Plasmalemma Voltage Noise in Chara corallina

Voltage noise analysis is applied to plasmalemma ion transport in Chara corallina. There is a component in the noise power spectrum that is probably associated with current fluctuations within passive transport channels, and another component that may be associated either with fluctuations in the number of open channels, or with active transport. The data allow the calculation of time constants that may be attributable to molecular level events in these transport processes.     

Cooperative behavior of K+ channels in the tonoplast of Chara corallina.

Spontaneous cooperatively of K+ channels is studied in excised patches of Chara corallina tonoplasts. Bar histograms (dwell time versus number of open channels) are constructed from the time series of current by means of the higher-order Hinkley detector (R. Schultze and S. Draber. 1993. J. Membr. Biol. 132:41-52). A statistical test, based on these bar histograms, shows that the channels are not independent. Further analysis reveals that the channels are cooperatively changing their open probability, which leads to the idea of cooperative mode shifting.     

Intercellular Transport and Photosynthetic Differentiation in Chara corallina

Intercellular transport of ¹⁴C-labelled photoassimilates, bothin isolated upper shoots and in isolated internode-branchletcomplexes of Chara corallina, was measured. The isolated uppershoots were composed of a primary apex, two mature internodes,and three branchlet whorls. A 10 min loading of the isolatedupper shoot with H¹⁴CO₃^– resulted in a greater accumulationof ¹⁴C in the apical complex and branchlets than in the internodes,while a subsequent 50 min chase with unlabelled solution inthe light resulted in a greater accumulation of ¹⁴C in internodesthan in other parts of the shoot. In the isolated internode-branchlet complex, when the apex wasnot detached, the amount of ¹⁴C transported from branchletsto internodes was about fives times that transported from internodesto branchlets. Removal of the apex resulted in a decrease intransport from branchlets to internodes and an increase in transportin the opposite direction. In an attempt to explain the mechanism of the polar transportof photosynthetically fixed carbon between branchlets and internodes,photosynthetic activities of both types of cells were investigated.Detached branchlets have higher photosynthetic ¹⁴C-fixationactivities than those of internodes. Chlorophyll contents, measuredin terms of surface area, in internodes and branchlets werealmost identical. The ribulose-l,5-bisphosphate carboxylase(RuBPCase) activity of branchlets was 1.6 times that of internodes,and the rate of ferricyanide-dependent evolution of oxygen inbranchlets was 1.4 times that in internodes. Key words: Chara, internode, branchlet, polar transport, photosynthesis     

Calcium-salinity interactions affect ion transport in Chara corallina

Detached internodes of Chara corallina survived in solutions containing 100 mol m^?3 NaCl when the external concentration of Ca²⁺ was greater than 1 mol m^?3. Na⁺ influx was roughly proportional to external Na⁺ up to 100 mol m^?3 NaCl. Na⁺ influx involved two components: a Ca²⁺-insensitive influx which allowed the passage of Na⁺ independently of external Ca²⁺; and a Ca²⁺-inhibitable mechanism where Na⁺ influx was inversely proportional to external Ca²⁺. The Ca²⁺-inhibitable Na⁺ influx was similar to the Ca²⁺-inhibitable K⁺ influx. Mg²⁺ and Ba²⁺ were able to substitute for Ca²⁺ in partially inhibiting Na⁺ influx in the absence of external Ca²⁺. The effect of Ca²⁺ appears specific to Na⁺ and K⁺ influx since the effects of a Ca²⁺-free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na⁺ influx via the Ca²⁺-inhibitable mechanism represents Na⁺ leakage through K⁺ channels and that cell death at high salinity occurs due to a cytotoxic Na⁺ influx via this mechanism.     

Quantitative Analysis of Intercellularly-Transported Photoassimilates in Chara corallina

Using a thin-layer chromatographic technique, we have identifiedthe photoassimilates that are transported intercellularly frombranchlets to internodes in Chara corallina. An internode-branchletcomplex having a primary apex was used in these experiments.After feeding 1 mol m³ NaH¹⁴CO₃ to a branchlet for 10 min, the¹⁴C-labelled photoassimilates (¹⁴C-photoassimilates) found inthe sol endoplasm of the branchlet were composed of sucrose,amino acids, malate, and sugarphosphates. The composition ofthe ¹⁴C-photoassimilates transported from the source branchletto the sink internode in 10 min was the same as that in thesol endoplasm of the source branchlet. From the proportion ofeach ¹⁴C-photoassimilate in both the source branchlet and thesink internode, it was deduced that the main photoassimilatesinvolved in the intercellular transport were sucrose and aminoacids. We found previously that polar transport of photoassimilatesoccurs from a branchlet to an internode with an apex. Determinationof the amount of sucrose, amino acids, glucose-6-phosphate,and malate in both branchlet and internode with or without anapex revealed that there were gradients in the concentrationsof sucrose, serine, and glutamic acid between the sol endoplasmof the two cells. The levels were higher in the branchlet andlower in the internode and the gradients decreased when theapex was detached. Therefore, it is concluded that sucrose andthese amino acids are the compounds involved in the polar intercellulartransport. Key words: Chara corallina, intercellular transport, photoassimilates, ¹⁴C     

The Uptake of Sugars by Chara corallina

The influx of several monosaccharides into Chara corallina wasstudied under varying conditions of temperature, in the presenceof inhibitors, and by the use of competition experiments. Itwas found that the mechanism of transport was stereospecific,and the complex interactions between the sugars were characteristicof a carrier-mediated system. There is reason to believe thatthe carrier involved in this system has a narrower range ofaffinities than is usually attributed to a sugar carrier, forglucose and fructose appear to have different sites of entry,though each can influence the uptake of the other. In additionthe carrier system is situated in the plasmalemma and maintainedby metabolic energy.     

Effects of oleic acid on ionic channels of plasma membranes of green alga Chara corallina

Extracellular application of 5–30 μM oleic acid (C_18:1:9cis) produced a significant decrease in the amplitude of Ca²⁺ currents and Ca²⁺-activated chloride currents in plasma membrane of green alga Chara corallina. Introduction of 10 μM oleic acid intracellularly suppressed the development of the Ca²⁺ current. This inhibition of the Ca²⁺ current was reversible, and the Ca²⁺ conductivity of the cell membrane was restored after the removal of oleic acid. There were no changes in the non-specific leakage current, which indicates the intactness of the lipid component of the membrane. An increase of the oleic acid concentration up to 50–100 μM in the extracellular solution induced almost complete and irreversible suppression of inward currents and caused a substantial increase in the non-specific leakage current and conductivity of plasma membrane, suggesting its disruption. We suggest that the mechanism of cytotoxicity of oleic acid may be related to transport across plasma membrane of oleic acid and its complexes with α-lactalbumin (HAMLET and La-OA).     

Calcium influx at the plasmalemma of Chara corallina

Influx of ⁴⁵Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La³⁺-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca²⁺ externally was 0.25–0.5 pmol·cm^-2·s^-1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K⁺. In cells in which the control flux was less than about 0.5 pmol·cm^-2·s^-1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm^-2·s^-1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K⁺), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm^-2·s^-1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm^-2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca²⁺ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6 artificial pondwater, pH 6, containing 0.4 mM KCl - 20 K-APW6 artificial pondwater, pH 6, containing 20 mM KCl - Ca_o external Ca²⁺     

Intracellular measurement of ammonium in Chara corallina using ion-selective microelectrodes

The study of ammonium (NH₄ ⁺) transport across plant cell membranes requires accurate measurement of NH₄ ⁺ gradients across subcellular gradients. We have developed an ammonium-selective microelectrode based on the ionophore nonactin. This electrode can detect NH₄ ⁺ activities (a_NH4) in vivo in the millimolar range in the presence of cytosolic levels of potassium, the main interfering ion. The electrode was used to measure intracellular a_NH4 in internodal cells of the giant alga Chara corallina. Results from cells incubated in media supplemented with 1 mM NH₄ ⁺ produced two populations, with means of 7.3 and 30.8 mM, respectively. HPLC analysis of vacuolar sap suggests the higher population represents vacuolar impalements, and the lower population can thus be assumed to be cytosolic. These results suggest a four-fold accumulation of NH₄ ⁺ in the vacuolar compartment of Chara. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tonoplast Anion Channel Activity Modulation by pH in Chara corallina

The patch-clamp technique was used to investigate regulation of anion channel activity in the tonoplast of Chara corallina in response to changing proton and calcium concentrations on both sides of the membrane. These channels are known to be Ca²⁺-dependent, with conductances in the range of 37 to 48 pS at pH 7.4. By using low pH at the vacuolar side (either pH_vac 5.3 or 6.0) and a cytosolic pH (pH_cyt) varying in a range of 4.3 to 9.0, anion channel activity and single-channel conductance could be reversibly modulated. In addition, Ca²⁺-sensitivity of the channels was markedly influenced by pH changes. At pH_cyt values of 7.2 and 7.4 the half-maximal concentration (EC ₅₀) for calcium activation was 100–200 μm, whereas an EC ₅₀ of about 5 μm was found at a pH_cyt of 6.0. This suggests an improved binding of Ca²⁺ ions to the channel protein at more acidic cytoplasm. At low pH_cyt, anion channel activity and mean open times were voltage-dependent. At pipette potentials (V _p) of +100 mV, channel activity was approximately 15-fold higher than activity at negative pipette potentials and the mean open time of the channel increased. In contrast, at pH_cyt 7.2, anion channel activity and the opening behavior seemed to be independent of the applied V _p. The kinetics of the channel could be further controlled by the Ca²⁺ concentration at the cytosolic membrane side: the mean open time significantly increased in the presence of a high cytosolic Ca²⁺ concentration. These results show that tonoplast anion channels are maintained in a highly active state in a narrow pH range, below the resting pH_cyt. A putative physiological role of the pH-dependent modulation of these anion channels is discussed. Received: 14 March 2001/Revised: 16 July 2001     

Fracture mechanics of the cell wall of Chara corallina

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K _1c was estimated to be 0.63 ± 0.19 MNm^−3/2, comparable to published values for grasses. Received: 4 April 2000 / Accepted: 21 July 2000     

Proteomic identification of putative plasmodesmatal proteins from Chara corallina

Plasmodesmata are channels that bridge the cell walls of plant cells, allowing regulated transport of molecules between neighbouring cells. We have used a proteomic strategy to identify putative plasmodesmata-associated proteins in the giant-celled green alga Chara corallina. Proteins were extracted from the plasmodesmata-rich nodal complexes and the middle of the long internodal cells, which do not contain plasmodesmata. Comparison of protein spot patterns generated by two-dimensional gel electrophoresis of both the soluble and cell wall fractions from the two cell types was done. Fifty-eight spots that were common to the nodal and internodal soluble fractions were analysed by matrix assisted laser desorption/ionisation-time of flight mass spectrometry, and peptide mass fingerprint data were used to search the database. Matches were made to four of these spots, in each case to housekeeping proteins. Further, a number of nodal specific spots were identified, 11 from the soluble fraction and nine from the wall fraction. These spots were excised from the gels and analysed by liquid chromatography tandem mass spectrometry to obtain peptide sequence. Database searches suggest that these spots include homologues to previously identified plasmodesmata-associated proteins cp-wap13 and heat shock cognate 70, as well as RNA-binding proteins, eukaryotic initiation factor 4A and a beta-1,3-glucanase. Several spots remained unidentified providing exciting new candidate plasmodesmata-associated proteins.     

Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata-associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.     

Cortical Microtubule Organization and Internodal Cell Maturation in Chara corallina

The arrangement of cortical microtubules, as documented by immunofluorescence and immunogold-silver enhancement, changes during the growth and maturation of giant internodal cells of Chara corallina, a process taking approximately 45 days. Transverse microtubules are found throughout growth along with a subset of distinctly non-transverse microtubules. During the second half of the growing period, when relative growth rates are diminishing, these non-transverse microtubules become more abundant but a few days prior to growth cessation, they are mostly absent. At about the time of growth cessation the microtubules, while retaining their locally parallel alignment, begin to show increasing deviation from the transverse axis. Eventually, a mosaic of locally parallel yet variably oriented fields of microtubules forms. Many days after growth stops, microtubules become shorter and less numerous and lose parallel alignment, leading to the formation of a random MT pattern.     

Intercellular Transport and Subcellular Distribution of Photoassimilates in Chara corallina

The mechanism of polar transport of ¹⁴C-photoassimilates betweenChara corallina branchlets and internodes was investigated usingan intracellular perfusion procedure. When the apex remained,more ¹⁴C-photoassimilates were transported from branchlet tointernode than in the opposite direction. When the apex wasdetached, this polarity disappeared. To study the polar transportmechanism, we examined the subcellular distribution of ¹⁴C-photoassimilatesin the three main intracellular compartments, the cortical chloroplastlayer, streaming sol endoplasm and vacuole. When the internode or branchlet of an internode-branchiet complexwas exposed to ¹⁴C-bicarbonate for 10 min, 70 to 80% of thetotal fixed carbon was found in the sol endoplasm. In the subsequent3 h chase, the ¹⁴C-photoassimilates in the sol endoplasm ofthe source cell decreased greatly to 20 to 40% of the totallabelled substances. When the apex remained, the branchlet showed higher photosyntheticactivity than the internode, which resulted in a concentrationgradient of 13·5 mol m^–3 in the sol endoplasm frombranchlet to internode after photosynthesis for 10 mm. In the3 h chase period, this gradient decreased to about 1·0mol m^–3. In the other three cases, involving transportfrom branchlet to internode when the apex was detached, or transportfrom internode with or without apex to branchlet, the intercellulargradient of ¹⁴C after 10 mm photosynthesis was about 70 molm^–3 in the sol endoplasm. Thus, when the apex remains,the greater gradient of photoassimilates between the sol endoplasmof branchlet and internode seems to be one of the reasons forpolar intercellular transport. Key words: Chara corallina, polar transport, ¹⁴C photoassimilates     

Calcium pectate chemistry controls growth rate of Chara corallina

Pectin, a normal constituent of cell walls, caused growth rates to accelerate to the rates in living cells when supplied externally to isolated cell walls of Chara corallina. Because this activity was not reported previously, the activity was investigated. Turgor pressure (P) was maintained in isolated walls or living cells using a pressure probe in culture medium. Pectin from various sources was supplied to the medium. Ca and Mg were the dominant inorganic elements in the wall. EGTA or pectin in the culture medium extracted moderate amounts of wall Ca and essentially all the wall Mg, and wall growth accelerated. Removing the external EGTA or pectin and replacing with fresh medium returned growth to the original rate. A high concentration of Ca2+ quenched the accelerating activity of EGTA or pectin and caused gelling of the pectin, physically inhibiting wall growth. Low pH had little effect. After the Mg had been removed, Ca-pectate in the wall bore the longitudinal load imposed by P. Removal of this Ca caused the wall to burst. Live cells and isolated walls reacted similarly. It was concluded that Ca cross-links between neighbouring pectin molecules were strong wall bonds that controlled wall growth rates. The central role of Ca-pectate chemistry was illustrated by removing Ca cross-links with new pectin (wall "loosening"), replacing vacated cross-links with new Ca2+ ("Ca2+-tightening"), or adding new cross-links with new Ca-pectate that gelled ("gel tightening"). These findings establish a molecular model for growth that includes wall deposition and assembly for sustained growth activity.     

Generation of action potentials in Chara corallina by turgor pressure changes

A depolarisation of the membrane potential difference (about-170 mV) of Chara corallina is observed in response to changes in cell turgor pressure using the pressure probe technique. The depolarisation occurs in phase with the pressure pulse (0.2 s duration) and is independent of the direction of the applied pressure gradient. This latter finding is in contradiction to results predicted on the basis of electro-kinetic phenomena. Pressure induced electrical leakages were ruled out by measuring the depolarisation in response to pressure in individual segments of the internode which were electrically isolated from one another. The changes in potential were recorded by external electrodes and an internal electrode which was positioned close to the micropipette of the pressure probe inserted through one of the electrically isolated nodes. The depolarisation in response to increasing positive or negative pressure gradients in the intact node region and in the intact middle segments was comparable to that monitored in the node region containing the pressure probe. Action potentials were initiated when the pressure gradients exceeded at least 2 bar. The action potentials were elicited at random in one of the two compartments adjacent to the node regions, but were never found to be initiated in the node regions themselves. The pressure-induced action potentials are explained in terms of an electro-mechanical compression (or expansion) of a local membrane area and discussed in their relevance to the propagation of pressure signals in response to water and salt stress in higher plants.Abbreviations PD potential difference     

Cytoplasmic streaming in Chara corallina studied by laser light scattering.

An apparatus is described by means of which the power versus frequency spectrum of the photomultiplier current can be obtained for laser light scattered by streaming cytoplasm in the algal cell Chara corallina. A Doppler peak is noted in the spectrum which is abolished when cytoplasmic streaming is arrested by electrical stimulation. For 5 cells of Chara, this simple laser-Doppler velocimeter gave streaming velocities (46-7 mum s-1, S.D. +/- 4-8 at 20 degrees C) similar to those obtained for the same cells using the light microscope (44-3 mum s-1, S.D. +/- 5-3 at 20 degrees C). A narrow distribution of streaming velocities is indicated. The technique described provides a rapid, quantitative assay of the in vivo rheological properties of cytoplasm.     

Through pore diameter in the cell wall of Chara corallina.

Determination of pore size of the cell wall of Chara corallina has been made by using the polyethylene glycol (PEG) series as the hydrophilic probing molecules. In these experiments, the polydispersity of commercial preparation of PEGs was allowed for. The mass share (gamma(p)) of polyethylene glycol preparation fractions penetrating through the pores was determined using a cellular 'ghost', i.e. fragments of internodal cell walls filled with a 25% solution of non-penetrating PEG 6000 and tied up at the ends. In water, such a 'ghost' developed a hydrostatic pressure close to the cell turgor which persisted for several days. The determination of gamma(p), for polydisperse polyethylene glycols with different average molecular mass (M) was calculated from the degree of pressure restoration after water was replaced by a 5-10% polymer solution. Pressure was recorded using a dynamometer, which measures, in the quasi-isometric mode, the force necessary for the partial compression of the 'ghost' in its small fragment. By utilizing the data on the distribution of PEG 1000, 1450, 2000, and 3350 fractions over molecular mass (M), it was found that gamma(p), for these polyethylene glycols corresponded to the upper limit of ML=800-1100 D (hydrodynamic radius of molecules, r(h)=0.85-1.05 nm). Thus, the effective diameter of the pores in the cell wall of Chara did not exceed 2.1 nm.