Background: Auxin (IAA) is a central player in plant cell growth. In contrast to the well-established function of the plasma membrane in plant cell expansion, little is known about the role of the vacuolar membrane (tonoplast) in this process. + 2+ Results: It was found that under symmetrical 100 mM K and 100 μM cytoplasmic Ca the macroscopic currents showed a typical slow activation and a strong outward rectification of the steady-state currents. The addition of IAA at a final concentration of 1 μM to the bath medium stimulated the SV currents, whereas at 0.1 and 10 μM slight inhibition of SV currents was observed. The time constant, τ, decreased in the presence of this hormone. When single channels were analyzed, an increase in their activity was recorded with IAA compared to the control. The single-channel recordings that were obtained in the presence of IAA showed that auxin increased the amplitude of the single-channel currents. Interestingly, the addition of IAA to the bath medium with the same composition as the one that was used in the patch-clamp experiments showed that auxin decreased the volume of the vacuoles. Conclusions: It is suggested that the SV channels and the volume of red beet taproot vacuoles are modulated by auxin (IAA). Keywords: Beta vulgaris L., IAA (indole-3-acetic acid), SV channels, Vacuole, Vacuolar volume Background channels are activated by a hyperpolarizing membrane Auxins, particularly indole-3-acetic acid (IAA), play an potential and by extracellular apoplastic protons. essential role in the regulation of plant cell extension. Significantly less is known about the role of the vacu- According to the so-called “acid growth theory”, auxin olar membrane, the tonoplast, in the auxin-mediated activates the PM H -ATPase, which acidifies the apo- growth of plant cells. Plant cells contain a large central plast and causes the activation of the enzymes that are vacuole that occupies up to 95% of the total cell volume involved in cell wall loosening (for a review see ). It is in many mature plant cells. Plant cell expansion is also well established, at least in maize coleoptile cells, driven by a combination of the osmotic uptake of water that auxin-induced growth involves K uptake through into the vacuoles and altered cell wall extensibility. To voltage-dependent, inwardly rectifying K channels maintain the turgor pressure of expanding cells, solutes (ZMK1, Zea mays K channel 1), the activity of which must be transported into the vacuole to maintain its contributes to water uptake and consequently to cell ex- osmolarity. Vacuoles are very dynamic organelles, whose pansion [2, 3]. It has been shown that apart from the morphology changes during plant growth and develop- posttranslational, auxin-dependent up-regulation of the ment [4, 5]. It has been shown that auxin (IAA) and its K uptake channels, auxin also regulates the expression metabolites are present in plant vacuoles and that auxin of the maize K uptake channel gene ZMK1 . ZMK1 transport across the tonoplast plays essential roles in maintaining auxin homeostasis . It is also well known * Correspondence: firstname.lastname@example.org that auxin stimulates or inhibits the growth of plant cells Department of Plant Physiology, Faculty of Biology and Environmental depending on its concentration as well as the cell type Protection, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland . Recently, it has been shown that auxin altered the Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Burdach et al. BMC Plant Biology (2018) 18:102 Page 2 of 10 appearance of the vacuoles in the root epidermal cells of , two groups of researchers revealed the essential struc- Arabidopsis thaliana so that they became smaller . At tural and functional properties of TPC1/SV channels in the same time, as these authors showed, auxin also Arabidopsis thaliana based on their crystal structure [17, inhibited the growth of the root epidermal cells. This 18]. Soon after, Jaślan et al.  published a paper in which finding was used by Dünser and Kleine-Vehn to the structural determinants of the voltage- and propose the “acid growth balloon theory” according to calcium-dependent channel gating of AtTPC1 were which plant growth is the interplay between the intracel- described. For their analysis, these authors built a lular space-filling “vacuolar balloon” and the required three-dimensional homology model of AtTPC1 that extracellular cell wall acidification/loosening. was based on the crystal structure of the bacterial Taking into account that plant vacuoles are highly dy- voltage-gated Na channel Na Ab. To the best of our namic organelles and are essential for growth and devel- knowledge, no research has been reported on the opment, we performed experiments in which the effect effects of IAA on the TPC1/SV channels in plant of auxin (IAA) on the slow vacuolar (SV) channels and cells. We hypothesize that SV channels representing the the volume of red beet taproot vacuoles were studied. In major cations conductance are involved in auxin-induced the plant vacuoles, slow vacuolar (SV) channels are volume changes of the vacuoles. 2+ Ca -permeable cation channels that are coregulated 2+ by voltage and Ca . These SV channels are ubiquitous and Results abundant in the vacuolar membrane of terrestrial plants. Effect of IAA on the volume of red beet taproot vacuoles The SV channel from Arabidopsis, TPC1, is encoded by the Red beet vacuoles were mechanically isolated directly single-copy gene AtTPC1. Structurally, TPC1 onto glass slides by rinsing the surface of fresh tissue represents a dimer of two Shaker-like monomers that are slices with a medium containing various K concentra- linked via a cytoplasmic loop that contains two EF hand tions (0, 20 and 100 mM). As Fig. 1 indicates, the vol- motifs ([11, 12] for a review see ). The activity of ume of the vacuoles that were incubated in the bath voltage-dependent TPC1 channels can be regulated by both medium without K and with 1 μM IAA increased after 2+ 2+ cytosolic and vacuolar Ca . Cytosolic Ca promotes chan- 60 min up to 8% of their initial value (at 0 min). In the 2+ nels opening [12, 14], whereas luminal Ca prevents their presence of IAA, the volume of the vacuoles that had opening . Three decades after the discovery of the SV been incubated in bath medium without K increased by channels by Hedrich et al. and Hedrichand Neher 20%. When the vacuoles were incubated in the presence Fig. 1 Effect of 1 μM indole-3-acetic acid (IAA) on the volume changes of vacuoles. The vacuoles had been incubated in the presence of K at 0, 20 and 100 mM. IAA was added to the incubation medium at time 0 min. The data points are the means (± SE) from nine independent experiments. The volume of individual vacuoles was calculated from the diameter of the individual vacuoles in a photographic image. The diameter of the vacuoles was measured at the indicated times and converted to a percentage of the initial value (fixed as 100%). The inset on the right shows the volume of vacuoles after one hour of the experiment. Bars indicate means ± SEs. Means followed by the same letter are not significantly different from each other (LSD test P <0.05) Burdach et al. BMC Plant Biology (2018) 18:102 Page 3 of 10 of 20 or 100 mM K , their volume was about 3% lower section clearly showed that the effect of IAA on the compared to the first value. The addition of IAA to the volume of red beet vacuoles depends on the potas- bath solution with 20 mM K slightly increased (by 3% sium concentration. over 60 min) the volume of the vacuoles, while its addition to the medium with 100 mM K decreased (by Electrophysiological experiments 10% over 60 min) their volume. Interestingly, in the Using the patch-clamp technique, we examined the ef- presence of both IAA and 100 mM K , a decrease in the fect of IAA on the slow vacuolar (SV) channel activity in volume of the vacuoles was observed from the begin- red beet (Beta vulgaris L.) taproot vacuoles. Both macro- ning of the experiment. The data obtained in this scopic currents (whole-vacuole configuration) and Fig. 2 Effect of cytosolic IAA on the slow vacuolar (SV) channels in red beet taproot vacuoles. a An example of an SV current recording for a single vacuole in the control bath (control at 0 time, recorded immediately after the establishment of the whole-vacuole configuration as well as 5 min later) and in the presence of IAA at 1 μM (auxin was added to the bath immediately after the current was recorded in the control at 0 time; however, the current in the presence of IAA was recorded 5 min after the control at 0 time). SV currents elicited by a series of voltage steps ranging from − 100 to + 100 mV in 10 mV steps; holding potential 0 mV. b Steady-state currents (normalized to the current amplitude at + 100 mV under control at 0 min) were determined in the control medium (control at 0 and 5 min) and in the presence of 0.1, 1 and 10 μM IAA. The current traces were fitted with the exponential function: i(t)= a + b (1-exp(−t/τ)), where a - current at t =0, b - current at saturation (plateau), t - time and τ - time constant. The steady state is the difference between current at saturation (plateau) and current at time “0” (leak). Data points are the means (± SE) from at least seven experiments performed with different vacuoles. The significance of the results was analyzed for voltage + 100 mV using the post hoc least significant difference (LSD) test. Means followed by the same letter are not significantly different from each other (LSD test P < 0.05) Burdach et al. BMC Plant Biology (2018) 18:102 Page 4 of 10 single-channel currents (cytosolic side-out configuration) phenomenon described in numerous publications [20– 2+ were recorded in a symmetrical 100 mM KCl and Ca 27]. However the opposite effect i.e. the increase of SV gradient (0.1 mM in the bath and 0 mM in the pipette). channel activity in time was also observed . Fig. 3 It should be added that we decided to use symmetrical presents the time constants, τ, of the monoexponential 100 mM KCl for two reasons – firstly, because at sym- function fitted to the time courses of the macroscopic SV metrical 100 mM KCl, which is very often used in currents that were recorded in the presence and absence patch-clamp experiments, the K current flows from the of IAA. These constants can be interpreted as the rate of cytosol to the vacuole and secondly because at this con- the SV channel activation after the application of a voltage centration of KCl, auxin, immediately (3 min) after its pulse. Fig. 3 indicates that at voltages between + 60 and + addition, causes a decrease in the volume of the vacu- 90 mV, the time constants, τ, decrease in the presence of oles. The macroscopic current recordings showed slow IAA by ca. 30% (for example at + 80 mV, τ =1.146 ±0.067 activation (Fig. 2a, control) and strong outward rectifica- SE for control 5 min and τ =0.884 ±0.09 SE for IAA tion of the steady-state currents at voltages that were 5 min, at + 70 mV, τ =1.298 ±0.077 SE for control 5 min more positive than + 20 mV (Fig. 2b, control). When and τ = 0.851 ± 0.066 SE for IAA 5 min,), thus suggesting IAA at a final concentration of 1 μM was added to the faster channel activation with IAA. At 40, 50 and 100 mV bath solution, the SV currents increased at all potentials the time constant did not depend on IAA. When consid- between + 20 and + 100 mV compared to the control at ering the microscopic currents in the cytosolic side-out 5 min (Fig. 2b). For example, in the whole-vacuole con- configuration (Fig. 4), channels that had a higher current figuration, the addition of 1 μM IAA resulted in a 60% amplitude could be recorded in the presence of IAA com- increase in the current amplitudes at 100 mV compared pared to the control at 5 min. This is evident in Fig. 5, to the control at 5 min (I = 0.49 ± 0.07 SE for control which shows that at voltages between 80 and 100 mV norm 5 min and I = 0.79 ± 0.1 SE for 1 μM IAA 5 min). auxin significantly increased the amplitude of the SV cur- norm Interestingly, at concentrations 0.1 and 10 μM IAA rents compared to the control at 5 min (for example at + cause only slight changes of steady-state current as com- 100 mV I = 1.957 ± 0.388 SE for control 5 min and I = pared to the control at 5 min. Therefore for further elec- 2.762 ± 0.124 SE for IAA 5 min). Taking into account the trophysiological experiments 1 μM concentration of IAA density of the SV channels calculated as whole-vacuole was chosen. As is presented in Fig. 2, the activity of ion current divided by current of the single channel and sur- channels can be lost during patch-clamp experiments, face area of the vacuole, IAA increased number of active which is known as “run down”.The “run down” defined SV channels as compared to the control after 5 min. For as inhibition of SV channel activity in time, particularly example, the density of the channels in control at 0 min visible in the whole-vacuole configuration, is a common amounted 795 channels per 1000 μm while 5 min later Fig. 3 Effect of IAA at 1 μM on activation time, τ, as a function of voltage. Data points are the means (± SE) from at least seven experiments performed with different vacuoles. The significance of the results was analyzed for every voltage (+ 40 mV to + 100 mV) using the post hoc least significant difference (LSD) test. Means followed by the same letter are not significantly different from each other (LSD test P < 0.05) Burdach et al. BMC Plant Biology (2018) 18:102 Page 5 of 10 Fig. 4 An example of the microscopic SV current traces at selected voltages. Single channel openings in the control bath (a) (control at 0 time and 5 min later, see explanation in Fig. 2) and in the presence of IAA at 1 μM(b) (the current in the presence of IAA was recorded 5 min after the control at 0 time) are shown. Single channel fluctuations were recorded at + 60, + 80 and + 100 mV. The solid line indicates closed state of the channels, the dashed line – open state. The values of open probabilities for single traces are presented as P this parameter was 600 channels per 1000 μm .In the compared to the control at 5 min, while at the remaining presence of IAA 5 min after its addition the density of the voltages, it was similar to the control values. channels was equal 724 per 1000 μm (the density values Taken together, our electrophysiological data suggest were calculated for + 100 mV). All of the points in the that in a whole-vacuolar configuration, (1) auxin at scatter plots (Fig. 6), which show the distribution of the 1 μM enhanced the SV channel activity compared to the times of the different current state events as a function of control, (2) the time constant, τ, in the range 40–90 mV the amplitude of the current, indicate the events of the decreased in the presence of IAA at 1 μM, (3) auxin at closing or opening of one, two, three and four SV chan- 1 μM increased the amplitude of the SV currents com- nels. As can be seen at Fig. 6a the current amplitude of pared to the control and (4) the open probability of sin- single channels in the presence of IAA is maintained at gle channels was only significantly higher at 80 mV the level comparable with that recorded for the control at compared to the control. 0 min. Fig. 6b and c, which show the average values of the times and the number of events (closed, open) versus the Discussion current level, respectively do not show the significant dif- The tonoplast regulates the traffic of ions and metabo- ference between control and IAA after 5 min. As can be lites between the cytosol and the vacuole, which are ne- seen in Fig. 7, the open probability of single channels at cessary for plant cell growth. In recent few years the 80 mV was threefold higher in the presence of IAA interest in vacuoles increased also in the aspect of auxin Burdach et al. BMC Plant Biology (2018) 18:102 Page 6 of 10 Fig. 5 Current-voltage relationships for the microscopic SV currents. The currents were recorded in the control bath (control at 0 time and 5 min later) and in the presence of IAA at 1 μM. Points represent the means (± SE, n = 8) for the events of “open 1” that were recorded at selected voltages. The significance of the results was analyzed for every voltage (+ 60 mV to + 100 mV) using the post hoc least significant difference (LSD) test. Means followed by the same letter are not significantly different from each other (LSD test P < 0.05) action in plant cell growth and development. The identi- performed experiments in which the effect of auxin (IAA) fication of tonoplast permease WAT1 transporting auxin on the SV channel activity in the vacuolar membrane of out of the vacuole and the inverse correlation between red beet vacuoles was studied. It is well established that auxin content in plant cell and its vacuolation status give these channels and H pumps represent the major con- new insight on role of vacuoles in auxin homeostasis [6, ductance of the vacuolar membrane (reviewed in [13, 32]). 29]. As hitherto interest focused on the role of vacuoles Here, we demonstrate that under the experimental condi- 2+ in auxin redistribution in the cell, our goal was to deter- tions of this study (symmetrical 100 mM KCl and Ca mine the auxin impact on vacuole volume changes and gradient), vacuoles that had been isolated from the red probable contribution of tonoplast cation channels beet taproot were characterized by SV channels whose (TPC1/SV) in this process. electrical properties, such as slow activation and outward In our experiments, the vacuoles that had been incu- rectification, are close to those that were previously de- bated in a medium without K and 1 μM IAA were found scribed in Beta vulgaris taproots [14, 33, 34]. The addition to swell (about 8%) over first 60 min, while a significantly of 1 μM IAA to the bath solution enhanced the SV cur- faster increase in the volume of the vacuoles (ca. twofold) rents compared to the control (Fig. 2). It is suggested that was observed in the presence of 1 μM IAA (Fig. 1). The the stimulation of the macroscopic SV currents that were fact that the increase or decrease of the volume of the vac- observed in the presence of IAA may indicate that auxin uoles is sensitive to IAA and depends on the K concen- either acts directly as a channel activator or that it indir- tration suggests that the electrical potential of the ectly alters the kinetics of the transition between the tonoplast may play a role in this phenomenon. It was pre- closed and open state of a channel. Analysis of the kinetics viously shown that at high external potassium concentra- of the relaxation of macroscopic current may be a source tions, which are comparable to cytosolic values, the of information on channel gating. Comparing the data membrane potential of the vacuoles that had been isolated that was obtained from the whole-vacuole and single from the storage roots of red beet was almost completely channel recordings, it might be suggested that IAA at abolished, whereas at 0 mM K , it was around + 75 mV 1 μM enhanced the SV currents as a result of the faster [30, 31]. The electrical potential and the pH gradient channel activation, the increased amplitude of SV currents across the tonoplast provide the driving forces for the and a higher open probability of single channels at 80 mV. transport and accumulation of metabolites and ions in the In order to explain the mechanism of the modulation vacuolar lumen. However, to date, it is not clear how of the SV channel by auxin, two scenarios are possible: auxin changes the ion transport across the vacuolar mem- (1) at the pH of the incubation medium used in the ex- brane of plant cells. Taking the above into account, we periments (pH 7.5), the anionic form of IAA (IAA ) Burdach et al. BMC Plant Biology (2018) 18:102 Page 7 of 10 Fig. 6 Distribution of the times of different current state events. The events (closed, open 1, 2, 3 and 4) were presented as a function of the amplitude of current events (i.e. its average value during an event) in the control (control at 0 min and 5 min later) and in the presence of IAA at 1 μM. All events were collected from eight current traces (each of 12 s duration) that were obtained at a voltage of + 100 mV (a). The average values of the times of the current events versus the current level (± SE, n = 8) (b). The average values of the number of events versus the current level (± SE, n = 8) (c). FitMaster software was used to analyze the opening events predominates, which can interact with the voltage sens- When comparing our electrophysiological experiments ing S10 domain, thus causing changes in the gating kin- with ones in which the diameters of vacuoles were mea- etics of the SV channel (for the structure of the TPC1 sured, it should be concluded that IAA at 1 μM increased channel, see [17–19] and (2) because auxins are able to the SV channel currents while it decreased the volume of interact with lipids and change the properties of the lipid the vacuoles. Taking the above into account, it might be bilayer [35–37], they may disturb the interaction be- hypothesized that in the presence of IAA, the SV channels tween lipids and proteins and therefore indirectly modu- play a role in the intracellular space-filling function of the late the activity of the SV channels. vacuole (“vacuolar balloon”). In agreement with our Burdach et al. BMC Plant Biology (2018) 18:102 Page 8 of 10 Fig. 7 Open probability of the slow vacuolar (SV) channels as a function of voltage. The open probability was calculated (using FitMaster software) as the sum of the channel open time in the current traces that were normalized to the total time of the traces and divided by the number of active channels in the patch. Data points are the means (± SE) from eight independent experiments. The significance of the results was analyzed for every voltage using the post hoc least significant difference (LSD) test. The significance of the results was analyzed for every voltage (+ 20 mV to + 100 mV) using the post hoc least significant difference (LSD) test. Means followed by the same letter are not significantly different from each other (LSD test P < 0.05) hypothesis, the IAA-induced uptake of K and activity were measured, the vacuoles were mechanically probably Cl ( and references therein) into the isolated directly onto glass slides or into an electro- cytoplasm of plant cells might be partly (apart from physiological chamber (1 ml in volume) by rinsing the maintenance of turgor pressure of expanding cell) surface of fresh tissue slices with the bath solution. compensated for by a decrease in the vacuole volume in order to maintain cytoplasm homeostasis. Never- Vacuole volume measurements theless the results presented in this paper indicate Vacuole diameters were measured using an Ax70 micro- that there is no simple interrelation between SV chan- scope (Olympus Provis) with a fully automatic nels activity and volume changes of the vacuoles in photomicrography that was connected to a camera the presence of IAA. (Hammamatsu, Japan). The volume of the vacuoles was measured in a bath solution containing: (1) 100 mM K (100 mM KCl, 2 mM MgCl ,0.1 mM CaCl , 2 mM DTT, 2 2 Conclusions 5 mM MES, 5 mM Tris and 400 mM sorbitol, pH 7.5, Taken together, our results suggest that auxin enhances osmolality 650 mOsm), (2) 20 mM K (20 mM KCl, the SV currents in red beet vacuoles as a result of a fas- 2 mM MgCl , 0.1 mM CaCl , 2 mM DTT, 5 mM MES, 2 2 ter channel activation, an increased amplitude of SV cur- 5 mM Tris and 460 mM sorbitol, pH 7.5, osmolality rents and a higher open probability of single channels at 650 mOsm) and (3) 0 mM K (0 mM KCl, 2 mM MgCl , 80 mV, thus simultaneously causing a decrease in vacu- 0.1 mM CaCl , 2 mM DTT, 5 mM MES, 5 mM Tris and ole volume. It is suggested that auxin (IAA)), at least at 600 mM sorbitol, pH 7.5, osmolality 650 mOsm). 1 μM, modulate the SV channels and the volume of red beet taproot vacuoles. Patch-clamp measurements The electrophysiological experiments were performed in Methods whole-vacuole and excised cytosolic side-out patch con- Plant material and vacuole isolation figuration. The recordings were made using an EPC-7 Red beet (Beta vulgaris L.) taproots vacuoles were iso- Plus amplifier (List-Medical-Electronic, Darmstadt, lated using the nonenzymatic method that was previ- Germany), as was recently described by Trela et al. . ously described by Coyaud et al. . In the experiments For signal filtration a five-pole Bessel filter was used with in which the diameter of the vacuoles or SV channel sampling frequency of 1 to 100 kHz. The patch pipettes Burdach et al. BMC Plant Biology (2018) 18:102 Page 9 of 10 were prepared from borosilicate glass tubes (Kimax-51, Authors’ contributions Design of work: WK. Performed experiments: ZB, AS. Data analysis: ZB, AS, ZT, Kimble Products, Toledo, Ohio, USA) in accordance RK. Write paper: WK, ZB. All authors read and approved the final manuscript. with the procedure previously described by us . Volt- age pulse within a range of 300 to 900 mV combined Ethics approval and consent to participate with gentle suction let gain the access to the vacuole in- No permission is needed for the collection of plant materials used in this study. terior. The current and voltage signs were in accordance with the convention proposed by Bertl et al. . Competing interests The authors declare no competing interests. The control bath solution in the patch-clamp experiments was the same as the one that was used for the vacuole Author details diameter measurements (solution number 1). The pipettes Department of Plant Physiology, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland. were filled with a solution containing 100 mM KCl, 2 mM Department of Physics and Biophysics, Wrocław University of Environmental MgCl , 5 mM MES, 5 mM Tris and pH 5.5, which was ad- and Life Sciences, Norwida 25, 50-375 Wrocław, Poland. justed to an osmolality of 580 mOsm with sorbitol. The Received: 20 October 2017 Accepted: 24 May 2018 osmolality of all of the media used during the measure- ments was adjusted by a cryoscopic osmometer (Semi-Mi- cro Osmometer K-7400, Knauer, Germany). It is well References established that under symmetrical 100 mM K and micro- + 1. Hager A. Role of the plasma membrane H -ATPase in auxin-induced 2+ + molar cytosolic Ca concentrations, the SV channels K elongation growth: historical and new aspects. J Plant Res. 2003;116: 483–505. current is outwardly directed. Nevertheless at a calcium gra- 2. 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BMC Plant Biology – Springer Journals
Published: Jun 4, 2018
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