RESUMO
Immobilized cultured tobacco cells become polarized upon the addition of naphthalene-1-acetic acid and start to elongate from an initial spherical shape. The question as to how a diffuse-growing cell forms a polar axis is addressed here with approaches successfully applied to the study of tip growth. With two kinds of vibrating probes the electric current flow and proton fluxes were mapped around such elongating cells. No consistent polar pattern of ion fluxes, which is typical for actively tip-growing cells, was detected. Therefore, other signals must provide the positional information needed for polar axis formation. Furthermore, neither a specific pattern of intracellular Ca(2+) concentration nor a polar distribution of putative ion-channel antagonist-binding sites were found in elongating tobacco cells. Auxin flux, on the other hand, was found to be important as TIBA, an inhibitor of polar auxin transport, clearly inhibited elongation in a concentration-dependent way. Cross-linking of arabinogalactan-proteins with the beta-Yariv reagent also resulted in inhibition of elongation. A model is proposed for the induction of polar growth where localized auxin efflux starts a signal cascade that triggers molecules that reorient microtubules. These then guide cellulose deposition in the cell wall, which in turn alters cell wall mechanics and leads to elongation. In this scheme, arabinogalactan-proteins are not causal agents but are probably important regulators of growth and survival of the cell.
Assuntos
Ácidos Indolacéticos/fisiologia , Transporte de Íons/fisiologia , Nicotiana/metabolismo , Floroglucinol/análogos & derivados , Sítios de Ligação , Ligação Competitiva , Transporte Biológico/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Relação Dose-Resposta a Droga , Glucosídeos/farmacologia , Modelos Biológicos , Ácidos Naftalenoacéticos/farmacologia , Floroglucinol/farmacologia , Caules de Planta/efeitos dos fármacos , Caules de Planta/fisiologia , Protoplastos/efeitos dos fármacos , Protoplastos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Nicotiana/citologia , Nicotiana/efeitos dos fármacos , Ácidos Tri-Iodobenzoicos/farmacologiaRESUMO
The effects of mechanical wounding on membrane voltage, endogenous ion currents, and ion fluxes were investigated in primary roots of maize (Zea mays) using intracellular microelectrodes, a vibrating probe, and ion-selective electrodes. After a wedge-shaped wound was cut into the proximal elongation zone of the roots, a large inward current of approximately 60 [mu]A cm-2 was measured, together with a change in the current pattern along the root. The changes of the endogenous ion current were accompanied by depolarization of the membrane voltage of cortex cells up to 5 mm from the wound. Neither inhibitors of ion channels nor low temperature affected the large, wound-induced inward current. The fluxes of H+, K+, Ca2+, and Cl- contributed only about 7 [mu]A cm-2 to the wound-induced ion current. This suggests the occurrence of a large mass flow of negatively charged molecules, such as proteins, sulfated polysaccharides, and galacturonic acids, from the wound. Natural wounding of the root cortex by developing lateral roots caused an outwardly directed current, which was clearly different in magnitude and direction from the current induced by mechanical injury.
RESUMO
This brief review summarizes gravity-induced changes in bioelectric parameters and evaluates their contribution to our understanding of the sensing of gravity, and the transduction and transmission of the gravity stimulus in plants. During the last few decades, information has accumulated demonstrating gravity-induced changes in surface potentials, membrane voltages, endogenous electric currents and ion fluxes. These changes point to the plasma membrane as the site of perception and transduction of the gravity signal. To date, it is reasonable to assume that gravity affects the state of ion channels (in particular, Ca2+ channels) and the activity of ion pumps (in particular, the electrogenic H(+)-ATPase) in the plasma membrane leading to intracellular and apoplasmic changes in ion activities and in membrane voltages. The flow of H+ and Ca2+ currents is probably the means by which information about gravity is amplified and transmitted from sensing to responding cells. No data are available so far about the effect of microgravity on bioelectric parameters. However, it would be interesting to learn if plants become hypersensitive to gravity during a prolonged stay in microgravity. If so, such plants might fire action potentials after return to earth, because more Ca2+ channels than usual may be activated by 1 g in microgravity-adapted plants.
Assuntos
Eletricidade , Gravitação , Sensação Gravitacional/fisiologia , Fenômenos Fisiológicos Vegetais , Brotos de Planta/fisiologia , Transdução de Sinais/fisiologia , Animais , Canais de Cálcio/fisiologia , Corrente Citoplasmática , Eletrofisiologia , Eucariotos , Magnoliopsida , Potenciais da Membrana , Raízes de Plantas/fisiologia , PrótonsRESUMO
Intact and decapped primary roots of maize (Zea mays L.) were exposed to DC electric fields of 0.5 to 8.0 V/cm in low-salinity media to resolve conflicting results about the direction of electrotropism. In DC fields of 0.5 V/cm or 1.0 V/cm, intact roots always curved toward the cathode. In a field of 8.0 V/cm, intact roots curved toward the anode and stopped growth. Decapped roots also curved toward the anode both in weak and strong fields. The results indicate that growth toward the cathode is the true response of healthy roots.
RESUMO
A novel, three-dimensional recording, vibrating probe was used for measuring the density and direction of the endogenous ionic current of cress roots (Lepidium sativum L.) bathed in low salt media (artificial pond water, APW). Roots submerged in regular APW and growing vertically show the following current pattern. Current of 0.7 microampere/square centimeter density enters or leaves the root cap; the current changes direction frequently. Current of 1.6 microamperes/square centimeter enters the meristem zone most of the time. Maximum current with a density of 2.2 microamperes/square centimeter enters the apical elongating zone, i.e. between 0.8 and 1.2 millimeters behind the root tip. The current density decreases to 1.4 microamperes/square centimeter at 2 millimeters, i.e. in the central elongating zone, and to 1.0 microampere/square centimeter at 3 millimeters, i.e. in the basal elongating zone. The current direction changes from inward to predominantly outward between 1.2 and 3 millimeters behind the tip. Measurements on opposite flanks of the roots indicate that the current pattern is fairly symmetrical. After placing the roots horizontally, the density of the endogenous current remains stable, but the current direction changes at the root cap and in the meristem zone. The current leaves the root on the upper side and enters on the lower side, causing a highly asymmetrical current pattern at the very tip. The current pattern at the upper and lower side further away from the tip remains the same as in vertical roots. Roots submerged in low Ca(2+) APW show a very different current pattern, no gravitropism, and no change of the current pattern after horizontal orientation. In these roots current enters the root cap and the basal elongating zone and leaves the apical elongating zone. Three conclusions are drawn from these results: First, plant roots elongate by two different modes of growth that are correlated with different current directions. They grow by cytoplasmic enlargement at sites of inward current and by turgor-driven elongation at sites of outward current. Second, a change in the current pattern at the root cap and in the meristem zone is a clear indicator of later gravitropism. Third, Ca(2+) ions are involved in the gravistimulated change in the current pattern, probably affecting the activity of plasmalemma H(+)-ATPases.
RESUMO
The distribution of intracellular free calcium ions ([Ca2+]i) was measured in pollen tubes of Lilium longiflorum using video imaging microscopy and the calcium sensitive indicators fura-2 and quin-2. The mean [Ca2+]i in growing pollen tubes measured with fura-2 shows a maximum of 1.7 to 2.6 microM in the tube tip and decreases almost exponentially to 60 to 100 nM at 100 microns behind the tip. Using quin-2, the maximum [Ca2+]i was also found in the tube tip but with a lower Ca2+ concentration, namely 1 microM. Addition of the calcium channel blocker La3+ caused a decrease of the [Ca2+]i maximum in the tube tip, indicating a heterogeneous distribution of Ca2+ channels along the plasma membrane of pollen tubes. The [Ca2+]i increased after addition of vanadate or compound 48/80. This suggests an involvement of a calmodulin-dependent Ca2+ pump in generation of the Ca2+ gradient in lily pollen tubes. The high [Ca2+]i found in the tube tip with fura-2 seems to indicate the real Ca2+ concentration and is probably responsible for vesicle fusion, fragmentation of actin filaments, and inhibition of cytoplasmic streaming.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Calmodulina/antagonistas & inibidores , Polaridade Celular/fisiologia , Pólen/metabolismo , Aminoquinolinas/metabolismo , Polaridade Celular/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Processamento de Imagem Assistida por Computador , Lantânio/farmacologia , Pólen/efeitos dos fármacos , Vanadatos/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The polarity of fucoid eggs is fixed either when tip growth starts or a bit earlier. A steady flow of calcium ions into the incipient tip is thought to establish a high calcium zone that is needed for its localization and formation. To test this hypothesis, we have injected seven different 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-type calcium buffers into Pelvetia eggs many hours before tip growth normally starts. Critical final cell concentrations of each buffer prove to block outgrowth (as well as cell division) for up to 2 weeks. This critical inhibitory concentration is lowest for two buffers with dissociation constants or Kd values of 4-5 x 10(-6) M and increases steadily as the buffers' Kd values shift either below or above this optimal value to ones as low as 4 x 10(-7) M or as high as 9.4 x 10(-5) M. To analyze these results, we have derived an equation (based on the concept of facilitated diffusion) for the effects of diffusable calcium buffers on steady-state calcium gradients. The data fit this equation quite well if it is assumed that cytosolic free calcium at the incipient tip is normally kept at about 7 microM and, thus, far above the general cytosolic level.
Assuntos
Cálcio/metabolismo , Ácido Edético/análogos & derivados , Ácido Edético/farmacologia , Sementes/fisiologia , Soluções Tampão , Difusão , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Indicadores e Reagentes , Matemática , Modelos Teóricos , Sementes/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The polarity of fucoid eggs is fixed when rhizoidal growth starts. A steady flow of calcium ions into the incipient tip is thought to establish a high calcium zone, which is needed for tip formation and outgrowth. To test this model, we injected six different BAPTA-type calcium buffers into Pelvetia eggs at about 3 to 7 h before outgrowth normally starts. Critical final cell concentration of each buffer proves to block outgrowth (as well as cell division) for up to two weeks. The critical inhibitory concentration falls as the calcium dissociation constant, KD, rises (and the buffers weaken) in the series of five buffers from 5,5' dimethyl-bapta (KD 0.4 µM) to 4,4' difluoro-bapta (KD = 5 µM); then finally rises again with the weakest buffer tested, 5,5' methylnitro-bapta (KD = 60 µM). Thus calcium buffers with a KD of about 5 µM prove most effective in blocking development. The effects of injecting a buffer do not depend upon the amount of coinjected calcium. To analyze these results, we imagine that each buffer acts to facilitate free calcium diffusion and thus suppresses gradient formation. This model leads to an exact equation and prediction of the concentrations of various buffers needed to inhibit development. The data fit this equation rather well if it is assumed that the free calcium concentration in the incipient tip is normally kept at about 10 µM and thus far above the general cytosolic level.
RESUMO
Irradiation of the growing apex of the algaVaucheria terrestris Götz var.terrestris with blue light (BL), which causes a transient acceleration of growth, also causes a large transient increase in inwardly directed current, which was monitored with a vibrating probe. The growing apex is normally the site of an inward current, and the surface of the non-growing, basal part of the coenocytic cell the site of an outward current. Irradiation of the apex causes only a slight increase in current efflux at the basal part of the cell. The BL-promoted current influx at the apex (BLCI) usually starts within 10 s after the onset of irradiation, preceding the light-growth response. With BL pulses shorter than 3 min, the BLCI reaches a maximum in about 3 min, and then declines to its original value over the next 3 min. If the BL pulse is longer than 3 min, the BLCI continues until the light is turned off. The threshold energy of the BLCI with broad-band BL is 2-5 J·m(-2), i.e. smaller than for both the light-growth response and phototropic response. The maximum BLCI reaches a value of approx. 5 µA·cm(-2), equivalent to an influx of 50 pmol·cm(-2)·s(-1) of monovalent cations. The effect of red light (RL) is completely different from that of BL: it either causes increases in the inward current of less than 0.3 µA·cm(-2), or a transient decrease of current. Furthermore, the direction of the RL-induced change is always the same at the apex and trunk, indicating the participation of photosynthesis. Our results indicate that the BLCI is kinetically and spatially related to the light-growth response and the phototropic bending ofVaucheria. It seems to be a necessary step for the phototropic bending.
RESUMO
Electric currents were measured around Trichoderma harzianum (Rifai) hyphae using an extracellular vibrating electrode. A steady current enters growing hyphal tips and along the side of the apical millimeter. In addition, outward currents were detected at about one-ninth of the locations tested, 60 to 150 minutes after illumination but not in dark controls. This sporadic, localized outward current pattern might be an early biophysical response to blue light.
RESUMO
Using a highly sensitive vibrating electrode, the pattern of naturally occurring electric currents around 1-day-old primary roots of Lepidium sativum L. growing vertically downward and the current pattern following gravistimulation of the root has been examined. A more or less symmetrical pattern of current was found around vertically oriented, downward growing roots. Current entered the root at the root cap, the meristem, and the beginning of the elongation zone and left the root along most of the elongation zone and in the root hair zone. After the root was tilted to a horizontal position, we observed current flowing acropetally at the upper side of the root cap and basipetally at the lower side within about 30 seconds in most cases. After a delay of several minutes, acropetally oriented current was also found flowing along the upper side of the meristematic zone. The apparent density of the acropetal current in the root cap region increased and then decreased with time. Gravitropic curvature was first visible approximately 10 minutes after tilting of the root to the horizontal position. Since the change in the pattern of current in the root cap region precedes bending of the root and is different for the upper and lower side, a close connection is suggested between the current and the transduction of information from the root cap to the elongation zone following graviperception in the cap.
RESUMO
Self generated electric currents were studied in protoplasmic drops and small plasmodia of Physarum polycephalum with the aid of an extracellularly measuring vibrating electrode. Ionic currents up to 15 microamperemetercm-2 density were found to traverse the objects. In protoplasmic drops current always enters the numerous protrusions and leaves areas with a smooth surface. In monopodial plasmodia current enters the strand and leaves both the advancing front and the retracting end. This result points toward large changes in membrane arrangement or properties occurring during development of plasmodia from protoplasmic drops.
Assuntos
Physarum/fisiologia , Citoplasma/fisiologia , Condutividade Elétrica , Eletrofisiologia , MovimentoRESUMO
Local irradiation of the alga Vaucheria sessilis (Vauch.) D.C. with blue light, which stimulates cortical fiber reticulation and chloroplast aggregation (M.R. Blatt and W.R. Briggs, 1980, Planta 147, 355-362), also induces an outward-directed current from the irradiated region of the cell. This current appears in conjunction with cortical fiber reticulation and precedes chloroplast aggregation. The current is not photosynthetic in origin, as indicated by experiments with 3(3,4-dichlorophenyl)-1,1-dimethyl urea and carbonyl-cyanide-m-chlorophenylhydrazone (CCCP). It shows a wavelength-dependence similar to that of chloroplast aggregation and reaches a maximum of 500 nA cm(-2) with saturating light intensities. The current is not dependent upon the presence of Na(+), K(+), or Cl(-) in a test medium containing only Na(+), K(+), Ca(2+), and Cl(-), but is inhibited, apparently nonspecifically, in the absence of external calcium. Both the light-induced current and chloroplast aggregation are stimulated by increases in the external KCl concentration and are inhibited by sub-micromolar concentrations of CCCP or by external pHs below approximately 5.5. We suggest that blue light stimulates the local extrusion of cations, possibly of protons, at the plasma membrane, an event which may act to destabilize the cortical fibers in Vaucheria, disrupt cytoplasmic streaming, and eventually lead to organelle aggregation in the light.
RESUMO
With the aid of an extracellular vibrating electrode, natural electric fields were detected and measured in the medium near growing roots and root hairs of barley seedlings. An exploration of these fields indicates that both the root as a whole, as well as individual root hairs, drive large steady currents through themselves. Current consistently enters both the main elongation zone of the root as well as the growing tips of elongating root hairs; it leaves the surface of the root beneath the root hairs. These currents enter with a density of about 2 microamperes per square centimeter, leave with a density of about 0.5 to 1 microampere per square centimeter, and total about 30 nanoamperes.Responses of the natural fields to changes in the ionic composition of the medium as well as observations of the pH pattern in the medium near the roots (made with bromocresol purple) together indicate that much of the current consists of hydrogen ions. Altogether, H(+) ions seem to leak into growing cells or cell parts and to be pumped out of nongrowing ones.
RESUMO
Ca(2+) is proposed to function as a messenger in such phytochrome-mediated responses as localized cell growth, intracellular movements, and control of plasma membrane properties. To test this hypothesis, the uptake of Ca(2+) in irradiated and non-irradiated regions of individual threads of the green alga Mougeotia was studied with the aid of (45)Ca(2+) and low temperature autoradiography: 10-20 cells within 40-60 cell-long threads were irradiated for up to 1 min, transferred to darkness for 3 to 10 min, submersed in a radioactive medium for 1 min, washed in an unlabelled medium for 30 min, and then autoradiographed at-80° C for several days.The autoradiographs show that those cells which had been pre-irradiated with red light did take up 2-10 times more Ca(2+) than the adjacent non-irradiated cells of the same thread. Cells pre-irradiated with farred light or red light followed by far-red light showed no enhanced uptake of Ca(2+). These results might be interpreted to indicate, firstly, that phytochrome-Pfr is involved in the enhanced uptake of Ca(2+) and secondly, that the accumulation of radioactive Ca(2+) in red light irradiated cells is an expression of an increased intracellular concentration of Ca(2+). This interpretation is based on the data that (i) the dark interval between irradiation and labelling precluded the involvement of photosynthesis, (ii) the effect of red light was reversible with far-red light, and (iii) the accumulation of Ca(2+) persisted during the long wash-out period. We speculate, that the red light-enhanced accumulation of Ca(2+) in Mougeotia cells is caused by a Pfr-mediated increase of the Ca-permeability of the plasma membrane, and perhaps by a Pfr-impeding of an active Ca(2+)-extrusion.
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Isolated internodes of Nitella (N. opaca, N. flexilis) and Nitellopsis spec. were punctured with single microelectrodes and their membrane potentials were recorded continuously during various light treatments. In red light the initial response was always a depolarization. This depolarization began with a lag-time of 0.4-3.5s and reached a steady state within 1-2 min of continuous illumination. Repolarization began within several seconds after turning off the light. The magnitude of the red-light-induced depolarization increased with the Ca(2+)-concentration of the medium. The largest depolarizations were recorded in 5 m mol l(-1) Ca(2+). Ca(2+) could not be replaced in this function by Na(+), Mg(2+), La(3+) or mannitol. Far-red light alone had no effect on the resting membrane potential. Far-red light applied immediately after red light accelerated the repolarization of the membrane potential. Far-red light applied simultaneously with red light reduced the amount of depolarization and increased the rate of repolarization. The results indicate that phytochrome and Ca(2+) are involved in the light-induced depolarization of the membrane. They are consistent with the hypothesis that phytochrome may act by triggering a Ca(2+)-influx at the plasma membrane.
RESUMO
Growing lily (Lilium longiflorum Thunb.) pollen always drive a current into their tubes and out of their grains. The only external ions needed for growth (and the growth current) are K(+), H(+), and Ca(2+). Increases in K(+) immediately stimulate the current; while decreases in K(+) immediately inhibit it. Comparable changes in H(+) have the opposite effect; while those in Ca(2+) have very little effect. We infer that most of the steady growth current is carried in by a potassium leak and out by a proton pump; but other considerations indicate that a minor, but controlling, component of the inward current consists of calcium ions.
RESUMO
Pollen of L. longiflorum was grown in 45Ca-labeled medium and washed with nonradioactive medium. Whole, labeled pollen was then frozen and autoradiographed at -78 degrees C. The autoradiographs show striking accumulations of 45Ca in the growing tips of the pollen tubes. This result is obtained when the pollen is labeled for times as short as 1 min, or as long as 5 h. In most cases, the tip concentration is about two to four times greater than that in the bulk of the pollen tube, and extends for a length of about 20 mum. In autoradiographs of tubes longer than 1 mm, a small fraction of cells show a distinctly larger 45Ca accumulation, the tip containing more than 100 times that in the rest of the cell. The 1- to 5-h labeling experiments show that calcium is relatively concentrated within the cytoplasm of the growing tip. The 1- to 3-min labeling experiments suggest that calcium may enter the tip faster than it enters other regions. These patterns of calcium accumulation and flux may be related to the localized secretion of vesicles at the grow;ng tip.
Assuntos
Cálcio/metabolismo , Plantas/análise , Pólen/análise , Cálcio/análise , Plantas/metabolismoRESUMO
Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B. After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink. In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.