Electromotive force of Nitella membrane during excitation

Excitation of the Nitella membrane is analysed by assuming themembrane to be an electromotive force in series with a resistance,both being variables of time and of membrane potential. Duringstep depolarization beyond a threshold, conductance and electromotiveforce increase transiently, finally reaching their respectivesteady state levels. The conductance increase peak is attainedearlier than the peak for electromotive force increase. Wheneverelectromotive force increases beyond the level of clamped membranepotential, the ionic current flows inward. This is consideredto be the origin of the apparent negative resistance characteristicof the excitable membrane. Anodal break response and spontaneousfiring of Nitella membrane are also caused by transient increasesin electromotive force and conductance irrespective of whetherthe membrane potential is being held at its resting level. Thetransient increase in electromotive force reflects changes,like a phase transition, occurring during excitation. (Received May 6, 1968; )     

Oscillations in Extracellular Current, External pH and Membrane Potential and Conductance in the Alkaline Bands of Nitella and Chara

Oscillations with a period of approximately 2 min were observedin the membrane potential of Chara and Nitella upon illuminationof dark-treated cells, as well as in the extracellular currentpattern and pH values. A 2-min oscillation in the membrane potentialwas observed when the voltage electrode was placed close tothe border of an alkaline and acid region. Comparison of resultsfrom Chara and Nitella revealed an identical control mechanismfor external pattern stabilization in the effect of light onmembrane potential and conductance. Vibrating probe experimentsindicated that oscillations in the extracellular current occurredonly at the border of the alkaline band. Ion-specific pH micro-electrodesplaced within the alkaline band detected oscillations associatedwith light reactivation of the banding phenomenon. These resultsindicate that the oscillations represent a localized phenomenoninvolving spatially-dependent time-constants. More evidencefor the spatial dependence of time constants is gained fromsingle active acid regions in Nitella. Using this combinationof techniques, we have established that a light-dependent H⁺transporter is involved in this oscillation. Current-voltagecurves taken during these oscillations and relaxation, afterchanging the light intensity, confirmed this identification. Key words: Oscillation, vibrating probe, pH micro-electrode, time-constant. I/V curve, Chara, Nitella     

ACTION POTENTIAL OF NITELLA INTERNODES

The ionic current during a non-propagating action potentialis analysed from the voltage clamp experiments. The shape ofthe action potential of the Nitella internode can be reconstructedfrom the data of the voltage clamp experiments. The N-shapedcurrent-voltage characteristics (I-V curve) of the Nitella membraneis not constant with time as it is in the tunnel diode, butdecays with time, converging finally into a delayed rectificationcurve. The temporal locus of the potential at which each I-Vcurve crosses the voltage axis coincides almost exactly withthe action potential. The membrane resistance which is calculatedfrom the slope of the I-V curve at each intersection with thevoltage axis also changes in parallel to the action potential.Such correlations are found in the Nitella not only in the pondwater, but also in high Na, high Ca or high Mg medium, wherethe shape of the action potential is modified in various ways.It is highly probable that the action potential is a locus ofthe change of the membrane potential so that the net membranecurrent may be maintained at zero after the transient modificationof the membrane structure by stimulation. (Received June 30, 1966; )     

Uptake of Radioactivity by Aquatic Plants and Location in the Cells: II. UPTAKE OF CERIUM-144 BY THE FRESHWATER PLANT NITELLA OPACA

The uptake of cerium-144 by Nitella from pond water at pH 7.7without added carrier was recorded over a period of 207 days,separate measurements being made for each of the main componentsof the cell. Some of the Nitella cells were then transferredto water which contained no radiocerium, for measurements overa further 54 days. A cerium-144 equilibrium was approached afterabout 100 days, with the whole plant having 26,000 times theactivity of the water. Under these conditions most of the ceriumwas located in the cell wall, though small but significant amountsentered the protoplasm and cell sap. It was apparent that cerium,freshly taken up either into the cell wall or into the interior,was freely exchangeable, but after a short period (some 10 days)the cerium became more firmly bound to the plant, and was notreleased again.Outstanding differences noticed between the locationof cerium in Nitella opaca and of yttrium in Ulva lactuca arediscussed in chemical and biological contexts.     

The Influence of the Cell Membrane on some Toxicity Effects of Growth Substances in Nitella

When cells of Nitella are placed in a solution of some plantgrowth substances there is a profound increase in the membraneresistance as measured by means of an internal silver/silverchloride micro-electrode. This impedance effect is accompaniedby some marked visible changes within the cell cytoplasm, thesechanges being dependent upon the concentration of undissociatedgrowth substance in the vicinity of the membrane, and upon thepH of the external environment. A detailed study is made of these visible changes and, takinginto account the negatively charged membrane, one possible interpretationof the results is that only undissociated molecules of the growthsubstance may enter the cytoplasm of Nitella from the environment,the diffusion obeying a simple Fick Law relationship. The substances are used in sufficient quantity to kill the Nitellacell, death following the visible effects, and they appear tobe unique in that their toxic effect produces a system of highmembrane impedance.     

Tonoplast Action Potential of Characeae

The plasmalemma action potential was found to be indispensableto the production of the tonoplast action potential. In a solutionlacking Ca²⁺ and containing other divalent cations such as Ba²⁺,Mg²⁺ or Mn²⁺, the plasmalemma excited in Nitella but did notin Chara. In Nitella, however, both the tonoplast action potentialand EC-coupling were abolished due to depletion of Ca²⁺ fromthe external medium. Ca²⁺ ions injected into the cytoplasmiclayer caused a transient change in both plasmalemma and tonoplastpotentials. These results suggest that a transient rise in Ca²⁺concentration during excitation of the plasmalemma may triggerthe tonoplast action potential. (Received February 14, 1986; Accepted August 29, 1986)     

STUDIES ON NITELLA HAVING ARTIFICIAL CELL SAP I. REPLACEMENT OF THE CELL SAP WITH ARTIFICIAL SOLUTIONS

A method for replacing the cell sap of Nitella with an artificialsolution was introduced. The technique, which is a modificationof KAMIYA and KURODA'S (1, 2), is applicable not only for isotonicbut also for hypertonic or hypotonic solutions. Photometricdeterminations of K⁺, Na⁺, Ca⁺⁺ and Cl^– proved that thereplacement of the cell sap with the present method is satisfactory.The internodal cell of Nitella, whose cell sap was replacedwith an isotonic solution with a simple composition such asa mixture of KCl, NaCl and CaCl₂, can be kept living at leastfor several days, sometimes even for more than one month. (Received September 6, 1963; )     

The effect of external pH on the membrane potential of Nitella and its linkage to metabolism

The membrane potential of Nitella is highly sensitive to externalpH in the neutral region (pH 5.5 to 8) in the light. We foundthat the sensitivity usually was reduced by darkness, metabolicinhibitors (DCMU, DNP) or cooling. Under some circumstancesthe high pH-sensitivity of the membrane potential was observedeven in the dark. However, even in this case, it was reducedby DNP or cooling. These results indicate that the externalpH-dependence of the membrane potential of Nitella is closelylinked to metabolism. (Received May 8, 1973; )     

Action of Mono- and Difluorodinitrobenzene on Induced Electrical Modifications by External pH Changes in Nitella

The action of mono- (FNB) or difluorodinitrobenzene (DFNB) onion permeability is mainly attributed to its interactionwithamino groups of the membrane by dinitrophenylation. Nitella cells were dimtrophenylated at pH 7.3 and the membranepotential and electrical resistance were then measured in acidicor basic solutions. No matter what the pH value was, FNB andDFNB induced a depolarization of the membrane potential andcaused a diminution of resistance. However these effects ofFNB and DFNB were more drastic at alkaline pH and in the presenceof a weak concentration of potassium. Neither the addition of0.1 mM calcium nor the substitution of chlorides by nitratesmodified the DFNB effect. These results are compatible withthe assumption that the DFNB binding to the membrane leads toan augmentation of the negative charges of the membrane bringingabout an increased cation conductance and a modification ofthe affinity of a K⁺/H⁺exchange pump. The transient responseof the membrane potential at the time of dinitrophenylationwas used to roughly estimate the total density of amino groupsof the membrane of Nitella.     

Characterization of Two Proton Transport Systems in the Tonoplast of Plasmalemma-Permeabilized Nitella Cells

ATP-dependent and PP_i-dependent H⁺-transport systems of thetonoplast were characterized in plasmalemma-permeabilized Nitellacells, where direct access to the protoplasmic surface of thetonoplast was possible. Since H⁺ transport across the tonoplastcan be measured in situ, the identity of the membrane responsiblefor H⁺ pumping is unequivocal. H⁺ transport was evaluated bythe accumulation of neutral red. While both transport systemswere obligately dependent on Mg²⁺, the two transport systemsshowed completely different sensitivity to NO₃^– and K⁺,suggesting the presence of two types of H⁺-pumps in Nitellatonoplast. NO₃^– applied to the protoplasmic surface, completelyand reversibly inhibited ATP-dependent transport but had noeffect on PP_i-dependent transport. By contrast, NO₃^– appliedinto the vacuole by the vacuolar perfusion technique did notinhibit ATP-dependent or PP_i-dependent H⁺ transport. Replacementof K⁺ with the organic cation, BTP, inhibited PP_i-dependenttransport but not the ATP-dependent one, indicating that PP_i-dependenttransport is K⁺ dependent. The sensitivities of the H⁺ transportsystems found in the tonoplast of Nitella are quite similarto those of higher plant tonoplasts. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received February 21, 1987; Accepted May 27, 1987)     

Effect of pH on the Membrane Potential and Electrical Resistance of Nitella flexilis in the Presence of Calcium

Effects of pH on the membrane potential and electrical resistanceof Nitella were investigated in a bathing medium with or withoutcalcium. The membrane potential became more negative as theexternal pH was raised, at a faster rate in the presence ofcalcium than in its absence. The value then achieved by thepotential could be reversed by restoring the original pH whilstin a Ca-free medium the cell remained ‘hyperpolarized’.Tenfold changes of the external concentration of potassium broughtabout larger modifications of the membrane potential when thepH of the solution was high and calcium concentration low. Theelectrical resistance was lowest in alkaline and calcium-freesolutions. We conclude that calcium prevents the mediation ofsome changes in the membrane structure by lowering the concentrationof external H⁺ ions, and that the permeability of Nitella topotassium increases with rising pH.     

The Uptake and Transport of 14C in Cells of Conocephalum conicum L. in Light

Dissolved inorganic ¹⁴carbon, fed to a small portion of thetissue of Conocephalun conicum, travelled along the tissuesfrom cell to cell at rates of about 2.0 to 1.7 µm 5^–1,which is of the same order of magnitude as streaming rates inthese cells. Trans-cellular transport can take place againsta pressure gradient, but pressure gradients of 200 kPa or morein either direction reduced the trans-cellular transport significantlywithout reducing streaming rate and are, therefore, thoughtto have induced ‘valving’ effects at the plasmodesmata.Ammonium ion reduced or inhibited transport without reducingstreaming rates and is, therefore, thought to have inhibitedan active component in the plasmodesmata. Thus the results obtainedfrom tandem cells of Nitella can be transferred to tissues. Key words: Conocephalum, transport, plasmodesmata     

HYPERPOLARIZING RESPONSE IN NITELLA INTERNODES

The ionic aspect of the hyperpolarizing response in the internodalcell of Nitella is reported in some detail. The response wasobserved by passing a large inward current through the Nitellamembrane, the resistance of which had been decreased by a concentratedalkali metal ion. It was not possible to demonstrate the responsein a concentrated solution of CaCl₂, MgCl₂, BaCl₂, ZnCl₂ orAlCl₂ or AlCl₃. After hundreds of the spontaneous repetitiveaction potentials, which occurred in a single solution of concentratedNaCl or LiCl or caused by an application of 1–2 mM EDTAin the artificial pond water, the Nitella cell showed the hyperpolarizingresponse. Almost the same size of the response was observedfor change in pH of the external KC1 solution from 6.7 to 10.0,but it decreased markedly for pH lower than 4.7. It seems tobe an essential condition for the response to remove the divalentcations from the cell surface, having a concentrated monovalentcation in the external medium. (Received April 22, 1966; )     

Oriented structure of matrix polysaccharides in and extension growth of Nitella cell wall

Infrared evidence indicated that the oriented structure of matrixpolysaccharides changed on mechanical extension as well as duringextension growth of Nitella cell walls. The importance of theultrastructure of polysaccharide gels in controlling extensiongrowth is discussed. (Received July 3, 1974; )     

Separate Oscillations of the Electrogenic Pump and of a K+ -Channel in Nitella as Revealed by Simultaneous Measurement of Membrane Potential and of Resistance

In Nitella oscillations of membrane potential with periods ofabout 15 min and of about 1 h can be observed. The comparisonof the related changes in resistance and in potential showsthat the 1-h oscillation controls a carrier-type transporter,probably an electrogenic pump with a stoichiometry of 1 H⁺/1ATP. The control loop related to the 1-h oscillation can bestimulated by internal pH as changed by butyrate or procaine.IV-curve studies show that the 15-min oscillation modulatesthe conductivity of a K⁺ -channel. No way has been found toinfluence the spontaneously occurring 15-min oscillation whichprobably is generated in the chioroplasts. The oscillationsseem to provide a means of exerting a selective influence onone transporter. Correspondence to: Institut für Angewandte Physik, NeueUniversität, Haus N61A, D-2300 Kiel, F.R.G. Key words: H⁺-pump, IV-curves, Nitella, oscillations, pH-regulation, potassium channel, stoichiometry     

Cycloprodigiosin Hydrochloride Inhibits Acidification of the Plant Vacuole

The effect of cycloprodigiosin hydrochloride (cPrG-HCl), obtainedfrom the marine bacterium Pseudoalteromonas denitnficans, onacidification of the vacuole was studied in Characeae cells.In internodal cells of Nitella, the plasma membrane of whichhad been permeabilized, cPrG-HCl inhibited both ATP- and pyrophosphate-dependentacidification of the vacuole. Application of cPrG-HCl to livinginternodal cells of Chara, induced a significant increase invacuolar pH. The role of cPrG-HCl as a tool for studying thephysiological role of acidic vacuole is discussed. (Received August 26, 1998; Accepted November 17, 1998)     

The Influence of Temperature on a K+-Channel and on a Carrier-Type Transporter in Nitella

Fisahn, J. and Hansen, U-P. 1986. The influence of temperatureon a K⁺ -channel and on a carrier type transporter in Nilella—J.exp. Bot. 37. 440–460. In Nitella, the effects of temperature on membrane potentialand on resistance consist of several components. The evaluationof their associated time-constants measured in linear(ized)temperature responses at a resting potential of–120 mVprovides an approach to their identification. For changes slowerthan c. 1 s, the temperature effect on membrane potential andresistance does not originate from temperature action on theinvolved transporter, but is mediated by signals from temperaturesensitive metabolic processes. In the case of potential, theseprocesses seem to be identical to those which also mediate thelight effect: pH-regulation, and two direct signals from photosynthesis,as indicated by the similarities of the related time-constants( respectively). The temperature effect on resistance displays only one time-constant of 40 sinmost experiments. The related process is unknown. The non-coincidenceof the time-constants of the effect on potential and on resistanceimplies the involvement of a carrier-type transporter (H+-pumpor cotransporter) in the effect on potential, and of a K+channelin the effect on resistance. The K+-channel is identified bythe reversal potential of the effect on membrane potential measuredin cells depolarized or hyperpolarized by an injected electricalcurrent Under these conditions the temperature effect on resistancedominates the effect on potential. Key words: H⁺-pump, K⁺-channel, kinetic analysis, Nitella, oscillation, pH-regulation, reversal, potential, temperature, time-constants     

23Na and 7Li NMR study of Nitella cell walls before and after an ion-induced loss of the cationic exchange capacity

²³Na- and ⁷Li-NMR studies were conducted on isolated cell wallsof Nitella to monitor the changes induced in the pectin matrixafter the cation exchange capacity was reduced to about 60%of its initial value by a pretreatment with a 500 mM LiCl solution.Walls in Ca²⁺ form were exchanged with NaCl and LiCl solutionsbefore and after this pretreatment. In the whole cell wall equilibratedwith 10, 35 and 100 mM NaCl solutions or mixtures at the sametotal concentrations of Na⁺ and Li⁺ in the same proportions,the ²³Na- and ⁷Li-NMR spectra were all characterized by onlyone signal. The Na⁺ and Li⁺ relaxation times were drasticallyshorter than those recorded in aqueous solution but the twoNa⁺ relaxation rates increased linearly with the Na⁺ concentrationin the external solutions. In contrast, in the cell wall wherethe CEC had been reduced, the Na⁺ relaxation times were longerand no correlation with external concentrations was obtained.The ⁷Li spectra in walls equilibrated with 500 mM LiCl displayedtwo distinct lines suggesting different Li⁺ bonding affinitiesfor the wall polyuronides. These data are interpreted in thescheme proposed by Gillet et al. (1994) to explain the competitiveeffects of alkaline ions on the wall pectin disorganizationvia disruption of divalent cation crosslinks, but also of solvation-likebonds between cell wall polymers. Key words: ²³Na, ⁷Li, NMR, Nitella, pectic polysaccharides, cell wall     

Evidence for a Structural Role of Protein in Algal Cell Walls

Load-extension measurements were made on three filamentous algaebefore and after either digestion with proteolytic enzymes ortreatment with dithiothreitol. Large differences in tensileproperties of the walls were observed, particularly after pronasedigestion, in two algae, Cladophora and Chaetomorpha, whichcontain hydroxyproline in the wall. Pronase had little or noeffect on a third alga, Nitella, lacking hydroxyproline. A smallerdifference was found on treatment with dithiothreitol, a specificreducing agent for disulphide bonds. These results suggest thata hydroxyproline containing protein is a structural componentof these algal walls, and that hydroxyproline itself is involvedin the carbohydratepeptide linkage.     

The electrogenic ion pump revealed by the external pH effect on the membrane potential of Nitella. Influences of external ions and electric current on the pH effect

The influences of external ions and electric current on thepH effect of the membrane potential of Nitella are described.Results are explained in terms of a pH-dependent electrogenicion pump. The reduction of membrane resistance with a pH rise is attributedto the pump current being potential-dependent. (Received July 9, 1974; )