Anomalous gravitropic response of Chara rhizoids during enhanced accelerations

Centrifugal accelerations of 50-250 g were applied to rhizoids of Chara globularis Thuill. at stimulation angles (alpha) of 5-90 degrees between the acceleration vector and the rhizoid axis. After the start of centrifugation, the statoliths were pressed asymmetrically onto the centrifugal flank of the apical cell wall. In contrast to the well-known bending (by bowing) under 1 g, the rhizoids responded in two distinct phases. Following an initial phase of sharp bending (by bulging), which is similar to the negatively gravitropic response of Chara protonemata, rhizoids stopped bending and, in the second phase, grew straight in directions clearly deviating from the direction of acceleration. These response angles (beta) between the axis of the bent part of the rhizoid and the acceleration vector were strictly correlated with the g-level of acceleration. The higher the acceleration the greater was beta. Except for the sharp bending, the shape and growth rate of the centrifuged rhizoids were not different from those of gravistimulated control rhizoids at 1 g. These results indicate that gravitropic bending of rhizoids during enhanced accelerations (5 degrees < or = alpha < or = 90 degrees) is caused not only by subapical differential flank growth, as it is the case at 1 g, but also by also by the centripetal displacement of the growth centre as was recently discussed for the negative gravitropism of Chara protonemata. A hypothesis for cytoskeletally mediated polar growth is presented based on data from positive gravitropic bending of Chara rhizoids at 1 g and from the anomalous gravitropic bending of rhizoids compared with the negatively gravitropic bending of Chara protonemata. The data obtained are also relevant to a general understanding of graviperception in higher-plant organs.     

Breakdown phenomena in the Chara membrane

The breakdown phenomenon in the Chara internodal cell was studiedusing the voltage clamp technique. When a slowly hyperpolarizingramp potential pulse was applied to the Chara membrane, thebreakdown occurred with hyperpolarization of about 220 mV. Thebreakdown was observed by less hyperpolarization, if the externalK⁺ concentration was increased. Such a breakdown phenomenonin the Chara membrane was caused principally by a large shiftof the membrane electromotive force toward depolarization. Thisshift frequently exceeded the peak level of the action potential. (Received July 26, 1976; )     

Electromotive force of Nitella membrane during excitation

Excitation of the Nitella membrane is analysed by assuming themembrane to be an electromotive force in series with a resistance,both being variables of time and of membrane potential. Duringstep depolarization beyond a threshold, conductance and electromotiveforce increase transiently, finally reaching their respectivesteady state levels. The conductance increase peak is attainedearlier than the peak for electromotive force increase. Wheneverelectromotive force increases beyond the level of clamped membranepotential, the ionic current flows inward. This is consideredto be the origin of the apparent negative resistance characteristicof the excitable membrane. Anodal break response and spontaneousfiring of Nitella membrane are also caused by transient increasesin electromotive force and conductance irrespective of whetherthe membrane potential is being held at its resting level. Thetransient increase in electromotive force reflects changes,like a phase transition, occurring during excitation. (Received May 6, 1968; )     

Further analysis of spontaneous membrane potential activity and the hyperpolarizing response to parathyroid hormone in osteoblastlike cells

Whole cell voltage clamp measurements using the patch technique on well-attached and well-spread cells of an osteoblastlike line (ROS 17/2.8) show the same spontaneous membrane potential activity as measurements with inserted microelectrodes. Furthermore, membrane potential measurements during the first 80 milliseconds (ms) following microelectrode penetration of the cell membrane usually show no decay. There is also good agreement between values of cell membrane resistance obtained by the microelectrode technique, the whole cell patch clamp technique, and the single channel patch clamp technique. These results indicate that our microelectrode measurements are not dominated by leak-induced artifacts, and that the spontaneous membrane potential activity is not induced by Ca2+ leakage around the microelectrode. The spontaneous membrane potential activity is eliminated in the presence of the Ca2+ ionophore A23187, also in serum-free medium, and by K+ and Ca2+ channel blockers, but it is not affected by the hyperpolarizing responses to parathyroid hormone (PTH) and dibutyryl cAMP, which persist under all of these conditions. These results support the hypothesis that the spontaneous membrane potential activity is related to repeated fluctuations of internal [Ca2+] and that such fluctuations result from a feedback loop involving Ca2+ channels or Ca2+ pumps in the cell membrane.     

The force exerted by the membrane potential during protein import into the mitochondrial matrix

The force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated on the basis of continuum electrostatics. The force is found to vary from 3.0 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 6.5 to 12 A, its measured range. In the present model, the decrease in force with increasing pore width arises from the shielding effect of water. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a purely membranous pore without water gives a force of 3.2 pN per unit charge, which should represent an upper limit. When applied to mitochondrial import experiments on the protein barnase, these results imply that forces between 11 +/- 2 pN and 13.5 +/- 2.5 pN catalyze the unfolding of barnase in those experiments. A comparison of these results with unfolding forces measured using atomic force microscopy is made.     

The plasma membrane of young Chara internodal cells revealed by rapid freezing

Young elongating internodal cells of Chara globularis var. capillacea (Thuill.) Zanev. were rapidly frozen and freze-fractured in order to observed transient events occurring within the plasma membrane. Several structures have been observed. Relatively small depressions, varying in depth, are prolific and scattered at random over the plasma membrane. Charasomes and clusters of particle rosettes are common. Arrays of intramembrane particle lines are a characteristic feature of the internodal cell plasma membrane. The charasomes and the arrays of particle lines occupy a considerable proportion of the plasma membrane. In these young cells, substantial movement must take place across this membrane and its basic structure must fluctuate accordingly. The innumerable small depressions may represent pinocytotic and secretory processes. The array of intramembrane particle lines may represent stages in fusion between the membranes of vesicles within the cytoplasm and the plasma membrane. The technique of ultra-rapid freezing allows these events and their intermediate stages to be visualised; some features of the membrane may only be seen by this method.     

Particle rosettes and microtubular ridges on the plasma membrane of Chara

We have examined the plasma membrane of cortical and internodal cells of Chara globularis var. capillacea (Thuill.) Zanev. using rapid-freezing and freeze-fracturing techniques. The non-etched image of the protoplasmic face reveals particle rosettes, and also reveals ridges which can be correlated with cortical microtubules. Since both rosettes and microtubular ridges can thus be viewed simultaneously, we have looked for a spatial relationship between them, but have found them to be essentially independent. We have found that the density of particle rosettes is a function of age and of type of cell. Groups of pits may represent the complementary exoplasmic component of the rosettes.     

Control of phosphate transport across the plasma membrane of Chara corallina

This paper examines the control of phosphate uptake into Chara corallina. Influxes of inorganic phosphate (Pi) into isolated single internodal cells were measured with ³²Pi. Pretreatment of cells without Pi for up to 10 d increased Pi influx. However, during this starvation the concentrations of Pi in both the cytoplasm and the vacuole remained quite constant. When cells were pre-treated with 0.1 mM Pi, the subsequent influx of Pi was low. Under these conditions the Pi concentrations in the cytoplasm was almost the same as that of Pi-starved cells, but vacuolar Pi increased with time. Transfer of cells from medium containing 0.1 mM Pi to Pi-free medium induced an increase of Pi influx within 3 d irrespective of the concentration of Pi in the vacuole.During Pi starvation, neither the membrane potential nor the cytoplasmic pH changed. Manipulation of the cytoplasmic pH by weak acids or ammonium decreased the Pi influx slightly.Pi efflux was also measured, using cells loaded with ³²Pi. Addition of a low concentration of Pi in the rinsing medium rapidly and temporarily induced an increase in the efflux.The results show that Pi influx is controlled by factors other than simple feedback from cytoplasmic or vacuolar Pi concentrations or thermodynamic driving forces for H⁺-coupled Pi uptake. It is suggested that uptake of Pi is controlled via the concentration of Pi in the external medium through induction or repression of two types of plasma membrane Pi transporters.Key words: Chara corallina, membrane transport, phosphate influx, phosphate starvation      

Salinity response of a freshwater charophyte, Chara vulgaris

Abstract. Chara vulgaris L. growing in an oligohaline lake was adapted to laboratory conditions and subjected to long-term salinity treatments ranging from 0 to 350 mol m ³ NaCl added to the lake water (40–680 mosmol kg ¹). Osmotic potential and concentration of the main osmotically active solutes (K⁺, Na⁺, Mg²⁺, Cl and sucrose) in the vacuolar sap of the central internodal cells were estimated. C. vulgaris did regulate turgor but incompletely. Turgor decreased from 335 mosmol kg ¹ under control conditions to 52–111 mosmol kg ¹ at 350 mol m ³ NaCl. The enhancement of πⁱ was achieved by increase in both ions and sucrose. Sterile and fertile plants differed in their response to osmotic stress. In sterile plants, the ions accounted for about 87% of the vacuolar osmotic potential. The increase of πⁱ under osmotic stress was exclusively due to an accumulation of Na⁺ and Cl^-. In fertile plants, sucrose accounted for about 35% of πⁱ and ions for about 51% Under osmotic stress, sucrose content increased together with the ionic content of Na⁺ and Cl^-.     

The response to gravity is correlated with the number of statoliths in Chara rhizoids.

In contrast to higher plants, Chara rhizoids have single membrane-bound compartments that appear to function as statoliths. Rhizoids were generated by germinating zygotes of Chara in either soil water (SW) medium or artificial pond water (APW) medium. Differential-interference-contrast microscopy demonstrated that rhizoids form SW-grown plants typically contain 50 to 60 statoliths per cell, whereas rhizoids from APW-grown plants contain 5 to 10 statoliths per cell. Rhizoids from SW are more responsive to gravity than rhizoids from APW because (a) SW rhizoids were oriented to gravity during vertical growth, whereas APW rhizoids were relatively disoriented, and (b) curvature of SW rhizoids was 3 to 4 times greater throughout the time course of curvature. The growth rate of APW rhizoids was significantly greater than that of SW-grown rhizoids. This latter result suggests that APW rhizoids are not limited in their ability for gravitropic curvature by growth and that these rhizoids are impaired in the early stages of gravitropism (i.e. gravity perception). Plants grown in APW appeared to be healthy because of their growth rate and the vigorous cytoplasmic streaming observed in the rhizoids. This study is comparable to earlier studies of gravitropism in starch-deficient mutants of higher plants and provides support for the role of statoliths in gravity perception.     

Occurrence of ubiquitin during spermiogenesis in Chara vulgaris

Experiments with an anti-ubiquitin antibody proved the presence of ubiquitin in spermatids at all spermiogenesis stages in Charta vulgaris. Its level increased before marked ultrastructural changes of spermatids correlated with disappearance of somatic proteins (histones) and appearance of protamine-type generative proteins. The obtained results seem to confirm our earlier hypotheses concerning a significant role of ubiquitin-proteasome system in Chara spermatozoid differentiation.     

Calcium/aluminium interactions in the cell wall and plasma membrane of Chara

The proposal that aluminium (Al) toxicity in plants is caused by either inhibition of Ca²⁺ influx or by displacement of Ca²⁺ from the cell wall, was examined. For this study the giant alga Chara corallina Klein ex Will. em. R.D. Wood was selected because it shows a similar sensitivity to Al as in roots of higher plants and, more importantly, it is possible to use the large single internodal cells to make accurate and unambiguous measurements of Ca²⁺ influx and Ca²⁺ binding in cell walls. Growth of Chara was inhibited by Al at concentrations comparable to those required to inhibit growth of roots, and with a similar speed of onset and pH dependence. At Al concentrations which inhibited growth, influx of calcium (Ca²⁺) was only slightly sensitive to Al. The maximum inhibition of Ca²⁺ influx at 0.1 mol·m^–3 Al at pH 4.4 was less than 50%. At the same concentration, lanthanum (La³⁺) inhibited influx of Ca²⁺ by 90% but inhibition of growth was similar for both La³⁺ and Al. Removal of Ca²⁺ from the external solution did not inhibit growth for more than 8 h whereas inhibition of growth by Al was apparent after only 2.5 h. Ca²⁺ influx was more sensitive to Al when stimulated by addition of high concentrations of potassium (K⁺) or by action potentials generated by electrical stimulation. Other membrane-related activities such as sodium influx, rubidium influx and membrane potential difference and conductance, were not strongly affected by Al even at high concentrations. In isolated cell walls equilibrated in 0.5 mol·m^–3 Ca²⁺ at pH 4.4, 0.1 mol·m^–3 Al displaced more than 80% of the bound Ca²⁺ with a half-time of 25 min. From the poor correlation between inhibition of growth and reduction in Ca²⁺ influx, it was concluded that Al toxicity was not caused by limitation of the Ca²⁺ supply. Short-term changes in other membrane-related activities induced by Al also appeared to be too small to explain the toxicity. However the strong displacement, and probable replacement, of cell wall ca²⁺ by Al may be sufficient to disrupt normal cell development.Abbreviations CPW artificial pond water - PD potential difference The technical assistance of Dawn Verlin is gratefully acknowledged. This work was supported by the Australian Research Council.     

Plasma membrane domains participate in pH banding of Chara internodal cells

We investigated the identity and distribution of cortical domains, stained by the endocytic marker FM 1-43, in branchlet internodal cells of the characean green algae Chara corallina and Chara braunii. Co-labeling with NBD C(6)-sphingomyelin, a plasma membrane dye, which is not internalized, confirmed their location in the plasma membrane, and co-labelling with the fluorescent pH indicator Lysotracker red indicated an acidic environment. The plasma membrane domains co-localized with the distribution of an antibody against a proton-translocating ATPase, and electron microscopic data confirmed their identity with elaborate plasma membrane invaginations known as charasomes. The average size and the distribution pattern of charasomes correlated with the pH banding pattern of the cell. Charasomes were larger and more frequent at the acidic regions than at the alkaline bands, indicating that they are involved in outward-directed proton transport. Inhibition of photosynthesis by DCMU prevented charasome formation, and incubation in pH buffers resulted in smaller, homogenously distributed charasomes irrespective of whether the pH was clamped at 5.5 or 8.5. These data indicate that the differential size and distribution of charasomes is not due to differences in external pH but reflects active, photosynthesis-dependent pH banding. The fact that pH banding recovered within several minutes in unbuffered medium, however, confirms that pH banding is also possible in cells with evenly distributed charasomes or without charasomes. Cortical mitochondria were also larger and more abundant at the acid bands, and their intimate association with charasomes and chloroplasts suggests an involvement in carbon uptake and photorespiration.     

The Tonoplast Impedance of Chara

The capacitance and conductance of the plasmalemma and tonoplastof Chara were measured simultaneously in space-clamped cells.At a frequency of 5 Hz the capacitance and conductance of thetonoplast were 60 ± 5 mF m^–2 (i. e. 6.0 µF cm^–2) and 6.5 ± 0.6 S m^–2 respectively.These values were respectively 2.9 ± 0.3 and 3.7 ±0.4 times greater than those of the plasmalemma. It is shownthat any leakage of current around the cytoplasmic electrodewill not drastically affect the calculated area-specific valuesof the tonoplast parameters under the experimental conditionsused, providing that the cytoplasm possesses a reasonable longitudinalconductivity. An examination of the relative measured impedancesof the plasmalemma and tonoplast supports this conclusion. Key words: Chara tonoplast: Plasmalemma, Capacitance/ conductance     

The Vacuolar pH of Chara Braunii

The intravacuolar pH of penultimate branchlet segments of Charabraunii Gm. was measured using intracellular antimony electrodes.This pH was shown to vary somewhat irregularly with extracellularpH and frequently to be so low that the entire vacuolar potentialcould be accounted for by the Nernst potential of hydrogen.     

Immediate response of Chara braunii exposed to zinc and hydrogen peroxide

The immediate effect of zinc (Zn) and hydrogen peroxide (H₂O₂) in Chara braunii was analyzed in short-time exposure experiments. The exposure concentrations were 12.3, 18.4, and 24.5 μmol L^?1 H₂O₂, 12, 60, and 120 mg L^?1 Zn, and 12.3 μmol L^?1 H₂O₂ + 12 mg L^?1 Zn, 12.3 μmol L^?1 H₂O₂ + 60 mg L^?1 Zn, and 18.4 μmol L^?1 H₂O₂ + 12 mg L^?1 Zn. The stress response of C. braunii was analyzed by measuring photosynthetic photosystem II activity, chlorophyll a and b and carotenoid contents, the H₂O₂ concentration, and antioxidant enzyme activities of ascorbic peroxidase, catalase, and guaiacol peroxidase. The short-term addition of Zn reduced pigment contents in C. braunii. Chlorophyll a and b and carotenoid contents in H₂O₂-exposed C. braunii were as high as in control plants. Photosynthesis was reduced in H₂O₂-treated C. braunii and the short-term addition of Zn did not affect the electron transport rate. H₂O₂ concentration and antioxidant enzyme activities in C. braunii were not significantly different between control and exposed plants. Trends of enzymatic adaptation were described: the H₂O₂-induced stress response was characterized by increased antioxidant enzyme activities, whereas Zn inactivated catalase in C. braunii.