Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO 2 signal transduction in guard cell
2011; Springer Nature; Volume: 30; Issue: 8 Linguagem: Inglês
10.1038/emboj.2011.68
ISSN1460-2075
AutoresShaowu Xue, Honghong Hu, Amber Ries, Ebe Merilo, Hannes Kollist, Julian I. Schroeder,
Tópico(s)Ion channel regulation and function
ResumoArticle18 March 2011free access Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO2 signal transduction in guard cell Shaowu Xue Shaowu Xue Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA Institute of Molecular Science, Shanxi University, Taiyuan, China Search for more papers by this author Honghong Hu Honghong Hu Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Amber Ries Amber Ries Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Ebe Merilo Ebe Merilo Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Hannes Kollist Hannes Kollist Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Julian I Schroeder Corresponding Author Julian I Schroeder Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Shaowu Xue Shaowu Xue Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA Institute of Molecular Science, Shanxi University, Taiyuan, China Search for more papers by this author Honghong Hu Honghong Hu Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Amber Ries Amber Ries Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Ebe Merilo Ebe Merilo Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Hannes Kollist Hannes Kollist Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Julian I Schroeder Corresponding Author Julian I Schroeder Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Shaowu Xue1,2,‡, Honghong Hu1,‡, Amber Ries1, Ebe Merilo3, Hannes Kollist3 and Julian I Schroeder 1 1Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA 2Institute of Molecular Science, Shanxi University, Taiyuan, China 3Institute of Technology, University of Tartu, Tartu, Estonia ‡These authors contributed equally to this work *Corresponding author. Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA 92093-0116, USA. Tel.: +1 858 534 7759; Fax: +1 858 534 7108; E-mail: [email protected] The EMBO Journal (2011)30:1645-1658https://doi.org/10.1038/emboj.2011.68 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Plants respond to elevated CO2 via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO2 response. It remains unclear whether CO2, HCO3− or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO3−]i with low [CO2] and [H+] activated S-type anion currents, whereas low [HCO3−]i at high [CO2] and [H+] did not. Bicarbonate enhanced the intracellular Ca2+ sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO2 signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO2-induced stomatal closing and HCO3− activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO2/HCO3− signalling, redefining the convergence point of CO2 and ABA signalling. A new working model of the sequence of CO2 signalling events in gas exchange regulation is presented. Introduction Plants control CO2 influx and water loss through stomatal pores, formed by guard cells in aerial tissues. Guard cells respond to abscisic acid (ABA), auxin, blue light, plant pathogens and CO2 and have been developed as a powerful system for plant cell signal transduction research (Blatt, 2000; Evans and Hetherington, 2001; Schroeder et al, 2001; Sirichandra et al, 2009). Elevated intercellular CO2 concentrations (Ci) that occur in leaves due to respiration in darkness and the continuing rise in atmospheric CO2 concentrations due to anthropogenic fossil fuel burning cause a reduction in stomatal apertures (Medlyn et al, 2001; Frommer, 2010). Stomatal closing is regulated by ion channel-mediated anion and K+ efflux from guard cells and parallel organic solute metabolism (Schroeder et al, 1987; Schroeder and Hagiwara, 1989; Blatt and Armstrong, 1993; MacRobbie, 1998). Elevated CO2 activates anion channels and K+out efflux channels in Vicia faba guard cells (Brearley et al, 1997; Raschke et al, 2003; Roelfsema et al, 2004) and triggers chloride release from guard cells causing guard cell depolarization in leaves (Hanstein and Felle, 2002; Roelfsema et al, 2002). Furthermore, cytosolic pH does not change in response to physiological [CO2] shifts in V. faba guard cells (Brearley et al, 1997). Recently, we have shown that the β-carbonic anhydrases, βCA1 and βCA4, function in CO2 regulation of stomatal movements. ca1;ca4 double-mutant plants show impaired CO2 induction of stomatal closing, whereas ABA-induced stomatal closing is functional (Hu et al, 2010). CO2 is reversibly catalysed by carbonic anhydrases (CAs) into bicarbonate ions and protons. Cytoplasmic high CO2 together with high HCO3− concentrations activates S-type anion channel currents in wild-type Arabidopsis guard cells (Hu et al, 2010). However, the mechanisms by which high CO2 and/or HCO3− mediate this response were not further investigated. Whether high [CO2] and [HCO3−] are able to activate anion channels in ca1;ca4 double-mutant plants remains unknown. The concentrations of HCO3− and CO2 required for channel regulation in patch-clamped guard cells are relatively high, necessitating genetic analyses to determine whether the high [CO2] plus [HCO3−] activation are physiologically relevant. Moreover, genetic mechanisms downstream of this high [HCO3−] plus [CO2] response and their position in the signalling cascade remain unknown and are dissected, with unexpected results, in the present study. Activation of S-type anion channels at the plasma membrane of guard cells has been regarded as a critical step in stomatal closure (Schroeder and Hagiwara, 1989; Schmidt et al, 1995; Grabov et al, 1997; Pei et al, 1997). Mutations in the SLAC1 anion channel cause greatly reduced S-type anion current activities, whereas R-type anion channels and ABA-activated Ca2+ permeable channels remain intact in slac1 mutants (Negi et al, 2008; Vahisalu et al, 2008). SLAC1 functions as an anion channel with permeabilities to Cl− and NO3− when heterologously expressed in Xenopus oocytes (Geiger et al, 2009; Lee et al, 2009), consistent with in vivo Cl− and NO3− permeabilities of S-type anion channels (Schmidt and Schroeder, 1994). The concentration of intracellular free calcium ions ([Ca2+]i) has been shown to function as a key signalling molecule in plants and mediates CO2 signal transduction in guard cells of several plant species (Schwartz, 1985; Webb et al, 1996; Hetherington and Woodward, 2003; Young et al, 2006; Kim et al, 2010). Elevation of [Ca2+]i in guard cells causes activation of S-type anion channels, downregulation of inward (rectifying) K+in channels and downregulation of proton ATPases, providing central mechanisms that mediate stomatal closing and inhibition of stomatal opening (Schroeder and Hagiwara, 1989; Kelly et al, 1995; Kinoshita et al, 1995; Grabov and Blatt, 1999; Siegel et al, 2009; Chen et al, 2010). Recent studies have suggested that the stomatal closing signals, CO2 and ABA, enhance the [Ca2+]i sensitivity of stomatal closing mechanisms (Young et al, 2006; Siegel et al, 2009) (for review see Hubbard et al, 2010). However, whether CO2 activation of S-type anion channels requires [Ca2+]i is not known. Furthermore, whether CO2 primes Ca2+ regulation of ion channels remains unknown and no genetic mutants and mechanisms are known that mediate CO2/HCO3− regulation of ion channels. The HT1 protein kinase was identified as a major negative regulator of high CO2-induced stomatal closure (Hashimoto et al, 2006) and is genetically epistatic to βCA1 and βCA4 in CO2 response pathway (Hu et al, 2010). However, the cellular signalling mechanisms of HT1 have not yet been investigated and whether the HT1 kinase affects S-type anion channel regulation and/or Ca2+ signalling remains unknown. The OST1 protein kinase, also named SnRK2.6 and SnRK2E, was identified as a key mechanism mediating ABA signal transduction (Mustilli et al, 2002; Yoshida et al, 2002; Vlad et al, 2009), but has no effect on low CO2-induced stomatal opening (Mustilli et al, 2002). Recent findings have shown that OST1 activates SLAC1 anion currents when OST1 and SLAC1 are coexpressed in Xenopus oocytes (Geiger et al, 2009; Lee et al, 2009). In the present study, we show that elevated bicarbonate, more so than elevated CO2, acts as intracellular signalling molecule to activate SLAC1-mediated anion channels. Elevated bicarbonate enhances (primes) the [Ca2+]i sensitivity of SLAC1 channel activation. The ht1-2 kinase mutant is found to enhance the HCO3− sensitivity of anion channel activation but also requires cytosolic Ca2+ for S-type anion channel activation, further defining the placement of HT1 effects on the CO2 signalling cascade. Surprisingly, our analyses of OST1 on CO2 regulation of stomatal movements and anion channels demonstrate that the OST1 protein kinase is a major regulator of CO2-induced stomatal closing and CO2 activation of anion channels in guard cells, leading to a new model for CO2 control of gas exchange in plants. Results Bicarbonate activates S-type anion currents in ca1;ca4 double-mutant guard cell protoplasts The βCA1 and βCA4 CAs act as upstream regulators in CO2-induced stomatal movements in guard cells (Hu et al, 2010). Elevated CO2 together with bicarbonate concentrations activate S-type anion channel currents in wild-type Arabidopsis guard cells. Previous studies of CO2 regulation of anion channels have only been pursued in wild-type guard cells (Brearley et al, 1997; Raschke et al, 2003; Hu et al, 2010). Therefore, we investigated whether elevated bicarbonate and intracellular CO2 can by-pass the ca1;ca4 mutant and activate S-type anion currents in ca1;ca4 mutant guard cells. Addition of 13.5 mM total bicarbonate to the pipette solution (equivalent to 11.5 mM free bicarbonate ([HCO3−]i)/2 mM free [CO2] at pH 7.1) activated anion currents in patch-clamped ca1;ca4 guard cells (Figure 1B and C), compared with control currents in the absence of added intracellular bicarbonate (Figure 1A). Free [HCO3−]i and [CO2] were calculated using the Henderson–Hasselbalch equation as described in Materials and methods. These findings are consistent with CAs acting as upstream regulators of CO2 signalling and that elevated bicarbonate and CO2 together can activate S-type anion channel in ca1;ca4 double-mutant guard cells. Figure 1.High intracellular [CO2] and [HCO3−] activate S-type anion channel currents in Arabidopsis ca1;ca4 double-mutant guard cells but do not activate S-type anion currents in slac1 mutant guard cells with 2 μM [Ca2+]i. (A) Whole-cell currents without HCO3−/CO2 and (B) with 11.5 mM free [HCO3−]i/2 mM free CO2 in the pipette solution (pH 7.1) in ca1;ca4 double-mutant guard cells. (C) Steady-state current–voltage relationships of the whole-cell currents recorded in ca1;ca4 mutant guard cells as in (A) (open circles, n=4 guard cells) and (B) (filled circles, n=9 guard cells). (D) Steady-state current–voltage relationships of whole-cell currents recorded in slac1-1 mutant guard cells (open circles: 0 mM added [HCO3−]i, n=6; filled circles: 11.5 mM free [HCO3−]i and 2 mM free [CO2], n=6) and (E) in slac1-3 mutant guard cells (open circles: 0 mM added [HCO3−]i, n=4; filled circles: 11.5 mM free [HCO3−]i and 2 mM free [CO2], n=8). Liquid junction potential was +1 mV. Data are mean±s.e. Download figure Download PowerPoint Bicarbonate-activated S-type anion currents are greatly impaired in slac1 mutant guard cell protoplasts The reversal potential of CO2+HCO3− activated whole-cell currents was +24.0±3.6 mV (n=8), which was close to the imposed chloride equilibrium potential of +31.1 mV, supporting the hypothesis that CO2+HCO3− activate guard cell anion channels. The bicarbonate and CO2 concentrations used for anion current activation were very high (Figure 1B and C) (Hu et al, 2010), giving rise to the question whether these anion currents correspond to physiological guard cell anion channel currents. SLAC1 is required for Arabidopsis ABA and Ca2+ activation of guard cell S-type anion channel function (Vahisalu et al, 2008). Therefore, we analysed whether high bicarbonate- and CO2-activated anion currents are mediated by SLAC1. Guard cell protoplasts from the recessive slac1-1 and slac1-3 mutants displayed small anion currents in the presence of 11.5 mM free [HCO3−]i and 2 mM [CO2] in the pipette solution, similar to control currents in the absence of added bicarbonate (Figure 1D and E, P>0.05). These data suggest that the high intracellular [HCO3−]+[CO2]-mediated anion currents are largely mediated by the physiologically relevant SLAC1 anion channel (Figure 1). Next, we analysed whether these anion currents show a clear HCO3− permeability in wild-type guard cells. The total bicarbonate was elevated to 50 mM in the pipette solution at pH 7.1 (corresponding to 43.4 mM free [HCO3−]i and 6.6 mM free [CO2]). Under such high [HCO3−], the reversal potential of whole-cell currents was +26.0±0.9 mV (Supplementary Figure S2, n=4). A relative permeability ratio of PHCO3−/PCl−=0.06±0.01 was estimated using the Goldman equation. This Cl− over HCO3− selectivity of whole-cell anion currents is consistent with the anion selectivity of SLAC1 channels found in heterologous expression experiments in Xenopus laevis oocytes (Geiger et al, 2009). High [CO2] and protons do not activate S-type anion currents in the absence of high bicarbonate levels in guard cells CAs reversibly catalyse the conversion of CO2 into bicarbonate ions and free protons (Supuran, 2008; Chandrashekar et al, 2009). Whether high [CO2], [HCO3−], [H+] or a combination of these mediates activation of S-type anion channels in Arabidopsis guard cells remains to be investigated (Hu et al, 2010). We investigated whether intracellular acidification is capable of activating S-type anion currents in wild-type guard cell protoplasts. Intracellular acidification at pH 6.1 alone did not significantly activate S-type anion channel currents compared with control recordings at pH 7.1 (Figure 2A, P>0.05, Student's t-test). Interestingly, when the intracellular free [CO2] was at a high concentration of 2 mM in the pipette solution (with 1.1 mM free [HCO3−]i) at pH 6.1, S-type anion channel currents were not activated in wild-type guard cell protoplasts, despite the applied high [CO2] and high proton concentrations (Figure 2B, P>0.05, Student's t-test). Figure 2.Elevated [H+] (pH 6.1) together with 2 mM intracellular free [CO2] did not activate S-type anion channel currents in wild-type Col-0 guard cells when bicarbonate levels were lower. (A) Steady-state current–voltage relationships of whole-cell currents recorded in guard cells at 2 μM [Ca2+]i without bicarbonate in the pipette solution at pH 7.1 (open circles, n=6) and pH 6.1 (filled circles, n=5). (B) Steady-state current–voltage relationships of whole-cell currents at pH 6.1 without bicarbonate (open circles, n=5) and with 2 mM intracellular free [CO2] and 1.1 mM free [HCO3−]i (filled circles, n=7) in the pipette solution. Data are mean±s.e. Liquid junction potential was +1 mV. (C) Example image of ratiometric pH sensitive Pt-GFP expressed guard cells. (D) Average fluorescence ratio time series of six guard cells expressing pH-sensitive reporter Pt-GFP during extracellular perfusion with buffers of different pH as indicated by the top bar. (E) Average fluorescence ratio time series of Pt-GFP expressed in guard cells perfused with MES buffer (10 mM MES, 10 mM KCl, 50 μM CaCl2, pH 5.6) and supplemented with sodium butyrate at mM concentrations as indicated by the top bar of the graph. Data are mean±s.e. The error bars presented in (D, E) were computed for the middle data points during each treatment, with the illustrated traces showing the averaged responses. (F) Fluorescence ratio time series of Pt-GFP-expressing guard cells perfused with extracellular buffers bubbled with 0 p.p.m. CO2 and 800 p.p.m. CO2. Raw data of two individual cells are depicted. GC denotes ratiometric fluorescence of guard cells and the ratio of non-guard cell background fluorescence (bg) is shown for the same experiments in (D–F). Download figure Download PowerPoint Previous research has shown no intracellular pH shifts in V. faba guard cells in response to [CO2] changes (Brearley et al, 1997). To further investigate whether cytosolic pH is affected in Arabidopsis guard cells in response to [CO2] shifts, a ratiometric pH indicator Pt-GFP (Schulte et al, 2006) under the control of a strong guard cell preferential promoter pGC1 (Yang et al, 2008) was transformed into Arabidopsis guard cells (Figure 2C). In control experiments, in vivo recordings of pH in fluorescent pGC1::Pt-GFP transgenic guard cells showed clear reversible shifts in ratiometric intracellular pH fluorescence when the extracellular pH was repeatedly shifted from pH 5.0 to pH 7.5 and back (Figure 2D; Supplementary Figure S3). Weak acids can control intracellular pH while maintaining a constant extracellular pH (Blatt and Armstrong, 1993; Grabov and Blatt, 1997). Therefore, the weak acid sodium butyrate was used to analyse whether Pt-GFP can report intracellular pH. Ratiometric fluorescence recordings of Pt-GFP-expressing guard cells showed clear shifts, when intact plant epidermes were perfused with defined concentrations of sodium butyrate-containing MES buffers (Figure 2E), indicating intracellular pH changes were easily detected in guard cells (Figure 2D and E). However, no clear shifts in guard cell intracellular pH fluorescence were observed when the concentration of CO2 bubbled in the extracellular perfusion buffers was repeatedly shifted from 0 to 800 p.p.m. (Figure 2F), consistent with the findings in V. faba guard cells using a pH-sensitive dye (Brearley et al, 1997). Average changes in intracellular pH in response to extracellular pH changes appeared to be relatively rapid (Figure 2D), and slightly slower in response to weak acid treatments and without clearly discernable overshoots upon weak acid removal under the imposed conditions (Figure 2E). In conclusion, protons alone or in combination with elevated CO2 could not activate S-type anion channels (Figure 2A and B) and [CO2] changes did not cause measurable changes in intracellular pH of Arabidopsis guard cells (Figure 2F) (Brearley et al, 1997). Bicarbonate activates S-type anion currents at low free CO2 in guard cells To analyse whether elevated intracellular [HCO3−] is sufficient to activate anion currents at low [H+] and low [CO2], 13.5 mM total CsHCO3 was applied to the pipette solution and the free [HCO3−] was calculated as 13.04 mM with 0.46 mM free [CO2] at pH 7.8. These analyses clearly showed that compared with the control recordings (Figure 3A), S-type anion currents were activated by the presence of high free HCO3− in the pipette solution (Figure 3B and C, P<0.05 at voltages from −146 to −26 mV, Student's t-test). Together, the above analyses show that elevated intracellular HCO3− is the main molecule that mediates activation of S-type anion currents in guard cells. Figure 3.High intracellular [HCO3−] at low [H+] and low free [CO2] activates S-type anion channel currents in wild-type Col-0 guard cells with 2 μM [Ca2+]i. (A) Typical recording of whole-cell currents in guard cell protoplasts without bicarbonate and (B) with 13.5 mM total bicarbonate (equivalent to 13.04 mM free [HCO3−]i/0.46 mM free [CO2]) added to the pipette solution at pH 7.8. (C) Average steady-state current–voltage relationships of whole-cell currents recorded as in (A) (open circles, n=3) and (B) (filled circles, n=5). Liquid junction potential was +1 mV. Data are mean±s.e. Download figure Download PowerPoint Extracellular bicarbonate was next tested on activation of S-type anion currents in wild-type guard cells. After obtaining whole-cell recordings in wild-type guard cells, the bath solution (200 μl) was perfused for 2 min at 1 ml/min with a solution that contained 11.5 mM free [HCO3−]i and 2 mM [CO2] at pH 7.1 (Supplementary Figure S1A). No large S-type anion currents were activated (Supplementary Figure S1B and C). A small increase in average anion current magnitude was not statistically significant and was not comparable to the clear activation of S-type anion currents by the same concentration of applied intracellular HCO3− (Supplementary Figure S1B and C). Elevated intracellular [Ca2+] is required for bicarbonate activation of S-type anion channel currents in guard cells The above analyses of activation of S-type anion currents were all conducted at 2 μM cytosolic free Ca2+ ([Ca2+]i) (Figures 1, 2, 3). We investigated whether the elevated [Ca2+]i (2 μM) was necessary for bicarbonate activation of S-type anion channel currents in Arabidopsis guard cells. At 2 μM [Ca2+]i, anion currents were not strongly activated in the absence of added [HCO3−]i (Figure 4A and G), consistent with previous studies (Allen et al, 2002; Siegel et al, 2009). In contrast, 11.5 mM free [HCO3−]i activated strong S-type anion channels (Figure 4C and G, P 0.05, Student's t-test). When [Ca2+]i was buffered to a baseline level of 0.15 μM even with high 11.5 mM free [HCO3−]i and 2 mM free [CO2] in the pipette solution (pH 7.1), S-type anion currents were not activated (Figure 4E and G). There was no significant difference between the average amplitudes of current recordings at 0.15 μM free [Ca2+]i with or without added 11.5 mM free [HCO3−]i (Figure 4G, P>0.05, at voltages from −146 to +34 mV). In addition, an elevated cytosolic free [Ca2+]i of 0.6 μM together with high 11.5 mM free [HCO3−]i and 2 mM free [CO2] in the pipette solution (pH 7.1) activated anion currents of intermediate average amplitudes (Figure 4F and G). A summary of cytosolic free Ca2+ and HCO3− activation of S-type anion channels are shown in Supplementary Table I. These data demonstrate a requirement for an elevated [Ca2+]i in HCO3−-mediated activation of guard cell anion channels and provide direct and mechanistic evidence for the model that CO2-induced stomatal closing enhances the ability of [Ca2+]i to activate stomatal closing mechanisms (Young et al, 2006). Figure 4.Both [Ca2+]i and elevated bicarbonate are required for activation of S-type anion channel currents in wild-type (Col-0) guard cells. (A) Whole-cell currents in guard cell protoplasts at 2 μM [Ca2+]i without bicarbonate, (B) with 5.75 mM intracellular free [HCO3−]i/1 mM free [CO2] (6.75 mM total bicarbonate added) and (C) with 11.5 mM intracellular free [HCO3−]i/2 mM free [CO2] (13.5 mM total bicarbonate added) in the pipette solution at pH 7.1. (D) Whole-cell currents in guard cell protoplasts at 0.15 μM [Ca2+]i without bicarbonate, (E) with 11.5 mM free [HCO3−]i/2 mM free [CO2] (13.5 mM total bicarbonate) in the pipette solution at pH 7.1. (F) Whole-cell currents in guard cell protoplasts with 0.6 μM [Ca2+]i and 11.5 mM intracellular free [HCO3−]i/2 mM free [CO2] in the pipette solution at pH 7.1. (G) Steady-state current–voltage relationships of whole-cell currents as recorded in (A) (open triangles, n=6), (B) (open square, n=7), (C) (filled triangles, n=10), (D) (open circles, n=5), (E) (filled circles, n=7), and (F) (filled squares, n=7). Average data shown by dashed lines in (G) with or without of 5.75 mM and 11.5 mM free [HCO3−]i at 2 μM [Ca2+]i correspond to data reported in Hu et al (2010) and are included for comparison to 0.15 μM and 0.6 μM [Ca2+]i data. Liquid junction potential was +1 mV. Data are mean±s.e. Download figure Download PowerPoint Lower [bicarbonate] is sufficient for activation of S-type anion channel currents in ht1-2 guard cells The Arabidopsis HT1 protein kinase functions as a negative regulator of CO2-induced stomatal closing (Hashimoto et al, 2006). To test whether HT1 functions in the CO2/HCO3− SLAC1 signalling pathway (Figures 1, 2, 3), the effects of bicarbonate on S-type anion currents in recessive ht1-2 mutant guard cells were analysed. Whole-cell currents were recorded in guard cell protoplasts at lower intracellular [HCO3−]i, 5.75 mM free [HCO3−]i and 1 mM free [CO2] at pH 7.1, compared with the above experiments (Figure 5A and B). In wild-type control guard cells, these intermediate [HCO3−]i+[CO2] together with 2 μM free [Ca2+]i showed small whole-cell current amplitudes that were slightly larger than wild-type guard cells in the absence of added HCO3− (Figure 5A, B and E, P>0.05, Student's t-test) (Hu et al, 2010). However, significant activation of S-type anion currents by intracellular addition of 5.75 mM free [HCO3−]i and 1 mM free [CO2] (pH 7.1) was observed in ht1-2 guard cells (Figure 5D and E) compared with the control currents (Figure 5A–C and E, P 0.05 at voltages from −146 to −26 mV, Student's t-test). Thus, ht1-2 guard cells show an enhanced sensitivity to intracellular HCO3−, but this enhanced activation cannot by-pass the requirement for [Ca2+]i in HCO3− activation of S-type anion currents. Figure 5.Enhanced bicarbonate sensitivity of S-type anion channel activation in ht1-2 mutant guard cells only at elevated [Ca2+]i. (A) Whole-cell currents in wild-type Col-0 guard cells at 2 μM [Ca2+]i without bicarbonate and (B) with 6.75 mM total bicarbonate (equivalent to 5.75 mM free [HCO3−]i/1 mM free [CO2]) added to the pipette solution. (C) Whole-cell currents in ht1-2 mutant guard cells at 2 μM [Ca2+]i without bicarbonate and (D) with 6.75 mM bicarbonate (equivalent to 5.75 mM free [HCO3−]i/1 mM free [CO2]) in the pipette solution. (E) Average steady-state current–voltage relationships of whole-cell currents as recorded in (A) (open triangles, n=6), (B) (filled triangles, n=7), (C) (open circles, n=5) and (D) (filled circles, n=9). Average data for wild-type Col-0 controls (WT) shown by dashed lines in (E) with 0 and 6.75 mM total bicarbonate (5.75 mM free [HCO3−]) with 2 μM [Ca2+]i correspond to data reported in Hu et al (2010) and are included for comparison to ht1-2 mutant data. (F) Whole-cell currents in ht1-2 mutant guard cell protoplasts at low 0.15 μM [Ca2+]i without bicarbonate and (G) with 6.75 mM bicarbonate (equivalent to 5.75 mM free [HCO3−]i/1 mM free [CO2]) added to the pipette solution. (H) Average steady-state current–voltage relationships of whole-cell currents as recorded in (F) (open circles, n=5) and (G) (filled circles, n=5). Liquid junction potential was +1 mV. Data are mean±s.e. Download figure Download PowerPoint The OST1 kinase functions in bicarbonate activation of S-type anion currents in guard cell protoplasts and CO2-induced stomatal closure The OST1 protein kinase was previously demonstrated to mediate ABA-induced stomatal closing. Recessive ost1 mutants disrupt ABA-induced stomatal closure as well as ABA inhibition of light-induced stomatal opening, but low CO2 induction of stomatal opening remained unaffected in the ost1-2 mutant, indicating that OST1 does not participate in CO2 signalling (Mustilli et al, 2002; Yoshida et al, 2002). Here, the effect of OST1 on bicarbonate activation of S-type anion channels was investigated. Using the same recording solutions as in Figure 1B, high [HCO3−]i (11.5 mM) and [CO2] (2 mM) activated only small S-type anion currents in Landsberg erecta (Ler) ost1-2 mutant guard cells (Figure 6A, B and F). Similar to Col-0 wild-type guard cells (Figures 1, 3 and 4), high HCO3− activated S-type anion channel currents in Ler wild-type guard cells (Figure 6D, E and F). While HCO3−-activated S-type anion currents in Ler wild-ty
Referência(s)