Ion Fluxes to the Vacuole of Nitella translucens

The time course of the appearance in the vacuole of Nitellatranslucens and of Tolypella intricata of tracer from the outsidesolution has been studied over short periods of uptake. Thereare two components of chloride transfer to the vacuole, a fastcomponent linear with time and a second component at longertimes whose behaviour is reasonably well described in termsof a single rate constant for exchange; a constant fractionof the total entry is in the fast component and the apparentrate constant for the second component is proportional to theinflux. In Nitella the path of rapid transfer involves chlorideand sodium, and may also involve a small but variable amountof potassium, but in Tolypella potassium has a significant componentof rapid transfer; these correspond to the cations for whichchloride-linked components of cation influx have been shownby another worker. Over both parts of the time course the level of activity inthe cytoplasm specifies, not the rate of transfer to the vacuoleas would be expected, but the rate as a fraction of the influx;the processes of influx to the cell and transfer to the vacuoleare intimately linked. It is difficult to explain the results in terms of static membranesand fixed compartments. An explanation in terms of the sequence,entry of salt by pino-cytotic vesicles at the plasmalemma, fusionof these vesicles with the endoplasmic reticulum after someloss of tracer to the surrounding cytoplasm, and transfer tothe vacuole in minivacuoles formed from the endoplasmic reticulum,is consistent with the time course found. A model of this kind,involving transport by a dynamic membrane system, seems necessaryto explain the results.     

Ion transport in Nitellopsis obtusa

The distribution and rates of exchange of the ions sodium, potassium, and chloride in single internodal cells of the ecorticate characean, Nitellopsis obtusa, have been studied. In tracer experiments three kinetic compartments were found, the outermost "free space" of the cell, a compartment we have called "protoplasmic non-free space", and the cell sap. The concentrations in the vacuole were 54 mM Na(+), 113 mM K(+), and 206 mM Cl(-). The steady state fluxes across the vacuolar membrane were 0.4 pmole Na(+)/cm.(2) sec., 0.25 pmole K(+)/cm.(2) sec., and 0.5 pmole Cl(-)/cm.(2) sec. The protoplasmic Na/K ratio is equal to that in the vacuole but protoplasmic chloride is relatively much lower. Osmotic considerations suggest a layer 4 to 6 micro thick with sodium and potassium concentrations close to those in the vacuole. The fluxes between protoplasm and external solution were of the order of 8 pmoles Na(+)/cm.(2) sec. and 4 pmoles K(+)/cm.(2) sec. We suggest that the protoplasm is separated from the cell wall by an outer protoplasmic membrane at which an outward sodium transport maintains the high K/Na ratio of the cell interior, and from the vacuole by the tonoplast at which an inward chloride transport maintains the high vacuolar chloride. The tonoplast appears to be the site of the principal diffusion resistance of the cell, but the outer protoplasmic membrane probably of the main part of the potential.     

Quantized Fluxes of Chloride to the Vacuole of Nitella translucens

It has been shown previously that the transfer of tracer chloridefrom the outside solution to the vacuole of Nitella translucensis initially linear with time. In this paper the relations betweenthe initial rate of chloride transfer to the vacuole and thetotal influx to the cell are further examined. In the individualcells in each experiment the ratio (initial rate of transferto the vacuole/rate of entry to the cell), M_ov/M_T, is quantized;that is, in each experiment the ratio takes values close to, 2, 3,... etc. Formation of pinocytotic vesicles is a processwhich could be imagined to be quantized, but the fact that itis a flux ratio, rather than a flux, which is quantized suggeststhat entry to a small cytoplasmic phase, such as the endoplasmicreticulum, must precede a quantized discharge to the vacuole.It is suggested that the kinetics of tracer movement to thevacuole are consistent with transfer in small vacuoles buddedoff the endoplasmic reticulum which then fuse with the centralvacuole.     

Effects of Light/Dark on Anion Fluxes in Isolated Guard Cells of Commelina communis L.

The effects of light/dark on anion fluxes in isolated guardcells of Commelina communis L. have been studied, using ⁸²Brand ³⁶Cl. Transfer of open guard cells from light to dark hasno effect on the ⁸²Br influx, but produces a marked transientstimulation of ⁸²Br or ³⁶Cl efflux, similar to the effect ofsuch transfer on the ⁸⁶Rb fluxes, and to the effects on both⁸⁶Rb and ⁸²Br fluxes of adding ABA. On return of guard cellsto light, after the transient, there is a further reductionin Cl/Br efflux. It is argued that control of a specific processof ion extrusion is important in regulating the ability of guardcells to stay open. In three out of four batches of steady-statetissue labelled with ⁸²Br, the plasmalemma fluxes were highenough, relative to the tonoplast fluxes, for the efflux kineticsto be separable into two exponential components, allowing estimationof bromide contents in cytoplasm and vacuole (Q_c and Q_v), andfluxes at plasmalemma and tonoplast. With opening in light,Q_c increased by 3.9 ± 0.4 pmol mm^–2 µm^–1and Qy by 5.2 ± 0.6 pmol mm^–2 µm^–1(change in content per mm² of epidermis perµm change inaperture). Using rough estimates for the volumes of cytoplasmand vacuole these figures suggest that at 6.1 µm in thedark the concentrations were about 63 mol m^–3 in the cytoplasmand 35 mol m^–3 in the vacuole, rising to about 185 molm^–3 in the cytoplasm and 125 mol m^–3 in the vacuole,at 16.7 µm aperture in light. Neither increase can providean adequate increase in salt concentration to account for theosmotic change required, and some solute other than potassiumsalt must also be involved. In one experiment with 82Br andin the only experiment with 36Cl the plasmalemma flux was lower,and not high enough relative to the tonoplast flux to allowseparation of two phases in the efflux curves, and calculationof cytoplasmic and vacuolar contents and fluxes. The effectsof transfer from light to dark were, nevertheless, similar inboth types of tissue. Key words: Commelina communis L., Light/dark effects, Anion fluxes, Guard cells     

The Effect of Dio-9 on Photosynthesis and Ion Transport in Nitella, Tolypella, and Hydrodictyon

The effect of Dio-9, on photosynthesis and active and passiveion transport at the plasmalemma has been investigated in Nitellatranslucens, Tolypella intricata, and Hydrodictyon africanum.The active K influx and the coupled active Na efflux were moreinhibited by this inhibitor of energy transfer in phosphorylationthan was photosynthesis. The active Cl influx, the associateddownhill cation influxes, and passive ion fluxes were inhibitedby Dio-9, to a smaller extent than was photosynthesis. Cl influxin the light was often stimulated at concentrations of Dio-9,which inhibited photosynthesis. It was concluded that the activeK influx and Na efflux require ATP, while the active Cl influxdoes not. Possible links between the Cl influx and electrontransport and intermediates of photophosphorylation, and possibleinhibition by Dio-9 of the transport ATPase in the plasmalemmaare discussed.     

Effects of Light/Dark on Cation Fluxes in Guard Cells of Commelina communis L.

The effects of light/dark on cation fluxes in isolated guardcells of Commelina communis L. have been studied, using ⁸⁶RbCland ²²NaCl. Transfer to the dark has no effect on ⁸⁶Rb influx,but produces a marked transient stimulation of ⁸⁶Rb efflux,similar to that seen previously on adding ABA. The ⁸⁶Rb effluxfalls on return to light only during the period of stimulatedflux; after the transient, return to light has no effect onefflux. The ability to produce this transient stimulation ontransfer to the dark is recovered in a subsequent light period.In general, in Na-loaded cells, the stimulated efflux is notseen. and the cells do not close in the dark. The results arenot consistent with a simple permeability or potential change,but suggest a specific ion excretion activated by the transferto the dark. Key words: Commelina communis L., Light/dark effects, Cation flux, Guard cells     

Comparison of Cytoplasmic pH and Cl- Influx in Cells of Chora corallina Following 'C1- Starvation'

Cytoplasmic pH and C1– influx in cells of Chara corallinahave been measured following pretreatment in C1– freesolutions (‘C1– starvation’) or in controlsolutions containing C1–. Influx of C1– was greatlyincreased following starvation for 3—24 h, but significantchanges in cytoplasmic pH were only detected after 23—24h. It is thus unlikely that increased cytoplasmic pH controlsCl-influx in the early stages of Cl—starvation. Additionof NH⁺⁴ also increased Cl- influx, both in non-starved cellsand (sometimes) in Cl-starved cells, and this effect was accompaniedby an increase in cytoplasmic pH. The possible significanceof the cytoplasmic pH and CI- concentration as factors controllingCI- influx in Chara are assessed.     

Effects of Fusicoccin in 'Isolated' Guard Cells of Commelina communis L

The effects of 3 ? 10^–2 mol m^–3 FC on rubidium fluxesand contents in isolated guard cells of Commelina communis L.have been studied using ⁸⁶RbCl. Fusicoccin causes a marked stimulationof influx and an immediate, apparently irreversible, decreasein efflux of ⁸⁶Rb. The effect on influx is usually more importantin determining the new net flux of Rb. Observed fluxes differmarkedly from those predicted by the Goldman-Hodgkin-Katz equation,suggesting that FC does not act solely via an effect upon theplasmalemma potential. Fusicoccin appears to have a more directeffect upon the ion movements associated with changes in stomatalaperture than either ABA or transfer to the dark. Observed changesin Rb content cannot account fully for the osmotic changes associatedwith aperture increase. Key words: Fusicoccin, Guard cells, Ion fluxes, Commelina communis     

Is Calmodulin Involved in Electrophysiology of Chara corallina?

The role of calmodulin in Chara was investigated using the antipsychoticdrug trifluoperazine (TFP), which is known to bind to the calmodulin/Ca⁺⁺complex preventing it from carrying out its normal functions. At low concentration (4.0 µM), TFP had profound and irreversibleeffects on the electrophysiology of Chara plasmalemma. The restingp.d. depolarized and the membrane conductance decreased suggestingan inhibition of the proton pump. After several hours of exposurethe membrane became leaky, as cells deteriorated. The excitation was also affected by TFP. Spontaneous repetitivefiring was observed. The excitation increased in duration andthe action potential peak depolarized to + 20 mV. The cytoplasmic streaming was unaffected by TFP; the streamingrate at the resting potential remained unchanged when TFP wasadded to the medium, stoppage occurred at the time of excitationand the streaming slowly resumed. Key words: Calmodulin, Chara corallina, Proton pump, Cytoplasmic streaming, Current, voltage characteristics     

Cl- Fluxes during Osmoregulation in Chara

Measurements of Cl^– influx in cells of Chara corallinashow that control of this flux contributes to the ability ofthis cell to regulate its osmotic pressure. Transcellular osmosiswas used to generate cell fragments with abnormally high ₁,(H-cells), and with abnormally low ₁, (L-cells). Plasmalemmainflux (_oc) was very high in L-cells, and markedly reduced inH-cells. Influx was not affected by the presence of sucrosein the pond water and the consequent reduction in turgor. InH-cells the chloride flux from cytoplasm to vacuole (_cv) wasalso strongly inhibited. It is suggested that control of Cl^–fluxes at both plasmalemma and tonoplast is involved in osmoregulationin these cells. Key words: Chara corallina, osmoregulation, Cl^– flux