Intracellular Distribution of Free Amino Acids between the Vacuolar and Extravacuolar Compartments in Internodal Cells of Chara australis

Distribution of free amino acids between the vacuolar and extra-vacuolar(cytoplasmic) compartments in internodal cells of Chara australiswas studied. Under the control conditions (14-h light : 10-hdark), most (90%) of the cellular free amino acids were foundin the extra-vacuolar compartment. The reverse was true forammonia. The major amino acids were isoasparagine, alanine,glutamic acid, aspartic acid, serine and glycine. The contentsof hydrophobic and basic amino acids were minor and relativelygreater proportions were found in the vacuole except when theircontents were extremely low. When cells were kept for 3 days under continuous light or incontinuous darkness, the total free amino acid content increasedto about 120% (light) and about 150% (dark) that of the control.These increases mainly took place in the vacuole, but the aminoacid species responsible for the increments differed with thelight conditions. In contrast, the cytoplasmic content was relativelyconstant (50–60 mM) even under continuous light or darkness.The results suggest that the vacuole acts in the homeostasisof the cytoplasmic amino acid content. As anion, amino acidsin the cytoplasm compensated for about 10–20% of the reported"anion deficiency" in the cytoplasm. (Received June 7, 1984; Accepted September 11, 1984)     

The Effects of pH on the Ionic and Electrical Properties of the Internodal Cells of Chara australis

The effects of pH on the resting and action potentials and onthe fluxes of potassium, sodium, and chloride across the membranesof internodal cells of Chara australis have been investigated. Experiments were carried out in an artificial pond water (A.P.W.)of standard composition: CaCl₂, 01 mM; KCl, 0.1 mM; NaCl, 1.0mM. The resting potential decreased as the pH was lowered from6.5, being depolarized by about 75 mV at pH 4.5. Measurementsof the ion fluxes as a function of pH suggested that this depolarizationwas caused by an increase in the permeability to sodium anda decrease in permeability to potassium at pH 4.5. Action potentialsof constant peak value can be elicited for some time at pH 4.5,but after 20 min or so the cell becomes refractory. All theseeffects on resting and action potentials are fully reversible.We briefly speculate about the mechanism of these pH effects.     

Malate Accumulates in the Vacuole of Chara australis During Uptake of Ammonium From Chloride-free Solution

Internodal cells of Chara australis can accumulate ammoniumto high concentrations (10 to 70 mol m^–3) in their vacuoles.When Cl^– is included in the bathing solution, changesin the cellular concentrations of ammonium, K⁺, Cl^– andNa⁺ have been shown to meet the requirements for electroneutralityand to account for the changes in vacuolar osmotic pressureassociated with ammonium uptake. If accumulation occurs in theabsence of external Cl^–, however; changes in the inorganicions do not meet these criteria. Malate is found in the vacuolesof cells accumulating amine in the absence of external Cl^–and its presence (at 0·5 to 8·5 mol m^–3)allows us to account for electroneutrality and for changes inthe osmotic potential. Key words: Malate, Chara, electroneutrality, ammonium     

Conduction Velocity and Blockage of Action Potential in Chara Internodal Cells

Conduction velocity of the action potential in Chara brauniiinternodal cells was 0.21 ?0.05 cms in moist air and 1.5?0.9cms in artificial pond water (APW). The action potential waspropagated at an almost constant velocity along the cell inmoist air except within 0.3 cm from an end of the cell, whereasin APW, the velocity increased to 5.7 ? 2.3 cms within 1.8 cmfrom the end of the cell. When part of the cell was put in moistair and the other part was immersed in APW, conduction of theaction potential in moist air decreased in velocity and sometimesstopped in the vicinity of the boundary between moist air andAPW. Some cells from the plants collected in late autumn towinter generated an action potential which could not propagatein moist air. In these cells, an increase in the threshold andpartial cessation of protoplasmic streaming were observed. ¹Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Seiryo-machi, Sendai 980 ²Present address: Biology Laboratory, Kyoritsu Women's University,Hachioji, Tokyo 193, Japan. (Received March 10, 1987; Accepted July 6, 1987)     

Entry of Methylammonium and Ammonium Ions into Chara Internodal Cells

Low concentrations of ammonia and methylamine greatly affectmembrane transport by, and the electrical properties of, cellsof Chara corallina (=C. australis). In the presence of theseamines, influx of Cl^– and efflux of K⁺ increase and alarge depolarizing current flows through the cell membrane. Measurements with [¹⁴C]methylamine show that methylamine isabsorbed rapidly over a wide pH range, and that the absorptionisotherm is complex. Methylamine influx is not affected by presenceor absence of Cl^–, K⁺, or Na⁺, but is decreased by additionof . The depolarizing current is associated with a small increase in membrane conductance, except at highpH, and both these effects are reversible. The current showssaturation with increasing amine concentration; when methylamineis 10–12 times more concentrated than ammonia, it producesa current of the same magnitude. The results show that the amines enter the cells as cations( or CH₃) except where external pH is high, and that a specific, selective electrogenicporter is involved. There is no need to invoke active transport,as there is no evidence that internal amine concentrations exceedthe equilibrium (Nernst) concentrations.     

Photosynthetic Fixation of 14Carbon by Internodal Cells of Chara corallina

Maximum fixation rates of 120 and 60 pmol cm^–2 s ^–1wereobtained when exogenous carbon was supplied as ¹CO₂ and H¹⁴CO₃^–respectively. These values are considerably higher than thosepreviously reported for this species. A kinetic analysis wasperformed on this data. Substrate saturation in the concentrationrange 1.0–1.5 mM was observed for both CO₂ and HCO₃^– In the presence of exogenous CO², a linear relationship wasobserved between light intensity and fixation while the HCO₃^–relationship was slightly sigmoidal. Fixation saturated at intensitiesof 15–20 W m^–2 and 13–15 W m^–2 for exogenous¹⁴CO² and H¹⁴CO₃^–respectively. The presence, in this species, of an extremely active HCO₃^–transport system, situated in the plasmalemma, demonstratesthat when alkaline solutions are employed the involvement ofthis ion cannot be ignored during electrical studies on thismembrane. The maximum H¹⁴CO₃^– influxes obtained duringthis study are the largest ionic fluxes measured for any Characeanspecies. It was demonstrated that CO² for fixation can be supplied simultaneouslyby gaseous diffusion and HCO₃^– transport (cf. Raven, 1968).Inhibition of H¹⁴CO₃^– influx was observed in the presenceof Tris, Tricine, and borate buffers, and CO₃^{2 –} alsoappeared to act as a strong inhibitor. The possible mechanism(s)by which this inhibition occurs is discussed.     

Autonomous Local Area Control over Membrane Transport in Chara Internodal Cells

Internodal cells of Chara were separated with a Plexiglas divider into two segments and the vibrating probe was used to investigate the extracellular current profiles that formed along these two surfaces. Treating one segment of the Chara cell with K⁺ concentrations greater than 2 millimolar caused a dramatic reduction in the extracellular current pattern in this compartment. Concentrations of 5, 10, and 20 millimolar K⁺ were used to establish that a normal current profile could be maintained along the cell surface in the control compartment, whereas the extracellular current profile was strongly reduced along the entire cell surface that was located in the second, high-K⁺ compartment. Simultaneous measurements of the membrane potential in the two segments of the divided Chara cell established that, in the presence of elevated K⁺ concentrations, a longitudinal voltage gradient of up to 60 millivolts was maintained. Experiments in which the pH value in one compartment was either decreased (pH 6.0) or increased (pH 11) gave rise to a reduced extracellular current profile along this segment of the cell, whereas the pattern in the control segment remained unaltered. These results are discussed in terms of the cellular spatial control system that must function to regulate the regions of outward and inward current, and the concept of autonomous local area (domain) control is presented.     

N-Ethylmaleimide Blocks the H+ Pump in the Plasma Membrane of Chara corallina Internodal Cells

The sulfhydryl (SH) modifying reagent N-ethylmaleimide (NEM)was applied to the internodal cells of Chara corallina to studythe role of SH residues in the activity of the plasma membraneH⁺ pump. NEM (1 µM) caused a marked depolarizing shiftof the resting potential by 6410mV (n=7) together with depressionof the conductance peak at around —200 mV, indicatinga marked depression of the H⁺ pump activity. This effect ofNEM was partly reversible, the membrane repolarized and theconductance peak was restored after extracellular washing. TheH⁺ pump inhibitor, dicyclohexylcarbodiimide (DCCD), caused noadditive membrane depolarization and/or depression of the H⁺pump conductance, in the presence of NEM. This suggests thatNEM blocks the H⁺ pump and that SH residues play a pivotal rolein maintaining the H⁺ pump activity in Chara corallina. (Received April 10, 1993; Accepted July 29, 1993)     

Subcellular Distribution of Free Amino Acids in Relation to Protein Synthesis in Cells of Chara corallina

The influence of protein-synthesis inhibitors on the subcellular distribution of free amino acids was studied in internodal cells of Chara corallina. Use of an intracellular perfusion technique allowed separate measurements of amino acids in the vacuole, in the flowing sol endoplasm and in the gel layer. The sol endoplasm predominantly represents the cytosol, while the gel layer is occupied, for the most part, by chloroplasts. When cells were treated with 0.5 mM chloramphenicol (CRP) in the dark, both the total concentration of amino acids and the subcellular distribution were almost the same as in cells without treatment. In the light, however, the subcellular distribution changed dramatically, although the total concentration of amino acids was unchanged. The vacuolar concentration of amino acids was 3 times greater in CRP-treated cells than in the control. The concentrations of amino acids in the sol endoplasm and in the gel layer were only half of those in the control. Amino acid permeability of the chloroplast envelope, measured using the perfused internodal cells, slightly increased after the CRP treatment in the light. Time-dependent changes in concentrations of amino acids in the CRP-treated cells were also measured in the light. The total concentration of amino acids in the cytoplasm gradually decreased, while that in the vacuole increased commensurately. The concentration and/or subcellular distribution of alanine, glutamine, glutamate and glycine changed dramatically. The concentration of alanine increased considerably both in the vacuole and in the cytoplasm. The cytoplasmic concentration of glutamine increased transiently within 1 ?2 h after treatment with CRP. The cytoplasmic concentrations of glutamate and glycine decreased. Although the concentrations of some amino acids changed so markedly both in the vacuole and cytoplasm, only small differences in the activities of glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and glutamine synthetase were detected between the control and the CRP-treated cells.     

Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

+ concentration ([K⁺]_o) on the membrane potential (E_m) of Chara corallina was studied. E_m more negative than -100 mV was maintained even at 100 mM [K⁺]_o. Addition of Ca²⁺ to the external medium further increased this tendency. However, E_m responded sensitively to the increase in [K⁺]_o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K⁺]_o. In the presence of 100 mM K⁺, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K⁺ at high concentration was discussed. Received 3 October 2000/ Accepted in revised form 6 January 2001     

Changes in the Subcellular Distribution of Free Amino Acids in Relation to Light Conditions in Cells of Chara corallina

Subcellular concentrations of free amino acids in internodal cells of a Characeae, Chara corallina, were measured in the dark and in the light. Using an intracellular perfusion technique, we measured concentrations of amino acids in the vacuole, in the flowing sol endoplasm and in the cortical gel layer. The sol endoplasm was predominantly the cytosol. On the basis of microscopic observations, the gel layer appeared to be occupied predominantly by a layer of chloroplasts, while the sol endoplasm was free from chloroplasts. Both in the light and in darkness, the major amino acids in the internodal cells were isoasparagine, glutamic acid, aspartic acid, serine, glycine and alanine, as reported by Sakano and Tazawa (1984). The same major amino acids are found in each of the three compartments. The pattern of distribution of amino acids in the vacuole was similar to that in the sol endoplasm, but quite different from that in the gel layer. The total level of amino acids in the light was lower than that in darkness. The amino acid composition did not change very much, but the subcellular distribution of amino acids differed significantly between cells subjected to illumination and those kept in the dark. Concentrations of amino acids in both the vacuole and the gel layer decreased, whereas those in the sol endoplasm were almost constant.     

Ultraviolet Microscopy of Purines and Amino Acids in the Vacuole of Candida albicans

Ultraviolet (UV) microscopy was used to study the capacity of yeast (ATCC 10231 and 10261) and filamentous (ATCC 10259) strains of Candida albicans to accumulate UV-absorbing materials from a medium supplemented with purines, pyrimidines, amino acids, or related compounds as the main nitrogen source. All strains accumulated UV-absorbing compounds when adenine, adenosine, isoguanine, xanthine, or uric acid was supplied as a nitrogen source, but they did not accumulate UV-absorbing compounds when pyrimidines were supplied. The filamentous strain accumulated UV-absorbing material from medium supplemented with hypoxanthine, but the yeast strains did not. In contrast, the yeast strains accumulated more UV-absorbing material than did the filamentous strain when guanine was the nitrogen source. Yeast strain 10231 not only accumulated UV-absorbing material from tyrosine-supplemented medium, but it became filamentous in form as well. Yeast strain 10261 and filamentous strain 10259 did not accumulate detectable amounts of UV-absorbing material, nor was their morphology noticeably affected by the supplement. The two yeast strains accumulated more lipid than the filamentous strain when they were incubated in a nitrogen-deficient medium.     

Electrotonic Coupling between Adjacent Internodal Cells of Chara braunii: Transmission of Action Potentials beyond the Node

Many nodal cells are interposed between two internodal cellsof Chara braunii. When an action potential conducted in an internodereached the node, no electrical activation in the nodal cellscould be found, although an area of the membrane bordering thenodal cells in this internode was partially activated (end-membraneaction potential). When the action potential approached thenode along the stimulated internode, an electrotonic potentialchange (depolarization) was produced in the other internode.This depolarization was greatly depressed by the end-membraneaction potential of the stimulated internode, so that hardlyany transmission took place. The ratio of the potential changein the surface membrane of the adjoining ("postsynaptic") internode(cell b) to that of the stimulated one (cell a), the couplingratio, e_b/e_a, can be estimated from a simple equivalent circuitof the nodal region composed of two surface-membrane resistances(R_a, R_b) and intercellular resistance (R_n). If R_n remains thesame, a higher ratio should be produced with a larger R_b, butthe ratio does not depend on any change in R_a, which could beproved experimentally. Transmission of the action potentialbeyond the node was frequent when the coupling ratio was increasedand when the threshold that elicits the action potential waslowered by immersing the node in a K or Na salt solution. ¹ Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Sendai 980, Japan. (Received December 1, 1980; Accepted January 23, 1981)     

Electrotonic Transmission of an Action Potential between Two Separated Internodal Cells of Chara through a Bridge

Two separated internodal cells of Chara braunii were broughtinto contact with each other longitudinally at their ends andconnected by another pathway composed of a metal bridge beyondthe region of intercellular contact. A conducted action potentialthat arrived at one foot of the bridge electrotonically depolarizedthe other foot of the bridge in the connected cell. The electriccoupling ratio (0.07?0.03), the ratio of the change in the membranepotential of one cell to that of the other cell, was too smallto allow transmission of an action potential. Two cells wereplaced in parallel and connected with two liquid bridges orpools, a' and b'. When the action potential of one cell wasconducted through one connecting pool (pool a)', the other cellwas depolarized electrotonically by the action current via theother connecting pool (pool b'). The coupling ratio was increasedto 0.26?0.07 by the solution bridge, but transmission of theaction potential was rarely observed. Application of 1 mM KC1to pools a' and/or b' slightly improved the frequency of transmissionof the action potential. When pool b' contained 5% urethane,the coupling ratio increased to 0.31?0.08 and transmission ofthe action potential was frequent. (Received August 24, 1989; Accepted March 14, 1990)     

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.     

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ¹-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na⁺-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means.     

Effects of Lanthanum on Calcium and Magnesium Contents and Cytoplasmic Streaming of Internodal Cells of Chara corallina

Biological and environmental effects of lanthanide series of elements have received much attention recently due to their wide applications. In this study, effects of La³⁺ treatments on calcium and magnesium concentrations as well as cytoplasmic streaming of internodal cells of Chara corallina were investigated. At all treatment concentrations (10, 100, and 1,000 μM), La³⁺ significantly decreased calcium concentrations in the cell-wall fractions after 5-h treatments. Calcium concentrations in the cell contents and magnesium concentrations in the cell-wall fractions were reduced by 100 and 1,000 μM La³⁺ treatments. However, cytoplasmic streaming as an indicator of [Ca²⁺]_cyt was only inhibited at the highest La³⁺ concentration (1,000 μM). The results suggest that La³⁺ may affect cellular calcium homeostasis by actions other than as a simple Ca²⁺ antagonist. La³⁺ could partially compensate for calcium deficiency at certain concentrations.     

The regulation of ammonia uptake in Chara australis

The regulation of ammonia uptake was investigated in internodalcells of the freshwater alga Chara australis. Ammonia uptakewas estimated by monitoring (i) its depletion from the bathingsolution, (ii) the uptake of radiolabelled methylamine, an analogueof ammonia, and (iii) depletion of ammonia in the unstirredlayer with the microelectrode ion-flux estimation technique(MIFE). Distribution of methylamine (¹⁴CH₃NH₃⁺) between thevacuole and cytoplasm was estimated with efflux analysis. Whencells were bathed continuously in solutions containing ammoniaor methylamine, the uptake rates of both amines decreased over12 to 48 h despite the continuing existence of a large electrochemicalgradient favouring influx of the NH⁺₄ and CH₃NH⁺₄ cations. Treatmentwith 1.0 to 10.0 mM MSX, an inhibitor of glutamine synthetase,caused the internal ammonia concentration to rise and reducedthe subsequent uptake of ammonia and methylamine by up to 70%within 2 h. These results suggest that the permease facilitatingNH⁺₄/CH₃NH⁺₄ influx is under feedback or kinetic regulationfrom either internal ammonia or an intermediate of nitrogenassimilation. Treatment with metabolic inhibitors (CCCP, azide and DCMU) andsome weak acids (DMO and butyric acid) for 30 to 60 min inhibitedmethylamine uptake, but the changes in the electrical potentialdifference across the plasma membrane could not account forthe magnitude of inhibition. The rate of cytopiasmic streaming,which is an indicator of the cellular ATP concentration in Chara,was inhibited by many of these treatments. However, under certainconditions of external pH and concentration, butyric acid couldreversibly inhibit ammonia and methylamine uptake without affectingcytoplasmic streaming, demonstrating that a decrease in cytoplasmicATP concentration was not responsible for the inhibition. Theeffect of butyric acid was rapid, causing a 60% inhibition ofuptake in 15 min. We conclude that weak acids can inhibit theNH⁺₄/CH₃NH⁺₄ permease by acidifying the cytoplasm and suggestthat this may also explain the effects of the metabolic inhibitorson ammonia and methylamine uptake. Key words: Ammonia, methylamine, uptake, regulation, Chara     

The Electrical Resistance of the Tonoplast of Chara australis

A critical investigation of the method usually used for measuringthe electrical resistance of the tonoplast shows that the valuethus obtained is always higher than the true value.     

Exogenous melatonin affects photosynthesis in characeae Chara australis

Melatonin was found in the fresh water characeae Chara australis. The concentrations (~4 μg/g of tissue) were similar in photosynthesizing cells, independent of their position on the plant and rhizoids (roots) without chloroplasts. Exogenous melatonin, added at 10 μM to the artificial pond water, increased quantum yield of photochemistry of photosystem II by 34%. The increased efficiency appears to be due to the amount of open reaction centers of photosystem II, rather than increased efficiency of each reaction center. More open reaction centers reflect better functionality of all photosynthetic transport chain constituents. We suggest that melatonin protection against reactive oxygen species covers not only chlorophyll, but also photosynthetic proteins in general.