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Received Date : 08-Dec-2014 Revised Date
: 01-Feb-2015
Accepted Date : 06-Feb-2015 Article type Editor
: Research Paper : R Mendel
Running head: Khokon et al. MPK9and MPK12 in guard cell MeJA signaling
Two guard cell MAPKs, MPK9 and MPK12, function in methyl jasmonate-induced stomatal closure in Arabidopsis thaliana
Md. Atiqur Rahman Khokon,1* Mohammad Abdus Salam,1* Fabien Jammes,2* Wenxiu Ye,1 Mohammad Anowar Hossain,1 Misugi Uraji,1 Yoshimasa Nakamura,1 Izumi C. Mori,3 June M. Kwak4&Yoshiyuki Murata1
1
Graduate School of Environmental and Life Science, Okayama University, 1-1-1
Tsushima-naka, Okayama 700-8530, Japan 2
Department of Biology, Pomona College, Claremont, 91711, CA
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/plb.12321 This article is protected by copyright. All rights reserved.
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3
Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama
710-0046, Japan 4
Center for Plant Aging Research, Institute for Basic Science, Department of New
Biology, Daegu Kyungbuk Institute of Science and Technology, Daegu 711-873 Republic of Korea * These authors contributed equally
Corresponding author: Yoshiyuki Murata Address: The Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan E-mail: [email protected] Tel: ++-81-86-251-8310 Fax: ++-81-86-251-8388
Key words: [Ca2+]cyt oscillations; cytosolic alkalization; guard cell, mitogen-activated protein kinase; methyl jasmonate; reactive oxygen species; S-type anion channels.
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ABSTRACT Methyl jasmonate (MeJA) and abscisic acid (ABA) signaling cascades share several signaling components in guard cells. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signaling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9-1 and mpk12-1 single mutants as well as wild-type plants but not in mpk9-1 mpk12-1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA-induced stomatal closure in the wild-type plants. MeJA elicited ROS production and cytosolic alkalization in guard cells of the mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants as well as those of the wild-type plants. Furthermore, MeJA triggered elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in the mpk9-1 mpk12-1 double mutant as well as the wild-type plants. Activation of S-type anion channels by MeJA was impaired in mpk9-1 mpk12-1. Altogether, these results indicate that MPK9 and MPK12 function upstream of S-type anion channel activation and downstream of ROS production, cytosolic alkalization, and [Ca2+]cyt elevation in guard cell MeJA signaling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signaling in guard cells.
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INTRODUCTION Methyl jasmonate (MeJA) plays a role in plant growth and developmental processes and mediates various plant defense responses (Liechti & Farmer 2002). MeJA induces stomatal closure in several plant species including Arabidopsis thaliana (Suhita et al. 2004; Munemasa et al. 2007; Saito et al. 2008; Akter et al. 2010; Hossain et al. 2011a,b; Munemasa et al. 2011), Commelina benghalensis (Raghavendra & Reddy 1987), Paphiopedilum Supersuk and P. tonsum (Gehring et al. 1997), and Nicotiana glauca (Suhita et al. 2003). Both MeJA and abscisic acid (ABA) induce production of ROS, cytosolic alkalization, elevation of cytosolic Ca2+ concentration ([Ca2+]cyt), and activation of S-type anion channels in guard cells (Kwak et al. 2003; Suhita et al. 2004; Munemasa et al. 2007; Saito et al. 2008; Islam et al. 2010a, b; Hossain et al. 2011a,b). CORONATINE INSENSITIVE 1 (COI1) is an F-box protein and functions as a jasmonate co-receptor with JASMONATE ZIM DOMAIN (JAZ), which recognizes the bioactive hormone (3R,7S)-jasmonoyl-L-isoleucine (JA-Ile) with high specificity (Fonseca et al. 2009; Yan et al. 2009). The conjugation of isoleucine to JA is catalyzed by JASMONATE RESISTANT 1 (JAR1) following wounding (Suza and Staswick 2008). MeJA does not promote the interaction of COI1 with JAZ (Yan et al. 2009) but induces stomatal closure which is mediated by COI (Munemasa et al. 2007). The coi1 mutation impairs the MeJA-induced stomatal closure but not the ABA-induced stomatal closure (Munemasa et al. 2007), indicating that the F-box COI1 protein functions in MeJA signaling in guard cells but not in ABA signaling. The calcium-dependent protein kinases (CDPKs) function in guard cell Ca2+ and anion channel activation in ABA signaling (Mori et al. 2006; Zhu et al. 2007). We This article is protected by copyright. All rights reserved.
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have recently demonstrated that in Arabidopsis guard cells, CPK6 functions in both MeJA and ABA signaling and that CPK3 functions in ABA signaling but not in MeJA signaling (Munemasa et al. 2011). Moreover, we have shown that endogenous ABA is required for MeJA signaling in Arabidopsis guard cells (Hossain et al. 2011a). These results indicate that ABA and MeJA signaling cascades crosstalk and share several signaling elements and also that each cascade employs unique signaling components. However, it is not fully understood how the crosstalk between MeJA and ABA signaling pathways is regulated in guard cells. Mitogen-activated protein kinases (MAPKs) are major components in cellular signal transduction pathways that mediate numerous biotic and abiotic stress responses (Ligterink 2000; Asai et al. 2002; Jonak et al. 2002; Pedley & Martin 2005). We have demonstrated that two members of the Arabidopsis MAPK family, MPK9 and MPK12, are preferentially expressed in guard cells and positively regulate ABA signaling downstream of ROS production and upstream of S-type anion channel activation (Jammes et al. 2009). Recently, Hettenhausen et al. (2012) reported that MPK4 is involved in ABA-induced stomatal closure. These studies imply that MAPKs are key components in ABA signal transduction in guard cells. However, to our knowledge, it has not been elucidated whether and how MPK9 and MPK12 act as regulators of MeJA signaling in Arabidopsis guard cells. In order to elucidate the involvement of these two MAPKs, MPK9 and MPK12 in MeJA signaling in Arabidopsis guard cells, we examined stomatal movements and ROS production, cytosolic alkalization, [Ca2+]cyt oscillations, and S-type anion channel activation in guard cells in response to MeJA using the
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mpk9-1 and mpk12-1 single mutants as well as the mpk9-1 mpk12-1 double mutant. MATERIALS AND METHODS Plant materials Arabidopsis (Arabidopsis thaliana) wild type, Columbia-erecta (Col-er), mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants were grown in a growth chamber at 22°C and 60% humidity in a cycle of a 16-h light with 80 μmol m-2 s-1 of photon flux density and 8-h dark. Twice or three times a week, 0.1% Hyponex solution was applied to the plant growth tray. A Ca2+-sensing fluorescent protein, Yellow Cameleon 3.6 (YC3.6), was used to measure [Ca2+]cyt (Mori et al. 2006). The mpk9-1 mpk12-1 double mutant was crossed with a Columbia-0 plant that had previously been transformed with YC3.6 to obtain YC3.6-expressing mpk9-1 mpk12-1 double mutant plants. These plants were also grown under the conditions as described above. Arabidopsis Genome Initiative numbers for the genes discussed in this study are MPK9 (At3g18040) and MPK12 (At2g46070).
Measurement of stomatal apertures Stomatal apertures were measured as previously described (Hossain et al. 2011a). Excised rosette leaves were floated on an assay solution consisting of 5 mM KCl, 50 µM CaCl2, and 10 mM MES adjusted to pH 6.15 with Tris for 2 h in the light to induce stomatal opening prior to addition of MeJA. An MAPKK inhibitor, PD98059, was added 30 min before MeJA application. Two hours after MeJA application, the
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leaves were shredded in a commercial blender for 30 s and were filtrated using nylon mesh to collect the remaining epidermal tissues. Twenty stomatal apertures were measured for each sample. Measurement of ROS production in guard cells Production of ROS in guard cells was examined using H2DCF-DA (Munemasa et al. 2011). The epidermal tissues were incubated in the light for 2 h in the assay solution containing 5 mM KCl, 50 µM CaCl2, and 10 mM MES adjusted to pH 6.15 with Tris, and then 50 µM H2DCF-DA was added to the assay solution. The epidermal tissues were kept at room temperature for 30 min and then were washed with the assay solution to remove the excess dye. After filtration using nylon mesh, the collected tissues were again incubated with the assay solution supplemented with 10 µM MeJA or ABA for 20 min at room temperature. The digital image was captured using a fluorescence microscope (model: Bio Zero BZ-8000, KEYENCE) and the pixel fluorescence intensity in guard cells was measured using ImageJ 1.42q (NIH).
Measurement of pHcyt Cytosolic pH in guard cells was evaluated using a pH-sensing dye, 2΄,7΄-bis-(2-carboxyethyl)-5,(6)-carboxyfluoresceinacetoxymethyl ester (BCECF-AM) (Islam et al. 2010a). The epidermal tissues were incubated in the light for 3 h in an assay solution containing 5 mM KCl and 10 mM MES adjusted to pH 6.5 with KOH, and then 20 µM BCECF-AM was added to the assay solution. The epidermal tissues were kept at room temperature in dark for 30 min and then were washed with the assay solution to remove the excess dye. After filtration using nylon This article is protected by copyright. All rights reserved.
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mesh, the collected tissues were treated with 10 µM MeJA or ABA for 20 min in dark. The digital image was obtained and analyzed as described above.
Measurement of [Ca2+]cyt in guard cells Four- to 6-week-old wild-type and mpk9-1 mpk12-1 double mutant plants expressing YC3.6 were used to measure [Ca2+]cyt in guard cells (Islam et al. 2010b). The abaxial side of an excised leaf was softly mounted on a glass slide with a medical adhesive (Stock No. 7730; Hollister Inc., Libertyville, Illinois, United States) and then the adaxial epidermis and the mesophyll tissue were gently removed with a razor blade. The remaining intact abaxial epidermis was incubated in a solution containing 5 mM KCl, 50 μM CaCl2, and 10 mM MES adjusted to pH 6.15 with Tris under light for 2 h at 22°C to induce stomatal opening. Turgid guard cells were used to measure [Ca2+]cyt. Guard cells were treated with MeJA or ABA using a peristaltic pump 5 min after monitoring. The cyan fluorescent protein (CFP) (F480 nm) and yellow fluorescent protein (YFP) (F535 nm) fluorescent intensities of guard cells were imaged and analyzed using W-View system equipped with a 440AF21 excitation filter, a 445DRLP dichroic mirror, a 480DF30 emission filter for CFP and a 535DF25 emission filter for YFP and AQUA COSMOS software (Hamamatsu Photonics). CFP and YFP fluorescence were simultaneously monitored.
Electrophysiology
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Arabidopsis guard cell protoplasts (GCPs) were enzymatically isolated from rosette leaves of 4- to 6-week-old plants according to Munemasa et al. (2007). Whole-cell currents were monitored using a patch clamp amplifier (model CEZ-2200; NIHON KOHDEN). Data were analyzed with a data acquisition and analysis software, pCLAMP 8.2 (Molecular Devices).The pipette solution contained 150 mM CsCl, 2 mM MgCl2, 6.7 mM EGTA, 5.58 mM CaCl2, 5 mM ATP, and 10 mM HEPES adjusted to pH 7.1 with Tris, and the bath solution consisted of 30 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, and 10 mM MES adjusted to pH 5.6 with Tris (Munemasa et al. 2007). Osmolarity of the pipette solution and bath solution were adjusted with D-sorbitol to 500 mmol kg-1 and 485 mmol kg-1, respectively. In order to examine the effect of MeJA, GCPs were treated with 10 μM MeJA for 2 h prior to recordings.
Statistical analysis Student’s t-test was used to assess the significance of differences between mean values of stomatal aperture, ROS production, and cytosolic alkalization and χ2 test was used to assess the frequency of [Ca2+]cyt oscillations. Differences were considered significant for P values < 0.05.
RESULTS MAPKK inhibitor PD98059 and mpk9-1 mpk12-1 mutation impair MeJA-induced stomatal closure
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To test whether the MAPK cascade functions in guard cell MeJA signaling, we tested the effect of the MAPKK inhibitor PD98059 on MeJA-induced stomatal closure. As previously reported, MeJA at 1 μM and 10 μM significantly induced stomatal closure (Fig. 1a). In contrast, pretreatment of rosette leaves with 100 μM PD98059 significantly inhibited the MeJA-induced stomatal closure (Fig. 1a). PD98059 alone did not affect stomatal apertures in the absence of MeJA (Fig. 1a). These results suggest that an MAPK cascade plays a role in MeJA-induced stomatal closure. Next, we examined whether the guard cell-preferential MPK9 and MPK12 are part of the MAPK cascade mediating MeJA signaling in guard cells. Epidermal strip analyses show that MeJA induced stomatal closure in the mpk9-1 and mpk12-1 single mutants (Fig. 1b). However, MeJA-induced stomatal closure was disrupted in mpk9-1 mpk12-1 double mutant (Fig. 1b), indicating that MPK9 and MPK12 redundantly function as positive regulators of MeJA signaling in Arabidopsis guard cells.
MeJA-induced ROS production is not impaired in mpk9 mpk12 Reactive oxygen species (ROS) function as second messengers in both ABA and MeJA signaling in guard cells (Kwak et al. 2008; Suhita et al. 2004; Munemasa et al. 2007; Saito et al. 2008; Islam et al. 2010a). To examine whether MPK9 and MPK12 act upstream or downstream of ROS, we monitored MeJA-triggered ROS production in guard cells of the mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants using 2΄,7΄-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Munemasa et al.
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2007). As shown in Fig. 2a, application of 10 μM MeJA induced ROS production in wild-type guard cells, as previously reported (Munemasa et al. 2007; Saito et al. 2008; Islam et al. 2010a; Munemasa et al. 2011). MeJA also induced ROS production in guard cells of the mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants (Fig. 2a). We next determined ABA-triggered ROS production in the mpk9-1 mpk12-1 double mutant. As shown in Fig. 2b, 10 μM ABA induced ROS production in guard cells of both wild type and the mpk9-1 mpk12-1 mutant. These results suggest that MPK9 and MPK12 function downstream of ROS production in stomatal response to MeJA and ABA.
MeJA and ABA cause cytosolic alkalization in mpk9 mpk12 guard cells Cytosolic alkalization in response to MeJA promotes stomatal closure (Suhita et al. 2004). To further determine the role of MPK9 and MPK12 in guard cell MeJA signaling, we analyzed MeJA-induced cytosolic alkalization in guard cells of the mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants using the pH indicator BCECF-AM. Figure 2c shows that application of 10 μM MeJA induced cytosolic alkalization in the wild type, mpk9-1, mpk12-1, and mpk9-1 mpk12-1. In addition, 10 μM ABA induced cytosolic alkalization in guard cells of the wild type and the mpk9-1 mpk12-1 double mutant (Fig. 2d). These results imply that MPK9 and MPK12 act downstream of cytosolic alkalization in response to MeJA and ABA in guard cells.
[Ca2+]cyt oscillations triggered by MeJA and ABA are not abolished in mpk9 mpk12 guard cells This article is protected by copyright. All rights reserved.
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MeJA as well as ABA induces elevation in [Ca2+]cyt, which in turn activates subsequent cellular events (Kwak et al. 2008; Islam et al. 2010b; Hossain et al. 2011a, b; Munemasa et al. 2011). Elevation in [Ca2+]cyt in guard cells often appear to be repetitive transient increases or oscillations. To examine whether the mutations in MPK9 and MPK12 affected MeJA- and ABA-induced [Ca2+]cyt oscillations, we conducted imaging analyses using plants expressing YC3.6 (Nagai et al. 2004). As shown in Fig. 3a-c, application of 10 μM MeJA induced one or more [Ca2+]cyt oscillations in 67% of the wild-type guard cells (n = 12 of 18 cells) and in 75% of the mpk9-1 mpk12-1 double mutant guard cells (n = 18 of 24 cells) over 60 min of monitoring. Moreover, ABA at 10 μM induced one or more [Ca2+]cyt oscillations in 85% of wild-type guard cells (n = 17 of 20 cells) and in 79% of the mpk9-1 mpk12-1 double mutant guard cells (n = 19 of 24 cells) (Fig. 3d-f). The frequencies of [Ca2+]cyt oscillations were not significantly different between the wild type and the mpk9-1 mpk12-1 double mutant (P = 0.55, 10 μM MeJA; P = 0.61, 10 μM ABA), suggesting that MPK9 and MPK12 function downstream of [Ca2+]cyt oscillations in guard cells in response to MeJA and ABA.
MeJA-induced S-type anion channels activation is impaired in mpk9 mpk12 guard cells Activation of S-type anion channels in the plasma membrane of guard cells is essential for stomatal closure (Schroeder & Hagiwara1989; Negi et al. 2008; Vahisalu et al. 2008). Like ABA and calcium, MeJA activates S-type anion channels in guard cells, resulting in stomatal closure (Schroeder & Hagiwara1989; Pei, Ward
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& Schroeder 1999; Munemasa et al. 2007, 2011). Using the whole-cell patch-clamp technique, we examined whether MeJA activates S-type anion channels in guard cells of the mpk9 and mpk12 mutant. As shown in Fig. 4a & 4c, MeJA at 10 μM activated S-type anion currents in wild-type guard cells (P < 0.001 at -145 mV) in agreement with our previous observations (Munemasa et al. 2007, 2011). To the contrary, MeJA activation of S-type anion currents was attenuated in the mpk9-1 mpk12-1 double mutant guard cells (P = 0.11 at -145 mV; Fig. 4b & 4d), which is consistent with the absence of MeJA-induced stomatal closure in the mpk9-1 mpk12-1 double mutant (Fig. 1b). These results suggest that MPK9 and MPK12 positively regulate MeJA signaling by acting upstream of the activation of S-type anion channels in guard cells.
DISCUSSION Mitogen-activated protein kinases are major components in various cellular signal transduction pathways in response to biotic and abiotic stresses (Ligterink 2000; Asai et al. 2002; Jonak et al. 2002; Pedley &Martin 2005). We have previously demonstrated that two members of the MAPK family, MPK9 and MPK12, are preferentially expressed in guard cells and positively regulate ABA signaling in guard cells (Jammes et al. 2009). In this study, we show that MPK9 and MPK12 are functionally redundant and positively regulate MeJA-induced stomatal closure. Stomata in mpk9-1 mpk12-1 double mutant but not in mpk9-1 or mpk12-1 single mutants were insensitive to MeJA (Fig. 1b). Furthermore, mpk9-1 mpk12-1 double mutant showed impaired S-type anion channel activation in response to MeJA (Fig.
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4). In contrast, upstream cellular events including MeJA-triggered ROS production, alkalization, and [Ca2+]cyt oscillations were not affected in mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants (Figs. 2 & 3), indicating that MPK9 and MPK12 act downstream of these cellular events. A simple model of the MeJA signaling in Arabidopsis guard cells is proposed to summarize the present study (Fig. 5). Although our previous study showed that MPK9 and MPK12 function downstream of ROS in guard cell ABA signaling (Jammes et al. 2009), it was unknown whether ABA can elicit ROS production and [Ca2+]cyt oscillations in mpk9-1 mpk12-1. We provide evidences that ABA induces ROS production, alkalization, and [Ca2+]cyt oscillations in mpk9-1 mpk12-1 mutant (Figs. 2 & 3). Taken together, these results demonstrate that MPK9 and MPK12 are common signaling components acting downstream of [Ca2+]cyt oscillations but upstream of S-type anion channel activation in ABA and MeJA signaling in guard cells. In guard cells, MeJA pathway shares several signaling components with ABA pathway such as ROS, [Ca2+]cyt oscillation/elevation, and cytosolic alkalization (Suhita et al. 2004; Munemasa et al. 2007; Saito et al. 2008; Islam et al. 2010a, b). On the other hand, stomata of jar1-1 and coi1 mutant plants are insensitive to MeJA (Suhita et al. 2004; Munemasa et al. 2007) but are sensitive to ABA, while stomata of ost1-2 are insensitive to ABA but are sensitive to MeJA (Suhita et al. 2004). Moreover, MeJA signal transduction requires endogenous ABA (Hossain et al. 2011a), whereas ABA signaling does not appear to require endogenous MeJA (Munemasa et al. 2007). These studies indicate that ABA and MeJA signaling possess unique signaling components in addition to common ones.
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Hydrogen peroxide activates MAPKs in Arabidopsis suspension cultured cells (Desikan et al. 1999; Kovtun et al. 2000; Desikan et al. 2001) and MPK12 kinase activity in Arabidopsis guard cells (Jammes et al. 2009). Activation of MPK8 needs binding of calmodulins in a Ca2+-dependent manner in Arabidopsis (Takahashi et al. 2011) and activation of MAPKs also requires an increase of [Ca2+]cyt in Nicotiana benthamiana (Segonzac et al. 2011). In addition, a 46-kDa MAPK is activated by ROS, which is involved in ABA-induced antioxidant defense in Zea mays (Zhang et al. 2006). These results suggest that MAPKs function downstream of ROS production and [Ca2+]cyt elevation, which is consistent with results presented in this study and our previous study (Jammes et al. 2009; Salam et al. 2012; Salam et al. 2013). Calcium-dependent protein kinases are unique enzymes considered as [Ca2+]cyt sensors found in plants and some protozoa. In Arabidopsis guard cells, MeJA-induced [Ca2+]cyt oscillation is impaired in the cpk6 mutant (Munemasa et al. 2011). Our study reveals that MeJA and ABA induced [Ca2+]cyt oscillation in the mpk9-1 mpk12-1 double mutant (Fig. 3). These CDPKs may therefore directly or indirectly activate MPK9 and MPK12 in MeJA and ABA signal cascades in guard cells. Both CDPKs and MAPKs are serine/threonine kinases. A broad-range serine/threonine kinase inhibitor, K252a, inhibits MeJA- and ABA-induced stomatal closure and [Ca2+]cyt oscillation (Hossain et al. 2011b). Hence, the inhibition of MeJA-induced stomatal closure by K252a may be at least in part due to inhibition of CPKs such as CPK6 and MAPKs such as MPK9 and MPK12.
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Both MeJA and ABA activate S-type anion channels in plasma membrane of guard cells, leading to stomatal closure (Munemasa et al. 2007; Jammes et al. 2009; Munemasa et al. 2011). The mpk9-1 mpk12-1 double mutation impairs the activation of S-type anion channels by MeJA and ABA (Fig. 4; Jammes et al. 2009), suggesting that MPK9 and MPK12 function upstream of S-type anion channel activation in ABA and MeJA signal cascades in guard cells. Recent studies have shown that the ABA-activated OST1 kinase and the CDPKs phosphorylate S-type anion channel protein SLAC1, resulting in stimulation of SLAC1-mediated anion currents in Xenopus oocytes (Lee et al. 2009; Geiger et al. 2009; Geiger et al. 2010). These results indicate that S-type anion channels are important molecular players in stomatal movements, thus their activity is highly regulated by protein phosphorylation under relevant physiological conditions. Since MPK9 and MPK12 act upstream of S-type anion channel activation in both MeJA and ABA signaling (Fig. 4; Jammes et al. 2009), it would be interesting to test whether these two MAPKs can phosphorylate SLAC1 proteins. In conclusion, MPK9 and MPK12 are key regulators of both MeJA and ABA signaling pathways by acting upstream of S-type anion channel activation and downstream of ROS production, cytosolic alkalization, and [Ca2+]cyt oscillation in Arabidopsis guard cells.
ACKNOWLEDGEMENTS This research was supported in part by The Asahi Glass Foundation, NOVARTIS Foundation (Japan) for the Promotion of Science, Grant-in-Aid for Young Scientists,
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and Grants for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Y.M., and in part by project IBS-R013-G2-2014-a00 to J.M.K.
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in guard cells of Arabidopsis thaliana. Journal of Plant Physiology, 168, 1901-1908. Hossain M.A., Munemasa S., Uraji M., Nakamura Y., Mori I.C., Murata Y. (2011a) Involvement of endogenous abscisic acid in methyl jasmonate-induced stomatal closure in Arabidopsis. Plant Physiology, 156, 430-438. Islam M.M., Hossain M.A., Jannat R., Munemasa S., Nakamura Y., Mori I.C., Murata Y.(2010b) Cytosolic alkalization and cytosolic calcium oscillation in Arabidopsis guard cells response to ABA and MeJA. Plant Cell and Physiology, 51, 1721-1730. Islam M.M., Munemasa S., Hossain M.A., Nakamura Y., Mori I.C., Murata Y. (2010a) Roles of AtTPC1, vacuolar two pore channel 1, in Arabidopsis stomatal closure. Plant Cell and Physiology, 51, 302-311. Jammes F., Song C., Shin D., Munemasa S., Takeda K., Gu D., Cho D., Lee S., Giordo R., Sritubtim S., Leonhardt N., Ellis B.E., Murata Y., Kwak J.M. (2009) MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling. Proceedings of the National Academy of Sciences of the United States of America, 106, 20520-20525. Jonak C., Okrész L., Bögre L., Hirt H. (2002) Complexity, cross talk and integration of plant MAP kinase signalling. Current Opinion in Plant Biology, 5, 415-424. Kovtun Y., Chiu W.L., Tena G., Sheen J. (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants.
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downstream of reactive oxygen species production in leaves of maize plants. Plant Physiology, 141, 475-487. Zhu S.Y., Yu X.C., Wang X.J., Zhao R., Li Y., Fan R.C., Shang Y., Du S.Y., Wang X.F., Wu F.Q., Xu Y.H., Zhang X.Y., Zhang D.P. (2007) Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis. Plant Cell, 19, 3019-3036.
FIGURE LEGENDS Figure 1. The effect of MAPKK inhibitor, PD98059, and mutation of MAPKs on MeJA-induced stomatal closure. (a) Rosette leaves of wild type were treated with MeJA (1 or 10 μM) in the presence or absence of 100 μM PD98059. PD98059 was applied to assay solution prior to MeJA treatment. (b) Rosette leaves of wild type (WT), mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants were treated without (white bars) or with 1 μM MeJA (black bars) or 10 μM MeJA (gray bars). Averages of three independent experiments (60 stomata per bar) are shown. Error bars represent standard deviation. Asterisks indicate significant differences (*, P < 0.05). Figure 2. The effects of MeJA (10 μM) and ABA (10 μM) on ROS production and cytosolic alkalization in wild type (WT) and mpk mutants. ROS production as shown by DCF fluorescence intensity in wild type and mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants in response to MeJA (a) and ABA (b). Cytosolic alkalization as shown by BCECF fluorescence intensity in wild type and mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants in response to MeJA (c) and ABA (d). Fluorescence
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intensities were normalized to the vehicle control values for each line with respect to each day. Averages of three independent experiments (50 guard cells per bar) are shown. Error bars represent standard deviation. Asterisks indicate significant differences (*, P < 0.05). Figure 3. MeJA-elicited [Ca2+]cyt oscillations in wild-type (WT) and mpk9 mpk12 guard cells. (a), (b), representative fluorescence emission ratios (F535 nm/F480 nm) showing [Ca2+]cyt oscillations in 10 µM MeJA-treated wild-type guard cells (n = 12 of 18 cells; 67%) (a) and 10 µM MeJA-treated mpk9-1 mpk12-1 double mutant guard cells (n = 18 of 24 cells; 75%) (b). (c) Stack column representation of MeJA-induced [Ca2+]cyt oscillations (%) in wild-type guard cells (n = 18) and mpk9-1 mpk12-1 guard cells (n = 24). ABA-elicited [Ca2+]cyt oscillations in wild-type (WT) and mpk9-1 mpk12-1 guard cells. (d), (e), representative fluorescence emission ratios showing [Ca2+]cyt oscillations in 10 µM ABA-treated wild-type guard cells (n = 17 of 20 cells; 85%) (d) and 10 µM ABA-treated mpk9-1 mpk12-1 guard cells (n = 19 of 24 cells; 79%) (e). (f) Stack column representation of ABA-induced [Ca2+]cyt oscillations (%) in wild-type guard cells (n = 20) and mpk9-1 mpk12-1 guard cells (n = 24). Figure 4. The effects of MeJA on S-type anion channel currents in wild-type guard cell protoplasts (GCPs) and mpk9-1 mpk12-1 double mutant GCPs. (a) S-type anion channel currents in wild-type GCPs treated without MeJA (top trace) and with 10 μM MeJA (bottom trace). (b) S-type anion channel currents in mpk9-1 mpk12-1 double mutant GCPs treated without MeJA (top trace) and with 10 μM MeJA (bottom trace). (c) Current-voltage relationships for effects of MeJA on S-type anion channel currents in wild-type GCPs as recorded in (a). (d) Current-voltage relationships for effects of MeJA on S-type anion channel currents in mpk9-1 This article is protected by copyright. All rights reserved.
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mpk12-1 double mutant GCPs as recorded in (b). The voltage protocol was stepped up from +35 mV to -145 mV in 30 mV decrements (holding potential, +30 mV). Each datum was obtained from at least 5 GCPs. Error bars represent SEM. Figure 5. A simple model of MeJA signaling in Arabidopsis guard cells. MeJA induces ROS production, cytosolic alkalization, and [Ca2+]cyt oscillations, which are mediated by COI1, and downstream of these signal events, MPK9 and MPK12 positively function in MeJA-induced stomatal closure by regulating activation of S-type anion channels.
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Received Date : 08-Dec-2014 Revised Date
: 01-Feb-2015
Accepted Date : 06-Feb-2015 Article type Editor
: Research Paper : R Mendel
Running head: Khokon et al. MPK9and MPK12 in guard cell MeJA signaling
Two guard cell MAPKs, MPK9 and MPK12, function in methyl jasmonate-induced stomatal closure in Arabidopsis thaliana
Md. Atiqur Rahman Khokon,1* Mohammad Abdus Salam,1* Fabien Jammes,2* Wenxiu Ye,1 Mohammad Anowar Hossain,1 Misugi Uraji,1 Yoshimasa Nakamura,1 Izumi C. Mori,3 June M. Kwak4&Yoshiyuki Murata1
1
Graduate School of Environmental and Life Science, Okayama University, 1-1-1
Tsushima-naka, Okayama 700-8530, Japan 2
Department of Biology, Pomona College, Claremont, 91711, CA
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/plb.12321 This article is protected by copyright. All rights reserved.
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3
Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama
710-0046, Japan 4
Center for Plant Aging Research, Institute for Basic Science, Department of New
Biology, Daegu Kyungbuk Institute of Science and Technology, Daegu 711-873 Republic of Korea * These authors contributed equally
Corresponding author: Yoshiyuki Murata Address: The Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan E-mail: [email protected] Tel: ++-81-86-251-8310 Fax: ++-81-86-251-8388
Key words: [Ca2+]cyt oscillations; cytosolic alkalization; guard cell, mitogen-activated protein kinase; methyl jasmonate; reactive oxygen species; S-type anion channels.
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ABSTRACT Methyl jasmonate (MeJA) and abscisic acid (ABA) signaling cascades share several signaling components in guard cells. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signaling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9-1 and mpk12-1 single mutants as well as wild-type plants but not in mpk9-1 mpk12-1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA-induced stomatal closure in the wild-type plants. MeJA elicited ROS production and cytosolic alkalization in guard cells of the mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants as well as those of the wild-type plants. Furthermore, MeJA triggered elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in the mpk9-1 mpk12-1 double mutant as well as the wild-type plants. Activation of S-type anion channels by MeJA was impaired in mpk9-1 mpk12-1. Altogether, these results indicate that MPK9 and MPK12 function upstream of S-type anion channel activation and downstream of ROS production, cytosolic alkalization, and [Ca2+]cyt elevation in guard cell MeJA signaling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signaling in guard cells.
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INTRODUCTION Methyl jasmonate (MeJA) plays a role in plant growth and developmental processes and mediates various plant defense responses (Liechti & Farmer 2002). MeJA induces stomatal closure in several plant species including Arabidopsis thaliana (Suhita et al. 2004; Munemasa et al. 2007; Saito et al. 2008; Akter et al. 2010; Hossain et al. 2011a,b; Munemasa et al. 2011), Commelina benghalensis (Raghavendra & Reddy 1987), Paphiopedilum Supersuk and P. tonsum (Gehring et al. 1997), and Nicotiana glauca (Suhita et al. 2003). Both MeJA and abscisic acid (ABA) induce production of ROS, cytosolic alkalization, elevation of cytosolic Ca2+ concentration ([Ca2+]cyt), and activation of S-type anion channels in guard cells (Kwak et al. 2003; Suhita et al. 2004; Munemasa et al. 2007; Saito et al. 2008; Islam et al. 2010a, b; Hossain et al. 2011a,b). CORONATINE INSENSITIVE 1 (COI1) is an F-box protein and functions as a jasmonate co-receptor with JASMONATE ZIM DOMAIN (JAZ), which recognizes the bioactive hormone (3R,7S)-jasmonoyl-L-isoleucine (JA-Ile) with high specificity (Fonseca et al. 2009; Yan et al. 2009). The conjugation of isoleucine to JA is catalyzed by JASMONATE RESISTANT 1 (JAR1) following wounding (Suza and Staswick 2008). MeJA does not promote the interaction of COI1 with JAZ (Yan et al. 2009) but induces stomatal closure which is mediated by COI (Munemasa et al. 2007). The coi1 mutation impairs the MeJA-induced stomatal closure but not the ABA-induced stomatal closure (Munemasa et al. 2007), indicating that the F-box COI1 protein functions in MeJA signaling in guard cells but not in ABA signaling. The calcium-dependent protein kinases (CDPKs) function in guard cell Ca2+ and anion channel activation in ABA signaling (Mori et al. 2006; Zhu et al. 2007). We This article is protected by copyright. All rights reserved.
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have recently demonstrated that in Arabidopsis guard cells, CPK6 functions in both MeJA and ABA signaling and that CPK3 functions in ABA signaling but not in MeJA signaling (Munemasa et al. 2011). Moreover, we have shown that endogenous ABA is required for MeJA signaling in Arabidopsis guard cells (Hossain et al. 2011a). These results indicate that ABA and MeJA signaling cascades crosstalk and share several signaling elements and also that each cascade employs unique signaling components. However, it is not fully understood how the crosstalk between MeJA and ABA signaling pathways is regulated in guard cells. Mitogen-activated protein kinases (MAPKs) are major components in cellular signal transduction pathways that mediate numerous biotic and abiotic stress responses (Ligterink 2000; Asai et al. 2002; Jonak et al. 2002; Pedley & Martin 2005). We have demonstrated that two members of the Arabidopsis MAPK family, MPK9 and MPK12, are preferentially expressed in guard cells and positively regulate ABA signaling downstream of ROS production and upstream of S-type anion channel activation (Jammes et al. 2009). Recently, Hettenhausen et al. (2012) reported that MPK4 is involved in ABA-induced stomatal closure. These studies imply that MAPKs are key components in ABA signal transduction in guard cells. However, to our knowledge, it has not been elucidated whether and how MPK9 and MPK12 act as regulators of MeJA signaling in Arabidopsis guard cells. In order to elucidate the involvement of these two MAPKs, MPK9 and MPK12 in MeJA signaling in Arabidopsis guard cells, we examined stomatal movements and ROS production, cytosolic alkalization, [Ca2+]cyt oscillations, and S-type anion channel activation in guard cells in response to MeJA using the
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mpk9-1 and mpk12-1 single mutants as well as the mpk9-1 mpk12-1 double mutant. MATERIALS AND METHODS Plant materials Arabidopsis (Arabidopsis thaliana) wild type, Columbia-erecta (Col-er), mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants were grown in a growth chamber at 22°C and 60% humidity in a cycle of a 16-h light with 80 μmol m-2 s-1 of photon flux density and 8-h dark. Twice or three times a week, 0.1% Hyponex solution was applied to the plant growth tray. A Ca2+-sensing fluorescent protein, Yellow Cameleon 3.6 (YC3.6), was used to measure [Ca2+]cyt (Mori et al. 2006). The mpk9-1 mpk12-1 double mutant was crossed with a Columbia-0 plant that had previously been transformed with YC3.6 to obtain YC3.6-expressing mpk9-1 mpk12-1 double mutant plants. These plants were also grown under the conditions as described above. Arabidopsis Genome Initiative numbers for the genes discussed in this study are MPK9 (At3g18040) and MPK12 (At2g46070).
Measurement of stomatal apertures Stomatal apertures were measured as previously described (Hossain et al. 2011a). Excised rosette leaves were floated on an assay solution consisting of 5 mM KCl, 50 µM CaCl2, and 10 mM MES adjusted to pH 6.15 with Tris for 2 h in the light to induce stomatal opening prior to addition of MeJA. An MAPKK inhibitor, PD98059, was added 30 min before MeJA application. Two hours after MeJA application, the
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leaves were shredded in a commercial blender for 30 s and were filtrated using nylon mesh to collect the remaining epidermal tissues. Twenty stomatal apertures were measured for each sample. Measurement of ROS production in guard cells Production of ROS in guard cells was examined using H2DCF-DA (Munemasa et al. 2011). The epidermal tissues were incubated in the light for 2 h in the assay solution containing 5 mM KCl, 50 µM CaCl2, and 10 mM MES adjusted to pH 6.15 with Tris, and then 50 µM H2DCF-DA was added to the assay solution. The epidermal tissues were kept at room temperature for 30 min and then were washed with the assay solution to remove the excess dye. After filtration using nylon mesh, the collected tissues were again incubated with the assay solution supplemented with 10 µM MeJA or ABA for 20 min at room temperature. The digital image was captured using a fluorescence microscope (model: Bio Zero BZ-8000, KEYENCE) and the pixel fluorescence intensity in guard cells was measured using ImageJ 1.42q (NIH).
Measurement of pHcyt Cytosolic pH in guard cells was evaluated using a pH-sensing dye, 2΄,7΄-bis-(2-carboxyethyl)-5,(6)-carboxyfluoresceinacetoxymethyl ester (BCECF-AM) (Islam et al. 2010a). The epidermal tissues were incubated in the light for 3 h in an assay solution containing 5 mM KCl and 10 mM MES adjusted to pH 6.5 with KOH, and then 20 µM BCECF-AM was added to the assay solution. The epidermal tissues were kept at room temperature in dark for 30 min and then were washed with the assay solution to remove the excess dye. After filtration using nylon This article is protected by copyright. All rights reserved.
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mesh, the collected tissues were treated with 10 µM MeJA or ABA for 20 min in dark. The digital image was obtained and analyzed as described above.
Measurement of [Ca2+]cyt in guard cells Four- to 6-week-old wild-type and mpk9-1 mpk12-1 double mutant plants expressing YC3.6 were used to measure [Ca2+]cyt in guard cells (Islam et al. 2010b). The abaxial side of an excised leaf was softly mounted on a glass slide with a medical adhesive (Stock No. 7730; Hollister Inc., Libertyville, Illinois, United States) and then the adaxial epidermis and the mesophyll tissue were gently removed with a razor blade. The remaining intact abaxial epidermis was incubated in a solution containing 5 mM KCl, 50 μM CaCl2, and 10 mM MES adjusted to pH 6.15 with Tris under light for 2 h at 22°C to induce stomatal opening. Turgid guard cells were used to measure [Ca2+]cyt. Guard cells were treated with MeJA or ABA using a peristaltic pump 5 min after monitoring. The cyan fluorescent protein (CFP) (F480 nm) and yellow fluorescent protein (YFP) (F535 nm) fluorescent intensities of guard cells were imaged and analyzed using W-View system equipped with a 440AF21 excitation filter, a 445DRLP dichroic mirror, a 480DF30 emission filter for CFP and a 535DF25 emission filter for YFP and AQUA COSMOS software (Hamamatsu Photonics). CFP and YFP fluorescence were simultaneously monitored.
Electrophysiology
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Arabidopsis guard cell protoplasts (GCPs) were enzymatically isolated from rosette leaves of 4- to 6-week-old plants according to Munemasa et al. (2007). Whole-cell currents were monitored using a patch clamp amplifier (model CEZ-2200; NIHON KOHDEN). Data were analyzed with a data acquisition and analysis software, pCLAMP 8.2 (Molecular Devices).The pipette solution contained 150 mM CsCl, 2 mM MgCl2, 6.7 mM EGTA, 5.58 mM CaCl2, 5 mM ATP, and 10 mM HEPES adjusted to pH 7.1 with Tris, and the bath solution consisted of 30 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, and 10 mM MES adjusted to pH 5.6 with Tris (Munemasa et al. 2007). Osmolarity of the pipette solution and bath solution were adjusted with D-sorbitol to 500 mmol kg-1 and 485 mmol kg-1, respectively. In order to examine the effect of MeJA, GCPs were treated with 10 μM MeJA for 2 h prior to recordings.
Statistical analysis Student’s t-test was used to assess the significance of differences between mean values of stomatal aperture, ROS production, and cytosolic alkalization and χ2 test was used to assess the frequency of [Ca2+]cyt oscillations. Differences were considered significant for P values < 0.05.
RESULTS MAPKK inhibitor PD98059 and mpk9-1 mpk12-1 mutation impair MeJA-induced stomatal closure
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To test whether the MAPK cascade functions in guard cell MeJA signaling, we tested the effect of the MAPKK inhibitor PD98059 on MeJA-induced stomatal closure. As previously reported, MeJA at 1 μM and 10 μM significantly induced stomatal closure (Fig. 1a). In contrast, pretreatment of rosette leaves with 100 μM PD98059 significantly inhibited the MeJA-induced stomatal closure (Fig. 1a). PD98059 alone did not affect stomatal apertures in the absence of MeJA (Fig. 1a). These results suggest that an MAPK cascade plays a role in MeJA-induced stomatal closure. Next, we examined whether the guard cell-preferential MPK9 and MPK12 are part of the MAPK cascade mediating MeJA signaling in guard cells. Epidermal strip analyses show that MeJA induced stomatal closure in the mpk9-1 and mpk12-1 single mutants (Fig. 1b). However, MeJA-induced stomatal closure was disrupted in mpk9-1 mpk12-1 double mutant (Fig. 1b), indicating that MPK9 and MPK12 redundantly function as positive regulators of MeJA signaling in Arabidopsis guard cells.
MeJA-induced ROS production is not impaired in mpk9 mpk12 Reactive oxygen species (ROS) function as second messengers in both ABA and MeJA signaling in guard cells (Kwak et al. 2008; Suhita et al. 2004; Munemasa et al. 2007; Saito et al. 2008; Islam et al. 2010a). To examine whether MPK9 and MPK12 act upstream or downstream of ROS, we monitored MeJA-triggered ROS production in guard cells of the mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants using 2΄,7΄-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Munemasa et al.
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2007). As shown in Fig. 2a, application of 10 μM MeJA induced ROS production in wild-type guard cells, as previously reported (Munemasa et al. 2007; Saito et al. 2008; Islam et al. 2010a; Munemasa et al. 2011). MeJA also induced ROS production in guard cells of the mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants (Fig. 2a). We next determined ABA-triggered ROS production in the mpk9-1 mpk12-1 double mutant. As shown in Fig. 2b, 10 μM ABA induced ROS production in guard cells of both wild type and the mpk9-1 mpk12-1 mutant. These results suggest that MPK9 and MPK12 function downstream of ROS production in stomatal response to MeJA and ABA.
MeJA and ABA cause cytosolic alkalization in mpk9 mpk12 guard cells Cytosolic alkalization in response to MeJA promotes stomatal closure (Suhita et al. 2004). To further determine the role of MPK9 and MPK12 in guard cell MeJA signaling, we analyzed MeJA-induced cytosolic alkalization in guard cells of the mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants using the pH indicator BCECF-AM. Figure 2c shows that application of 10 μM MeJA induced cytosolic alkalization in the wild type, mpk9-1, mpk12-1, and mpk9-1 mpk12-1. In addition, 10 μM ABA induced cytosolic alkalization in guard cells of the wild type and the mpk9-1 mpk12-1 double mutant (Fig. 2d). These results imply that MPK9 and MPK12 act downstream of cytosolic alkalization in response to MeJA and ABA in guard cells.
[Ca2+]cyt oscillations triggered by MeJA and ABA are not abolished in mpk9 mpk12 guard cells This article is protected by copyright. All rights reserved.
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MeJA as well as ABA induces elevation in [Ca2+]cyt, which in turn activates subsequent cellular events (Kwak et al. 2008; Islam et al. 2010b; Hossain et al. 2011a, b; Munemasa et al. 2011). Elevation in [Ca2+]cyt in guard cells often appear to be repetitive transient increases or oscillations. To examine whether the mutations in MPK9 and MPK12 affected MeJA- and ABA-induced [Ca2+]cyt oscillations, we conducted imaging analyses using plants expressing YC3.6 (Nagai et al. 2004). As shown in Fig. 3a-c, application of 10 μM MeJA induced one or more [Ca2+]cyt oscillations in 67% of the wild-type guard cells (n = 12 of 18 cells) and in 75% of the mpk9-1 mpk12-1 double mutant guard cells (n = 18 of 24 cells) over 60 min of monitoring. Moreover, ABA at 10 μM induced one or more [Ca2+]cyt oscillations in 85% of wild-type guard cells (n = 17 of 20 cells) and in 79% of the mpk9-1 mpk12-1 double mutant guard cells (n = 19 of 24 cells) (Fig. 3d-f). The frequencies of [Ca2+]cyt oscillations were not significantly different between the wild type and the mpk9-1 mpk12-1 double mutant (P = 0.55, 10 μM MeJA; P = 0.61, 10 μM ABA), suggesting that MPK9 and MPK12 function downstream of [Ca2+]cyt oscillations in guard cells in response to MeJA and ABA.
MeJA-induced S-type anion channels activation is impaired in mpk9 mpk12 guard cells Activation of S-type anion channels in the plasma membrane of guard cells is essential for stomatal closure (Schroeder & Hagiwara1989; Negi et al. 2008; Vahisalu et al. 2008). Like ABA and calcium, MeJA activates S-type anion channels in guard cells, resulting in stomatal closure (Schroeder & Hagiwara1989; Pei, Ward
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& Schroeder 1999; Munemasa et al. 2007, 2011). Using the whole-cell patch-clamp technique, we examined whether MeJA activates S-type anion channels in guard cells of the mpk9 and mpk12 mutant. As shown in Fig. 4a & 4c, MeJA at 10 μM activated S-type anion currents in wild-type guard cells (P < 0.001 at -145 mV) in agreement with our previous observations (Munemasa et al. 2007, 2011). To the contrary, MeJA activation of S-type anion currents was attenuated in the mpk9-1 mpk12-1 double mutant guard cells (P = 0.11 at -145 mV; Fig. 4b & 4d), which is consistent with the absence of MeJA-induced stomatal closure in the mpk9-1 mpk12-1 double mutant (Fig. 1b). These results suggest that MPK9 and MPK12 positively regulate MeJA signaling by acting upstream of the activation of S-type anion channels in guard cells.
DISCUSSION Mitogen-activated protein kinases are major components in various cellular signal transduction pathways in response to biotic and abiotic stresses (Ligterink 2000; Asai et al. 2002; Jonak et al. 2002; Pedley &Martin 2005). We have previously demonstrated that two members of the MAPK family, MPK9 and MPK12, are preferentially expressed in guard cells and positively regulate ABA signaling in guard cells (Jammes et al. 2009). In this study, we show that MPK9 and MPK12 are functionally redundant and positively regulate MeJA-induced stomatal closure. Stomata in mpk9-1 mpk12-1 double mutant but not in mpk9-1 or mpk12-1 single mutants were insensitive to MeJA (Fig. 1b). Furthermore, mpk9-1 mpk12-1 double mutant showed impaired S-type anion channel activation in response to MeJA (Fig.
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4). In contrast, upstream cellular events including MeJA-triggered ROS production, alkalization, and [Ca2+]cyt oscillations were not affected in mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants (Figs. 2 & 3), indicating that MPK9 and MPK12 act downstream of these cellular events. A simple model of the MeJA signaling in Arabidopsis guard cells is proposed to summarize the present study (Fig. 5). Although our previous study showed that MPK9 and MPK12 function downstream of ROS in guard cell ABA signaling (Jammes et al. 2009), it was unknown whether ABA can elicit ROS production and [Ca2+]cyt oscillations in mpk9-1 mpk12-1. We provide evidences that ABA induces ROS production, alkalization, and [Ca2+]cyt oscillations in mpk9-1 mpk12-1 mutant (Figs. 2 & 3). Taken together, these results demonstrate that MPK9 and MPK12 are common signaling components acting downstream of [Ca2+]cyt oscillations but upstream of S-type anion channel activation in ABA and MeJA signaling in guard cells. In guard cells, MeJA pathway shares several signaling components with ABA pathway such as ROS, [Ca2+]cyt oscillation/elevation, and cytosolic alkalization (Suhita et al. 2004; Munemasa et al. 2007; Saito et al. 2008; Islam et al. 2010a, b). On the other hand, stomata of jar1-1 and coi1 mutant plants are insensitive to MeJA (Suhita et al. 2004; Munemasa et al. 2007) but are sensitive to ABA, while stomata of ost1-2 are insensitive to ABA but are sensitive to MeJA (Suhita et al. 2004). Moreover, MeJA signal transduction requires endogenous ABA (Hossain et al. 2011a), whereas ABA signaling does not appear to require endogenous MeJA (Munemasa et al. 2007). These studies indicate that ABA and MeJA signaling possess unique signaling components in addition to common ones.
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Hydrogen peroxide activates MAPKs in Arabidopsis suspension cultured cells (Desikan et al. 1999; Kovtun et al. 2000; Desikan et al. 2001) and MPK12 kinase activity in Arabidopsis guard cells (Jammes et al. 2009). Activation of MPK8 needs binding of calmodulins in a Ca2+-dependent manner in Arabidopsis (Takahashi et al. 2011) and activation of MAPKs also requires an increase of [Ca2+]cyt in Nicotiana benthamiana (Segonzac et al. 2011). In addition, a 46-kDa MAPK is activated by ROS, which is involved in ABA-induced antioxidant defense in Zea mays (Zhang et al. 2006). These results suggest that MAPKs function downstream of ROS production and [Ca2+]cyt elevation, which is consistent with results presented in this study and our previous study (Jammes et al. 2009; Salam et al. 2012; Salam et al. 2013). Calcium-dependent protein kinases are unique enzymes considered as [Ca2+]cyt sensors found in plants and some protozoa. In Arabidopsis guard cells, MeJA-induced [Ca2+]cyt oscillation is impaired in the cpk6 mutant (Munemasa et al. 2011). Our study reveals that MeJA and ABA induced [Ca2+]cyt oscillation in the mpk9-1 mpk12-1 double mutant (Fig. 3). These CDPKs may therefore directly or indirectly activate MPK9 and MPK12 in MeJA and ABA signal cascades in guard cells. Both CDPKs and MAPKs are serine/threonine kinases. A broad-range serine/threonine kinase inhibitor, K252a, inhibits MeJA- and ABA-induced stomatal closure and [Ca2+]cyt oscillation (Hossain et al. 2011b). Hence, the inhibition of MeJA-induced stomatal closure by K252a may be at least in part due to inhibition of CPKs such as CPK6 and MAPKs such as MPK9 and MPK12.
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Both MeJA and ABA activate S-type anion channels in plasma membrane of guard cells, leading to stomatal closure (Munemasa et al. 2007; Jammes et al. 2009; Munemasa et al. 2011). The mpk9-1 mpk12-1 double mutation impairs the activation of S-type anion channels by MeJA and ABA (Fig. 4; Jammes et al. 2009), suggesting that MPK9 and MPK12 function upstream of S-type anion channel activation in ABA and MeJA signal cascades in guard cells. Recent studies have shown that the ABA-activated OST1 kinase and the CDPKs phosphorylate S-type anion channel protein SLAC1, resulting in stimulation of SLAC1-mediated anion currents in Xenopus oocytes (Lee et al. 2009; Geiger et al. 2009; Geiger et al. 2010). These results indicate that S-type anion channels are important molecular players in stomatal movements, thus their activity is highly regulated by protein phosphorylation under relevant physiological conditions. Since MPK9 and MPK12 act upstream of S-type anion channel activation in both MeJA and ABA signaling (Fig. 4; Jammes et al. 2009), it would be interesting to test whether these two MAPKs can phosphorylate SLAC1 proteins. In conclusion, MPK9 and MPK12 are key regulators of both MeJA and ABA signaling pathways by acting upstream of S-type anion channel activation and downstream of ROS production, cytosolic alkalization, and [Ca2+]cyt oscillation in Arabidopsis guard cells.
ACKNOWLEDGEMENTS This research was supported in part by The Asahi Glass Foundation, NOVARTIS Foundation (Japan) for the Promotion of Science, Grant-in-Aid for Young Scientists,
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and Grants for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Y.M., and in part by project IBS-R013-G2-2014-a00 to J.M.K.
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FIGURE LEGENDS Figure 1. The effect of MAPKK inhibitor, PD98059, and mutation of MAPKs on MeJA-induced stomatal closure. (a) Rosette leaves of wild type were treated with MeJA (1 or 10 μM) in the presence or absence of 100 μM PD98059. PD98059 was applied to assay solution prior to MeJA treatment. (b) Rosette leaves of wild type (WT), mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants were treated without (white bars) or with 1 μM MeJA (black bars) or 10 μM MeJA (gray bars). Averages of three independent experiments (60 stomata per bar) are shown. Error bars represent standard deviation. Asterisks indicate significant differences (*, P < 0.05). Figure 2. The effects of MeJA (10 μM) and ABA (10 μM) on ROS production and cytosolic alkalization in wild type (WT) and mpk mutants. ROS production as shown by DCF fluorescence intensity in wild type and mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants in response to MeJA (a) and ABA (b). Cytosolic alkalization as shown by BCECF fluorescence intensity in wild type and mpk9-1, mpk12-1, and mpk9-1 mpk12-1 mutants in response to MeJA (c) and ABA (d). Fluorescence
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intensities were normalized to the vehicle control values for each line with respect to each day. Averages of three independent experiments (50 guard cells per bar) are shown. Error bars represent standard deviation. Asterisks indicate significant differences (*, P < 0.05). Figure 3. MeJA-elicited [Ca2+]cyt oscillations in wild-type (WT) and mpk9 mpk12 guard cells. (a), (b), representative fluorescence emission ratios (F535 nm/F480 nm) showing [Ca2+]cyt oscillations in 10 µM MeJA-treated wild-type guard cells (n = 12 of 18 cells; 67%) (a) and 10 µM MeJA-treated mpk9-1 mpk12-1 double mutant guard cells (n = 18 of 24 cells; 75%) (b). (c) Stack column representation of MeJA-induced [Ca2+]cyt oscillations (%) in wild-type guard cells (n = 18) and mpk9-1 mpk12-1 guard cells (n = 24). ABA-elicited [Ca2+]cyt oscillations in wild-type (WT) and mpk9-1 mpk12-1 guard cells. (d), (e), representative fluorescence emission ratios showing [Ca2+]cyt oscillations in 10 µM ABA-treated wild-type guard cells (n = 17 of 20 cells; 85%) (d) and 10 µM ABA-treated mpk9-1 mpk12-1 guard cells (n = 19 of 24 cells; 79%) (e). (f) Stack column representation of ABA-induced [Ca2+]cyt oscillations (%) in wild-type guard cells (n = 20) and mpk9-1 mpk12-1 guard cells (n = 24). Figure 4. The effects of MeJA on S-type anion channel currents in wild-type guard cell protoplasts (GCPs) and mpk9-1 mpk12-1 double mutant GCPs. (a) S-type anion channel currents in wild-type GCPs treated without MeJA (top trace) and with 10 μM MeJA (bottom trace). (b) S-type anion channel currents in mpk9-1 mpk12-1 double mutant GCPs treated without MeJA (top trace) and with 10 μM MeJA (bottom trace). (c) Current-voltage relationships for effects of MeJA on S-type anion channel currents in wild-type GCPs as recorded in (a). (d) Current-voltage relationships for effects of MeJA on S-type anion channel currents in mpk9-1 This article is protected by copyright. All rights reserved.
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mpk12-1 double mutant GCPs as recorded in (b). The voltage protocol was stepped up from +35 mV to -145 mV in 30 mV decrements (holding potential, +30 mV). Each datum was obtained from at least 5 GCPs. Error bars represent SEM. Figure 5. A simple model of MeJA signaling in Arabidopsis guard cells. MeJA induces ROS production, cytosolic alkalization, and [Ca2+]cyt oscillations, which are mediated by COI1, and downstream of these signal events, MPK9 and MPK12 positively function in MeJA-induced stomatal closure by regulating activation of S-type anion channels.
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