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RESUMEN

White light (25 watts per square meter) induced an increase in plasma membrane K(+)-channel activity and a 30- to 70-millivolt transient membrane depolarization (completed in 2-3 minutes) in Arabidopsis thaliana leaf mesophyll cells. Transport characteristics of three types of ion channels in the plasma membrane were determined using inside-out patches. With 220 millimolar K(+) on the cytoplasmic side of the patch and 50 millimolar K(+) in the pipette, (220/50 K), the open-channel current-voltage curves of these channels were sigmoidal and consistent with an enzyme kinetic model. Two channel types were selective for K(+) over Na(+) and Cl(-). One (named PKC1) had a maximum conductance (G(max)) of 44 picosiemens at a membrane voltage (V(m)) of -65 mV in (220/50 K) and is stimulated by light. The other (PKC2) had G(max) = 66 picosiemens at V(m) = 60 millivolts in (220/50 K). The third channel type (PCC1) transported K(+) and Na(+) about equally well but not Cl(-). It had G(max) = 109 picosiemens at V(m) = 55 millivolts in (250/50 K) with 10 millimolar Ca(2+) on the cytoplasmic side. Reducing Ca(2+) to 0.1 millimolar increased PCC1 open-channel currents by approximately 50% in a voltage-independent manner. Averaged over time, PKC2 and PCC1 currents strongly outward rectified and PKC1 currents did so weakly. Reductants (1 millimolar dithiothreitol or 10 millimolar beta-mercaptoethanol) added to the cytoplasmic side of an excised patch increased the open probability of all three channel types.

RESUMEN

In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K(+)](o). When the membrane voltage was more positive than -50 millivolts, the membrane conductance in the whole-cell mode was low, and K(+) channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than -50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K(+) compared with Cs(+), Na(+), choline or Cl(-). Low concentrations of [Cs(+)](o) or [Na(+)](o) blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K(+) channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K(+)](o) about 10 millimolar, the influx through these channels is sufficient to increase the internal [K(+)] by 2 millimolar per minute. These K(+) channels are activated by membrane voltages in the normal physiological range and could contribute to K(+) uptake whenever the membrane is more negative than the K(+) equilibrium potential.

RESUMEN

We present here explicit mathematical formulas for calculating the concentration, mass, and velocity of movement of the center of mass of the plant growth regulator auxin during its polar movement through a linear file of cells. The results of numerical computations for two cases, (a) the conservative, in which the mass in the system remains constant and (b) the non-conservative, in which the system acquires mass at one end and loses it at the other, are graphically presented. Our approach differs from that of Mitchison's (Mitchison 1980) in considering both initial effects of loading and end effects of substance leaving the file of cells. We find the velocity varies greatly as mass is entering or leaving the file of cells but remains constant as long as most of the mass is within the cells. This is also the time for which Mitchison's formula for the velocity, which neglects end effects, reflects the true velocity of auxin movement. Finally, the predictions of the model are compared with two sets of experimental data. Movement of a pulse of auxin through corn coleoptiles is well described by the theory. Movement of auxin through zucchini shoots, however, shows the need to take into account immobilization of auxin by this tissue during the course of transport.

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RESUMEN

The temporal relations between early responses to indoleacetic acid (IAA), proton secretion, hyperpolarization of the membrane potential, and growth change during the incubation of segments of oat (Avena sativa L.) coleoptiles in a low salt medium. When IAA is added after pretreatment of several hours, proton secretion increases after a latency of 7 minutes and reaches its maximum 10 to 15 minutes later. This timing coincides with both the increase in growth of the segments and the hyperpolarization of the membrane potential of parenchyma cells, consistent with the hypothesis that the change in membrane voltage reflects the activity of an electrogenic proton pump. The extent of IAA-induced hyperpolarization is substantially reduced by elevating [KCl](0), most likely because this increases the passive conductance of the membrane. Neither growth nor proton secretion is affected by high [KCl](0) (30 millimolar), indicating that neither process is limited by the magnitude of the membrane potential. These results are consistent with the acid growth hypothesis. Following short incubation times, however, IAA-induced hyperpolarization and growth are detected within 10 minutes, while acidification of the medium is delayed for more than 40 minutes. This result is seemingly in conflict with the acid growth hypothesis, but in freshly cut tissue, the pH of the external medium may not reflect the pH of the epidermal cell walls. The temporal coincidence of auxin-induced growth and hyperpolarization suggests that in freshly isolated segments the hyperpolarization is a more sensitive indication of proton secretion than is acidification of the external aqueous environment.

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RESUMEN

We have compared the effects of the auxin, indole-3-acetic acid (IAA) with that of other weak acids on the plasma-membrane potential of oat (Avena sativa L.) coleoptile cells. Cells treated with 1 µM IAA at pH 6 depolarize 20-25 mV in 10-12 min, but they then repolarize, until by 20-25 min their potentials are about 25 mV more negative than the initial value. Similar concentrations of benzoic and butyric acids cause the initial depolarization, but not the subsequent hyperpolarization. The hyperpolarization is therefore specific to IAA. All the weak acids, including IAA, evoke a rapid hyperpolarization when their concentrations are raised to 10 mM. This result indicates that at high concentrations, the uptake of undissociated weak acids activates electrogenic proton pumping, most likely by lowering cytoplasmic pH. In contrast, the hyperpolarization observed with concentrations of IAA four orders of magnitude lower appears to be a specific hormonal effect. This specific, auxin-induced hyperpolarization occurs at the same time as the initiation of net proton secretion and supports the hypothesis that auxin initiates extension growth by increasing proton pumping.

RESUMEN

The velocity of transport and shape of a pulse of radioactive indole-3-acetic acid (IAA) applied to a section of maize (Zea mays L.) coleoptile depends strongly on the concentration of nonradioactive auxin in which the section has been incubated before, during, and after the radioactive pulse. A pulse of [(3)H]IAA disperses slowly in sections incubated in buffer (pH 6) alone; but when 0.5-5 µM IAA is included, the pulse achieves its maximum velocity of about 2 cm h(-1). At still higher IAA concentrations in the medium, a transition occurs from a discrete, downwardly migrating pulse to a slowly advancing profile. Specificity of IAA in the latter effect is indicated by the observation that benzoic acid, which is taken up to an even greater extent than IAA, does not inhibit movement of [(3)H]IAA. These results fully substantiate the hypothesis that auxin transport consists of a saturable flux of auxin anions (A(-)) in parallel with a nonsaturable flux of undissociated IAA (HA), with both fluxes operating down their respective concentration gradients. When the anion site saturates, the movement of [(3)H]IAA is nonpolar and dominated by the diffusion of HA. Saturating polar transport also results in greater cellular accumulation of auxin, indicating that the same site mediates the cellular efflux of A(-). The transport inhibitors napthylphthalamic acid and 2,3,5-triiodobenzoic acid specifically block the polar A(-) component of auxin transport without affecting the nonsaturable component. The transport can be saturated at any point during its passage through the section, indicating that the carriers are distributed throughout the tissue, most likely in the plasmalemma of each cell.

RESUMEN

Equations have been developed to describe the diffusional movement of a weak acid such as the auxin indoleacetic acid through a long file of vacuolated cells, where cellular accumulation is driven by the pH gradients across the cell membranes. If the permeability to the auxin anion is greater at one end of the cell than at the other, diffusional movement takes the form of polar transport, which exhibits: a nearly constant velocity either for the front or for a pulse of radioactive auxin, the capacity to move auxin against an external gradient of concentration, and a polar ratio that increases exponentially with the length of the section. The determinants of velocity include both diffusion through the vacuole and permeation steps at the cell membranes. Except for the permeabilities of the membranes to the anion, values are now available for all of the physical parameters in the equations. With reasonable estimates of permeability coefficients for the anion, the equations predict a velocity of transport of about 1 cm hr(-1), which agrees well with measured values. The analysis indicates, however, that the underlying cellular polarity may be greater than has been heretofore assumed. We thus demonstrate that the hypothesis of chemosmotic polar diffusion is capable of accounting quantitatively for the major features of auxin transport and provides a theoretical framework whose elements can be tested in future experiments.

RESUMEN

1. The predictions of a general kinetic model for the chemiosmotic uptake of auxin and other weak acids are compared with experimental results for the auxin indoleacetic acid. The proposed mechanism involves diffusional flux of undissociated acid, a saturable, voltage-sensitive flux of anion (A(-)), and a carrier-mediated symport of H(+) and A(-), all operating in parallel. During much of uptake, the electrochemical gradients are such that the net symport and the net anion flux are in opposition: the symport contributes more to influx; the anion path, to efflux. The voltage-sensitive flux of A(-) therefore constitutes a "leak". 2. The presence of a symport, whose carrier can distribute across the membrane in response to the internal and external concentrations of auxin, can speed the rate of uptake, but does not by itself alter the accumulation of auxin at equilibrium. 3. The accumulation ratio at equilibrium is less at low concentrations of auxin than at higher concentrations, indicating the presence of a saturable anion path. The concentration dependence of the transition depends on several factors, and is not a reliable indicator of the A(-)-carrier binding constant. 4. Observed uptake near neutral pH appears larger than is consistent with a voltage-sensitive anion flux being the only carrier-mediated path across the membrane. This observation provides indirect evidence for the presence of an auxin-proton symport in addition to a saturable A(-) carrier. 5. The change in kinetics of uptake of [(3)H]indole-3-acetic acid (IAA), observed as the total concentration of IAA is raised from 0.1 to 100 µM, is consistent with either (i) a symport that saturates at low concentrations, or (ii) activation of an A(-) efflux by intermediate concentrations of auxin. 6. The data on the concentration dependence of uptake of auxin are not consistent with a multi-proton symport.

RESUMEN

The validity of a chemiosmotic hypothesis for uptake of weak acids as an explanation for the accumulation of auxin by cells has been explored further by comparing the uptake of indole-3-acetic acid (IAA) by 1-mm segments of corn (Zea mays L.) coleoptiles with that of benzoic acid and two neutral indoles, indoleethanol and indoleacetonitrile, which do not ionize. These substances, while structurally related to IAA lack both auxin activity and polar transport. Uptake of IAA and benzoic acid increase with decreasing external pH, whereas the uptake of the two neutral indoles is independent of external pH.Although metabolism of IAA, during 90 min or less, is minimal and without significant effect on its uptake, metabolism of benzoic acid appears responsible for the apparent saturation of benzoic acid uptake at high concentrations. An inhibitor of auxin transport, N-1-naphthylphathalamic acid (NPA), stimulates uptake of IAA but has no effect on uptake of either benzoic acid or the two neutral indoles. Thus, NPA does not affect the driving forces for accumulation of weak acids but probably specifically decreases the flux of the auxin anions relative to undissociated auxin. Since the electrochemical potential of auxin anions is usually higher in than outside cells, blocking the anion flux with NPA would enhance auxin uptake. Azide, which abolishes accumulation of both IAA and benzoic acid, may simply collapse the pH gradient across the plasma membrane.In the absence of NPA, increasing concentrations of auxins or the analogoue ß-naphthaleneacetic acid (ß-NAA) exert two opposing effects on the uptake of IAA-depression and stimulation. Stimulation results from saturating the anion flux. With uptake fully stimulated by NPA, however, increasing concentrations of auxins or analogues only depress uptake of [(3)H]IAA. These results are consistent with more than one path for auxin transport each with a different dependence on concentration. In depressing NPA-stimulated IAA uptake, the effectiveness of ß-NAA≧IAA≫α-NAA≫ benzoic acid, a specificity similar to that of an auxin binding site in vitro that has been implicated by others in auxin transport. The results support the general hypothesis that cellular auxin uptake and polar transport through tissues are chemiosmotically coupled to the electrochemical potential of auxin and protons.

RESUMEN

Using both 1-mm segments of corn (Zea mays L.) coleoptiles and a preparation of membranes isolated from the same source, we have compared the effectiveness of several inhibitors of geotropism and polar transport in stimulating uptake of auxin (indole-3-acetic acid, IAA) into the tissue and in competing with N-1-naphthylphthalamic acid (NPA) for a membrane-bound site. Low concentrations of 2,3,5-triiodobenzoic acid (TIBA), NPA, 2-chloro-9-hydroxyfluorene-9-carboxylic acid (morphactin), and fluorescein, eosin, and mercurochrome all stimulated net uptake of [(3)H]IAA by corn coleoptile tissues while higher concentrations reduced the uptake of both [(3)H]IAA and another lipophilic weak acid, [(14)C]benzoic acid. Since low concentrations of fluorescein and its derivatives competed for the same membrane-bound site in vitro as did morphactin and NPA, the basis for both the specific stimulation of auxin accumulation and the inhibition of polar auxin transport by all these compounds may be their ability to interfere with the carrier-mediated efflux of auxin anions from cells. At higher concentrations, the decrease in accumulation of weak acids was nonspecific and thus may be the result of acidification of the cytoplasm and a general decrease in the driving force for uptake of the weak acids. Triiodobenzoic acid was an exception. Low concentration of TIBA (0.1-1 µM) were much less effective than NPA in competing for the NPA receptor in vitro, but little different from NPA in ability to stimulate auxin uptake. One possibility is that TIBA, a substance which is polarly transported, may compete with auxin for the polar transport site while NPA, morphactin, and the fluorescein derivatives may render this site inactive.

RESUMEN

Conditions for obtaining reproducible light-induced reduction of a b-type cytochrome in membrane fractions from coleoptiles of dark-grown Zea mays L. include a glucose-glucose oxidase system that lowers O(2) tension and generates H(2)O(2), substrate amounts of ethylenediaminetetraacetic acid which, in some manner, facilitates photoreduction by both added flavin and the endogenous photoreceptor and a sample temperature below 10 C. Cytochrome reduction could be obtained by photoexcitation of either a tightly bound endogenous receptor, which is probably a flavin, or added riboflavin, flavin mononucleotide, or flavin adenine dinucleotide. The latter flavin was the least effective. The endogenous photoreceptor appears to be rather firmly bound to the membranes, suggesting that this association may also exist in vivo. When any of the above four photoreceptors or methylene blue were used to sensitize the reaction, a cytochrome with a reduced alpha-band near 560 nanometers and a Soret difference peak near 429 nanometers was the electron acceptor. This cytochrome could be clearly distinguished spectrally from other cytochromes that predominated in the membrane preparations.

RESUMEN

The uptake of auxin by 1-mm slices of corn (Zea mays L.) coleoptiles, a tissue known to transport auxin polarly, depends on the pH of the medium. Short-term uptake of indole-3-acetic acid (IAA) in coleoptiles increases with decreasing pH of the buffer as would be expected if the undissociated weak acid, IAA·H, were more permeable than the auxin anion, IAA(-), and IAA(-) accumulates in the tissues because of the higher pH of the cytoplasm. Although uptake of [(3)H]IAA is reduced in neutral buffers, it is greater than expected if it were limited to just the extracellular space of the tissue. The radioactivity accumulated by the tissue can be quantitatively extracted by organic solvents and identified as IAA by thin-layer chromatography. The tissue radioactivity is freely mobile and can efflux from the tissue. Thus these cells in pH 5 buffer are able to retain an average internal concentration of mobile IAA that is at least several times greater than the external concentration. A prominent feature of auxin uptake from acidic buffers is enhanced accumulation at high auxin concentration. This indicates that, in addition to fluxes of IAA·H, a saturable site is involved in auxin uptake. Whenever the auxin-anion gradient is directed outward, saturating the efflux of auxin anions increases accumulation. Furthermore, the observed slowing of short-term uptake of radioactive IAA by increasing concentrations of IAA or K(+) indicates either an activation of the presumptive auxin leak or saturation of another carrier-mediated uptake system such as a symport of auxin anions with protons. By contrast in neutral buffers, effects of concentration on uptake rates disappear. This implies that at neutral pH the anion leak is decreased and influx depends on the symport.

RESUMEN

The cytoplasm of subepidermal parenchyma cells of Avena sativa L. coleoptiles was collected at one end of the cell by centrifugation. The electrical properties of both plasmalemma and tonoplast were then examined with microelectrodes inserted into both cytoplasm and vacuole of the same cell. The input resistance of the cytoplasm measured with either electrode was 7.5±0.8 MΩ while that of the vacuole measured with the single vacuolar electrode and a bridge circuit was 29.2±3.1 MΩ. The latter value was not significantly different from that of control, uncentrifuged cells. The resistance of the tonoplast is therefore several times larger than the input resistance of the cytoplasm, but the specific resistance of the plasma membrane cannot be calculated without knowledge of the extent and pattern of intercellular coupling. Electrical coupling of the cytoplasms of adjacent cells was observed in only two out of eight experiments. The mean potential of the vacuoles,-77.8±6.4 mV, was not significantly different from that of the cytoplasm; however, all the available evidence indicates that variable tip potentials in impaled cells made absolute determination of the membrane potential uncertain. In fusicoccin, the cells hyperpolarized by 20 mV within 10 min. This reponse occurred entirely at the plasmalemma.

RESUMEN

Results of microelectrode impalements of parenchymal cells of coleoptiles made in several different laboratories differ widely. The highest membrane potentials correlate with lower input resistance and the presence of intercellular coupling, whereas high input resistance seems to be associated with an absence of measurable coupling and possibly lower membrane potentials. In this paper we demonstrate that these results are consistent with (1) a tonoplast resistance several times greater than the input resistance of the cytoplasmic compartment, and (2) the presence of variable amounts of shunting introduced by insertion of the microelectrode through the cell membranes. The general consequences of this hypothesis are developed quantitatively. If the ideas are applicable to other tissues of higher plants-and on this point the evidence is still insufficient to judgeboth the design of experiments and the interpretation of measurements made with microelectrodes will have to be reevaluated.

RESUMEN

Indole-3-acetic acid (IAA) is transported from a nearly mature leaf throughout an intact Coleus blumei Benth. plant in the phloem. A buffered solution of both (14)C-methylene-labeled indoleacetic acid ([(14)C]IAA) and [6-(3)H]glucose was supplied in a glass capillary to the distal end of a severed main lateral vein of the leaf. Both labeled sugar and auxin move rapidly through the plant at velocities of ca. 16-20 cm h(-1) with closely similar, exponential profiles. This translocation is nonpolar; both auxin and sugar move upwards to the apex and young expanding leaves as well as downwards to the base of the shoot. Neither tracer appears in mature leaves; this eliminates the possibility that they enter the xylem. At the end of the transport period, 80-90% of the radioactivity recovered from various portions of the plants supplied with [(14)C]IAA is still identical chromatographically with IAA. In microautoradiographs prepared by techniques that minimize loss and redistribution of soluble compounds, radioactivity from [(3)H]IAA is concentrated in the phloem of the midrib and petiole of the fed leaf. A ring of triiodobenzoic acid (TIBA) strongly inhibits the polar auxin transport in sections isolated from the ringed region but does not significantly affect auxin translocation in the phloem of intact plants. TIBA does, however, reduce the entry of auxin into the collecting veins of the leaf. Thus steps in auxin transport sensitive to TIBA may occur during transfer through the leaf or into the phloem, but not during long distance translocation in the phloem.

RESUMEN

When cytoplasmie streaming in oat and maize coleoptile cells is completely inhibited by cytochalasin B (CB), polar transport of auxin (indole-3-acetic acid) continues at a slightly reduced rate. Therefore, cytoplasmic streaming is not required for polar transport. Auxin induces elongation in CB-inhibited coleoptile and pea stem segments, but elongation rate is reduced about 40% by CB. Therefore, stimulation of cytoplasmic streaming cannot be the means by which auxin promotes cell elongation, but streaming may be beneficial to elongation growth although not essential to it. A more severe inhibition of elongation develops after several hours in CB. With coleoptiles this could be due to inhibition of sugar uptake; in pea tissue it may be due to permeability changes and cytoplasmic degeneration. CB does not disorganize or disorient microfilament bundles when it inhibits streaming in maize, but appears instead to cause hypercondensation of microfilament material.

RESUMEN

The cytoplasm of maize coleoptile cells was displaced to either the apical or basal ends of the cells by centrifuging (1750xg for 10 min) segments in which protoplasmic streaming had been stopped by pretreatment with cytochalasin B. Centrifugation toward the base of the segment promotes the subsequent basipetal transport of indole-3-acetic acid, whereas apical centrifugation dramatically inhibits this transport. Apical centrifugation neither promotes acropetal transport nor reverses the polarity of auxin transport. Experiments in which the amyloplasts were separated from the bulk of the cytoplasm indicate that the basipetal transport is independent of both the position and pressure exerted by the amyloplasts but is strongly dependent on the amount of cytoplasm at the basal end of the cells. These effects of centrifugation on auxin transport lead to the conclusion that the metabolic component of the transport is a polar secretion of auxin localized in the basal plasma membrane of each cell.

RESUMEN

Parenchymal cells of oat (Avena sativa) coleoptiles had an osmotic concentration of 410 mM (determined by plasmolysis); of this only 22 mM was K(+) and 1 mM Na(+) (flame photometry). Cells were impaled with micropipette electrodes. Iontophoretic injection of the dye Niagara sky-blue from the micropipette showed that the tip of the electrode penetrated the vacuole. When sections of tissue were immersed in a solution of 22 mM KCl, 1 mM CaCl2, and 50 mM glucose, average membrane potential was found to be 38.5 mV inside negative specific membrane resistance was ∼510 Ω cm(2), and specific membrane capacitance, ∼2 µf cm(-2). The cell membranes showed <25% retification and no electrical excitability. Electrotonic coupling of adjacent cells could not be demonstrated.