Intercellular Transport and Subcellular Distribution of Photoassimilates in Chara corallina

The mechanism of polar transport of ¹⁴C-photoassimilates betweenChara corallina branchlets and internodes was investigated usingan intracellular perfusion procedure. When the apex remained,more ¹⁴C-photoassimilates were transported from branchlet tointernode than in the opposite direction. When the apex wasdetached, this polarity disappeared. To study the polar transportmechanism, we examined the subcellular distribution of ¹⁴C-photoassimilatesin the three main intracellular compartments, the cortical chloroplastlayer, streaming sol endoplasm and vacuole. When the internode or branchlet of an internode-branchiet complexwas exposed to ¹⁴C-bicarbonate for 10 min, 70 to 80% of thetotal fixed carbon was found in the sol endoplasm. In the subsequent3 h chase, the ¹⁴C-photoassimilates in the sol endoplasm ofthe source cell decreased greatly to 20 to 40% of the totallabelled substances. When the apex remained, the branchlet showed higher photosyntheticactivity than the internode, which resulted in a concentrationgradient of 13·5 mol m^–3 in the sol endoplasm frombranchlet to internode after photosynthesis for 10 mm. In the3 h chase period, this gradient decreased to about 1·0mol m^–3. In the other three cases, involving transportfrom branchlet to internode when the apex was detached, or transportfrom internode with or without apex to branchlet, the intercellulargradient of ¹⁴C after 10 mm photosynthesis was about 70 molm^–3 in the sol endoplasm. Thus, when the apex remains,the greater gradient of photoassimilates between the sol endoplasmof branchlet and internode seems to be one of the reasons forpolar intercellular transport. Key words: Chara corallina, polar transport, ¹⁴C photoassimilates     

Intercellular Transport and Cytoplasmic Streaming in Chara hispida

The correlation between the velocities of cytoplasmic streamingand of translocation of ¹⁴C-photosynthate and ³²P-phosphateassociated radioactivity has been investigated in whole plantsof the green freshwater alga Chara hispida L. Tracer was suppliedto the plant's rhizoid system in a split-chamber. The velocityof cytoplasmic streaming of 52±3.3 µm s^–1compares with 57±10 µm s^–1 found for ¹⁴C-transportand 32±20 µm s^–1 found for ³²P-transport.There was no indication of intercellular translocation at avelocity faster than visible streaming. Cytochalasin B inhibitedthe translocation of ³²P and cytoplasmic streaming. CytochalasinB becomes fully effective in inhibiting streaming and transportafter an incubation time of at least 5 h. Key words: Chara hispida, Cytoplasmic streaming, Intercellular transport     

Photosynthetic Fixation of 14Carbon by Internodal Cells of Chara corallina

Maximum fixation rates of 120 and 60 pmol cm^–2 s ^–1wereobtained when exogenous carbon was supplied as ¹CO₂ and H¹⁴CO₃^–respectively. These values are considerably higher than thosepreviously reported for this species. A kinetic analysis wasperformed on this data. Substrate saturation in the concentrationrange 1.0–1.5 mM was observed for both CO₂ and HCO₃^– In the presence of exogenous CO², a linear relationship wasobserved between light intensity and fixation while the HCO₃^–relationship was slightly sigmoidal. Fixation saturated at intensitiesof 15–20 W m^–2 and 13–15 W m^–2 for exogenous¹⁴CO² and H¹⁴CO₃^–respectively. The presence, in this species, of an extremely active HCO₃^–transport system, situated in the plasmalemma, demonstratesthat when alkaline solutions are employed the involvement ofthis ion cannot be ignored during electrical studies on thismembrane. The maximum H¹⁴CO₃^– influxes obtained duringthis study are the largest ionic fluxes measured for any Characeanspecies. It was demonstrated that CO² for fixation can be supplied simultaneouslyby gaseous diffusion and HCO₃^– transport (cf. Raven, 1968).Inhibition of H¹⁴CO₃^– influx was observed in the presenceof Tris, Tricine, and borate buffers, and CO₃^{2 –} alsoappeared to act as a strong inhibitor. The possible mechanism(s)by which this inhibition occurs is discussed.     

The Effect of Darkness on Active and Passive Transport in Chara corallina

In Chara corallina, the membrane potential may stay much morenegative than the equilibrium potential for potassium in thedark, indicating that the proton pump is operative. The highproton conductance which occurs at high external pH, as indicatedby a high membrane conductance and a membrane potential nearthe equilibrium potential for protons, is not seen in the darkat pH 11. This effect is likely to be related to inhibitionof photosynthesis since DCMU has the same effect. The effectis similar but not identical to the effect of a decreased internalpH. Key words: Light, dark, membrane potential, Chara     

Water channels in Chara corallina

Water relations parameters ofChara corallina inter-nodes weremeasured using the single cell pressure probe. The effect ofmercurials, which are recognized as non-specific water channelinhibitors, was examined. HgCl₂ concentrations greater than5 mmol m^–3 were found to inhibit hydraulic conductivity{Lp) close to 90%, whereas pCMPS was found to have no effecton Lp. The activation energy of water flow was increased significantlyfrom 21.0 kJ mol^–1 to 45.6 kJ mol^–1, following theapplication of HgCl₂. These results are in accordance with evidencefor Hg²⁺sensitive water channels in the plasma membrane of charophytes(Henzler and Steudle, 1995; Tazawa et al., 1996). The metaboliceffects must, however, be considered in view of the rapid inhibitionof respiration and the depolarization of the membrane potentialwith HgCl₂ concentrations lower than those found to affect Lp.It was possible to measure simultaneously water relations andmembrane PD, in order to examine the contribution of potassiumchannels to Lp. Cells were induced into a K⁺ permeable state.The K⁺ channels, assumed to be open, were subsequently blockedby various blockers. No significant difference in Lp was foundfor any of these treatments. Finally, the permeability of C.corallina membranes to ethanol was examined. HgCl₂ was foundto cause a decrease in reflection coefficient, coinciding witha decrease in Lp, but there was no change in the ethanol permeabilitycoefficient. This has been interpreted in terms of both thefrictional model and composite model of non-electrolyte membranetransport. Key words: Water channels, Chara, hydraulic, conductivity, membrane transport models, reflection coefficient     

Fluorescence and Photosynthetic Activity of Chloroplasts in Acid and Alkaline Zones of Chara corallina

Continuous profiles of local pH near the cell surface of Chara corallinawere recorded during uniform longitudinal movement of an internodal cell relative to a stationary pH microelectrode. Under illumination, the pH profile consisted of alternating acid and alkaline bands with a pH difference of up to 3 pH units. After darkening, the bands disappeared and pH became uniformly distributed along the cell length. Chlorophyll fluorescence of chloroplasts was measured by microfluorometry at different locations within one cell, and significant differences were observed in close relation to light-dependent pH banding. The chlorophyll fluorescence yield was lower in zones of low external pH than in alkaline zones both under actinic and saturating light. The fluorescence parameters Fand F" _m and the quantum yield of photosystem II (PSII) displayed variations along the cell length in accordance with pH changes in unstirred layers of the medium. The results show that PSII photochemical efficiency and the rate of noncyclic electron transport are higher in the chloroplasts of acid zones (zones of H⁺extrusion from the cell) than in alkaline zones. The dependence of photosynthetic electron transport on local pH near the cell surface may result from different contents of CO₂in acid and alkaline regions. The acid zones are enriched with CO₂that readily permeates through the membrane providing the substrate for the Calvin cycle. Conversely, a poorly permeating form, HCO^– ₃is predominant in alkaline zones, which may restrict the dark reactions and photosynthetic electron flow.     

Chloride Transport in Chara corallina and the Electrochemical Potential Difference for Hydrogen Ions

The pH of the cytoplasm of Chara corallina cells has been measuredwith the weak acid 5,5-dimethyloxazolidine-2,4-dione (DM0).Over an external pH range 4·5–9·5 the resultsfit the regression equation pH_cytoplasm=6·28+0·22pH_out. Using measured values of the electric potential difference acrossthe plasmalemma we have calculated the electrochemical potentialdifference across this membrane for H⁺ and Cl^–. Thesedata are used to test the hypothesis that the inward transportof Cl^– is coupled to the inthix of H⁺ or, which comesto the same thing, efflux of OH^–. One-for-one couplingwill not give net Cl^– uptake from solutions with pH greaterthan about 7·2, unless the cytoplasmic Cl^– concentrationis lower than 10 mM, or the pH just outside the membrane islower than that in the bulk solution. It is shown that net Cl^–uptake proceeds from solutions with pH up to 9. The alternative possibility is that Cl^– transport is broughtabout by co-transport of two H⁺ for each Cl^–; this isnot ruled out by the results reported. Such a mechanism mightbe detectable by its electrogenic effect: although such effectshave not been detected, it is shown that they would be smallunder most conditions. Other possible mechanisms are discussed.     

Plasmalemma Voltage Noise in Chara corallina

Voltage noise analysis is applied to plasmalemma ion transport in Chara corallina. There is a component in the noise power spectrum that is probably associated with current fluctuations within passive transport channels, and another component that may be associated either with fluctuations in the number of open channels, or with active transport. The data allow the calculation of time constants that may be attributable to molecular level events in these transport processes.     

Plasmalemma Transport of HCO3- and OH- in Chara corallina: Non-antiporter Systems

An experimental system was designed to test the obligate couplingbetween HCO₃^– and OH^– fluxes (i. e. a ‘Mitchell-type’antiporter) proposed by Lucas and Smith (1973). The resultsof these experiments demonstrated categorically that the OH^–efflux process can function in the absence of exogenous HCO₃^–at the actual OH^– efflux site. Hence, the obligate couplinghypothesis is invalid. It is proposed that HCO₃^– and OH^–are transported across the plasmalemma ‘independently’,on quite distinct carriers. It is possible, therefore, thatthese fluxes contribute towards determining the electrical propertiesof this membrane when the bathing solution pH value is 6.5.It was also found that HCO₃^– can be transported acrossthe dark segment of a partly illuminated cell. The observedrates were always much less than those obtained in the illuminatedcell segment. The significance of this result is discussed.     

Hydroxyl- and Bicarbonate-Associated Transport Processes in Chara corallina: Studies on the Light-Dark Regulation Mechanism

Lucas, W. J. and Ogata, K. 1985. Hydroxyl– and bicarbonate–associatedtransport processes in Chara corallintr. Studies on the light–darkregulation mechanism.—J. exp. Bot. 36: 1947–1958.Experiments were undertaken on the fresh water alga Chara corallinato investigate the nature of the coupling between the chloroplastsand the light–dependent OH^– and -associated plasmalemma transport systems. Electrophysiologicalexperiments, in which DCMU was employed, revealed that thischemical could elicit a hyperpolarization of the membrane potentialthat was identical to that normally obtained by turning offthe light. This DCMU–induced hyperpolarization was obtainedunder control () and phosphate–decoupled conditions (). Measurements of the extracellular electric potentialswhich are associated with the acidic () and alkaline (OH^–) regions, indicated that, in the presenceof control ()or phosphate–decoupled conditions, normal profiles were established under air, oxygenor nitrogen environments. These results indicate that the generationof the control signal(s) is related to events associated withchloroplast electron transport, rather than to changes in theflow or levels of carbon intermediates within the reductivepentose phosphate or photorespiratory cycles. Although the levelof oxygen was found to have no effect on the light–inducedactivation of the OH regions, we found that in pure oxygen thedark–induced inactivation of the OH^– efflux systemwas delayed, and that partial transport function could be maintainedin the dark. The possible involvement of changes in either theratio of oxidized to reduced ferridoxin or NADP^? to NADPH, aspart of this light–mediated control signal, is discussed. Key words: Chara corallina, Plasma membrance transport, OH^– and , regulation     

Sulfhydryl Group Involvement in Plasmalemma Transport of HCO(3) and OH in Chara corallina

The effect of the sulfhydryl reagents (—SH) p-chloromercuribenzene-sulfonic acid (PCMBS), N-ethylmaleimide (NEM), and inorganic mercury on H¹⁴CO₃⁻ assimilation in Chara corallina is reported. Commercial grade PCMBS caused severe inhibition of H¹⁴CO₃⁻ assimilation. Results obtained using purified PCMBS (stock solution passed through a chelating resin) indicated that inhibition observed using unpurified PCMBS was due predominantly to the presence of inorganic mercury (as a contaminant). The inhibitory role of inorganic mercury was verified using HgCl₂. This chemical caused a dramatic inhibition of H¹⁴CO₃⁻ assimilation, while it had little effect on cellular ¹⁴CO₂ fixation. Reversal of the Hg²⁺ inhibition of H¹⁴CO₃⁻ assimilation (in presence of 1.0 millimolar dithioerythritol) was extremely slow, requiring 2 to 3 hours for the reestablishment of control rates. This slow recovery may reflect de novo synthesis of transport proteins.     

Plasmalemma Chloride Transport in Chara corallina: Inhibition by 4,4'-Diisothiocyano-2,2'-Disulfonic Acid Stilbene

Chloride transport, presumably via a Cl⁻-2H⁺ co-transport system, was investigated in Chara corallina. At pH 6.5, the control influx (3.1 picomoles per centimeter² per second) was stimulated 4-fold by an 18-hour Cl⁻ starvation. The stimulated influx was inhibited to 4.7 picomoles per centimeter² per second after a 60-minute pre-exposure to 0.5 millimolar 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). This compares with a nonsignificant inhibition of the control under similar conditions. At 2 millimolar DIDS, both stimulated and control influx were inhibited to values of 1.1 and 2.2 picomoles per centimeter² per second, respectively; in all cases, DIDS inhibition was reversible. Over the pH range 4.8 to 8.5, the control and DIDS-inhibited influx showed only slight pH sensitivity; in contrast, the stimulated flux was strongly pH dependent (pH 6.5 optimum). Inasmuch as changes in pH alter membrane potential, N-ethylmaleimide was used to depolarize the membrane; this had no effect on Cl⁻ influx. A transient depolarization of the membrane (about 20 millivolts) was observed on restoration of Cl⁻ to starved cells. The membrane also depolarized transiently when starved cells were exposed to 0.5 millimolar DIDS, but the depolarization associated with Cl⁻ restoration was inhibited by a 40-minute pretreatment with DIDS. Exposure of control cells to DIDS caused only a small hyperpolarization (about 7 millivolts). DIDS may have blocked Cl⁻ influx by inhibiting the putative plasmalemma H⁺-translocating ATPase. Histochemical studies on intact cells revealed no observable effect of DIDS on plasmalemma ATPase activity. However, DIDS application after fixation resulted in complete inhibition of ATPase activity.

The differential sensitivity of the stimulated and control flux to inhibition by DIDS may reflect an alteration of transport upon stimulation, but could also result from differences in pretreatment. The stimulated cells were pretreated with DIDS in the absence of Cl⁻, in contrast to the presence of Cl⁻ during pretreatment of controls. The differential effect could result from competition between Cl⁻ and DIDS for a common binding site. Our histochemical ATPase results indicate that Cl⁻ transport and membrane ATPase are separate systems, and the latter is only inhibited by DIDS from the inside of the cell.

     

Plasmalemma Transport of HCO-3 and OH- in Chara corallina: Inhibitory Effect of Ammonium Sulphate

The influence of (NH₄)₂SO₄ on ¹⁴C assimilation and cyclosisin internodal cells of Chara corallina was investigated. Severeinhibition of ¹⁴C assimilation was found at pH values above7·0, this inhibition being correlated with the exogenouslevel of NH3 rather than NH⁺₄. Cyclosis was also affected athigher concentrations of (NH₄)₂SO₄. This effect was similarlycorrelated with exogenous levels of NH₃. ¹⁴C assimilation was inhibited non-competitively by (NH₄)₂SO₄,the apparent Km being increased from 0·55 to 1·5mM. The results suggest that the site(s) of inhibition is locatedat the plasmalemma, rather than at the chloroplasts. (Evidencein support of in vivo uncoupling of photophosphorylation, bylow concentrations of (NH₄)₂SO₄, was not obtained). Significant perturbation of the OH^– efflux pattern wasobserved as the level of (NH₄)₂SO₄ was increased. Induced migrationof efflux sites indicates that NH₃ may interfere with the cellularmechanism that controls OH^– transport. Using a cell-segmentisolating chamber it was shown that (NH₄)₂SO₄ inhibited OH^–efflux rather than HCO^–₃ transport. This inhibitory effectwas readily reversible. These data are discussed in terms of a possible relationshipbetween the observe NH₄)²SO₄ stimulation of ³⁶Cl^– influxand the effect of this compound on ¹⁴C assimilation.     

Calcium-salinity interactions affect ion transport in Chara corallina

Detached internodes of Chara corallina survived in solutions containing 100 mol m^?3 NaCl when the external concentration of Ca²⁺ was greater than 1 mol m^?3. Na⁺ influx was roughly proportional to external Na⁺ up to 100 mol m^?3 NaCl. Na⁺ influx involved two components: a Ca²⁺-insensitive influx which allowed the passage of Na⁺ independently of external Ca²⁺; and a Ca²⁺-inhibitable mechanism where Na⁺ influx was inversely proportional to external Ca²⁺. The Ca²⁺-inhibitable Na⁺ influx was similar to the Ca²⁺-inhibitable K⁺ influx. Mg²⁺ and Ba²⁺ were able to substitute for Ca²⁺ in partially inhibiting Na⁺ influx in the absence of external Ca²⁺. The effect of Ca²⁺ appears specific to Na⁺ and K⁺ influx since the effects of a Ca²⁺-free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na⁺ influx via the Ca²⁺-inhibitable mechanism represents Na⁺ leakage through K⁺ channels and that cell death at high salinity occurs due to a cytotoxic Na⁺ influx via this mechanism.     

Comparison of the Effects of Ammonia and Methylamine on Chloride Transport and Intracellular pH in Chara corallina

Ammonium and methylammonium ions greatly increase the rate ofCl^– transport in Chara corallian. This effect is dependenton the pH of the bathing solution. The amine-stimulated Cl^–influx is small at pH 5·5, increases to a maximum atpH 6·5–7·5, and decreases again as the pHis raised to 8·5. Increased Cl^– influx is accompaniedby an increase in cytoplasmic pH, as calculated from the distributionof DMO. When the external pH lies between 5·5 and 7·3,cytoplasmic pH in the absence of amine is 7·65–7·70,with an increase of 0·15–0·25 in the presenceof amine. As external pH is increased above 7·3, cytoplasmicpH also increases, with progessively less effect of amine. Although the relationship between Cl^– influx and cytoplasmicpH is not simple, the results provide evidence in accord withthe hypothesis that Cl^– transport in Chara involves H⁺—Cl^–symport, or the equivalent OH^–—Cl^– antiport.The possible role of cytoplasmic pH as a factor involved inthe regulation of membrane transport in Chara is discussed.     

Quantitative Analysis of Intercellularly-Transported Photoassimilates in Chara corallina

Using a thin-layer chromatographic technique, we have identifiedthe photoassimilates that are transported intercellularly frombranchlets to internodes in Chara corallina. An internode-branchletcomplex having a primary apex was used in these experiments.After feeding 1 mol m³ NaH¹⁴CO₃ to a branchlet for 10 min, the¹⁴C-labelled photoassimilates (¹⁴C-photoassimilates) found inthe sol endoplasm of the branchlet were composed of sucrose,amino acids, malate, and sugarphosphates. The composition ofthe ¹⁴C-photoassimilates transported from the source branchletto the sink internode in 10 min was the same as that in thesol endoplasm of the source branchlet. From the proportion ofeach ¹⁴C-photoassimilate in both the source branchlet and thesink internode, it was deduced that the main photoassimilatesinvolved in the intercellular transport were sucrose and aminoacids. We found previously that polar transport of photoassimilatesoccurs from a branchlet to an internode with an apex. Determinationof the amount of sucrose, amino acids, glucose-6-phosphate,and malate in both branchlet and internode with or without anapex revealed that there were gradients in the concentrationsof sucrose, serine, and glutamic acid between the sol endoplasmof the two cells. The levels were higher in the branchlet andlower in the internode and the gradients decreased when theapex was detached. Therefore, it is concluded that sucrose andthese amino acids are the compounds involved in the polar intercellulartransport. Key words: Chara corallina, intercellular transport, photoassimilates, ¹⁴C     

The Uptake of Sugars by Chara corallina

The influx of several monosaccharides into Chara corallina wasstudied under varying conditions of temperature, in the presenceof inhibitors, and by the use of competition experiments. Itwas found that the mechanism of transport was stereospecific,and the complex interactions between the sugars were characteristicof a carrier-mediated system. There is reason to believe thatthe carrier involved in this system has a narrower range ofaffinities than is usually attributed to a sugar carrier, forglucose and fructose appear to have different sites of entry,though each can influence the uptake of the other. In additionthe carrier system is situated in the plasmalemma and maintainedby metabolic energy.     

Plasmalemma Transport of OH in Chara corallina: III. FURTHER STUDIES ON TRANSPORT SUBSTRATE AND DIRECTIONALITY

The identity of the plasmalemma-transported species that develops the alkaline bands of Chara corallina was investigated. The effect of fusicoccin on the rate of HCO₃⁻ assimilation, and on the time-dependent alkaline band pH buildup following low pH flushing, was found to be small, with no stimulatory effect. Computer simulation of the flushing experiments showed that in the experimental situation the alkaline band transport system was slowed down, rather than speeded up, by low pH flushing. A detailed theoretical examination of the maximum rate of proton production from water showed that measured alkaline band fluxes are too large to be explicable in terms of an H⁺ influx system. The experimental and theoretical results indicate that the plasmalemma transport of OH⁻ ions is responsible for the measured negative external electric potential and alkalinity flux which are associated with the alkaline band phenomenon. Consequently, HCO₃⁻ influx across the characean plasmalemma must be charge-balanced by the efflux of OH⁻ ions.