in plants, which had previously been demonstrated only in alkalophilic species,may not have evolved recently as an alternative to H -coupled transport in high pHenvironments, and might therefore be more widely distributed than has hitherto beenrecognized.3). K -ATPaseThe ability of wild-type strains of E. coil to grow in medium of low Kconcentration is due to the expression of Kdp, a K -transport ATPase with high affinity forK-’- (Epstein, 1985). Such a high affinity transport system may be operating in other plants aswell. In higher plants (Cheeseman and Hanson, 1980; Newman et al., 1987) and algal cells(Kannan, 1971) it appears that at low external K levels, uptake is a thermodynamically activeprocess. Thus, the high affinity 1(1- uptake system must either be an ATPase that transports Kdirectly (K’- -ATPase or KIH’- exchange ATPase) or a K!H’- cotransport system that couplesactive K influx to the passive movement of protons into the symplasm (Kochian et al., 1989).These authors have speculated that K’- influx in higher plants could be mediated by a K’- -ATPase of the kind that has been found in E. coli (Epstein 1985) and Saccharomyces (Gaberet aL, 1988). Villalobo (1982) has reconstituted an H’- -ATPase of yeast plasma membrane in13proteoliposomes and showed that the ATPase itself has K transport activity. Thus, it wassuggested that such a H -ATPase may have a channel-like structure mediating K uptake(Ltittge and Clarkson, 1989).4). K ChannelsIt is now well established that K channels occur in the membranes of plant cells.The channels have been found in giant algal cells (Keifer and Lucas, 1982; Coleman andFindlay, 1985; Luhring, 1986; Homblè et al., 1987; Beilby, 1986), in yeast (Gustin et al.,1986) and in cells of higher plants (Moran et al, 1984; Kolb et al., 1987; Schroeder et al.,1987; Schauf and Wilson, 1987a and b; Ketchum et al., 1989; White and Tester, 1992).Tetraethylammonium (TEA) is a specific K channel blocker and has been used todemonstrate K channels in several organisms (Keifer and Lucas, 1982; Kochian et al., 1985;Tester, 1988). A13 ions have been shown to block K (inward) channels in guard cells ofViciafaba ( Schroeder, 1988).The technique of patch clamping, involving the electrophysiological study of a verysmall patch of membrane, has been applied to plant cells and is capable of resolving the activityof single ion channels (MacRobbie, 1988). Some channels have been found to open inresponse to changes in membrane potential produced by modulation of the electrogenic pump(Schroeder et al., 1987) whereas others may open in response to ions such as Ca2.Potassium channels in the plasma membrane of Chara open in response to membranedepolarization or by increase in external K/Ca2(see, Sokolik and Yurin, 1981; Beilby,141985). The high affinity K transport system of Neurospora has been suggested to be madeup of two components, a K channel and a H pump (Hedrich and Schroeder, 1989).Membrane potentials as low as -305 mV in Neurospora (Rodriguez-Navarro et aL, 1986), and-300 mV in barley roots (Glass et al., 1992) have been reported, in the absence of K in theexternal medium. Thus, it has been suggested that even at very low external [K], in the rangeof 1-10 jiM, K uptake might be passive (Hedrich and Schroeder, 1989).1.4 Kinetics of K Fluxes in Plant CellsThe rapid absorption of K by plant cells led several researchers to hypothesize thatspecial structures must exist that would facilitate K uptake across the plasma membrane(Jacobson et aL, 1950; Osterhout, 1952; Epstein and Hagen, 1952). The carrier-mediatedprocess of ion transport through membranes is characterized by saturation kinetics, assumingthat the number of carriers in the membrane is limited. Epstein and Hagen (1952) developedthe concept that specific carriers were responsible for K uptake across the plasma membrane.They regarded the kinetics of ion transport through membranes of plant cells as equivalent tothe binding of a substrate to its enzyme. They were the first to apply Michadis-Menten enzymekinetics to ion transport in plants. Considering this analogy, we can write the equation for thebinding and dissociating of substrate and enzyme as follows:k1 k3E+ S =====? ES ====— E+ Pk2 k415Where, S = substrate, E = enzyme, ES = enzyme-substrate complexand P = product.k1 is the rate constant for the formation of ES, k2 and k3 are the rate constants forits dissociation. k4 is the rate constant for the reverse reaction (formation of ES complex fromE plus P) is generally considered to be zero.The ratio of these constants (k1/2-i-k3),known as the Michaelis constant (Km), is ameasure of the affmity of the enzyme for its substrate. The amount of enzyme present and itsrate of turnover determines the maximum velocity (Vm) at which the reaction occurs.Using barley seedlings grown in very dilute nutrient solutions, Epstein and Hagen(1952) reported that the uptake of K(86Rbj showed a typical hyperbolic relationship at lowexternal concentrations (< 1 mM). Epstein referred to this saturable transport process as“System 1”. At such low external [K], active K transport is usually required (Cheesemanand Hanson, 1979) because K moves against its electochemical potential gradient in thisconcentration range. It is therefore generally accepted now that “System 1” is an activetransport process (but see Hedrich and Schroeder, 1989). K uptake at low external [K] ishighly selective for K and largely insensitive to the nature of the accompanying ion (seeGlass, 1990).In Epstein’s experiments, when the external K concentration was increasedbeyond 1 mM, another system became evident, which was referred to as “System II”. Thissystem was found to be saturable, had low affinity for K and a high velocity of K transport.Later, Epstein and coworkers reported that “System II” consisted of a series of hyperbolae16(Elzam et at, 1964; Epstein and Rains, 1965). These biphasic kinetics were referred to as the“dual isotherm of uptake”.According to Nissen (1973, 1980), the uptake isotherms for uptake of K and othersolutes, when replotted as 1/V verses 1/S (Lineweaver-Burk), plots are made up of a series ofadjacent linear segments. He claimed that this type of analysis is strong evidence for theoperation of a single complex system located in the plasma membrane. This system is thoughtto undergo concentration-dependent phase changes and to mediate transport at high externalconcentration by facilitated transport. This approach has been seriously challenged by manyworkers (Borstiap, 1981, 1983). According to Kochian and Lucas (1982a), the kinetics forK uptake could be resolved into saturable and non-saturating components. The saturablecomponent was similar to Epstein’s “System I”, whereas the linear component was specificallyinhibited by a K channel blocker. Thus, these authors, proposed that K transport at highexternal [Ki is mediated by K channels in corn roots. Potassium channels have also beendetected in corn roots by electrophysiological methods (Ketchum et al., 1989). Recently, twoputative K channels have been identified from Arabidopsis by transforming a K -transportmutant of yeast (Anderson et al., 1992; Sentenac et at, 1992).Numerous investigations have been conducted over the last 40 years to resolve thecontroversy concerning the dual or multiple transport processes across the plasma membrane.As Kochian and Lucas (1988) have rightly put it “Despite the surfeit of literature, it can be saidwith some confidence that the processes by which K ions are transported into and across theplant cells are still far from being fully resolved”.171.5 Regulation of K TransportNumerous workers have reported that cytoplasmic K is maintained at a constantlevel despite large variations in biological and environmental factors (Leigh and Wyn Jones,1984; Memon et al, 1985). It has also been observed that when plants are deprived of K, theyshow an increased rate of K uptake (Glass, 1976; Jensen and Pattersson, 1978; Glass andSiddiqi, 1984). This suggests that K uptake is regulated but the signals responsible forincreasing K uptake and maintaining [K] close to an optimum value, are only poorlyunderstood. Considerably more work needs to be done to understand this aspect of K uptake.The relationship between internal [Ki and K influx has been explained in terms ofdirect negative feedback regulation of K influx by internal [K] (Young and Sims, 1972;Glass, 1976; Jensen and Pettersson, 1978). Negative feedback regulation may be exertedthrough allosteric effects or other kinds of kinetic control, or repression and derepression ofcarrier synthesis.From the values of Km and Vmax for K influx in low salt roots and the relationshipbetween influx and root [Kj, it was suggested that influx was regulated allosterically by directfeedback from cytoplasmic K on the K transporter (Glass, 1976). Later, however, it becameevident that change in root [K] was largely the result of changes of vacuolar [Ki rather thancytoplasmic [K]. In vacuolated cells, vacuolar K may serve as a reservoir for themaintenance of a constant cytoplasmic [K] (Leigh and Wyn Jones, 1984; Memon et al.,1985). However, vacuolar [K] will not fall below a certain minimum level which has been18suggested to be 10 to 20 mM K. Once this value is reached and cytoplasmic K begins todecline, metabolic processes such as protein synthesis and photosynthesis would be disrupted.K influx into the xylem has been suggested to be regulated by shoot growth,root:shoot ratio or shoot [K]. Pitman (1972a and b) recorded correlations between K uptakeby the roots and shoot K status. Thus, potassium fluxes into xylem may regulate fluxes at theplasma membrane of root epidermal and cortical cells (Pitman, 1972a and b; Drew and Saker,1984; Siddiqi and Glass, 1987). Likewise, Drew and Saker (1984), using split rootexperiments under K starved and K replete conditions, suggested that the negative feedbackcontrol of K influx by internal [K] of the root was absent and that it was the shoot demandthat determined the rate of uptake of K. Siddiqi and Glass (1987) have suggested that such anexception should not be used as the basis for rejecting the relationship between K influx androot [K]. They acknowledged that K influx may be influenced by the shoot, but argued thatthese effects may be indirect, resulting from modification of root factors, mainly root [K] as aresult of transport of K to the shoot.Clearly, we are far from understanding the complexities of K regulation and morestudy at the biochemical level needs to be done. According to Yildiz et al. (1994),Chiamydomonas appears to have multiple sulfate transporters that are regulated by sulfuravailability. McClure et al. (1987) and Dhugga et al. (1988) have reported that N03 inductionis associated with changes in membrane polypeptides in maize. Similarly, Fernando et al.(1990, 1992) in barley and Hawkesford and Beicher (1991) in cultured tomato roots, haveobserved that transfer of plants to a low [K] , low [PO4Ior low [S041medium, was19associated with enhanced expression of membrane polypeptides. Recently, Basu et al. (1994)have demonstrated the induction of microsomal membrane proteins in roots of wheat underconditions of aluminum stress.It has been proposed that cytoplasmic [Kj is maintained at a constant level duringK-’- deprivation (Leigh and Wyn Jones, 1984). Does this mean [K] is invariant ? If it is, thenwhere does the signal to increase K influx come from? An interruption of K supply mustinevitably lead to short-term perturbations of cytoplasmic [K’-] (Fernando et al., 1992). Thus,[K-’-] may be oscillating about its optimum value when the cell is subjected to varying [Kj0and a small fluctuation in [K] during this process may be sufficient to trigger changes in thetransport systems involved in K transport across the plasma membrane. These changes maythen be responsible for increasing the influx. Therefore, the signal to increase K influx acrossthe plasma membrane may have come from the cytoplasm itself.In summary, despite extensive kinetic studies of the absorption of K’- in higherplants, beginning with the pioneering investigations of Epstein and coworkers in the 1950’s,mechanisms of K’- transport based upon kinetic data remain controversial (Bange, 1973;Goring, 1976; Borstlap, 1981). Details of energy coupling are uncertain (Cheeseman andHanson, 1980; Poole, 1974; Behi and Raschlce, 1987; Marre, 1977; Pitman et al., 1975) andthe biochemistry of K’- transport constitutes virtually unexplored territory. Contributing factorsto the difficulties experienced by higher plant workers include the tissue heterogeneity whichis characteristic of plant roots as well as their dual role in the absorption and secretion ofinorganic ions.20To avoid these difficulties I have selected a unicellular alga, Chiamydomonasreinhardtii for both physiological and biochemical studies of membrane transport of K. Thismicroalga has been the subject of extensive genetic studies and has provided a convenientsystem for the isolation of transport mutants (Polley and Doctor, 1985). Only a few transportstudies have been carried out on microalgal cells such as Chiorella (Barber, 1968; Kannan,1971), Mougeotia (Wagner, 1974), Dunaliella (Carandang et al., 1992), Skeletonema (Serraet al., 1978) and Chiamydomonas (Polley and Doctor, 1985; Yildiz et al., 1994). Clearly,more work concerning energy sources for K transport, induction and repression of transportsystems in microalgae is needed.Objectives:1). To characterize K transport in Chiamydomonas reinhardtii in terms of its concentrationdependence, energy dependence and patterns of regulation. 2). To explore the mechanism(s)responsible for K uptake and their regulation through comparative studies between the wildtype strain of Chiamydomonas reinhardtii (CCl25) and a mutant defective in K transport(trkl).The studies reported here make use of 86Rb and 42K to examine the kinetics ofK transport over a wide range of external K concentrations ([K]0) and to examine changesof K influx and cellular [K-i associated with K deprivation. Metabolic inhibitors andspecific inhibitors of protein synthesis were employed in order to characterize the observedtransport systems and their responses to K deprivation.To determine the electochemical potential difference across the plasma membrane,21values of the following were needed: 1) membrane electrical potential across the plasmamembrane 2) [K] in the cytoplasm, and 3) external [K]. Membrane electrical potentialswere calculated from the equilibrium distribution of a lipophiic cation,tetraphenylphosphonium (TPPj; [KJ in the cytoplasm was determined from compartmentalanalysis. Concentration dependence of the membrane potential was studied.Prior to doing any influx studies, it was important to estimate the half-lives ofexchange for K for the different compartments. This was required to correct for tracer in thecell wall and also to make sure that influx was being measured across the plasma membrane,rather than fluxes across other cellular membranes. Thus, compartmental analysis wasperformed and the number of cellular compartments, their half-lives and tracer fluxes acrossthese compartments were calculated in the wild type as well as in mutant cells. Estimates of[KJ of a large subcellular compartment were compared with estimates of the [K] of isolatedchloroplasts.To study the correlation between K influx and internal [K], time courseexperiments were undertaken. In these studies, cells were deprived of K for varying durationsof time, and their internal [K] and K influx were measured using 86Rb and 42K as thetracers.To study membrane transport at the biochemical level, microsomes and plasmamembranes were isolated and the membrane proteins were separated on polyacrylamide gels byelectrophoresis (SDS-PAGE). These studies were undertaken to understand the responses ofChiamydomonas cells to K deprivation. To see if the mutant cells (trkl) were different from22the wild type cells at the biochemical level, patterns of their polypeptides were compared usingSDS-PAGE.23II. GENERAL MATERIALS AND METHODS2.1. Strain, Growth Conditions and SynchronizationChiamydomonas reinhardtii strain CC 125(mt) was used for all the experimentsof this research project. Standing cultures were maintained on 1.5% agar slants in 10 mM K(10K) medium in 50 mL culture tubes. These standing cultures were kept at 18°C under lowlight intensity (80 to 100 jiE rn-2 s-1). Stock cultures were started by inoculating a smallvolume of liquid 10K medium with cells from the standing cultures by means of a sterileplatinum loop.Cells used for experimental purposes were grown in 6 L or I L culture flasks(depending on the volume of the culture needed) in TAPM (Tris acetate phosphate medium).The composition of TAPM medium is given in Table I; this is a form of TAP medium (Gormanand Levine 1965) modified by Polley and Doctor (1985) for growing Chlainydomonas. Thegrowth medium was buffered at pH 7.0 and did not change significantly during the growthcycle of the cells. Cell division was synchronized by using a 16 h light /8 h dark regime andcells were sampled for the experiments at the same time (after 48 h of inoculation) in thegrowth cycle. Irradiance was maintained at 200 tE rn-2 s1 at 20°C in a growth cabinet. Allexperiments were carried out when cells were between the mid-log and late-log phases ofgrowth at a cell concentration of 1.5-3.0 x106 cells m11. The cultures were grownmixotrophically with constant stirring and aeration throughout the study period. Theexperiments were conducted in a walk-in environment room having the same conditions of24Table I. Composition of the standard TAPM medium according toPolley and Doctor (1985).NH4C1 7.5 mMCaC12 0.4 mMMgSO4 1mMK2HPO4 4.1 mMKH2PO4 2.7 mMEDTA 0.5mMFeSO4 20 p.MZnSO4 5jiMMnSO4 50 IIMH3B0 5OpMNaMoO4 0.5 tMCoC12 0.5 jiMCuSO4 1jiM;Acetic acid 17.4 mM2-amino-2-(hydroxymethyl)- 1 ,3-propanediol 20 mM25temperature and light as described above.2.2. Growth Media and Modifications for Different [Kj.Cells were normally grown at 10 mM K in TAPM medium which is the standardformulation of TAPM, referred to as 10K. Potassium concentrations in the medium werechanged by adding different concentrations of K (as KC1) to TAPM medium containing noadded potassium (OK). The basic medium was the same as TAPM except thatK2HPO4andKH2PO4were omitted, 7 mM NH4H2POwas substituted for NH4C1; 1.5 mM NH4O and0.5 mM EDTA were substituted for disodium EDTA and the acetate concentration was adjustedto 13.1 mM. The pH of this standard medium was 7.0.All media and glassware were autoclaved and then cooled to the growthtemperature (20°C) before inoculation with the standing cultures. Glassware for growing andmaintaining OK cultures was soaked in detergent and then rinsed with tap water for severalhours. This glassware was finally rinsed with deionized water 5 to 10 times. OK medium waschecked for any traces of K in the flame photometer and found to contain 1-5 p.M K. 3 L ofmedium were inoculated with a small volume (approximately 10 mL) of a log phase stockculture (taken from an agar slant) and an equal volume of the medium was removed beforeadding the inoculum to the flask. These cultures were kept in a temperature and light controlledchamber (Conviron E8) for further growth. Cell samples for different experiments werewithdrawn axenically with a sterile syringe. I attempted to maintain the cultures axenic byautoclaving the glassware and checking periodically for any bacterial contamination with a26phase contrast microscope. No bacterial contamination was detected in these cultures.2.3 Methods to Monitor Cell Growth1) Coulter CounterThe Coulter Counter (Model TAIl) was calibrated with 5.11 p.m microspheres. Astandard volume (10 mL) of cell culture was removed from all flasks as described in theprevious section. These samples (usually quite concentrated) were diluted with ifitered 3%NaC1 solution (which served as an electrolyte) before counting in the Coulter Counter, using anaperture of 70 p.m. Dilutions were from 1:15 to 1: 50 (cells and 3% NaCl); 3% NaCl solutionswere used as blanks.2) HemocytometerCells were also counted with a hemocytometer according to Harris (1985). Cellswere stained by adding 20 p.L of IKI mL’ of solution (1 g‘2’ 0.5 g KI in 100 mL deionizedwater) to the cell suspension before introducing a 1 mL sample into the hemocytometerchamber.3) FluorescenceIn vivo fluorescence of the cells was measured with a fluorometer (Turner DesignsInc. Model 10-000R). TAPM medium was used as a blank and the machine was set at zerobefore reading the actual sample. Five mL samples (4 replicates) were withdrawn from theexperimental culture and transferred to 5 mL glass tubes and read by the fluorometer.274) Chlorophyll ContentChlorophyll content of the cells was estimated using the method given by Harris(1988) with a modification for smaller volumes of cultures using 1.5 mL microcentifuge tubes.One mL of cell suspension (4 replicates) for each sample was pipetted into a 1.5 mLmicrocentrifuge tube and centrifuged at 10,000 rpm for 2.5 mm. The clear supernatant wasdiscarded and 0.1 mL of 0.1% NH4O was added to the pellet followed by 0.9 mL of 90%acetone. This mixture was vortexed for a few seconds to dissolve the pellet and extracted onice for 10 to 15 mm. The tubes were then centrifuged at 14,000 rpm for 5 mm to pellet the celldebris. The absorbance of this chlorophyll solution in the supernatant was read at AM5 (forchlorophyll b) and A663 (for chlorophyll a) in a spectrophotometer (Philips, PU 8820UV/VIS). Acetone served as a blank. Total chlorophyll content (jtg mL1)was calculated asfollows:Total Chlorophyll = (20.2 x AM5) + (8.02 x A663)This equation was taken from Harris (1988). A standard curve was prepared by plotting cellnumber against chlorophyll content.After experimenting with the four methods of determining cell growth, it was decidedto use cell numbers determined with the Coulter Counter throughout this research because thismethod was more convenient, needed no sample preparation and also gave cell volumes inaddition to cell number.282.4 Determination of K Content of the CellsIn all cases, cells in their log phase of growth were used to determine K contentand corrections were made for extracellular K. Several methods were employed to determinethe K content of the cells:1) Centrifugation through silicone oil: Samples were withdrawn axenically from the cultureflasks and 1 ml samples (4 replicates for each sample) were layered on top of 200 pL siliconeoil (1:1 AR 20 /AR 200; Wacker Chemie, MUnchen) in a 1.5 ml microcentrifuge tube. The 1:1ratio of AR 20 fAR 200 silicone oil was chosen because with this mixture, a minimum amountof culture medium and a maximum number of cells (determined by Coulter Counter) passedthrough the oil layer during sedimentation. Only 5% of the cells remained in the supematantand most of these were broken cells which tended to remain at the culture medium and siliconeoil interface. The cell suspension was centrifuged, in an Eppendorf centrifuge (Brinkmann5415), for 20 s at 10,000 rpm, through the silicone oil. The bottom 5 mm of themicrocentrifuge tube, containing the pellet, was cut (Cole.Parmer tube cutter) and immersed in1 M perchioric acid. The resulting solution was diluted with distilled water before aspirationinto the flame photometer (Instrumentation Laboratory 443). Potassium content was expressedas moles cell’. Amount of extracellular K carried through the silicone oil centrifugation wasdetermined as described below:29for 20 to 30 mm. Control cells were kept at 22°C during this incubation period. One mL cellsuspension (4 replicates) from each vial were layered on 200 jiL silicone oil mixture (seedetails in the section dealing with influx measurements). An equal volume of stock 86Rbsolution was added to each sample which was incubated on a reciprocating shaker at 60 rpmfor 10 mm. At the end of this influx period, cells were centrifuged through the silicone oilmixture for 20 s. Counts in the cell pellet were determined by Cerenkov counting in ascintillation counter (Beckman, LS60001C). Radioactivity associated with the heat-treatedcells was presumed to be associated with the cell wall and used to correct for the extracellularK or 86Rb of the cells.b) SDS Treated CellsIntact cells were treated with 1% SDS for a period of 30 mi 86Rb was added tothese preparations at the same radioactivities as present in suspensions of intact cells. TheSDS treated cells were incubated on a shaker for 10 mm with 86Rb as described in Method 1,above, and then filtered through silicone oil as in the influx experiments. The radioactivityremaining in the pelleted material was determined by dissolving the pellet in 1 M perchioric acidand counting by Cerenlcov counting as described in the previous section for the heat-treatedcells. The presumption of this method was that all cell membranes would be completelysolubilized by the SDS treatment and hence radioactivity retained by the cells would beprincipally associated with the cell wall.30c)‘4C-Inulin and3H-Water TreatmentThis method was performed to obtain the cell wall and the intracellular volumes ofthe cells. Since inulin does not penetrate the plasma membrane, the volume of the cell wall andthe rest of the extracellular space can be calculated by incubating the cells in “'C-labeled inulinand determining the radioactivity associated with the cells. From a knowledge of14C-inulintsspecific activity in bulk solution, the volume of the extracellular space can be calculated.Tntiated water is distributed uniformly inside and outside the cells, hence the radioactivityassociated with cells treated with3H-water provides an estimate of the total cell volume.intracellular space can then be obtained by subtracting the extracellular space (derived from the14C-inulin treatment) from the total space (derived from the3H-tritiated water treatment). OnemL cell suspensions (4 replicates) at a cell density of 2.5x106cells mL1 were layered onsilicone oil. “C inulin (at a final specific activity of 2 j.tCi mL’) and tritiated water (at a finalspecific activity of 2 gCi mL’) were added to the cell suspensions which were incubated on ashaker for 10 mm. Cell suspensions were centrifuged for 20 s at 10,000 rpm andradioactivities in the pellets were determined by scintillation counting. The values for the cellwall plus intracellular volumes by all the above methods were very comparable (10-15%). Theinulin method was found to be more convenient and reproducible than the other two methodsand thus, used thoughout. The other two methods (the heat and SDS) were used as acomparison.312) Filter Paper MethodA sample of cells of known cell number was placed on a Miffipore filter (pore size1.2 pm) in a filtering funnel (Nalgene). The growth medium was filtered by applying lowsuction from a tap aspirator. Cells were washed three times with OK for 5 mm in order toremove extracellular K. A wash period of 5 mm was chosen on the basis of a previousexperiment in which the cells were washed on the same Miffipore filter and the filtrate wasanalysed for [K]. It was observed that after 5 mm of washing, virtually all of the cell-wallassociated K had been exchanged and negligible amounts were released subsequently. Thisresult was also confirmed by the compartmental analysis which gave a half-life of 1 mm for theexchange of cell wall K. Thus in 5 mm (5 half- lives) > 95% of cell wall K should havebeen exchanged. Cells on the filter paper were digested with 1 M perchioric acid and analysedfor K by flame photometry as described in part 1 of this section.3) Low Speed CentifugationA 25 mL cell suspension (4 replicates for each sample) at a cell density of 2.5x106cells mL1,was centrifuged at 2000 rpm for 5 mm. The pellet was washed twice with the samevolume of OK medium. The washed pellet was dissolved in 1 M perchioric acid and aliquotsof the resulting solution were analysed for K in the flame photometer as described in part 1 ofthis section.324) Compartmental AnalysisFor compartmental analysis, cells were loaded with 86Rfr or 42K for 24 h at anexternal [K] of 0.1 mM or 10 mM and eluted at an external [K] of 0.1 or 10 mM in thewashing medium. Details of the procedures followed for compartmental analysis (includingthe estimation of pooi sizes of subcellular compartments and K contents) are given inChapter VII.Based on the K content data derived from several methods, the K content per 106cells (nmoles 106 cells-’) was determined. Cell volume was calculated assuming the cells to bespherical. The average [K] (mM) of cells was then expressed on the basis of cell watercontent. Water content of the cells was determined by drying a known wet weight of cells at80°C; the difference between the wet weight and the dry weight was taken as the cell waterplus the cell wall water. To correct for the extracellular space, 10% was subtracted from thetotal water volume (cell water plus cell wall water) of the cell. The value of 10% for theextracellular space was derived from the 14C-inulin and3H-water experiments (described inSection 2.4).5) Determination of K Content of Cells during Time Course StudiesCells were grown to mid-log phase in 10 mM K and centrifuged in 250 mL bottlesin a GSA rotor (2000 rpm for 2.5 mm) at 20°C in a Sorvall centrifuge (model RC5B). Thepellet was washed with OK TAPM medium and resuspended into OK or 100 jiM K TAPMmedium at equal cell densities in order to evaluate the effect of the duration of K deprivation33on cell [K]. Cultures were stirred and aerated during their subsequent growth periods.Samples were withdrawn axenically from the flasks at hourly intervals and 1 mL samples(4 replicates for each time period) were layered on top of 200 iL of silicone oil (1:1 AR 20/AR 200) in a 1.5 ml microcentrifuge tube. These subsamples were centrifuged through thesilicone oil as described above. The pellets were dissolved in 1 M perchloric acid and dilutedwith distilled water before flame photometry. Corrections for extracellular K carried by thecells through the silicone oil were made as described under Section 2.4 (c) of this chapter.2.5 Depletion of Potassium From the Growth MediaDepletion of K from the growth media containing 0.1 mM or 0.5 mM K1- wasmonitored by withdrawing 1 mL samples (4 replicates) from the cell culture at prescribedintervals during the growth cycle. These samples were layered on a small volume of siliconeoil and centrifuged as described previously. Supematant (0.5 mL) was analysed for K byflame photometry.2.6 Potassium DeprivationFor experiments in which K influx had to be measured in the starved and thereplete cells, Chiamydoinonas was grown at 10 mM K to a cell density of 2.5x106cellsmL’. Forty eight hours after inoculation, cells were centrifuged in 250 mL bottles in a GSArotor at 2000 rpm for 2 mm. The cell pellet was washed with OK medium and centrifugedagain. This final pellet was resuspended into OK medium at a cell density of 1.5x 106 cells34mL1 and incubated with aeration and stirring for 24 h in a growth chamber under the samegrowth conditions as described under “General Materials and Methods’tin Chapter II. Thus,these cells were deprived of K for 24 h. Replete cells, growing in 10 mM K, werecentrifuged and washed with 10 mM K and resuspended into 10 mM K for 24 h in parallelwith the starved cells.2.7 Determination of Cell Diameters and Volumes1) Coulter CounterCell volumes were calculated from the volumes of cells in all channels of theCoulter Counter. There are sixteen channels in the Coulter Counter according to cell size.Chiamydomonas cells were counted in channels 5 to 15 using the 70 im aperture of thesample collector. Counts in the blank sample were subtracted from the total counts in thetreatment sample. Total volume of the cells in a sample was expressed as volume ceTh1 orvolume 106 cells1. The volume of the chloroplasts was also determined by use of the CoulterCounter.2) Light MicroscopyCell diameters were obtained by examining cells under a light microscope (Ziess)using a micrometer. Twenty cells for each sample (4 replicates) were examined for eachexperiment.353) Kontron Imaging AnalysisCells grown at different [K9 in the external medium were stained with IKI andmounted on a glass slide. This slide was then examined under the phase contrast microscope atx 450 magnification. Thirty to 50 cells were selected in a random manner to obtain theirminimum and maximum diameters for each replicate (3 replicates for each treatment).The volume of the chloroplasts was also determined by Confocal Imaging Microscopy.Areas and volumes of the cells were calculated from their diameters on the basis of therelationships:A =V =4/3 it r3where, A is the area (jim2)V is the volume (jim3)36III GROWTH3.1 INTRODUCTIONCell division in algal cells is routinely synchronized by periodic reductions in lightintensity (Kates and Jones, 1964). Periodic temperature reductions have also been observed tosynchronize Chiamydomonas reinhardtii division in batch culture at low cell density (Rooneyet al., 1971). Cultures of the unicellular green alga Chiamydomonas can be synchronized bylight/dark cycling under both phototrophic and mixotrophic growth conditions (Voigt andMUnzer, 1987). Under suboptimal growth conditions, e.g. alternating periods of low-intensitylight and dark, cells enter the division phase during the dark period, even though they have notreached the critical cell mass (Mihara and Hase, 1971). Thus, in Chiamydomonas, celldivision is not only under size control, but it can also be initiated by another trigger, which iseffective under suboptimal growth conditions. Two possible hypotheses for cell cycle controlwere proposed: first, that synchrony results from metabolic events which are either light-ordark-dependent, or second, that the light:dark alternation entrains an endogenous circadianrhythm (Spudich and Sager, 1980).The cell cycle in Chiamydomonas is marked by the expressions of discretecytological and biochemical events. Synchronized cultures in light are useful in studying thetiming of these events. The synthesis of chioroplast DNA, and chlorophyll, and othercomponents of the photosynthetic system occur during the light period, while cell division andsubsequent cell separation occur during the dark period (Kates and Jones, 1967). The37accumulation of cellular protein is continuous during the light period, but ceases when the lightis turned off (see Lang and Chrispeels, 1976).When plants are starved of nutrients, the influx of the limiting nutrient increasesseveral fold when it is added back to the medium. Such a negative relationship has beenobserved in the case of K. This relationship between the K concentrations of root cells andK-’- influx into these cells is believed to be due to direct negative feedback regulation of K’-influx by internal K’- (Young and Sims, 1972; Glass, 1976). It has been demonstrated thattransfer of barley piants to low K’- resulted in increased uptake of this ion (Fernando et at,1992). Likewise, increased uptake of [P04-] or [S041 was reported when plants were starvedof these ions (Hawkesford and Beicher, 1991). The above authors also demonstrated thatthese treatments induced the synthesis of plasma membrane associated polypeptides. Only afew such studies have been made in microalgal cells. For example, when grown under -Sconditions, Chlainydomonas reinhardtii responds by increasing sulfate transport andsynthesizing the enzyme and transport systems necessary to exploit alternative sources to sulfur(Davies et al., 1994).383.2 MethodsGrowth was monitored by determining cell number from measurements of in vivofluorescence using methods described under” General Materials and Methods”, section 2.3.a) Relative growth rates (day-’) were calculated using the relationship:K10 = log f.N..LN1t2- tlandk = log2 CN,..LN..o)t2 - tiwhere:K10 = growth constant, equivalent to the number of logarithm-to-base-1O’ units of increaseper day;k = growth constant expressed as ‘logarithm-to-base-2’ units of increase per dayt2 and t1 are the times over which the growth constants were determined.b) The generation time Td, equal to the days/division and Th (hours/division) were calculatedas follows:Td (days/division) = 1/kTh (hours/division) = 24/k393.3 Results3.3.1 Growth of Cells in Media Containing 0 to 10 mM Ka) Growth in 0, 10, 25 and 50 jiM KCell growth rates of cells previously grown at 10 mM K, as shown by cell numberand fluorescence, in media containing various concentrations of K, were comparable (Fig. 2).At very low [Kj, such as 0 p.M K (Fig. 2A), growth rates were slower after 24 h, than atother concentrations. In 10 p.M K, growth rate dropped after 44 h because there wasinsufficient K in the medium to match the cells’ capacity for absorption. Cells divided andgrew at the same rate in 25 and 50 p.M K and maximum cell numbers obtained at the end ofthe growth cycle were 2.5x106and 2.9x106cells mL1,respectively. The growth constants(divisions per day) in the exponential phases of their growth, were 1.6, 2.4, 3.6 and 3.6 in 0,10, 25 and 50 p.M K, respectively. The growth constants in 0 and 10 p.M K weresignificantly different from the values in 25 and 50 p.M K. These growth constants werecalculated over a period from 27 to 51 h in all cases except in 0 p.M K, Tn 0 p.M K cells werein the exponential phase during the first 27 h after which growth declined. In all other cases,cells were in the exponential phases between 27 and 51 h.In vivo fluorescence (IVF) of the cells grown at 0, 10, 25 and 50 p.M K hadmaximum values of 4, 61, 89 and 89 IVF units (Fig. 2B). These IVF values declined in thelate log phases of growth for 0 and 10 p.M K.40- 10SC)— 1Fig. 2 A and C: Cell growth shown by logarithm of cell number (x105)mL-1 ofculture containing various concentrations of K. B and D: Cell growth shown byin vivo fluorescence in media containing different concentrations of K. Arrowsindicate the period of exponential growth. Bars=S.E. of means of 4 replicates.100101.10 20 40 60 80Time (h)100Time (h).10 20 40 60 80Time (h)40Time (h)41b) Growth in 75, 100, 500 jiM and 10 mM KGrowth rates at 75 jiM K were not significantly different (p=0.05) from thegrowth rates at lower K (25 and 50 jiM). Growth rates at 100, 500 jiM and 10 mM K wereequivalent but significantly higher than at 10 p.M K. This was a batch culture and if K wasreplenished the cells would probably have grown at the same rate at 10 jiM as was observed athigher [K] such as 500 p.M or even 10 mM (Fig. 2C). The cells in 75 p.M K grew at thesame rate as in the standard growth medium with 10 mM K. A concentration of 75 p.M Kwas therefore taken as the minimum to sustain minimal growth rates for 60 h. The growthconstants were 3.6, 3.3, 3.5 and 3.3 d for the cells grown in 75 jiM, 100 jiM, 500 p.M and 10mM K in their exponential phases (27 to 51 h) of growth.The peak value for in vivo fluorescence of cells grown at 75, 100, 500 p.M and 10mM K-’- were 86, 82, 82 and 82 IVF units respectively (Fig. 2D). The cells grown at 75 p.MK’- reached this value of maximum IVF later than the cells grown at higher [Ki. The IVFvalues declined during the late log phases after 80 h in all cases (data not shown).3.3.2 Growth of Cells in the Light and Dark at 10 mM KCells in dark-grown cultures divided at a slower rate than in light-grown cultures.Cell numbers increased from 1.16x106to 7.05x106cells mL-1 in the light compared to1.08x106to 2.4x106cells mL-’ in the dark after two days of growth (Fig. 3A). Concomitantwith the increase of cell numbers, there was a large increase in the chlorophyll content in thelight-grown cells whereas it increased only slightly in the dark-grown cells (Fig. 3B). It42100 100A —a-—— light B—0—— light• dark• darkECl) c10. C.)C.) —:10 1 2 3 0 1 2 3Time (days) Time (days)Fig. 3 A: Cells (10) mL-1 B: Chlorophyll content (jig) mL-’ of culture grown (10 mMK) in either light or dark for 0, 1 and 2 days. Bars (smaller than the symbols) =S.E. of means of 4 replicates.43appears chlorophyll was not degraded in the dark, because even after two days, totalchlorophyll content remained the same.3.3.3 Growth of Cells in K-Replete and K-Deplete MediaFollowing transfer from 10 K to OK, cells grew at a rate comparable to that of cellswhich were maintained at 10 mM K for the first 24 h (Fig. 4A and 4B). In the first 24 h, OKcells underwent 1.71 divisions compared to 2 divisions for the 10K cells and these are notsignificantly different at p=O.05. During the next 24 h, OK cells showed no further divisions,but 10K cells divided 1.3 times.In OK solution, cellular [Kj declined from 25 nmoles 106 cells-1 to 8.6 nmoles 106cells-’ after 24 h (Fig. 4A). In the next 24 h there was only a slight decline in [K], andtherefore after 48 h of K starvation, cell [Kj was 7.4 nmoles 106 cells’. Under saturatingconditions (cells grown at 10 mM K throughout; Fig. 4B), K content fluctuated between 25nmoles 106 cells-’ (0 h) and 18 nmoles 106 cells-1 during the 48 h experiment. This fluctuationwas associated with cell divisions which occurred at 11 h due to synchrony of cell division.Control cells swam and divided normally for 48 h, but cells deprived of K grew and swamnormally for the first 24 h only, after which they stopped growing and their swimming sloweduntil, by 48 h no movement was evident.4430A___30B • ceilnumber• cell number020_20_—C_C.)_10.CC C.)C.)+‘ :I .0 10 20 30 40 50 0 10 20 30 40 50Time (h) Time (h)Fig. 4. A: Cells (x105)mL-’ of culture and K content (nmols 106 cells’) showingthe change in K content of cells during K deprivation for 48 h. Cells weregrown at 10 mM K and then transferred at t=O toO mM K for 48 h. B: whencells were grown at 10 mM [K] for 48 h. Bars=S.E. of means of 4 replicates.453.4 DiscussionCell growth, as shown by cell number and fluorescence, in media containingvarious concentrations of K, was essentially independent of [K]0 above 25 .tM K (Fig. 2).At lower [K] such as 0 and 10 jiM, there was not enough K available in the medium to matchthe cells’ demand for K-’- absorption. It has been shown that the transfer of barley plants(Fernando et al., 1992) or cultured tomato roots (Hawkesford and Beicher, 1991) to a low[K-’-], low [P04-] or low [SO4-] medium, resulted in substantial increases in the capacity forhigh affinity transport of the corresponding ions. In the case of K, the affinity for uptake(l/Km) also increased (Glass, 1976). Fernando et al. (1992) and Hawkesford and Beicher(1991) demonstrated that these treatments also induced the synthesis of novel polypeptides(putative transporters) which were associated with the plasma membrane. When the numberof such high affinity transporters increases, the capacity for K-’- absorption should increase.However, this capacity can only be exploited if sufficient K is present in the growth mediumas was the case for cells grown at 0.1 and 0.5 mM K (Fig. 2C) or for cells in turbidostatculture.Thus, when cells were grown at K concentrations in the range from 25 jiM to10 mM, rates of K-’- absorption and growth were comparable because there was sufficient K’available. These growth rates (3 divisions/day) were comparable to the values found in otheralgal cells (2-3 divisions/day). When cells were grown at [K] lower than 25 pM, their growthrate dropped because depletion of K’- in the growth medium showed that they had scavenged46all traces of K until no more K was available in the growth medium. This should not beinterpreted to imply that 25 j.iM is the lower limit for acclimation to [K]0. Rather, becausethese experiments used batch cultures, all available K was scavenged within the first 24 hwhen [K I was lower than 25 jiM. In chemostat-like experiments (because in theseexperiments growth is limited by supply of the limiting nutrient), Asher and Ozanne (1967) andSiddiqi and Glass (1983) demonstrated that acclimation to [K] in the range from 5-10 p.M canoccur in higher plants without a reduction in growth rate as long as the external [K] ismaintained by constant replacementIt is apparent from the cell number and fluorescence data that when cells reachedtheir late log phases (after 51 h in all cases except 10 p.M [K]), their cell numbers increasedsteadily whereas their fluorescence declined beyond this point. This is a common observationin other algal cells (Brand et al., 1981). Since the cells were grown in batch cultures, this couldbe the result of nutrients (possibly nitrogen or carbon) being in short supply at this late stage ofculture. Nitrogen deficiency in the medium in the late log phase would result in reducedsynthesis of chlorophyll. Another possible reason for this decline in fluorescence could be thedensity of cells in the culture causing distortion in the detection of total fluorescence emitted bythe cells.When 10 mM-grown cells were transferred to OK, they divided normally for thefirst 24 h by using internal K reserves. This explains the decline in their internal K content asthey grew to their maximum size and divided. After 24 h, cells stopped dividing in OK cultureand no further significant change could be detected in K content of these cells. Thus, the K47content of cells limited their growth when they were grown in OK for more than 24 h. In the10 mM-grown cells, K-’- content 106 cells-1 was maintained at a value of approximately 24nmoles 106 cells-’ throughout their growth period except for the fluctuations right after the celldivisions where daughter cells were released from their mother cells and had a smaller size anda lower K content (16.4 nmoles 106 cells-’).48IV. CHARACTERIZATION OF POTASSiUM TRANSPORT SYSTEMS4.1. INTRODUCTIONIntact membranes are effective barriers to the passage of ions and unchargedmolecules. On the other hand, they are also the sites of selectivity and transport against theconcentration gradient of solutes (Marschner, 1986). When Epstein and co-workers (1952)plotted K-’- uptake against low external concentrations of K, they obtained a rectangularhyperbola. These authors regarded the kinetics of ion transport through membranes of plantcells as equivalent to the relationship between an enzyme and its substrate. They also showedthat at higher external [K], the influx isotherm reached another level of saturation. This dualpattern of K’- uptake was proposed to be due to the presence of two separate transporters in theplasma membrane.It is widely accepted now that when K is withdrawn from external medium mostplants respond rapidly by increasing the capacity for influx of K across plasma membranes aswell as mobilizing vacuolar reserves (Leigh and Wyn Jones, 1984; Memon et aL, 1985; Glassand Fernando, 1993). Indeed, recent studies using barley roots have demonstrated that within6-12 h of withdrawing exogenous K, the synthesis of several polypeptides, including a 43 lcDintrinsic plasma membrane polypeptide, is increased several fold (Fernando et a!., 1992).Despite extensive kinetic studies of the absorption of K in higher plants, beginningwith the pioneering investigations of Epstein and coworkers in the 1950’s, mechanisms of K’-transport based upon kinetic data remain controversial (Bange, 1973; Epstein, 1976; Borstlap,1981; Per Nissen, 1989). Details of energy coupling are uncertain (Cheeseman and Hanson,1980; Poole, 1974; Behl and Raschke, 1987; Glass and Fernando, 1992 ) and the biochemistryof high affinity K transport continues to be an unexplored territory. Recently, two separategroups (Sentenac et a!., 1992; Anderson et a!., 1992) have cloned K’- channels from49Arabidopsis. These are considered to mediate low affmity K transport. However, the natureof the high affinity system (ATPase, K/H symport, K/H antiporter) is still unknown.Contributing factors to the difficulties experienced by higher plant workers include the tissueheterogeneity which is characteristic of plant roots as well as their dual role in the absorptionand secretion (to the xylem) of inorganic ions.To avoid such difficulties, we chose a unicellular alga, Chlamydomonas and madeuse of 86Rb and 42K to examine the kinetics of K transport over a wide range of external Kconcentrations ([K10). Metabolic inhibitors were employed in order to characterize theobserved transport systems and their responses to K deprivation, at several values of external[K].504.2. METHODS4.2.1 Strain and Culture ConditionsStrain and culture conditions were those described in the “General Materials andMethods”, section 2.1.4.2.2 Influx Determinations(a) General MethodsLog-phase cells were centrifuged, washed with OK TAPM medium andresuspended into OK TAPM before each influx experiment. After centrifugation, these cellswere allowed an adaptation period of 30 mm with constant aeration and stirring. A period of30 mm to recover from the trauma of centrifugation was determined at from previous work(Polley and Doctor, 1985) and from my own experiments. Cells were examined under thelight microscope at various time periods after centrifugation and the rate of oxygen evolutionwas measured (oxygen monitor YSI, model 53), before centrifugation and 30 mm aftercentrifugation. Light microscopy revealed that immediately after centrifugation the mobility ofthese cells was lower than in the undisturbed cells, but began to recover and was comparable tothe uncentrifuged cells after 30 min. The rate of 02 evolution in the cells after 30 min ofcentrifugation, was comparable to the rate in the uncentrifuged cells (61 EImols 02 mg Chlt h’and 63 llmols 02 mg Ch[’ fr1 respectively).One mL of resuspended cells, at a concentration of 2 to 4x106cells mL’51(4 replicates) was layered on silicone oil in the microcentrifuge tubes and, depending upon theexperiment, either K or Rb was added to the cells to bring the final concentration to thedesired level. Stock solutions of K-’- or Rb-’- were prepared so that an aliquot of 25 tL per mLof cell suspension would give the final required [KJ or [RbJ in all cases. Immediately afteradding Rb or K, 86Rb (0.25 IlCi / mL final volume) or 42K’- (0.25 pCi I mL final volume)was added to the microcenirifuge tubes. The microcentrifuge tubes were incubated on areciprocating shaker at 60 rpm for 10 minute-influx periods under the same conditions oftemperature and irradiance as had prevailed during the prior growth periods. After the influxperiod, the cells were centrifuged for 20 s at 10,000 rpm through the silicone oil layer and theresulting pellets were dissolved in 1 M perchioric acid. Radioactivities of the pellets wereestimated by Cerenkov counting in a scintillation counter (Beckman, LS60001C). Values forVm and Km for all the experiments were obtained from Hofstee plots (see Epstein, 1976).(b) Effects of pHTo study the effect of external pH on K(86Rb) influx, log phase cells werewashed with OK as described above. These cells were resuspended in 10 mL of OK mediumbuffered at various pHs by means of 20 mlvi Tris/acetate in 20 mL glass vials. These vialswere placed horizontally on a shaker for an adaptation period of 30 mm. K(86Rb) influxwas then measured as described in the previous section.52TABLE II. Concentrations and pretreatment times for various inhibitors employed forK(86Rb-’-) influx experiments. Cells were pretreated with the inhibitors for the timesshown and were exposed to the inhibitors during the 10 mm-uptake period.Inhibitors Concentration Pretreatment Time(mM) (mm)KCN 0.5 20mmCCCP 0.01 20mmTEA 20 120 mmPCMBS 1 20 mm534.2.3 Inhibitor Treatment and Influx DeterminationIn the experiments where metabolic inhibitors (KCN, CCCP), a sulfhydrylmodifier (pCMBS), a K channel blocker (tetraethylammonium ch1oride-TEA) and inhibitorsof cytoplasmic protein synthesis (cycloheximide and anisomycin) were used, cell suspensionswere pretreated with different concentrations of the inhibitors prior to the influx experiments.Concentrations of inhibitors used are as given in Table II. After the inhibitor pretreatment thecell suspension was transferred to the microcentrifuge tubes and K(86Rbj influx wasmeasured as described in Section 4.2. The inhibitors were also present in the cell suspensionsduring the 10 mm-influx periods.4.2.4 Light/Dark EffectsTo study the effects of dark treatment on K influx, cells, growing in their lightperiod, were washed and then resuspended into OK. After an adaptation period of 30 min,KC1 and 86Rb-’- were added to the cell suspension in the microcentrifuge tubes and these tubeswere immediately transferred to standard conditions of light or darkness and incubated on ashaker. After an influx period of 10 mm, radioactivities accumulated by the cells in the lightand dark were measured as described in the previous section.544.3 RESULTS4.3.1 K InfluxPotassium influx into cells of Chkzmydomonos reinhardtii was studied in theconcentration range from 0.005 to 30 mM using 86Rb and 42K (Fig. 5) as tracers. It is clearfrom the influx data (Fig. 6 and 7) that at low external [K], 42K and 86Rb influxdemonstrated saturation. At high external [K] (>1 mM) K influx showed no saturation andcontinued to increase in apparently linear fashion at [K]0even as high as 30 mM (Fig. 8 and9).a) High Affinity Transport System (HA TS)In the low concentration range from 0.005 to 0.75 mM K, K influx determinedby use of 42K and 86Rb, saturated at about 0.20 to 0.75 mM [K]0 in both starved as wellas unstarved cells (Fig. 6 and 7). A comparison of unstarved and starved cells, as shown inTable ifi, revealed that K(42K) influx was significantly higher in starved cells (Vm: 85nmols h’ 106 cells-’) than the replete cells (Vm: 50 nmols h’ 106 cells1). Km values,determined by Eddie-Hoffstee transformations, for K(42K) influx in unstarved cells werehigher, 0.25 mM compared to 0.162 mM in the starved cells (Table Ill). Similar trends wereobserved when 86Rb was used as a tracer for K but the fluxes were much lower than thosedetermined with 42K (Fig. 7).55300• KrepIeteC K4starved C200 Ccdr-,CE LIri•100 C0 I • I0 10 20 30 40[KJ (rp)Fig. 5 42K influx (nmoles h-1 106 cells’) from [K]0 in the range 0-30 mM inChiamydomonas reinhardtii cells grown in K replete medium (10K; circles)and K deficient medium (OK; squares). Bars=S.E. of means of 3 separateexperiments.5680 80C] starved C] starved• replete.‘ • replete—Q UV C.) 60C———rM40 40.E 20• 200.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2[K} (mM) [KJ (mM)Fig. 6 K-’-(42- ) influx (nmoles h-1 106 cells-’) from [KJ0 in the range 0-0.75 mMshowing he saturable high affinity system in cells grown in K replete and Kdeficient media. Symbols are the same as were used in Fig. 5.Fig. 7 K(86Rb) influx (nmoles h-’ 106 cells-1)from [K}0 in the range 0-0.75 mMshowing the saturable high affinity system in cells grown in K replete and Kdeficient media. Bars (smaller than the symbols) =S.E. of means of 3 separateexperiments57TABLE ifi. Vmax (nifioles h1 106 cells-’), Km (mM) and r2 values for linear regressions forthe starved (0 mM K for 24 h) and unstarved (10 mM K for 24 h) cells. K-’- ( 86Rb-’- or 42K-1-)influx was determined from [K’]0 in the range from 0 to 0.75 mM.86Rb÷unstarved starved unstarved starvedVm 10.69±0.91 16.75±1.57 50.58±4.42 85±0.92Km 0.17±.02 0.16±.01 0.25±0.04 0.16±0.02r2 0.99 0.93 0.94 0.96TABLE IV Effects of various inhibitors on K’-(86Rbj influx (nmoles h’ 106 cell-’)from TAPM with [K]0 at 0.005 and 0.1 mM (HATS) and 4.0 and 10.0 mM (LATS).Values given for % inhibition are the means for % inhibition at the twO HATS and twoLATS concentrations.Inhibitors % InhibitionHATS LATSKCN 59 30CCCP 40 7TEA 17 19pCMBS 28 0Dark treatment 50 1858b) Low Affinity Transport System (LATS)When [K} was increased beyond 1.0 mM, K(42K) influx increased linearlywith [K]0 (Fig. 8). Such a linear trend was also evident in experiments where 86Rb wasused as a tracer for K (Fig. 9) but this linearity was not apparent in experiments where 86Rbwas used to label RbC1 (Fig. 10). Rather, the plot of influx against external Rb concentrationresembled that of a saturable system, although the absolute values of fluxes were comparable tothose measured with 42K.4.3.2 Metabolic Dependence of the Transport SystemsMetabolic dependence of the transport systems was examined by exposing cells toCCCP or to KCN (Table IV). CCCP (10 .iM) inhibited K(86Rb) uptake more (40% ) in thelow concentration range than in the high concentration range (7%). KCN (0.5 mlvi) alsodemonstrated greater inhibition (59%) of the saturable system than the linear system (30%).Influx experiments performed in the dark also showed a greater inhibition of the lowconcentration system (50%) than the high concentration system (18%). TEA, which has beenshown to be a specific K channel blocker in Chara (Tester, 1988 ) and in corn ( Kochian etal., 1985 ) caused a relatively small inhibition of K(86Rb) influx throughout the whole rangeof [K] (0.005 to 30 mM). Sulfhydryl binding reagent, pCMBS was found to inhibit the lowconcentration system by 28% while the high concentration system was unaffected.59I—c)——II100-I IFig. 8. K-I-(42t ) influx (nmoles h-1 106 cells-1)showing the linear system (LATS) obtainedby subtracting the Vmax of the HATS from observed fluxes in the range 1.5-30mM [K]0.Fig. 9. K(86Rb-’-) influx (nmoles h-’ 106 cells-’) showing the linear system (LATS)obtained by subtracting the Vmax of the HATS from observed fluxes in therange 1.5-30 mM [K]0.Bars (smaller than the symbols) =S.E. of means of 3separate experiments.200y = 6.16 + 4.06x RA2 = 0.99y = 13.41+ 4.88x RA2 = 0.98y = 2.97 + 2.30x RA2 = 0.99y=O.61 +3.06x R’2=0.9800 10 20[K] (mM)30 40 0 10 20[K] (mM)30 400C+6040030020010000 10 20 30[RbY (mM)40 50Fig. 10 86Rb influx (nmoles 1r 106 cells-1)from [Rb]0 in the range 0-40 mM showingthe saturable pattern in cells grown in K replete and K deficient media. Insetshows the plot at low external [Rb]. Symbols are the same as were used in Fig.5. Bars=S.E. of means of 3 separate experiments.614.3.3 Effect of External pH on K(86Rb) InfluxK(86Rbj influx demonstrated a strong dependence upon external pH with a clearoptimum at pH 6.0 (Fig. 11). There was a 51% and 32% reduction of K influx at pH 5.0and at pH 7.0, respectively compared to the value at pH 6.0. K(86Rb) influx at all other pHvalues was lower than at pH 6.0.4.3.4 Influx in Cells Grown in the DarkWhen K(86Rbj influx was measured in the light, in light-grown cells, influx wasdetermined to be 2.75 nmoles h-’ 106 cells-’. This influx was reduced by 50% when light-grown cells were transferred to dark for a 10 mm-influx period. When cells were grown in thedark for a period of 1 day, 2 days and 3 days prior to measuring the influx in the dark, K(86Rbj influx was reduced by 52%, 45% and 40% respectively compared to light-grown cells(Fig. 12).628- 7Cl)C.)C::pHFig. 11. Effect of external pH in the range 3.5 to 9.0 on K(86Rb) influx (nmoles h’ 106cells-1).Each symbol represents the average of two separate experiments (fourreplicates for each treatment). Bars=S.E. of means of 2 separate experiments.I • I • I • I • I3 4 5 6 7 8 963injiuxo 2CLTreatmentFig. 12 K(86Rb) influx (nmoles h 106 cells-1)grown in light (L) and dark (D).L: Influx measured in light in light-grown cells; D: Influx measured in dark indark-grown cells after 0 day (OD), 1 day (1D), 2 days (2D) and 3 days (3D) indarkness. Bars=S.E. of means of 4 replicates.644.4 DISCUSSIONData from the influx experiments using either86Rb or as tracers indicated thatK-’- influx was mediated by two distinct transport systems. At low external [K], 42K and86Rb influx occurred by means of a saturable system (Fig. 6 and Fig. 7). At high external[K] (>1 mM), K-’- influx showed a linear pattern (Fig. 8 and Fig. 9).Thus, influx isotherms for K influx in Chiamydomonas reindardtii were resolvedinto saturating and non-saturating components. This corresponds to earlier observations of thepatterns of K uptake by corn roots (Kochian and Lucas, 1982), NO3-uptake in barley roots(Siddiqi et al., 1990) and NH4 uptake by rice roots (Wang et al., 1993). The saturablesystem corresponds to Epstein’s Mechanism 1. It dominates in the low K concentrationrange (0 to 1 mM) and shows Michaelis-Menten kinetics, saturating in the range from 0.20 to0.75 mM [K]0. When K(42K) influx was measured after Chiamydomonas had beendeprived of K for 24 h, Vmax for this influx system increased from 50 nmol h’ 106 cells-’ to85 nmol h-1 106 cells-’ and the K decreased from 0.25 mM to 0.16 mM. The increased Vmvalues may be interpreted to suggest that increased K influx associated with K deprivation isthe result of increased synthesis of the high affinity K carriers. In barley and corn roots, Kdeprivation was also associated with increased Vm values (Glass 1976; Kochian and Lucas,1982). Decreased Km values, indicating an increased affmity for K was first observed inbarley roots during K’- deprivation (Glass, 1976) and was interpreted as evidence for aflostericregulation of K’- influx (see Discussion in Section 1.5) by root [K].In Arabidopsis, the high affmity, saturable system for K’- influx is considered tomediate an active transport step (Polley and Hopkins, 1979). In Chiamydomonas, K’-65(86Rb) influx in the HATS range of [K]0demonstrated a greater inhibition in the presence ofCCCP and KCN and thus, shows its metabolic dependence to a larger extent than the LATS.Transferring the cells to the dark caused a 50% reduction of K(86Rb) influx (compared to18% in the LATS) which again points to a metabolic dependence of the HATS.The non-saturable pattern observed at high [K]0,conforms to the linear kineticsfor K absorption in corn roots reported by Kochian and Lucas (1982). These authorsinterpreted these kinetics as a result of channel- mediated transport. Similar uptake kineticshave been observed for NO3-influx in barley (Mellis, 1982; Siddiqi et al., 1990), maize (Paceand McClure, 1986) and in the diatom Skeletonema costatum (Serra et aL, 1978). A dualpattern of K influx was also reported in Chiorella (Kannan, 1971) but influx at high [K]0appeared to occur via a second saturable system. Likewise, NH influx in rice is mediated bysaturable and linear transport systems (Wang et al., 1993).Although TEA inhibits the majority of known plant K channels (see Bentrup,1990), some TEA -insensitive K channels have been reported, e.g. the Ca2 -dependent Kchannel found in Haemanthus and Clivia endosperm (Stoeckel and Takeda, 1989a) and theplasma membrane 49 pS channel from rye roots (White and Tester, 1992). The failure of theLATS to respond to TEA in Chiamydomonas, may be due to reasons related to the structureand characteristics of this low affinity transporter. Firstly, it is possible that the 2 h treatmentperiod may not have been enough for TEA ions to get into the cell and be accessible to blockthe channel pore. Secondly, the pore structure of this channel responsible for transporting Kmay be different and thus have a different active site from the channels in corn and Chara.Thirdly, this transporter may not be a channel and the linear kinetics we observed in this alga66represent the linear part of a saturable system with a veiy high Km value. In Chiamydomonascells, pCMBS inhibited the saturable component by 28% whereas the linear component wasnot affected at all. In high-salt corn roots, the linear component remained unaffected but inlow-salt roots it was stimulated by 40% in the presence ofpCMBS. Nevertheless, inChiamydomonas it is evident that the saturable and the linear components are respondingdifferently to this non-penetrating sulthydryl reagent and are probably the result of twoindependent mechanisms of K transport. The failure of the linear component to respond topCMBS may be explained by the absence of sufficient sulthydryl groups on this transporterexposed to the extracellular surface.Considering that the half-life for cytoplasmic K exchange was determined to be13 min and the choice of a short influx period (10 min; see Chapter VI) ensures that the fluxesmeasured in these experiments were plasma membrane fluxes. Hence the saturable and linearphase are characteristic of plasma membrane fluxes of K. This finding in a single-celledorganism suggests that in higher plants it is unnecessary to invoke spatially separate membranesystems (plasma membrane and tonoplast) or separate tissue systems (e.g; epidermis andcortex) as the locations of the saturable and linear transport processes as suggested by LUttgeand Laties (1967), Goring et al. (1973) and Bange (1973).K-’-(86Rb) influx showed considerable sensitivity to external pH with an optimumat pH 6.0 (Fig. 4). Between pH 3.5 and 6.0, K influx increased as pH increased. However,between pH 6.0 and 8.0, influx decreased. Thus pH may be exerting an effect on thetransporter activity by causing a conformational change. Such a response to pH was alsodemonstrated in Neurospora crassa (Rodriguez-Navarro et al., 1986). Because of anoptimum for K’- influx at pH 6.0 and evidences from other electrophysiological data in67Neurospora, a K/H symport into the cells has been proposed at low [K]0. InChiamydomonas, the observed effect of external pH on K influx is different from thatreported in red beet slices and maize roots, where K uptake increased as the external pH wasincreased (Lin, 1979; Poole, 1974) or in roots of barley (Glass and Siddiqi, 1982) or corn(Kochian et al., 1989) where K influx showed very low pH sensitivity. This insensitivity ofK uptake to external pH lead these authors to propose that the two fluxes were not coupled.However, Behi and Raschke (1987) reported a K :H flux stoichiometry close to one andhave suggested that the two fluxes are indirectly coupled, via the electrical component of theproton motive force generated by the H -ATPase.Recently, two putative K channels have been isolated from Arabidt,psis bytransforming a K - transport mutant of yeast (Anderson et aL, 1992; Sentenac et aL, 1992).The gene coding for this polypeptide appears to share considerable homology with the ShakerK channels of Drosophila. Surprisingly, transformation of the yeast mutant appeared toconfer both saturable and linear transport capacity in a single gene product (Sentenac et al.,1992). However, the published data are difficult to interpret because the transformed cellsappear to yield linear uptake patterns at low external [Ii rather than the anticipated saturatingpatterns. These results may indicate that traditional interpretation of dual kinetics in terms ofdiscrete transport systems (presumably coded for by distinct genes) may need to be revised.However, Maathius and Sanders (1993) have cautioned against the interpretation of data fromheterologously expressed transport systems. Their electrophysiological data leave little doubtthat in Arabidopsis the channel could not (on thermodynamic grounds) achieve uptake of K atlow external [Ki.68In order to evaluate the effectiveness of 86Rb as a tracer for K, parallelexperiments were undertaken with 86Rb and 42K as tracers for K. Estimates of K influxincreased almost 4 to 5 fold when 42K was substituted for 86Rb. The present study hasestablished that 86Rb gives a good qualitative correspondence to the uptake by 42K butfluxes were reduced quantitatively. This discrimination against 86Rb in Chlainydomonaswas earlier reported by Polley and Doctor (1985). Such discrimination against Rb has beenreported for some higher plants (Jacoby, 1975), algae (West and Pitman 1967; Keifer andSpanswick, 1978) and bacteria (Rhoades et al., 1977). Thus, 86Rb may serve as asatisfactory tracer for K fluxes under certain conditions, specifically, when K status isunchanged. However, experiments to examine the concentration dependence of influx using86Rb÷ in Rb÷ solutions revealed that the pattern of influx was qualitatively different from thatobserved with 86Rb in K÷ or 42K in K solutions. Therefore 86Rb÷ in Rb÷ solution is not asatisfactory tracer for K fluxes in Chiamydomonas.69V. MEMBRANE POTENTIAL DETERMINATIONS5.1 INTRODUCTIONMembrane electrical potential is a property of the whole system and is independent ofthe spatial location of the electrodes. If the measured membrane potential is more negative thanthe value of membrane potential calculated from the Nernst equation (see Methods) when Kconcentrations are inserted, it is feasible that an electrogenic ion flux is occurring across theplasma membrane. It is now accepted that the membrane potential difference has threecomponents, the Donnan potentials, the diffusion potentials and the electrogenic potentials.The Donnan potential is generally quite small, due to asymmetry of non-mobile charged groupssuch as proteins or cell wall charged groups. Diffusion potential is generated due to a gradientof ion concentration across the plasma membrane which is differentially permeable to the cationand anion species present. Electrogenic potential originates due to electrogenic (charge-generating) processes such as H extrusion from the cell. The existence of the electrogenicpotential has been suggested on the basis of experiments in which metabolic inhibitors or lowtemperature were used. Both membrane potential and ATP content decreased concomitantlywhen Neurospora hyphae were treated with azide, CN- or N2 (Slayman et al., 1973).The most direct method for the determination of membrane potential is the use ofmicroelectrodes but their use in small cells is very difficult. The two main problems with themicroelectrode technique are, “tip potentials” of the intracellular pipette in contact with the70cytoplasmic polyelectrolytes and current leakage round the point of microelectrode insertionwhere the plasmamembrane has not resealed properly. These will have a significant effect onthe measured potential difference, tending to lower it. A further problem is the large size ofthe chioroplast in Chiamydomonas. It is very difficult to make sure that the tip is in thecytoplasm rather than the chioroplast (Raven 1980, 1989). Despite these problems,microelectrodes have been used to measure membrane potentials in small cells such asChiorella and Mougeotia (Barber, 1968; Wagner and Bentrup, 1973).Remis et al. (1992) found that the results from the microelectrode technique and fromthe equilibrium distribution of the lipophiic ions such as TPP were in agreement. Thus, itwas decided to use tetraphenyiphosphonium to determine the membrane potential inChiamydomonas, although the values obtained from such indirect methods may be in errordue to internal adsorption, accumulation in subcellular compartments or metabolism (Gimmierand Greenway, 1983; Remis et al., 1992).The positively charged monovalent ion tetraphenyiphosphonium has been usedpreviously in several algal cells (Chiorella: Komor and Tanner, 1976; Dunaliella: Gimmier etal., 1990). This ion penetrates passively into the cells and is distributed between the cell andthe external medium according to the electrical potential difference (z\v) across the plasmamembrane. It was used to measure A’ii in Chiamydomonas in the work described here.715.2. METHODS5.2.1 ChemicalsThe tritiated monovalent organic ion tetraphenylphosphonium (TPP) was obtainedas the bromide salt from Amersham; the non-labeled tetraphenyiphosphonium bromide wasobtained from Aldrich; DNP was obtained from Sigma.5.2.2 Accumulation of TPPCells were grown at 10 mM K, under light and temperature conditions asdescribed in Section 2.1. Log phase cells were centrifuged at 2000 rpm for 2.5 mm and cellpellets were washed with OK medium and resuspended into OK at a cell density of 2.5x 106cells mL-’. This cell suspension was aerated and stirred during an adaptation period of 30minutes.3H-TPP (final radioactivity, 0.5 iCi mL-1)and TPPBr (14.4 tM) were added to thiscell suspension in a 125 mL flask. The external concentration of TPPBr was kept low to avoidaltering the potential difference due to substantial permeation of the ion (Raven, 1980) and outof a concern for decreasing the photosynthetic rate of the cells (Carandang et a!., 1992). Thecell suspensions were incubated with constant stirring and aeration under the same temperatureand light conditions as had prevailed during the prior growth period. One mL samples(4 replicates for each sample) were withdrawn from this cell suspension at intervals (0 to 4 h)and centrifuged through silicone oil as described in section 4.2. This method of cell separation72was chosen because previous work (Komor and Tanner, 1976) had shown that some TPPwas adsorbed by membrane filters during filtration. Cell pellets were first dissolved in 1Mperchioric acid and then mixed with 10 mL of the scintillation fluid (Ecolume). Radioactivitieswere determined by scintillation counting.5.2.3 Calculation of electrical potential difference across the plasmamembrane from TPP+ accumulationThe membrane electrical potential difference (AW) across the plasma membrane ofChiamydomonas was estimated by means of the equilibrium distribution of TPP (Komor andTanner, 1976). It is assumed that at equilibrium, the distribution of ‘fl)p+ will be in accordwith the electrical potential difference across the plasma membrane, as given by the followingequation:AWN = n FTPP10zF [TPP]1This is the Nemst potential for TPP distribution. With this value of AN,, togetherwith the intracellular and extracellular [K], it is possible to estimate ALK and hence ascertainwhether K is moving into the cell actively or passively. Values for cytoplasmic Kconcentration ([K-’-J) were obtained by compartmental analysis (Chapter VI).The Nemst electrical potential difference for K at equilibrium was calculated as follows:AN,N=RT1nKzF K1Subtituting values for constants at a temperature of 20°C in this equation we get73= 58 log,0 IK]O[K]1The electrochemical potential differences LILK, (U mol-’) for K between the cytoplasm andoutside medium were calculated from the relationship:4tK = zF (, - iiN)where L$tK = electrochemical potential difference between cytoplasm andoutside medium (kJ mol’).z = charge on the ionF = Faraday constant (96.5 kJ mol’ V’)R = gas constant (8.3 Joules mo!-’ degree-’)T = absolute temperature in degrees CelsiusAN, = electrical potential difference across the membrane (mV)AN = equilibrium electrical potential difference or Nernst potential(mV)[K]0 = K concentration in the external medium (mM)[K]1 = K concentration of K in the cytoplasm (mM)745.2.4 Effects of external [Kj in the range from 0 to 200 mM on membraneelectrical potentialCells were resuspended in OK medium as described in the previous section. Afteran adaptation period of 30 mm, 10 mL cell suspensions, at a concentration of 2.5x106cellsmL-1,were transferred to 20 mL glass vials.3H-TPP (0.5 iCi mL’ final volume), TPPBr(14.38 pM) and K (as potassium citrate) were added to the cell suspensions to generate fmalconcentrations in the range from 0 to 200 mM. The glass vials were incubated horizontally ona shaker for a period of 3 h. The incubation time of 3 h was derived from the time course ofTPP accumulation described above; by 3 h the accumulation of TPP appeared to havereached equilibrium. At the end of the 3 h incubation period, one mL subsamples (4 replicates)were withdrawn from the glass vials and layered on top of the silicone oil layers. The siliconeoil was a mixture of AR 20 and AR 200 (Wacker Chemie, MUnchen) at a ratio of 1:1 for all[K] except 100 and 200 mM K. In the case of the latter, a mixture of the ratio 1:3 was usedbecause 1:1 mixture of silicone oil was lighter than the TAPM medium (plus cells) with 100and 200 mM K and thus stayed on top of the medium after centrifugation. The procedurefollowed after this was the same as that given in Section 4.2.5.2.5 Effect of DNP on membrane potentialTo determine the effect of DNP on electrical differences (Ai), cell suspensions inOK were incubated with3H-TPP (0.5 pCi mL1 fmal volume), TPPBr (14.4 pM) and DNP(250 pM) in glass vials on a shaker for a period of 3 h. The radioactivities in the cells at the end75of the incubation period were determined as described in the previous section. K was omittedso as to optimize the value of z, to facilitate measuring the effect of DNP.5.2.6 Effects of external pH on membrane potentialTAPM medium (OK) was prepared at a range of pH values (3.5 to 9.0) by using20 mM Tris/acetate as a buffer. Cells growing at 10 mM K were washed and resuspended ina volume of 10 mL OK (in 20 mL glass vials) at different pH values (pH 3.5 to pH 9.0). Afteran adaptation period of 30 mm at these pH values,3H-TPP (0.5 pCi mL-’) and TPPBr (14.38ItM) were added to all the cell suspensions in the glass vials. In this experiment also, no Kwas added to the suspensions to prevent depolarization of the membrane potential so as toamplify the response to pH changes. The glass vials were incubated on a shaker in a horizontalposition for a period of 3 h. At the end of 3 h, radioactivities associated with the cells weredetermined as described in the previous section using the silicone oil filtration method.5.2.7 Effects of Oligomycin and Light/Dark treatments on TPPAccumulation(a) Membrane PotentialsTo measure the membrane potential in the dark, cells were transferred to the glassvials. 3H-TPP (0.5 pCi mL-1),TPPBr (14.38 pM) and potassium citrate (25 pM) were addedto this culture and incubated immediately in the dark (control cells were kept in light) on ashaker for a period of 3 h. After the incubation period, cell suspensions were centrifuged76through the silicone oil and radioactivities of the cells were measured using the scintillationcounter. In order to measure the membrane potential in the presence of oligomycin, 75 .tgmL-1 oligomycin was added to the cell suspension at the same time as3H-TPP was added.Cells were then incubated either in dark or in light for 3 h. One mL samples (4 replicates) ofthe cell suspension were spun through silicone oil and radioactivities in the cells were estimatedas described above.b) Effect of Oligomycin on RespirationThe effect of oligomycin on oxygen uptake was studied by the use of an oxygenelectrode and an oxygen monitor (YS1 model 53) at 20°C. Light intensity was 200 !i.E rn-2 s.The electrode and the chart recorder were calibrated with air-saturated water at 20°C. Air waspassed through the water with constant stirring. Nitrogen gas from a cylinder was bubbledthrough water in the chamber to obtain the zero level of oxygen. One mL of the cell suspensionwas poured into a temperature-controlled chamber. This chamber was covered with analuminum foil to exclude light. The rate of oxygen consumption was recorded on the chartrecorder. From 2.5 to 15 iL of oligomycin stock was added carefully to the cell suspension inthe dark to give final concentrations of 12.5, 25, 50 and 75 jig mL1 and the rate of oxygenconsumption was recorded.c) Effect of Darkness on Oxygen Liberation by PhotosynthesisOne rnL of cell suspension was placed in the electrode chamber and light wassupplied from a lamp at a photon flux density of 200 jiE m2 s, measured at the electrodechamber. A cold water bath was placed in the path of the light to avoid raising the temperature77of the electrode chamber. The rate of oxygen evolution during photosynthesis was recorded onthe chart recorder. The rate of oxygen liberation was then measured in the dark using the sameprocedure. The electrode was calibrated again before recording the rate of oxygen liberation fora replicate.785.3. Results5.3.1. Time Course of Tetraphenylphosphonium (TPP) DistributionIt is clear from Fig. 13 that equilibration of TPP distribution between the cells andthe external medium was not achieved until 2 to 3 h had elapsed. This ion was talcen up by thecells until, at equilibrium, TPP was concentrated about 150 to 200 times the externalconcentration. For all subsequent experiments in which this agent was used to measure Mu,therefore, a 3 h incubation of cells with TPP was used.5.3.2. Effects of External [K] in the Range from 0 to 200 mM on MembranePotentialMembrane potential failed to respond to external [KJ in the range from 0 to0.1 mM and the values remained at about -136 mV (Fig. 14B). At 0.5 and 1 mM K,respectively, there were hyperpolarizations of membrane potential to -149 mV and -156 mV. At[K-’-]0 higher than 1 mM, membrane potential demonstrated a linear dependence on [Kj0,andwas depolarized from a value of -156 mV at 1 mM [Kfl to -70 mV at 200 mM [K] (Fig.14A). The value of depolarization per decade of K’- was 38.7 mV per log [K’] compared to avalue of 58 mV predicted on the basis of a pure Nemstian system.5.3.3. Response of Membrane Potential to External pHMembrane potential responded to pH changes (from pH 3.5 to pH 9.0) in a linear7980006000IU.4000EzU200000 1 2 3 4 5 6Time (h)Fig. 13 Time course for TPP accumulation over a period of 5 h. Counts accumulatedare for 1 mL cell suspensions containing about 3 million cells. Bars=S.E. ofmeans of 4 replicates.I • I • I • I • Ia)Va)a)C1Ea)80-60-100-140-1800 100[K1 (mM)Fig. 14 Effects of external [1(j in the range from 1 to 200 mM on the membranepotential. Cells were grown at 0.1 mM K and briefly exposed to differingconcentrations of [K]0. Inset B shows the membrane potential at low (0 to1000 .tM) [Kj0. Bars=S.E. of means of 3 separate experiments.1 1081fashion (Fig. 15). At low external pH, the membrane potential was more positive. As externalpH increased, there was a hyperpolarization of membrane potential. From pH 3.5 to pH 9.0,M.ji hyperpolarized by 66 mV (-69 mV to -135 mV). Thus, at alkaline pH values themembrane potential was always more negative than at acidic pH values.5.3.4 Metabolic Dependence of AN!Based on TPP accumulation in the light (control), membrane potential wasestimated to be -136 mV in solutions containing 0.1 mM K. This value decreased to-101.9 mV (a depolarization of 34 mV) when TPP accumulation was measured in the dark(Fig. 16). Membrane potential was also measured in the presence of 75 jig mL’ oligomycin.This concentration of oligomycin was found to be most effective in inhibiting respiration(based upon measurements of oxygen consumption in the dark) while having only a smalleffect on photosynthesis. When membrane potential was measured in light in the presence ofoligomycin, a value of -123 mV was obtained (depolarization of 13 mV). This value wasdiminished a further 27 mV when TPP accumulation was measured with oligomycin in thedark (membrane potential of 95.8 mV). The presence of DNP in the uptake solution caused adepolarization of 34 mV, from the control values of -124 mV to -90 mV.a) Effect of Oligomycin on RespirationThis experiment was performed to determine the minimum concentration ofoligomycin required to inhibit respiration completely. Oligomycin concentrations in the range82Table V. Effect of oligomycin on respiration was determined by measuring rate of 02consumption (jimols mg Chi1 h1) in the dark. One mL cell suspension in TAPMmedium (pH 7.0) at 20°C was used.Rate of 02 consumption %Control 100Oligo (50 jig mL’) 60Oligo (75 jig mL-’) 25Oligo (100 jig mL-’) 25Oligo (150 jig mL1) 25Table VI. Effect of darkness and oligomycin on oxygen liberation by photosynthesis.Rate of 02 liberation was determined in one mL cell suspensions in TAPM medium(pH 7.0) at 20°C. Cells were treated with oligomycin for 2 h before transferring themto the electrode chamber. The light intensity was 200 jiE m2s1. Rate of 02consumption was determined under same conditions in the dark.Rate of 02 liberation % Rate of 02 consumption %Control 100Oligo (50 jig mL-1) 93Oligo (75 jig mL’) 91.45Dark treatment 85.883from 5 to 100 jig mL-1 (5, 12.5, 25, 50, 75 and 100 jig mL-’) had no effect on the rate of oxygenliberation in the light. Only at 75 and 100 jig mL’ was photosynthesis affected. To separaterespiration from photosynthesis, this experiment was repeated, under the same conditions, in thedark. Even in the dark, there was no change in the rate of oxygen consumption at lowconcentrations of oligomycin (5 to 25 jig mL-1)but the rate began to decline at 50 jig mL-’ (60%of controls) and it declined to 25% of the control value at 75 jig mL-’. No further decrease ofrespiration was observed when oligomycin concentration was increased to 100 jig mL1 (Table V).b) Effect ofDarkness and Oligomycin on Oxygen Liberation by PhotosynthesisWhen cells were incubated in the dark, oxygen liberation stopped completely and itwas replaced by oxygen consumption due to respiration (Table VI).84-60E -80—.—1If—iv’.,o 0-120E-1406 7 8 9 10pHFig. 15 Effect of external pH in the range 3.5 to 9.0 on membrane potential. Cells weregrown at 0.1 mM K and exposed to differing pH in the external medium.Each symbol represents the average of two separate experiments.Fig. 16 Membrane potential difference (mV) in the presence (+oligo) and absence(-oligo) of oligomycin, measured in light and dark.345Treatment855.4 DiscussionAccording to Remis et aL (1992), the membrane potential values obtained fromTPP may be in eor due to internal adsorption, accumulation in subcellular compartments ormetabolism. Of the organelles, although the chioroplast may occupy up to 50% of cell volume,it is generally considered not to generate an electrical potential difference between the stromaand cytoplasm. Mitochondria are considered capable of generating ANt between the matrix andcytoplasm but their volume is generally quite small. To check the relative contributions ofinternal TPP-’- accumulation due to subcellular compartments or adsorption, effects of externalpH on the measured AN! were estimated. The rationale for this approach was that if Ai acrossthe plasma membrane was the principal cause of TPP accumulation, pH changes in theexternal medium might strongly affect A across the plasma membrane, and thus the amount ofTPP accumulated. By contrast, changes of external pH would not be expected to alter Aicacross internal organelles because of the constancy of cytoplasmic pH (Smith and Raven,1979). It is evident from the data that AN! was very sensitive to external pH. From this result itis concluded that TPP accumulation in Chlainydomonas was largely due to A across theplasma membrane. In addition to this, A measurements in the presence of oligomycin showedthat mitochondria were contributing only about 10% to the value of AN!.The failure of membrane potential to respond to the [K]0 in the lower range (0 to100 tM K) is similar to that observed in Arabidopsis (Maathuis and Sanders, 1993) and isquite typical of algal cells (Vorobeiv, 1980 and personal communication) as opposed to the86situation in corn (Kochian et at., 1989) and Neurospora cells (Rodriguez-Navarro et aL,1986), where large depolarizations occur with the addition of micromolar K to the externalmedium. In Neurospora, at low [K]0 current measurements have shown that two chargesenter the cell for each K ion which enters. This was taken as evidence for a K-’-IW symport asthe primary mechanism for the high affmity uptake of K. For Chiamydomonas, trans-plasmamembrane ALK at low [K÷]0 (12.5 p.M and 100 jiM), had positive values (7.3 and 2.7 kJmol-’ respectively) which means that K is at a higher electrochemical potential inside the cell.Thus, K÷ would move actively into such a cell, against the electrochemical potential gradient.Glass and Fernando (1993) and Maathuis and Sanders (1993) obtained essentially similarfindings based upon measured values of Mi and [K+j in barley and Arabidopsis thaliana,respectively. From their AJ.LK values, they argued that at low external [KJ, K is activelyabsorbed and could not be channel-mediated as suggested earlier (Hedrich and Schroeder,1989; Sentenac et al., 1992).At higher [K]0 (ito 200 mM), membrane potential depolarized from -156 mV to-70 mV in Chiamydomonas. At such high [K]0,K is at a lower electrochemical potentialinside the cell than outside and could move into such a cell passively. Thus, thesedepoarizations may have been due to K entry via K -specific channels, down theelectrochemical potential gradient. These linear depolarizations observed at high [K]0 confirmsimilar observations of Komor and Tanner (1976) in Chiorella and Maathuis and Sanders(1993) in Arabidopsis. Membrane potential is primarily a function of membrane pemìeabiity,ion activity and electrogenic transport processes across the plasmarnembrane. At high [Kj0,the value of depolarization per decade of K was large (38 mV per log [K]). This comparesfavorably with a value of 58 mV for a pure Nernstian system. The lower value (38 mV87compared to 58 mY) may be due to a lower ratio of K to Cl- permeability of the plasmamembrane at such high [K]0.Increasing external pH of the medium had a hyperpolarizing effect on the membranepotential. A similar response of membrane potential to external pH was observed in Charaaustralis (Kawamura et al., 1980), Chiorella (Komor and Tanner, 1976), red beet cells(Poole, 1974) and corn (Kochian et al., 1989).In Chlainydomonas, membrane potential hyperpolarized in light which is inagreement with observations in Chara (Gouta et a!., 1980) and in Nitella (Spanswick, 1974).In these green cells, light may be activating the H pump which acts electrogenically. InNitella, illumination caused a hyperpolarization of 60 mY whereas in Chiamydomonas, ahyperpolarization of 34 mV was detected. DNP, an uncoupler, also depolarized the cellmembrane potential to the dark level (90 mV). When oligomycin was used in illuminated cells,membrane potential depolarized by 13 mV compared to 40 mV depolarization in cells treatedwith oligomycin in the dark. This value of 95 mV in the presence of oligomycin in the dark isprobably obtained when membrane potential is reduced to the diffusion potential for K.The concentration of oligomycin required to inhibit respiration was >50 jig mL’ ata cell density of 3x106cells mL-’. Even at concentrations as high as 100 jig mL-’, only 75%of the oxygen consumption was inhibited in contrast to previous reports (Mottley and Griffiths1977) where the minimum inhibitory concentration of oligomycin has been found to be >25 jigmL-1,but the actual % inhibition was not reported. The difference in the effectiveconcentration of oligomycin may be due to the growth of cells on a solid medium in their studycompared to cells grown in liquid culture in the present study. In addition, the number of cells88mL-’ used in Mottley and Griffith’s experiments must have been lower than used in the presentexperiments.89VI. COMPARTMENTAL ANALYSIS6.1 INTRODUCTIONPotassium is probably found in all compartments of plant cells but its distribution androles have not been well characterized. Its concentrations in cytoplasm and vacuole have beendetermined by using several techniques (Pitman, 1963; Pitman et al., 1981; Harvey et al.,1981; Rona et a!., 1982). Potassium is not replacable in its cytoplasmic functions and plantsappear to maintain cytoplasmic K concentration at a constant level. Leigh and Wyn Jones(1984) proposed a model in which, as the concentration of K in the tissue declines, theconcentration in the cytoplasm is initially maintained constant, while that in the vacuoledecreases. Turgor is maintained by accumulating alternative solutes in the vacuole. Thishypothesis explains the functions of K in cytoplasm and vacuole in vacuolated cells.However, certain microalgae, such as Chiamydomonas lack a large vacuole and have a singlelarge chioroplast as an additional compartment. Potassium concentrations in chioroplasts havebeen reported to be quite high (Larkum, 1968; Robinson and Downton, 1984) but its functionsin this compartment are not well understood. Furthermore, the extent to which intactchioroplasts are able to maintain a constant K concentration independent of the externalmedium is unknown. If the K of the chioroplast is maintained at a constant level, it seemsthere must be some form of ionic control at the outer boundary of the chloroplast.Potassium is believed to play an important role in photosynthesis in the chioroplast(Kaiser et a!., 1980; Demmig and Gimmler, 1983; Pier and Berkowitz, 1987). When intactchioroplasts were assayed in a medium containing only low concentrations of mono- and90divalent cations, CO2 fixation was strongly inhibited. Addition of K, Rb, or Na (50-100mM) fully restored photosynthesis. Similar to its role in cytoplasm (Leigh and Wyn Jones,1984), K-- may be involved in enzyme activation and protein synthesis.Efflux analysis of K has been performed in algal cells (Barber, 1968; Wagner, 1974)and higher plants (Memon et al., 1985; Benirup and Pfruner, 1978) to determine the number ofintracellular compartments and subcellular distribution of K. Compartmental analysis ofChiorella cells showed the presence of two cellular compartments (cell wall and cytoplasm)and that of Mougeotia showed three compartments (cell wall, cytoplasm and vacuole). InMougeotia most of the cytoplasmic compartment is occupied by the large chioroplast.Nevertheless, compartmental analysis showed only three compartments.Values for K concentrations and membrane potentials across the chioroplast envelopehave been determined in isolated chioroplasts (Robinson and Downton, 1984; Bulychev et al.,1972). K has been suggested to move across the chioroplast envelope by passive H/Kexchange (Maury et al., 1981) or through K’- channels (Wu and Berkowitz, 1992). Yet nosingle hypothesis is adequate to explain the mechanism and regulation of K’- movement acrossthis barrier. Clearly, more work needs to be done in order to explain K’- transport intochloroplasts.In the experiments described here, K’- concentrations were determined in the majorsubcompartments of Chiamydomonas by use of compartmental analysis. Both wild typestrains and a mutant (ti-k]) defective in K-’- transport (referred to as kdp4 by Polley and Doctor,1985) were used to probe the basis of defects in the mutant and to explore the mechanism(s) ofK-’- uptake in normal cells. The trkl strain has a specific requirement for high external K91concentrations in the external medium and failed to absorb K or to show enhanced K uptakeafter K deprivation. A model is proposed which explains the mechanism(s) of K transport inwild type cells under conditions of adequate and inadequate K supply.926.2 METHODS-Efflux experiments were conducted using the wild type (CC125) and the mutant(trkl) strains of Chiamydomonas reinhardtii.6.2.1 Efflux from 10 mM KC1 labeled with 86Rb in WT and trkl cellsIn preliminary experiments, 86Rb was used for the efflux analysis. In order tocheck these data in light of the reported discrimination against Rb (Polley and Doctor, 1985),some experiments were repeated using 42K. The experimental set up and the procedure aredescribed in Section 6.2.2.6.2.2 Efflux using 42K in wild type cellsEfflux experiments were conducted using the wild type (CC125) strain ofChiamydomonas. The cells were grown either in 10 mM K throughout (grown, loaded andeluted at 10 mM Kj or grown to log phase at an external KC1 concentration of 0.1 mM andresuspended into fresh 0.1 mM K medium before adding the tracer for loading the cells.These cells were allowed to accumulate 42K from TAPM solution containing KC1 (0.1 mM;pH 7.0) and 42K (2 jiCi mL’ final volume) for 24 h. After loading the cells for 24 h, a smallvolume —10 mL of cell suspension, containing approximately 30-40 x 106 cells was layered onto a Mihipore filter paper (pore size 1.2 jim) in the efflux apparatus (Nalgene filtering funnel).Millipore filters were used for all the experiments because they bound the least amount of93(•. Ree•Washing MediumPlow Pe Couro1 V8irel’Talgene Filring FurmelWashixg SolutionMflhipore1-2 111M layer of mediumFilerP1e1 Snd/___ L.\\‘Bell Jar/ Sroe ThbPlJmpSuction Con13o1. ValveScrilillaliori VialFig. 17 Filtering device used for compartmental analysis ofChiamydomonas cells.94ocJoeFig. 18 Symbols used for various K fluxes across the cellular compartments and their[K9. oc, co: influx and efflux at the plasma membrane; cchl, chic: influx andefflux at the chioroplast envelope; Q. Qcffl: cytoplasmic and chioroplast Kcontent; [K]c, [K]cffl: K concentration of cytoplasm and chioroplast.95radioactivity(42Kj, compared to other filters tested, such as glass fiber and Whatman #1 filterpaper. These cells were eluted with TAPM medium in the filtering device. TAPM mediumsupplied from a reservoir through a fme silicone tube having a screw valve to control the flowrate of the medium (Fig. 17). The flow rate through the Millipore filter was maintained at2 mL mm-’ throughout the experiment, except towards the end of the experiment when theMillipore filter became slightly clogged, probably from small amounts of particulate material inthe wash medium. Vials, to collect the filtrate (eluate), were placed in a bell jar which wasconnected to a vacuum pump having a two-way valve to maintain a reproducible suction forreplicate experiments. By applying low suction, the cells were washed briefly (30 s) withunlabelled TAPM medium (pH 7.0), to remove the radiolabeled medium adhering to thesurface of the cells, as rapidly as possible. The cells were then continuously washed with thewash solution from the reservoir containing 0.1 mM K (for cells loaded at 0.1 mM K) or 10mM K (for cells loaded at 10 mM Kj, and the filtrate was removed by gravity filtrationthroughout the experiment, which typically lasted for 7 h. The rate of delivery of the TAPMmedium was adjusted so that a constant level of -4-2 mm of medium covered the cells at alltimes. The filtrate from this elution was collected in glass vials during the earlier intervals. Atthe longer periods, when samples were taken after 30 to 60 mm intervals, eluates werecollected in flasks and subsampled for radioactivity detenmnations.966.2.3 Efflux using 42K and KC1 (10 mM) in wild type (CC125) and themutant (trkl) cellsEfflux experiments were undertaken using the wild type (CC125j and mutant(trkl) strains of Chiamydomonas. The cells were grown to log phase at an external Kconcentration of 10 mM and loaded with 42K for 24 h at [K]o of 10 mM. It was necessary togrow and load the mutant cells in 10 mM [K]o because these cells failed to grow at lowerconcentrations of K. Wild type cells grew very well at very low [K]o but they were grownand loaded at 10 mM K to conduct proper controls. Unfortunately, the low specific activity ofin 10 mM K resulted in low counts released during efflux analysis. To deal with thisproblem, the radioactivity in the loading solution was increased 5 times and efflux analysiswas then undertaken using 10 mM K for elution.6.2.4 Efflux in the dark using 86Rb and KC1 (10 mM) in wild type cellsCells, grown in 10 mM K were transferred to a 250 mL flask and placed indarkness. The presumption of this experiment was that the characteristics of ion fluxes to andfrom the chioroplast would be altered in dark-grown cells. When these cells were grown in thedark for as long as 10 days, cells swam very slowly even when light was provided to them.Cells grown in the dark for 4 days swam as fast as cells grown in the light and appeared tohave the same shape. Thus, a dark period of 4 days was selected as preconditioning prior toconducting the efflux experiment. These cells were incubated in 10 mM K in the dark for 4days after which 100 p.L of 86Rfr (2 pCi mL1;0.2 pCi pmol-’) was added to the flask. All97steps were performed in the dark. The cells were allowed to grow in the dark for another 24 hin order to label the cells before being eluted for? h, with a washing solution containing10 mM K-’-. The 10 mM potassium concentration was chosen for purposes of comparison withthe data of other experiments which had been performed at this concentration of K-’-. Theefflux experiments were conducted in complete darkness using the procedure described above.A green safe light was used only to check the flow rate and to collect eluates in the collectingvials. In order to make valid comparisons between these dark grown and light grown cells,parallel experiments were conducted at the same time using cells drawn from the same batchculture but exposed to light according to the standard procedure, described in Section 6.2.2.The results shown (Section 6.3.5.) are the mean of 3 independent experiments.6.2.5 Subcellular distribution of K in wild type and mutant cellsUnidirectional fluxes, net fluxes and subcellular distributions of K’- in wild typeand mutant cells were calculated using standard methods (Walker and Pitman, 1976). Thesuperscripts and the subscripts used for fluxes across various compartments are shown inFig. 18. An automated computer methodology for calculations and graphic analysis (Rygieviczet al., 1984) was modified to provide estimates of fluxes between subcompartments. K’concentrations were estimated by assuming the size of the cytoplasm to be 40% of the total cellvolume (Harris, 1989). The volume of each cell was estimated to be 200 pm3 cell-’ on thebasis of light microscope data.98Table VII. Preparation of a 20,45 and 65% gradient mixture (volume 25 mL) forchioroplast isolation.Chemical 20% 45% 65%GRMix5x(mL) 7.0 7.0 7.0Isoascorbate buffer (mL) 0.25 0.25 0.25Solid glutathione (mg) 4.28 4.28 4.28PCBFmixture(mL) 5 11.25 16.25Deionized water (mL) 12.75 6.5 1.5Table Vifi. Solutions for the separation of Chlamydonwnas chloroplasts5 x GR Mix (g needed to prepare 500 mL stock solution)Amount (g) Final conc’nNa4P2O7 1.112 5mMHEPES 29.78 0.25 MSorbitol 150.3 1.65MNa2EDTA 1.86 10mMMgC12 .6H20 0.507 5 mMMnC12 0.495 5mM996.2.6 Chioroplast IsolationIntact chioroplasts were isolated from Chiamydomonas strain CC-400 (cw-15)according to the methods of Mason et al. (1991) and Price and Reardon (1982). Cells, grownunder the same conditions of light and temperature as described in Section 2.1, were harvestedin the late log phase and centrifuged at 2000 rpm for 2 mm. The pellets were washed in 20mM Hepes-KOH (pH 7.5) buffer and resuspended in ice-cold breaking buffer (300 mMsorbitol, 50 mM Hepes-KOH (pH 7.5), 2 mM Na-EDTA, 1 mM MgC12, 1% BSA) at a celldensity of 20 x 10 cells mL1. These cells (10 mL) were broken immediately by one passagethrough a 27 gauge stainless steel needle at a flow rate of 0.5 mL s (Mason et al., 1991). Thebroken cells were then centrifuged in a Sorvall HB-4 rotor for 2 mm at 2000 rpm to pelletwhole cells and intact chioroplasts. The pellet was resuspended in 2 mL breaking buffer andlayered on top of discontinuous Percoll gradients in 30 mL Corex tubes. Compositions of 5 xGR Mix (Gradient mixture; see Price and Reardon, 1982 for details) and Percoll gradients aregiven in Tables VII and VIII. To prepare 5 x GR Mix, pyrophosphate was dissolved in 10 mLboiling water before adding to the rest of the mixture. pH was adjusted to 6.8 with NaOH andthe mixture was brought to its final volume with dd water. The isoascorbate buffer wasprepared with isoascorbic acid (0.5 M) and Hepes (0.05 M) and the mixture was titrated to pH7.0 using NaOH. The PCBF mixture consisted of 35 mL Percoll (Pharmecia Fine Chemicals),1.05 g polyethylene glycol (Sigma 8000), 0.35 g bovine serum albumin and 0.35 g Ficoll.The gradients were centrifuged in a Sorvall HB-4 rotor at 5000 rpm for 15 min. The 45 and65% interface, containing intact chloroplasts, was collected and diluted four-fold with breaking100buffer. Intact chioroplasts were concentrated by centrifugation at 2000 rpm for 1 mm, andresuspended in 0.5 mL 50 mM Hepes-KOH buffer (0.3 M sorbitol, pH 8.0).Oxygen evolution from these intact chioroplasts was measured in a temperaturecontrolled chamber using an oxygen electrode and an oxygen monitor (YSI). Intactness of theisolated chioroplasts was detennined by using the ferricyanide exclusion test. Ferricyanide,which is widely used as an oxidant, does not cross the intact chioroplast envelope at anappreciable rate (Lilley et aL, 1975) and will therefore only react freely with thylakoids. Ratesof oxygen evolution were monitored in the intact chioroplasts (control) and in the chloroplastssubjected to osmotic shock (buffer with no sorbitol), so that their envelopes would rupture torelease the thylakoids, in the presence of 1 mM ferricyanide (Mason et aL, 1991).Comparisons of the rates of 02 evolution observed before and after osmotic shock were usedas an intactness assay. The assay mixture contained 250 ig chlorophyll (intact chioroplasts) in150 mM sorbitol, 50 mM Hepes-KOH (pH 7.5), 2 mM EDTA, 1 mM MgCl2, 1 mM MnCl2,3 mM KC1, 0.25 mM KH2PO4,4 mM glycerate-3-P, 2 mM HCO3-and 1 mM ATP in a finalvolume of 10 mL. The light intensity was 400 lIE rn2s1. This light intensity was used inorder to be able to compare our results with those of previous workers (Mason et a!., 1991).The rate of 02 evolution was also measured in isolated chioroplasts and whole cellsunder the same conditions used to estimate the integrity of intact chioroplasts.6.2.7 Potassium Concentrations of Intact ChloroplastsPotassium concentrations were determined in these intact chioroplasts to compare101them with the values obtained from compartmental analysis. Isolated chloroplasis weredisrupted in 1 M HC1O4and run through the flame photometer. Values of [K] estimated bythis method were significantly lower than the estimates of compartment ifi (considered to bethe chioroplast) derived from compartmental analysis. However, it has been documented that[K] of the chloroplasts declines during isolation due to its mobility across the chioroplastenvelope (Robinson and Downton, 1984). To minimize this loss, potassium was precipitatedin the cells by treatment with sodium cobaltinitrite before isolation. To prepare the stocksolution of sodium cobaltinitrite, 2.5 g of this salt was dissolved in 10 mL dd water andfiltered through a Whatman #1 filter paper. Sodium cobaltinitrite stock solution was added to1 L cell culture (at a final concentration of 2.5 g L-1) and the culture was incubated in a 3 Lflask on a shaker, for 5 mm. This concentation of sodium cobaltinitrite caused all of the cells tostop moving due to precipitation of K. The cells were centrifuged at 2000 rpm for 2 mm,washed in 20 mM Hepes-KOH (pH 7.5) buffer to remove extra salt and resuspended in thebreaking buffer. Intact chioroplasts were then isolated from these cells as described above.Chlorophyll contents of the whole cells and the isolated chloroplasts were estimated accordingto the method described in section 2.3 under “General Materials and Methods”.1026.3 Results6.3.1 Efflux using KC1 (0.1 or 10 mM) Labeled with 86Rb in Wild TypeCellsCompartmental analysis of WT (CCl25) cells grown at 0.1 mM and 10mM Kand labelled with 86Rb under the same conditions of K supply (steady state conditions)revealed the presence of three compartments (1,11 and ifi) with half-lives for exchange of0.7±0.06 mm, 15±1.12 mm and 3.3±0.5 h respectively for 100 p.M grown cells and0.56±0.03 mm, 12.8±1.61 mm and 3.50.83 h, respectively for 10 mM K grown cells(Fig. 19 and Table IX).6.3.2 Efflux using KC1 (10 mM) Labeled with 86Rb in the Mutant (trkl)CellsCompartmental analysis of trkl cells also showed three compartments with half-lives of 1.1±0.5 mm, 16.7±1.73 mm and 8.6±1.59 h, respectively (Fig. 20 and Table IX).According to Students’ t-test analysis, the half lives of compartment ifi in WT and trkl cellswere significantly different, but those of compartment I and compartment II had the same half-lives.6.3.3 Efflux using KC1 (10 mM) Labeled with 42K in WT and the Mutant(trkl) CellsHalf-lives of the three compartments were similar to those determined from 86RbFig. 191033.0,-J3.3: •...3.5-L)-S•Q30 50 30 50TIME (,.dn)Linear regression (as dots) on semi-log plot of 86Rb elution data from wildtype Chiamydomonas cells grown at 10 mM K used to detennine the slowcompartment half-life; inset shows linear regression on log cpm rnin1remaining in cells after subtraction of slow compartment.3.0 —2.92. 8(Ti-J-JwLi0II-J-I(1)0LiCD0-J2. 72. 62.5 —±•.2.4 —2.31I I I 1 I I t i •i I I I I i i2 3I I t4.TIME (h)I I I i i , I5 6 7Fig. 201044.0 —cT) .S..+z+0 •U +( ++z—3.0 -w(_::2.! — I I I i I t•t0 20 40 80. TIME (mm)Linear regression (as dots) on semi-log plot of 86Rb elution data from trklChiamydomonas cells grown at 10 mM K used to determine the slowcompartment half-life; inset shows linear regression on log cpm mm-1remaining in cells after subtraction of slow compartment.4.24. 14.03.9(nC)CDuJCD-JF++3. 83. 7.+.3.6—3. - I I --- I I - I I II 2 3I 1 ; .4TIME (h)5 6 7•105Table IX. Compartmental analysis using K4(86Rb) efflux in 10 mM [K4]0inChiamydomonas reinhardtii. Half lives of exchange (t05) for the three compartments(1,11 and ifi) are given in minutes, minutes and hours, respectively, and include 1S.E. of means of 4 separate experiments. Values followed by the same letter are notsignificantly different at p=0.05 level of probability.Compartment I Compartment II Compartment IllHalf life (mm) Half life (mm) Half life (h)WT trkl WT trkl WT trkl0.56±0.03a 1. 10.5 12.8±1.6 lb l6.7±l3b 3.5±0.83c 8.6±1.6dTable X. Compartmental analysis of Chiamydomonas reinhardtii conducted by use ofK4(42K) efflux at 10 mM [K4]0in wild type (WT) and mutant (trkl) strains. Halflives of exchange (t05) of the three compartments (1, II and ifi) are given in minutes,minutes and hours, respectively, and include ±1 S.E. of means of 4 separateexperiments. Values followed by the same letter are not significantly different atp=0.O5 level of probability.Compartment I Compartment II Compartment ifiHalf life (min) Half life (min) Half life (h)WT trkl WT trkl WT trkl1.07±0.07a 0.93±0.2a 12.8±0.90b 14.75±1.5” 2.9±0.62c 9.8±1.9d106efflux as is shown in Table X.6.3.4 Subcellular Distribution and Fluxes of K in Wild Type and MutantCellsThe subcellular distribution and fluxes of K (Fig. 18) in cells of WT and mutant(trkl) strains grown at 10 mM external K ([K]0)were estimated by compartmental analysis.At low external K, trkl cells failed to grow whereas the WT cells grew very well. From thecompartmental analysis data (Table XI), it is evident that the [IC] of compartment ifi in WTcells is much higher (223±31.4 mM) than the corresponding compartment of trkl cells(127±14.2 mM) even in cells growing at 10 mM K. Compartments II [K] for the WT andtrkl cells were almost the same (WT: 77.6±13.8; trkl: 71.2±4.96). Total K contents ofcompartment II and Ill ([K] + [K]Cffl) were calculated to be 24.15±1.42 nmoles 106 cells-’ inWT cells and 16.03±1.64 nmoles 106 cells-’ in trkl cells. These differences were confirmedby independent K analyses by flame photometry, which indicated that the K contents of WTand mutant cells were 24 nmoles 106 cells-’ and 15.43 nmoles 106 cells’, respectively. For10 mM K-’- grown cells, potassium(86Rb) influx from 0.1 mM [K]0 was 2.78±0.07 nmolesh1 106 cells-1 in the WT cells compared to 2.12±0.5 1 nmoles h” 106 cells-’ in trkl cells. Netfluxes of K’-(86Rbj were also lower (0.42 nmoles h’ 106 cells-’) in trkl cells compared tothe WT cells (0.81 nmoles h-’ 106 cells-’).In the WT cells, decreasing [K’-]0 from 10 mM to 0.1 mM provided during theloading period (Table XII), caused a 3 to 4 fold decrease in [K] of compartment ifi (223 mMto 64 mM ) but compartment II [Kj decreased only slightly (77.6 mM to 65.1 mM). The data107Table XI. Unidirectional K fluxes 0 (nmoles h’ 106 cells-’), net flux (J and Jeehi),K-’- concentrations ([K] in mM) and K contents (Q and Q, in nmoies 106 cells-’)calculated from compartmental analysis at 10 mM [K]0in wild type and trkl cells.Subscripts oc and co refer to fluxes from outside to cytoplasm and cytoplasm tooutside, respectively, while Subscripts cchl and chic refer fluxes from cytoplasm tochioroplast and chloroplast to cytoplasm respectively.K fluxes Wi’ trkl0 11.24±3.96 9.1±2.760 10.38±3.85 8.55±2.7J 0.865±0.11 0.6±0.05Occhl 7.77±2.8 1.51±0.22Ochlc 7.01±2.68 1.06±0.21cchl 0.81±0.15 0.42±0.04Q, 6.25±1.10 5.72±0.42Qchl 17.9±2.53 10.44±1.34Qr 24.15±1.42 16.03±1.64[K’-]c 77.6±13.8 71.2±4.96[K’-Jchl 223.5±3 1.4 127±14.2Table XII. K-’- concentrations ([K] in mM) calculated from compartmental analysis performedat 10 mM and 0.1 mM K’- in wild type cells of Chiamydomonas. Subscripts c and chl refer to[K’-] of cytoplasm and chloroplast respectively.[Kj 10mM K’- 0.1 mM K[K]c 77.6±13.8 65. 1±3.09[K]cffl 223.5±31.4 64±6.14108for compartment II ([K]0)and compartment III ([K]0M) for trkl cells in 0.1 mM K were notobtained because these cells failed to grow at 0.1 mM [K]0.6.3.5 Efflux in the Dark using 86Rb and KC1 (10 mM) in Wild Type CellsWhen the efflux experiments were conducted in the dark using wild type cells, thesame three compartments became evident. The half-lives of compartment 1(1.15 mm) andcompartment 11(13.8 mm) were rather similar to those detennined in the light (Table Xffl).The fluxes between these two compartments also remained unchanged (Table XIV). Theaverage half-life of exchange of compartment Ill (based on 3 independent experiments),however, was 2.4 h compared to 4.15 h in the light. Potassium(86Rb) fluxes to compartmentIII also diminished in the dark and resulted in a lower net flux in the dark (0.41 nmoles h1 106cells1)than in the light (0.81 nmoles h’ 106 cells-’). Total K content in the dark-grown cellswas lower (15.93 nmoles 106 cells-1)than the light-grown cells (24.15 nmoles 106 cells-’).Potassium concentrations in compartment II remained unchanged at a value of 75.38±2.46mM, but the [K] of compartment UI ([K]0ffl) decreased to a value of 124±2 1.22 mM(compared to 223.5±3 1.4 mM in the light grown cells: Table XIV).109Table XIII. Compartmental analysis of K(86Rb) efflux at 10 mM [K]0inChiamydomonas reinhardtii in light and in dark. Half lives of exchange (t05) of thethree compartments (1,11 and III) are given in minutes, minutes and hours respectivelyand include ± 1 S.E. of means of 3 separate experiments. Values followed by the sameletter are not significantly different at p=O.O5 level of probability.Compartment I Compartment II Compartment IllHalf life (mm) Half life (mm) Half life (h)L D L D L D0.6±0.lOa 1.15±0.6a 14±0.05” 13.8±o.7b 4.15±o.65c 2.4±0.3dTable XIV. Unidirectional K fluxes 0 (nmoles h-1 106 cells-’), net flux (J and Jeehi),K-’- concentrations ([K-’-] in mM) and K content (Q and Q, in nmols 106 cells-1)calculated from compartmental analysis in wild type cells of Chiamydomonas in thelight and the dark. Subscripts oc and co refer to fluxes from outside to cytoplasm andcytoplasm to outside, respectively. Subscripts cchl and chlc are fluxes from cytoplasmto chioroplast and chioroplast to cytoplasm respectively.K fluxes Light Dark0 11.24±3.96 13.5±3.4Oco 10.38±3.85 12.9±3.53J 0.865±0.11 0.58±0.06Occhl 7.77±2.8 2.99±0.31Ochic 7.01±2.68 2.57±0.24cchl 0.81±0.15 0.41±0.06Q 6.25±1.10 6.03±0.19Qchl 17.9±2.53 9.95±1.67Qr 24.15±1.42 15.93±1.44[Kflc 77.6±13.8 75.38±2.46[K]chi 223.5±31.4 124±21.22110Fig. 21. Isolated chioroplast of Chiamydomonas reinhardtii under a fluorescent lightmicroscope at 600x magnification.1116.3.6 Intactness and Yield of Isolated ChioroplastsThat isolated chloroplasts retained their ability to photosynthesize was shown bytheir capacity for 02 liberation. The rate of 02 evolution was 22 pmols 02 mg’ Chi h1 inintact chioroplasts. According to the ferricyanide exclusion test, 93% of the chioroplasts wereintact. The isolated chioroplasts were also examined under light microscope (x 600magnification) and were found to retain their cup shape (Fig. 30). The yield of intactchioroplasts was 12% (9156 i’g chlorophyll contained in intact cells yielded 1050 igchlorophyll in chioroplasts). This is in agreement with earlier published results of Moroney etal. (1991) who also used the syringe-method to isolate chioroplasts.6.3.7 Potassium Concentration of Isolated ChioroplastsIn an attempt to prevent K loss from chioroplasts during isolation, K wasprecipitated in whole cells by use of sodium cobaltinitrite prior to disruption of the cells andchioroplast fractionation. The yield of intact chloroplasts by this method was lower than forthe standard isolation method, but estimates of the [K] were much higher (165±2 1 mMcompared to 53±3.09 mM). This value compared favorably with the estimates obtained bycompartmental analysis (223±3 1.4 mM). These values were still somewhat lower thanobtained from compartmental analysis. This may be due to some K loss during isolation ofthe chloroplasts, inspite of the precautions taken.1121000-I-100 --200225[K] (mM)Fig. 22 Electrochemical potential differences for K (z.tKj were calculated for cellsgrown at 0.1 mM K, using the value of -135 mV as the Aji across the plasmamembrane. Inset B shows AJ.LK+ at low [K+]0.Each symbol represents theaverage of three separate experiments.A0.5—— . I • I0 25 75 125 175113WT trkl(c) (d)Fig. 23. Electrochemical potential differences, 4u. (U mo11)for K across the plasmamembrane and chioroplast envelope at 0.1 mM and 10 mM [K]0 in wild type (aand c) and trkl (b and d) cells of Chiamydomonas.1146.3.8 Electrochemical Potential Difference Across the Plasma MembraneIn order to evaluate the electrochemical potential gradients for K between the majorcompartments of wild type and trkl cells, AN! values were taken from Chapter V together withvalues for [K] from the present paper. For wild type cells two values of external [K],0.1 mM and 10 mM, were used. For trkl cells, it was possible only to use 10 mM becausethis strain failed to grow at 0.1 mM.Under these conditions, cytoplasmic [K] was estimated to be 65.1 and 77.6 mM,respectively for WT cells grown at 0.1 and 10 mM [K], while corresponding AN! values were-135.57 and -129.52 mV. The electrochemical potential difference for K (A$.tK) had apositive value at low [KJ0 in the range of 0 to 200 p.M while from 0.2 to 200 mM, theelectrochemical potential difference assumed a negative value (Fig. 22; Table XV). At 0.1 mM[K]0,AP.K across the plasma membrane was 2.7 U mol-’ whereas at 10 mM [K-’-]0,Ap.was —7.5 U mo!-1 in WT cells. Assuming that AN! in trkl cells was the same as in WT cellswe estimated Ap.+ for trkl cells (Fig. 23). Since cytoplasmic [K’-] were extremely similar intwo strains, this assumption appears to be valid. The electrochemical potential differences forK’- (Ap.) across the chioroplast envelope were also calculated on the assumption that therewas no electrical potential difference between chioroplast and cytoplasm (Fig. 23). Across thechioroplast envelope, Ap.+ was calculated to be 2.6 U mol-’ in the WT cells and 1.4 U mol-’in trkl cells when [K]0was 10 mM.115Table XV. Elecirochemical potential differences (U mo!-1)for K between cytoplasm andexternal media at different values of external K concentration.[K]0 (mM) [K]1(mM) 1,N (mV) b, (mV) Aj.t (U mo11)0.0125 65.1 -215.6 -139.7 7.30.025 65.1 -198.1 -137.7 5.80.05 65.1 -180.6 -136.4 4.30.075 65.1 -170.4 -135.4 3.40.1 65.1 -163.2 -135.6 2.70.5 77.6 -127.1 -149.5 -2.21.0 77.6 -109.6 -156.4 -4.510 77.6 -51.6 -129.5 -7.5100 77.6 6.4 -71 -6.2200 77.6 23.8 -70 -4.41166.4 DiscussionThree compartments detected by compartmental analysis in Chiamydomonasreinhardtii probably correspond to the cell wall, cytoplasm and chioroplast, respectively.Half-lives of the first and the second compartments were very close to values that have beenreported for the cell wall and the cytoplasm in other organisms (Pfruner and Bentrup, 1978).It is evident that the half-lives of the first and the second compartments are similarin WT type and trkl. By contrast, the half-life of exchange for the third compartment of trklcells was significantly longer than that in WT cells. The half-life of the third compartment wasseveral hours which is similar to the vacuolar half-life of exchange in vacuolated cells (Pfrunerand Bentrtip, 1978). Chiamydomonas does not contain a large vacuole. However, it doescontain a huge chioroplast which occupies about 40 to 50 % of the cell volume (Harris, 1988).Thus, the most likely candidate for compartment ifi was the chioroplast. This hypothesis wastested in two ways: 1) by comparing the [K’] of isolated chioroplasts to the [K’] ofcompartment ifi estimated by compartmental analysis; and 2) by estimating the [Ki ofcompartment ifi in darkness. The rationale for this approach was that this treatment wasanticipated to affect chioroplast development and hence the characteristics of K distribution inthis organelle.The [K] of isolated chloroplasts, immobilized by sodium cobaltinitrite, was foundto be 165±2 1 mM, which is reasonably close to the [1(1 obtained for compartment III fromcompartmental analysis (223±3 1.4) and is consistent with the hypothesis that the thirdcompartment is the chloroplast. These values are in the range of those reported in the literature117for the [K] of isolated chloroplasts. For example, Robinson and Downton (1984) reportedvalues which were up to 200 mM for chioroplasts from spinach, sugarbeet and pea leaves.Half-lives of K-’- exchange for compartment Ill and fluxes of K+ to and from this compartmentwere significantly lower in dark-grown than in light-grown cells. It was observed that whenChiamydomonas cells were grown in the dark, their structure was severely affected. Thus,our observation that growth of Chlainydomonas cells in the dark altered the characteristics ofcompartment ifi but not of the other compartments, is consistent with the hypothesis that thethird compartment is, indeed, the chloroplast. In the following discussion, therefore,compartments 1,11 and III will be refeffed to as the cell wall, cytoplasm and the chloroplast,respectively.Compartmental analysis of WT and trkl cells also showed differences in theallocation of K’- between compartment II (cytoplasm) and compartment ifi (chioroplast). It isevident that the [K’-] of chioroplasts of WT cells is much higher (223±31.4 mM) than that oftrkl cells (127±14.2 mM). Potassium fluxes across the chioroplast were diminished in thetrkl cells compared to the WT cells which may account for the lower [K]ffl in the trkl cells(Table XI).At low external K (0.1 mM to 1.0 mM), ti-k] cells failed to grow whereas WTcells grew normally. Since the half-life of K’- exchange for chloroplasts of trkl cells was verymuch longer than that of WT cells, the failure of the mutants to grow at low external K’- mayhave been due to their inability to load and unload K’- at an appreciable rate to and from thiscompartment. It is evident from the data of Table XII that compartment III was, indeed,118serving as a reservoir for K when WT cells were grown at low [K]0. When cells weregrown at 0.1 mM K, cytoplasmic [Kj was maintained at an almost constant level, whilechioroplast [K] dropped from 223 mM to 64 mM.Because these chioroplasts were isolated from wall-less mutants (cw 15), it wasnecessary to check the subcellular compartmentation of K and half-lives of K exchange inthis strain using efflux analysis. The half-lives of these compartments in cw 15 cells wereidentical to those of the respective compartments of WT cells (data not shown). It wassurprising that even the half-life of the cell wall did not differ significantly in these cells(0.5±0.12 mm ) from that of the wild type cells (0.56±0.03 mm) although it has been shownthat these wall-less mutants produce a much less cell wall material (Davies and Plaskitt, 1971).It may be that the part of cell wall lacking in these wall-less mutants (W2-W6 layers) does notbind any significant amounts of K•I was concerned that the two types of cells (WT and trkl) might discriminate todifferent extents against 86Rb and that this difference might account for the different half-livesobserved in WT and the trkl cells. Thus, the efflux experiments were repeated using 42K asthe tracer in the two types of cells. It is clear that the half-lives of the three compartments werevery similar, whether estimated by the use of 86Rb or 42K and that the differences in thehalf-lives observed between the WT and trkl cells are real.I4.LK+ values across the major cell membranes (shown in Fig. 23) were calculated,using the estimated value of cytoplasmic [K4] and the ANt value obtained from TPPdistribution (see Chapter V). In WT cells, AI.LK+ across the plasma membrane at 0.1 mM119[K]0,was calculated to have a positive value (2.7 U mol’) which means that K is at ahigher electrochemical potential inside the cell. Thus, it must move actively into such a cell,against the electrochemical gradient and a channel-mediated transport at or below thisconcentration is unlikely (ef. Hedrich and Schroeder, 1989). At higher external (0.2 mM)[K-’-], K’- is at a lower electrochemical potential inside the cell and could move into such a cellpassively or down its electrochemical potential via some K’- specific channels. For trkl cells,AJIK+ across the plasma membrane was calculated for cells grown at 10 mM only. At thisconcentration, IS4IK+ was -7.7 U mol’. Thus, in the mutant, as in WT cells, K’- entry may bepassive.A$IK+ across the chioroplast envelope was calculated to be -0.04 U mol’ and 2.5U mol’ respectively, in WT cells grown in 0.1 and 10 mM [K]0. It was assumed that noelectrical potential difference occurred across the chloroplast envelope. In trkl cells L4IK+ wascalculated only for cells grown at 10 mM [K]0. Under these conditions AI.LK+ was 1.4 Umo!-’ (Fig. 23). Thus, at 10 mM [K]0,K is at a higher electrochemical potential in thechioroplast and has to move against the electrochemical potential gradient. Considering Ap+across the plasma membrane and the chioroplast, a model is proposed to explain the movementof K in WT and trkl cells (Fig. 24). It is proposed that at 10 mM [K’-]0,WT as well as trklcells grow normally because K’- is moving into the cells of both strains through K’--specificchannels, down the electrochemical potential gradient. Under these conditions cytoplasmic[K’-] reaches 70-80 mM in both WT and trkl strains, and the transfer of K’- to the chioroplastbrings chloroplast [K’-] to 224 and 127 mM, respectively. The maintenance of these high120wr trklFig. 24 A model proposed to explain the mechanisms of K transport across the plasmamembrane and chioroplast envelope at 0.1 mM and 10 mM [K10 in wild typeand trkl cells.K = K channel= K- symporter121concentrations requires that the flux to the chioroplast be active, mediated perhaps by a KIHsymporter. was calculated to be -8.4 U mo11 for both strains. Thus in energetic termsthere is adequate p. m. f. to sustain this flux.When the cells are transferred to 0.1 mM [K]0,WT cells grow normally whereastrkl cells failed to grow at all. Indeed continued growth of WT cells at 0.1 mM K led toincreased activity of the HATS, possibly as a result of derepression of the gene coding for theHATS. Also, at this low external concentration of K, the chioroplast appeared to serve as areservoir of K. Under these conditions, K was transferred from the chloroplast to thecytoplasm, probably moving “downhill” via K channels. WT cells can withdraw K from thechioroplast at an appreciable rate (hence the shorter half-life of exchange) so that cytoplasmic[K-’-] is maintained at an optimum level. Hence, at steady state cytoplasmic [K9 was slightlyreduced (from 77 to 65 mM), while chioroplast [K’-] was substantially lowered (from 224 to64 mM). By contrast, trkl cells appeared to withdraw K from the chioroplast at a muchlower rate (as indicated by the longer half-life of exchange) which is perhaps inadequate tomaintain the cytoplasmic [K’-] at a level required for protein synthesis (see Memon et al.,1985a, b; Wyn Jones et al., 1984). Alternatively, at low external [Ki the flux to thechloroplast may be insufficient to sustain chioroplast function. Even at 10 mM [Kj0,the K’-flux to the chloroplast was much lower in trkl cells than in WT cells (Table XI), andchloroplast [K’-] was roughly 50% of the WT value. However, our inability to grow trklcells at 0.1 mM [K]0 prevented determination of [Kj and [Kjffl for these mutants, so theirvalues remain uncertain.122Conclusions: (1) Efflux analysis revealed the existance of three compartments inWT as well as in trkl mutants of Chlamydômonas. These compartments are considered torepresent the cell wall (I), cytoplasm (II) and chioroplast (ifi), respectively. Evidence isprovided to suggest that compartment ifi is the chioroplast, based upon comparisons of theexchange kinetics of dark-grown versus light-grown cells and by direct measurement of the Kconcentration of isolated intact chioroplasts. (2) [K] and fluxes across the chioroplastenvelope were much lower in trkl cells than in wild type cells. This may account for the lackof growth of trkl at low [K]0. In WT cells, the chloroplast may serve as a reservoir for K,from which K may be withdrawn into the cytoplasm to maintain [K] when [K-’-]0 isinadequate. (3) The [K-’-] of WT chloroplasts, estimated from compartmental analysis wasclose to that of isolated chioroplasts. (4) Estimates of [K-’-] by compartmental analysis inconjuction with my previously reported measurements of A were used to calculate Aji+ atvarious [K]0. These calculations show that at <0.2 mM [K]0,K influx has to be activeand that only at concentrations > 0.2 mM was a passive channel-mediated entry of K-’-possible.123VII. REGULATION7.1 INTRODUCTIONWhen plants are deprived of ions, they show increased rates of ion uptake. These ionfluxes are believed to be regulated in response to feedback or feedforward from internal signalssuch as cellular ion concentrations and relative growth rate (see Glass, 1990). But the cellularcompartment which controls the signals responsible for this increased influx is not known forcertain. This signal is believed to come from the vacuole (Leigh and Wyn Jones, 1984) or thecytoplasm itself (Fernando and Glass, 1992). It is also widely accepted that cytoplasmic [K]is maintained at a constant level and vacuolar [K] declines when cells are deprived of K’-(Leigh and Wyn Jones, 1984; Memon et a!., 1985). In cells like Chiamydomonas, thechioroplast (rather than the vacuole) may be involved in regulating K-’- uptake.Despite extensive kinetic studies of the absorption of K in higher plants, the regulationof K transport remains poorly understood. Studies using barley roots have demonstrated thatwithin 6-12 h of withdrawing exogenous K, the expression of several polypeptides, includinga 43 kD intrinsic plasma membrane polypeptide, is enhanced (Fernando et a!., 1992).Likewise, McClure et a!. (1987) and Dhugga et al. (1988) have reported that N03 induction isassociated with changes in membrane polypeptides in maize.Potassium channels have been found in giant algal cells (Keifer and Lucas, 1982;Coleman and Findlay, 1985; Luhring, 1986; Homble et al., 1987; Beilby, 1986), in yeast124(Gustin et aL, 1986) and in cells of higher plants (Moran et aL, 1984; Koib et al., 1987;Schroeder et aL, 1987; Schauf and Wilson, 1987; Ketchum et al., 1989; White and Tester,1992). These channels are strongly regulated by the membrane potential (Hedrich andSchroeder, 1989). Recently, two separate groups (Sentenac et aL, 1992; Anderson et aL,1992) have cloned K-’- channels from Arabidopsis. These are considered to be responsible forlow affinity K transport.To understand the regulation of K influx, [K’-] and K’- influx were determined in wildtype and mutant cells of Chiamydomonas. Inhibitors of protein synthesis were used to studythe role of de novo protein synthesis in the development of increased K’- influx associatedwith K’- deprivation.1257.2 METHODS7.2.1 Effects of Varying K Concentrations in the Growth Media on KUptake and Cell K Concentrationsa) Time Course of K DeprivationCells were grown, under conditions as described in Section 2.1, to mid-log phasein 10 mM K and centrifuged in 250 mL bottles in a GSA rotor (2000 rpm for 2.5 mm) at22°C in a Sorvall centrifuge. The pellet was washed with OK TAPM medium andresuspended into OK or 100 p.M K TAPM medium at equal cell densities in order to evaluatethe effect of the duration of K deprivation on cell [K]. Samples were withdrawn axenicallyfrom the flasks at hourly intervals and 1 ml samples (4 replicates for each time period) wereused to analyze the K content of the cells by the procedure given in section 2.5.b) Influx DeterminationsTo measure 86Rb or 42K influx in cells starved of K for different durations, cellswere transferred to OK medium in 500 mL culture flasks at various intervals prior to influxmeasurement. Influx was measured in all samples at the same time, according to the methoddescribed in section 4.2. This method avoided any problems associated with diurnal variationin uptake rates. Correction for tracers carried through the silicone oil in the apparent free space(A.F.S ) during centrifugation was achieved by using the methods given in Section 2.4.Immediately after the influx experiments, K content was also determined in these cultures bythe slow centrifugation method (method 3) described in Section 2.41267.2.2 Potassium Influx and K Content Determinations of Wild Type andtrkl CellsPotassium content in trkl cells was determined by using the slow centrifugationmethod (“General Materials and Methods”) and K influx was determined by the use of 86Rb.7.2.3 Effects of Protein Synthesis Inhibitors on K InfluxThese experiments were undertaken to study the effects of protein synthesisinhibitors on the developement of enhanced K(86Rb) influx which resulted fromwithholding K from the growth medium. In these experiments, inhibitors of cytoplasmicprotein synthesis (cycloheximide=10 jig mL’ and anisomycin=3.5 jig mL1)were employed.Cell suspensions in 20 mL glass vials were deprived of K and pretreated (for 0, 3 and 6 h)with different concentrations of the inhibitors prior to the uptake experiments. These glassvials were incubated on a shaker during the period of pretreatment prior to influxdetermination. After the inhibitor pretreatment, 1 mL cell suspensions (4 replicates) weretransfeffed to the microcentrifuge tubes (1.5 ml) and K(86Rb) influx was measured asdescribed in Section 4.2. The inhibitors were also present in the cell suspensions during 10min-influx periods. Control treatments were also deprived of K to bring about increased Kinflux. Effects of these inhibitors on K influx in cells starved of K for longer durations werenot evaluated out of the concern that prolonged exposure to these inhibitors would eventuallyinhibit all cellular processes rather than protein synthesis per Se.1277.3 Results7.3.1 Effects of Varying K Concentrations in the Growth Media on KUptake and Cell K ConcentrationsFollowing resuspension in culture medium lacking K (OK), the K content ofwild type Chiamydomonas declined from 25 nmoles 106 cells-’ to 8.7 nmoles 106 cells-’ by24 hours (Fig. 25). In the next 24 h there was only a slight decline in [K], so that by 48 hafter removing K, cell [K] was 7.4 nmoles 106 cells-’. Under control conditions (cellsgrown at 10 mM K throughout) K content fluctuated between 25 nmols 106 cells-’ (0 h) and18 nmoles 106 cells-’ during the 48 h of the experiment, according to the stage of the growthcycle. The control cells moved and divided normally for 48 h but cells deprived of K grewand moved normally for the first 24 h only, after which they stopped growing and theirmovement slowed until by 48 h no movement was evident. K influx was measured at variousintervals during the 48 h of K deprivation using both 42K and 86Rb as tracers. K(42Kjinflux increased by 40% when cells were starved of K for 3 to 6 h (Fig. 25) and by 60% incells starved of K for 10 h, reaching a peak value (70% increase) by 18 h. Furtherdeprivation caused influx to decline. Deprivation of K for 40 and 47 h caused the fluxes todiminish to very low values (4.4±0.1 nmoles h-’ 106 cells-’ cells as compared to 12.43±0.31nmoles h-’ 106 cells-’ in control cells). A negative correlation was observed between internalK content and K(42K ) influx only during the first 18 h of K deprivation. Similarpatterns of K uptake were observed when 86Rb was used as a tracer (data not shown) butFig. 25 K+ content (nmoles 106 cells-1)and K+ influx (nmoles h 106 cells-1)from 0.1 mM [K]0cells deprived of K for different durations. Bars=S.E. ofmeans of 3 independent experiments.12 B0.‘o.-4CS)0E+302010000-4Cl)0E.1)00 .10 20 30 40Time of K deprivation (h)050129influx values were much lower than those estimated by use of 42K.7.3.2. Potassium Influx and K Content of Wild Type and trkl CellsIn K-replete trkl cells, K(86Rb ) influx from 0.1 mM [Kb, was 2.12±0.51nmoles h-’ 10-6 cells compared to 2.78±0.07 nmoles h-’ 106 cells-1 in wild type cells. Whenwild type cells were resuspended in medium without K, K(86Rb ) influx was negativelycorrelated with internal K content until influx peaked in cells starved for 18 h (10.07 nmolesh-’ 106 cells-1), after which K influx declined significantly (Fig. 26). trkl cells did notshow any rise in K(86Rb) influx and at 24 h, influx was 1.81 nmoles h-1 106 cells-’, afterwhich K influx declined. K(86Rb) influx values determined by compartmental analysis intrkl cells were 9. 1±2.76 nmoles h 106 cells-’ compared to 11.24±3.96 nmoles h’ 106cells-’ in wild type cells at 10 mM [K]o.Analysis of the K content of these two types of cells gave a lower value(15.43±0.02 nmoles 106 cells-’) for trkl cells compared to the wild type cells (24±2.3 nmoles106 cells-’) in unstarved cells. When transferred to medium lacking K, there was a decline inthe total K-’- content of wild type cells as described above, but K’- content of trkl cellsremained unchanged during this period of starvation (Fig. 27).7.3.3. Effects of Protein Synthesis Inhibitors on K InfluxStudies of K’-(86Rb’-) influx following transfer to OK conditions showed that13020‘0CC,,0Fig. 26 K influx (nmoles h1 106 cells-1) from 0.1 mM [KJ0 in wild type and trklcells deprived of K for different durations. Bars=S.E. of means of 3separate experiments.Fig. 27. K content (nmoles 106 cells-1 ) in wild type and trkl cells deprived of K fordifferent durations. Bars=S.E. of means of 3 separate experiments.1210300‘0C0+0 10 20 30 40Time of K deprivation (h)5000 10 20 30 40Time of K deprivation (h)50131.6.0a.)4.00Ez0.00 3Time of K deprivation (h)Fig. 28 Effect of inhibitors of protein synthesis cycloheximide (CHX; 10 tg nfr1) andanisomycin (Aniso; 3.5 j.tg m11) on development of increasedK(86Rb) influxduring K4 deprivation. Cells were grown at 10 mM K4 and fluxes weredetermined at 0.1 mM K. Influx values are in nmoles h 106 cells-1 andinclude ± I S.E. of means of 4 replicates.-CHX(-K)+CHX (-K)B +ANTSO(.K)6132influx increased by 43% when cells were deprived of K for 3 h (Fig. 28). By 6 h this valuehad increased to 56.2%. Potassium influx, measured by means of 86Rb or as tracersrevealed that influx had peaked (70% increase) by 18 h, but further deprivation caused influx todecline compared to the peak value. Inhibitors of protein synthesis, cycloheximide (Clix) andanisomycin (ANISO) prevented the increase of K influx normally associated with Kdeprivation. Exposure of cells to CHX for 3 h caused a 36% reduction in K(86Rb) influx inK-’--deprived (+CHX, -K) cells. Potassium concentration in the uptake solution was 0.1 mM.When these cells were treated with CHX for 6 h, K(86Rbj influx was inhibited by 60% incells deprived of K for 6 h. ANISO also caused a reduction in K(86Rb) influx (amountingto 54% in cells deprived of K for 6 h).1337.4 DiscussionWhen cells were resuspended in culture medium lacking K (OK), their K contentdeclined significantly. This decline in internal K’- content was accompanied by an increased K’(42K-i-) influx (Fig. 33) from 14 nmoles h-’ 106 cells-’ (in K replete cells) to 45 nmoles h-1 106cells-1 (in cells starved of K for 20 h). These cells demonstrated a clear relationship betweeninternal [K-’-] and K-’- influx. Similar negative feedback relationships between cell [K] and K’-influx have been reported in Lemna minor (Young and Sims, 1972) and barley roots(Fernando et al., 1990) in time course deprivation experiments.Since cytoplasmic [K’-] ([K]) in this alga was shown to be maintained at a constantlevel following reduction of external K-’- concentrations, the regulatory signal responsible forinitiating the increase of K-’- influx across the plasma membrane following the removal ofexogenous K’-, may have come from another subcellular compartment of the cell. From thecompartmental analysis of this alga at two different [K]0,it is evident that lowering theexternal [K’-] from 10 mM to 0.1 mM, resulted in a decline of chloroplast [K-’-]. It has beenreported previously by several workers (Leigh and Wyn Jones, 1984; Memon et al., 1985) thatcytoplasmic [K] is held close to its optimum value at the expense of the vacuole. Invacuolated cells, it is believed that the regulatory signal responsible for increasing K’- influxacross the plasma membrane comes from the vacuole (Leigh and Wyn Jones, 1984). InChiamydomonas, this reservoir appears to be the chioroplast which may also be involved inthe regulation of K influx. It is also possible that the signal responsible for increasing K134influx across the plasma membrane may have come from the cytoplasm itself. An interruptionof K-’- supply must inevitably lead to short-term perturbations of cytoplasmic [K’-] (Fernando etaL, 1992). Thus, [K-’-]0may be oscillating about its optimum value when the cell is subjectedto varying [K-’-]0 and a small fluctuation in [K]0 during this process may be sufficient totrigger the transport mechanisms across the plasma membrane. Thus, it is more reasonable thatsignals should arise from both the cytoplasm and the vacuole/chioroplast.When trkl cells were resuspended in culture medium lacking K (OK), their K’content did not decline with K’- deprivation, rather it remained the same. These cells alsofailed to show the increased K-’-(86Rb-’-) influx on K’- deprivation (Fig. 26) which may be dueto the absence of transport mechanisms in the plasma membrane and chioroplast envelope thatare found in the WT cells.The maintenance of the high level of K influx in K’- -deprived cells depends oncontinued protein synthesis. Cycloheximide and anisomycin prevented the increase of K’-influx associated with K-’- deprivation (Fig. 28). A similar response of K(86Rb) infux toprotein synthesis inhibitors was also reported for barley (Glass, 1976; Fernando, 1991).Thus, protein synthesis is necessary for the enhanced K’- influx in Chlamydonwnas cellsunder conditions of K’- deprivation.135Vifi. Biochemical Aspects of K Transport in Chiamydomonas8.1. INTRODUCTIONIon transport systems in plants have been characterized by using many strategies.Some workers have tagged the transporters with covalent protein binding reagents (Clarksonand Saker, 1989; Clarkson et al., 1989; Ruiz-Cristin and Briskin, 1991) while others haveused the differential expression in response to nutrient deprivation to identify specificallysynthesised polypeptides (McClure et al., 1987; Dhugga et al.,1988; Hawkesford and Beicher,1991 Fernando et al., 1992). The above studies have shown that when plants are deprived ofions, their influx increases. Such a correlation has been reported for several ions (NO3-,K,P04NH, Si04 and SO4). In parallel with the increased ion influx, expression of somemembrane polypeptides has been reported to increase. A 31 kDa polypeptide was found to beinduced by NO3-in a microsomal fraction from corn roots (McClure et al., 1987). LikewiseDhugga et al. (1988) reported the synthesis of 4 plasma membrane polypeptides with molecularweight (Mr) 165, 95, 70 and 40 kD after N03 treatment. In barley roots, several plasmamembrane polypeptides were synthesized in plants following removal of exogenous K.These polypeptides may form part of high affinity transporter (Fernando et al., 1992).In E. coli, the high affinity K transporter has been suggested to be a K-ATPase(Epstein et al., 1984) while in Neurospora it was suggested to be a K/H cotransport system(Blatt, 1987; Rodriguez-Navarro et al., 1986). Only a few attempts have been made so far toidentify the high affinity transporters in plants (Dhugga et al., 1988; Hawkesford and Beicher,1361991; Fernando et al., 1992). In corn roots, the high affinity transport system was suggestedto be a K-ATPase (Kochian et al., 1989).The low affinity transport is relatively better understood. In corn, it is thought to be aK channel, mediating K uptake at high external [K] (Ketchum et al., 1989; Kochian et al.,1985). The presence of K channels has been demonstrated in several other organisms (Keiferand Lucas, 1982; Coleman and Findlay, 1985; Beilby, 1986; Gustin et al., 1986; Schroeder etal., 1987; Schauf and Wilson, 1987; Ketchum et al., 1989; White and Tester, 1992). Hedrichand Schroeder (1989) have suggested that K channels may operate even in the p.M range ofexternal [K]. Consideration of the electrochemical potential gradient for K from low externalK-’-, however, led Maathuis and Sanders (1993) for Arabidopsis and Glass and Fernando(1992) for barley to conclude that in plants inwardly directed K channels could function in Kabsorption only at elevated K concentrations. Gassmann et al. (1993) also suggest that Kmchannels can serve as major components of low affinity K uptake and/or as a backup systemwhen the high affinity system is non-functional. Thus in addition to channel-mediatedtransport, a high affinity active transport system is also necessary to account for biphasic Ktransport.The kinetics and regulation of K’- transport in plants have been studied extensively, but,the details of the mechanisms of K’- transport are controversial. Several different mechanismsof K influx have been proposed (see “General Introduction” and Luttge and Clarkson, 1989;Glass and Fernando, 1993). In the work presented here, I have attempted to identify proteinswhich participate in membrane transport of K’-. Membrane polypeptides were isolated from K137-replete and K -deplete cells and stained with coomassie or silver stain. This strategy wasused, anticipating that K -deprivation would lead to increased expression of proteins involvedin K transport. Cells were also labeled with‘4C-arginine after the period of K deprivation.The similarity between the appearance of these polypeptides and observed rapid increase of Kinflux following K deprivation indicate a possible involvement of these polypeptides as a partof the high affinity K transporter.1388.2 MATERIALS and METHODS8.2.1 Culture and Growth ConditionsCulture and growth conditions were identical to those described in Section 2.1.Cells were grown either in 6 L or 3 L culture flasks, depending on the quantity of membranesrequired. The cultures were grown mixotrophicafly with constant stirring and aerationthroughout the study period. The duration of K deprivation varied according to the goals ofthe experiments. Potassium deprivation was usually applied when cells were in their mid-logphases, after being in 10 mM K-’- for 48 h. For the control cells, complete TAPM at 10 mMK, was used throughout their growth. For the cells to be deprived of K, cell suspensionswere first centrifuged at 2000 rpm for 2.5 mm and then washed with OK medium before beingresuspended into new OK medium. This washing step was necessaiy to avoid carrying overK-’- from the high K-’- medium and to minimize the addition of K’- to OK by K efflux.Generally the increase in [K-’-] in the OK medium was negligible as shown by flamephotometry. The cells were always resuspended at a lower cell density than the original cultureso that they could divide during the deprivation period. The mutant cells were grown at 10 mMK’- throughout because they failed to grow at lower concentrations of K’-.8.2.2. Cell harvestingCells were grown at 10 mM K or deprived of K’- for various durations. Thesecells were either labeled with‘4C-arginine before harvesting or harvested unlabeled. Cell139cultures were then centrifuged in precooled and preweighed 250 mL bottles at 5000 rpm in thecase of unlabeled cells and in 50 mL tubes in the case of labeled cells at 4°C. Cell pellets wereresuspended into ice cold homogenizing buffer and centrifuged again to remove isotope fromthe extracellular space. This cell pellet was dissolved in homogenizing buffer at a cell densityof 2x109cells in 5 mL or 1 g (wet weight) of cells in 5 mL. The composition of thehomogenizing buffer is given in Section 8.4. These cells were disrupted immediately afterharvesting or 1 mL aliquots were transferred to the microcentrifuge tubes (1.5 mL) and frozenat -90°C in a deep freezer until used for membrane preparations.8.2.3.35S-methionine labelingControl and K-deprived cells were incubated in 500 mL flasks for further growthunder identical light and temperature conditions as had prevailed before K deprivation.35S-methionine (1 pCi mL-’ final volume; 0.1 pCi j.tmol-’) was added to these cellsuspensions 6 h before harvesting them. As a preliminary experiment, 1 mL samples(4 replicates) were withdrawn from a culture at various intervals after adding the tracer tocheck the amount of35S-methionine being accumulated. It revealed that very low counts wereaccumulated. To increase the accumulation of isotope, pH and NH cencentrations (asNH4H2PO)of the medium before and during labelling were lowered. Medium withoutnitrogen (N) was prepared by substituting Na3PO4forNH4H2PO.The cell cultures weregrown in the standard TAPM medium at pH 7.0, and 24 h before the labeling time, cells weretransferred to various concentrations of NH (from 0.1 mM to 9 mM) at pH 7.0 under the140Table XVI. Effect of different concentrations of NHI (0.05 mM to 9.0 mM) and pH (5.5 and7.0) in the external medium on35S-methionine accumulation (cpm 106 cells-’). Cells weregrown in TAPM medium at pH 7.0 and 24 h before labeling, transferred to various [NH4-’-] forthe times shown. Cells were then transferred and exposed to35S-methionine in the mediumwith [NH4-’-] and pH values shown.[NH4+] (mM) [NH4](mM) pH cpm24 h before labeling 0.5 h before labeling during 6 h labeling accumulated9.0 9.0 7.0 5863.0 3.0 7.0 8023.0 .05 7.0 10153.0 .05 5.5 8320.1 .05 7.0 21500.1 .05 5.5 2398141same conditions of light and temperature described in Section 8.1. Half an hour before thelabeling period these cell suspensions were transferred to even lower concentrations of NH(0.05 mM) at either pH 7.0 or pH 5.0. Such [NH4Jwere considered to be inappropriate forthe 24 h exposure as they would have resulted in severe N deprivation. However, this shortexposure was designed to lower cell N quota sufficiently to stimulate amino acid uptake. Thecells were then labelled for 6 h before harvesting. Immediately after centrifugation. controlcells were allowed to grow at pH 7.0 in high NH4 (9 mM) throughout the experimentalperiod. The pH of the medium was adjusted by the use of Tris/acetate buffer. Ammoniumconcentrations were varied between 50 p.M and 9 mM during the course of the experiment asshown in Table XVI. Unfortunately, the radioactivity incorporated into the microsomalfraction (as shown by gel electrophoresis and autoradiography of microsomes) was still toolow and thus,35S-methionine labelling was abandoned for further labeling experiments.8.2.4 14C-arginine labelingBefore labeling the cells, it was important to determine the length of labeling periodrequired to obtain maximum accumulation of radioactivity. This was achieved by adding theradioisotope to the cells (3 x 106 cells mL-’) and withdrawing 1 mL samples (4 replicates) fromthis suspension to measure the radioactivity accumulated by the cells using the silicone oilcentrifugation method as described in Section 2.4. Control and K-deprived cells weretransferred to 250 mL glass flasks and incubated in a growth chamber under conditions142described in Section 8.1.‘4C-arginine (0.1 jiCi pmol-’) was added to all the cultures 1 hbefore the harvest. At the end of a 1 h labeling period, the flasks containing the labeledcultures were placed on ice and then transferred to precooled and preweighed 50 mL centrifugetubes. All subsequent steps were carried out at 4°C and cells were harvested as described inSection 8.2.To check the specificity of the response to K deprivation, and to rule out thepossibility that differential synthesis of polypeptides during the diurnal cell cycle might beresponsible for differential expression of the polypeptides rather than the K deprivation, cellswere labeled at different stages of the growth cycle (early light, late light, early dark and latedark phases). Cells growing in high K medium were labeled with‘4C-arginine for 2 h duringthese 4 phases as described above and used for membrane isolation and protein analysis bySDS-PAGE.1) Isolation of Membrane FractionsCells were harvested and resuspended in the homogenizing buffer as describedabove. Homogenizing buffer consisted of 50 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) - NaOH (pH 7.5), 0.5 M sucrose, 5 mM ascorbic acid, 3 mMEDTA, 10 mM dithiothreitol (DTT’), 1 mM phenylmethylsulfonyffluoride (PMSF) and 0.6%(W/V) polyvinylpolypyrrolidone (PVP). Twentyfive mL of this slurry of cells was transferredto an ice-cold stainless steel cell (capacity 30 mL) of a French press (SLM AMINCO, SLMinstruments Inc.) and the lid was placed on this cell and pressed down to displace any air143trapped in the cell. The cell suspension was passed through the French press at a pressure of5000 psi. The disrupted cells were collected in chilled 50 mL centrifuge tubes (Polycarbonate)while maintaining the pressures at 5000 psi. In parallel experiments, cells at the sameconcentration were subjected to sonication according to the procedure used by Dolle (1988).Briefly, the cells resuspended in the homogenizing buffer were transferred to 20 mL plasticvials and placed on ice. The sonicator probe was cooled and then used for disrupting the cellsby three lOs applications at 60 kHz. The resulting slurry of cells was then poured into chilledcentrifuge tubes and centrifuged (Sorvall centrifuge, RC5B) at 10,000 x g8 for 20 mm in aSorvall SS 34 rotor to pellet cell wall debris, unbroken cells, mitochondria and nuclei. Thispellet was discarded and the supematant was centrifuged in an ultracentrifuge (Becicinan,L8-M) at 80,000 x gayg for 45 mm in a Ti 70 rotor to obtain the microsomal pellet. Thismicrosomal pellet was resuspended using a camel hair brush in ice-cold resuspension buffer(1 g wet weight microsomes in 5 mL resuspension buffer). In‘4C-arginine labelingexperiments where only a small number of cells was used because of the high cost of‘4C-arginine, the final microsomal pellet was resuspended in 100 to 200 p1 of resuspensionbuffer. Resuspension buffer contained 250 mM Tris-Mes (pH 7.8), 0.33 mM sucrose, 5 mMNaCl, 10 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF. Stock solution of PMSF was addedfreshly to the homogenizing and the resuspension buffers to obtain the required concentrations.DTT was also added fresh as a solid and mixed carefully before using the buffer. Theseresulting microsomal preparations were washed with new resuspension buffer and stored asaliquots of 1 mL each in Eppendorf microcentrifuge tubes at -90°C until ready for protein144determinations and gel electrophoresis.2) Sucrose Step GradientsAfter washing in resuspension buffer, microsomes were used for furtherpurification by means of sucrose step gradients. These step gradients were chosen because thismethod had been successfully used for purification of microsomal membranes in several higherpiants (Hodges and Mills, 1986; Fernando, 1991) and algal systems. This method is also fastand provides good yields of plasma membranes. The composition of the gradient buffer usedfor preparing the sucrose gradient mixtures was the same as that of resuspension buffer(composition given in part 1 of this section) except that it contained no sucrose. Mixtures ofvarious concentrations (22, 29, 34 and 45%) of sucrose were prepared by diluting a 60%(wlw) sucrose stock (prepared according to Griffith, 1986) with gradient buffer. Two mL of45% sucrose mixture was overlayed with 3 mL each of 34, 29 and 22% sucrose mixtures,respectively. One mL of each sample were then layered on top of the 22% layer. The tubeswere centrifuged at 80,000 x gavg in a Beckman SW 27 rotor for 2 h. The well formed bandswhich were green in color, were collected at the 22-29% and 34-45% interphases with apasteur pipette. These fractions were diluted with resuspension buffer 3 to 5 times and spun at80,000 X avg for 45 min in a Ti 70 rotor. The resulting pellets were resuspended in 0.5 mL ofresuspension buffer, transferred to microcenthfuge tubes and frozen at -90°C. The 34-45%interphase has been shown to be enriched in the plasma membranes in higher plants (Leonardand Hodges, 1974; Fernando, 1991) and was used for protein determinations and gelelectrophoresis. This method was used to isolate microsomal fractions enriched for plasma145membrane.3) Aqueous Polymer Two Phase PartitioningPlasma membranes were also isolated from the microsomal pellet by means of theaqueous polymer two-phase partitioning method according to Dolle (1988). This was carriedout in order to obtain pure and chlorophyll-free plasma membranes. The two phase systemconsisted of dextran, polyethylene glycol 3350,60 mM NaC1, 0.33 M sucrose and 5 mMpotassium phosphate (pH 7.8). Four different concentrations of the two polymers (6, 6.5, 7and 7.5% of each polymer) were tried in order to get optimum phase composition withmaximum yield of plasma membranes. The microsomal preparation (15 mg protein) wasapplied to the phase mixtures to give phase systems (50 g) with final concentrations of 6.0 to7.5% (wlw) of both dextran T500 and PEG 3350. This phase system also contained 5 mMpotassium phosphate (pH 7.8), 0.33 M sucrose, 5 mM NaCl, 0.1 mM EDTA and 0.1 mMPMSF. The phase mixture was mixed by several inversions and centrifuged at 400 x gavg for3 mm. After phase setting, the upper phase was removed and repartitioned by mixing with anew lower phase. The top phases from all samples were diluted with 5 volumes ofresuspension buffer and centrifuged at 80,000 x avg for 60 mm. The pellets wereresuspended in the same resuspension buffer and stored at -90°C until used for polyacrylamidegel electrophoresis.1468.2.5 Protein DeterminationsSeveral methods (Lowry et aL, 1951; Bradford, 1976; Markwell et al., 1978 andPeterson 1977) were used to determine protein. Chemicals present in the buffers interferedwith most of the assays. SDS, which was used to solubilize the membrane proteins, caused thegreatest interference. Two methods were chosen for their reproducibility, Bradford (1976) andPeterson (1977).Bradford’s method was used in the presence of 0.1% Triton X 100 for estimationof membrane proteins. This method was fast and needed less sample preparation than othermethods. Bovine serum albumin was used to prepare the standard curve. Ten p.L of sample (inresuspension buffer) was made up to 70 .tL with distilled deionized water (ddwater) in themicrocentrifuge tubes. Standards were prepared from 1mg mL1 BSA stock to obtain 0,5, 10,15, 20, 30, 40 and 50 pg protein and 10 tL of resuspension buffer with appropriate amountsof ddwater added to make up to a final volume of 70 liL. To these tubes, 30 pL of Triton X100 (final concentration 0.1 %) was added to bring the reaction mixture to a final volume of100 IlL. Distilled deionized water (0.9 mL) was added to bring the volume to 1 mL followedby the addition of 0.2 mL of the dye (Biorad). The absorbance of the samples was measured at595 nm in a spectrophotometer (Philips, 8820 UVIVIS).To perform Peterson’s (1977) assay, 20 IlL of the sample was made up to 0.5 mLwith cidwater. To each set of standards (0, 10, 20, 30, 40, 50, and 70 pg protein), 20 pL ofresuspension buffer and ddwater was added to bring the volume to 0.5 mL. To these tubes,50 pL deoxycholate (stock 0.15%) was added and allowed to stand for 10 mm at room147temperature. This was followed by the addition of 50 j.LL of trichioroacetic acid (72%), mixingand spinning at 14,000 rpm for 5 to 10 mm in an Eppendorf microcentrifuge. After discardingthe supernatant, the pellet was immersed in 0.5 mL ddwater. Half a mL of Reagent A (1 partof “CTC”-Na-tartarate, 0.2%; CuSO4,0.1%; Na2CO310% with 2 parts of 5% SDS and 1part of 0.8 M NaOH) was added and mixed and kept for 10 mm at room temperature. To thismixture, 0.25 mL Reagent B (1 part 2N folin reagent in 5 parts ddwater) was added, vortexedand allowed to stand at room temperature. After 30 mm, this mixture was centrifuged at12,000 rpm for 15 mm. The pellet was discarded and the absorbance of the supematant wasread at 750 nm. Bradford’s method was the best of all the methods and was used to estimateprotein content in each microsomal preparation.8.2.6 Electrophoresis and AutoradiographySDS Polyacrylamide Gel Electrophoresis was performed according to the procedureused by Rochaix et al. (1988).1) Gel Casting and Buffer PreparationGradient gels (7.5 to 15%) were prepared using a gradient maker ( Biorad). Thecomposition of the 7.5 and 15% mixture is given in Table XVII. All stock solutions werefiltered through Millipore filters (pore size 0.45 pm) and stored at 4°C. The gel was cast in theBiorad gel system using clean glass gel plates and spacers of width 0.75 mm. For casting onegradient gel, 15 mL x 2 resolving gel mixture was prepared (15 mL for 7.5% and 15 mL for148Table XVII. Volumes of various stock solutions (see text for composition and concentrationsof the stock solutions) used to prepare the gradient gel mixtures.Stock solution 7.5% 15%30% acrylamide, 0.2% bisacrylamide 5.0 mL 10 mL5x resolving buffer 4.0 mL 4.0 mL60% sucrose 1.7 mL 5.75 mL10% SDS 0.2 mL 0.2 mLWater 9.OmLTEMED 9iL 4.5 IlL10%APS 67 IlL 671.tL14915%). After degassing the mixture for 5 mm TEMED (N,N,N’,N’-tetramethylethylenediamine) was added to initialize polymerization and the mixture was poured into thegradient maker (heavier mixture into the left chamber of the gradient maker). Ammoniumpersulfate (NH4)2S08) and TEMED were added to the flask last to avoid polymerization ofthe mixture in the gradient maker. The gel was poured between the glass plates by means of afine silicone tubing. A thin layer of iso-butanol was carefully layered over the gel to avoidexposure to air. After about an hour, when polymerization was complete, iso-butanol wasdecanted and the gel was washed with ddwater. Stacking gel solution (5 mL) was poured ontop of the resolving gel and allowed to polymerize. The stacking gel (10 mL) was composedof 30% acrylamide, 0.8% bisacrylamide (2 mL); 2 M TrisH2S04,pH 6.1(2.5 mL); water(5.6 mL); 10% SDS (100 pL); TEMED (10 jiL) and 10% ammonium persulfate(100 jiL).2) Sample Preparation, Electrophoresis and AutoradiographyMicrosomes or plasma membrane preparations in resuspension buffer wereremoved from the deep freezer and allowed to thaw on ice. These membranes were used forpolyacrylamide gel electrophoresis. Ten to 20 liL (100 to 200 pg protein/lane for Coomassiestained and 20 pg/lane for silver-stained gels) or equal 14C counts (100,000 cpmllane) of thesemicrosomes were used for each sample. These microsomes were either subjected to 80%acetone precipitation or used as such for solubilization. The precipitated microsomes wereimmersed in 50 pL of sample buffer to solubilize the membrane proteins. This bufferconsisted of 2.5% SDS; 10 mM DTT; 0.2 M Tris-HC1, pH 8.3; 0.1 M sucrose; 4 M urea and1500.1% bromophenol blue. DT was always added fresh to the thawed sample buffer to generatethe required concentration. This mixture was incubated in a water bath at 700C for 20 mm.The sample mixture was vortexed at the end of the incubation period and loaded into the wellsof the gel apparatus (Biorad). Standard molecular weight markers (Sigma) were also loaded inparallel (to determine the apparent molecular weights). The solubilized samples were loadedinto the wells by means of a Hamilton syringe. The syringe was washed 5 to 7 times withddwater after loading each sample. Gel elecrophoresis was carried out overnight at a constantcurrent of 13 mA in upper buffer (Tris, SDS, boric acid, pH 8.64) and resolving buffer (TrisHC1, pH 9.18). Temperature was controlled at about 10°C by running tap water through thesystem surrounding the gel. Gels were either stained with Coomassie blue or silver stain(Wayne Wray et al., 1981) to visualize the proteins. Radiolabeled gels were soaked in“AJ4pL]fy” (Amersham) for 20 mm, dried in a Biorad gel drier and exposed to Kodak XOMAT 2R film. These X-ray films were developed and fixed by means of an automaticprocessor (Kodak, M35A X-Omat).1518.3 Results8.3.1 35S-Methionine LabelingWhen Chiamydomonas reinhardtii cells were labeled with35S-methionine for 3and 6 h in TAPM medium (9 mM [NH4fl at pH 7.0, the radioactivity accumulated was verylow (Table XVI). This increased from 586 to 823 cpm 106 cells-’ when the cells were grownat 3 mM NH4for 24 h, incubated at 0.05 mM NH for 0.5 h and then labeled with35S-methionine at 0.05 mM [NH] and pH 5.5 for 6 h instead of the normal 9 mM NH andpH 7.0 for the labeling period. The accumulated cpm increased to 2098 when cells weregrown at 0.1 mM NH for 24 h, incubated at 0.05 mM NH for 0.5 h and then labeled with35S-methionine at pH 7.0 for 6 h in the same medium. When the pH was lowered (pH 5.5)during the labeling period, 2391 cpm 106 cells-’ were accumulated. Thus, depriving the cellsof NH and lowering the pH of the culture medium increased35S-methionine uptake fourfold.Unfortunately, even at this low pH and [NH4I, accumulated radioactivity was not sufficientto adequately label the microsomal polypeptides.8.3.2 14C-Arginine LabelingWhen Chiamydomonas reinhardtii cells were labeled with “C-arginine, most ofthe radioactivities (91%) were taken up during the first hour of labeling.152II—45—36—24I—20—14Fig. 29A Silver-stained polypeptides of microsomal proteins (20 pgf1ane) obtained fromwild type Chiamydomonas cells (lane 1) and trkl cells (lane 2). Molecularweights are given in kD and 21 kD polypeptide is marked with an arrow.—6612153—3624I---20Fig. 29B Close up of silver-stained polypeptides of microsomal proteins (20 jig/lane)obtained from wild type Chiamydomonas cells (lane 1) and trkl cells (lane 2).Molecular weights and and 21 kD polypeptide are marked as in Fig. 29A.--141 21548.3.3 Recovery of Plasma Membranes by Various TechniquesVesicles at the 34-45% interphase in a sucrose step gradient contained about 10% ofthe microsomal protein. Plasma membranes were also isolated from the microsomal pellet bymeans of the aqueous polymer two-phase partitioning method and the yield was about 1 to 5%of the total microsomal protein applied to the phase mixture. Several workers (Widell andLarsson, 1981; Kjelbom and Larsson, 1984; Bérczi and MØller, 1986) have reported that theaqueous polymer two phase partitioning method gives about 95% pure plasma membranevesicles but results in significant reduction in yield.8.3.4 Comparison of Coomassie Blue and Silver Stained Polypeptides ofMicrosomal Membranes and Plasma Membranes of Wild Type and Mutant(trkl) CellsIn WT cells, several microsomal polypeptides were visible on silver- or coomassieblue-stained gels. The most prominent bands were observed at M 72, 60, 50, 45, 39, 32, 30,25, 24, 23, 22, 21, 20, 18 and 16 kD as shown in this silver stained gel (Fig. 29A).There were only a few differences observed in the expression of microsomal andplasma membrane polypeptides in the wild type and trkl cells. The most obvious change wasevident at Mr of 21 kD (Fig. 29A). A polypeptide with this apparent molecular weight waspresent in the wild type cells, but was absent from trkl microsomes (Fig. 29B).155—66—45—“36—24—20Fig. 30A Changes in the expression of microsomal polypeptides in wild type cells whengrown at 10 mM K (lane 1), deprived of K for 4 h (lane 2) 20 h (lane 3).Polypeptides at molecular weights 22 H) and 18 H) are shown with arrows.—141 23156—45—24—20Fig. 30B. Close up of Fig. 30A to show the changes in the expression of microsomalpolypeptides in wild type cells when grown at 10 mM K (lane 1), deprived of Kfor 4 h (lane 2) 20 h (lane 3). Polypeptides of interest are marked as in Fig. 30A.—141 231578.3.5 Time Course of‘4C-Arginine Labelling of Membrane Polypeptides inRelation to K Deprivation1) Polypeptides ofInterestWhen cells were deprived of K, changes in the expression of several polypeptidespresent in microsomal fractions were evident in14C-arginine labeled gels (Fig. 30A and 30B).Lane 1 shows the microsomal polypeptides in control cells which had been grown in high Kthroughout. When cells were deprived of K for 4 h, a polypeptide of Mr 22 kD showedincreased expression (lane 2; arrow) while the expression of another polypeptide having a Mrof 18 kD had decreased in these microsomes (lane 2; arrow). Studies of K(42K as well as86Rb) influx under the same conditions showed a 40% increase of influx when cells werestarved of K for 3 to 6 h (Fig. 25). When the cells were starved of K for 20 or 28 h (Fig.31; lanes 4 and 5), a polypeptide of apparent Mr 51 kD appeared which may be a K stress-related protein.2) Polypeptides ofinterest are Specific in Response to K DeprivationWhen microsomes were isolated from cells under control conditions (10 mM K) indifferent stages of their growth cycle, either in their light or dark phases, the 22 kD polypeptidewas not apparent (Fig. 32). Some polypeptides, such as a 32 kD polypeptide and 24 kDpolypeptide (lanes 1, 2; arrows), were specific to light and were visible in samples taken in thelight phases. These polypeptides were not seen when cells were obtained from their darkphases of growth.21583 4—66I—45—36—24—20Fig. 31 Microsomal polypeptides in wild type Chlamydomona.s cells when grown at 10mM K (lane 1), deprived of K for 8 h (lane 2), 12 h (lane 3), 20 h (lane 4)and 28 h (lane 5).1—145159—45—36-—20—14Fig. 32 Microsomal polypeptides in wild type cells grown at 10 mM K throughout.Cells were harvested in their early light phase (lane 1), late light phase(lane 2) and mid dark phase (lane 3).1 2 31608.4 DiscussionA polypeptide with apparent M 21 kD was absent from the trkl microsomalfraction. This polypeptide may be a component of the high affinity transport system for K inChiamydomonas. trkl cells failed to grow at low external [Kj, whereas WT cells grew verywell. WT cells probably grew well at low external [K] due to the presence of this highaffmity transporter in their plasma membrane. trkl cells may have been defective in thistransporter due to the absence of this 21 kD protein.A major polypeptide that appeared after 4 h of K deprivation had M 22 lcD. Therapidity of appearance of this 22 kD polypeptide correlates well with the observed rapidincrease of K influx following K deprivation. This polypeptide may form part of the highaffinity K transporter, which may be a K -ATPase or a K/W symporter. Physiological,biochemical and genetic studies have provided evidence for the existence of the Kdp system inE. coil (Epstein et al., 1984) and the TRK system in Saccharomyces (Rodriguez-Navarro andRamos, 1984).The high affinity K uptake system in E. coil has been shown to be a K ATPase(Epstein et al., 1984) while in Neurospora the high affinity uptake system was suggested to bea K-’-/H cotransport system (Blatt et al., 1987; Rodriguez-Navarro et al., 1986). K transportis driven by ATP hydrolysis in the first case and by the the energy inherent in a large protongradient across the plasma membrane generated by the plasma membrane lI -ATPase in thelatter case. if a K/H cotransport system is operating in Chiamydomonas, the movement of161K into the cell would be coupled to the passive H movement down a strong proton gradientgenerated by the proton ATPase. Such a system would be expected to be sensitive to changesin the pH gradient. In Chiamydomonas, K influx did not increase with decreasing pH(increasing pmf) but showed an optimum at pH 6.0. However, Glass and Siddiqi (1982) andKochian et al. (1989) demonstrated a lack of sensitivity of the high affinity K transport systemin barley and corn roots, respectively, to changes in the pH gradient. In the case of a K/Hcotransport system, it would be expected that at high external pH, the pmf may be insufficientto drive K uptake. Based on this, Kochian et al. (1989) speculated that the high affinity Ktransport system in corn roots might be a K -ATPase, of the kind that has been found in E.coli. A similar K -ATPase may also be present in this unicellular alga.Another possibility for the K uptake system in Chlainydomonas is a channel.Potassium channels have been identified in Chara, (Beilby, 1985; Keifer and Lucas, 1982)Nitella (Sokolik and Yurin, 1986) and in guard cells of higher plants (Schroeder et al., 1987).In corn protoplasts, Ketchum et al. (1989) have interpreted the low affmity transport system asan inwardly directed K channel and have demonstrated that the K channel blocker TEA iseffective in inhibiting the passage of K current. Kochian et al. (1985) demonstrated earlierthat only the low affinity K transport system (the linear flux component) could be inhibited byTEA in corn roots. Thus, at high external [K], when the high-affinity transport system(HATS) is repressed, K might move into the cell down the electrochemical gradient throughK channels. Anderson et al. (1992) and Sentenac et al. (1992) have isolated two K channelsfrom Arabidopsis by transfonning a K -transport mutant of yeast. The transformed yeast162mutant restored the capacity of both saturable and linear transport in a single gene product(Sentenac et aL, 1992). This may suggest that the saturable and linear kinetics can beaccounted for by a single transport system. The existence of channels capable of mediating Kuptake from low external concentrations has also been suggested by Heidrich and Schroeder(1989). In Neurospora, where membrane potentials have been reported to be as low as -305mV (Rodriguez-Navarro et al., 1986), the former authors proposed that the electrical potentialgradient would be more than sufficient to drive passive K entry even when the external K isat jiM concentrations. Glass et al. (1992) have recently measured electrical potential values aslow as -300 mV in barley in the absence of K in the external medium. Under such conditionsentry of K into the cells even from very low concentrations might occur passively via Kchannels. Nevertheless, when K is available, ANr is strongly depolarized. Therefore,continued K uptake through channels may not be possible. The electrophysiological data inArabidopsis (Maathius and Sanders, 1993) shows very clearly that the channel could notmediate K uptake at low external [K].Polypeptides related to K deprivation appeared as early as 4 h after K deprivationand this correlates quite well with the increased K influx after 3 h of K deprivation. Thus,the increased K-’- influx following K deprivation may be due to the derepresssion of synthesisof K-’- transport polypeptides. There may have been some kind of allosteric control of theexisting carriers during the first hour or so of K’- deprivation, similar to that suggested forbarley roots (Fernando, 1991). That K’- uptake may be regulated allosterically by cytoplasmic[K-’-] was suggested several years ago by Glass (1976). It may also be possible that both163derepression of K carriers and allosteric effects operate together in parallel and are responsiblefor the increased K influx on K deprivation.In parallel with the derepression of expression of the 22 kD polypeptide, anotherpolypeptide of apparent Mr 18 kD was absent from microsomes from cells deprived of K for4 h. Thus, the expression of this polypeptide appears to have been turned off during the initialperiods of K deprivation. This may indicate that K deprivation results in both the repressionand derepression of specific membrane polypeptides. Synthesis of the 22 kD polypeptide wasstrongly reduced by 20 h by which time K influx had dropped to a very low value.In summary, it can be said that K influx during K deprivation may be controlledby repression as well as derepression of synthesis of membrane polypeptides. During theinitial hours, K influx may be regulated by exerting allosteric control of existing carriers in theplasma membrane. These high affinity transport carriers enable the WT cells to grow at verylow external [K-’-]. The mutant cells (trkl) fail to grow under these conditions, because theylack the regulatory component(s) of this high affinity transporter. At high external [Kfl, bothWT and trkl cells grow normally because of the presence of K channels which permit K’entry down the electrochemical potential gradient.164IX. SUMMARYKinetics of potassium transport have been studied extensively in plants but themechanisms responsible for K transport remain controversial. In addition to this, muchprogress has been made in defming the nature of the H -translocating ATPase of the plasmamembrane and tonoplast but the details of energy coupling are poorly understood and thebiochemical aspects of the high affinity transport system have not been explored yet. We knowthat when K is withdrawn from the external medium, plants respond by increasing K influx.The regulatory mechanism responsible for this increased uptake is poorly understood.The present work was conducted using Chiamydomonas. This study describes thekinetic, biophysical and biochemical aspects of K transport in Chiamydomonas. Thisorganism has several advantages such as a short generation time; it can be grown andmaintained in axenic cultures; it has a huge chloroplast which can be easily isolated and studied;mutants defective in K transport are available which could be used to study K transport; Ktransport can be studied in a single cell. Potassium transport was studied using 86Rb andas the tracers and a comparison is made between the two.The first part of this work deals with the kinetics of K transport. Potassium influxwas mediated by two transport systems. At low external [KJ, 42K and 86Rb influx occursby means of a saturable system. At high external [K], K influx showed a linear pattern.These two transport systems were further characterized by using different inhibitors. The highaffmity transport system (HATS) was found to demonstrate greater metabolic dependence thanthe low affinity transport system (LATS). The two transport systems also responded165differently to a sulfhydryl reagent and to light/dark treatments. This indicates that the HATSand the LATS are two independent mechanisms of K transport.Membrane electrical potential difference across the plasma membrane wasdetermined by measuring TPP accumulation. This method was found to the best formeasuring AN, in a small cell with a large chioroplast. From TPP accumulation, it wasconcluded that TPP accumulation in Chiamydomonas was largely due to AN, across theplasma membrane rather than intracellular organelles such as chioroplast and mitochondria.Experiments in the presence of oligomycin showed that mitochondria were contributing onlyabout 10% to the value of AN,.Values for A were obtained at several external [K], and AIK were calculated.From AtK values it can be concluded that K enters the cell against its electrochemicalpotential gradient at low external [Ki (<0.2 mM) and is probably moving down theelectrochemical gradient at high [K] (>1 mM).A unique set-up was designed to perform compartmental analysis ofChlainydomonas. Three compartments detected by compartmental analysis probablycorrespond to the cell wall, cytoplasm and chloroplast, respectively. The [Kj of isolatedchioroplasts was 165+2 1 mM which is close to the [KJ obtained for compartment ifi from thecompartmental analysis (223+31.4mM). Half-lives of exchange for compartment ifi in dark-grown cells were shorter than in light-grown cells and the fluxes across this compartment weremuch lower in dark-grown than in light-grown cells. These findings suggest that the thirdcompartment is the chloroplast. Comparisons of the wild type and trkl cells show that166compartment ifi could serve as a reservoir for K, particularly at low external [K].When Chiamydomonas cells were deprived of K, their internal [K] declined andK influx increased up to 18 h of K deprivation. Thus, there was a clear relationship betweeninternal [KJ and K influx. The origin of the regulatory signal responsible for the increasedK influx on K deprivation is not known. Since [K] in the cytoplasm is maintained at aconstant level following deprivation of external [K], the regulatory signal must have comefrom another subcellular compartment. Compartmental analysis of Chiamydoinonas revealedthat lowering external [K] from 10 mM to 0.1 mM resulted in a decline of the chloroplast[K]. Thus, in these cells, the chioroplast appears to be involved in initiating this regulatorysignal. On the other hand, it is more reasonable that signals should arise from both thecytoplasm and the chioroplast.Studies of effects of protein synthesis inhibitors on K influx were carried out tothe role of de novo protein synthesis in K influx. In the presence of cyclohexirnide andanisomycin, cells failed to show the increase of K influx associated with the K deprivation.Thus, protein synthesis is necessary for the enhanced K influx in Chlainydomonas underconditions of K deprivation.Microsomes were isolated from WT and trkl cells and further separated on SDSpolyacrylamide gel electrophoresis. A polypeptide with Mr 21 kD was absent from the trklmicrosomal as well as plasma membrane fraction. Wild type cells probably grow well at lowexternal [KJ due to the presence of this polypeptide in their plasma membranes. I suggestthat this polypeptide may be a component of the high affinity transport system for K in167Chiamydomonas. When cells were deprived of K for 4 h, a polypeptide of Mr 22 lCDappeared. This polypeptide is another likely candidate as a part of the high affinity Ktransporter. It may also be possible that the 21 kD and 22 kD polypeptides are the same. Thishigh affmity transporter for K may be a K -ATPase or a K/H symporter. Anotherpossibility for the K uptake system in Chiamydomonas is a channel. K channels in cornhave been suggested to operate only at high external [K]. Thus, at high external [K], whenthe high affmity transport system (HATS) is repressed, K might move into the cell down theelectochemical gradient through K channels. Thus, the increased K influx following Kdeprivation may be due to the derepression of K transport polypeptides. 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