The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate... Abstract Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize. Introduction Stomata formed by paired guard cells are the main tunnels for CO2 intake for photosynthesis, release of O2 and water loss in plants. Light, including blue light and red light, can induce stomatal opening by triggering the influx of osmotic ions into guard cells (Roelfsema and Hedrich 2005, Shimazaki et al. 2007, Ward et al. 2009, Kollist et al. 2014), and diverse stimuli, including drought stress and ABA, ROS, darkness and the increases of ambient [CO2], can induce stomatal closure by triggering the release of the osmotic ions out of guard cells (Ward et al. 2009, Kollist et al. 2014, Song et al. 2014, Murata et al. 2015). It has been well established that K+ is the main osmotic cation for stomatal opening and closure (Humble and Hsiao 1969, Sawhney and Zelitch 1969, Outlaw and Lowry 1977, Schnabl and Raschke 1980, MacRobbie 1982, Hosy et al. 2003, Wang and Wu 2013). Guard cells absorb K+ mainly through inward K+ ( Kin+) channels during stomatal opening and release K+ mainly through outward K+ ( Kout+) channels for stomatal closure. The activity, K+ selectivity and voltage dependence of the Kin+ and Kout+ channels of Vicia faba guard cell protoplasts have been analyzed in detail in early studies (Schroeder et al., 1987, Schroeder 1988, Schroeder 1989). The Kin+ channels are composed of several members of the Shaker family, including KAT1, KAT2, AKT1, AKT2 and KC1 in Arabidopsis (Szyroki et al. 2001, Véry and Sentenac 2003, Lebaudy et al. 2007, Ward et al. 2009, Hedrich 2012, Wang and Wu 2013, Ronzier et al. 2014). The Kin+ channels are assembled as diverse hetero- or homotetramers, and each subunit has six transmembrane domains, a C-terminus and an N-terminus (MacKinnon 1991, Daram et al. 1997, Lebaudy et al. 2008). KAT1, KAT2, AKT1 and AKT2 are capable of forming homotetramers alone. But, KC1 is unable to do so, and functions as an inhibitory subunit of the Kin+ channels by integrating itself into the tetramers as one of the four subunits (Reintanz et al. 2002, Geiger et al. 2009a, Jeanguenin et al. 2011). For stomatal closure, the K+ efflux is mediated by a single Kout+ GORK, which is also a member of the Shaker family (Véry and Sentenac 2003, Lebaudy et al. 2007, Ward et al. 2009, Kim et al. 2010, Hedrich 2012). During stomatal opening, blue light activates H+-ATPase in the plasma membrane of guard cells to pump H+ out of guard cells, and K+ influx mediated by the Kin+ channels is then triggered by the subsequent hyperpolarization of the guard cell plasma membrane (Roelfsema and Hedrich 2005, Roelfsema et al. 2012, Kollist et al. 2014). For stomatal closure, GORK-mediated K+ efflux is activated by the depolarization of the guard cell plasma membrane caused by the efflux of osmotic anions (Ward et al. 2009, Kollist et al. 2014). The positive charges of K+ need to be counterbalanced by anions in guard cells. Malate and chloride have been identified as important osmotic anions for the regulation of charge balance and stomatal movement in early studies (Outlaw and Lowry 1977, Schnabl 1978, MacRobbie 1980, Schnabl and Kottmeier 1984, Talbott and Zeiger 1993), and each of them may take about 50% of the responsibility for positive charge balancing in guard cells (Roelfsema and Hedrich 2005). Later on, it was reported that nitrate is also essential for stomatal movement in Arabidopsis (Guo et al. 2003). For anion flux through the plasma membrane of guard cells, the nitrate transporter NRT1.1 is responsible for nitrate influx (Guo et al. 2003), and S-type anion channels AtSLAC1 and AtSLAH3 as well as the R-type anion channel AtALMT12 are responsible for anion efflux in Arabidopsis (Negi et al. 2008, Vahisalu et al. 2008, Meyer et al. 2010, Hedrich and Geiger 2017). Loss-of-function mutations of AtSLAC1 dramatically impair stomatal closure induced by multiple stimuli, whereas mutations in either AtSLAH3 or AtALMT12 only impair stomatal closure partially, demonstrating that AtSLAC1 is the main anion channel for osmotic anion efflux and stomatal closure in Arabidopsis. SLAC1 is a conserved S-type anion channel for stomatal closure in the whole plant kingdom because SLAC1-like sequences can be identified in diverse species from algae to higher plants (Lind et al. 2015). AtSLAC1 exhibits a high permeability to nitrate, a lower permeability to chloride and a minor permeability to malate (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012, Maierhofer et al. 2014). Interestingly, OsSLAC1, the ortholog of AtSLAC1 in rice, is highly nitrate selective, and its permeability to both chloride and malate is negligible relative to that of nitrate (Sun et al. 2016). A recent study revealed that SLAC1 from a desert plant Phoenix dactylifera is a nitrate-regulated chloride-permeable channel (Müller et al. 2017). These studies indicate that the anion selectivity and permeability of SLAC1 are complex, and could be different between diverse plant species. Maize is an important crop worldwide, and its global annual yield has exceeded that of rice and wheat. Drought stress and shortage of water are the main environmental factors to limit the yield of maize. Therefore, it is important to identify the essential components for stomatal movement in maize. The plasma membrane Kin+ channel ZmKZM1 in maize guard cells for stomatal opening has been identified (Philippar et al. 2003). However, the genetic identity as well as the anion selectivity and permeability of the main S-type anion channel ZmSLAC1 in maize remains to be addressed. In this study, we identified the gene encoding the main S-type anion channel ZmSLAC1 in maize, and further found that ZmSLAC1 is involved in stomatal closure mainly by specifically mediating the efflux of nitrate rather than that of chloride and malate. Results The identification of the gene encoding the main S-type anion channel ZmSLAC1 for stomatal closure in maize To identify and clone the gene encoding ZmSLAC1, we conducted a blast search using the AtSLAC1 amino acid sequence, and 10 candidate proteins and their encoding DNA sequences from the maize genome were identified (Supplementary Fig. S1, left panel). Phylogenetic analysis showed that four of them, namely GRMZM2G106921, GRMZM2G061469, GRMZM2G447657 and GRMZM2G169951, exhibited similarities of 67.31, 51.64, 51.21 and 51.25% to AtSLAC1, respectively. The four proteins each contain an N-terminal domain, 10 transmembrane domains and a C-terminal domain, being similar to AtSLAC1 (Supplementary Fig. S1, right panel). We next analyzed the gene expression profiles of the four close orthologs of AtSLAC1 in maize using qRT–PCR. The expression of the four genes was observed in maize leaves, and the expression level of GRMZM2G106921 was much higher than that of the other three genes (Supplementary Fig. S2A). We then analyzed the expression of GRMZM2G106921 in diverse maize tissues, and found that the expression level of this gene in epidermal strips was clearly higher than in other tissues, including roots, shoots and whole leaves (Supplementary Fig. S2B). These results are consistent with the gene chip data in MaizeGDB (www.maizegdb.org), and demonstrate that GRMZM2G106921 is the gene encoding ZmSLAC1. To investigate further whether GRMZM2G106921 was the gene encoding ZmSLAC1, two maize mutant lines zmslac1-1 (UFMu-04043) and zmslac1-2 (mu-illumina_231212.3) with a Mu transposon insertion in GRMZM2G106921/ZmSLAC1 were obtained (Supplementary Fig. S2C), and the insertions of this transposon in ZmSLAC1 were confirmed by RT–PCR and qRT–PCR (Supplementary Fig. S2D, E). We next analyzed the stomatal movement phenotype of these mutants. We measured the temperature of maize seedlings, and found that the temperature at the leaf surface was approximately 20.0°C for zmslac1-1 and zmslac1-2 mutants at room temperature (25°C), which was noticeably lower than the 24.0°C found for the wild type (W22) (Fig. 1A). We then analyzed the water loss of detached maize leaves, and found that the detached leaves of zmslac1-1 and zmslac1-2 mutants lost 36.44% and 41.01% of their initial fresh weight in 60 min, respectively, which was significantly faster than for the wild type (11.41%) (Fig. 1B;Supplementary Fig. S3A). We next analyzed ABA-induced stomatal closure using SEM technique, and found that the stomatal closure of zmslac1-1 and zmslac1-2 mutants exhibited a strong insensitivity to ABA compared with the wild type (Fig. 1C;Supplementary Fig. S3B). We analyzed the transpirational conductance of maize seedling leaves, and observed a strong insensitivity of the transpirational conductance of zmslac1-1 to the changes of [CO2], darkness, ABA (200 μM) and H2O2 (5 mM) compared with the wild type (Fig. 1D–G). We also analyzed stomatal density on the surface of maize leaves, and did not observe any significant difference for the two mutants relative to the wild type (Fig. 1H, P-values of 0.815 and 0.196 for zmslac1-1 and zmslac1-2, respectively, vs. the wild type), demonstrating that the stomatal movement phenotypes of zmslac1-1 and zmslac1-2 resulted from the impaired stomatal movement rather than the changes of stomatal density. The strong stomatal movement phenotypes of zmslac1-1 and zmslac1-2 mutants are quite similar to those of the Arabidopsis mutant atslac1 and the rice mutant osslac1 (Negi et al. 2008, Vahisalu et al. 2008, Vahisalu et al. 2010, Kusumi et al. 2012). Thus we revealed that the main anion channel ZmSLAC1 of maize guard cells is encoded by GRMZM2G106921. Fig. 1 View largeDownload slide ZmSLAC1 is essential for stomatal closure in maize. (A) Thermal images of WT, zmslac1-1 and zmslac1-2 seedlings. (B) Water loss of detached leaves. (C) ABA-induced stomatal closure. (D–G) Time courses of normalized transpirational conductance of intact leaves in response to the changes of [CO2] (D), darkness (E), 200 μM ABA (F) and 5 mM H2O2 (G). (H) Stomatal density analysis of the WT, zmslac1-1 and zmslac1-2. n = 3 biological replicates for all experiments. Error bars indicate means ± SE. **P < 0.01 vs. the WT by Students t-test. Fig. 1 View largeDownload slide ZmSLAC1 is essential for stomatal closure in maize. (A) Thermal images of WT, zmslac1-1 and zmslac1-2 seedlings. (B) Water loss of detached leaves. (C) ABA-induced stomatal closure. (D–G) Time courses of normalized transpirational conductance of intact leaves in response to the changes of [CO2] (D), darkness (E), 200 μM ABA (F) and 5 mM H2O2 (G). (H) Stomatal density analysis of the WT, zmslac1-1 and zmslac1-2. n = 3 biological replicates for all experiments. Error bars indicate means ± SE. **P < 0.01 vs. the WT by Students t-test. ZmSLAC1 is an OST1-activated nitrate-selective anion channel To investigate the functions of ZmSLAC1, we analyzed the protein–protein interaction between ZmSLAC1 and either AtOST1 or ZmOST1. We fused split YFP to the C-terminus of ZmSLAC1 and the C-termini of AtOST1 and ZmOST1, respectively. We observed a clear fluorescent signal in the peripheral area of Xenopus oocytes co-expressing ZmSLAC1:: YFCc and either AtOST1:: YFPN or ZmOST1:: YFPN (Supplementary Fig. S4A). We analyzed the BiFC signal intensity, and found that the BiFC signal intensity of AtSLAC1 + AtOST1 was slightly stronger than that of either ZmSLAC1 + AtOST1 or ZmSLAC1 + ZmOST1 (Supplementary Fig. S4B). We also transiently expressed ZmSLAC1 with a GFP fused to its C-terminus in the epidermal cells of Nicotiana benthamiana leaves, and a plasma membrane-localized protein KAT2 was used as a positive control. We observed the merged fluorescent signal in the periphery of epidermal cells (Supplementary Fig. S4C). These data suggest a protein–protein interaction between ZmSLAC1 and either AtOST1 or ZmOST1 as well as a plasma membrane localization of ZmSLAC1 in vitro. We next conducted an in vitro phosphorylation assay, and observed clear phosphorylation of the N-terminus (amino acids 1–193) of ZmSLAC1 by AtOST1 (Supplementary Fig. S5). We then conducted electrophysiological analysis in Xenopus oocytes by transiently co-expressing ZmSLAC1 and either AtOST1 or ZmOST1 using a nitrate-based bath solution containing 50 mM NO3−, and observed a strong activation of ZmSLAC1 by AtOST1, and a weaker activation of ZmSLAC1 by ZmOST1 relative to AtOST1 (Fig. 2A, B). We did not observe obvious currents in negative control oocytes, into which water or only the cRNA of the kinase or channel was injected (Fig. 2A, B). For convenience for analysis of the activity of ZmSLAC1 in oocytes, we used AtOST1 for the activation of ZmSLAC1 in further experiments. Further voltage clamp experimental results showed that the increases of extracellular NO3− [NO3−]ext from 3 mM to 10 mM, 30 mM and further to 100 mM, led to larger anion channel currents, and the reversal potential was shifted from about −2.8 mV to −38.0 mV upon the increase of [NO3−]ext from 3 mM to 100 mM (Fig. 2C). The shift of the reversal potential is consistent with the change of equilibrium potential of NO3− according to the Nernst equation, demonstrating a clear [NO3−] dependence of ZmSLAC1-mediated channel currents. We then replaced NO3− in the bath solution with chloride, sulfate and malate, respectively, at the same concentration, and failed to observe obvious ZmSLAC1-mediated anion channel currents (Fig. 2D). Fig. 2 View largeDownload slide ZmSLAC1 is a nitrate-selective anion channel. (A, B) Typical whole-oocyte anion current recordings using standard nitrate-based bath solution (A), and the average current amplitudes of instantaneous anion currents at −100 mV (B). The numbers of oocytes tested were four for control, AtOST1 and ZmSLAC1, seven for ZmOST1, six for ZmSLAC1 + AtOST1 and 11 for ZmSLAC1 + ZmOST1. (C, D) Average current–voltage curves of steady-state anion channel currents recorded in diverse external solutions as indicated. The numbers of oocytes tested were five, 10, 15 and five for 3, 10, 30 and 100 mM NO3− bath solutions, respectively (C), and eight, 12, six, seven and five for control, NO3−, Cl–, SO42− and malate– bath solutions, respectively (D). Error bars indicate means ± SE. **P < 0.01 by Student’s t-test vs. control (B), 3 mM NO3− (C) and NO3− (D), respectively. Fig. 2 View largeDownload slide ZmSLAC1 is a nitrate-selective anion channel. (A, B) Typical whole-oocyte anion current recordings using standard nitrate-based bath solution (A), and the average current amplitudes of instantaneous anion currents at −100 mV (B). The numbers of oocytes tested were four for control, AtOST1 and ZmSLAC1, seven for ZmOST1, six for ZmSLAC1 + AtOST1 and 11 for ZmSLAC1 + ZmOST1. (C, D) Average current–voltage curves of steady-state anion channel currents recorded in diverse external solutions as indicated. The numbers of oocytes tested were five, 10, 15 and five for 3, 10, 30 and 100 mM NO3− bath solutions, respectively (C), and eight, 12, six, seven and five for control, NO3−, Cl–, SO42− and malate– bath solutions, respectively (D). Error bars indicate means ± SE. **P < 0.01 by Student’s t-test vs. control (B), 3 mM NO3− (C) and NO3− (D), respectively. We calculated ZmSLAC1’s NO3−/Cl– permeability ratio (PNO3/PCl) using the Goldman–Hodgkin Katz equation. We estimated that the cytosolic Cl− concentration in oocytes could be no less than 100 mM because a large amount of Cl− could diffuse from the pipette solution (3 M KCl) into the cytoplasm of the oocytes. PNO3/PCl was approximately 7.22. If the cytosolic Cl− concentration in Xenopus oocytes was much higher than 100 mM, then the PNO3/PCl of ZmSLAC1 could be doubled a few times. It has been reported that the PNO3/PCl was 5.4 and 20 for AtSLAC1 and AtSLAH3, respectively (Lee et al. 2009, Geiger et al. 2011). Therefore, the nitrate selectivity of ZmSLAC1 over chloride is clearly larger than that of AtSLAC1, and similar to that of AtSLAH3. Together, these results demonstrate that ZmSLAC1 is a nitrate-selective anion channel, and its ion selectivity is similar to that of AtSLAH3 and OsSLAC1 (Geiger et al. 2011, Sun et al. 2016), but its selectivity to nitrate is more specific than that of AtSLAC1 (Geiger et al. 2009b, Lee et al. 2009). ZmSLAC1 is not a nitrate-activated chloride-permeable channel AtSLAC1 is permeable to both nitrate and chloride, and its permeability to nitrate is clearly greater than that to chloride (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012). It is also known that nitrate can function as a ligand for the activation of AtSLAH3 and PdSLAC1, and low nitrate can trigger AtSLAH3- and PdSLAC1-mediated chloride currents (Geiger et al. 2011, Müller et al. 2017). To investigate whether ZmSLAC1 has a nitrate-activated permeability to chloride, we analyzed ZmSLAC1-mediated anion channel currents using a group of combinations of nitrate and chloride in external solutions as described (see the Materials and Methods) (Sun et al. 2016). We first tested the ion selectivity of AtSLAC1, and observed small AtSLAC1-mediated anion channel currents in a 3 mM NO3− + 5 mM Cl− bath solution, but larger currents in a 3 mM NO3− + 50 mM Cl− bath solution (Fig. 3A, B). The reversal potential was shifted by 19.6 mV from −5.8 mV to −25.4 mV upon the increase of the external chloride concentration from 5 mM to 50 mM (Fig. 3B). These results are consistent with previous reports (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012). We next analyzed the characterization of ZmSLAC1. Interestingly, we only observed similar small anion channel currents in either 3 mM NO3− + 5 mM Cl− or 3 mM NO3− + 50 mM Cl− bath solution in the oocytes co-expressing ZmSLAC1 and AtOST1, and no shift in reversal potential was observed upon the increase of Cl− concentration from 5 mM to 50 mM (Fig. 3A, B). Thus ZmSLAC1 showed no nitrate-activated permeability to chloride. This character of ZmSLAC1 is obviously different from AtSLAC1 (Lee et al. 2009), but similar to OsSLAC1 (Sun et al. 2016). Considering the strong stomatal movement phenotype of zmslac1 mutants (Fig. 1), these data also suggest that ZmSLAC1 is involved in stomatal closure mainly by mediating nitrate efflux. Fig. 3 View largeDownload slide ZmSLAC1 exhibits no NO3−-activated chloride currents. (A, B) Typical whole-oocyte recordings in oocytes (A), and the average current–voltage curves of steady-state anion channel currents (B). For AtSLAC1, the numbers of oocytes tested were seven for the 3 mM NO3− + 5 mM Cl– bath solution and six for the 3 mM NO3− + 50 mM Cl– bath solution. For ZmSLAC1, the numbers of oocytes tested were 12 for the 3 mM NO3− + 5 mM Cl– bath solution and 15 for the 3 mM NO3− + 50 mM Cl– bath solution. Error bars indicate means ± SE. **P < 0.01 for AtSLAC1 vs. 3 mM NO3− + 5 mM Cl– conditions by Students t-test. Fig. 3 View largeDownload slide ZmSLAC1 exhibits no NO3−-activated chloride currents. (A, B) Typical whole-oocyte recordings in oocytes (A), and the average current–voltage curves of steady-state anion channel currents (B). For AtSLAC1, the numbers of oocytes tested were seven for the 3 mM NO3− + 5 mM Cl– bath solution and six for the 3 mM NO3− + 50 mM Cl– bath solution. For ZmSLAC1, the numbers of oocytes tested were 12 for the 3 mM NO3− + 5 mM Cl– bath solution and 15 for the 3 mM NO3− + 50 mM Cl– bath solution. Error bars indicate means ± SE. **P < 0.01 for AtSLAC1 vs. 3 mM NO3− + 5 mM Cl– conditions by Students t-test. ZmSLAC1 is involved in stomatal closure not mainly by releasing chloride To investigate whether ZmSLAC1 is involved in stomatal closure mainly by mediating nitrate efflux, we generated two transgenic Arabidopsis lines by expressing ZmSLAC1 in the Arabidopsis mutant atslac1-3 under an AtSLAC1 promoter. Two transgenic lines ZMS-4 (ZmSLAC1 expressed in the single mutant atslac1-3) and ZMS-6 were selected. The expression of ZmSLAC1 in ZMS-4 and ZMS-6 was confirmed by RT–PCR (Supplementary Fig. S6A). We next analyzed the water loss of detached leaves. The experimental results showed that the detached leaves of the atslac1-3 mutant wilted quickly, and lost 63.05% of their fresh weight in 60 min, whereas the detached leaves of ZMS-4 and ZMS-6 lost 28.78% and 26.59%, respectively, of their initial fresh weight in 60 min, which were similar to the wild type (24.48%, P-value = 0.072 and 0.396 for ZMS-4 and ZMS-6, respectively, vs. the wild type), but significantly less than in the atslac1-3 mutant (Fig. 4A, P < 0.01). We then conducted an ABA-induced stomatal closure assay employing KNO3-based solution (see the Materials and Methods) using epidermal strips of leaves. The experimental results showed that the stomata were mostly closed in the wild type, atslac1-3, ZMS-4 and ZMS-6 upon the application of 10 μM ABA (Fig. 4B, P < 0.01 for the wild type, atslac1-3, ZMS-4 and ZMS-6 vs. the ABA-free condition, but P > 0.05 for ZMS-4 and ZMS-6 vs. the wild type and atslac1-3). We further conducted an ABA-induced stomatal closure assay using chloride-based solution (see the Materials and Methods), and observed normal ABA-induced stomatal closure in the wild type, ZMS-4 and ZMS-6, but the atslac1-3 mutant showed a strong ABA insensitivity (Supplementary Fig. S7). The strong ABA insensitivity of atslac1-3 in chloride-based solution is consistent with previous reports (Negi et al. 2008, Vahisalu et al. 2008), but the normal ABA-induced stomatal closure in atslac1-3 relative to the wild type in NO3–-based solution is a novel observation. These data suggest an important role for ZmSLAC1-mediated NO3- efflux in stomatal closure. Fig. 4 View largeDownload slide ZmSLAC1 did not fully rescue the stomatal closure phenotypes of the atslac1-3 mutant by restoring chloride-carried anion channel currents of guard cells in Arabidopsis. (A) Water loss analysis of detached leaves (n = 3 replicates). (B) ABA-induced stomatal closure assay using KNO3-based solution (n = 3 replicates). (C, D) Typical whole-cell anion channel current recordings of Arabidopsis guard cells (C), and the average current–voltage curves of the WT (n = 6), atslac1-3 (n = 6), ZMS-4 (n = 10) and ZMS-6 (n = 13) using chloride-based bath and pipette solutions (D). –ABA and +ABA denote the conditions without and with ABA added, respectively. Error bars indicate means ± SE. **P < 0.01 by Student’s t-test vs. the atslac1-3 mutant (A, B) and the wild type (D), respectively. Fig. 4 View largeDownload slide ZmSLAC1 did not fully rescue the stomatal closure phenotypes of the atslac1-3 mutant by restoring chloride-carried anion channel currents of guard cells in Arabidopsis. (A) Water loss analysis of detached leaves (n = 3 replicates). (B) ABA-induced stomatal closure assay using KNO3-based solution (n = 3 replicates). (C, D) Typical whole-cell anion channel current recordings of Arabidopsis guard cells (C), and the average current–voltage curves of the WT (n = 6), atslac1-3 (n = 6), ZMS-4 (n = 10) and ZMS-6 (n = 13) using chloride-based bath and pipette solutions (D). –ABA and +ABA denote the conditions without and with ABA added, respectively. Error bars indicate means ± SE. **P < 0.01 by Student’s t-test vs. the atslac1-3 mutant (A, B) and the wild type (D), respectively. We next analyzed S-type anion channel currents of Arabidopsis guard cell protoplasts using chloride-based pipette and bath solutions as described (see the Materials and Methods) (Vahisalu et al. 2008). We observed large S-type anion channel currents in wild-type guard cells (Fig 4C, D), but only low conductance was observed in the guard cells of atslac1-3, ZMS-4 and ZMS-6 (Fig. 4C, D, P < 0.01 for atslac1-3, ZMS-4 and ZMS-6 vs. the wild type at −145 mV). The average anion channel current amplitudes showed no obvious difference between the atslac1-3 mutant and the two transgenic lines ZMS-4 and ZMS-6 (Fig. 4D, P = 0.740 and 0.654 for ZMS-4 and ZMS-6, respectively, vs. atslac1-3 at −145 mV), demonstrating that the expression of ZmSLAC1 did not noticeably restore chloride-carried anion channel currents of guard cells in the atslac1-3 mutant. Considering normal stomatal closure induced by ABA in the atslac1-3 mutant in NO3−-based solution (Fig. 4B) and the lack of Cl–-carried S-type anion channel currents in ZMS-4 and ZMS-6 (Fig. 4C, D), we did not test anion channel currents of guard cells using NO3–-based pipette and bath solutions because AtSLAH3 is present in the guard cells, and could produce obvious S-type anion channel currents in atslac1-3, ZMS-4 and ZMS-6. ZmSLAC1 is involved in stomatal closure mainly by releasing nitrate To avoid the disturbance by AtSLAH3 of the analysis of ZmSLAC1-mediated anion channel currents of Arabidopsis guard cells, we generated a double mutant atslac1-3atslah3-2 by crossing the single mutants atslac1-3 and atslah3-2, and further generated transgenic lines by expressing ZmSLAC1 in the double mutant atslac1-3atslah3-2 under an AtSLAC1 promoter. Two transgenic lines ZMD-6 (ZmSLAC1 expressed in the double mutant atslac1-3atslah3-2) and ZMD-8 were selected, and the expression of ZmSLAC1 in ZMD-6 and ZMD-8 was confirmed by RT–PCR (Supplementary Fig. S6B). We then performed patch clamp experiments in guard cell protoplasts using nitrate-based bath and pipette solutions (see the Materials and Methods). We observed a very low background conductance in the double mutant atslac1-3atslah3-2, but similar large anion channel currents were observed in the wild type, ZMD-6 and ZMD-8 (Fig. 5A, B;P < 0.01 for ZMD-6, ZMD-8 and the wild type vs. slac1-3slah3-2 at −145, −115, −85 and −55 mV, respectively), demonstrating that the expression of ZmSLAC1 successfully restored nitrate-carried anion channel currents of Arabidopsis guard cells in the atslac1-3atslah3-2 mutant. We next analyzed the water loss of detached leaves at room temperature (25°C), and found that ZMD-6, ZMD-8 and the wild type lost 33.03, 26.69 and 17.42%, respectively, of their initial fresh weight in 60 min, whereas the atslac1-3atslah3-2 double mutant lost 54.44% of its initial fresh weight in 60 min (Fig. 5C, P < 0.01 for the wild type, ZMD-6 and ZMD-8 vs. atslac1-3atslah3-2 at 120 min; P =0.024 and 0.011 for ZMD-6 and ZMD-8 at 120 min, respectively, vs. the wild type), demonstrating a strong but partial rescue of the stomatal movement phenotype of the double mutant atslac1-3atslah3-2 by ZmSLAC1. We also analyzed ABA-induced stomatal closure in KNO3-based solution. The epidermal strips were exposed to light for 2.5 h to allow stomata to open, and then exposed to 10 μM ABA for an additional 2 h under light. The double mutant atslac1-3atslah3-2 showed a strong ABA-insensitive phenotype, whereas the stomata of ZMD-6, ZMD-8 and the wild type were mostly closed upon the application of 10 μM ABA (Fig. 5D, P < 0.01 for the wild type, ZMD-6 and ZMD-8 vs. atslac1-3atslah3-2). These results together demonstrate that ZmSLAC1 rescued the stomatal closure phenotype of atslac1-3atslah3-2 mainly by restoring nitrate-carried anion channel currents of guard cells, i.e. the efflux of nitrate is an essential driving force for stomatal closure. Fig. 5 View largeDownload slide ZmSLAC1 strongly but partially rescued the stomatal movement phenotypes of the atslac1-3atslah3-2 double mutant by restoring nitrate-carried anion channel currents of guard cells in Arabidopsis. (A, B) Typical whole-cell anion channel recordings of guard cells (A), and the average current–voltage curves of the WT (n = 12), slac1-3slah3-2 double mutant (n = 8), ZMD-6 (n = 9) and ZMD-8 (n = 10) using nitrate-based solutions (B). (C) Water loss analysis of detached leaves (n = 3 replicates). (D) ABA-induced stomatal closure assay using KNO3-based solution (n = 3 replicates). –ABA and +ABA denote the conditions without and with ABA added, respectively. Error bars indicate means ± SE. **P < 0.01 vs. the slac1-3slah3-2 mutant by Student’s t-test. Fig. 5 View largeDownload slide ZmSLAC1 strongly but partially rescued the stomatal movement phenotypes of the atslac1-3atslah3-2 double mutant by restoring nitrate-carried anion channel currents of guard cells in Arabidopsis. (A, B) Typical whole-cell anion channel recordings of guard cells (A), and the average current–voltage curves of the WT (n = 12), slac1-3slah3-2 double mutant (n = 8), ZMD-6 (n = 9) and ZMD-8 (n = 10) using nitrate-based solutions (B). (C) Water loss analysis of detached leaves (n = 3 replicates). (D) ABA-induced stomatal closure assay using KNO3-based solution (n = 3 replicates). –ABA and +ABA denote the conditions without and with ABA added, respectively. Error bars indicate means ± SE. **P < 0.01 vs. the slac1-3slah3-2 mutant by Student’s t-test. BrSLAC1 and SlSLAC1 are permeable to both nitrate and chloride For monocots, ZmSLAC1 and OsSLAC1 (Sun et al., 2016) show a strong selectivity to nitrate over chloride, but PdSLAC1 is a nitrate-activated chloride-permeable channel (Müller et al. 2017). For dicots, only AtSLAC1 was characterized as a nitrate- and chloride-permeable S-type anion channel (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012). To test more SLAC1s from dicots plants, BrSLAC1 and SlSLAC1 were selected for further experiments (Supplementary Fig. S8A). Split YFP was fused to the C-terminus of the two SLAC1 channels and AtOST1, respectively, and expressed in oocytes. We observed obvious YFP fluorescence in the periphery of oocytes co-expressing AtOST1 and either BrSLAC1 or SlSLAC1 (Supplementary Fig. S8B), suggesting a protein–protein interaction between the kinase and the two channels in Xenopus oocytes. We analyzed the relative BiFC signal intensity, and found it was similar for SlSLAC1, BrSLAC1 and AtSLAC1 (Supplementary Fig. S8C), suggesting a similar protein–protein interaction strength between the three SLAC1 channels and AtOST1. We next performed voltage clamp experiments in Xenopus oocytes, and observed obvious anion channel currents in either nitrate- or chloride-based bath solution (Supplementary Fig. S8D, E). However, BrSLAC1 showed smaller S-type anion channel currents than BrSLAC1 (Supplementary Fig. S8D, E). These data indicate that the characteristics of BrSLAC1 and SlSLAC1 are similar to those of AtSLAC1. Discussion The anion permeability and selectivity of SLAC1 could be variable in higher plants It has been well established that SLAC1 is the main anion channel for stomatal closure induced by diverse stimuli because loss-of-function mutations in AtSLAC1 dramatically impair ABA-, darkness-, ozone- and CO2-induced stomatal closure in Arabidopsis (Negi et al. 2008, Vahisalu et al. 2008, Vahisalu et al. 2010, Kusumi et al. 2012, Yamamoto et al. 2016). Further research show that Arabidopsis SLAC1 is permeable to both nitrate and chloride (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012), rice SLAC1 is nitrate elective (Sun et al. 2016) and PdSLAC1 from Phoenix dactylifera is a nitrate-activated chloride-permeable channel (Müller et al. 2017). In this study, we characterized ZmSLAC1 from maize as a nitrate-selective anion channel with a larger permeability ratio PNO3/PCl relative to AtSLAC1 (Figs. 2, 3) (Lee et al. 2009), and also found that the ion selectivity and permeability of BrSLAC1 and SlSLAC1 to nitrate and chloride are close to those of AtSLAC1. Therefore, it seems that the ion selectivity and permeability of SLAC1 channels from diverse plant species are variable, and we cannot draw conclusions on, or even imply, any general difference between monocot and dicot SLAC1. Furthermore the strong nitrate permeability of ZmSLAC1 relative to chloride does not exclude the importance of other anions, such as chloride and malate. In fact, the expression of ZmSLAC1 partially, but not fully, rescued the stomatal movement phenotypes of the atslac1-3atslah3-2 mutant (Fig. 4), suggesting that the efflux of other anions is also required for full stomatal closure. It is possible that one or a few ZmSLAC1 homologs are responsible for the release of chloride during stomatal closure in maize. The anion selectivity of ZmSLAC1 could be determined by a few key residues and the pore geometry of ZmSLAC1 A recent study revealed that Phe231 and Ser228 of AtSLAH2 and Phe276 of AtSLAC1 are essential sites for the anion selectivity of the S-type anion channels (Maierhofer et al. 2014). The point mutation of either F231A or S228V in AtSLAH2 converts AtSLAH2 from a nitrate-specific channel to a chloride-permeable SLAC1-like channel (Maierhofer et al. 2014). The point mutation of F276A in AtSLAC1 corresponding to F231A in AtSLAH2 can increase AtSLAC1’s chloride permeability relative to that of nitrate (Maierhofer et al. 2014). However, the reverse point mutation of V273S in AtSLAC1, a site corresponding to Phe228 of AtSLAH2, cannot convert AtSLAC1 into an AtSLAH2-like nitrate-selective channel (Maierhofer et al. 2014). Further analysis revealed that the change of transmembrane domain 3 in AtSLAC1 can convert AtSLAC1 into an SLAH2-like nitrate-selective channel (Maierhofer et al. 2014). It seems that the anion selectivity profile of an S-type anion channel is determined not only by a few key residues, but also by the pore geometry of the channel. We thus compared the amino acid sequences of AtSLAC1, AtSLAH2 and ZmSLAC1, and found that the amino acid residue at position 285 of ZmSLAC1 corresponds to and is the same as Phe231 of AtSLAH2 and Phe276 of AtSLAC1, but the amino acid residue sequences of ZmSLAC1 in this fragment are different from those of both AtSLAH2 and AtSLAC1 (Supplementary Fig. S9). Considering that maize is a monocot plant, we also compared ZmSLAC1 with OsSLAC1 and PdSLAC1, which are from two monocot plants. We found that the three anion channels have the same conserved phenylalanine residue in the corresponding site, but the amino acid sequences of this fragment of ZmSLAC1 are similar to those of OsSLAC1 and more different from PdSLAC1 (Supplementary Fig. S9). OsSLAC1 is known as a nitrate-selective anion channel (Sun et al. 2016), which is similar to ZmSLAC1, and PdSLAC1 has been reported to be a nitrate-activated chloride-permeable channel (Müller et al. 2017). Thus it is very likely that the high nitrate selectivity of ZmSLAC1 is determined by both a few key amino acid residues and the pore geometry, similar to AtSLAH2, AtSLAC1 and OsSLAC1, but could be different from PdSLAC1. Detailed electrophysiological analysis of diverse mutated versions of the multiple S-type anion channels will be needed and helpful for understanding the anion selectivity of ZmSLAC1 channel as well as its orthologs. Nitrate could be a ‘smart’ choice for stomatal movement for plants It could be a smart choice for plants to use nitrate as an essential osmotic anion for stomatal closure and opening. First, it is known that leaves function as nitrate sinks, and a large amount of nitrate is translocated from roots to leaves (Chiu et al. 2004, Fan et al. 2009, Hsu and Tsay 2013). Thus nitrate could be abundantly available to guard cells in leaves. Secondly. chloride is toxic to plant cells at high concentrations (Teakle and Tyerman 2010), and the concentration of chloride in guard cells can be reduced when nitrate is involved in stomatal movement as an alternative. Thirdly, nitrate could also be a regulator and facilitator of stomatal closure considering that nitrate can activate the chloride permeability of AtSLAH3 and PdSLAC1 (Geiger et al. 2011, Müller et al. 2017). Materials and Methods Plant material and growth conditions Arabidopsis thaliana (Columbia ecotype) plants were grown in soil (Sunrise, Canada) in a growth room under a 16 h light/8 h dark cycle at a photon fluence rate of approximately 75 μmol m–2 s–1 during the day time at a temperature of 21 ± 0.5°C, as described (Sun et al. 2016). Maize (Zea mays L.) plants were grown in soil (Sunrise, Canada) in a growth room with a 16 h light (25°C)/8 h dark (18°C) cycle at a photon fluence rate of approximately 210 μmol m–2 s–1 during the day time, as described (Philippar et al. 2003). The generation of transgenic Arabidopsis lines expressing ZmSLAC1 For the generation of transgenic Arabidopsis lines, ProAtSLAC1::ZmSLAC1 was constructed by introducing the full-length CDS (coding sequence) of ZmSLAC1 into the binary vector pCAMBIA1304 under an AtSLAC1 promoter (see Supplementary Table S1 for primers). The construct ProAtSLAC1::ZmSLAC1-pBIAM1304 was transformed into the single mutant atslac1-3 and the double mutant atslac1-3atslah3-2 with Agrobacterium (strain GV3101) using a floral dip method (Clough and Bent 1998). Homozygous lines from the T3 generation were selected for experiments. RT–PCR and qRT–PCR For ZmSLAC1 expression analysis in maize and Arabidopsis, total RNA was extracted from leaves of 2-week-old maize seedlings and 1-week-old Arabidopsis seedlings using Trizol reagent (Invitrogen) following the manufacturer’s instruction. cDNA was synthesized from DNase I-digested total RNA using M-MLV reverse transcriptase (Promega). qRT–PCR was performed using TransStart Tip Green qPCR Super Mix (TransGen Biotech) on a Bio-Rad CFX Connect™ Real-Time System according to the manufacturer’s protocols, as described (Zhang et al. 2016). RT–PCR and qRT–PCR were performed using Easy-Taq DNA polymerase (TransGen)_ENREF_48 (see Supplementary Table S1 for primers). Protein purification and in vitro protein phosphorylation assay To generate glutathione S-transferase (GST) fusion proteins, AtOST1 and the N-terminus of ZmSLAC1 (amino acids 1–193) were cloned into the pGEX4T-1 vector (see Supplementary Table S1 for primers), then transformed into the Escherichia coli BL21 (DE3) strain. Protein purification and in vitro phosphorylation assay were conducted as described (Xu et al. 2006). Stomatal movement-related analysis Transpirational conductance of intact maize leaves was measured using a LI-6400 XT Portable Photo-Synthesis System (LI-COR Biosciences) as described (Hu et al. 2010). Briefly, the conductance measurements were conducted at noon in a growth room at a temperature of 25 ± 1°C. The surface of the gas exchange analyzer chamber was fully covered by an intact maize leaf, the conductance was stabilized for at least 30 min, then the transpirational conductance of the leaf surface was continuously measured, and data were recorded during the whole period of treatment. For CO2 responses, leaf surface conductance measurement and the injection of 2,000 p.p.m. CO2 into the gas exchange analyzer chamber were started simultaneously, and the CO2 concentration was then shifted to 400 p.p.m. as indicated. For darkness response, lights were turned off immediately after conductance measurement, and data recording was started. For ABA and H2O2 responses, leaf surface conductance measurements were started immediately after the spraying of either 200 µM ABA or 5 mM H2O2. The data presented are the mean ± SE of at least three leaves per treatment. Transpirational conductance was divided by the conductance value at 0 min for data normalization. For the assay of ABA-induced stomatal closure in maize, young leaves of 3-week-old maize seedlings were harvested and sprayed with 200 µM ABA solution. After 15 min, the leaves were cut into small pieces, then the samples were fixed in FAA solution (50% ethanol, 5% acetic acid, 3.7% formaldehyde) for 24 h and dehydrated with a series of ethanol solutions (30, 50, 70, 85, 95 and 100%). The samples were then CO2 critical point-dried, sputter coated with gold and observed by SEM (JSM-6360LV, JEOL) (Zheng et al. 2013). Thermal images of maize plants were captured from 4-week-old maize seedlings using a FLIR A655sc infrared camera (FLIR®) with an InfRec Analyzer NS9500 Standard as described (Merlot et al. 2002). For the assay of stomatal movement in Arabidopsis, the KCl-based solution contained 5 mM KCl, 50 μM CaCl2 and 10 mM MES-Tris, pH 6.15 (Vahisalu et al. 2008). KNO3-based solution was modified from the KCl-based solution, and contained 5 mM KNO3, 50 μM CaCl2 and 10 mM MES-Tris, pH 6.15. Rosette leaves from 3- to 4-week-old plants were cut in the morning, and floated in the solution for 2.5 h with the abaxial side facing up under white light at a photon fluence rate of approximately 200–250 μmol m–2 s–1 (Sun et al. 2016). The epidermal strips were then peeled off the leaves using a razor blade, and incubated under white light for an additional 2 h with 10 μM ABA. Pictures of stomata were captured within 2 min before and after exposure to ABA under an inverted microscope (Model D1, Carl Zeiss) equipped with a neo CCD camera (Andor™ Technology) as described (Zhang et al. 2016). The length of stomatal apertures was measured after the experiments using an image software (Digimizer). More than 50 stomata were measured in each experiment, and the experiments were repeated at least three times. Figures were graphed using the average length of stomatal apertures by SigmaPlot software (version 11.0). For water loss measurement of detached maize and Arabidopsis leaves, the leaves were placed in a weighing dish with the abaxial side facing up at room temperature (25 ± 1°C), and weighed at various time points as indicated. The rate of water loss was calculated as the percentage of the initial fresh weight of the detached leaves. Electrophysiological analysis The glass pipettes were prepared using a glass capillary puller (model PC-10, Narishige). The software pClamp10.0 (Axon) was used for data acquirement and analysis. For the voltage clamp experiment in Xenopus oocytes, the coding sequences of AtSLAC1, ZmSLAC1, BrSLAC1 and SlSLAC1 were cloned into the pGEMHE-YFPC vector, and AtOST1 and ZmOST1 were cloned into thje pGEMHE-YFPN vector (Geiger et al. 2009b, Brandt et al. 2012) (see Supplementary Table S1 for primers). The cRNAs were prepared in vitro using a T7 RiboMAX™ large-scale RNA production system (Promega). The oocytes were isolated from Xenopus laevis, and injected with cRNAs. Oocytes injected with water were used as a negative control. Co-expressing mixtures were injected into oocytes with 6 ng for each gene in a total volume of 50 nl (Sun et al. 2016). The injected oocytes were incubated at 16°C in a modified bath solution supplemented with gentamycin (0.1 mg ml–1) for 2–3 d as described (Zhang et al. 2016). For anion channel current recordings, the standard NO3–-based bath solution contained (in mM) 1 Ca-gluconate, 1 Mg-gluconate, 1 K-gluconate, 50 NaNO3, 51 Na-gluconate and 10 MES-Tris (pH 5.6), and other anion-based solutions were modified from the standard NO3–-based solution by replacing NO3– with the anions at the same concentration as indicated. For the analysis of nitrate-activated chloride channel currents, the bath solution was modified from NO3–-based bath solution by replacing 50 mM NaNO3 with a combination of NaNO3 and NaCl, and contained 3 mM NaNO3 plus either 5 or 50 mM NaCl as described (Sun et al. 2016). Whole-oocyte currents were recorded using a step voltage protocol with 7 s voltage pulses from +40 to −140 mV with a 20 mV decrement, and the holding potential was 0 mV (Brandt et al. 2012). The channel currents were recorded using a 900 A two-electrode voltage clamp amplifier (Axon) connected to a personal computer through a 1440 A interface (Axon). Glass pipettes were filled with 3 M KCl as pipette solution (Brandt et al. 2012). For patch clamp experiments in Arabidopsis guard cells, Arabidopsis guard cell protoplasts were isolated enzymatically as described (Vahisalu et al. 2008). For anion channel current measurements, the Cl–-based standard bath solution contained (in mM) 30 CsCl, 1 CaCl2, 2 MgCl2 and 10 MES-Tris (pH 5.6), and the Cl–-based standard pipette solution contained (in mM) 150 CsCl, 2 MgCl2, 6.7 EGTA, 5 Mg-ATP (freshly added daily) and 10 HEPES-Tris (pH 7.1), with 5.864 CaCl2 added to give 2 μM free Ca2+ as described (Vahisalu et al. 2008). The NO3–-based standard bath solution contained (in mM) 30 CsNO3, 1 CaCl2, 2 Mg(NO3)2 , 10 MES-Tris (pH 5.6), and the NO3–-based standard pipette solution contained (in mM) 150 CsNO3, 2 Mg(NO3)2, 1 CaCl2, 6.7 EGTA, 5 Mg-ATP (fresh added daily) and 10 HEPES-Tris (pH 7.1) with 5.864 Ca(NO3)2 added to result in 2 μM free Ca2+. The osmolalities of the standard bath and pipette solutions were adjusted with d-sorbitol to 485 and 500 mmol kg–1, respectively. Guard cell protoplasts were pre-incubated for 30 min before patch clamp experiments in the bath solution with the Ca2+ concentration adjusted to 40 mM using CaCl2 for the Cl–-based bath solution or Ca(NO3)2 for the NO3–-based bath solution, and S-type anion channel currents were measured 7–10 min after accessing to the whole-cell configuration (Vahisalu et al. 2008). The membrane voltage was stepped from +35 mV to −145 mV with a 30 mV decrement as described (Vahisalu et al. 2008), the holding potential was 0 mV, the voltage pulse duration was 7 s and the voltage inter-pulse period was 12 s. No liquid junction potential correction was applied. BiFC experiments and fluorescence observation For BiFC experiments in Xenopus oocyts, vectors were the same as for the voltage clamp experiments (see the method for electrophysiological analysis). For GFP and OFP observation in N. benthamiana leaves, GFP and OFP were fused to the C-terminus of ZmSLAC1 and AtKAT1, and ZmSLAC1–GFP and AtKAT2 were each cloned into pCAMBIA2300 as described (Liu et al. 2013). Fluorescent images were captured under a Zeiss LSM510 META confocal microscope (Carl Zeiss). Accession numbers Gene sequence information in this article can be found in the MaizeGDB and GenBank databases using accession numbers GRMZM2G106921 (ZmSLAC1), GRMZM2G061469, GRMZM2G447657, GRMZM2G169951, ACG36261 (ZmOST1), AT1G12480 (AtSLAC1), AT4G33950 (AtOST1), AT3G18780 (ACTIN2), NC_024800.1 (BrSLAC1) and NC_015445.2(SlSLAC1). Statistical analysis Significance of difference of means between data sets was assessed by Student’s t-test. A difference of P < 0.05 was regarded as significant. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Natural Science Foundation of China [91635301, 31300214, 31570262 and 31770292]; the National Key Research and Development Program of China [2016YFD0100600]; the Science and Technology Commission of Shanghai Municipality [basic research key project 13JC1406100]; and the State Key Laboratory of Crop Stress Biology for Arid Areas [the Open Project (grant No. CSBAA2017005)]. Acknowledgments We thank Mingjie Cao from Jian-Kang Zhu’s group (SIPPE, SIBS, CAS) for assistance in temperature analysis of maize plants. zmslac1-1 and zmslac1-2 mutants were kindly provided by the Maize Genetics COOP Stock Center (http://maizecoop.cropsci.uiuc.edu/). Disclosures The authors have no conflict of interest to declare. References Brandt B., Brodsky D.E., Xue S., Negi J., Iba K., Kangasjärvi J., et al.   ( 2012) Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proc. Natl. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations ALMT aluminum-activated malate transporter BiFC bimolecular fluorescence complementation GFP green fluorescent protein GORK guard cell outward rectifying K+ channel NRT1.1 nitrate transporter 1 OFP orange fluorescent protein qRT–PCR quantitative RT–PCR R-type rapid type ROS reactive oxygen species RT–PCR reverse transcription–PCR SEM scanning electron microscopy SLAC1 slow anion channel-associated 1 SLAH SLAC1 homolog S-type slow type YFP yellow fluorescent protein © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize

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Abstract

Abstract Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize. Introduction Stomata formed by paired guard cells are the main tunnels for CO2 intake for photosynthesis, release of O2 and water loss in plants. Light, including blue light and red light, can induce stomatal opening by triggering the influx of osmotic ions into guard cells (Roelfsema and Hedrich 2005, Shimazaki et al. 2007, Ward et al. 2009, Kollist et al. 2014), and diverse stimuli, including drought stress and ABA, ROS, darkness and the increases of ambient [CO2], can induce stomatal closure by triggering the release of the osmotic ions out of guard cells (Ward et al. 2009, Kollist et al. 2014, Song et al. 2014, Murata et al. 2015). It has been well established that K+ is the main osmotic cation for stomatal opening and closure (Humble and Hsiao 1969, Sawhney and Zelitch 1969, Outlaw and Lowry 1977, Schnabl and Raschke 1980, MacRobbie 1982, Hosy et al. 2003, Wang and Wu 2013). Guard cells absorb K+ mainly through inward K+ ( Kin+) channels during stomatal opening and release K+ mainly through outward K+ ( Kout+) channels for stomatal closure. The activity, K+ selectivity and voltage dependence of the Kin+ and Kout+ channels of Vicia faba guard cell protoplasts have been analyzed in detail in early studies (Schroeder et al., 1987, Schroeder 1988, Schroeder 1989). The Kin+ channels are composed of several members of the Shaker family, including KAT1, KAT2, AKT1, AKT2 and KC1 in Arabidopsis (Szyroki et al. 2001, Véry and Sentenac 2003, Lebaudy et al. 2007, Ward et al. 2009, Hedrich 2012, Wang and Wu 2013, Ronzier et al. 2014). The Kin+ channels are assembled as diverse hetero- or homotetramers, and each subunit has six transmembrane domains, a C-terminus and an N-terminus (MacKinnon 1991, Daram et al. 1997, Lebaudy et al. 2008). KAT1, KAT2, AKT1 and AKT2 are capable of forming homotetramers alone. But, KC1 is unable to do so, and functions as an inhibitory subunit of the Kin+ channels by integrating itself into the tetramers as one of the four subunits (Reintanz et al. 2002, Geiger et al. 2009a, Jeanguenin et al. 2011). For stomatal closure, the K+ efflux is mediated by a single Kout+ GORK, which is also a member of the Shaker family (Véry and Sentenac 2003, Lebaudy et al. 2007, Ward et al. 2009, Kim et al. 2010, Hedrich 2012). During stomatal opening, blue light activates H+-ATPase in the plasma membrane of guard cells to pump H+ out of guard cells, and K+ influx mediated by the Kin+ channels is then triggered by the subsequent hyperpolarization of the guard cell plasma membrane (Roelfsema and Hedrich 2005, Roelfsema et al. 2012, Kollist et al. 2014). For stomatal closure, GORK-mediated K+ efflux is activated by the depolarization of the guard cell plasma membrane caused by the efflux of osmotic anions (Ward et al. 2009, Kollist et al. 2014). The positive charges of K+ need to be counterbalanced by anions in guard cells. Malate and chloride have been identified as important osmotic anions for the regulation of charge balance and stomatal movement in early studies (Outlaw and Lowry 1977, Schnabl 1978, MacRobbie 1980, Schnabl and Kottmeier 1984, Talbott and Zeiger 1993), and each of them may take about 50% of the responsibility for positive charge balancing in guard cells (Roelfsema and Hedrich 2005). Later on, it was reported that nitrate is also essential for stomatal movement in Arabidopsis (Guo et al. 2003). For anion flux through the plasma membrane of guard cells, the nitrate transporter NRT1.1 is responsible for nitrate influx (Guo et al. 2003), and S-type anion channels AtSLAC1 and AtSLAH3 as well as the R-type anion channel AtALMT12 are responsible for anion efflux in Arabidopsis (Negi et al. 2008, Vahisalu et al. 2008, Meyer et al. 2010, Hedrich and Geiger 2017). Loss-of-function mutations of AtSLAC1 dramatically impair stomatal closure induced by multiple stimuli, whereas mutations in either AtSLAH3 or AtALMT12 only impair stomatal closure partially, demonstrating that AtSLAC1 is the main anion channel for osmotic anion efflux and stomatal closure in Arabidopsis. SLAC1 is a conserved S-type anion channel for stomatal closure in the whole plant kingdom because SLAC1-like sequences can be identified in diverse species from algae to higher plants (Lind et al. 2015). AtSLAC1 exhibits a high permeability to nitrate, a lower permeability to chloride and a minor permeability to malate (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012, Maierhofer et al. 2014). Interestingly, OsSLAC1, the ortholog of AtSLAC1 in rice, is highly nitrate selective, and its permeability to both chloride and malate is negligible relative to that of nitrate (Sun et al. 2016). A recent study revealed that SLAC1 from a desert plant Phoenix dactylifera is a nitrate-regulated chloride-permeable channel (Müller et al. 2017). These studies indicate that the anion selectivity and permeability of SLAC1 are complex, and could be different between diverse plant species. Maize is an important crop worldwide, and its global annual yield has exceeded that of rice and wheat. Drought stress and shortage of water are the main environmental factors to limit the yield of maize. Therefore, it is important to identify the essential components for stomatal movement in maize. The plasma membrane Kin+ channel ZmKZM1 in maize guard cells for stomatal opening has been identified (Philippar et al. 2003). However, the genetic identity as well as the anion selectivity and permeability of the main S-type anion channel ZmSLAC1 in maize remains to be addressed. In this study, we identified the gene encoding the main S-type anion channel ZmSLAC1 in maize, and further found that ZmSLAC1 is involved in stomatal closure mainly by specifically mediating the efflux of nitrate rather than that of chloride and malate. Results The identification of the gene encoding the main S-type anion channel ZmSLAC1 for stomatal closure in maize To identify and clone the gene encoding ZmSLAC1, we conducted a blast search using the AtSLAC1 amino acid sequence, and 10 candidate proteins and their encoding DNA sequences from the maize genome were identified (Supplementary Fig. S1, left panel). Phylogenetic analysis showed that four of them, namely GRMZM2G106921, GRMZM2G061469, GRMZM2G447657 and GRMZM2G169951, exhibited similarities of 67.31, 51.64, 51.21 and 51.25% to AtSLAC1, respectively. The four proteins each contain an N-terminal domain, 10 transmembrane domains and a C-terminal domain, being similar to AtSLAC1 (Supplementary Fig. S1, right panel). We next analyzed the gene expression profiles of the four close orthologs of AtSLAC1 in maize using qRT–PCR. The expression of the four genes was observed in maize leaves, and the expression level of GRMZM2G106921 was much higher than that of the other three genes (Supplementary Fig. S2A). We then analyzed the expression of GRMZM2G106921 in diverse maize tissues, and found that the expression level of this gene in epidermal strips was clearly higher than in other tissues, including roots, shoots and whole leaves (Supplementary Fig. S2B). These results are consistent with the gene chip data in MaizeGDB (www.maizegdb.org), and demonstrate that GRMZM2G106921 is the gene encoding ZmSLAC1. To investigate further whether GRMZM2G106921 was the gene encoding ZmSLAC1, two maize mutant lines zmslac1-1 (UFMu-04043) and zmslac1-2 (mu-illumina_231212.3) with a Mu transposon insertion in GRMZM2G106921/ZmSLAC1 were obtained (Supplementary Fig. S2C), and the insertions of this transposon in ZmSLAC1 were confirmed by RT–PCR and qRT–PCR (Supplementary Fig. S2D, E). We next analyzed the stomatal movement phenotype of these mutants. We measured the temperature of maize seedlings, and found that the temperature at the leaf surface was approximately 20.0°C for zmslac1-1 and zmslac1-2 mutants at room temperature (25°C), which was noticeably lower than the 24.0°C found for the wild type (W22) (Fig. 1A). We then analyzed the water loss of detached maize leaves, and found that the detached leaves of zmslac1-1 and zmslac1-2 mutants lost 36.44% and 41.01% of their initial fresh weight in 60 min, respectively, which was significantly faster than for the wild type (11.41%) (Fig. 1B;Supplementary Fig. S3A). We next analyzed ABA-induced stomatal closure using SEM technique, and found that the stomatal closure of zmslac1-1 and zmslac1-2 mutants exhibited a strong insensitivity to ABA compared with the wild type (Fig. 1C;Supplementary Fig. S3B). We analyzed the transpirational conductance of maize seedling leaves, and observed a strong insensitivity of the transpirational conductance of zmslac1-1 to the changes of [CO2], darkness, ABA (200 μM) and H2O2 (5 mM) compared with the wild type (Fig. 1D–G). We also analyzed stomatal density on the surface of maize leaves, and did not observe any significant difference for the two mutants relative to the wild type (Fig. 1H, P-values of 0.815 and 0.196 for zmslac1-1 and zmslac1-2, respectively, vs. the wild type), demonstrating that the stomatal movement phenotypes of zmslac1-1 and zmslac1-2 resulted from the impaired stomatal movement rather than the changes of stomatal density. The strong stomatal movement phenotypes of zmslac1-1 and zmslac1-2 mutants are quite similar to those of the Arabidopsis mutant atslac1 and the rice mutant osslac1 (Negi et al. 2008, Vahisalu et al. 2008, Vahisalu et al. 2010, Kusumi et al. 2012). Thus we revealed that the main anion channel ZmSLAC1 of maize guard cells is encoded by GRMZM2G106921. Fig. 1 View largeDownload slide ZmSLAC1 is essential for stomatal closure in maize. (A) Thermal images of WT, zmslac1-1 and zmslac1-2 seedlings. (B) Water loss of detached leaves. (C) ABA-induced stomatal closure. (D–G) Time courses of normalized transpirational conductance of intact leaves in response to the changes of [CO2] (D), darkness (E), 200 μM ABA (F) and 5 mM H2O2 (G). (H) Stomatal density analysis of the WT, zmslac1-1 and zmslac1-2. n = 3 biological replicates for all experiments. Error bars indicate means ± SE. **P < 0.01 vs. the WT by Students t-test. Fig. 1 View largeDownload slide ZmSLAC1 is essential for stomatal closure in maize. (A) Thermal images of WT, zmslac1-1 and zmslac1-2 seedlings. (B) Water loss of detached leaves. (C) ABA-induced stomatal closure. (D–G) Time courses of normalized transpirational conductance of intact leaves in response to the changes of [CO2] (D), darkness (E), 200 μM ABA (F) and 5 mM H2O2 (G). (H) Stomatal density analysis of the WT, zmslac1-1 and zmslac1-2. n = 3 biological replicates for all experiments. Error bars indicate means ± SE. **P < 0.01 vs. the WT by Students t-test. ZmSLAC1 is an OST1-activated nitrate-selective anion channel To investigate the functions of ZmSLAC1, we analyzed the protein–protein interaction between ZmSLAC1 and either AtOST1 or ZmOST1. We fused split YFP to the C-terminus of ZmSLAC1 and the C-termini of AtOST1 and ZmOST1, respectively. We observed a clear fluorescent signal in the peripheral area of Xenopus oocytes co-expressing ZmSLAC1:: YFCc and either AtOST1:: YFPN or ZmOST1:: YFPN (Supplementary Fig. S4A). We analyzed the BiFC signal intensity, and found that the BiFC signal intensity of AtSLAC1 + AtOST1 was slightly stronger than that of either ZmSLAC1 + AtOST1 or ZmSLAC1 + ZmOST1 (Supplementary Fig. S4B). We also transiently expressed ZmSLAC1 with a GFP fused to its C-terminus in the epidermal cells of Nicotiana benthamiana leaves, and a plasma membrane-localized protein KAT2 was used as a positive control. We observed the merged fluorescent signal in the periphery of epidermal cells (Supplementary Fig. S4C). These data suggest a protein–protein interaction between ZmSLAC1 and either AtOST1 or ZmOST1 as well as a plasma membrane localization of ZmSLAC1 in vitro. We next conducted an in vitro phosphorylation assay, and observed clear phosphorylation of the N-terminus (amino acids 1–193) of ZmSLAC1 by AtOST1 (Supplementary Fig. S5). We then conducted electrophysiological analysis in Xenopus oocytes by transiently co-expressing ZmSLAC1 and either AtOST1 or ZmOST1 using a nitrate-based bath solution containing 50 mM NO3−, and observed a strong activation of ZmSLAC1 by AtOST1, and a weaker activation of ZmSLAC1 by ZmOST1 relative to AtOST1 (Fig. 2A, B). We did not observe obvious currents in negative control oocytes, into which water or only the cRNA of the kinase or channel was injected (Fig. 2A, B). For convenience for analysis of the activity of ZmSLAC1 in oocytes, we used AtOST1 for the activation of ZmSLAC1 in further experiments. Further voltage clamp experimental results showed that the increases of extracellular NO3− [NO3−]ext from 3 mM to 10 mM, 30 mM and further to 100 mM, led to larger anion channel currents, and the reversal potential was shifted from about −2.8 mV to −38.0 mV upon the increase of [NO3−]ext from 3 mM to 100 mM (Fig. 2C). The shift of the reversal potential is consistent with the change of equilibrium potential of NO3− according to the Nernst equation, demonstrating a clear [NO3−] dependence of ZmSLAC1-mediated channel currents. We then replaced NO3− in the bath solution with chloride, sulfate and malate, respectively, at the same concentration, and failed to observe obvious ZmSLAC1-mediated anion channel currents (Fig. 2D). Fig. 2 View largeDownload slide ZmSLAC1 is a nitrate-selective anion channel. (A, B) Typical whole-oocyte anion current recordings using standard nitrate-based bath solution (A), and the average current amplitudes of instantaneous anion currents at −100 mV (B). The numbers of oocytes tested were four for control, AtOST1 and ZmSLAC1, seven for ZmOST1, six for ZmSLAC1 + AtOST1 and 11 for ZmSLAC1 + ZmOST1. (C, D) Average current–voltage curves of steady-state anion channel currents recorded in diverse external solutions as indicated. The numbers of oocytes tested were five, 10, 15 and five for 3, 10, 30 and 100 mM NO3− bath solutions, respectively (C), and eight, 12, six, seven and five for control, NO3−, Cl–, SO42− and malate– bath solutions, respectively (D). Error bars indicate means ± SE. **P < 0.01 by Student’s t-test vs. control (B), 3 mM NO3− (C) and NO3− (D), respectively. Fig. 2 View largeDownload slide ZmSLAC1 is a nitrate-selective anion channel. (A, B) Typical whole-oocyte anion current recordings using standard nitrate-based bath solution (A), and the average current amplitudes of instantaneous anion currents at −100 mV (B). The numbers of oocytes tested were four for control, AtOST1 and ZmSLAC1, seven for ZmOST1, six for ZmSLAC1 + AtOST1 and 11 for ZmSLAC1 + ZmOST1. (C, D) Average current–voltage curves of steady-state anion channel currents recorded in diverse external solutions as indicated. The numbers of oocytes tested were five, 10, 15 and five for 3, 10, 30 and 100 mM NO3− bath solutions, respectively (C), and eight, 12, six, seven and five for control, NO3−, Cl–, SO42− and malate– bath solutions, respectively (D). Error bars indicate means ± SE. **P < 0.01 by Student’s t-test vs. control (B), 3 mM NO3− (C) and NO3− (D), respectively. We calculated ZmSLAC1’s NO3−/Cl– permeability ratio (PNO3/PCl) using the Goldman–Hodgkin Katz equation. We estimated that the cytosolic Cl− concentration in oocytes could be no less than 100 mM because a large amount of Cl− could diffuse from the pipette solution (3 M KCl) into the cytoplasm of the oocytes. PNO3/PCl was approximately 7.22. If the cytosolic Cl− concentration in Xenopus oocytes was much higher than 100 mM, then the PNO3/PCl of ZmSLAC1 could be doubled a few times. It has been reported that the PNO3/PCl was 5.4 and 20 for AtSLAC1 and AtSLAH3, respectively (Lee et al. 2009, Geiger et al. 2011). Therefore, the nitrate selectivity of ZmSLAC1 over chloride is clearly larger than that of AtSLAC1, and similar to that of AtSLAH3. Together, these results demonstrate that ZmSLAC1 is a nitrate-selective anion channel, and its ion selectivity is similar to that of AtSLAH3 and OsSLAC1 (Geiger et al. 2011, Sun et al. 2016), but its selectivity to nitrate is more specific than that of AtSLAC1 (Geiger et al. 2009b, Lee et al. 2009). ZmSLAC1 is not a nitrate-activated chloride-permeable channel AtSLAC1 is permeable to both nitrate and chloride, and its permeability to nitrate is clearly greater than that to chloride (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012). It is also known that nitrate can function as a ligand for the activation of AtSLAH3 and PdSLAC1, and low nitrate can trigger AtSLAH3- and PdSLAC1-mediated chloride currents (Geiger et al. 2011, Müller et al. 2017). To investigate whether ZmSLAC1 has a nitrate-activated permeability to chloride, we analyzed ZmSLAC1-mediated anion channel currents using a group of combinations of nitrate and chloride in external solutions as described (see the Materials and Methods) (Sun et al. 2016). We first tested the ion selectivity of AtSLAC1, and observed small AtSLAC1-mediated anion channel currents in a 3 mM NO3− + 5 mM Cl− bath solution, but larger currents in a 3 mM NO3− + 50 mM Cl− bath solution (Fig. 3A, B). The reversal potential was shifted by 19.6 mV from −5.8 mV to −25.4 mV upon the increase of the external chloride concentration from 5 mM to 50 mM (Fig. 3B). These results are consistent with previous reports (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012). We next analyzed the characterization of ZmSLAC1. Interestingly, we only observed similar small anion channel currents in either 3 mM NO3− + 5 mM Cl− or 3 mM NO3− + 50 mM Cl− bath solution in the oocytes co-expressing ZmSLAC1 and AtOST1, and no shift in reversal potential was observed upon the increase of Cl− concentration from 5 mM to 50 mM (Fig. 3A, B). Thus ZmSLAC1 showed no nitrate-activated permeability to chloride. This character of ZmSLAC1 is obviously different from AtSLAC1 (Lee et al. 2009), but similar to OsSLAC1 (Sun et al. 2016). Considering the strong stomatal movement phenotype of zmslac1 mutants (Fig. 1), these data also suggest that ZmSLAC1 is involved in stomatal closure mainly by mediating nitrate efflux. Fig. 3 View largeDownload slide ZmSLAC1 exhibits no NO3−-activated chloride currents. (A, B) Typical whole-oocyte recordings in oocytes (A), and the average current–voltage curves of steady-state anion channel currents (B). For AtSLAC1, the numbers of oocytes tested were seven for the 3 mM NO3− + 5 mM Cl– bath solution and six for the 3 mM NO3− + 50 mM Cl– bath solution. For ZmSLAC1, the numbers of oocytes tested were 12 for the 3 mM NO3− + 5 mM Cl– bath solution and 15 for the 3 mM NO3− + 50 mM Cl– bath solution. Error bars indicate means ± SE. **P < 0.01 for AtSLAC1 vs. 3 mM NO3− + 5 mM Cl– conditions by Students t-test. Fig. 3 View largeDownload slide ZmSLAC1 exhibits no NO3−-activated chloride currents. (A, B) Typical whole-oocyte recordings in oocytes (A), and the average current–voltage curves of steady-state anion channel currents (B). For AtSLAC1, the numbers of oocytes tested were seven for the 3 mM NO3− + 5 mM Cl– bath solution and six for the 3 mM NO3− + 50 mM Cl– bath solution. For ZmSLAC1, the numbers of oocytes tested were 12 for the 3 mM NO3− + 5 mM Cl– bath solution and 15 for the 3 mM NO3− + 50 mM Cl– bath solution. Error bars indicate means ± SE. **P < 0.01 for AtSLAC1 vs. 3 mM NO3− + 5 mM Cl– conditions by Students t-test. ZmSLAC1 is involved in stomatal closure not mainly by releasing chloride To investigate whether ZmSLAC1 is involved in stomatal closure mainly by mediating nitrate efflux, we generated two transgenic Arabidopsis lines by expressing ZmSLAC1 in the Arabidopsis mutant atslac1-3 under an AtSLAC1 promoter. Two transgenic lines ZMS-4 (ZmSLAC1 expressed in the single mutant atslac1-3) and ZMS-6 were selected. The expression of ZmSLAC1 in ZMS-4 and ZMS-6 was confirmed by RT–PCR (Supplementary Fig. S6A). We next analyzed the water loss of detached leaves. The experimental results showed that the detached leaves of the atslac1-3 mutant wilted quickly, and lost 63.05% of their fresh weight in 60 min, whereas the detached leaves of ZMS-4 and ZMS-6 lost 28.78% and 26.59%, respectively, of their initial fresh weight in 60 min, which were similar to the wild type (24.48%, P-value = 0.072 and 0.396 for ZMS-4 and ZMS-6, respectively, vs. the wild type), but significantly less than in the atslac1-3 mutant (Fig. 4A, P < 0.01). We then conducted an ABA-induced stomatal closure assay employing KNO3-based solution (see the Materials and Methods) using epidermal strips of leaves. The experimental results showed that the stomata were mostly closed in the wild type, atslac1-3, ZMS-4 and ZMS-6 upon the application of 10 μM ABA (Fig. 4B, P < 0.01 for the wild type, atslac1-3, ZMS-4 and ZMS-6 vs. the ABA-free condition, but P > 0.05 for ZMS-4 and ZMS-6 vs. the wild type and atslac1-3). We further conducted an ABA-induced stomatal closure assay using chloride-based solution (see the Materials and Methods), and observed normal ABA-induced stomatal closure in the wild type, ZMS-4 and ZMS-6, but the atslac1-3 mutant showed a strong ABA insensitivity (Supplementary Fig. S7). The strong ABA insensitivity of atslac1-3 in chloride-based solution is consistent with previous reports (Negi et al. 2008, Vahisalu et al. 2008), but the normal ABA-induced stomatal closure in atslac1-3 relative to the wild type in NO3–-based solution is a novel observation. These data suggest an important role for ZmSLAC1-mediated NO3- efflux in stomatal closure. Fig. 4 View largeDownload slide ZmSLAC1 did not fully rescue the stomatal closure phenotypes of the atslac1-3 mutant by restoring chloride-carried anion channel currents of guard cells in Arabidopsis. (A) Water loss analysis of detached leaves (n = 3 replicates). (B) ABA-induced stomatal closure assay using KNO3-based solution (n = 3 replicates). (C, D) Typical whole-cell anion channel current recordings of Arabidopsis guard cells (C), and the average current–voltage curves of the WT (n = 6), atslac1-3 (n = 6), ZMS-4 (n = 10) and ZMS-6 (n = 13) using chloride-based bath and pipette solutions (D). –ABA and +ABA denote the conditions without and with ABA added, respectively. Error bars indicate means ± SE. **P < 0.01 by Student’s t-test vs. the atslac1-3 mutant (A, B) and the wild type (D), respectively. Fig. 4 View largeDownload slide ZmSLAC1 did not fully rescue the stomatal closure phenotypes of the atslac1-3 mutant by restoring chloride-carried anion channel currents of guard cells in Arabidopsis. (A) Water loss analysis of detached leaves (n = 3 replicates). (B) ABA-induced stomatal closure assay using KNO3-based solution (n = 3 replicates). (C, D) Typical whole-cell anion channel current recordings of Arabidopsis guard cells (C), and the average current–voltage curves of the WT (n = 6), atslac1-3 (n = 6), ZMS-4 (n = 10) and ZMS-6 (n = 13) using chloride-based bath and pipette solutions (D). –ABA and +ABA denote the conditions without and with ABA added, respectively. Error bars indicate means ± SE. **P < 0.01 by Student’s t-test vs. the atslac1-3 mutant (A, B) and the wild type (D), respectively. We next analyzed S-type anion channel currents of Arabidopsis guard cell protoplasts using chloride-based pipette and bath solutions as described (see the Materials and Methods) (Vahisalu et al. 2008). We observed large S-type anion channel currents in wild-type guard cells (Fig 4C, D), but only low conductance was observed in the guard cells of atslac1-3, ZMS-4 and ZMS-6 (Fig. 4C, D, P < 0.01 for atslac1-3, ZMS-4 and ZMS-6 vs. the wild type at −145 mV). The average anion channel current amplitudes showed no obvious difference between the atslac1-3 mutant and the two transgenic lines ZMS-4 and ZMS-6 (Fig. 4D, P = 0.740 and 0.654 for ZMS-4 and ZMS-6, respectively, vs. atslac1-3 at −145 mV), demonstrating that the expression of ZmSLAC1 did not noticeably restore chloride-carried anion channel currents of guard cells in the atslac1-3 mutant. Considering normal stomatal closure induced by ABA in the atslac1-3 mutant in NO3−-based solution (Fig. 4B) and the lack of Cl–-carried S-type anion channel currents in ZMS-4 and ZMS-6 (Fig. 4C, D), we did not test anion channel currents of guard cells using NO3–-based pipette and bath solutions because AtSLAH3 is present in the guard cells, and could produce obvious S-type anion channel currents in atslac1-3, ZMS-4 and ZMS-6. ZmSLAC1 is involved in stomatal closure mainly by releasing nitrate To avoid the disturbance by AtSLAH3 of the analysis of ZmSLAC1-mediated anion channel currents of Arabidopsis guard cells, we generated a double mutant atslac1-3atslah3-2 by crossing the single mutants atslac1-3 and atslah3-2, and further generated transgenic lines by expressing ZmSLAC1 in the double mutant atslac1-3atslah3-2 under an AtSLAC1 promoter. Two transgenic lines ZMD-6 (ZmSLAC1 expressed in the double mutant atslac1-3atslah3-2) and ZMD-8 were selected, and the expression of ZmSLAC1 in ZMD-6 and ZMD-8 was confirmed by RT–PCR (Supplementary Fig. S6B). We then performed patch clamp experiments in guard cell protoplasts using nitrate-based bath and pipette solutions (see the Materials and Methods). We observed a very low background conductance in the double mutant atslac1-3atslah3-2, but similar large anion channel currents were observed in the wild type, ZMD-6 and ZMD-8 (Fig. 5A, B;P < 0.01 for ZMD-6, ZMD-8 and the wild type vs. slac1-3slah3-2 at −145, −115, −85 and −55 mV, respectively), demonstrating that the expression of ZmSLAC1 successfully restored nitrate-carried anion channel currents of Arabidopsis guard cells in the atslac1-3atslah3-2 mutant. We next analyzed the water loss of detached leaves at room temperature (25°C), and found that ZMD-6, ZMD-8 and the wild type lost 33.03, 26.69 and 17.42%, respectively, of their initial fresh weight in 60 min, whereas the atslac1-3atslah3-2 double mutant lost 54.44% of its initial fresh weight in 60 min (Fig. 5C, P < 0.01 for the wild type, ZMD-6 and ZMD-8 vs. atslac1-3atslah3-2 at 120 min; P =0.024 and 0.011 for ZMD-6 and ZMD-8 at 120 min, respectively, vs. the wild type), demonstrating a strong but partial rescue of the stomatal movement phenotype of the double mutant atslac1-3atslah3-2 by ZmSLAC1. We also analyzed ABA-induced stomatal closure in KNO3-based solution. The epidermal strips were exposed to light for 2.5 h to allow stomata to open, and then exposed to 10 μM ABA for an additional 2 h under light. The double mutant atslac1-3atslah3-2 showed a strong ABA-insensitive phenotype, whereas the stomata of ZMD-6, ZMD-8 and the wild type were mostly closed upon the application of 10 μM ABA (Fig. 5D, P < 0.01 for the wild type, ZMD-6 and ZMD-8 vs. atslac1-3atslah3-2). These results together demonstrate that ZmSLAC1 rescued the stomatal closure phenotype of atslac1-3atslah3-2 mainly by restoring nitrate-carried anion channel currents of guard cells, i.e. the efflux of nitrate is an essential driving force for stomatal closure. Fig. 5 View largeDownload slide ZmSLAC1 strongly but partially rescued the stomatal movement phenotypes of the atslac1-3atslah3-2 double mutant by restoring nitrate-carried anion channel currents of guard cells in Arabidopsis. (A, B) Typical whole-cell anion channel recordings of guard cells (A), and the average current–voltage curves of the WT (n = 12), slac1-3slah3-2 double mutant (n = 8), ZMD-6 (n = 9) and ZMD-8 (n = 10) using nitrate-based solutions (B). (C) Water loss analysis of detached leaves (n = 3 replicates). (D) ABA-induced stomatal closure assay using KNO3-based solution (n = 3 replicates). –ABA and +ABA denote the conditions without and with ABA added, respectively. Error bars indicate means ± SE. **P < 0.01 vs. the slac1-3slah3-2 mutant by Student’s t-test. Fig. 5 View largeDownload slide ZmSLAC1 strongly but partially rescued the stomatal movement phenotypes of the atslac1-3atslah3-2 double mutant by restoring nitrate-carried anion channel currents of guard cells in Arabidopsis. (A, B) Typical whole-cell anion channel recordings of guard cells (A), and the average current–voltage curves of the WT (n = 12), slac1-3slah3-2 double mutant (n = 8), ZMD-6 (n = 9) and ZMD-8 (n = 10) using nitrate-based solutions (B). (C) Water loss analysis of detached leaves (n = 3 replicates). (D) ABA-induced stomatal closure assay using KNO3-based solution (n = 3 replicates). –ABA and +ABA denote the conditions without and with ABA added, respectively. Error bars indicate means ± SE. **P < 0.01 vs. the slac1-3slah3-2 mutant by Student’s t-test. BrSLAC1 and SlSLAC1 are permeable to both nitrate and chloride For monocots, ZmSLAC1 and OsSLAC1 (Sun et al., 2016) show a strong selectivity to nitrate over chloride, but PdSLAC1 is a nitrate-activated chloride-permeable channel (Müller et al. 2017). For dicots, only AtSLAC1 was characterized as a nitrate- and chloride-permeable S-type anion channel (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012). To test more SLAC1s from dicots plants, BrSLAC1 and SlSLAC1 were selected for further experiments (Supplementary Fig. S8A). Split YFP was fused to the C-terminus of the two SLAC1 channels and AtOST1, respectively, and expressed in oocytes. We observed obvious YFP fluorescence in the periphery of oocytes co-expressing AtOST1 and either BrSLAC1 or SlSLAC1 (Supplementary Fig. S8B), suggesting a protein–protein interaction between the kinase and the two channels in Xenopus oocytes. We analyzed the relative BiFC signal intensity, and found it was similar for SlSLAC1, BrSLAC1 and AtSLAC1 (Supplementary Fig. S8C), suggesting a similar protein–protein interaction strength between the three SLAC1 channels and AtOST1. We next performed voltage clamp experiments in Xenopus oocytes, and observed obvious anion channel currents in either nitrate- or chloride-based bath solution (Supplementary Fig. S8D, E). However, BrSLAC1 showed smaller S-type anion channel currents than BrSLAC1 (Supplementary Fig. S8D, E). These data indicate that the characteristics of BrSLAC1 and SlSLAC1 are similar to those of AtSLAC1. Discussion The anion permeability and selectivity of SLAC1 could be variable in higher plants It has been well established that SLAC1 is the main anion channel for stomatal closure induced by diverse stimuli because loss-of-function mutations in AtSLAC1 dramatically impair ABA-, darkness-, ozone- and CO2-induced stomatal closure in Arabidopsis (Negi et al. 2008, Vahisalu et al. 2008, Vahisalu et al. 2010, Kusumi et al. 2012, Yamamoto et al. 2016). Further research show that Arabidopsis SLAC1 is permeable to both nitrate and chloride (Geiger et al. 2009b, Lee et al. 2009, Brandt et al. 2012), rice SLAC1 is nitrate elective (Sun et al. 2016) and PdSLAC1 from Phoenix dactylifera is a nitrate-activated chloride-permeable channel (Müller et al. 2017). In this study, we characterized ZmSLAC1 from maize as a nitrate-selective anion channel with a larger permeability ratio PNO3/PCl relative to AtSLAC1 (Figs. 2, 3) (Lee et al. 2009), and also found that the ion selectivity and permeability of BrSLAC1 and SlSLAC1 to nitrate and chloride are close to those of AtSLAC1. Therefore, it seems that the ion selectivity and permeability of SLAC1 channels from diverse plant species are variable, and we cannot draw conclusions on, or even imply, any general difference between monocot and dicot SLAC1. Furthermore the strong nitrate permeability of ZmSLAC1 relative to chloride does not exclude the importance of other anions, such as chloride and malate. In fact, the expression of ZmSLAC1 partially, but not fully, rescued the stomatal movement phenotypes of the atslac1-3atslah3-2 mutant (Fig. 4), suggesting that the efflux of other anions is also required for full stomatal closure. It is possible that one or a few ZmSLAC1 homologs are responsible for the release of chloride during stomatal closure in maize. The anion selectivity of ZmSLAC1 could be determined by a few key residues and the pore geometry of ZmSLAC1 A recent study revealed that Phe231 and Ser228 of AtSLAH2 and Phe276 of AtSLAC1 are essential sites for the anion selectivity of the S-type anion channels (Maierhofer et al. 2014). The point mutation of either F231A or S228V in AtSLAH2 converts AtSLAH2 from a nitrate-specific channel to a chloride-permeable SLAC1-like channel (Maierhofer et al. 2014). The point mutation of F276A in AtSLAC1 corresponding to F231A in AtSLAH2 can increase AtSLAC1’s chloride permeability relative to that of nitrate (Maierhofer et al. 2014). However, the reverse point mutation of V273S in AtSLAC1, a site corresponding to Phe228 of AtSLAH2, cannot convert AtSLAC1 into an AtSLAH2-like nitrate-selective channel (Maierhofer et al. 2014). Further analysis revealed that the change of transmembrane domain 3 in AtSLAC1 can convert AtSLAC1 into an SLAH2-like nitrate-selective channel (Maierhofer et al. 2014). It seems that the anion selectivity profile of an S-type anion channel is determined not only by a few key residues, but also by the pore geometry of the channel. We thus compared the amino acid sequences of AtSLAC1, AtSLAH2 and ZmSLAC1, and found that the amino acid residue at position 285 of ZmSLAC1 corresponds to and is the same as Phe231 of AtSLAH2 and Phe276 of AtSLAC1, but the amino acid residue sequences of ZmSLAC1 in this fragment are different from those of both AtSLAH2 and AtSLAC1 (Supplementary Fig. S9). Considering that maize is a monocot plant, we also compared ZmSLAC1 with OsSLAC1 and PdSLAC1, which are from two monocot plants. We found that the three anion channels have the same conserved phenylalanine residue in the corresponding site, but the amino acid sequences of this fragment of ZmSLAC1 are similar to those of OsSLAC1 and more different from PdSLAC1 (Supplementary Fig. S9). OsSLAC1 is known as a nitrate-selective anion channel (Sun et al. 2016), which is similar to ZmSLAC1, and PdSLAC1 has been reported to be a nitrate-activated chloride-permeable channel (Müller et al. 2017). Thus it is very likely that the high nitrate selectivity of ZmSLAC1 is determined by both a few key amino acid residues and the pore geometry, similar to AtSLAH2, AtSLAC1 and OsSLAC1, but could be different from PdSLAC1. Detailed electrophysiological analysis of diverse mutated versions of the multiple S-type anion channels will be needed and helpful for understanding the anion selectivity of ZmSLAC1 channel as well as its orthologs. Nitrate could be a ‘smart’ choice for stomatal movement for plants It could be a smart choice for plants to use nitrate as an essential osmotic anion for stomatal closure and opening. First, it is known that leaves function as nitrate sinks, and a large amount of nitrate is translocated from roots to leaves (Chiu et al. 2004, Fan et al. 2009, Hsu and Tsay 2013). Thus nitrate could be abundantly available to guard cells in leaves. Secondly. chloride is toxic to plant cells at high concentrations (Teakle and Tyerman 2010), and the concentration of chloride in guard cells can be reduced when nitrate is involved in stomatal movement as an alternative. Thirdly, nitrate could also be a regulator and facilitator of stomatal closure considering that nitrate can activate the chloride permeability of AtSLAH3 and PdSLAC1 (Geiger et al. 2011, Müller et al. 2017). Materials and Methods Plant material and growth conditions Arabidopsis thaliana (Columbia ecotype) plants were grown in soil (Sunrise, Canada) in a growth room under a 16 h light/8 h dark cycle at a photon fluence rate of approximately 75 μmol m–2 s–1 during the day time at a temperature of 21 ± 0.5°C, as described (Sun et al. 2016). Maize (Zea mays L.) plants were grown in soil (Sunrise, Canada) in a growth room with a 16 h light (25°C)/8 h dark (18°C) cycle at a photon fluence rate of approximately 210 μmol m–2 s–1 during the day time, as described (Philippar et al. 2003). The generation of transgenic Arabidopsis lines expressing ZmSLAC1 For the generation of transgenic Arabidopsis lines, ProAtSLAC1::ZmSLAC1 was constructed by introducing the full-length CDS (coding sequence) of ZmSLAC1 into the binary vector pCAMBIA1304 under an AtSLAC1 promoter (see Supplementary Table S1 for primers). The construct ProAtSLAC1::ZmSLAC1-pBIAM1304 was transformed into the single mutant atslac1-3 and the double mutant atslac1-3atslah3-2 with Agrobacterium (strain GV3101) using a floral dip method (Clough and Bent 1998). Homozygous lines from the T3 generation were selected for experiments. RT–PCR and qRT–PCR For ZmSLAC1 expression analysis in maize and Arabidopsis, total RNA was extracted from leaves of 2-week-old maize seedlings and 1-week-old Arabidopsis seedlings using Trizol reagent (Invitrogen) following the manufacturer’s instruction. cDNA was synthesized from DNase I-digested total RNA using M-MLV reverse transcriptase (Promega). qRT–PCR was performed using TransStart Tip Green qPCR Super Mix (TransGen Biotech) on a Bio-Rad CFX Connect™ Real-Time System according to the manufacturer’s protocols, as described (Zhang et al. 2016). RT–PCR and qRT–PCR were performed using Easy-Taq DNA polymerase (TransGen)_ENREF_48 (see Supplementary Table S1 for primers). Protein purification and in vitro protein phosphorylation assay To generate glutathione S-transferase (GST) fusion proteins, AtOST1 and the N-terminus of ZmSLAC1 (amino acids 1–193) were cloned into the pGEX4T-1 vector (see Supplementary Table S1 for primers), then transformed into the Escherichia coli BL21 (DE3) strain. Protein purification and in vitro phosphorylation assay were conducted as described (Xu et al. 2006). Stomatal movement-related analysis Transpirational conductance of intact maize leaves was measured using a LI-6400 XT Portable Photo-Synthesis System (LI-COR Biosciences) as described (Hu et al. 2010). Briefly, the conductance measurements were conducted at noon in a growth room at a temperature of 25 ± 1°C. The surface of the gas exchange analyzer chamber was fully covered by an intact maize leaf, the conductance was stabilized for at least 30 min, then the transpirational conductance of the leaf surface was continuously measured, and data were recorded during the whole period of treatment. For CO2 responses, leaf surface conductance measurement and the injection of 2,000 p.p.m. CO2 into the gas exchange analyzer chamber were started simultaneously, and the CO2 concentration was then shifted to 400 p.p.m. as indicated. For darkness response, lights were turned off immediately after conductance measurement, and data recording was started. For ABA and H2O2 responses, leaf surface conductance measurements were started immediately after the spraying of either 200 µM ABA or 5 mM H2O2. The data presented are the mean ± SE of at least three leaves per treatment. Transpirational conductance was divided by the conductance value at 0 min for data normalization. For the assay of ABA-induced stomatal closure in maize, young leaves of 3-week-old maize seedlings were harvested and sprayed with 200 µM ABA solution. After 15 min, the leaves were cut into small pieces, then the samples were fixed in FAA solution (50% ethanol, 5% acetic acid, 3.7% formaldehyde) for 24 h and dehydrated with a series of ethanol solutions (30, 50, 70, 85, 95 and 100%). The samples were then CO2 critical point-dried, sputter coated with gold and observed by SEM (JSM-6360LV, JEOL) (Zheng et al. 2013). Thermal images of maize plants were captured from 4-week-old maize seedlings using a FLIR A655sc infrared camera (FLIR®) with an InfRec Analyzer NS9500 Standard as described (Merlot et al. 2002). For the assay of stomatal movement in Arabidopsis, the KCl-based solution contained 5 mM KCl, 50 μM CaCl2 and 10 mM MES-Tris, pH 6.15 (Vahisalu et al. 2008). KNO3-based solution was modified from the KCl-based solution, and contained 5 mM KNO3, 50 μM CaCl2 and 10 mM MES-Tris, pH 6.15. Rosette leaves from 3- to 4-week-old plants were cut in the morning, and floated in the solution for 2.5 h with the abaxial side facing up under white light at a photon fluence rate of approximately 200–250 μmol m–2 s–1 (Sun et al. 2016). The epidermal strips were then peeled off the leaves using a razor blade, and incubated under white light for an additional 2 h with 10 μM ABA. Pictures of stomata were captured within 2 min before and after exposure to ABA under an inverted microscope (Model D1, Carl Zeiss) equipped with a neo CCD camera (Andor™ Technology) as described (Zhang et al. 2016). The length of stomatal apertures was measured after the experiments using an image software (Digimizer). More than 50 stomata were measured in each experiment, and the experiments were repeated at least three times. Figures were graphed using the average length of stomatal apertures by SigmaPlot software (version 11.0). For water loss measurement of detached maize and Arabidopsis leaves, the leaves were placed in a weighing dish with the abaxial side facing up at room temperature (25 ± 1°C), and weighed at various time points as indicated. The rate of water loss was calculated as the percentage of the initial fresh weight of the detached leaves. Electrophysiological analysis The glass pipettes were prepared using a glass capillary puller (model PC-10, Narishige). The software pClamp10.0 (Axon) was used for data acquirement and analysis. For the voltage clamp experiment in Xenopus oocytes, the coding sequences of AtSLAC1, ZmSLAC1, BrSLAC1 and SlSLAC1 were cloned into the pGEMHE-YFPC vector, and AtOST1 and ZmOST1 were cloned into thje pGEMHE-YFPN vector (Geiger et al. 2009b, Brandt et al. 2012) (see Supplementary Table S1 for primers). The cRNAs were prepared in vitro using a T7 RiboMAX™ large-scale RNA production system (Promega). The oocytes were isolated from Xenopus laevis, and injected with cRNAs. Oocytes injected with water were used as a negative control. Co-expressing mixtures were injected into oocytes with 6 ng for each gene in a total volume of 50 nl (Sun et al. 2016). The injected oocytes were incubated at 16°C in a modified bath solution supplemented with gentamycin (0.1 mg ml–1) for 2–3 d as described (Zhang et al. 2016). For anion channel current recordings, the standard NO3–-based bath solution contained (in mM) 1 Ca-gluconate, 1 Mg-gluconate, 1 K-gluconate, 50 NaNO3, 51 Na-gluconate and 10 MES-Tris (pH 5.6), and other anion-based solutions were modified from the standard NO3–-based solution by replacing NO3– with the anions at the same concentration as indicated. For the analysis of nitrate-activated chloride channel currents, the bath solution was modified from NO3–-based bath solution by replacing 50 mM NaNO3 with a combination of NaNO3 and NaCl, and contained 3 mM NaNO3 plus either 5 or 50 mM NaCl as described (Sun et al. 2016). Whole-oocyte currents were recorded using a step voltage protocol with 7 s voltage pulses from +40 to −140 mV with a 20 mV decrement, and the holding potential was 0 mV (Brandt et al. 2012). The channel currents were recorded using a 900 A two-electrode voltage clamp amplifier (Axon) connected to a personal computer through a 1440 A interface (Axon). Glass pipettes were filled with 3 M KCl as pipette solution (Brandt et al. 2012). For patch clamp experiments in Arabidopsis guard cells, Arabidopsis guard cell protoplasts were isolated enzymatically as described (Vahisalu et al. 2008). For anion channel current measurements, the Cl–-based standard bath solution contained (in mM) 30 CsCl, 1 CaCl2, 2 MgCl2 and 10 MES-Tris (pH 5.6), and the Cl–-based standard pipette solution contained (in mM) 150 CsCl, 2 MgCl2, 6.7 EGTA, 5 Mg-ATP (freshly added daily) and 10 HEPES-Tris (pH 7.1), with 5.864 CaCl2 added to give 2 μM free Ca2+ as described (Vahisalu et al. 2008). The NO3–-based standard bath solution contained (in mM) 30 CsNO3, 1 CaCl2, 2 Mg(NO3)2 , 10 MES-Tris (pH 5.6), and the NO3–-based standard pipette solution contained (in mM) 150 CsNO3, 2 Mg(NO3)2, 1 CaCl2, 6.7 EGTA, 5 Mg-ATP (fresh added daily) and 10 HEPES-Tris (pH 7.1) with 5.864 Ca(NO3)2 added to result in 2 μM free Ca2+. The osmolalities of the standard bath and pipette solutions were adjusted with d-sorbitol to 485 and 500 mmol kg–1, respectively. Guard cell protoplasts were pre-incubated for 30 min before patch clamp experiments in the bath solution with the Ca2+ concentration adjusted to 40 mM using CaCl2 for the Cl–-based bath solution or Ca(NO3)2 for the NO3–-based bath solution, and S-type anion channel currents were measured 7–10 min after accessing to the whole-cell configuration (Vahisalu et al. 2008). The membrane voltage was stepped from +35 mV to −145 mV with a 30 mV decrement as described (Vahisalu et al. 2008), the holding potential was 0 mV, the voltage pulse duration was 7 s and the voltage inter-pulse period was 12 s. No liquid junction potential correction was applied. BiFC experiments and fluorescence observation For BiFC experiments in Xenopus oocyts, vectors were the same as for the voltage clamp experiments (see the method for electrophysiological analysis). For GFP and OFP observation in N. benthamiana leaves, GFP and OFP were fused to the C-terminus of ZmSLAC1 and AtKAT1, and ZmSLAC1–GFP and AtKAT2 were each cloned into pCAMBIA2300 as described (Liu et al. 2013). Fluorescent images were captured under a Zeiss LSM510 META confocal microscope (Carl Zeiss). Accession numbers Gene sequence information in this article can be found in the MaizeGDB and GenBank databases using accession numbers GRMZM2G106921 (ZmSLAC1), GRMZM2G061469, GRMZM2G447657, GRMZM2G169951, ACG36261 (ZmOST1), AT1G12480 (AtSLAC1), AT4G33950 (AtOST1), AT3G18780 (ACTIN2), NC_024800.1 (BrSLAC1) and NC_015445.2(SlSLAC1). Statistical analysis Significance of difference of means between data sets was assessed by Student’s t-test. A difference of P < 0.05 was regarded as significant. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Natural Science Foundation of China [91635301, 31300214, 31570262 and 31770292]; the National Key Research and Development Program of China [2016YFD0100600]; the Science and Technology Commission of Shanghai Municipality [basic research key project 13JC1406100]; and the State Key Laboratory of Crop Stress Biology for Arid Areas [the Open Project (grant No. CSBAA2017005)]. Acknowledgments We thank Mingjie Cao from Jian-Kang Zhu’s group (SIPPE, SIBS, CAS) for assistance in temperature analysis of maize plants. zmslac1-1 and zmslac1-2 mutants were kindly provided by the Maize Genetics COOP Stock Center (http://maizecoop.cropsci.uiuc.edu/). Disclosures The authors have no conflict of interest to declare. References Brandt B., Brodsky D.E., Xue S., Negi J., Iba K., Kangasjärvi J., et al.   ( 2012) Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proc. Natl. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations ALMT aluminum-activated malate transporter BiFC bimolecular fluorescence complementation GFP green fluorescent protein GORK guard cell outward rectifying K+ channel NRT1.1 nitrate transporter 1 OFP orange fluorescent protein qRT–PCR quantitative RT–PCR R-type rapid type ROS reactive oxygen species RT–PCR reverse transcription–PCR SEM scanning electron microscopy SLAC1 slow anion channel-associated 1 SLAH SLAC1 homolog S-type slow type YFP yellow fluorescent protein © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com

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Plant and Cell PhysiologyOxford University Press

Published: Mar 1, 2018

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