Characean Cells Research Articles

Characean cells as a tool for studying electrophysiological characteristics of plant cells.

Characean cells have contributed significantly to various areas of plant cell biology such as cell motility and membrane transport. Since characean cells are very large, various kinds of operations can easily be applied to them. Development of techniques of intracellular perfusion and permeabilization of plasma membrane has facilitated studies on functions of the plasma membrane and the vacuolar membrane (or tonoplast) which is specific to plant cells. The present article is aimed at reviewing the contribution of characean cells to the study of electrophysiological characteristics of plant membranes. Our attention was mainly focused on experiments using plasma membrane-permeabilized cells and intracellularly perfused cells.

特集 植物細胞膜 植物細胞のイオンチャンネル

Although ionic and metabolic gradients across membranes in living cells are known to be vital to cell functioning, in plants both the stiffness of cell wall and complexity of the cell compartmentalization sometimes prevent the study of membrane transport. This difficulty has, however, largely been overcome with the aid of two techniques, one employing a vibrating electrode1) and the other a patch clamp (Hamill et al., Pflugers Arch., 391, 85 (1981)). The former technique has revealed the ionic currents which flow locally around various type of cells. In the introduction to the present article, the relation of local currents with several physiological phenomena, such as pollination and cell polarity, is reviewed. The patch-clamp technique has directly led to the development of research strategies, not only on the plasma membrane, but also on the vacuolar membrane and other intracellular organelles. In fact, various types of ion channels, such as voltage-dependent, Ca2+-regulated, and phytohormone-modulated channels, have been found in plant cells. In the latter half of this article, the properties of several ion channels in the plasma and vacuolar membranes of characean cells which were identified using the patch clamp are reviewed, and their physiological functions are summarized.

Calcium -regulated channels and their bearing on physiological activities in Characean cells

Calcium is involved in the regulation of cytoplasmic streaming, membrane excitation, turgor regulation and salt tolerance in the giant internodal cells of the Characeae. To analyse the mechanism of Ca 2+ action, model systems were used, namely, tonoplast-free and plasma m em brane-perm eabilized cells. In the former, the plasma membrane remained intact and its activity could be investigated by m anipulating the cytoplasmic and external media, whereas in the latter, the tonoplast remained intact and its activity could be studied by altering the bathing solution. Studies using these model systems have established the presence of voltage-dependent Ca 2+ -channels in the plasma membrane and Ca 2+ - dependent ion channels in both the plasma membrane and the tonoplast. To further analyse Ca 2+ action on the basis of single channel activities, patch-clam p techniques were applied to plasmolysed protoplasts and isolated cytoplasmic drops. Channel activities were measured using both cell-attached and excised membrane patch modes. A fresh-water member of the Characeae, Nitellopsis , becomes salt-tolerant if millimolar amounts of Ca 2+ are present in the external medium. Under these conditions, excised patches of the plasma membrane exhibit K + -channel activity with unitary conductances of 25-50 pS and a permeability ratio ( P Na / P K ) of 0.28. These K + channels were closed by external Ca 2+ when ATP was present on the cytoplasmic side of the membrane. ATP could be replaced with AMP, which suggests that ATP acted neither as an energy source nor as a substrate for protein phosphorylation, but rather as an effector. In the tonoplast, K + channels having a unitary conductance of 75 pS and a P Na / P K ratio of 0.2 were not activated by Ca 2+ when it was present on the cytoplasmic side of the excised patches, but these channels were activated by Ca 2+ injected into the cytoplasmic drop, which suggested the involvement of an unknown cytoplasmic factor(s) that mediates the Ca 2+ signal. The brackish water Characeae, Lamprothamnium , can regulate elevated turgor induced by hypotonic treatment only when millimolar amounts of Ca 2+ are present in the external medium. In this situation, elevated turgor may first activate Ca 2+ channels and the increased level of Ca 2+ in the cytoplasm may then activate K + and Cl - channels in both the plasma membrane and the tonoplast. In the cytoplasmic-drop-attached mode, single K + channel current-voltage measurements established that the K + channel exhibited a unitary conductance of 50 pS for negative shifts of the voltage, while under positive shifts in the voltage 100 pS channel conductance was observed. The channel with a P Na / P K of 0.02 is highly selective for K + against Na + and this channel is directly activated by Ca 2+ added to the cytoplasmic side of the excised patch. These results suggest that, in Nitellopsis and Lamprothamnium , Ca 2+ regulation of channels in both the plasma membrane and the tonoplast may form the molecular basis for Ca 2+ -regulated physiological functions such as salt tolerance and turgor regulation in characean cells. The mode of Ca 2+ regulation is discussed in light of current findings.

Light dependence of protoplasmic streaming in Nitella flexilis L. as measured by means of laser-velocimetry

Laser-velocimetry was applied in order to study the effect of light on the velocity of protoplasmic streaming (pps) in Characean cells. A change from dark to light (= 6 W · m(-2)) leads to an acceleration of streaming by about 15-30% with a time-constant of approx. 300 s. The transition from light to dark causes a transient decrease of velocity below the original dark level. This response occurs with a time constant of about 500 s. It returns to its initial value with a time-constant of about 2000 s. This may indicate that a control loop of cytosolic homeostasis takes a decrease in pCa more seriously than an increase. A possible involvement of temperature effects caused by illumination was excluded by measuring the influence of temperature. Steady-state velocity of streaming changed by 5% per 1° C. Irradiation with infra-red light (λ > 780 nm) did not cause a change in velocity. The absence of a light effect on streaming velocity in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) shows that photosynthesis and not phytochrome is involved. The role of light-induced changes of pCa is discussed, especially with respect to the hypothesis of Vanselow and Hansen (1989, J. Membr. Biol. 110, 175-187) that photosynthesis acts on the plasmalemma K(+)-channel via light-induced uptake of Ca(2+) into the chloroplasts.

Hydrostatic pressure mimics gravitational pressure in characean cells.

Hydrostatic pressure applied to one end of a horizontal Chara cell induces a polarity of cytoplasmic streaming, thus mimicking the effect of gravity. A positive hydrostatic pressure induces a more rapid streaming away from the applied pressure and a slower streaming toward the applied pressure. In contrast, a negative pressure induces a more rapid streaming toward and a slower streaming away from the applied pressure. Both the hydrostatic pressure-induced and gravity-induced polarity of cytoplasmic streaming respond identically to cell ligation, UV microbeam irradiation, external Ca2+ concentrations, osmotic pressure, neutral red, TEA Cl-, and the Ca2+ channel blockers nifedipine and LaCl3. In addition, hydrostatic pressure applied to the bottom of a vertically-oriented cell can abolish and even reverse the gravity-induced polarity of cytoplasmic streaming. These data indicate that both gravity and hydrostatic pressure act at the same point of the signal transduction chain leading to the induction of a polarity of cytoplasmic streaming and support the hypothesis that characean cells respond to gravity by sensing a gravity-induced pressure differential between the cell ends.

Formation of band-type electric patterns in Characean cells

It is observed by experiments that band patterns of alternating acid and alkaline zones are formed on the surface of Characean cells under illumination. In order to understand theoretically such pattern formation, we employ a reaction-diffusion equation model with activator-inhibitor interaction. We study the existence problem of band patterns and hysteresis phenomena such as patterns appearing and disappearing with changing light intensity by using singular perturbation methods.

The contribution of the extracellular matrix to gravisensing in characean cells.

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

Wall appositions induced by ionophore A 23187, CaCl2, LaCl3, and nifedipine in characean cells

The formation of wall appositions (plugs) by ionophore A 23187, CaCl2, LaCl3, and nifedipine was studied in mature internodal cells of characeaen algae. CaCl2 at concentrations above 10−2M induces thick fibrillar plugs without callose inNitella flexilis. InChara corallina andNitella flexilis ionophore A 23187 (1.25×10−5 to 5×10−5M) and LaCl3 (7.5×10−5 to 2.5×10−4M) cause flat appositions which contain callose and have a more granular structure. Plug formation by ionophore A 23187, CaCl2, and LaCl3 is pH-dependent and occurs beneath the alkaline regions of the cell. Nifedipine (10−4 to 10−5M) induces plugs inNitella flexilis after previous injury. These callose-containing wall appositions consist of a heterogeneous granular core which is covered by a fibrillar layer. The results of this work are compared with previous studies on wound wall formation and chlortetracycline (CTC)-induced plug formation which reveal that abundant coated vesicles occur only when a thick fibrillar wall layer is formed. Neither LaCl3 nor nifedipine inhibit the formation of CaCl2- or CTC-plugs. The unusual effects of these substances, which normally act as Ca2+ antagonists and therefore should prevent and not induce plug formation, are discussed. It is suggested that La3+ mimicks the effects of calcium and that nifedipine binding to the Ca2+ channels is altered in the alkaline regions of characean internodes and allows an influx of Ca2+.

Characterization of the translocator associated with pollen tube organelles

In pollen tubes, the motive force of cytoplasmic streaming is assumed to be generated by the sliding of the translocator associated with cell organelles along actin filaments. In the present study, the characteristics of the translocator were studied by reconstituting the movement of pollen tube organelles along characean actin bundles. Movement of pollen tube organelles proceeded from the pointed end to the barbed end of the actin filaments of the characean cells. The reconstituted movement was not inhibited by vanadate. KCL at higher concentrations inhibited the movement. Furthermore, heavy meromyosin (HMM) prepared from rabbit skeletal muscle myosin partially inhibited the reconstituted movement and pCMB-modified HMM inhibited it completely. The present results strongly support our previous conclusion that the translocator which generates the motive force of cytoplasmic streaming in pollen tube is myosin.

Inhibition of cytoplasmic streaming by sulfate in characean cells

To characterize the cytoplasmic streaming in characean cells, tonoplast-free cells were prepared and the chemical composition of the cytoplasm was modified by intracellular perfusion. Perfusion media containing sulfate inhibited the cytoplasmic streaming. The extent of inhibition was dependent on the concentration of ATP: the lower the concentration of ATP, the stronger the inhibition. It was suggested that inhibition by sulfate is competitive with respect to ATP. The effects of other anions were also examined.

Actomyosin and movement in the angiosperm pollen tube: an interpretation of some recent results

Recent confirmations of the presence of myosin in angiosperm pollen tubes indicate that an energy-transducing actomyosin system is involved in the motility system of the vegetative cells. Myosin has been localised by immunofluorescence on the surfaces of vegetative nuclei and generative cells. It has been shown to be associated with individual amyloplasts in grass pollen, and there are indications that it is present on other particulate bodies in the cytoplasm. The organelles in the leading part of the tube move along separate traffic lanes of acropetal and basipetal polarity, known from electron microscopy and phalloidin labelling to contain numbers of fibrils containing aggregates of actin microfilaments; in older segments the movement can be related to single, uniformly polarised, fibrils. Circulatory flow is maintained at the proximal end by the looping of the fibrils in the grain or at callose plugs. Such loops do not occur at the apex, where entering organelles undergo random movement before becoming associated with basipetal streams. Vegetative nuclei and generative cells interact with several fibrils, and it is suggested that they are held in the leading part of the protoplast in unstable equilibrium between acropetal and basipetal forces. Constantly changing form, especially of the vegetative nucleus, is one consequence of these varying stresses. Possible analogies with the intracellular motility system of the giant cells of the Characeae are noted, and it is suggested that lipid globuli and other nonorganellar bodies may be transported in the pollen tube by association with myosin-bearing membranes similar to those involved in endoplasm movement in the characean cells.

Bicarbonate utilization: Function and mechanism

A review is given of the occurrence, ecological significance and physiological mechanism of bicarbonate utilization by aquatic photosynthetic organisms. Following a short description of the carbon dioxide water system, the significance of the unstirred layer and the slow diffusion rate of CO 2 in water is discussed. Three experimental procedures are discussed to establish the use of bicarbonate: establishing the relationship between pH and rate of carbon assimilation; the kinetic-reaction approach, which uses the slow equilibration between CO 2 and HCO 3 − in water; the difference in discrimination between 12C and 13C during CO 2 and HCO 3 − assimilation. The ability to use HCO 3 − occurs in many different groups of aquatic photosynthetic organisms, with the notable exception of Bryophytes and Pteridophytes, and is related to growth conditions. Polarity, that is the spatial separation of HCO 3 − influx and resulting OH − efflux, is observed in higher plant species and Characeae that assimilate HCO 3 −. These polar leaves or banded Characean cells are often used to study the mechanism. Three mechanisms for HCO 3 − assimilation apparently exist: H +HCO 3 − symport, external acidification of HCO 3 − into CO 2 and an increased rate of conversion of HCO 3 − into CO 2 by the action of the enzyme carbonic anhydrase. The role of transfer cells or plasmalemmasomes, often observed in HCO 3 − assimilating leaves, is still unclear. Their role may be to create sufficient space for membrane-bound enzyme systems by increasing the membrane surface area or to confine an extracellular space with a low pH value. The ecological significance of photosynthetic HCO 3 − utilization seems to be that it compensates for slow supply of CO 2 by diffusion and/or suppresses photorespiration by creating a high intracellular CO 2 concentration.

Oscillations of electric spatial patterns emerging from the homogeneous state in characean cells

Electric spatial patterns of bands formed along the cell wall of the characean internode were studied using a multi-electrode measuring system. The electric potential near the surface of the cell was measured by arranging about 25 electrodes along the cell at approximately 1.6 mm intervals. Since the time required for one scan over the cell length is only 1 s, the temporal change in the spatial pattern of surface electric potential can be readily observed. Oscillations were sometimes found as the electric pattern started to appear after the cell was illuminated. Fourier analysis shows that a single spatial mode arises gradually and then becomes stabilized in an oscillatory manner. A simple electric circuit model comprising three variables, i.e., a membrane potential, an electric current across the membrane and an electromotive force, can simulate well the oscillatory rise of bands. These results imply that the electric spatial pattern observed in characean internodes is a self-organized structure emerging far from equilibrium, known as a dissipative structure. Biophysical mechanisms of band formation are discussed.

Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+.

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.

Theory of electric dissipative structure in Characean internode

A band-type alternating pattern of acidic and alkaline regions formed along the Characean cell wall is discussed theoretically. The model system is constructed from linear diffusion equations for the concentration of H + outside the internode and in the protoplasm. The plasmalemma is taken as a boundary transporting H + under energy supply by light. The sizes of the protoplasm and extracellular water phase are taken into account explicitly in the present model system to reproduce qualitatively the characteristics observed in various types of experiments. Theoretical analysis shows that the band pattern belongs to dissipative structures emerging far from equilibrium, and is stabilized through the electric current loops produced by locally activated electrogenic H + pumps and spatially separated passive H + influx (or OH − efflux) across the membrane. Both the numerical calculation and the theoretical analysis using a generalized time-dependent Ginzburg-Landau equation reveal the following points: (i) the intemodal cell with a larger vacuole in a smaller size of the extracellular water phase tends to exhibit a clearer band pattern; (ii) the increase in viscosity of the external aqueous medium makes the bands appear more easily and, furthermore, distinctly; (iii) the change in size of the extracellular water phase significantly affects the kinetics of the pattern- formation process. These results are interpreted reasonably by taking account of the electric current circulating between the acidic and alkaline regions.

Phosphorylation-dephosphorylation is involved in Ca2+-controlled cytoplasmic streaming of characean cells

The mechanism of Ca2+ regulation of the cytoplasmic streaming in characean cells was studied in relation to protein phosphorylation and dephosphorylation. A tonoplast-free cell model was developed which was sensitive to Ca2+. Protein phosphatase-1 and its inhibitor-1 were applied into the tonoplast-free cells. A synthetic inhibitor of protein phosphatase, α-naphthylphosphate, was applied either to tonoplast-free cells from inside or to the outside of plasmalemma-permeabilized cells which are known to be very sensitive to Ca2+. ATP-γ-S applied to permeabilized cells strongly inhibited the recovery of the streaming which had been stopped by 10 μ M Ca2+. Both inhibitor-l and α-naphthylphosphate inhibited the streaming even in the absence of Ca2+. On the other hand, protein phosphatase-l recovered the streaming even in the presence of Ca2+.

Cytoplasmic streaming in giant algal cells: A historical survey of experimental approaches

Various methods have been used to study cytoplasmic streaming in giant algal cells during the past three decades. Simple techniques can be used with characean internodal cells to modify the cell constitution in various ways to gain insight into the mechanism of cytoplasmic streaming. Another method involves isolatingin vitro a huge drop of uninjured endoplasm, to examine its physical and dynamic properties. The motive force responsible for streaming has been measured by three different techniques with similar results. Subcortical fibrils consisting of bundles of F-actin with the same polarity are indispensable for streaming. Differential treatment of the endoplasm and ectoplasm has shown that putative characean myosin is localized in the endoplasm. Studies of the roles of ATP, Mg2+, Ca2+, H+ etc. in the streaming have been conducted by cellular perfusion, which allows removal of the tonoplast, or by techniques permeabilizing the protoplasmic membrane. A slow version of the movement can even be artificially reproduced by combining characean actinin situ and exogenous myosin in the presence of Mg-ATP. The findings thus far obtained support the hypothesis that cytoplasmic streaming in characean cells is caused by an active shearing force produced by interaction of the actin filament bundles on the cortex with myosin in the endoplasm.

Growth and regrowth of actin bundles in Chara: bundle assembly by mechanisms differing in sensitivity to cytochalasin.

Cytochalasin is known to inhibit cytoplasmic streaming rapidly in characean cells without disassembling their actin bundles. Lower cytochalasin concentrations than those needed for streaming inhibition are now shown to disrupt bundle assembly and, over longer periods, assembled bundles. After local wounding, cytochalasin limited bundle regeneration to the production of polygons and straight, discontinuous bundles that rarely connected to bundles outside the wound. The regenerated bundles supported only scattered organelle movements, whereas long, oriented bundles of control cells were connected to those outside the wound and supported bulk endoplasmic streaming. Unwounded Chara plants cultured for up to 2 weeks in 1 microM-cytochalasin maintained normal bundle orientation and rapid cytoplasmic streaming, but the mean number of bundles per file of chloroplasts fell from 5.2 in controls to 2.0 in growing cells and 3.4 in nongrowing cells. These structural effects seem more likely than the streaming inhibition to reflect cytochalasin's in vitro effect of blocking extension at the barbed but not the pointed end of F-actin. In particular, cytochalasin inhibited the extension into the wound of bundles in which only the barbed ends of filaments would be exposed. However, short lengths of isolated bundles grew within the wound and bundle growth in the intact cell continued, albeit in modified form. It is suggested that these examples of continuing bundle growth involve cytochalasin-resistant mechanisms that are not wholly dependent on barbed-end filament growth.

Light-Induced Membrane Hyperpolarization and Adenine Nucleotide Levels in Perfused Characean Cells

Light-Induced Membrane Hyperpolarization and Adenine Nucleotide Levels in Perfused Characean Cells

Extremely Low Frequency Resistance Fluctuations of Chara Membrane

The membrane resistivity of resting characean cells was measured and found to fluctuate unpredictably about its resting level. The amplitude of these fluctuations relative to the mean resistivity was normally somewhat larger than the same ratio for the vacuolar voltage; further the resistivity and voltage fluctuations seemed not to be strongly correlated.