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J. Biol. Chem., Vol. 282, Issue 3, 1916-1924, January 19, 2007

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The ATP Binding Cassette Transporter AtMRP5 Modulates Anion and Calcium Channel Activities in Arabidopsis Guard Cells^*

Su Jeoung Suh¹², Yong-Fei Wang¹, Annie Frelet¹, Nathalie Leonhardt¹³, Markus Klein, Cyrille Forestier^¶, Bernd Mueller-Roeber^||, Myeon H. Cho^**, Enrico Martinoia⁴, and Julian I. Schroeder⁵

From the Institut für Pflanzenbiologie, University Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland, the Divison of Biological Sciences, Cell & Developmental Biology Section, University of California San Diego, La Jolla, California 92093-0116, the ^¶CEA Cadarache, UMR 163 CEA-CNRS, Départment d'Ecophysiologie Végétale et de Microbiologie (DEVM)-Laboratoire des Echanges Membrangire et Signalisation (LEMS), BP 1, F-13108 St. Paul Lez Durance, France, the ^||University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany, and the ^**Department of Biology, Yonsei University, Seoul, 120749 South Korea

Received for publication, August 18, 2006 , and in revised form, November 1, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Stomatal guard cells control CO₂ uptake and water loss between plants and the atmosphere. Stomatal closure in response to the drought stress hormone, abscisic acid (ABA), results from anion and K⁺ release from guard cells. Previous studies have shown that cytosolic Ca²⁺ elevation and ABA activate S-type anion channels in the plasma membrane of guard cells, leading to stomatal closure. However, membrane-bound regulators of abscisic acid signaling and guard cell anion channels remain unknown. Here we show that the ATP binding cassette (ABC) protein AtMRP5 is localized to the plasma membrane. Mutation in the AtMRP5 ABC protein impairs abscisic acid and cytosolic Ca²⁺ activation of slow (S-type) anion channels in the plasma membrane of guard cells. Interestingly, atmrp5 insertion mutant guard cells also show impairment in abscisic acid activation of Ca²⁺-permeable channel currents in the plasma membrane of guard cells. These data provide evidence that the AtMRP5 ABC transporter is a central regulator of guard cell ion channel during abscisic acid and Ca²⁺ signal transduction in guard cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Guard cells are highly specialized epidermal cells that in pairs form stomatal pores in aerial organs of plants. Each pair of guard cells forms a pore through which water and CO₂ exchange occurs. Guard cells modulate stomatal pore size by coordinating responses to environmental and physiological factors, including light, temperature, Ca²⁺, and the plant hormone abscisic acid. These factors induce dynamic changes in the intracellular concentrations of inorganic and organic ions. Stomatal opening occurs by osmotic swelling of guard cells, caused by K⁺ uptake through inward K⁺ channels, Cl^- uptake, and production of organic solutes (1–3). Stomatal closure results from anion and K⁺ release from guard cells and conversion of malate to starch. Previous studies have shown that cytosolic Ca²⁺ elevation and abscisic acid (ABA)⁶ activate S-type anion channels in the plasma membrane of guard cells (4–9). Anion efflux, Ca²⁺ influx, and H⁺-ATPase inhibition depolarize the membrane potential of guard cells, thus providing the driving force for K⁺ efflux through outward K⁺ channels (10–12). The present understanding of the guard cell signal transduction network and of the functions of individual genes in these processes has benefited from the analyses of mutants affected in stomatal physiology. First mutants that impair Ca²⁺-activation of anion channels have been recently identified, demonstrating that Ca²⁺-dependent protein kinases function in this response (9). However, the membrane proteins that function in channel regulation remain unknown.

The ATP binding cassette (ABC) superfamily is a large, ubiquitous, and diverse group of proteins, most of which mediate substrate transport across biological membranes (13, 14). ABC transporters have been shown to function not only as ATP-dependent pumps but also as ion channels and ion channel regulators. In animals, the cystic fibrosis transmembrane conductance regulator (CFTR) exhibits ion channel activity and CFTR and sulfonylurea receptors (SURs) also modulate the activity of associated ion channels. In addition, both of these membrane proteins are receptors for sulfonylureas (15, 16) and are blocked by glibenclamide in numerous tissues (17, 18). Leonhardt et al. (6, 19) showed that treatment with the sulfonylurea glibenclamide and other pharmacological compounds that interact with SURs and CFTR, modulate stomatal movements, and affect outward K⁺ and S-type anion channel properties.

Subsequently, it was shown that glibenclamide-induced stomatal opening is impaired in Arabidopsis plants carrying a T-DNA insertion in the AtMRP5 gene, which encodes a multidrug resistance-associated protein. Homozygous atmrp5-1 T-DNA insertion lines do not open their stomatal pores in response to glibenclamide (20), and AtMRP5 expressed in mammalian HEK293 cells is able to bind glibenclamide (21). These results suggest that AtMRP5 is responsible for glibenclamide-induced stomatal opening. In addition, detailed physiological analysis revealed that the stomata of atmrp5-1 mutant plants opened slightly less in the light and exhibited a reduced sensitivity to ABA and external calcium (22). Overall, atmrp5-1 mutant plants were less susceptible to drought stress. Different roles of AtMRP5 in guard cell regulation can be hypothesized: (i) AtMRP5 encodes a guard cell anion channel; (ii) AtMRP5 directly modulates a guard cell ion channel; and (iii) AtMRP5 does not modulate ion channels but affects regulation of stomatal movements via other structural or metabolic mechanisms. To analyze the functions of AtMRP5 in signal transduction during regulation of stomatal movements and to distinguish among these hypotheses, we localized AtMRP5 and performed patch clamp analyses of guard cell anion and Ca²⁺-permeable channels in atmrp5 mutant and wild-type guard cells, which reveal an interesting dual regulation of both ion channel types.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plant Growth—Arabidopsis thaliana plants, ecotype WS (Wassilewskija), were grown for 3–4 weeks in pots in standard soil (ED 73 + Bims, Einheitserde, Germany) in a plant growth chamber with an 8-h light period (100 µmol m^-2 s^-1 photosynthetic active radiation) at 22 °C and a 16-h dark period at 21 °C and relative humidity of 70%, or as described previously (23). Plants were watered daily.

Molecular Cloning of AtMRP5-GFP and Generation of Transgenic Plants—The AtMRP5 cDNA was cloned via NotI-NotI digestion from pN-AtMRP5 (20) into pRT-Not (24) resulting in pRT-MRP5. In this vector, AtMRP5 was placed under the control of CaMV (cauliflower mosaic virus) 35S promoter and the Cabb B-D polyadenylation signal. To construct a C-terminal in-frame fusion between AtMRP5 and GFP6, the terminal 1215 bases of AtMRP5 were amplified using primers 5Z-s (5'-ctt caa gag aat tgg) and 5Y-as (5'-cg tctaga gcggccgc cccggg taa ttc agg gat tcc) where XbaI, NotI, and SmaI sites in 5Y-as are underlined. In 5Y-as, the stop codon of AtMRP5 was omitted. The PCR fragment was subcloned into pGEM T-easy (Promega, Madison, WI) resulting in pG-5Y. AtMRP5 contains a unique XbaI site at position +3465 of the coding sequence. The entire gene of an enhanced GFP version (GFP6) (25) was amplified by PCR using primers GFP5SmaI-s (5'-cgg cccggg ggt ggc gga ggg atg agt aaa gga gaa gaa ctt ttc ac) and GFP6SmaI-as (5'-cgg cccggg gagctc tta ttt gta tag ttc atc cat gcc atg tg). In GFP5SmaI-s, a SmaI restriction site (underlined) and four glycine codons (bold) were introduced upstream of the EGFP coding sequence. In GFP6SmaI-as, SacI and SmaI sites (underlined) were introduced downstream of the stop codon of GFP6. The amplified GFP6 fragment was introduced into pG-6Y via SmaI. The resulting fragment fused to GFP6 was released by XbaI and cloned into XbaI-cut pRT-MRP5 resulting in pRT-MRP5GFP. Because pRT-MRP5 contains an additional XbaI site in the cassette cloning site 3' of AtMRP5, XbaI restriction allowed replacement of the native AtMRP5 3' end with the fragment carrying the GFP6 fusion. All PCR fragments and plasmids were verified by sequencing.

The cassette containing AtMRP5-GFP was excised using AscI and inserted into either pCambia1302 (www.cambia.org) or pGreen0179. Both vectors were modified to contain a unique AscI site in the multicloning site by insertion of a linker.⁷ The resulting constructs pC1302-AtMRP5-GFP and pG0179-AtMRP5-GFP were used to transform either Arabidopsis wild-type plants or mutant atmrp5-1 plants by floral dipping using Agrobacterium strain GV3101 (26). More than 25 T1 transformants for each construct were selected on half-strength MS (Murashigo and Skoog, Duchefa) medium agar plates containing hygromycin (Duchefa) for 7 days and were subsequently transferred on fresh sterile agar plates without antibiotic before transfer to soil pots.

Root Length of Complemented Lines—Homozygous transgenic Arabidopsis plants expressing AtMRP5-GFP, wild-type (Ws-2), and atmrp5-1 mutants were grown on half-strength MS (Sigma) medium in a vertical position for 9 days in continuous light. These plates were scanned using EPSON Perfection 2450 PHOTO, and the roots were measured using the measuring tool integrated in the Leica IM1000 software (Leica, Heerbrugg, Switzerland).

Analysis of Stomatal Apertures—For experiments at the University of Zurich stomatal apertures of rosette leaves of 7- to 8-week-old plants were measured according to the method of Lascève et al. (27). Measurements were started in the morning after 15–16 h of darkness. Leaves were detached and floated on a solution containing 10 mM KCl, 30 mM KOH, 10 mM MES-iminodiacetic acid, pH 6.0, in Petri dishes containing glibenclamide at the indicated concentrations. Glibenclamide-dependent stomatal apertures were analyzed after 2 h in the dark at 20 °C. Parts of abaxial epidermis were peeled off with sharp forceps, transferred into a drop of floating solution on a glass slide, and immediately processed for microscopic analysis of stomatal apertures. For experiments at UCSD, leaves of 4- to 5-week-old wild-type and atmrp5-1 plants were incubated in white light with a fluency rate of 125 µmol m^-2 s^-1 for 2.5 h in stomatal opening solution containing 5 mM KCl, 50 µM CaCl₂, and 10 mM MES/Tris, pH 6.15. Stomatal apertures were measured 2.5 h after glibenclamide or ABA were added. Plants were grown in growth chambers with a period of 16-h light with 8-h darkness. Standard errors were calculated relative to the number of epidermal peel experiments. Bright-field pictures of stomata were taken with a Leica DMR microscope equipped by a Leica DC300F charged coupled device camera and controlled by software (Leica) or with an inverted Nikon Diaphot microscope and a charged coupled device camera as previously described (23). The width of the stomatal pores was determined using the measuring tool integrated in imaging software.

Protoplast Isolation—For the analysis of AtMRP5-GFP, homozygous transgenic Arabidopsis plants were grown for 7 days on half-strength MS medium under continuous light. Protoplasts were prepared following the procedure described in Song et al. (28) with minor modifications (see supplemental "Materials and Methods"). Seedlings were incubated in medium A (0.5 M Sorbitol, 20 mM MES/KOH, pH 5.6, 1 mM CaCl₂) containing 0.03% (w/v) pectolyase Y-23 and 0.75% (w/v) cellulase Y-C for 2 h at 30 °C. The protoplasts were collected by centrifugation (250 x g, 5 min) on a cushion of osmotically stabilized Percoll (Amersham Biosciences, 500 mM sorbitol, 20 mM MES, pH 6.0). They were washed in medium B (500 mM betaine, 1 mM CaCl₂, 10 mM MES/KOH, pH 5.6) three times with a centrifugation at 180 x g, 5 min to recover them at the bottom of the tube and to remove Percoll in the medium. For patch clamp analyses at the University of Zurich, guard cell protoplasts were isolated by a two-step enzyme digestion as described by Wang et al. (29), except that protoplasts were resuspended in a 500 mM mannitol, 0.1 mM CaCl₂ in the final step.

For analysis of ABA regulation of I_Ca channel currents and S-type anion currents at UCSD, wild-type Ws-2 and mutant plants were grown in soil in a plant growth chamber with a 16-h light (75 µmol s^-1 m^-2 light fluence rate) and 8-h dark regime at 20 °C and watered every 2 days. Arabidopsis guard cell protoplasts were isolated enzymatically from leaf epidermal strips of 4- to 6-week-old plants as described previously (30).

Electrophysiological Measurements—Whole cell patch clamping was performed on guard cell protoplasts. S-type anion channel activity was measured in the absence or the presence of ABA and under high and low calcium concentrations. For experiments performed at the University of Zurich, the bath contained 40 mM CaCl₂ (23), 2 mM MgCl₂, 10 mM MES-Tris (pH 5.5); the pipette contained 150 mM tetraethyl-ammonium-Cl, 2 mM MgCl₂, 6.7 mM EGTA, 3.35 mM CaCl₂, 5 mM ATP (4 mM ATP and 1 mM GTP in ABA experiments), and 10 mM Hepes-Tris (pH 7.2), which results in a free intracellular calcium concentration of 0.26 µM. For high cytosolic Ca²⁺ ([Ca²⁺]_cyt) experiments, CaCl₂ was increased to 5.864 mM, which results in 2 µM free calcium. Free calcium concentration was calculated using "CALCIUM" software. Osmolality was adjusted to 500 mos·mol·kg^-1 with sorbitol. For ABA experiments, cells were preincubated with 50 µM ABA (50 mM stock solution in ethanol, and final ethanol concentration of 0.1%) for 90 min on ice, and ABA was included in the pipette and bath during the recording. After establishing the whole cell configuration, the membrane potential was held at 30 mV for 10 min, and subsequently the channel activity was measured by applying a voltage protocol consisting of seven pulses starting at -145 mV. The duration of each pulse was 40 s, and the increase in voltage was +30 mV (31). Current amplitudes were measured at the end of voltage pulses. At the University of Zurich electrophysiological measurements were performed with an EPC10 amplifier (HEKA, Lambrecht, Germany). Data acquisition and analysis were made using PULSE (HEKA). Patch pipettes were prepared from N-51A capillaries (Garner glass, Claremont, CA) by a DMZ-Universal puller (Zeitz Instruments GmbH, München, Germany). Currents were low pass filtered at 2 kHz and digitized at 1 kHz.

For experiments performed at UCSD, the pipette solution contained 5 mM ATP-Tris, 150 mM CsCl, 6.7 mM EGTA, 2 mM MgCl₂, 10 mM HEPES adjusted to pH 7.1 with Tris. The CaCl₂ concentration was calculated to obtain a concentration of free [Ca²⁺]_cyt equal to 2 µM as described previously (7). In addition, the bath contained 30 mM CsCl, 2 mM MgCl₂, 1 mM CaCl₂, and 10 mM MES adjusted to pH 5.6 with Tris. The osmolalities of pipette and bath solutions were adjusted using an osmometer (Model 5500, Wescor, Inc., Logan, UT) to 500 mmol/kg and 485 mmol/kg with D-sorbitol, respectively. The bath solution for guard cell protoplast preincubation was supplemented with 40 mM CaCl₂ (23). 12–15 min after establishment of the whole cell configurations, the bath solution containing 1 mM CaCl₂ was perfused, and voltage pulses were applied.

Whole cell patch clamp recordings of I_Ca currents and S-type anion currents at UCSD in Arabidopsis guard cell protoplasts were performed using an Axopatch 200A patch clamp amplifier (Axon Instruments, Union City, CA), which was connected to a microcomputer via an interface as described previously (5). Seal resistances were >10 G. The liquid junction potential was measured for I_Ca analyses and corrected as described previously (33). pClamp software (version 8.0, Axon Instruments) was used to acquire and analyze whole cell currents. Voltage ramps were applied each minute with a ramp speed of -210 mV s^-1. For I_Ca current analyses, the bath solution contained 100 mM BaCl₂, 0.1 mM dithiothreitol, and 10 mM MES adjusted to pH 5.6 with Tris, and the pipette solution contained 10 mM BaCl₂, 0.1 mM dithiothreitol, 4 mM EGTA, 10 mM Hepes adjusted to pH 7.1 with Tris. The osmolalities of bath and pipette solutions were adjusted to 485 and 500 mmol/kg with D-sorbitol, respectively. 1 mM NADPH was freshly added to the pipette solution before experiments each day. Approximately 20 min after establishment of the whole cell configuration, 50 µM ABA was added extracellularly by perfusion, and the ramp protocol was applied for 16 additional min after ABA application for each cell. Student t-tests were performed for statistical analysis.

Confocal Laser Scanning Microscopy Analysis: Image Acquisition and Processing—Homozygous transgenic Arabidopsis seedlings were grown for 10 days on half-strength MS medium under continuous light. Root pieces of seedlings were excised and incubated in the presence or absence of either 5 µM FM4-64 (Molecular Probes) (33) during one night or with 1 mM propidium iodide (Molecular Probes) just before visualization. They were subsequently analyzed by confocal laser scanning microscopy. The emitted fluorescence was captured between 500 and 530 nm for GFP (green channel) and 620 and 750 nm for FM4-64, propidium iodide, and autofluorescence of chlorophyll (red channel) following excitation with a 488 nm argon laser line and an RSP500 dichroic beam splitter. Laser intensities were set to 40–50%, and intensities, gain, and offset adjustments were not changed when the specimens obtained from AtMRP5-GFP-expressing plant material were compared with corresponding wild-type samples. Stored images were false-colored in green (GFP) or red (FM4-64, propidium iodide, and chlorophyll autofluorescence) using Adobe Photoshop.

View larger version (50K): [in this window] [in a new window]   FIGURE 1. CaMV35S::AtMRP5-GFP complements diverse phenotypes of atmrp5-1 knock-out mutant plants. A, CaMV35S::AtMRP5-GFP complements the short root phenotype in atmrp5-1 and restores it to wild-type root length (Ws-2). Photographs are shown for two different homozygous T3 lines, 54-1-1 and 54-6-1. Plants were grown for 9 days under continuous light on one-half MS 1% sucrose plates. Bar = 1 cm. B, comparison of root length between wild-type (Ws-2), atmrp5-1, and different atmrp5-1/CaMV35S::AtMRP5-GFP homozygous lines. Epithelial sodium channel < 0.005 using the Mann-Whitney t test. C, atmrp5-1 mutation impairs glibenclamide-induced stomatal opening. Stomatal apertures were measured in plants kept in the dark for 15 and 3 h after the addition of the indicated concentrations of glibenclamide to wild-type (open symbols) and atmrp5-1 mutant stomata (filled symbols) in darkness. Error bars represent standard errors relative to n = 3 independent experiments with 60 total stomata per data point. D, CaMV35S::AtMRP5-GFP complements the stomatal insensitivity phenotype in mrp5-1 mutant plants. Stomatal apertures after application of glibenclamide. E, atmrp5-1/CaMV35S::AtMRP5-GFP plants exhibit increased sensitivity to drought stress when compared with atmrp5-1 mutant and Ws-2 plants. 8-week-old plants were watered fully before irrigation was stopped completely. Pictures were taken after 8 days without water. Bar = 1 cm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
AtMRP5 Is Localized in the Plasma Membrane—Localization studies of plant MRPs have revealed that these transporters can be targeted to the vacuolar as well as to the plasma membrane (34–36). To determine the localization of AtMRP5, we used an enhanced version of GFP (GFP6) (25), which was inserted at the C-terminal position of the gene. This particular position was chosen to minimize the interference with functional transmembrane domains of AtMRP5. Furthermore, four glycine residues were added between the last amino acids of AtMRP5 and the methionine of GFP6 to provide a linker allowing flexible movements of the GFP. Stably transformed, independent homozygous plant lines were used for localization studies.

To determine whether AtMRP5 is correctly targeted, we investigated whether the expression of the AtMRP5-GFP fusion in the atmrp5-1 T-DNA disruption mutant restores the wild-type phenotype. The enhanced drought stress tolerance, the glibenclamide sensitivity of stomata, and shorter root length were analyzed. Deletion mutant lines expressing CaMV35S::AtMRP5-GFP exhibited a root length almost identical to the wild type and statistically different from atmrp5-1 plants (Fig. 1, A and B). Additionally, two complemented lines were used to analyze the response of stomata to the sulfonylurea compound glibenclamide. As previously observed, in atmrp5-1 plants, stomatal opening induced by glibenclamide was strongly impaired (Fig. 1C) (20). In contrast in four independent AtMRP5-GFP-expressing atmrp5-1 lines, glibenclamide-induced stomatal opening in the dark was restored (Fig. 1D). Finally, the AtMRP5-GFP-expressing atmrp5-1 lines showed drought-induced wilting similar to wild-type plants (Fig. 1E). These results demonstrate that AtMRP5-GFP fusion is functionally active and complements the atmrp5-1 disruption mutant phenotype.

The complementation of three phenotypes by the AtMRP5-GFP-expressing atmrp5-1 lines allowed us to investigate the cellular localization of AtMRP5-GFP. Confocal microscope analysis of seedlings transformed with AtMRP5-GFP showed fluorescence at the periphery of cells, suggesting that the AtMRP5 protein is targeted to the plasma membrane. GFP fluorescence could be observed in different tissues of transgenic lines, e.g. in root (Fig. 2, A–D), leaves, and petals. To determine whether the observed fluorescence was associated with the vacuolar membrane or the cell wall, we used specific markers for these compartments. In case of the vacuole we used the fluorescence dye FM4-64 (37, 38). It has been reported that, after 60-min incubation, FM4-64 reaches the vacuolar membrane of BY2 cells and that, between 3 and 10 h, all vacuolar membranes are stained (33, 39). As shown in Fig. 2 (A and B), green fluorescence due to AtMRP5-GFP and red fluorescence due to FM4-64 were not overlapping, and the green fluorescence was located between two vacuolar membranes of adjacent cells. These observations demonstrate that AtMRP5-GFP is not located in the tonoplast. To exclude that the green fluorescence is emitted by the cell wall, we used propidium iodide (40), a membrane-impermeant red fluorescent dye known to stain the cell wall. In this case the red fluorescence due to the propidium iodide and corresponding to the cell wall between neighboring cells was located between two layers of green fluorescence, due to AtMRP5-GFP and corresponding to the plasma membrane (Fig. 2, C and D). These data provide evidence that AtMRP5-GFP is targeted to the plasma membrane.

View larger version (45K): [in this window] [in a new window]   FIGURE 2. AtMRP5-GFP is targeted to the plasma membrane. A, merged image of root cells of mrp5-1/CaMV35S::AtMRP5-GFP seedlings after incubation with the styryl dye FM4-64 (red) labeling the tonoplast. Bar = 4 µm. B, enlarged view of root cells of mrp5-1/CaMV35S::AtMRP5-GFP seedlings after incubation with FM4-64. Bar = 4 µm. Green and red fluorescence is clearly separated. C, cells of epidermal layer of a rosette leaf of mrp5-1/CaMV35S::AtMRP5-GFP seedlings after incubation with propidium iodide labeling the cell wall in red. Bar = 8.5 µm. Red propidium iodide fluorescence of cell walls is between two GFP-labeled plasma membranes of neighboring cells. D, enlarged view of root cells of mrp5-1/CaMV35S::AtMRP5-GFP seedlings after incubation with propidium iodide. Bar = 6.5 µm. E–H, protoplasts isolated from Ws-2/CaMV35S::AtMRP5-GFP plants. Bar = 20 µm. E, fluorescence signal false color in green, emission em = 500–530 nm, corresponding to GFP. F, corresponding chlorophyll fluorescence images false colored in red (em = 620–750 nm). G, merged images of E and F, respectively. H, corresponding differential interference contrast images.

Electrophysiological Studies Reveal that AtMRP5 Modulates S-type Anion Channel Activity—Calcium is a second messenger that induces stomatal closure and calcium-dependent activation of guard cell S-type anion channels (4, 7, 9, 23, 41–43). It was previously shown that stomata of atmrp5-1 deletion mutants show impaired stomatal closing induced by extracellular Ca²⁺ (22). Expression of AtMRP5 in insect cells or Xenopus oocytes did not result in altered potassium or anion currents in these cells (data not shown). It is therefore possible that AtMRP5 itself may regulate the activity of other ion channel proteins. Thus we tested whether S-type anion channels are regulated by calcium in AtMRP5 disruption mutant and wild-type plants. In the presence of elevated free [Ca²⁺](2 µM) in the pipette solution, which dialyzes the cytoplasm, wild-type guard cells showed an activation of anion currents (-58 ± 9.6 pA steady-state current at -115 mV; n = 16), which is similar to published data in other Arabidopsis ecotypes (Fig. 3). In contrast, activation of S-type anion currents in atmrp5-1 deletion mutants was much less pronounced (-32 ± 5.0 pA at -115 mV; n = 21), corresponding to a decrease of 45% compared with wild-type guard cells (p < 0.05). Similar findings were observed in both of the participating laboratories (supplemental Fig. S1, n = 12 atmrp5-1 guard cells). These results show that AtMRP5 modulates Ca²⁺-dependent activation of S-type anion channels.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Anion channel activation by elevated cytoplasmic Ca2+ is impaired in atmrp5 knock-out mutant guard cells. A, current traces obtained in response to voltage pulses ranging from -145 mV to +35 mV with 30-mV intervals. Ws2, wild type, atmrp5-1 mutant. B, current-voltage curves of currents obtained at the end of voltage pulses. Filled circles, wild type, Ws-2; open circles, atmrp5-1 mutant guard cells, n = 16 and 21, respectively. Bars represent ± S.E.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Anion channel activation by abscisic acid is impaired in atmrp5 knock-out mutant guard cells. A, current-voltage curves were obtained from recordings in the presence and absence of ABA. Open circles, no ABA; filled circles, +ABA. (n = 13–15 guard cells). B, current at the end of -115 mV voltage pulses re-plotted from A to compare channel activities between three lines in the absence of ABA. Asterisks show significant difference between atmrp5-1 and the AtMRP5 overexpression line (5OE) (p = 0.002).

View larger version (23K): [in this window] [in a new window]   FIGURE 5. ABA activation of ICa currents is impaired in Arabidopsis atmrp5 mutant guard cell protoplasts. A and B, examples of whole cell currents in WS wild-type guard cell protoplasts in the absence (0 ABA) and presence (+ABA) of abscisic acid. A, an example of ABA activation of ICa in WS wild type. B, an example of lack of ABA activation of ICa in WS wild type (see "Results"). C, shows the average current-voltage curve for all WS wild-type guard cells (n = 14 guard cells). D and E, whole cell currents in atmrp5-1 mutant guard cell protoplasts in the absence and presence of ABA. D, an example of slight ABA activation of ICa in an atmrp5-1 guard cell. E, an example of lack of ABA activation of ICa in an atmrp5-1 guard cell. F, the average current-voltage curve for all atmrp5-1 mutant guard cell protoplasts in the absence and presence of ABA (n = 21).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the present study the membrane targeting and the question whether the ATP-binding cassette membrane protein AtMRP5 affects ion channel regulation in plants was analyzed. We show here that AtMRP5 is targeted to the plasma membrane. No AtMRP5-dependent ion current could be observed when AtMRP5 was expressed in insect cells and Xenopus oocytes. In contrast, both abscisic acid and cytoplasmic Ca²⁺ activation of S-type anion channels were impaired in atmrp5 mutant guard cells. Interestingly, ABA regulation of plasma membrane Ca²⁺-permeable I_Ca channel currents was also impaired in atmrp5 mutant guard cells. Mutants in the Ca²⁺-dependent protein kinase genes, CPK3 and CPK6 were recently shown to impair cytosolic Ca²⁺ activation of S-type anion channels (9). The impairment in regulation of both S-type anion channels and Ca²⁺-permeable I_Ca channels in atmrp5 mutant guard cells (Figs. 3, 4, 5) suggests that AtMRP5 functions as a central plasma membrane regulator of several types of ion channels. Interestingly, cpk6 and cpk3,as well as atmrp5 mutant guard cells showed impairment in ABA activation of both S-type anion channels and I_Ca channels (Figs. 3, 4, 5) (9), indicating that the CDPK and AtMRP5 proteins may function in a common signal transduction pathway that regulates both types of plasma membrane ion channels in guard cells.

AtMRP5 encodes a membrane protein with typical features of MRP-type ABC transporters, constituted by an N-terminal hydrophobic extension, followed by the transmembrane domain TMD1, a first hydrophilic nucleotide binding fold (NBD1), the second transmembrane domain TMD2 and a second nucleotide binding fold (NBD2). The expression of AtMRP4 and AtMRP5 in Arabidopsis guard cells, together with previous analyses of corresponding T-DNA null mutants, revealed that the regulation of stomatal movements depends on plant MRP-like ABC proteins (20, 22, 36). However, the physiological characterization of atmrp4 and atmrp5 mutants established that their mode of action is clearly distinguishable: atmrp5 mutant plants are impaired in hormone- and calcium-induced stomatal movements, whereas atmrp4 mutants are more sensitive to water stress coinciding with increased transpiration and constitutively more open stomata in the light and dark (20, 22, 36). It is therefore possible that both ABC transporters regulate different targets in guard cells.

The observation that atmrp5 mutants are impaired in calcium- and ABA-induced stomatal closure suggested that AtMRP5 may in some way be linked to guard cell ion channel regulation. This suggestion was supported by pharmacological experiments that showed that sulfonylurea induces opening of stomata in a different species (6, 19). To determine in which membrane we should focus our electrophysiological analyses we investigated the location of AtMRP5. AtMRP1 and AtMRP2, members of clade I (49) of Arabidopsis MRPs have been localized on the vacuolar membrane (34, 35). Our results clearly show that AtMRP5, which is a member of clade II, is targeted to the plasma membrane. The finding that the AtMRP5-GFP fusion protein can complement atmrp5 disruption mutant phenotypes provides evidence for the correct targeting of the AtMRP5-GFP construct.

No AtMRP5-dependent currents could be detected in insect cells and Xenopus oocytes. Therefore, we hypothesized that AtMRP5 might regulate guard cells ion channels. To identify the functions or targets of AtMRP5, electrophysiological studies were performed on guard cells by comparing ion currents of guard cells isolated from wild-type and mutant plants. The most striking differences were observed for the S-type anion channel in the presence of elevated cytoplasmic calcium. Ca²⁺ activation of S-type anion channel activity was strongly reduced in atmrp5 mutant guard cells compared with the wild type. This finding correlates with findings that atmrp5 mutant plants show impaired Ca²⁺-dependent stomatal closing in Arabidopsis.

In contrast, in the presence of lower calcium concentrations, atmrp5 mutant guard cells exhibited a slightly larger S-type anion current activity, and this difference was even more pronounced between T-DNA insertion and AtMRP5 overexpression guard cells. This observation is in agreement with the observed drought-resistant phenotype and might indicate that the reduced water loss observed for atmrp5 plays a more pronounced role than the missing stomatal closure in response to ABA under our growth conditions. However, to verify this hypothesis and to determine whether AtMRP5 has water transpiration-related functions in other cell types, plants expressing AtMRP5 only in guard cells have to be generated and analyzed for stomatal behavior and water loss. The difference between anion channel activities in the absence of ABA show a slight, but not statistically significant tendency that atmrp5 guard cells may exhibit a higher anion channel activity. This result may reflect that, depending on the growth conditions, slight differences in stomata aperture can be observed in the absence of ABA (22) (supplemental Fig. S2). However, more importantly and in agreement with the results observed in planta and with epidermal strips (22), S-type channel activity was consistently increased after ABA application in WT and overexpression plants but not in atmrp5 mutants. The impairment in both ABA activation and cytoplasmic Ca²⁺ activation of S-type anion channels provides genetic evidence for a linkage of ABA and cytoplasmic Ca²⁺ signaling in guard cells. AtMRP5 may also play a role in a recently proposed pathway in which ABA primes cytosolic Ca²⁺ sensors, thus facilitating Ca²⁺-dependent stomatal closure (50).

The above findings would be consistent with two non-exclusive hypotheses that (i) AtMRP5 functions as an S-type anion channel subunit or that (ii) AtMRP5 functions as a membrane regulator of ion channels. Two animal MRP-like counterparts are known to be involved in mediating or controlling ion fluxes. The CFTR is a Cl^- channel that is modulated by its phosphorylation state. In addition, CFTR is also a regulator of epithelial Na⁺ channels and the so-called outwardly rectifying chloride channel. The outwardly rectifying chloride channel, however, has not been molecularly identified, and a detailed analysis of the role of CFTR in its regulation is still missing (51). The SUR is the regulatory subunit of the ATP-sensitive potassium (K_ATP) channel. Each K_ATP channel is composed of four subunits of both the SUR protein and the pore-forming inwardly rectifying K⁺ channel Kir6 (52). The examples described so far therefore indicate that ABC transporters are either ion channels (CFTR) or modulators of ion channels (CFTR and SUR). To further investigate the hypothesis that AtMRP5 functions as a membrane regulator of ion channels, we analyzed abscisic acid regulation of Ca²⁺-permeable cation (I_Ca) currents in the plasma membrane of guard cells (46–48). ABA regulation of S-type anion currents and I_Ca currents was weaker in the Ws-2 wild-type background compared with previous studies in the L. erecta background (5, 30). These patch clamp data are consistent with generally weaker ABA responses in Ws-2 in both stomatal movements and seed germination compared with the L. erecta ecotype. Nevertheless, in the present study we resolved a statistically significant impairment in ABA regulation of both S-type anion and I_Ca currents in atmrp5 mutant guard cells. These data suggest that AtMRP5 can function as a regulator of more than one ion channel type in the plasma membrane of guard cells. Thus the present study supports a model in which AtMRP5 may function as a parallel regulator of several types of ion channels in the plasma membrane in guard cells, including Ca²⁺ and anion channels, rather than a model with sequential regulation of the ion channel types. We attempted to extend our analyses to inward K⁺ channel currents in guard cells. However, patch clamp experiments in wild-type guard cells showed inward K⁺ channel currents that were too variable in their magnitudes in both of our laboratories. Given that K⁺ channels are generally considered to be less strongly regulated by ABA compared with anion and I_Ca channels, the variability found in wild-type guard cell K⁺ currents precluded a robust analysis (5). Transgenic tobacco guard cell protoplasts overexpressing AtMRP5 were also affected in anion channel regulation, but K⁺ channels were not affected, which may also be related to the larger range in physiological anion channel regulation compared with K⁺ channels.⁸

In conclusion we present evidence that the ABC transporter AtMRP5 functions in anion and Ca²⁺ channel regulation in the plasma membrane of guard cells. The mechanism of this interaction remains to be elucidated. There might be some common features with the well characterized cystic fibrosis transmembrane conductance regulator CFTR, which functions not only as a Cl^- channel but fulfills several other cellular functions, particularly by regulating other membrane conductance pathways through interactions with the amiloride-sensitive epithelial sodium channel and the outwardly rectifying chloride channel (53–56). Interestingly, outwardly rectifying chloride channel regulation has been shown to depend on conformation of the second nucleotide binding domain 2 by ATP (57). Future identification of the molecular identities of guard cell S-type anion channels and Ca²⁺-permeable I_Ca channels will allow analyses of the mechanisms by which AtMRP5 mediates regulation of both of these ion channel types.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant R01GM060396 and National Science Foundation Grant MCB0417118 (to J. I. S.), by the Swiss National Foundation (Grant 3100A0-103911 to E. M.), and partly by Grant R01-2006-000-11026-0 from KOSEF (to M. H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ These authors contributed equally to this work.

² Present address: Dept. of Biological Sciences, Seoul National University, Seoul 151-747, Korea.

³ Present address: CEA Cadarache, UMR 163 CEA-CNRS, DEVM-LEMS, BP 1, F-13108 St. Paul Lez Durance, France.

⁴ To whom correspondence may be addressed. Tel.: 41-1-634-8222; Fax: 41-44-634-8204; E-mail: enrico.martinoia{at}botinst.unizh.ch. ⁵ To whom correspondence may be addressed. Tel.: 858-534-7759; Fax: 858-534-7108; E-mail: julian{at}biomail.ucsd.edu.

⁶ The abbreviations used are: ABA, abscisic acid; ABC, ATP binding cassette; CFTR, cystic fibrosis transmembrane conductance regulator; SUR, sulfonylurea receptor; GFP, green fluorescent protein; MES, 4-morpholineethanesulfonic acid; UCSD, University of California San Diego; CaMV, cauliflower mosaic virus.

⁷ M. Klein, unpublished.

⁸ S. J. Suh, A. Frelet, H. Grob, N. Gaedeke, U. Schmidt, B. Mueller-Roeber, E. Martinoia, and M. Klein, manuscript in preparation.

	ACKNOWLEDGMENTS

 
We thank Bo Burla for the help in preparing some parts of the manuscript, and Mohammad H. Maktabi (UCSD) and Fabien Dalmas for reproducing oocyte experiments.

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