Internodal Cells Of Chara Corallina Research Articles

Age dependence of the hydraulic resistances of the plasma membrane and the tonoplast (vacuolar membrane) in cells of Chara corallina.

Hydraulic resistances (reciprocals of hydraulic conductivities) of the cell (Lp-1), the cell wall (Lpw-1), the membrane (Lpm-1), the plasma membrane (Lppm-1), and the tonoplast (Lptp-1) were determined in individual internodal cells of Chara corallina and their dependence on the cell age was studied. The thickness of the cell wall (d) was adopted as an index of the cell age, since the cell wall of spring-grown young cells (sg-cells) was found to be significantly thinner than that of winter-spent old cells (ws-cells). Both Lpw-1 and Lpm-1 were found to increase with cell age. Since Lpm-1 is the sum of Lppm-1 and Lptp-1, their dependence on the wall thickness was studied. It was found that both Lppm-1 and Lptp-1 increase with cell age using d as a proxy and that the former is distinctly higher than the latter. The ratio Lppm-1/Lptp-1 amounts to 30 for 5μm of d, indicating that the tonoplast is a negligible barrier to osmotic water flow. The ratio decreases with the increase in d and amounts to 5.0 for 11μm of d, showing that the tonoplast ages faster than the plasma membrane. The physiological meaning of the age dependence of hydraulic resistance of the tonoplast was discussed in terms of the role of the vacuole in the osmoregulation of the cytoplasm.

Prolonged oxygen depletion in microwounded cells of Chara corallina detected with novel oxygen nanosensors.

Primary physicochemical steps in microwounding of plants were investigated using electrochemical nano- and microprobes, with a focus on the role of oxygen in the wounding responses of individual plant cells. Electrochemical measurements of cell oxygen content were made with carbon-filled quartz micropipettes with platinum-coated tips (oxygen nanosensors). These novel platinum nanoelectrodes are useful for understanding cell oxygen metabolism and can be employed to study the redox biochemistry and biology of cells, tissues and organisms. We show here that microinjury of Chara corallina internodal cells with the tip of a glass micropipette is associated with a drastic decrease in oxygen concentration at the vicinity of the stimulation site. This decrease is reversible and lasts for up to 40 minutes. Membrane stretching, calcium influx, and cytoskeleton rearrangements were found to be essential for the localized oxygen depletion induced by cell wall microwounding. Inhibition of electron transport in chloroplasts or mitochondria did not affect the magnitude or timing of the observed response. In contrast, the inhibition of NADPH oxidase activity caused a significant reduction in the amplitude of the decrease in oxygen concentration. We suggest that the observed creation of localized anoxic conditions in response to cell wall puncture might be mediated by NADPH oxidase.

Longitudinal profiles of the vacuolar pH in internally perfused cells of characean alga

Activities of ion pumps and H+-conducting channels in the plasmalemma of illuminated characean algae are distributed inhomogeneously along the internode, which accounts for the shifts of surface pH up to 3.5 units between various cells regions. Spatial variations in cytoplasmic properties provide the basis for uneven distribution of photosynthetic activity along the cell length and might affect the operation of H+-transporting systems at the tonoplast. In order to visualize the longitudinal distribution of the vacuolar pH in Chara corallina internodal cells, the pH microelectrode was inserted into the vacuole and the cell sap was gradually displaced along the cell during intracellular perfusion with an artificial medium. Fluorescein was added to the perfusion medium as a fluorescent marker to detect the arrival of the replacing medium into the area of pH and fluorescence measurements. In light-adapted cells, nonuniform longitudinal pH profiles were observed, with pH shifts as large as 2–2.5 units. In dark-adapted cells, the pH shifts in longitudinal profiles did not exceed 0.5 pH units. The occurrence of large pH changes within the vacuole of individual internodes indicates the possibility of nonuniform distribution of the tonoplast H+-transporting systems in different regions of the illuminated cell.

Polar auxin transport: an early invention

In higher plants, cell-to-cell polar auxin transport (PAT) of the phytohormone auxin, indole-3-acetic acid (IAA), generates maxima and minima that direct growth and development. Although IAA is present in all plant phyla, PAT has only been detected in land plants, the earliest being the Bryophytes. Charophyta, a group of freshwater green algae, are among the first multicellular algae with a land plant-like phenotype and are ancestors to land plants. IAA has been detected in members of Charophyta, but its developmental role and the occurrence of PAT are unknown. We show that naphthylphthalamic acid (NPA)-sensitive PAT occurs in internodal cells of Chara corallina. The relatively high velocity (at least 4–5 cm/h) of auxin transport through the giant (3–5 cm) Chara cells does not occur by simple diffusion and is not sensitive to a specific cytoplasmic streaming inhibitor. The results demonstrate that PAT evolved early in multicellular plant life. The giant Chara cells provide a unique new model system to study PAT, as Chara allows the combining of real-time measurements and mathematical modelling with molecular, developmental, cellular, and electrophysiological studies.

Effects of cyclosis on chloroplast–cytoplasm interactions revealed with localized lighting in Characean cells at rest and after electrical excitation

Cytoplasmic streaming in Characean internodes enables rapid intracellular transport and facilitates interactions between spatially remote cell regions. Cyclosis-mediated distant interactions might be particularly noticeable under nonuniform illumination, in the vicinity of light–shade borders where metabolites are transported between functionally distinct cell regions. In support of this notion, chlorophyll fluorescence parameters assessed on a microscopic area of Chara corallina internodal cells (area of inspection, AOI) responded to illumination of nearby regions in asymmetric manner depending on the vector of cytoplasmic streaming. When a beam of white light was applied through a 400-μm optic fiber upstream of AOI with regard to the direction of cytoplasmic streaming, non-photochemical quenching (NPQ) developed after a lag period in AOI exposed to moderate intensity light. Conversely, no NPQ was induced in the same cell area when the beam position was shifted to an equal distance downstream of AOI. Light–response curves for the efficiency of photosystem II electron transport in chloroplasts differed markedly depending on the illumination pattern (whole-cell versus small area illumination) but these differences were eliminated after the inhibition of cytoplasmic streaming with cytochalasin B. Localized illumination promoted chloroplast fluorescence responses to electrical plasmalemma excitation at high light intensities, which contrasts to the requirement of low to moderate irradiances for observation of the stimulus–response coupling under whole-cell illumination. The results indicate that different photosynthetic capacities of chloroplasts under general and localized illumination are related to lateral transport of nonevenly distributed cytoplasmic components between the cell parts with dominant photosynthetic and respiratory metabolism.

Cyclosis-related asymmetry of chloroplast–plasma membrane interactions at the margins of illuminated area in Chara corallina cells

Cytoplasmic streaming in plant cells is an effective means of intracellular transport. The cycling of ions and metabolites between the cytosol and chloroplasts in illuminated cell regions may alter the cytoplasm composition, while directional flow of this modified cytoplasm may affect the plasma membrane and chloroplast activities in cell regions residing downstream of the illumination area. The impact of local illumination is predicted to be asymmetric because the cell regions located downstream and upstream in the cytoplasmic flow with respect to illumination area would be exposed to flowing cytoplasm whose solute composition was influenced by photosynthetic or dark metabolism. This hypothesis was checked by measuring H(+)-transporting activity of plasmalemma and chlorophyll fluorescence of chloroplasts in shaded regions of Chara corallina internodal cells near opposite borders of illuminated region (white light, beam width 2mm). Both the apoplastic pH and chlorophyll fluorescence, recorded in shade regions at equal distances from illuminated area, exhibited asymmetric light-on responses depending on orientation of cytoplasmic streaming at the light-shade boundary. In the region where the cytoplasm flowed from illuminated area to the measurement area, the alkaline zone (a zone with high plasma membrane conductance) was formed within 4-min illumination, whereas no alkaline zone was observed in the area where cytoplasm approached the boundary from darkened regions. The results emphasize significance of cyclosis in lateral distribution of a functionally active intermediate capable of affecting the membrane transport across the plasmalemma, the functional activity of chloroplasts, and pattern formation in the plant cell.

Effects of Lanthanum on Calcium and Magnesium Contents and Cytoplasmic Streaming of Internodal Cells of Chara corallina

Biological and environmental effects of lanthanide series of elements have received much attention recently due to their wide applications. In this study, effects of La(3+) treatments on calcium and magnesium concentrations as well as cytoplasmic streaming of internodal cells of Chara corallina were investigated. At all treatment concentrations (10, 100, and 1,000 μM), La(3+) significantly decreased calcium concentrations in the cell-wall fractions after 5-h treatments. Calcium concentrations in the cell contents and magnesium concentrations in the cell-wall fractions were reduced by 100 and 1,000 μM La(3+) treatments. However, cytoplasmic streaming as an indicator of [Ca(2+)](cyt) was only inhibited at the highest La(3+) concentration (1,000 μM). The results suggest that La(3+) may affect cellular calcium homeostasis by actions other than as a simple Ca(2+) antagonist. La(3+) could partially compensate for calcium deficiency at certain concentrations.

Facilitated permeation of methyl viologen into chloroplasts in situ during electric pulse generation in excitable plant cell membranes

Generation of action potential (AP) in plasma membranes of characean algae has a strong impact on photoreactions occurring in chloroplasts. Under physiological conditions, AP suppresses electron transport in alkaline and acidic regions, although to a different extent; these changes are transient and reversible. In the presence of the artificial electron acceptor, methyl viologen (MV2+), AP-induced changes in electron transport in photosystem II become irreversible. Incubation of Chara corallina internodal cells with MV2+ has no effect on the chlorophyll P700 photooxidation kinetics in photosystem I reaction centers, suggesting that MV2+ is inaccessible for interactions with photosystem I, because its permeation into chloroplasts of a resting cell is hindered by membrane barriers. At the same time, AP generation in the presence of MV2+ is accompanied by irreversible modification of P700 photooxidation kinetics, as can be evidenced from differences in absorption changes at 810 and 870 nm (ΔA810 signals). These findings suggest that MV2+ permeation into chloroplasts in situ is facilitated during or after the AP generation. Similar to the ΔA810 signals, light-induced changes in membrane potential do not depend on the presence of MV2+ in the external medium until the first excitatory stimulus is applied. Electric photoresponses of the cell are irreversibly modified by AP generated in the presence of MV2+ at the expense of non-cyclic photosynthetic electron transport redirected to the MV2+ reduction. It is concluded that AP effects on chloroplast photosynthesis in situ are complex and involve permeability changes for MV2+ in membrane barriers of the “plasmalemma-chloroplast envelope” system.

Involvement of Membrane Potential in Alkaline Band Formation by Internodal Cells of Chara corallina

Internodal cells of Chara corallina form alkaline bands on their surface upon illumination via photosynthesis. In the present study, the effect of KCl on alkaline band formation was analyzed. When the extracellular KCl concentration was increased, alkaline band formation was extensively inhibited. Electrophysiological analysis unequivocally showed the need for inner negative membrane potential for alkaline band formation.

Direct measurement of lanthanum uptake and distribution in internodal cells of Chara

Rare earth elements (REEs) influence many physiological processes in plants, but the mechanism is still unknown. The question of whether REEs can enter plant cells is crucial in assessing the biological effects of REEs. In this paper, a method for determination of REE lanthanum (La) in ultratrace level in cell contents of giant algal cells was established. The uptake and subcellular distribution of La in internodal cells of Chara corallina were investigated. After incubation with 6 μM LaCl 3 for 72 h, about 0.523% of total lanthanum in the whole cell was internalized. Approximately 55% of the total intracellular lanthanum was in the cytoplasm, and lanthanum concentration in the cytoplasm was much higher than that in the vacuole. The concentration dependence of La 3+ uptake occurred in a biphasic manner, which was tentatively attributed to the operation of two separate transport systems, a high-affinity system which is saturated at 10 μM and a low affinity system which showed a linear dependence on concentration up to at least 100 μM. Only the high-affinity system was studied in detail. Effects of channel activator, blocker and metabolic inhibition on lanthanum uptake through this system were investigated. The results suggest that lanthanum transport across plasma membrane was a metabolically mediated process and membrane potential difference was independent of lanthanum uptake.

Intracellular Axial Current in Chara corallina Reflects the Altered Kinetics of Ions in Cytoplasm under the Influence of Light

Recent experiments demonstrate that the concentration of Ca 2+ in cytoplasm of Chara corallina internodal cells plays important role in electrical excitation of the plasma membrane. The concentration of free Ca 2+ in the cytoplasm −[Ca 2+] c is also sensitive to visible light. Both phenomena were simultaneously studied by noninvasive measuring action potential (AP) and magnetic field with a superconducting quantum interference device magnetometer in very close vicinity of electrically excited internodal C. corallina cells. A temporal shift in the depolarization maximum, which progressively occurred after transferring cells from the dark into the light, can be explained by the extended Othmer model. Assuming that the change in membrane voltage during the depolarization part of AP is the direct consequence of an activation of [Ca 2+] c sensitive Cl − channels, the model simulations compare well with the experimental data. We can say that we have an example of electrically elicited AP that is of biochemical nature. Electric and magnetic measurements are in good agreement.

Studies on semipermeability and electrical characteristics in membranes of Chara cells fixed with glutaraldehyde.

When internodal cells of Chara corallina were incubated in a solution containing 1% glutaraldehyde, an increase in intracellular osmolality was observed, and this indicates that the plasma membrane maintained its semipermeability. The effect on the membrane potential was studied. Although the active component generated by the electrogenic proton pump was lost, the passive component (more negative than -100 mV) was maintained during glutaraldehyde treatment for more than 1-2 h. The membrane resistance increased significantly. It was found that the tonoplasts also maintained their semipermeability during glutaraldehyde treatment.

Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells.

In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin filaments into bundles throughout the plant kingdom.

Induction of a new alkaline band at a target position in internodal cells of Chara corallina.

Characean cells develop alternating alkaline and acid bands on their surface upon illumination. However, the mechanism of band formation is not fully understood. In the present study, we succeeded in inducing a new alkaline band at an original acid band in internodal cells of Chara corallina. Chloroplasts in an acid band were locally removed by wounding the cell in the absence of the cell turgor pressure. The chloroplast-removed area was observed as a white belt in a green cylindrical internodal cell. This internodal cell developed a new alkaline band on the surface at the chloroplast-removed area. The narrower the chloroplast-removed area, the less significant the extent of OH(-) extrusion. This is the first success in inducing a new alkaline band at a target position in Characeae.

Regulation of the H+ Pump Activity in the Plasma Membrane of Internally Perfused Chara corallina

The role of cytoplasm for the maintenance of the H+ pump activity in Chara corallina internodal cells was examined by the intracellular perfusion technique. Cytoplasm-rich and -poor states were obtained by changing the perfusion time, short-term (less than 2 min) and long-term (more than 5 min), respectively. A large portion of cytoplasm was left by short-term perfusion but most of the cytoplasm was removed by long-term perfusion. The activities of the H+ pump of these two different conditions were examined by measuring current-voltage relation (I-V curve) and conductance-voltage relation (G-V curve) under voltage clamp conditions. The H+ pump conductance decreased to 37%, 9% and zero by short-term, long-term and hexokinase perfusion, respectively, whereas the passive channel conductance decreased to 71%, 39% and 73% by short-term, long-term and hexokinase perfusion, respectively. On the other hand, the electromotive-force of the H+ pump (approximately -260 mV) and the passive channel (approximately -130 mV) were not affected by either short- or long-term perfusion. It is indicated that the cytoplasm plays an essential role to regulate the activity of both the H+ pump and the passive channel together with ATP.

Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

concentration ([K+]o) on the membrane potential (Em) of Chara corallina was studied. Em more negative than -100 mV was maintained even at 100 mM [K+]o. Addition of Ca2+ to the external medium further increased this tendency. However, Em responded sensitively to the increase in [K+]o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K+]o. In the presence of 100 mM K+, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K+ at high concentration was discussed.

Effects of aluminum on plasma membrane as revealed by analysis of alkaline band formation in internodal cells of Chara corallina.

To study the mechanism of aluminum toxicity in plant cells, the effects of aluminum on alkaline band formation were analyzed in the internodal cells of Chara. After cells were treated with AlCl3, they were examined for their capacity to develop alkaline bands. Treating cells with AlCl3 medium at pH 4.5 completely inhibited alkaline band formation. When either CaCl2 or malic acid was added to the AlCl3 medium (pH 4.5), it did not produce an ameliorative effect, whereas addition of both CaCl2 and malic acid induced a significant ameliorative effect. It was found that treatment at pH 4.5 in the absence of AlCl3 strongly inhibited alkaline band formation. This inhibition by the low pH (4.5) treatment was effectively ameliorated by CaCl2. At higher pH (5.0), malic acid alone produced a significant ameliorative effect on aluminum inhibition of alkaline band formation, but CaCl2 did not. Recovery from aluminum inhibition was also studied. When cells treated with AlCl3 at pH 4.5 were incubated in artificial pond water, they could not recover the capacity to develop alkaline band. When either malic acid or CaCl2 was added to artificial pond water, cells recovered their alkaline band formation. It was concluded that one of the primary targets of aluminum is the plasma membrane and that aluminum affects the plasma membrane from the cell exterior at the beginning of the treatment (within 24 h). It was also suggested that the aluminum treatment impairs the HCO3- influx mechanism but not the OH- efflux mechanism.

A New Turgor/Membrane Potential Probe Simultaneously Measures Turgor and Electrical Membrane Potential

Abstract: A new combined turgor/membrane potential probe (T‐EP probe) monitored cell turgor and membrane potential simultaneously in single giant cells. The new probe consisted of a silicone oil‐filled micropipette (oil‐microelectrode), which conducted electric current. Measurements of turgor and hydraulic conductivity were performed as with the conventional cell pressure probe besides the membrane potential. In internodal cells of Chara corallina, steady state turgor (0.5‐0.7 MPa) and resting potentials (‐200 to −220 mV) in APW, and hydraulic conductivity (0.07 to 0.21 × 10∼5 m s−1 MPa−1) were measured with the new probe, and cells exhibited healthy cytoplasmic streaming for at least 24 h during measurements. When internodal cells of Chara corallina were treated with 30, 20, 10, and 5 mM KCI, turgor responded immediately to all concentrations, and the osmotic changes in the medium were measured. Action potentials, which brought the membrane potential to a steady depolarization that measured the concentration difference of K+ in the medium, were induced in a concentration — dependent delay and occurred only 30, 20, and 10 mM of KCl. When the solution was changed back to APW, the repolarization of membrane potential consisted of a quick and a following slow phase. During the quick phase, which took place immediately and lasted 1 to 3 min, the plasma membrane remained activated. The membrane was gradually deactivated in the slow phase, and entirely deactivated when the membrane potential recovered to the resting potential in APW. Although the activated plasma membrane was permeable to K+, no major ion channels were activated on the tonoplast, and therefore, internodal cells of Chara corallina did not regulate turgor when osmotic potential changed in the surrounding medium.

Examination of the contribution of vacuolar proteases to intracellular protein degradation in Chara corallina.

The contribution of proteases in the central vacuole of Chara corallina internodal cells to overall cellular protein degradation was examined. I measured the decrease in the trichloroacetic acid (TCA)-precipitable radioactivity in the cell for a 6-d chase period after labeling cellular proteins with [3H]leucine. The kinetics of [3H]leucine-labeled protein disappearance showed that the half-life of the cellular soluble proteins was 4 to 5 d. This value did not change when cells were treated with (2S,3S)-trans-epoxysuccinyl-L-leucylamido- 3-methyl-butane ethyl ester, a permeant inhibitor of cysteine proteases. This inhibitor mostly inhibited bovine serum albumin-degrading activity in the vacuole. I also measured the release of TCA-soluble radioactivity from the TCA-insoluble fraction in the cell. This experiment showed that 13% of [3H]leucine-labeled cellular proteins were degraded in 1 d. This value agreed well with the half-life obtained for soluble proteins in the above experiment. This value did not change even when both trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, a cysteine protease inhibitor, and pepstatin A, an aspartic protease inhibitor, were introduced into the vacuole. With this operation, bovine serum albumin-degrading activity in the vacuole was almost completely inhibited. These data suggest that the cytoplasmic but not the vacuolar proteases contribute to cellular protein turnover in Chara internodal cells.

Reversible closing of water channels inCharainternodes provides evidence for a composite transport model of the plasma membrane

Treatment of internodal cells of Chara corallina with the water channel blocker HgCl 2 caused a decrease of the hydraulic conductivity of the membrane (Lp) by a factor of three to four. In the presence of (practically) non-permeating solutes such as sugars or salts, the osmotic responses were similar to those found in controls, i.e. reflection coefficients ( s ) were close to unity. However, when treating the internodes with osmotic solutions of rapidly permeating lipophilic substances such as low molecular weight alcohols or acetone, the pirture of biphasic pressure relaxations due to the exchange of both water and solutes changed considerably. In the presence of HgCl 2 , reflection coefficients were substantially reduced and even became negative for some solutes (anomalous osmosis). Different from reflection coefficients, permeability coefficients (P s ) remained constant upon treatment. When using heavy water (HDO) as a small hydrophilic solute which should cross the membrane largely via water channels, results were different: the reflection coefficient of HDO increased and permeability decreased. Treating the cells with 5 mM 2-mercaptoethanol to remove the mercury from transport proteins (water channels) reversed changes in Lp and s . The results disagree with conventional membrane models, which imply a homogeneous membrane. They are readily explained by a composite model of the membrane in which proteinaceous arrays with specific water channels are arranged in parallel with lipid arrays. The latter account for most of the permeability of the small organic test solutes used and are not affected by the channel blocker. From the data, detailed information is obtained about transport properties of the arrays (water channels).