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The hydraulic resistance (the reciprocal of the hydraulic conductivity Lp) Lp -1 was measured in cells of Chara corallina by the method of transcellular osmosis. Treatment of cells with 100 mM KCl decreased Lp -1 significantly. Subsequent treatment of the cells with 70 mM CaCl 2 recovered the decreased Lp -1 to the original value. To know whether K + or Ca 2+ /Mg 2+ acts on the cell wall and/or the membrane, the hydraulic resistances of the cell wall (Lp w -1 ) and that of the membrane (Lp m -1 ) were determined in one and the same cell. For this, a pair of cells (twin cells) were made from an internodal cell, one used for measurement of Lp -1 and the other used for the measurement of Lp w -1 . From Lp -1 and Lp w -1 , Lp m -1 was calculated. Both Lp -1 and Lp w -1 were decreased by K + , while Lp m -1 was not affected by K + . The same result was obtained with 5 mM EGTA. Lp w -1 was decreased more than it was by KCl but Lp m -1 remained constant after EGTA treatment. The recovery of the K + -decreased Lp -1 with Ca 2+ can be explained exclusively by the recovery of Lp w -1 with Ca 2+ . The Ca 2+ recovery of Lp w -1 was observed in the intact cell wall but not in the cell wall tube isolated from an internodal cell. The different response to Ca 2+ between the intact cell wall and the isolated cell wall was discussed in relation to the tension in the cell wall which may be an important factor for the ionic regulation of hydraulic conductivity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Symplastic interconnections of plant cells via perforations in adjoining cell walls (plasmodesmata) enable long-distance transport of photoassimilates and signaling substances required for growth and development. The pathways and features of intercellular movement of assimilates are often examined with fluorescent tracers whose molecular dimensions are similar to natural metabolites produced in photosynthesis. Chlorophyll fluorescence was recently found to be a sensitive noninvasive indicator of long-distance intracellular transport of physiologically produced photometabolites in characean internodes. The present work shows that the chlorophyll microfluorometry has a potential for studying the cell-to-cell transport of reducing substances released by local illumination of one internode and detected as the fluorescence increase in the neighbor internode. The method provides temporal resolution in the time frame of seconds and can be used to evaluate permeability of plasmodesmata to natural components released by illuminated chloroplasts. The results show that approximately one third of the amount of photometabolites released into the streaming cytoplasm during a 30-s pulse of local light permeates across the nodal complex with the characteristic time of ~ 10 s. The intercellular transport was highly sensitive to moderate elevations of osmolarity in the bath solution (150 mM sorbitol), which contrasts to the view that only transnodal gradients in osmolarity (and internal hydrostatic pressure) have an appreciable influence on plasmodesmal conductance. The inhibition of cell-to-cell transport was reversible and specific; the sorbitol addition had no influence on photosynthetic electron transport and the velocity of cytoplasmic streaming. The conductance of transcellular pores increased in the presence of the actin inhibitor cytochalasin D but the cell-to-cell transport was eventually suppressed due to the deceleration and cessation of cytoplasmic streaming. The results show that the permeability of plasmodesmata to low-molecular photometabolites is subject to upregulation and downregulation.

The restoration capability of charophyte Chara braunii was studied in arsenic-polluted water in the context of biogenic calcium and manganese depositions on the plant. In addition to calcite encrustation, formation of craterlike shape deposits of manganese oxides (MnO x ) with diameters of 5-10 μm was detected on the cell walls of the plants grown in Mn-rich media. Relative proportions of arsenic taken up by the plant biomass to those incorporated into the calcium and manganese biominerals were determined using a modified sequential chemical extraction method. The mean total arsenic recovery from water reached its highest value at 375 mg kg -1 in treatment with HCO 3 - and high concentrations of Ca and Mn (40 and 2 mg L -1 , respectively). The percentage of arsenic associated with the manganese deposits in the plants exposed to 0.5 mg L -1 As(III) increased from 16.3% to 51.7% of the total arsenic accumulation at low and high Mn levels (<0.05 and 2 mg L -1 , respectively), that accounted for the highest Mn-bound arsenic contribution. Surface oxidation of As(III) by MnO x and subsequent precipitation-adsorption of the formed As(V) onto the evolving structure of MnO x could be a plausible mechanism for arsenic removal. The presence, and in some cases dominance of arsenic bound to the biogenic Ca and Mn deposits on the studied aquatic plant may contribute to preservation of arsenic in sediments in a less bioavailable form upon its senescence and decomposition., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The role of macroalgal allelopathy in aquatic systems has received increasing attention as a potential means of controlling cyanobacterial blooms. However, the allelopathic activity of Chara sp. on coexisting and bloom-forming picocyanobacteria is still largely unknown. Therefore, the laboratory experiments were conducted to investigate the allelopathic activity of extracts of Chara aspera, C. baltica, and C. canescens on the growth, the fluorescence parameters: maximum and effective quantum yield of photosystem II (PSII) photochemistry (F v /F m and ΦPSII, respectively) and photosynthesis parameters such as the initial slope of photosynthesis-irradiance (P-E) curves (alpha) and photosynthetic capacity (P m ) of the picocyanobacterium Synechococcus sp. Batch cultures of picocyanobacterium were exposed to three concentrations of extracts originating from three charophyte cultures and the effect was followed at three sampling times. Dried specimens of C. aspera, C. baltica, and C. canescens were extracted in the water-based matrix and the initial Synechococcus sp. inoculum, derived from unialgal culture media, was used. We found both negative and positive allelopathic effects of all tested Chara extracts on Synechococcus sp. The strongest adverse impact of picocyanobacterium growth was caused by C. baltica. This study clearly demonstrated that the allelopathic effect depends on the Chara species identity. Our results also suggested that some allelopathic Chara sp. have the potential to mitigate harmful cyanobacterial blooms in systems dominated by Synechococcus sp.

Two basic ecological relationships, herbivory and competition, distinctively influence terrestrial ecosystem characteristics, such as plant cover, species richness and species composition. We conducted a cage experiment under natural conditions in an aquatic ecosystem to test the impacts of two treatments combined in a factorial manner: (i) a pulse treatment - removal of dominant competitors among primary producers (macroalgae Chara sp. and Vaucheria sp.), and (ii) a press treatment - preventing herbivore (fish, crayfish) access to caged plots. The plots were sampled once before the treatments were established and four more times within two years. Both treatments had a significantly positive impact on macrophyte cover and species richness and changed the macrophyte species composition. The effect of the macroalgae removal was immediate with the highest species richness occurrence during the first post-treatment monitoring, but the positive effect vanished with time. In contrast, preventing herbivore access had a gradual but long-lasting effect and reached a more steady-state over time. Two of the most common species showed contrasting responses, the palatable Potamogeton pectinatus was most supported by caging, while the distasteful Myriophyllum spicatum preferred open plots. Our findings may be applicable during the revitalisation of aquatic ecosystems that aims to increase macrophyte biodiversity.

To understand salt stress, the full impact of salinity on plant cell physiology has to be resolved. Electrical measurements suggest that salinity inhibits the proton pump and opens putative H + /OH - channels all over the cell surface of salt sensitive Chara australis (Beilby and Al Khazaaly 2009; Al Khazaaly and Beilby 2012). The channels open transiently at first, causing a characteristic noise in membrane potential difference (PD), and after longer exposure remain open with a typical current-voltage (I/V) profile, both abolished by the addition of 1 mM ZnCl 2 , the main known blocker of animal H + channels. The cells were imaged with confocal microscopy, using fluorescein isothiocyanate (FITC) coupled to dextran 70 to illuminate the pH changes outside the cell wall in artificial fresh water (AFW) and in saline medium. In the early saline exposure, we observed alkaline patches (bright fluorescent spots) appearing transiently in random spatial distribution. After longer exposure, some of the spots became fixed in space. Saline also abolished or diminished the pH banding pattern observed in the untreated control cells. ZnCl 2 suppressed the alkaline spot formation in saline and the pH banding pattern in AFW. The osmotic component of the saline stress did not produce transient bright spots or affect banding. The displacement of H + from the cell wall charges, the H + /OH - channel conductance/density, and self-organization are discussed. No homologies to animal H + channels were found. Salinity activation of the H + /OH - channels might contribute to saline response in roots of land plants and leaves of aquatic angiosperms.

Background: It is a common incident in nature, that two waves or pulses run into each other head-on. The outcome of such an event is of special interest, because it allows conclusions about the underlying physical nature of the pulses. The present experimental study dealt with the head-on meeting of two action potentials (AP) in a single excitable plant cell (Chara braunii internode)., Methods: The membrane potential was monitored with multiple sensors along a single excitable cell. In control experiments, an AP was excited electrically at either end of the cell cylinder. Subsequently, stimuli were applied simultaneously at both ends of the cell in order to generate two APs that met each other head-on., Results: When two action potentials propagated into each other, the pulses did not penetrate but annihilated (N=26 experiments in n=10 cells)., Conclusions: APs in excitable plant cells did not penetrate upon meeting head-on. In the classical electrical model, this behavior is specifically attributed to relaxation of ion channel proteins. From an acoustic point of view, annihilation can be viewed as a result of nonlinear material properties (e.g. a phase change)., General Significance: The present results suggest that APs in excitable animal and plant cells belong to a similar class of nonlinear phenomena. Intriguingly, other excitation waves in biology (intracellular waves, cortical spreading depression, etc.) also annihilate upon collision and are thus expected to follow the same underlying principles as the observed action potentials., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The month-to-month variability of biomass and CaCO 3 precipitation by dense charophyte beds was studied in a shallow Chara-lake at two depths, 1 and 3 m. Charophyte dry weights (d.w.), the percentage contribution of calcium carbonate to the dry weight and the precipitation of CaCO 3 per 1 m 2 were analysed from May to October 2011. Physical-chemical parameters of water were also measured for the same sample locations. The mean dry weight and calcium carbonate precipitation were significantly higher at 1 m than at 3 m. The highest measured charophyte dry weight (exceeding 2000 g m -2 ) was noted at 1 m depth in September, and the highest CaCO 3 content in the d.w. (exceeding 80 % of d.w.) was observed at 3 m depth in August. The highest CaCO 3 precipitation per 1 m 2 exceeded 1695 g at 1 m depth in August. Significant differences in photosynthetically active radiation (PAR) were found between 1 and 3 m depths; there were no significant differences between depths for other water properties. At both sampling depths, there were distinct correlations between the d.w., CaCO 3 content and precipitation and water properties. In addition to PAR, the water temperature and magnesium and calcium ion concentrations were among the most significant determinants of CaCO 3 content and d.w. The results show that light availability seems to be the major factor in determining charophyte biomass in a typical, undisturbed Chara-lake. The study results are discussed in light of the role of charophyte vegetation in whole ecosystem functioning, with a particular focus on sedimentary processes and the biogeochemical cycle within the littoral zone.

The plant hormone auxin has been widely studied with regard to synthesis, transport, signaling and functions among the land plants while there is still a lack of knowledge about the possible role for auxin regulation mechanisms in algae with "plant-like" structures. Here we use the alga Chara corallina as a model to study aspects of auxin signaling. In this respect we measured auxin on membrane potential changes and different ion fluxes (K(+), H(+)) through the plasma membrane. Results showed that auxin, mainly IAA, could hyperpolarize the membrane potential of C. corallina internodal cells. Ion flux measurements showed that the auxin-induced membrane potential change may be based on the change of K(+) permeability and/or channel activity rather than through the activation of proton pumps as known in land plants., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Histone acetylation is one of the epigenetic modifications which play a significant role in chromatin remodeling during spermiogenesis. Acetylation of the histone H4 makes the exchange of nucleoproteins easy. Research on mouse spermatogenesis showed that H4 histone acetylated at Lys 12 (H4K12ac) was specific only to spermatids. Immunocytochemical studies of Chara vulgaris spermatids with the use of antibodies against the histone H4K12ac revealed positive reactions in spermatid nuclei at stages I-VII. This reaction, connected with nuclear condensation, was much stronger at the early stages of spermiogenesis than later on. Moreover, it showed that at the stages V-VII in spermatid nuclei the presence of the histone H4K12ac corresponded with DNA double-strand breaks. Electron microscopy studies with the use of immunogold technique revealed an almost twofold difference between the mean total numbers of gold grains in the examined chromatin in both stages. This study showed nearly equal distribution of gold grains on condensed and non-condensed chromatin of spermatids at the stage III/IV (48.11% and 51.89%, respectively). In the later stage-VI, when chromatin condensation proceeded, labeling of condensed chromatin reached 57.27%, while in the case of non-condensed chromatin it dropped to 42.73%. The percentage analysis also revealed an increase (above 9%) in condensed chromatin labeling in relation to the stage III/IV. Intensive acetylation of histone H4 at the early stages is correlated with DNA DSBs and transcriptional activity. It facilitates chromatin loosening, which enables the correct course of chromatin remodeling at a later stage. Histone γH2AX also influences chromatin structure in many biological processes in different cell types. Current studies reveal other similarities regarding histone H4 acetylation, not only between Chara and mammals but between invertebrates (molluscs) and vertebrates (bony fishes) as well., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Microperforation of characean cell wall with a glass micropipette in the absence of the tonoplast impalement was found to cause rapid alkalinization of the apoplast by 2-3 pH units, which may rigidify the cell wall structure, thus protecting the cell from further injury. A similar but a deeper insertion of a microneedle, associated with piercing the tonoplast and with an action potential generation, led to a considerable delay in the apoplast alkalinization without affecting the amplitude of the eventual increase in pH. The retardation by the mechanically elicited action potential of the incision-mediated pH transients in the apoplast contrasted sharply to the enhancement of these pH transients by the action potential triggered electrically before the microperforation. Hence, the delay of the apoplast alkalinization was not related to basic ionic mechanisms of plant action potentials. Measurements of the vacuolar pH after mechanical elicitation of an action potential indicate that the tonoplast piercing was accompanied by leakage of protons from the vacuole into the cytoplasm, which may strongly acidify the cytoplasm around the wounded area, thus collapsing the driving force for H(+) influx from the medium into the cytoplasm. The lag period preceding the onset of external alkalinization was found linearly related to the duration of temporal cessation of cytoplasmic streaming. The results suggest that the delayed alkalinization of the apoplast in the region of tonoplast wounding reflects the localized recovery of the proton motive force across the plasmalemma during replacement of the acidic cytoplasm with fresh portions of unimpaired cytoplasm upon restoration of cytoplasmic streaming.

DNA topoisomerase II plays an essential role in animal spermiogenesis, where changes of chromatin structure are connected with appearance of transient DNA breaks. Such topo II activity can be curtailed by inhibitors such as etoposide and suramine. The aim of the present study was to investigate, for the first time, the effect of etoposide on spermatid chromatin remodeling in the green alga Chara vulgaris. This inhibitor prolonged the early spermiogenesis stages and blocked the formation of the phosphorylated form of histone H2AX at stages VI-VII. The lack of transient DSBs at these stages impairs the elimination of supercoils containing nucleosomes which lead to disturbances in nucleoprotein exchange and the pattern of spermatid chromatin fibrils at stages VI-VIII. Immunofluorescent and ultrastructural observations revealed that during C. vulgaris spermiogenesis topo II played an important role similar to that in mammals. Some corresponding features had been pointed out before, the present studies showed further similarities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The RAB5 GTPase ARA6 (AtARA6) of Arabidopsis thaliana is known to be involved in endosomal trafficking by targeting vesicles to the plasma membrane. During this process AtARA6 is working in close relationship with the SNARE protein VAMP727 (vesicle associated membrane protein 727). Recently, ARA6 of the characean green algae Chara australis (CaARA6) was shown to have properties similar to AtARA6, pointing to similar trafficking pathways. In order to gain further insight into the vesicle trafficking machinery of characeae, C. australis was analyzed for homologous proteins of the VAMP72-family. A CaVAMP72 protein was detected and classified by protein sequence alignment and phylogenetic analyses.

Increased ammonium (NH4-N) concentrations in water bodies have been reported to adversely affect the dominant species of submersed vegetation in meso-eutrophic waters worldwide. However calcareous plants were lowly sensitive to NH4-N toxicity. In order to make clear the function of calcification in the tolerance of calcareous plants to NH4-N stress, we studied the effects of increased HCO3(-) and additional NH4-N on calcification and utilization of dissolve inorganic carbon (DIC) in Chara vulgaris Linn in a 7-d sub-acute experiment (light:dark 12:12h) carried out in an open experimental system in lab. Results revealed that calcification was dependent of utilization of dissolve inorganic carbon. Additional HCO3(-) significantly decreased the increase of pH while additional NH4-N did not. And additional HCO3(-) significantly improved calcification while NH4-N did in versus in relation to the variation of DIC concentration. However, addition of both HCO3(-) and NH4-N increased utilization of DIC. This resulted in calcification to utilization of DIC ratio decreased under additional NH4-N condition while increased under additional HCO3(-) conditions in response to the variation of solution pH. In the present study, external HCO3(-) decreased the increase of solution pH by increasing calcification, which correspondingly mitigated the toxic effect of high NH4-N. And we argue that the mitigating effect of increased HCO3(-) on NH4-N toxicity is dependent of plant calcification, and it is a positive feedback mechanism, potentially leading to the dominance of calcareous plants in meso-eutrophic water bodies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

By taking advantage of large cell size of Chara corallina, we analyzed the membrane depolarization induced by decreased turgor pressure (Shimmen in J Plant Res 124:639-644, 2011). In the present study, the response to increased turgor pressure was analyzed. When internodes were incubated in media containing 200 mM dimethyl sulfoxide, their intracellular osmolality gradually increased and reached a steady level after about 3 h. Upon removal of dimethyl sulfoxide, turgor pressure quickly increased. In response to the increase in turgor pressure, the internodes generated a transient membrane depolarization at its nodal end. The refractory period was very long and it took about 2 h for full recovery after the depolarizing response. Involvement of protein synthesis in recovery from refractoriness was suggested, based on experiments using inhibitors.

In the previous paper, I reported that 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil) depolarizes the plasma membrane by inhibiting the electrogenic proton pump and discussed that the inhibition is caused by cytosol acidification due to influx of protonated bromoxynil and following release of proton (Shimmen in J Plant Res 123:715-722, 2010). However, a possibility of direct inhibition of the proton pump by bromoxynil flowed into the cell could not be excluded. In the present study, the direct effect of bromoxynil on the proton pump was unequivocally excluded.

This work describes the characean internodal cell as a model system for the study of wound healing and compares wounds induced by certain chemicals and UV irradiation with wounds occurring in the natural environment. We review the existing literature and define three types of wound response: (1) cortical window formation characterised by disassembly of microtubules, transient inhibition of actin-dependent cytoplasmic streaming and chloroplast detachment, (2) fibrillar wound walls characterised by exocytosis of vesicles carrying wall polysaccharides and membrane-bound cellulose synthase complexes coupled with endocytosis of surplus membrane and (3) amorphous, callose- and membrane-containing wound walls characterised by exocytosis of vesicles and endoplasmic reticulum cisternae in the absence of membrane recycling. We hypothesize that these three wound responses reflect the extent of damage, probably Ca(2+) influx, and that the secretion of Ca(2+) -loaded endoplasmic reticulum cisternae is an emergency reaction in case of severe Ca(2+) load. Microtubules are not required for wound healing but their disassembly could have a signalling function. Transient reorganisation of the actin cytoskeleton into a meshwork of randomly oriented filaments is required for the migration of wound wall forming organelles, just as occurs in tip-growing plant cells. New data presented in this study show that during the deposition of an amorphous wound wall numerous actin rings are present, which may indicate specific ion fluxes and/or a storage form for actin. In addition, we present new evidence for the exocytosis of FM1-43-stained organelles, putative endosomes, required for plasma membrane repair during wound healing. Finally, we show that quickly growing fibrillar wound walls, even when deposited in the absence of microtubules, have a highly ordered helical structure of consistent handedness comprised of cellulose microfibrils., (© 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.)

The parthenogens of Chara canescens (Charophyceae) occupy broader geographical and ecological ranges than their sexual counterparts. Two possible hypotheses explain the ubiquity of parthenogens: the occurrence of one or several parthenogens with wide niches, or of many parthenogens that are restricted to narrow ecological niches. For the purposes of this study, C. canescens individuals from two neighbouring populations of the Baltic Sea (Bodstedter Bodden = BB; Salzhaff = SH), which differed significantly in water transparency and salinity, were investigated for significant differences in physiological capacity. Individuals of both habitats acclimated quickly to daily changes in irradiances in the field, but the photosynthetic efficiency of PS II showed a significant decrease with increasing daily irradiance in the habitat BB, which has lower levels of salinity and water transparency. In addition to the field study, individuals were reared under different levels of environmental factors in the laboratory: four irradiances (70-600 μmol m(-2) s(-1)) and five salinity levels (0-24 psu). The individuals of both habitats grew almost equally well at intermediate salinity levels. Growth under the artificial light supply was highest at levels corresponding to the in situ conditions for each population. Total chlorophyll was highest at intermediate salinities (BB), or hardly changed with salinity (SH). The physiological capacity for individuals from SH clearly depends upon changing growth irradiance, whereas the capacity for individuals from BB was relatively independent of salinity and irradiance. These findings indicate that both parthenogenetic C. canescens populations are locally adapted to light. However, to test adaptive potential of the parthenogens, more than two populations should be tested in future.

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., (2011 Elsevier B.V. All rights reserved.)

Upon addition of sorbitol to the external medium of an internodal cell of Chara corallina, a transient depolarization is induced at its nodal end (Shimmen in Plant Cell Physiol 44:1215-1224, 2003). In the present study, refractory period was found to be very long, 2-4 h. Recovery from refractoriness was completely inhibited by inhibitors of eukaryote-type protein synthesis, cycloheximide or anisomysin, but not by inhibitors of prokaryote-type protein synthesis. This suggested that proteinous factor(s) responsible for generation of the depolarization is lost or inactivated upon depolarization and synthesized during the resting state. Low temperature, which is supposed to inhibit protein synthesis, also inhibited recovery from refractoriness. When unstimulated internodal cells were incubated in the medium containing an inhibitor of eukaryote-type protein synthesis, generation of the depolarization was almost completely inhibited. This result suggested that the factor is slowly turning over even in the absence of osmotic stimulation.

We investigated the behaviour of organelles stained with FM1-43 (putative endosomes) and/or LysoTracker Red (LTred; acidic compartments) and of the endoplasmic reticulum (ER) during healing of puncture and UV-induced wounds in internodal cells of Nitella flexilis and Chara corallina. Immediately after puncture, wounds were passively sealed with a plug of solid vacuolar inclusions, onto which a bipartite wound wall was actively deposited. The outer, callose-containing amorphous layer consisted of remnants of FM1-43- and LTred-labelled organelles, ER cisternae and polysaccharide-containing secretory vesicles, which became deposited in the absence of membrane retrieval (compound exocytosis). During formation of the inner cellulosic layer, exocytosis of secretory vesicles with the newly formed plasma membrane is coupled to endocytosis via coated vesicles. Migration of FM1-43- and LTred-stained organelles, ER and secretory vesicles towards the cell cortex and deposition of a bipartite wound wall could also be induced by spot-like irradiation with ultraviolet light. Cytochalasin D reversibly inhibited the accumulation and deposition of organelles. Our study indicates that active actin-dependent deposition of putative recycling endosomes is required for wound healing (plasma membrane repair) and supports the hypothesis that deposition of ER cisternae helps to restore wounding-disturbed Ca(2+) metabolism., (© 2010 German Botanical Society and The Royal Botanical Society of the Netherlands.)

In a previous paper, we proposed that the primary action of the herbicide bromoxynil (BX; 3,5-dibromo-4-hydroxybenzonitrile) is cytosol acidification, based on the fact that bromoxynil induced the inhibition of cytoplasmic streaming and cell death of Chara corallina in acidic external medium (Morimoto and Shimmen in J Plant Res 121:227-233, 2008). In the present study, electrophysiological analysis of the BX effect was carried out in internodal cells of C. corallina. Upon addition of BX, a large and rapid pH-dependent depolarization was induced, supporting our hypothesis. Ioxynil, which belongs to the same group as bromoxynil, also induced a large and rapid membrane depolarization in a pH-dependent manner. On the other hand, four herbicides belonging to other groups of herbicides did not induce such a membrane depolarization. Thus, BX has a unique cellular effect. The decrease in the electro-chemical potential gradient for H(+) across the plasma membrane appears to result in inhibition of cell growth and disturbance of intracellular homeostasis in the presence of BX.

The action potential (AP) of excitable plant cells is a multifunctional physiological signal. Its generation in characean algae suppresses the pH banding for 15-30 min and enhances the heterogeneity of spatial distribution of photosynthetic activity. This suppression is largely due to the cessation of H(+) influx (OH(-) efflux) in the alkaline cell regions. Measurements of local pH and membrane conductance in individual space-clamped alkaline zones (small cell areas bathed in an isolated pool of external medium) showed that the AP generation is followed by the transient disappearance of alkaline zone in parallel with a large decrease in membrane conductance. These changes, specific to alkaline zones, were only observed under continuous illumination following a relaxation period of at least 15 min after previous excitation. The excitation of dark-adapted cells produced no conductance changes in the post-excitation period. The results indicate that the origin of alkaline zones in characean cells is not due to operation of electroneutral H(+)/HCO(3)(-) symport or OH(-)/HCO(3)(-) antiport. It is concluded that the membrane excitation is associated with inactivation of plasmalemma high conductance in the alkaline cell regions.

Cytoplasmic streaming circulates the contents of large eukaryotic cells, often with complex flow geometries. A largely unanswered question is the significance of these flows for molecular transport and mixing. Motivated by "rotational streaming" in Characean algae, we solve the advection-diffusion dynamics of flow in a cylinder with bidirectional helical forcing at the wall. A circulatory flow transverse to the cylinder's long axis, akin to Dean vortices at finite Reynolds numbers, arises from the chiral geometry. Strongly enhanced lateral transport and longitudinal homogenization occur if the transverse Péclet number is sufficiently large, with scaling laws arising from boundary layers.

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.

Spermiogenesis in Chara vulgaris and in animals share many common features, including exchange of nucleohistones into nucleoprotamines, remodeling and extreme condensation of chromatin, formation of flagellae and of microtubule manchette, and decrease in cytoplasm volume. In C. vulgaris, spermiogenesis is not preceded by meiosis since this alga is a haplobiont. In the present work we showed that in early spermiogenesis characterized by a significant metabolic activity of spermatids, the inhibitors of proteasomes did not visibly change their ultrastructure but significantly prolonged this process. At late stages of spermiogenesis, MG-132 and epoxomicin dramatically changed the structure of nuclei: regular fibrillar and lamellar structure of chromatin was disturbed and clusters of grains corresponding to aggresomes appeared, but the nucleus shape and cytoplasm structure were the same as in the controls. Immunocytochemical studies revealed that these inhibitors blocked disappearance of histones from nuclei while the structures corresponding to aggresomes were clusters of undegraded ubiquitinated histones, since they gave positive immunosignals indicating the presence of ubiquitin and histones.

Electrical characteristics of the node were analyzed in comparison with those of the flank of the internodal cell in Chara corallina. The dependence of the membrane potential of the node on pH and K+ concentration was almost the same as that of the flank. In the flank, the increase in the Ca2+ concentration stopped the depolarization in the presence of 100 mM KCl. In the node, however, Ca2+ could not stop the depolarization induced by 100 mM KCl. It has been reported that the node has a function to tranduce the signal of osmotic shock into a transient depolarization. In combination with osmotic shock, 10 mM K+ could induce a long-lasting depolarization of the node. These electrical characteristics of the node were suggested to be responsible for the electrical response to wounding in Characeae.

Characean internodal cells generate receptor potential (DeltaE (m)) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated DeltaE (m) at the moment of both compression and decompression, and the amplitude of DeltaE (m) at the moment of decompression, (DeltaE (m))(E), was larger than that at compression. The long-lasting stimulus caused a membrane deformation (DeltaD (m)) having two components, a rapid one, (DeltaD (m))(rapid), at the moment of compression and a slower one, (DeltaD (m))(slow), during the long-lasting compression. We assumed that (DeltaD (m))(slow) might have some causal relation with the larger DeltaE (m) at (DeltaE (m))(E). We treated internodal cells with either HgCl(2) or ZnCl(2), water channel inhibitors, to decrease (DeltaD (m))(slow). Both inhibitors attenuated (DeltaD (m))(slow) during compression. Cells treated with HgCl(2) generated smaller (DeltaE (m))(E) compared to nontreated cells. On the other hand, cells treated with ZnCl(2) never attenuated (DeltaE (m))(E) but, rather, amplified it. Thus, the amplitude of (DeltaD (m))(slow) did not always show tight correlation with the amplitude of (DeltaE (m))(E). Furthermore, when a constant deformation was applied to an internodal cell in a medium with higher or lower osmotic value, a cell having higher turgor always showed a larger (DeltaE (m))(E). Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca(2+) channel.

Chara myosin, two-headed plant myosin belonging to class XI, slides F-actin at maximally 60 microm s(-1). To elucidate the mechanism of this fast sliding, we extensively investigated its mechanochemical properties. The maximum actin activated ATPase activity, Vmax, was 21.3 s(-1) head(-1) in a solution, but when myosin was immobilized on the surface, its activity was 57.6 s(-1) head(-1) at 2 mg ml(-1) of F-actin. The sliding velocity and the actin activated ATPase activity were greatly inhibited by ADP, suggesting that ADP dissociation was the rate limiting step. With the extensive assay of motility by varying the surface density, the duty ratio of Chara myosin was found to be 0.49-0.44 from velocity measurements and 0.34 from the landing rate analysis. At the surface density of 10 molecules microm(-2), Chara myosin exhibited pivot movement under physiological conditions. Based on the results obtained, we will discuss the sliding mechanism of Chara myosin according to the working stroke model in terms of its physiological aspects. aspects., ((c) 2006 Wiley-Liss, Inc.)

Transient Ca2+ and Ca2+-dependent Cl- currents of plasmatic membranes of voltage-clamped Chara corallina freshwater alga cells were studied. Our earlier described method was used for rapid (approximately 10 ms) injection of Ca2+ ions into the cell during the deactivation period of calcium channels following their activation by a positive voltage pulse (injection by "tail" Ca2+ current). This procedure allowed one to determine the amplitude of the Ca2+ component, as well as the amplitude and kinetics of the submembrane Ca2+ concentration-dependent Cl- component for the transient current. Calculations based on the cell model allowing for Ca2+ diffusion, the Ca2+-buffering properties of the cytoplasm, and the nonlinear dependency of iCl on [Ca2+]cyt, as well as the presence of chloroplasts agreed well with the experimentally observed behavior of the transient current. The slow stage of the [Ca2+]cyt relaxation to the resting level (approximately 10(-7) M), related to the functioning of Ca2+-ATPases, was shown to take approximately 10(2) s. We assume this stage to determine the duration of the refractory period after the generation of action potential.

We review electrophysiological measures of turgor regulation in some siphonous green algae, primarily the giant-celled marine algae, Valonia and Ventricaria, with particular comparison to the well studied charophyte algae Chara and Lamprothamnium. The siphonous green algae have a less negative plasma membrane potential, and are unlikely to have a proton-based chemiosmotic transport system, dominated by active electrogenic K(+) uptake. We also make note of the unusual cellular structure of the siphonous green algae. Hypertonic stress, due to increased external osmotic pressure, is accompanied by positive-going potential difference (PD), increase in conductance, and slow turgor regulation. The relationship between these is not yet resolved, but may involve changes in K(+ )conductance (G (K)) or active K(+) transport at both membranes. Hypotonic turgor regulation, in response to decreased external osmotic pressure, is approximately 3 times faster than hypertonic turgor regulation. It is accompanied by a negative-going PD, although conductance also increases. The conductance increase and the magnitude of the PD change are strongly correlated with the magnitude of hypotonic stress.

The impact of unstirred layers (USLs) during cell pressure probe experiments with Chara corallina internodes has been quantified. The results show that the hydraulic conductivity (Lp) measured in hydrostatic relaxations was not significantly affected by USLs even in the presence of high water flow intensities ('sweep-away effect'). During pressure clamp, there was a reversible reduction in Lp by 20%, which was explained by the constriction of water to aquaporins (AQPs) in the C. corallina membrane and a rapid diffusional equilibration of solutes in arrays where water protruded across AQPs. In osmotic experiments, Lp, and permeability (Ps) and reflection (sigma s) coefficients increased as external flow rate of medium increased, indicating some effects of external USLs. However, the effect was levelling off at 'usual' flow rates of 0.20-0.30 m s(-1) and in the presence of vigorous stirring by air bubbles, suggesting a maximum thickness of external USLs of around 30 microm including the cell wall. Because the diameters of internodes were around 1 mm, internal USLs could have played a significant or even a dominating role, at least in the presence of the rapidly permeating solutes used [acetone, 2-propanol and dimethylformamide (DMF)]. A comparison of calculated (diffusion kinetics) and of measured permeabilities indicated an upper limit of the contribution of USLs for the rapidly moving solute acetone of 29%, and of 15% for the less rapidly permeating DME The results throw some doubt on recent claims that in C. corallina, USLs rather than the cell membrane dominate solute uptake, at least for the most rapidly moving solute acetone.

When the nodal end of an internodal cell of Chara corallina was subjected to a pulse treatment with artificial cell sap, a depolarization lasted in artificial pond water. This depolarization could also be induced by pulse treatment with KCl solution, indicating that K+ in the artificial cell sap is responsible (K+ -induced depolarization). The depolarization was prolonged in the presence of 2 mM KCl and the prolonged depolarization was terminated by supplementing with either CaCl2, MgCl2 or NaCl. These results supported the hypothesis that K+ released from the killed cell is responsible for generation of the wound-induced membrane depolarization.

The voltage-clamp technique was used to study Ca(2+) and Cl(-) transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about -140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10(3) ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca(2+) tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca(2+) injection into tonoplast-free and intact cells, to activate the chloride channels by Ca(2+) . When cells are perfused with EDTA-containing medium in the presence of excess Mg(2+), this method of injection allows the free submembrane Ca(2+) concentration, [Ca(2+)](c), to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to [see text]. When Ca(2+) is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation-inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m(-2) and tau(inact)~0.5 s at -200 mV). Analysis of our data and theoretical considerations indicate that the [Ca(2+)](c) rise during cell excitation is caused mainly by Ca(2+) entry through plasmalemma Ca channels rather than by Ca(2+) release from intracellular stores.

It is known that illumination of the algae Chara corallina results in the formation along the membrane of regions with inhomogeneous distribution of pH. It was shown that, in a particular range of illumination intensities, two states with different pH distribution are realized at one and the same value of light intensity: an entirely homogeneous state and completely formed structures (pattern). The transition from the homogeneous state to the pattern formation takes place at one value of light intensity, and the back transition, at another light intensity, i.e., the hysteresis is observed. This phenomenon was studied by mathematical modeling. The mechanism of hysteresis is discussed.

Early processes underlying plant gravity sensing were investigated in rhizoids of Chara globularis under microgravity conditions provided by parabolic flights of the A300-Zero-G aircraft and of sounding rockets. By applying centrifugal forces during the microgravity phases of sounding rocket flights, lateral accelerations of 0.14 g, but not of 0.05 g, resulted in a displacement of statoliths. Settling of statoliths onto the subapical plasma membrane initiated the gravitropic response. Since actin controls the positioning of statoliths and restricts sedimentation of statoliths in these cells, it can be calculated that lateral actomyosin forces in a range of 2 x 10(-14) n act on statoliths to keep them in place. These forces represent the threshold value that has to be exceeded by any lateral acceleration stimulus for statolith sedimentation and gravisensing to occur. When rhizoids were gravistimulated during parabolic plane flights, the curvature angles of the flight samples, whose sedimented statoliths became weightless for 22 s during the 31 microgravity phases, were not different from those of in-flight 1g controls. However, in ground control experiments, curvature responses were drastically reduced when the contact of statoliths with the plasma membrane was intermittently interrupted by inverting gravistimulated cells for less than 10 s. Increasing the weight of sedimented statoliths by lateral centrifugation did not enhance the gravitropic response. These results provide evidence that graviperception in characean rhizoids requires contact of statoliths with membrane-bound receptor molecules rather than pressure or tension exerted by the weight of statoliths.

A mathematical model of potencial-dependent proton transfer across the membrane of Chara corallina cells is considered. To construct the model, partial differential equations describing the system dynamics in time and in space were used. The variables of the model are the proton concentration and membrane potential. The model describes the experimentally observed inhomogeneous distribution of transmembrane potential and pH along the membrane and oscillations of the potential and pH in time. A mechanism of the distribution of pH and membrane potential along the Chara corallina cell is suggested.

In Chara corallina cells exposed to continuous light, external pH (pH(o)) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H+-pumping and H+-conductance are coupled in the different bands. First, PM H+-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H+ fluxes across the PM. Next, H+-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pH(o) by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H+-pump upon the detachment of 14-3-3 proteins from the H+-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pH(o) and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H+-ATPase disrupts the balanced operation of PM H+-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.

Both parts of the actin-myosin complex involved in cytoplasmic streaming could be regulated by mineral ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall and plasma membrane. The transport of K(+) and Ca(2+) along pH bands in Chara branchlet internodal cells was characterized by using the MIFE system for non-invasive microelectrode measurement of ion fluxes. Branchlets formed acidic and alkaline bands with the pH ranging from 5 to 8. Different pH patterns were observed for different sides of the branchlets. Sides with cyclosis streaming acropetally generally showed greater variation in the profiles of pH and H(+) fluxes. Although a high correlation was not found between pH bands and Ca(2+) or K(+) fluxes, there was a positive correlation between Ca(2+) and K(+) fluxes themselves for both sides of the branchlets. Application of cytochalasin D, an inhibitor of cyclosis, had no immediate effect on pH and ion fluxes, however, the time of cyclosis cessation corresponded with a dramatic change in Ca(2+) and K(+) fluxes; pH profiles and H(+) fluxes were affected within 2 h. The evidence suggests that, in Chara branchlets, pH band formation and Gd(3+)-insensitive Ca(2+) transport systems are linked to the cyclosis machinery: (i) the pH band amplitude for the acropetally streaming side was larger than that for the basipetally streaming side; (ii) cessation of cytoplasmic streaming after cytochalasin D application resulted in changed pH banding profiles and H(+), Ca(2+) and K(+) fluxes; and (iii) the application of GdCl(3) or incubation in GdCl(3) solutions did not lead to the cessation of cytoplasmic streaming, although external Ca(2+) fluxes changed.

A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.

Using pH microelectrodes and a Microscopy PAM (pulse-amplitude modulated) chlorophyll fluorometer, it is shown that a propagation of an action potential in Chara corallina leads to transient suppression of spatially periodic pH profiles along the illuminated cell. The suppression was manifested as a large pH decrease in the alkaline zones and a slight pH increase in the acid zones. The propagating action potential diminished the maximum yield of chlorophyll fluorescence (F(m)') in the alkaline cell regions, as well as the quantum yield of photosystem II photochemistry, without affecting F(m)' in the acid cell regions. The results indicate an interference of membrane excitation in the mechanisms responsible for pH banding patterns in Characean algae. Apparently, the electrical excitation of the plasma membrane in the alkaline cell regions initiates a pathway that can modulate membrane events at the thylakoid membrane.

We studied the effects of 2,3-butanedione monoxime (BDM) on the cytoplasmic streaming of Chara corallina and on the motility of myosin prepared from the same plant to examine whether this reagent really affects the plant class XI myosin. It was found that BDM inhibited both cytoplasmic streaming and the motility of myosin at a very similar concentration range (10-100 mM). BDM introduced directly into tonoplast-free cells also inhibited cytoplasmic streaming. These results suggested that effect of BDM on cytoplasmic streaming was exerted through myosin and not through ion channels at least in Chara corallina, though a very high concentration of BDM was required.

Isolated internodes of Chara corallina have been used to study the gating of aquaporins (water channels) in the presence of high concentrations of osmotic solutes of different size (molecular weight). Osmolytes were acetone and three glycol ethers: ethylene glycol monomethyl ether (EGMME), diethylene glycol monomethyl ether (DEGMME), and triethylene glycol monoethyl ether (TEGMEE). The 'osmotic efficiency' of osmolytes was quite different. Their reflection coefficients ranged between 0.15 (acetone), 0.59 (EGMME), 0.78 (DEGMME), and 0.80 (TEGMEE). Bulk water permeability (Lp) and diffusive permeabilities (Ps) of heavy water (HDO), hydrogen peroxide (H2O2), acetone, and glycol ethers (EGMME, DEGMME, and TEGMEE) were measured using a cell pressure probe. Cells were treated with different concentrations of osmotic solutes of up to 800 mM ( approximately 2.0 MPa of osmotic pressure). Inhibition of aquaporin activity increased with both increasing concentration and size of solutes (reflection coefficients). As cell Lp decreased, Ps increased, indicating that water and solutes used different passages across the plasma membrane. Similar to earlier findings of an osmotic gating of ion channels, a cohesion/tension model of the gating of water channels in Chara internodes by high concentration is proposed. According to the model, tensions (negative pressures) within water channels affected the open/closed state by changing the free energy between states and favoured a distorted/collapsed rather than the open state. They should have differed depending on the concentration and size of solutes that are more or less excluded from aquaporins. The bigger the solute, the lower was the concentration required to induce a reversible closure of aquaporins, as predicted by the model.

The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 microm/s at 30 degrees C and a CCM motor domain with an artificial lever arm consisting of two alpha-actinin repeats moved actin filaments at 16.2 microm/s. Both constructs displayed high actin-activated ATPase activities ( approximately 500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.

During the development of the antheridia of Chara species, dynamic changes in the occurrence and ultrastructure of plasmodesmata are observed which are closely correlated to particular developmental phases and presumably regulate the morphogenetic events in the antheridia. The disappearance of plasmodesmata between shield cells and between shied cells and the basal cell leads to a cessation in symplasmic transport around the antheridum and determines its concentric or centrifugal character via centrally situated capitular cells. Unplugged plasmodesmata are present between fully synchronously developing antheridial filament cells and obviously coordinate the development of the cells. In the middle phase of spermiogenesis, rough endoplasmic reticulum in antheridial filaments passes uncompressed through wide plasmodesmata and provides an additional transport pathway for developmental control factors. Plugged plasmodesmata link cells of different types or cells of the same type which are at different phases of cell cycle and guarantee their individual development. The plugging of plasmodesmata is a reversible process that depends on the morphogenetic situation. Plasmodesmata connecting the basal cell and the subbasal cell as well as the basal cell and capitular cells are transformed successively from the simple into the complex type and might be the pathways for an import of gibberellins and nutrients into the strong sink tissues of the developing antheridium. There is a symplasmic connection between the antheridum and the thallus via a basal cell. Prior to the initiation of spermatozoid differentiation (spermiogenesis), plasmodesmata connecting the basal cell with a subbasal cell and the basal cell with capitular cells are spontaneously broken, resulting in symplasmic isolation of the antheridium that is probably a signal which triggers the induction of spermatozoid differentiation. Premature plasmolytically evoked symplasmic isolation of the antheridium leads to the elimination of 1 to 2 cell cycles from the proliferative stage of spermatogenesis. Autoradiographic studies demonstrate that both natural and induced symplasmic isolation drastically decreases the entry of isotopically labeled gibberellic acid into antheridia of Chara species that may be the consequence of the elimination of the hormone's transport through plasmodesmata.

Although the decrease in cell turgor by application of sorbitol to the external medium did not inhibit the alkaline band formation in Chara corallina, recovery of normal turgor severely inhibited it. Alkaline-loading analysis suggested that the inhibition of alkaline band formation was caused by inhibition of HCO(3)(-) influx but not that of OH(-) efflux. In the presence of 10 mM CaCl(2), the capacity of alkaline band formation was maintained during osmotic treatment. Cells could not form alkaline bands, when plasmolysis was induced by application of sorbitol at a higher concentration. Addition of 10 mM CaCl(2) could ameliorate the inhibition caused by plasmolyis.