Brefeldin A inhibits clathrin-dependent endocytosis and ion transport in Chara internodal cells.

BACKGROUND: The Characeae are multicellular green algae, which are closely related to higher plants. Their internodal cells are a convenient model to study membrane transport and organelle interactions. RESULTS: In this study, we report on the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on internodal cells of Chara australis. BFA induced the commonly observed agglomeration of Golgi bodies and trans Golgi network into 'brefeldin compartments' at concentrations between 6 and 500 µM and within 30-120 min treatment. In contrast to most other cells, however, BFA inhibited endocytosis and significantly decreased the number of clathrin-coated pits and clathrin-coated vesicles at the plasma membrane. BFA did not inhibit secretion of organelles at wounds induced by puncturing or local light damage but prevented the formation of cellulosic wound walls probably because of insufficient membrane recycling. We also found that BFA inhibited the formation of alkaline and acid regions along the cell surface ('pH banding pattern') which facilitates carbon uptake required for photosynthesis; we hypothesise that this is due to insufficient recycling of ion transporters. During long-term treatments over several days, BFA delayed the formation of complex 3D plasma membranes (charasomes). Interestingly, BFA had no detectable effect on clathrin-dependent charasome degradation. Protein sequence analysis suggests that the peculiar effects of BFA in Chara internodal cells are due to a mutation in the guanine-nucleotide exchange factor GNOM required for recruitment of membrane coats via activation of ADP-ribosylation factor proteins. CONCLUSIONS AND SIGNIFICANCE: This work provides an overview on the effects of BFA on different processes in C. australis. It revealed similarities but also distinct differences in vesicle trafficking between higher plant and algal cells. It shows that characean internodal cells are a promising model to study interactions between seemingly distant metabolic pathways.

Bioaccumulation and toxicity studies of macroalgae (Charophyceae) treated with aluminium: Experimental studies in the context of lake restoration.

The objective of this study was to examine the impact of aluminium on the perennial macroalgae Chara hispida L. and its bioaccumulation capacities. Aluminium (Al) was introduced into the environment in the form of polyaluminium chloride, an agent utilized in the restoration of waterbodies. Research was conducted in an experimental setting using mesocosms (volume 0.8m3) placed in the littoral zone of a lake with C. hispida. Three doses of the coagulant were applied, each with a different volume: low - 6.1g Al m-3, medium - 12.2gm-3 and high - 24.5g Al m-3. A significant acidification of environment was determined, which would imply the presence of toxic Al3+ ions. It has been demonstrated that aluminium penetrates and accumulates in the cells of the charophyte. This caused damage to the thalli, which manifested itself in chloroses, necroses, flaking of the cortex cells and softening of the thallus, whose severity was proportionate to the dose of the coagulant. The first negative signs were observed after 24h. The study shows that C. hispida is a poor accumulator of aluminium (bioconcentration factor < 200), while bioaccumulation capacity was inhibited at the concentration of approx. 2.0mg Al g-1 d.w. Accumulation in the thalli of the charophytes accounted for 58% of variation following removal of aluminium from the environment. The results of the experiment demonstrate a negative impact of aluminium on charophytes at concentrations used in aggressive restoration of lakes.

The defense substance allicin from garlic permeabilizes membranes of Beta vulgaris, Rhoeo discolor, Chara corallina and artificial lipid bilayers.

BACKGROUND: Allicin (diallylthiosulfinate) is the major volatile- and antimicrobial substance produced by garlic cells upon wounding. We tested the hypothesis that allicin affects membrane function and investigated 1) betanine pigment leakage from beetroot (Beta vulgaris) tissue, 2) the semipermeability of the vacuolar membrane of Rhoeo discolor cells, 3) the electrophysiology of plasmalemma and tonoplast of Chara corallina and 4) electrical conductivity of artificial lipid bilayers. METHODS: Garlic juice and chemically synthesized allicin were used and betanine loss into the medium was monitored spectrophotometrically. Rhoeo cells were studied microscopically and Chara- and artificial membranes were patch clamped. RESULTS: Beet cell membranes were approximately 200-fold more sensitive to allicin on a mol-for-mol basis than to dimethyl sulfoxide (DMSO) and approximately 400-fold more sensitive to allicin than to ethanol. Allicin-treated Rhoeo discolor cells lost the ability to plasmolyse in an osmoticum, confirming that their membranes had lost semipermeability after allicin treatment. Furthermore, allicin and garlic juice diluted in artificial pond water caused an immediate strong depolarization, and a decrease in membrane resistance at the plasmalemma of Chara, and caused pore formation in the tonoplast and artificial lipid bilayers. CONCLUSIONS: Allicin increases the permeability of membranes. GENERAL SIGNIFICANCE: Since garlic is a common foodstuff the physiological effects of its constituents are important. Allicin's ability to permeabilize cell membranes may contribute to its antimicrobial activity independently of its activity as a thiol reagent.

Salinity-induced noise in membrane potential of Characeae Chara australis: effect of exogenous melatonin.

Salt sensitive Characeae Chara australis responds to 50 mM NaCl by a prompt appearance of noise in the trans-membrane potential difference (PD). The noise diminishes with time in saline and PD depolarization, leading to altered current-voltage characteristics that could be modeled with H(+)/OH(-) channels. Beilby and Al Khazaaly (JMB 230:21-34, 2009) suggested that the noise might arise from cooperative transient opening of H(+)/OH(-) channels. Presoaking cells in 10 µM melatonin over 24 h abolished the noise in some cells, postponed its appearance in others or changed its characteristics. As melatonin is a very effective antioxidant, we postulated opening of H(+)/OH(-) channels by reactive oxygen species (ROS). Measurement of ROS using dihydrodichlorofluorescein diacetate confirmed substantial reduction in ROS production in melatonin-treated cells in saline and sorbitol media. However, ROS concentration decreased as a function of time in saline medium. Possible schemes for activation of H(+)/OH(-) channels under salinity stress are considered.

Instant rerouting of photosynthetic electron transport to O2 reduction after the plasma membrane excitation of Chara in the presence of methyl viologen.

-Action potential (AP) of excitable plant cells is an important signaling event that can differentially alter physicochemical and physiological processes in various parts of the same cell. In giant cells of characean algae, the AP propagation has minor effect on photosynthetic electron transport in areas with high activity of plasmalemmal H+-pump but inhibits linear electron flow in regions featuring high passive H+/OH- conductance of the plasma membrane (PM). Uneven spatial distributions of local periplasmic and cytoplasmic pH facilitate the operation of distinct (CO2-dependent and O2-mediated) pathways of photoinduced electron flow, which presumably accounts for differential influence of AP on photosynthesis. The excitation of Chara australis cell in the presence of methyl viologen (MV), a redox mediator with the prooxidant action, provides a convenient model system to clarify the influence of voltage-dependent ion fluxes across PM on photosynthetic activity of chloroplasts. This study shows that permeation of MV to their target sites in chloroplasts is restricted by PM in resting cells, but MV easily passes through ionic channels opened during the PM depolarization. This gated permeation of MV gives rise to strong non-photochemical quenching, decrease in the effective quantum yield of linear electron flow, apparent O2 uptake, and, finally, the enhanced ROS production, as detected by the fluorescent probe dichlorofluorescein. Taken together, the results indicate that the AP generation in the presence of MV acts as trigger for instant redirection of photosynthetic linear electron flow from CO2-dependent route to the path of O2 reduction with the eventual formation of H2O2 as a dominant and most stable ROS form.

Transduction of pressure signal to electrical signal upon sudden increase in turgor pressure in Chara corallina.

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.

Combined effects of cadmium and zinc on growth, tolerance, and metal accumulation in Chara australis and enhanced phytoextraction using EDTA.

Chara australis (R. Br.) is a macrophytic alga that can grow in and accumulate Cd from artificially contaminated sediments. We investigated the effects of Zn independently and in combination with Cd on C. australis growth, metal tolerance, and uptake. Plant growth was reduced at concentrations ≥ 75 mg Zn (kg soil)⁻¹. Zn also increased the concentration of glutathione in the plant, suggesting alleviation of stress. Phytotoxic effects were observed at ≥ 250 mg added Zn (kg soil)⁻¹. At 1.5mg Zn (kg soil)⁻¹, the rhizoid bioconcentration factor (BCF) was >1.0 for both Cd and Zn. This is a criterion for hyperaccumulator status, a commonly used benchmark for utility in remediation of contaminated soils by phytoextraction. There was no significant interaction between Cd and Zn on accumulation, indicating that Chara should be effective at phytoextraction of mixed heavy metal contamination in sediments. The effects of the chelator, ethylenediaminetetraacetic acid (EDTA), were also tested. Moderate levels of EDTA increased Cd and Zn accumulation in rhizoids and Cd BCF of shoots, enhancing Chara's potential in phytoremediation. This study demonstrates for the first time the potential of macroalgae to remove metals from sediments in aquatic systems that are contaminated with a mixture of metals.

Bioconcentration and intracellular storage of hexachlorobenzene in charophytes and their potential role in monitoring and remediation actions.

It has been hypothesized that highly hydrophobic substances (LogK(OW) > 5) including many persistent organic pollutants cannot overtake protective tissues and diffuse inside the body of plants due to steric hindrance or very slow diffusion. We investigated the bioaccumulation of hexachlorobenzene (HCB, LogK(OW) = 5.5) in a benthic charophycean macro-alga: Chara rudis. Chara species are a group of common freshwater algae with a complex body structure encompassing a protective layer of cortex cells surrounding large internode cells. The charophyte cell wall has many features in common with that of higher plants; therefore, they are useful models to investigate bioaccumulation mechanisms in general. We found that HCB diffused through the cortex and reached the cytoplam of internode cells. More than 90% of the HCB mass found in the organism was in the cortex and 10% in the internode cell cytoplasm. The cortex reached a pseudoequilibrium partitioning with water, and the bioconcentration factor was in the same range as that of lower aquatic organisms such as phytoplankton. Charophytes are therefore very efficient accumulators of hydrophobic compounds. Based on the structural and ecological features of charophytes, we speculated on their possible use as biomonitors and bioremediation tools.

Further electrophysiological studies on cellular effect of herbicide, bromoxynil, using characean cells.

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.

Cd tolerance and accumulation in the aquatic macrophyte, Chara australis: potential use for charophytes in phytoremediation.

We investigated the potential use of the alga Chara australis (R. Br.) forphytore mediation of Cd-contaminated sediments in aquatic systems. Chara tolerated up to 20 mg added Cd (kg soil)⁻¹ in laboratory culture. Chlorophyll a and b levels were not affected even at Cd concentrations that suppressed growth. Levels of glutathione were suppressed at 2-35 mg added Cd (kg soil)⁻¹ to 200-350 nmol GSH (g DW)⁻¹, while control levels were 660 nmol GSH (g DW)⁻¹). Histochemical studies showed Cd occurred throughout cell walls and cytoplasm in plants grown in 5-20 mg Cd (kg soil)⁻¹. Quantification using ICP-MS showed the maximum concentration in shoots was 72 mg Cd (kg DW)⁻¹ at 35 mg added Cd (kg soil)⁻¹, while the maximum in rhizoids was 116 mg Cd (kg DW)⁻¹ at 25 mg added Cd (kg soil)⁻¹. The bioconcentration factor (BCF, concentration in plant/concentration in soil) exceeded 1.0, the critical value for hyperaccumulators, for shoots exposed to 35 mg Cd (kg soil)⁻¹ and rhizoids exposed to ≥25 mg Cd (kg soil)⁻¹. Translocation factors (TF, shoot concentration/rhizoid concentration) did not exceed 1.0 for any treatment. While Chara cannot be considered a hyperaccumulator, it shows promise for use in phytoremediation efforts.

Involvement of protein synthesis in recovery from refractory period of electrical depolarization induced by osmotic stimulation in Chara corallina.

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.

Unique cellular effect of the herbicide bromoxynil revealed by electrophysiological studies using characean cells.

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.

Iron-induced behavioural and biochemical responses of charophytes in consequence of phosphates coagulant addition: Threats to lake ecosystems restoration.

The study aimed to evaluate the impact of iron (Fe) on the physiological and behavioural reaction of Chara tomentosa L. Fe was introduced into the environment in the form of iron chloride, the most common coagulants used in the restoration of water bodies. The investigations concerned the oxidative stress comprising phenolic compounds content, antioxidant activity and photosynthetic pigments concentration. Research was conducted as a laboratory microcosm experiment with one-off application of Fe at the level of 26.8 mg dm-3. Coagulant application caused short-term acidification, increased salinity and deterioration of light conditions. The shading resulted initially from the increase of water colour and turbidity and was followed by covering of the charophytes with a precipitated suspension. C. tomentosa did not activate defensive mechanisms to prevent the shading effect such as intensive elongation and elevated concentration of chlorophylls. Neither oxidative stress nor production of stress-specific phenolic metabolites was found. It was a result of iron coagulant toxicity, which led to cell membrane damage and leakage of cell contents to the water environment. Charophyte growth was significantly impaired, and thalli suffered numerous chlorotic and necrotic spots which extended gradually during experiment and finally caused death of specimens.

Blocking the Bromodomains Function Contributes to Disturbances in Alga Chara vulgaris Spermatids Differentiation.

Bromodomain containing (BRD) proteins play an essential role in many cellular processes. The aim of this study was to estimate activity of bromodomains during alga Chara vulgaris spermatids differentiation. The effect of a bromodomain inhibitor, JQ1 (100 µM), on the distribution of individual stages of spermatids and their ultrastructure was studied. The material was Feulgen stained and analysed in an electron microscope. JQ1 caused shortening of the early stages of spermiogenesis and a reverse reaction at the later stages. Additionally, in the same antheridium, spermatids at distant developmental stages were present. On the ultrastructural level, chromatin fibril system disorders and significantly distended endoplasmic reticulum (ER) cisternae already at the early stages were observed. Many autolytic vacuoles were also visible. The ultrastructural disturbances intensified after prolonged treatment with JQ1. The obtained data show that JQ1 treatment led to changes in the spermatid number and disturbances in chromatin condensation and to cytoplasm reduction. The current studies show some similarities between C. vulgaris and mammals spermiogenesis. Taken together, these results suggest that JQ1 interferes with the spermatid differentiation on many interdependent levels and seems to induce ER stress, which leads to spermatid degeneration. Studies on the role of bromodomains in algae spermiogenesis have not been conducted so far.

The role of H(+)/OH(-) channels in the salt stress response of Chara australis.

We investigate the electrophysiological salt stress response of the salt-sensitive charophyte Chara australis as a function of time in saline artificial pond water (saline APW) containing 50 mM NaCl and 0.1 mM CaCl(2). The effects are due to an increase in Na(+) concentration rather than an increase in Cl(-) concentration or medium osmolarity. A previous paper (Shepherd et al. Plant Cell Environ 31:1575-1591, 2008) described the rise in the background conductance and inhibition of proton pumping in saline APW in the first 60 min. Here we investigate the shift of membrane potential difference (PD) to levels above -100 mV and the change of shape of the current-voltage (I/V) profiles to upwardly concave. Arguing from thermodynamics, the I/V characteristics can be modeled by channels that conduct H(+) or OH(-). OH(-) was chosen, as H(+) required an unrealistic increase in the number/permeability of the channels at higher pH levels. Prolonged exposure to saline APW stimulated opening of more OH(-) channels. Recovery was still possible even at a PD near -50 mV, with partial return of proton pumping and a decrease in OH(-) current following APW wash. Upon change of pH from 7 to 9, the response was consistent with previously observed I/V characteristics of OH(-) channels. For a pH change to 6, the response was transient before channel closure but could still be modeled. The consequences of opening of H(+) or OH(-) channels while the cell is under salt stress are discussed.

Membrane potential fluctuations in Chara australis: a characteristic signature of high external sodium.

We have studied fluctuations in membrane PD in Chara australis at frequencies between 1 and 500 mHz, by classical noise analysis and by inspection of the PD time-course. The former shows (1) a quasi-Lorentzian (1/f (2)) rise of noise power as frequency falls, and (2) a marked increase in noise power when the cell is exposed to high salinity (Chara australis is a salt-sensitive species). The latter shows that, as well as initiating depolarization, exposure to 50 mM Na as either chloride or sulfate usually initiates a continuous but random series of small depolarizations which gives rise to the increase in noise and whose mechanism is discussed.

Cyclosis-mediated intercellular transmission of photosynthetic metabolites in Chara revealed with chlorophyll microfluorometry.

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.

Electrophysiological characterization of the node in Chara corallina: functional differentiation for wounding response.

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.

Toxic effects of linear alkylbenzene sulfonate on Chara vulgaris L.

Linear alkylbenzene sulfonate (LAS) is a common organic pollutant in freshwater environments. Studies have shown that the toxicity of LAS to aquatic plants is directly related to the LAS concentration and depends on the plant species. A 2-week exposure experiment was designed to investigate the toxicity of LAS for the submerged plant Chara vulgaris L. and focused on the effects on growth, photosynthetic pigment content, and antioxidant enzyme activity. The results showed that when exposed to lower LAS doses (≤ 1.0 mg l-1), the dry weight of C. vulgaris was significantly reduced. Compared to those of the control group, superoxide dismutase (SOD) and peroxidase (POD) activities significantly increased, while no significant effect was observed for catalase (CAT) activity. Malondialdehyde (MDA) content significantly increased in the LAS treatment groups except for the LAS concentration of 1.0 mg l-1. The content of carotenoids was significantly lower in plant groups exposed to lower concentrations of LAS, while carotenoid content significantly increased at the highest concentration of LAS (5.0 mg l-1). LAS treatment did not significantly affect chlorophyll a and b or total chlorophyll content. The results showed that 5.0 mg l-1 causes some oxidative damage to C. vulgaris but that this concentration was far below the lethal concentration of LAS to C. vulgaris and did not produce severe effects on growth. C. vulgaris plants had some resistance to LAS stress (in the group with ≤ 5.0 mg l-1). SOD, POD, and carotenoids were more sensitive to the effects of LAS stress and may be considered as response indicators for LAS stress.

Long-range interactions of Chara chloroplasts are sensitive to plasma-membrane H+ flows and comprise separate photo- and dark-operated pathways.

Local illumination of the characean internode with a 30-s pulse of white light was found to induce the delayed transient increase of modulated chlorophyll fluorescence in shaded cell parts, provided the analyzed region is located downstream in the cytoplasmic flow at millimeter distances from the light spot. The fluorescence response to photostimulation of a remote cell region indicates that the metabolites produced by source chloroplasts in an illuminated region are carried downstream with the cytoplasmic flow, thus ensuring long-distance communications between anchored plastids in giant internodal cells. The properties of individual stages of metabolite signaling are not yet well known. We show here that the export of assimilates and/or reducing equivalents from the source chloroplasts into the flowing cytoplasm is largely insensitive to the direction of plasma-membrane H+ flows, whereas the events in sink regions where these metabolites are delivered to the acceptor chloroplasts under dim light are controlled by H+ fluxes across the plasma membrane. The fluorescence response to local illumination of remote cell regions was best pronounced under weak background light and was also observed in a modified form within 1-2 min after the transfer of cell to darkness. The fluorescence transients in darkened cells were suppressed by antimycin A, an inhibitor of electron transfer from ferredoxin to plastoquinone, whereas the fluorescence response under background light was insensitive to this inhibitor. We conclude that the accumulation of reduced metabolites in the stroma leads to the reduction of photosystem II primary quinone acceptor (QA) via two separate (photochemical and non-photochemical) pathways.