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The chloroplast genome sequence of Chara vulgaris sheds new light into the closest green algal relatives of land plants.

The phylum Streptophyta comprises all land plants and six monophyletic groups of charophycean green algae (Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales, and Charales). Phylogenetic analyses of four genes encoded in three cellular compartments suggest that the Charales are sister to land plants and that charophycean green algae evolved progressively toward an increasing cellular complexity. To validate this phylogenetic hypothesis and to understand how and when the highly conservative pattern displayed by land plant chloroplast DNAs (cpDNAs) originated in the Streptophyta, we have determined the complete chloroplast genome sequence (184,933 bp) of a representative of the Charales, Chara vulgaris, and compared this genome to those of Mesostigma (Mesostigmatales), Chlorokybus (Chlorokybales), Staurastrum and Zygnema (Zygnematales), Chaetosphaeridium (Coleochaetales), and selected land plants. The phylogenies we inferred from 76 cpDNA-encoded proteins and genes using various methods favor the hypothesis that the Charales diverged before the Coleochaetales and Zygnematales. The Zygnematales were identified as sister to land plants in the best tree topology (T1), whereas Chaetosphaeridium (T2) or a clade uniting the Zygnematales and Chaetosphaeridium (T3) occupied this position in alternative topologies. Chara remained at the same basal position in trees including more land plant taxa and inferred from 56 proteins/genes. Phylogenetic inference from gene order data yielded two most parsimonious trees displaying the T1 and T3 topologies. Analyses of additional structural cpDNA features (gene order, gene content, intron content, and indels in coding regions) provided better support for T1 than for the topology of the above-mentioned four-gene tree. Our structural analyses also revealed that many of the features conserved in land plant cpDNAs were inherited from their green algal ancestors. The intron content data predicted that at least 15 of the 21 land plant group II introns were gained early during the evolution of streptophytes and that a single intron was acquired during the transition from charophycean green algae to land plants. Analyses of genome rearrangements based on inversions predicted no alteration in gene order during the transition from charophycean green algae to land plants.

Base Sequence↗

The motility of Chara corallina myosin was inhibited reversibly by 2,3-butanedione monoxime (BDM).

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

Chara↗

Growth towards light as an adaptation to high light conditions in Chara branches.

Growth of plants or plant organs towards more light is commonly interpreted as an adaptation to low light conditions. Here, we show for the first time, in a study of charophyte branches, a growth-based orientation towards light functioning as a mechanism to protect the plant from excessive light. Two Chara species were exposed to five different intensities of photosynthetically active radiation and species traits and pigmentation were measured. Branches of plants exposed to higher light intensities were convergent and pointed steeply upwards, whereas those exposed to lower light intensities grew nearly straight and were less inclined. Only branches that increased in length during the experiments reacted to differences in light intensity. This indicates that branch orientation is determined by a light-dependent growth reaction. Orientation of charophyte branches towards light is accompanied by a decrease in chlorophyll a (Chla) content and a lower Chla : carotenoid ratio, which clearly indicates that the plant is taking protective measures against potentially damaging excess light conditions. We suggest that the growth-based orientation of Chara branches towards light may protect sexual organs, which grow on adaxial branch sides, from light damage. In addition, the upward orientation of branches might lead to increased light transmission within dense charophyte beds, thus enabling an enhanced gross production.

Adaptation, Physiological↗

[Modelling of pattern formation and oscillations in pH and transmembrane potential near the cell membrane of Chara corallina].

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

Algal Proteins↗

Light-triggered pH banding profile in Chara cells revealed with a scanning pH microprobe and its relation to self-organization phenomena.

When exposed to light, Characean cells develop a pattern of alternating alkaline and acid bands along the cell length. The bands were identified with a tip-sensitive antimony pH microelectrode positioned near one end of Chara internode at a distance of 50-100 microm from the cell wall. The stage with Chara cell was moved along its longitudinal axis at a computer-controlled speed (100 or 200 microm s(-1)) relative to the pH probe over a distance of 50 mm. Under sufficient uniform illumination of the cell (from 100 to 2.5 Wm(-2)), the homogeneous pH distribution becomes unstable and a banding pattern is formed, the spatial scale of which decreases with the light intensity. If the cell is locally illuminated, bands are formed only in the region of illumination. It is shown that the inhibition of cyclosis by cytochalasin B leads to the disappearance of the banding pattern. The addition of ammonium (weak base) inhibited the banding pattern, whereas acetate (weak acid) alleviated the inhibitory effect of ammonium and restored the pH banding. A model explaining the observed phenomena is formulated in terms of proton concentration outside and bicarbonate concentration inside the cell. It contains two diffusion equations for the corresponding ions with nonlinear boundary conditions determined by ion transport processes across the cell membrane. The model qualitatively explains most of the experimental observations. It describes the dependence of the pattern characteristics on the light intensity and reveals the role of cyclosis in this phenomenon.

Antimony↗

Negative gravitropism in Chara protonemata: a model integrating the opposite gravitropic responses of protonemata and rhizoids.

The unicellular protonema of Chara fragilis Desv. was investigated in order to establish a reaction chain for negative gravitropism in tip-growing cells. The time course of gravitropic bending after stimulation at angles of 45 degrees or 90 degrees showed three distinct phases of graviresponse. During the first hour after onset of stimulation a strong upward shift of the tip took place. This initial response was followed by an interval of almost straight growth. Complete reorientation was achieved in a third phase with very low bending rates. Gravitropic reorientation could be completely abolished by basipetal centrifugation of the cells, which lastingly removed conspicuous dark organelles from the protonema tip, thus identifying them as statoliths. Within minutes after onset of gravistimulation most or all statoliths were transported acropetally from their resting position 20-100 micrometers from the cell apex to the lower side of the apical dome. This transport is actin-dependent since it could be inhibited with cytochalasin B. Within minutes after arrival of the statoliths, the apical dome flattened on its lower side and bulged on the upper one. After this massive initial response the statoliths remained firmly sedimented, but the distance between this sedimented complex and the cell vertex increased from 7 micrometers to 22 micrometers during the first hour of stimulation and bending rates sharply declined. From this it is concluded that only statoliths inside the apical dome convey information about the spatial orientation of the cell in the gravitropic reaction chain. After inversion of the protonema the statoliths transiently arranged into a disk-shaped complex about 8 micrometers above the vertex. When this statolith complex tilted towards one side of the apical dome, growth was shifted in the opposite direction and bending started. It is argued that the statoliths intruding into the apical dome may displace a growth-organizing structure from its symmetrical position in the apex and may thus cause bending by bulging. In the positively gravitropic Chara rhizoids only a more stable anchorage of the growth-organizing structure is required. As a consequence, sedimented statoliths cannot dislocate this structure from the vertex. Instead they obstruct a symmetrical distribution of cell-wall-forming vesicles around the structure and thus cause bending by bowing.

Actin Cytoskeleton↗

Microscopic elements of electrical excitation in Chara: transient activity of Cl- channels in the plasma membrane.

The plasma membrane of Chara corallina was made accessible for patch pipettes by cutting a small window through the cell wall of plasmolyzed internodal cells. With pipettes containing Cl- as Ca2+ or Ba2+ (50 or 100 mM), but not as Mg2+ or K+ salt, it was possible to record in the cell-attached mode for long periods with little channel activity, randomly interspersed with intervals of transient activation of two Cl- channel types (cord conductance at +50 mV: 52 and 16 pS, respectively). During these periods of transient channel activity, variable numbers (up to some 10) of the two Cl- channel types activated and again inactivated over several 100 msec in a coordinated fashion. Transient Cl- channel activity was favored by voltages positive of the free running membrane voltage (> -45 mV); but positive voltage alone was neither a sufficient nor a necessary condition for activation of these channels. Neither type of Cl- channel was markedly voltage dependent. A third, nonselective 4 pS channel is a candidate for Ca2+ translocation. The activity of this channel does not correlate in time with the transient activity of the Cl- channels. The entire set of results is consistent with the following microscopic mechanism of action potentials in Chara, concerning the role of Ca2+ and Cl- for triggering and time course: Ca2+ uptake does not activate Cl- channels directly but first supplies a membrane-associated population of Ca2+ storage sites. Depolarization enhances discharge of Ca2+ from these elements (none or few under the patch pipette) resulting in a local and transient increase of free Ca2+ concentration ([Ca2+]cyt) at the inner side of the membrane before being scavenged by the cytoplasmic Ca2+ buffer system. In turn, the transient rise in [Ca2+]cyt causes the transient activity of those Cl- channels, which are more likely to open at an elevated Ca2+ concentration.

Cell Membrane↗

The uptake and metabolism of urea by Chara australis: IV. Symport with sodium--a slip model for the high and low affinity systems.

We have previously investigated the electrogenic influx of urea in Chara, and the urea- and sodium-dependent membrane current. We have shown that there is a sodium-stimulated component of urea influx and a urea-stimulated component of sodium influx, and that these are of the same size. We conclude that the electrogenic inward transport of urea, and of its analogues acetamide and acrylamide, is by sodium symport, with a stoichiometric ratio of 1:1. The kinetics of the fluxes and currents show two different KM values for sodium in different cells and two different kinds of kinetics for the effect of urea on membrane current, one of which fits the Michaelis-Menten equation, while the other shows a maximum and fits the difference of two Michaelis-Menten terms, suggesting a phenomenon like cis-inhibition. Similarities in kinetic characteristics between the inhibitory site and the electrically silent uptake site (System II) lead us to suggest that the same protein may be responsible for both the low-KM, electrogenic influx of urea (System I) and the high-KM, electrically silent influx by System II. We suggest a "slip" model for urea uptake in Chara.

Acetamides↗

Ca2+-dependent deposits at the plasmalemma of Chara internodal cells.

Electron dense deposits (EDD) were observed on the extracellular side of the plasmalemma of Chara internodal cells by a calcium-glutaraldehyde fixation technique. The number and size of EDD were greatly increased when cells had been preincubated in Ca2+-enriched medium before fixation. The addition of Na+, Mg2+, or La3+ instead of Ca2+ in incubation and fixation media produced no deposits at all, Sr2+ caused deposits with similar distribution to those formed by Ca2+, and Ba2+ addition resulted in deposits localized at different sites within the cell. Microprobe analysis of single EDD from Ca2+ incubated cells ascertained the presence of calcium in these deposits. Possible functions of the Ca2+-binding sites at the plasma membrane of Chara cells are discussed.

Calcium↗

Electrical tolerance (breakdown) of the Chara corallina plasmalemma: II. Inductive property of membrane and effects of pHo and impermeable monovalent cations on breakdown phenomenon.

Changes in the chord conductance G and the membrane electromotive force Em in the so-called breakdown region of large negative potential of the Chara plasmalemma were analyzed in more detail. In addition to the increase in G, the voltage sensitivity of the change in G increased, which was the cause of marked inductive current in the breakdown region. The break-down potential, defined as a critical potential at which both low and high slope conductances of the I-Vm relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pHo in a range between 5 and 9. The Chara plasmalemma was electrically most tolerant around pHo 7. In some cells Em shifted to a positive level as large as +50 approximately +70 mV during the breakdown phenomenon. Such a large positive shift of Em is caused mainly by the increase in conductance of Cl- and partly Ca2+ and K+.

Cations, Monovalent↗

Voltage dependence of the Chara proton pump revealed by current-voltage measurement during rapid metabolic blockade with cyanide.

It is generally agreed that solute transport across the Chara plasma membrane is energized by a proton electrochemical gradient maintained by an H(+)-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H(+)-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the true I-V profile for the pump when treatments alter the thermodynamic pressure on transport. We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cell I-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl3, so that steady-state currents could be recorded under voltage clamp between -400 and +100 mV. Exposures to 1 mM NaCN (CN) and 0.4 mM salicylhydroxamic acid (SHAM) depolarized (positive-going) Chara membrane potentials by 44-112 mV with a mean half time of 5.4 +/- 0.8 sec (n = 13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3 +/- 0.9 sec [( ATP]t = 0, 0.74 +/- 0.3 mM; [ATP]t = infinity, 0.23 +/- 0.02 mM). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses of dI-V curves (+/- CN + SHAM) as well as of families of I-V curves taken at times during CN + SHAM exposures indicated a stoichiometry for the pump of one charge (H+) transported per ATP hydrolyzed and an equilibrium potential near -420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60-75% of the total membrane conductance near Vm. Complementary results were obtained also in fitting previously published I-V data gathered over the external pH range 4.5-7.5. Kinetic features deduced for the pump were dominated by a slow step preceding H+ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops from Chara australis.

Patch-clamp studies of cytoplasmic drops from the charophyte Chara australis have previously revealed K+ channels combining high conductance (170 pS) with high selectivity for K+, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K+ greater than Rb+ greater than NH4+ much greater than Na+ and Cl-. Divalent cytosolic ions reduce the K+ conductance of this channel and alter its K+ gating in a voltage-dependent manner. The order of blocking potency is Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. The channel is activated by micromolar cytosolic Ca2+, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca2+ needed to open the channel. The possible role of the Ca(2+)-activated K+ channel in the tonoplast of Chara is discussed.

Calcium↗

Cytoplasmic streaming and gravity sensing in Chara internodal cells.

Since the nineteenth century, the merits of two alternate models for explaining the mechanism of plant gravity perception have been discussed. The gravitational pressure model states that plant cells perceive gravity by sensing their relative buoyancy to that of the surrounding medium, whereas the more popular starch-statolith model states that intracellular sedimenting particles act as gravity sensors. Vertically-oriented Chara internodal cells exhibit a gravity dependent polarity of cytoplasmic streaming such that the downwardly-directed stream moves ca. 10% faster than the upwardly-directed stream. This polarity of cytoplasmic streaming is not simply a consequence of gravity acting directly on the cytoplasm but is rather under physiological control. When Chara internodal cells are placed in a medium more dense than themselves, the gravity-induced polarity of cytoplasmic streaming is reversed. This phenomenon cannot be explained by a model which relies on intracellular sedimenting particles as gravity sensors but is consistent with the gravitational pressure model for gravity sensing. We propose that gravity causes the internodal cells to settle within the confines of the extracellular matrix resulting in a tension between the plasma membrane and the extracellular matrix at the top of the cell and a compression between the plasma membrane and the extracellular matrix at the bottom of the cell. These stresses are proposed to act upon peptides which span the plasma membrane/extracellular matrix interface at the ends of the cells and which subsequently activate Ca2+ channels which in turn may induce a polarity of cytoplasmic streaming.

Calcium↗

Effect of avermectins on Ca2+-dependent Cl- currents in plasmalemma of Chara corallina cells.

A natural complex of avermectins, aversectin C, and a component of this complex, avermectin A1, were shown to change the conductivity of Ca2+-dependent Cl- channels of plasmalemma of Chara corallina cells by acting from the outer side of the cellular membrane. Low concentrations of aversectin C and avermectin A1 increased the Cl- current: K1/2 = 35 ng/ml for the whole complex and K1/2 = 21 pg/ml for A1. Relatively high concentrations of the compounds suppressed the Cl- current: K1/2 = 2.2 microg/ml for aversectin C and K1/2 = 4.2 ng/ml for A1. The Hill coefficient for the interaction of avermectin A1 with the corresponding targets was identical for stimulation and suppression of the Cl- current: 2.8 and 2.5, respectively. Bicuculline, a nonspecific inhibitor of the GABAa receptors, did not influence stimulation of Cl- currents caused by low concentrations of avermectins, but at the same time blocked suppression of the Cl- currents by high concentrations of avermectins. Avermectins A2, B1, B2, abamectin and 22,23-dihydroavermectin B1 (ivermectin) did not affect the Cl- currents of Chara corallina cells.

Calcium↗

Ca2+ dynamics during membrane excitation of green alga Chara: model simulations and experimental data.

Kinetic investigations of stimulus response coupling in the green alga Chara have revealed that an intermediate second messenger is formed in the process of membrane excitation. This second messenger links electrical stimulation to the mobilization of Ca2+ from internal stores. In the present work, the experimentally based kinetic model, which describes the stimulus-dependent production of the second messenger and Ca2+ mobilization, is combined with a model for inositol 1,4,5-trisphosphate (IP3)-and Ca2+-sensitive gating of a Ca2+-release channel in endomembranes of animal cells. The combination of models allows a good simulation of experimental data, including the all-or-none-type dependence of the Ca2+ response on stimulus duration and complex phase locking phenomena for the dependence of the Ca2+ response on stimulation frequency. The model offers a molecular explanation for the refractory phenomenon in Chara, assigning it to the life time of an inactive state of the Ca2+-release channel. The model furthermore explains the steep dependence of excitation on strength/duration of electrical stimulation as a consequence of an interplay of the dynamical variables in the model.

Calcium↗

Transient activity of excitatory Cl- channels in Chara: evidence for quantal release of a gating factor.

In attached patches on the plasma membrane of nonexcited Chara corallina cells, randomly activating, transient Cl- currents with variable amplitudes were recorded. The peak amplitudes of these currents could be grouped into distinct populations with approximately equidistant mean peak currents. Generally, the mean current of the smallest population measured about half of the distance between the means of subsequent populations. Currents of the smallest population occurred most frequently at all voltages; the frequency of observations decreased with increasing amplitudes of the currents. At all voltages transient currents from different populations were similar in duration with the exception of the smallest currents, which lasted only 0.6 times as long as larger currents. Furthermore, transient currents were most frequent at positive voltages, but once initiated at a positive conditioning pulse they were also observed during subsequent pulses to negative voltages. The results are consistent with the idea that Chara contains Ca2+ stores in the vicinity of the plasma membrane, which are indirectly filled from the external medium. Upon quantal Ca2+ discharge from adjacent stores, a process independent of membrane voltage, the concentration of Ca2+ in the cytoplasm increases transiently. Depending on the number of discharging stores, distinct numbers of Ca2+-stimulated Cl- channels activate, giving rise to the macroscopic excitatory Cl- current in these cells.

Biological Transport↗

Discontinuities in the temperature function of transmembrane water transport in Chara: relation to ion transport.

The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30 degreesC of diffusional water permeability (Pd) were observed delimiting a region of low activation energy (Ea around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region water transport was of a different type with high Ea (about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at 15-30 degreesC and lipid matrix-mediated transport beyond this region. The K+ channel inhibitor, tetraethylammonium as well as the Cl- channel inhibitor, ethacrynic acid, diminished Pd in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate) the water transport inhibitor in erythrocytes also known to affect K+ transport in Chara, only increased Pd below 15 degreesC. In high external potassium ('K-state') water transport minima were pronounced. The role of K+ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence of the conductance of a single K+ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K+ single channel conductance at around 15 degreesC, unlike the one at 30 degreesC, was sensitive to changes of growth temperature underlining membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at 13 and 29 degreesC.

4-Chloromercuribenzenesulfonate↗

The Double-Water-Film Electrode: A Device for Measuring the Resistance and the Capacitance of the Internode/Node Interface of Chara as Functions of Time and Temperature.

A "double-water-film electrode technique" has been developed for the long-term characterization of the electrical properties across the interface between the nodal (N) and internodal (A or B) cells and the vacuole along the length of an internode of Chara as a function of time and temperature. The electrode unit consisted of a pair of the water-film electrodes described elsewhere (Chilcott 1988; Chilcott and others 1983; Coster and others 1984; Lucas 1985; and Ogata 1983). The distance between two water-film probes was fixed at 1.0 cm. By scanning the electrode unit, the spatial variations in electrical resistance and capacitance along the longitudinal axis of Chara were observed. Analysis was performed by applying an electrical equivalent circuit for the biomembrane (Philippson 1921). Across the internode (-A or -B)/central nodal cells interface, the specific parallel resistance (Rm) and the parallel capacitance (Cm) at 20 degrees C were 30 +/- 5 x 10(-3) Omegam(2) and 1.5 +/- 0.5 x 10(-1)Fm(-2) (at 30 Hz), respectively. And the series resistance, corresponding to the vacuole of the internode was 8 x 10(-3) Omegam(2). Study of temperature dependencies of Rm and Cm suggested that a dynamic homeostatic regulation was operating at the interface where numerous plasmodesmata were observed with an electron microscope (Pickett-Heaps 1967; Spanswick and Costerton 1967). Assuming that the individual cylinder of plasmodesma was filled only with cytoplasm, the number of plasmodesma per interface was estimated at 2.6 x 10(5).

Journal Article↗