PH-dependent cell-cell interactions in the green alga Chara.

Characean internodal cells develop alternating patterns of acid and alkaline zones along their surface in order to facilitate uptake of carbon required for photosynthesis. In this study, we used a pH-indicating membrane dye, 4-heptadecylumbiliferone, to study the kinetics of alkaline band formation and decomposition. The differences in growth/decay kinetics suggested that growth occurred as an active, autocatalytic process, whereas decomposition was due to diffusion. We further investigated mutual interactions between internodal cells and found that their alignment parallel to each other induced matching of the pH banding patterns, which was mirrored by chloroplast activity. In non-aligned cells, the lowered photosynthetic activity was noted upon a rise of the external pH, suggesting that the matching of pH bands was due to a local elevation of membrane conductance by the high pH of the alkaline zones of neighboured cells. Finally, we show that the altered pH banding pattern caused the reorganization of the cortical cytoplasm. Complex plasma membrane elaborations (charasomes) were degraded via endocytosis, and mitochondria were moved away from the cortex when a previously acid region became alkaline and vice versa. Our data show that characean internodal cells react flexibly to environmental cues, including those originating from neighboured cells.

Action of tannin on cellular membranes: Novel insights from concerted studies on lipid bilayers and native cells.

Hydrolyzable tannin (3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-ß-d-glucose) has a dual effect on the cell membrane: (1) it binds to a plasmalemmal protein of the Chara corallina cell (C50 = 2.7 ±â€¯0.3 µM) and (2) it forms ionic channels in the lipid membrane. Based on these facts, a molecular model for the interaction of tannins with the cell membrane is proposed. The model suggests that the molecules of hydrolyzable tannin bind electrostatically to the outer groups of the membrane protein responsible for the Ca2+-dependent chloride current and blocks it. Some tannin molecules penetrate into the hydrophobic region of the membrane, and when a particular concentration is reached, they form ion-conducting structures selective toward Cl-.

Arsenic Removal of Contaminated Soils by Phytoremediation of Vetiver Grass, Chara Algae and Water Hyacinth.

This research has been carried out for assessing phytoremediation of contaminated soils with 4 concentrations of arsenic by three plants, namely Vetiver grass (Vetiveria zizanioides), Chara algae (Chara vulgaris) and Water hyacinth (Hyacintus orientalis). The experimental results showed that at least two sampling times were significantly different. In addition, at least two plants were also significantly different in terms of percentages of total arsenic that were removed from the soil of the pots, as well as significant interactions between plant and arsenic concentrations. The results obtained from the thermodynamic studies show that, obtained by zero Gibbs free-energy, the process reached an equilibrium on the 60th day of the experiment, and, in fact, the adsorption of arsenic after the 60th day would be negligible.

Allelopathic effects of Chara species (C. aspera, C. baltica, and C. canescens) on the bloom-forming picocyanobacterium Synechococcus sp.

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 (Fv/Fm and ΦPSII, respectively) and photosynthesis parameters such as the initial slope of photosynthesis-irradiance (P-E) curves (alpha) and photosynthetic capacity (Pm) 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.

The seasonal effects on the encrustation of charophytes in two hard-water lakes.

Encrustation and element content of six charophyte species from two hard-water lakes were investigated monthly for a period of 1 year. Seasonal patterns were analyzed for the interaction of water chemistry. Encrustation followed a seasonal pattern for Chara contraria, Chara subspinosa, and Nitellopsis obtusa in Lake Krüselin and for Chara globularis and Chara tomentosa in Lake Lützlow. However, no seasonality in the precipitated CaCO3 was observed for C. subspinosa in Lake Lützlow and for C. tomentosa in Lake Krüselin, indicating a lake-specific dependency. Species-specific encrustation was found. Chara contraria and N. obtusa encrusted the most in June and August, whereas C. subspinosa and Nitella flexilis/opaca exhibited lowest encrustation in March and April. The precipitated CaCO3 of charophytes correlated negatively to the concentration of total inorganic carbon in both lakes. Element content of plant dry weight was species-specific for Ca and K, and lake-specific for Mg. No specific pattern was found for the TP and Fe contents. The results showed seasonal, species, and lake-specific influences on the encrustation of charophytes.

Influence of drying of chara cellulose on length/length distribution of microfibrils after acid hydrolysis.

Chara is a genus of freshwater alga that is evolutionarily observed at the aquatic-terrestrial boundary, whose cellulose microfibrils are similar to those of terrestrial plants regarding the crystallinity and biosynthesis of cellulose. Oven-dried and never-dried celluloses samples were prepared from chara. Terrestrial plant cellulose samples were used as references. The lengths and length distributions of oven-dried and never-dried chara cellulose microfibrils after acid hydrolysis with or without pretreatment by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, which was used for efficient fibrillation of acid-hydrolyzed products, were observed by transmission electron microscopy. All terrestrial plant celluloses and oven-dried chara cellulose had short nanocrystal-like morphologies of 100-300 nm in length after acid hydrolysis. In contrast, the never-dried chara cellulose had much longer microfibrils of ∼970 nm in length after acid hydrolysis. These results indicated that disordered regions present periodically along the cellulose microfibrils, which cause the formation of cellulose nanocrystals after acid hydrolysis, are not present in inherent chara cellulose microfibrils in water, but are formed artificially under drying or dehydration conditions.

Performance of packed bed column using Chara aculeolata biomass for removal of Pb and Cd ions from wastewater.

Biosorption of Pb and Cd from aqueous solution by biomass of Chara aculeolata was studied in a continuous packed bed column. C. aculeolata in the fixed bed column is capable of decreasing Pb and Cd concentrations from 10 mg/L to a value below the detection limit of 0.02 mg/L. Selective uptake of Pb and Cd in a binary solution resulted in Pb having much higher relative affinity than Cd. The experiments were conducted to study the effects of column design parameters, bed depth, and flow rate on the metal biosorption. Pb uptake capacity of C. aculeolata increased with increased bed depth and decreased flow rate, while Cd uptake capacity increased with increased bed depth but remained constant at any flow rate. The Thomas model was found in a suitable fitness with the experiment data for Pb and Cd (R2 > 0.90). The efficiency of biosorbent regeneration achieved by 0.1 M HCl was very high, that was, 98% for Pb and 100% for Cd in the third reused cycle. It can be concluded that C. aculeolata is a good biosorbent for treating wastewater having low concentrations of Pb and Cd contamination.

Seasonality of water chemistry, carbonate production, and biometric features of two species of Chara in a shallow clear water lake.

The objective of this study was to analyze the temporal variability of biometric features and the carbonate production of two charophytes: Chara polyacantha A. Braun and Chara rudis A. Braun against the background of the physical-chemical properties of water. The investigation was carried out in a small, mid-forest Lake Jasne (western Poland). It is a polymictic, mesotrophic, hardwater ecosystem dominated by charophyte vegetation. Each month, 10 individuals of each species were characterized in terms of morphometric features, fresh and dry weight, and the percentage of calcium carbonate. Additionally, physical-chemical parameters of the water were studied. The results of physical-chemical analyses indicated similar habitat conditions for both species. Despite smaller dry weight C. polyacantha was characterized by greater morphological variability and higher rates of growth and percentage share of calcium carbonate in dry mass than C. rudis. The percentage of calcium carbonates in dry mass did not differ significantly between the species and exceeded 60%, reaching the maximum (76% in C. polyacantha) in July and August. For both species, distinct correlations between the structure of biomass and morphological features were found. The obtained results show the great importance of charophyte vegetation in carbon cycling and functioning of lake ecosystems.

Remote control of myosin and kinesin motors using light-activated gearshifting.

Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.

Engineering myosins for long-range transport on actin filaments.

Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s(-1).

Membrane viscosity determined from shear-driven flow in giant vesicles.

The viscosity of lipid bilayer membranes plays an important role in determining the diffusion constant of embedded proteins and the dynamics of membrane deformations, yet it has historically proven very difficult to measure. Here we introduce a new method based on quantification of the large-scale circulation patterns induced inside vesicles adhered to a solid surface and subjected to simple shear flow in a microfluidic device. Particle image velocimetry based on spinning disk confocal imaging of tracer particles inside and outside of the vesicle and tracking of phase-separated membrane domains are used to reconstruct the full three-dimensional flow pattern induced by the shear. These measurements show excellent agreement with the predictions of a recent theoretical analysis, and allow direct determination of the membrane viscosity.

Strong alkalinization of Chara cell surface in the area of cell wall incision as an early event in mechanoperception.

Mechanical wounding of cell walls occurring in plants under the impact of pathogens or herbivores can be mimicked by cell wall incision with a glass micropipette. Measurements of pH at the surface of Chara corallina internodes following microperforation of cell wall revealed a rapid (10-30s) localized alkalinization of the apoplast after a lag period of 10-20s. The pH increase induced by incision could be as large as 3 pH units and relaxed slowly, with a halftime up to 20min. The axial pH profile around the incision zone was bell-shaped and localized to a small area, extending over a distance of about 100µm. The pH response was suppressed by lowering cell turgor upon the replacement of artificial pond water (APW) with APW containing 50mM sorbitol. Stretching of the plasma membrane during its impression into the cell wall defect is likely to activate the Ca(2+) channels, as evidenced from sensitivity of the incision-induced alkalinization to the external calcium concentration and to the addition of Ca(2+)-channel blockers, such as La(3+), Gd(3+), and Zn(2+). The maximal pH values attained at the incision site (~10.0) were close to pH in light-dependent alkaline zones of Chara cells. The involvement of cytoskeleton in the origin of alkaline patch was documented by observations that the incision-induced pH transients were suppressed by the inhibitors of microtubules (oryzalin and taxol) and, to a lesser extent, by the actin inhibitor (cytochalasin B). The results indicate that the localized increase in apoplastic pH is an early event in mechanoperception and depends on light, cytoskeleton, and intracellular calcium.

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.

Characteristics of light chains of Chara myosin revealed by immunological investigation.

Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Ca⁺⁺-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively.

Chlorophyll fluorescence images demonstrate variable pathways in the effects of plasma membrane excitation on electron flow in chloroplasts of Chara cells.

Chlorophyll fluorescence Imaging and Microscopy PAM fluorometry were applied to study spatial dynamics of photosystem II quantum yield (ΔF/F'(m)) and non-photochemical quenching (NPQ) in resting and electrically stimulated Chara corallina cells in the absence and presence of the hydrophilic electron acceptor methyl viologen (MV) in the external medium. Electrical excitation of the plasma membrane temporarily enhanced the heterogeneity of photosynthetic patterns under physiological conditions (in the absence of MV), but irreversibly eliminated these patterns in the presence of MV. These findings suggest that the action potential (AP) of the excitable plant cell affects the spatial patterns of photosynthesis and chlorophyll fluorescence through different pathways operated in the absence and presence of MV. Based on the extent of NPQ as an indicator of MV-dependent electron flow, it is supposed that MV cannot permeate into the chloroplasts of photosynthetically active "acid cell regions" but gains an immediate access to the stroma of these chloroplasts after triggering of an AP. The AP-triggered MV-dependent non-photochemical quenching in the chloroplasts of acidic cell regions was routinely observed at 0.1 mM Ca(2+) in the medium but not at elevated (2 mM) external Ca(2+) concentration. The results are interpreted in terms of competition between two permeant divalent ion species, Ca(2+) and MV(2+), for their passage through the voltage-gated calcium channels of the plasma membrane. It is proposed that the herbicidal activity of MV in characean cells, here serving as model object, can be manipulated by triggering AP and varying Ca(2+) concentration in the environmental medium.

The mechanism of boron mobility in wheat and canola phloem.

Low-molecular-weight borate complexes were isolated from canola (Brassica napus) and wheat (Triticum aestivum) phloem exudates, as well as the cytoplasm of the fresh-water alga Chara corallina, and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phloem exudate was collected from field-grown canola inflorescence stalks by shallow incision, while wheat phloem exudate was collected by aphid stylectomy. Chara cytoplasm was collected by careful manual separation of the cell wall, vacuole, and cytosolic compartments. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed the presence of isotopic borate complexes, at mass-to-charge ratio of 690.22/691.22 in the canola and wheat phloem and at 300.11/301.11 in canola phloem and Chara cytoplasm. Using reference compounds, the borate complexes with mass-to-charge ratio 690.22/691.22 was identified as a bis-sucrose (Suc) borate complex in which the 4,6-hydroxyl pairs from the two alpha-glucopyranoside moieties formed an [L(2)B](-1) complex. Further investigation using liquid chromatography electrospray ionization triple quadrupole mass spectrometry analysis confirmed the presence of the bis-Suc borate complex in wheat phloem with a concentration up to 220 microm. The 300.11/301.11 complex was putatively identified as a bis-N-acetyl-serine borate complex but its concentration was below the detection limits of the liquid chromatography electrospray ionization triple quadrupole mass spectrometer so could not be quantified. The presence of borate complexes in the phloem provides a mechanistic explanation for the observed phloem boron mobility in canola and wheat and other species that transport Suc as their primary photoassimilate.

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin.

Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected V(max) and K(app) of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected K(app) and the velocity, although it did not affect V(max). Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.

[Allelopathic effect of Corallina pilulifera on Heterosigma akashiwo and its responses to UV-B irradiation].

By the method of co-culture and using cell density as the main indicator, this paper studied the allelopathic effect of Corallina pilulifera on Heterosigma akashiwo and its responses to UV-B irradiation. Under normal condition, the fresh tissue and aqueous extracts of C. pilulifera had significant inhibitory effects on the growth of H. akashiwo (P < 0.05), indicating their allopathic effect on H. akashiwo, while the dry power and culture media filtrate of C. pilulifera had less effect (P > 0.05). After pre-treated with different dose UV-B radiation and then co-cultured with H. akashiwo, C. pilulifera had some changes in the allelopathic activity of its fresh tissue, dry powder, and aqueous extracts. High-dose UV-B radiation (3.0 J x m(-2)) induced the decrease of the allelopathic effect, whereas low-dose UV-B radiation (0.9 J x m(-2)) was in adverse (P < 0.05).

Epiphytic bacterial community composition on two common submerged macrophytes in brackish water and freshwater.

BACKGROUND: Plants and their heterotrophic bacterial biofilm communities possibly strongly interact, especially in aquatic systems. We aimed to ascertain whether different macrophytes or their habitats determine bacterial community composition. We compared the composition of epiphytic bacteria on two common aquatic macrophytes, the macroalga Chara aspera Willd. and the angiosperm Myriophyllum spicatum L., in two habitats, freshwater (Lake Constance) and brackish water (Schaproder Bodden), using fluorescence in situ hybridization. The bacterial community composition was analysed based on habitat, plant species, and plant part. RESULTS: The bacterial abundance was higher on plants from brackish water [5.3 x 10(7) cells (g dry mass)-1] than on plants from freshwater [1.3 x 10(7) cells (g dry mass)-1], with older shoots having a higher abundance. The organic content of freshwater plants was lower than that of brackish water plants (35 vs. 58%), and lower in C. aspera than in M. spicatum (41 vs. 52%). The content of nutrients, chlorophyll, total phenolic compounds, and anthocyanin differed in the plants and habitats. Especially the content of total phenolic compounds and anthocyanin was higher in M. spicatum, and in general higher in the freshwater than in the brackish water habitat. Members of the Cytophaga-Flavobacteria-Bacteroidetes group were abundant in all samples (5-35% of the total cell counts) and were especially dominant in M. spicatum samples. Alphaproteobacteria were the second major group (3-17% of the total cell counts). Betaproteobacteria, gammaproteobacteria, and actinomycetes were present in all samples (5 or 10% of the total cell counts). Planctomycetes were almost absent on M. spicatum in freshwater, but present on C. aspera in freshwater and on both plants in brackish water. CONCLUSION: Bacterial biofilm communities on the surface of aquatic plants might be influenced by the host plant and environmental factors. Distinct plant species, plant part and habitat specific differences in total cell counts and two bacterial groups (CFB, planctomycetes) support the combined impact of substrate (plant) and habitat on epiphytic bacterial community composition. The presence of polyphenols might explain the distinct bacterial community on freshwater M. spicatum compared to that of M. spicatum in brackish water and of C. aspera in both habitats.

[Allelopathic effects of Corallina pilulifera on red tide microalgae Heterosigma akashiwo].

Different concentration methanol-, acetone-, ether-, and chloroform extracts of Corallina pilulifera were used to study their growth inhibitory effects on red tide microalgae Heterosigma akashiwo. The results showed that methanol extract at relatively higher concentrations had the highest growth inhibitory activity and killed all H. akashiwo cells, while the other three kinds of organic solvent extracts had no apparent inhibitory effects, suggesting that the growth inhibitory substances in C. pilulifera had relatively high polarity. The methanol extract was partitioned to petroleum ether phase, ethyl acetate phase, butanol phase, and distilled water phase by liquid-liquid fractionation, and the bioassays on the activity of each fraction were carried out on H. akashiwo. It was found that petroleum ether phase and ethyl acetate phase had strong algicidal effects on H. akashiwo, suggesting that the fatty acids in C. pilulifera tissues might be one of the main allelochemicals.