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Plant Cell Advance Publication. Published on June 7, 2016, doi:10.1105/tpc.16.00078 1 CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and 2 Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor 3 4 Shuangshuang Zhaoa, b, Yuxiang Jiangc, Yang Zhaob, Shanjin Huangc,d, Ming 5 Yuanb, Yanxiu Zhaoa, 1, and Yan Guob, 1 6 7 8 9 10 11 12 13 14 a Key Laboratory of Plant Stress, Life Science College, Shandong Normal University, Jinan 250014, China b State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China c Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Science, Beijing 100093, China d Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China 15 16 1 To whom correspondence should be addressed. 17 18 Keywords: Arabidopsis, Actin filaments, Phosphorylation, Stomatal closure 19 20 For correspondence: 21 Yan Guo 22 State Key Laboratory of Plant Physiology and Biochemistry 23 College of Biological Sciences 24 China Agricultural University 25 Beijing 100193 26 P.R. China 27 E-mail: [email protected] 28 Phone: 86-10-62732030 29 Fax: 86-10-62732030 30 1 ©2016 American Society of Plant Biologists. All Rights Reserved. 31 Yanxiu Zhao 32 Key Laboratory of Plant Stress 33 Life Science College 34 Shandong Normal University 35 Jinan 250014 36 P.R. China 37 E-mail: [email protected] 38 Phone: 86-531-86180002 39 Fax: 86-531-86180002 40 Distribution statement 41 The author responsible for distribution of materials integral to the findings presented 42 in this article in accordance with the policy described in the Instructions for Authors 43 (www.plantcell.org) is Yan Guo ([email protected]). 44 45 Synopsis 46 Arabidopsis CASEIN KINASE1-LIKE PROTEIN2 associates with actin filaments 47 and phosphorylates Actin Depolymerizing Factors, and this is required for actin 48 filament reorganization and stomatal closure. 2 49 50 Abstract 51 The opening and closing of stomata are crucial for plant photosynthesis and 52 transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, 53 but the underlying mechanism for this cytoskeletal reorganization remains largely 54 unclear. In this study, we identified and characterized Arabidopsis thaliana casein 55 kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and 56 participates in ABA- and drought-induced stomatal closure. Although CKL2 does not 57 bind to actin filaments directly and has no effect on actin assembly in vitro, it 58 co-localizes with and stabilizes actin filaments in guard cells. Further investigation 59 revealed that CKL2 physically interacts with and phosphorylates actin 60 depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. 61 During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin 62 reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas 63 deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal 64 closure to be more sensitive to ABA. Deletion of ADF4 in the ckl2 mutant partially 65 recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs 66 from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 67 regulates actin filament reorganization and stomatal closure mainly through 68 phosphorylation of ADF. 69 3 70 INTRODUCTION 71 72 Stomata regulate the uptake of CO2 for photosynthesis, water loss through 73 transpiration, and defense responses during pathogen attack (Kim et al., 2010; Du et 74 al., 2014). To cope with changes in environmental conditions, such as light, 75 temperature, humidity, CO2, and salt in soil, plants must tightly regulate the opening 76 and closing of stomata (Roelfsema and Hedrich, 2005; Vavasseur and Raghavendra, 77 2005; Israelsson et al., 2006). Many cellular signals (e.g. abscisic acid [ABA], H2O2, 78 Ca2+, CO2, and NO) regulate stomata by influencing the activities of H+, K+, Ca2+, and 79 anion transporters and channels (Pei et al., 2000; Schroeder et al., 2001; Hosy et al., 80 2003; Desikan et al., 2004; Hirayama and Shinozaki, 2007; Wang and Song, 2008; 81 Gayatri et al., 2013; Kollist et al., 2014). Actin filament reorganization occurs during 82 stomatal closure. The actin cytoskeleton in the guard cells changes from 83 well-organized cortical filaments in the guard cells of open stomata, to randomly 84 distributed filaments, and then finally reorganizes into highly bundled long cables in 85 the longitudinal direction in the guard cells of closed stomata (Hwang and Lee, 2001; 86 Zhao et al., 2011). This regulatory process involves actin-binding proteins such as 87 SCAB1 and the Arp2/3 complex (Zhao et al., 2011; Jiang et al., 2012; Li et al., 2014). 88 SCAB1 stabilizes actin filaments and loss of SCAB1 in plants causes defects in 89 stomatal closure (Zhao et al., 2011). The Arp2/3 complex mediates stomatal closure in 90 response to external stimuli and regulates actin reorganization in guard cells (Jiang et 91 al., 2012; Li et al., 2014). However, how such actin filament reorganization in guard 92 cells is regulated remains an open question. 93 Actin filaments are highly dynamic, undergoing rapid reorganization and turnover 94 regulated by actin-binding proteins such as ADF/cofilin, villin, profilin, fimbrin, and 95 capping protein (Wasteneys and Galway, 2003; Hussey, et al., 2006; Staiger and 96 Blanchoin, 2006; Higaki, et al., 2007; Thomas, et al., 2009; Li et al., 2010; Su et al., 97 2012; Qu et al., 2013; Wang et al., 2015). ADF/cofilin proteins function as key 98 regulators of actin filament dynamics and reorganization through binding to both 99 globular and filamentous actin. ADF/cofilin proteins promote actin filament severing 4 100 and depolymerization and inhibit nucleotide exchange on actin monomers 101 (Hotulainen et al., 2005; Andrianantoandro and Pollard, 2006; Henty et al., 2011). 102 The Arabidopsis thaliana genome encodes 11 ADF proteins, which play important 103 roles in various biological processes. ADF4 is involved in innate immune signaling 104 (Tian et al., 2009; Henty-Ridilla et al., 2014); ADF7 promotes pollen tube growth 105 (Zheng et al., 2013); ADF2 is required for cell growth, development, and root-knot 106 nematode infection (Clement et al., 2009). In addition, the 14-3-3 λ protein interacts 107 with phosphorylated ADF1 to regulate actin dynamics during hypocotyl elongation 108 (Zhao et al., 2015). Overexpression of ADF1 causes disruption of F-actin cables in 109 guard cells and results in a stomatal closure-defect phenotype following ABA 110 treatment, suggesting that ADF proteins might function in this process (Dong et al., 111 2001). 112 In animals and plants, many factors regulate the F-actin disassembling activity of 113 ADF/cofilin. Two proteins, actin-interacting protein-1 (AIP1) and cyclase-associated 114 protein (CAP), enhance the F-actin disassembling activity of ADF/cofilin (Moriyama 115 and Yahara, 2002; Ono, 2003; Ketelaar et al., 2004; Shi et al., 2013). The F-actin 116 disassembling activity of ADF/cofilin can also be enhanced by increased intracellular 117 pH (Bernstein et al., 2000; Allwood et al., 2002). The F-actin disassembling activity 118 of ADF/cofilin is decreased by phosphoinositide and cortactin binding (Yonezawa et 119 al., 1990; Allwood et al., 2002; Maciver and Hussey, 2002) as well as by 120 phosphorylation at the serine residue 3 of animal cofilin (Agnew et al., 1995). 121 Changes in the Ser3 phosphorylation level are tightly associated with extracellular 122 stimuli, actin rearrangement, and cell activities, which implies a critical role of such 123 phosphorylation for modulation of cofilin activity (Mizuno, 2013). Ser6 of plant ADFs 124 has been considered to function analogously to Ser3 of animal cofilin based on amino 125 acid sequence similarity searches and tertiary structure prediction (Chen et al., 2002). 126 Similar to Ser3 phosphorylation in cofilin, the phosphorylation of the Ser6 in ADFs 127 results in a reduction of their F-actin disassembling activity (Agnew et al., 1995; 128 Moriyama et al., 1996; Nagaoka et al., 1996; Smertenko et al., 1998). In yeast, casein 129 kinase 1 (CK1) Hrr25 phosphorylates a number of actin-binding proteins, including 5 130 the cofilin Cof1 (Peng et al., 2015). Two kinase families, LIM (Lin-11/Isl-1/Mec-3) 131 and TES (testicular protein), specifically phosphorylate and deactivate ADF/cofilin 132 (Toshima et al., 2001). However, mammalian LIMK does not phosphorylate plant 133 ADFs, and the plant LIMK homologs do not phosphorylate ADFs (Bernard et al., 134 2007). Rapid dephosphorylation of ADF/cofilin occurs in response to various stimuli 135 in animal cells and results in changes of F-actin organization and assembly (Davidson 136 and Haslam, 1994; Samstag et al., 1994; Kanamori et al., 1995). In plants, the 137 phosphorylation of the wheat (Triticum aestivum) ADF is regulated by low 138 temperature (Ouellet et al., 2001). A protein fraction purified from cell suspension 139 cultures of French bean (Phaseolus vulgaris L. cv. Immuna 1.1) and enriched in 140 calmodulin-like domain protein kinase(s) (CDPKs) can phosphorylate maize ADF3 at 141 Ser6 and this phosphorylation is inhibited by the addition of anti-CDPK antibodies 142 (Smertenko et al., 1998; Allwood et al., 2001). Recently, Arabidopsis CDPK6 protein 143 has been shown to phosphorylate ADF1 in vitro (Dong and Hong, 2013). However, it 144 remains to be determined whether these plant CDPKs associate with F-actin and what 145 the physiological function of the phosphorylation is. 146 Casein kinase1 (CK1) is a family of serine/threonine protein kinases highly 147 conserved in eukaryotic organisms. CK1 is involved in biological processes including 148 circadian rhythm establishment, vesicular trafficking, DNA repair, the cell cycle, and 149 morphogenesis (Gross et al., 1995; Akashi et al., 2002; Cheong et al., 2011). A 150 number of cytoskeleton-related proteins have been identified as targets of CK1 151 kinases; these targets include cofilin, twinfilin, myosin, tropin, spectrin3, dynein, 152 α-/β-tubulin, microtubule-associated protein, and kinesin-like protein (Boesger et al., 153 2014; Knippschild et al., 2014; Peng et al., 2015). In addition to the other 154 cytoskeleton-related proteins, CK1 directly phosphorylates actin protein in vitro 155 (Shibayama et al., 1986; Knippschild et al., 2005). Arabidopsis casein kinase 1-like 156 protein 6 (CKL6) phosphorylates tubulin and regulates microtubule organization 157 (Ben-Nissan et al., 2008). 158 In this study, we show that casein kinase 1-like protein 2 (CKL2) and actin 159 depolymerizing factor 4 (ADF4) are involved in stomatal closure in response to 6 160 drought stress and ABA treatment. Although CKL2 does not bind to and stabilize 161 actin filaments in vitro, it decorates and stabilizes actin filaments in guard cells. 162 Interestingly, we found that CKL2 physically interacts with ADF4 and that 163 phosphorylation of ADF4 decreases its F-actin disassembling activity in vitro. In 164 terms of ABA-induced stomatal closure, CKL2 deletion and ADF4 deletion have 165 opposite effects. Importantly, deletion of ADF4 in the ckl2 mutant background 166 partially rescues its ABA-insensitive stomatal closure phenotype. Considering that 167 CKL2 non-selectively phosphorylates Arabidopsis ADFs from subclass I in vitro, we 168 propose that CKL2 stabilizes actin filaments by phosphorylating ADFs and inhibiting 169 their F-actin disassembling activity in guard cells to promote the reassembly of actin 170 filaments during drought/ABA-induced stomatal closure. 171 172 7 173 RESULTS 174 175 The ckl2 Mutant Exhibits Rapid Water Loss and Impaired ABA-Induced 176 Stomatal Closure 177 To obtain mutants that lost water faster and wilted earlier than the wild type, we 178 performed a genetic screen of T-DNA insertion lines ordered from the Arabidopsis 179 Biological Resource Center (ABRC, Ohio State University). Rosette leaves of 180 3-week-old plants were detached and the rate of water loss was monitored. After 181 identification of mutants with both more rapid and less rapid water-loss phenotypes, 182 we further performed a stomatal aperture assay with an ABA stimulus as a standard 183 during the screening. We identified a mutant (SALK_104209) that showed faster 184 water loss and was less sensitive to ABA-induced stomatal closure than the wild type 185 (Figures 1A-1C). The T-DNA insertion in the Arabidopsis gene At1g72710 was 186 confirmed by polymerase chain reaction (PCR) using the T-DNA left border and the 187 gene-specific primers. Because At1g72710 encodes casein kinase 1-like protein 2 188 (CKL2), SALK_104209 was designated ckl2. Reverse transcription (RT) PCR 189 analysis revealed that transcript accumulation of CKL2 was significantly decreased in 190 the ckl2 homozygous mutant (Supplemental Figure 1A and 1B). To determine the 191 transpiration rates of shoots, we used an infrared camera to measure the leaf 192 temperature of 4-week-old Col-0 and ckl2 mutant plants (Merlot et al., 2002). The leaf 193 temperature of the ckl2 mutant was significantly lower than that of Col-0, suggesting 194 that the mutant leaves display a higher transpiration rate and lose more water than the 195 wild type (Figures 1D and 1E). 196 To rescue the water-loss and stomatal closure phenotypes of the mutant, we 197 transformed the ckl2 mutant with a construct harboring a 5.09-kb CKL2 genomic 198 DNA fragment including 1.06-kb promoter and 0.5-kb 3’ untranslated region. The 199 expression level of CKL2 in the transgenic lines was similar to that in the wild type 200 (Supplemental Figure 1B). The water-loss and stomatal closure phenotypes were fully 201 rescued in the transgenic lines (Figures 1B and 1C), suggesting the phenotype is 202 indeed caused by the loss of function of CKL2. 8 203 204 CKL2 Expression Is Induced by Water Loss and ABA Treatment 205 To determine whether the expression of CKL2 is regulated by drought stress and ABA, 206 total RNA was extracted from 7-d-old wild-type seedlings treated with 20 μM ABA 207 for 0, 0.5, and 1 h, or water loss treatment until the seedlings lost 20% of their fresh 208 weight. Real-time RT-PCR analysis showed that CKL2 expression was induced by 209 both water-loss and ABA treatments (Figure 1F and 1G). As a positive control, the 210 expression of RESPONSIVE TO DESICCATION 29A (RD29A) was confirmed to be 211 induced by both treatments; RD29A is an abiotic stress/ABA-responsive gene (Ishitani 212 et al., 1997). For the negative control, we monitored the expression of a 213 salt-responsive gene, SOS3-LIKE CALCIUM BINDING PROTEIN 8 (SCaBP8) (Quan 214 et al., 2007; Lin et al., 2009), which showed no obvious changes under these 215 conditions. The induction of CKL2 under water-loss and ABA treatments is consistent 216 with microarray data from AtGeneExpress 217 (http://jsp.weigelworld.org/expviz/expviz.jsp, Supplemental Figure 1C) and a 218 previous study (Cui et al., 2012). 219 CKL2 is widely expressed in a variety of tissues throughout Arabidopsis 220 development (Gene Express Map of Arabidopsis, 221 http://jsp.weigelworld.org/expviz/expviz.jsp, Supplemental Figure 2A). To determine 222 the tissue-specific expression pattern of CKL2, real-time RT-PCR was performed 223 using total RNA extracted from various tissues of 4-week-old Col-0 plants. Consistent 224 with results obtained in the TAIR website, expression of CKL2 was detected in roots, 225 stems, cauline leaves, rosette leaves, flowers, and siliques (Supplemental Figure 2B). 226 227 CKL2 Co-localizes with Actin Filaments in Cells, but Does Not Directly Interact 228 with Actin Filaments in Vitro 229 To determine the subcellular localization of CKL2, the GFP (Green Fluorescent 230 Protein)-CKL2 construct driven by the CKL2 native promoter that was used in the 231 genetic rescue experiment was transformed into Col-0 and the ckl2 mutant. The 232 stomatal closure phenotype of the ckl2 mutant was rescued by the 9 233 CKL2pro:GFP-CKL2 transgene (Figure 2A) and the expression level of CKL2 in the 234 transgenic lines was similar to that in the wild type (Supplemental Figure 2C). 235 Although the GFP signal formed filamentous structures in the cells of these transgenic 236 plants, it was very weak, and it was difficult to obtain high-quality images. The 237 GFP-labeled CKL2 expressed from the CKL2 native promoter was observed in 238 filamentous structures in the cytoplasm of hypocotyl epidermal cells, leaf epidermal 239 cells, guard cells, root epidermal cells and root hairs of the Col-0 transgenic line 240 (Figure 2B). These results suggest that GFP-CKL2 accurately indicates the 241 intracellular localization of CKL2. To further investigate the subcellular localization 242 of CKL2, we fused GFP to the N terminus of CKL2 under the control of the CaMV 243 35S promoter. This construct was used to transform Col-0 and the ckl2 mutant. The 244 GFP-CKL2 fusion gene rescued the stomatal-closure defect and drought-sensitive 10 245 phenotypes of the ckl2 mutant. GFP-labeled CKL2 in the transgenic plants formed 246 fine fibrous networks in the cytoplasm of hypocotyl epidermal cells (Supplemental 247 Figure 3A), leaf epidermal cells (Supplemental Figure 3B), guard cells (Supplemental 248 Figure 3C), root epidermal cells (Supplemental Figure 3D), and root hairs 249 (Supplemental Figure 3E). 250 To determine if CKL2 was associated with actin filaments or microtubules, a 251 suspension cell line was generated from the rosette leaves of the GFP-CKL2 252 transgenic plants. The GFP-labeled CKL2 co-localized with 253 rhodamine-phalloidin-stained actin filaments in the suspension cells (Supplemental 254 Figure 3F). Co-localization was analyzed by plotting GFP-CKL2 and actin filament 255 signal intensities using ImageJ software. The Pearson’s correlation coefficient value 256 was 0.83 in the indicated regions of interest, suggesting a strong correlation between 257 the spatial localizations of GFP-CKL2 and the actin filaments (Supplemental Figure 258 3G). To further confirm the association of CKL2 with actin filaments, the GFP-CKL2 11 259 transgenic plants were treated with latrunculin A (Lat A), an inhibitor of actin 260 polymerization that disrupts actin filaments by binding actin monomers, or oryzalin, a 261 microtubule-disrupting reagent. After 0.5 h treatment with 200 nM Lat A, the 262 filamentous network of GFP-CKL2 was disrupted in most of the hypocotyl cells. As a 263 control, GFP-fABD2-GFP-labeled actin filaments were also disrupted (Supplemental 264 Figure 3H). However, after 1 h treatment with 10 μM oryzalin, the filamentous 265 structure of GFP-CKL2 remained intact in most of the hypocotyl cells, whereas 266 MBD-GFP-labeled microtubules were disrupted (Supplemental Figure 3I). Disruption 267 of filamentous structures by Lat A was also observed in guard cells of 268 CKL2pro:GFP-CKL2;ckl2 (Figure 2C). These results indicate that CKL2 co-localizes 269 with actin filaments but not microtubules in cells. 270 To test whether CKL2 binds to actin filaments directly in vitro, we performed an 271 actin co-sedimentation assay by high-speed centrifugation. We found that most of the 272 CKL2 remained in the supernatant in both the absence and presence of F-actin; 273 however, a significant amount of the actin-binding protein Fim1 (Su et al., 2012) was 274 pulled down by F-actin (Supplemental Figure 4A). These results indicate that CKL2 275 does not directly bind to actin filaments. 276 To test if CKL2 has another effect on actin filaments in vitro, we directly 277 visualized actin filaments stained with Alexa-488-phalloidin by epifluorescence light 278 microscopy. The length of F-actin showed no significant differences in the presence or 279 absence of His-CKL2, indicating that CKL2 has no effect on the length distribution of 280 actin filaments in vitro (Supplemental Figures 4B and 4C). To determine whether 281 CKL2 is involved in actin assembly, we determined the effect of CKL2 on 282 spontaneous actin assembly and found that CKL2 had no such activity in vitro 283 (Supplemental Figure 4D). Thus, these results suggest that CKL2 neither interacts 284 with actin filaments nor affects actin assembly and disassembly in vitro. 285 286 CKL2 Stabilizes Actin Filaments and Regulates Stomatal Closure 287 To test if CKL2 affects actin filaments in cells, we measured the F-actin stability in 288 ckl2 and wild-type cells. To visualize the actin filaments, we generated transgenic 12 289 plants harboring 35S:GFP-fABD2-GFP (containing the second actin-binding domain 290 of At-Fim1) in the Col-0 and ckl2 background, and examined the stability of actin 291 filaments in the guard cells of the ckl2 mutant and Col-0. After the transgenic lines 292 were treated with 200 nM Lat A for 30 min, the actin filaments became more 293 fragmented and less abundant in both Col-0 and ckl2 guard cells compared with the 294 untreated guard cells (Figure 3A). However, the actin filaments were disrupted more 295 rapidly in ckl2 guard cells than in wild-type guard cells. In the ckl2 mutant, more 296 GFP-labeled dot-like structures were detected after Lat A treatment (Figure 3A). 297 When the GFP-fABD2-GFP signal was monitored and quantified by measuring 298 average GFP fluorescence pixel intensity, the GFP signal was similar between ckl2 299 and Col-0 in widely opened stomata. However, the GFP signal in the ckl2 guard cells 13 300 decreased to a greater extent than in wild-type guard cells after the Lat A treatment 301 (Figure 3B). These results indicate that CKL2 plays a role in stabilizing actin 302 filaments in guard cells. 303 We next determined the physiological relevance of the actin-filament-stabilizing 304 activity of CKL2 during stomatal closure. Since it has been shown that the actin 305 cytoskeleton in guard cells is reorganized during ABA-induced stomatal closure 306 (Lemichez et al., 2001; Zhao et al., 2011) and the ckl2 mutant displayed impaired 307 ABA-induced stomatal closure, we examined whether the reorganization of actin 308 filaments during ABA-induced stomatal closure was altered in ckl2 by the exogenous 309 application of ABA. When the stomata were in an opened state, the actin filaments 310 were present as well-organized cortical filaments in both Col-0 and ckl2 guard cells. 311 After 2 μM ABA treatment for 0.5 h, approximately 73% of stomata were closed and 312 the actin filaments were randomly rearranged and then bundled preferentially as long 313 cables in Col-0. However, the stomatal closure was less sensitive to ABA treatment in 314 the ckl2 mutant and 71% of stomata were not closed; the actin filaments failed to form 315 bundled cables and were trapped in a randomly distributed state (Figure 3C). To 316 quantify the actin architecture in the guard cells, skewness and density (Higaki et al., 317 2010) parameters were measured to determine the extent of actin filament bundling 318 and the percentage of occupancy of actin filaments. In widely opened guard cells, the 319 ckl2 mutant had a lower density value than wild type (Figure 3D); however, the 320 skewness value showed no significant difference in the ckl2 mutant compared with 321 wild type (Figure 3E). After the plants were treated with 2 μM ABA for 0.5 h, 73% of 322 stomata were closed in Col-0 and actin filaments became bundled with higher 323 skewness in these guard cells; however, 71% of stomata were not closed in the ckl2 324 mutant and actin displayed no obvious change in skewness in these guard cells. The 325 density value decreased in Col-0 and increased in the ckl2 mutant after the treatment 326 (Figure 3D). These results suggest that the actin-filament-stabilizing activity of CKL2 327 is required for the subsequent construction of longitudinal actin cables and therefore 328 facilitates actin filament reorganization during stomatal closure. 329 14 330 CKL2 Regulates the Activity of Actin Filament Severing in Guard Cells 331 Observation of a single actin filament lifetime, including elongation rate and 332 depolymerizing rate, in living cells has been employed to study actin dynamics in 333 vivo (Smertenko et al., 2010; Henty-Ridilla et al., 2013; Li et al., 2013; Qin et al., 334 2014; Li et al., 2014). To examine the actin filament dynamics in the guard cells of 335 Col-0 and the ckl2 mutant, we performed time-lapse imaging using spinning disk 336 confocal microscopy to determine single actin filament dynamics (Figure 4A, see 337 Supplemental Movie 1 and Supplemental Movie 2). Several parameters of actin 338 filament dynamics in guard cells were significantly different between wild type and 339 the ckl2 mutant (Figure 4B). The average lifetime of single actin filaments in Col-0 340 was about 2-fold longer than that in ckl2 guard cells. Most of the single actin 341 filaments in Col-0 guard cells kept growing for more than 10 s before the first 342 severing event occurred. However, most of the single actin filaments kept growing 343 less than 8 s in ckl2 guard cells. In Col-0 guard cells, the severing rate was 0.027 ± 344 0.002 breaks μm-1 s-1 and a single actin filament elongated to an average maximum 345 filament length of 4.3 ± 0.2 μm. In the ckl2 mutant, however, the severing rate was 346 increased to 0.039 ± 0.05 breaks μm-1 s-1, and the average maximum actin filament 347 was 3.4 ± 0.2 μm. There was about a 1.5-fold increase in severing frequency in the 348 ckl2 mutant compared to wild type. These results demonstrate that loss of CKL2 led 349 to more severing events in the ckl2 guard cells, and suggest that CKL2 plays an 350 important role in controlling actin filament severing in guard cells. 351 352 CKL2 Interacts with and Phosphorylates ADF4 353 Our in vitro analyses indicated that CKL2 did not have any effect on the actin 354 cytoskeleton on its own; these results suggest that an actin-binding protein (ABP) 355 might mediate the regulatory effect of CKL2 on the actin cytoskeleton in vivo. 356 Furthermore, our findings regarding actin filament dynamics and severing in Col-0 357 and ckl2 guard cells suggest that an ABP that could sever actin filaments and be 358 regulated by phosphorylation might be relevant, especially considering that CKL2 is a 359 protein kinase. ADF family members function as important regulators of actin 15 360 dynamics via severing actin filaments and promoting monomer dissociation from the 361 pointed end of actin filaments, and phosphorylation of ADFs is critical for regulating 362 their activity. We reasoned that ADFs with similar expression patterns to CKL2 might 363 act as substrates for CKL2 and mediate the regulation of actin dynamics by CKL2. 364 ADF4 belongs to subclass I, members of which are expressed abundantly in all 365 vegetative tissues and reproductive tissues, and its expression pattern is similar to that 366 of CKL2 (Supplemental Figures 2A-2C, Ruzicka et al., 2007; Henty et al., 2011). 367 Both CKL2 and ADF4 are expressed in guard cells. However, the expression of CKL2 368 was induced by ABA treatment, whereas that of ADF4 was not (Supplemental Figure 369 5). The adf4 mutant displays different F-actin dynamic behavior from that of the wild 370 type, including decreased severing frequency and increased maximum filament length 371 and filament lifetime (Henty et al., 2011). We therefore selected ADF4 for further 16 372 analysis as a potential target of CKL2 kinase to mediate its regulatory effects on the 373 actin cytoskeleton. 374 First, we determined the interaction between ADF4 and CKL2 in vitro. His-CKL2 375 and GST-ADF4 recombinant proteins were therefore purified. Pull-down assays 376 showed that GST-ADF4 co-precipitated with His-CKL2 (Figure 5A), suggesting that 377 CKL2 interacts with ADF4. To determine which domain of CKL2 interacts with 378 ADF4, we fused the CKL2 N-terminal domain (catalytic domain, from 1 to 295 379 amino acids) and CKL2 C-terminal domain (regulatory domain, including 296 to 464 380 amino acids) to His tag, respectively, purified both recombinant proteins, and 381 performed pull-down assays with ADF4. GST-ADF4 co-precipitated with the 382 His-CKL2 N-terminus but not the C-terminus. Immunoblotting analyses further 383 confirmed that the full-length CKL2 and the N-terminus of CKL2 interacted with 384 ADF4 (Figure 5A). 385 To explore the interaction between CKL2 and ADF4 in vivo, we performed 386 co-immunoprecipitation assays in vivo. Anti-ADF4 antibodies were generated. They 387 recognized endogenous ADF1 and ADF4, but were not specific to ADF4 388 (Supplemental Figure 6A). For co-immunoprecipitation assays in vivo, total protein 389 was isolated from the transgenic plants expressing the construct 390 Pro35S:Flag-HA-CKL2. CKL2 was immunoprecipitated with anti-Flag antibody 391 conjugated agarose, and ADFs were detected in the pull-down products by anti-ADF 392 antibodies. PKS5, a cytoplasmic localized protein kinase (Fuglsang et al., 2007), was 393 used as a control and showed no interaction with ADFs (Supplemental Figure 6B). We 394 further used split-luciferase (split-LUC) complementation assays in Nicotiana 395 benthamiana to determine the interaction of ADF4 and CKL2 (Figure 5B). ADF4 was 396 fused to the C terminus of LUCIFERASE (ADF4-cLUC) and CKL2 was fused to the 397 N terminus of LUCIFERASE (nLUC-CKL2). The split-LUC assays showed that 398 transient co-expression of nLUC-CKL2 and ADF4-cLUC in N. benthamiana yielded 399 strong fluorescence signals, but no fluorescence signal was detected in the control 400 leaves co-expressing nLUC-CKL2 and cLUC or nLUC and ADF4-cLUC, which 401 further confirmed that CKL2 interacts with ADF4 in vivo (Figure 5B). In addition, 17 402 403 this interaction was induced by ABA treatment (Figure 5B). To detect whether ADF4 is a substrate of CKL2, we conducted in vitro kinase 404 assays using the His-CKL2 and His-ADF4 proteins. A phosphorylated ADF4 signal 405 was detected (Figure 5C). However, CKL2 did not phosphorylate the actin-binding 406 protein SCAB1 in vitro (Zhao et al., 2011), suggesting that phosphorylation of ADF4 407 by CKL2 is relatively specific. 18 408 To determine if CKL2 phosphorylates ADF4 in vivo, the construct 409 Pro35S:3Flag-ADF4 was used to transform Col-0 and the ckl2 mutant. Flag-ADF4 410 was immunoprecipitated with anti-Flag antibody-conjugated agarose, and eluted using 411 a Flag peptide. Equal amounts of the Flag-ADF4 protein were used for two dimension 412 (2D) immunoblotting assays with anti-Flag antibody to detect the phosphorylation 413 level of ADF4. Two ADF4 spots with different pI values were detected in the 2D gel 414 from both the wild type and the ckl2 mutant. Spot A, close to the low pI region, was 415 significantly decreased, and spot B, close to the high pI region, was increased in the 416 ckl2 mutant. When the wild-type sample was treated with Lambda Protein 417 Phosphatase, spot A almost disappeared (Figures 5D and 5E), suggesting that spot A 418 represented phosphorylated ADF4. These results suggest that ADF4 is phosphorylated 419 in plant cells and that CKL2 is required for this phosphorylation. 420 Arabidopsis ADF subclass I family proteins contain a conserved amino acid Ser6 421 (Supplemental Figure 6C). Phosphorylation at Ser6 has been observed in all tested 422 plant ADF subclass I family proteins (Allwood et al., 2001; Allwood et al., 2002; 423 Chen et al., 2002). To determine whether Ser6 is required for the phosphorylation by 424 CKL2, we generated a mutated form of ADF4 with Ser6 exchanged for Ala (ADF4S6A). 425 Recombinant proteins His-ADF4 and His-ADF4S6A were purified and used for the 426 kinase assay. The phosphorylation level of ADF4 by CKL2 was significantly reduced 427 by the mutation at Ser6 although this point mutation did not abolish phosphorylation 428 (Figures 5F and 5G). Taken together, our data suggest that CKL2 phosphorylates 429 ADF4 and that Ser6 is one of the phosphorylation sites. 430 Given that ADF proteins are highly conserved and the ADF family in Arabidopsis 431 consists of 11 members that are phylogenetically divided into four subclasses (Mun et 432 al., 2000; Ruzicka et al., 2007), we wondered whether CKL2 could phosphorylate 433 other ADFs besides ADF4 in Arabidopsis. To explore this, we selected other ADF 434 proteins from subclass I (ADF1-4) and found that all 4 ADFs in the subclass I were 435 phosphorylated by CKL2 (Supplemental Figure 6D), suggesting that at least the 436 subclass I ADFs are potential targets of CKL2 in Arabidopsis. 437 19 438 Phosphorylation of ADF4 by CKL2 Inhibits the Activities of ADF4 439 Phosphorylation of ADFs at the N terminal serine (Ser3 in animals and Ser6 in plants) 440 is conserved in both animal and plant cells, and plays an important role in regulating 441 ADF actin binding and disassembly activity (Ressad et al., 1998; Chen et al., 2002). 442 To further detect the effects of CKL2-mediated phosphorylation on the actin 443 disassembling activity of ADF4, we performed bulk actin disassembly assays and 444 found that CKL2-mediated phosphorylation decreased ADF4-induced actin 445 disassembly (Figure 6A). We next monitored the effects of the phosphorylation of 446 ADF4 by CKL2 on the actin filament-severing activity in real time using total internal 447 reflection fluorescence (TIRF) microscopy. In the absence of ADF proteins, few 448 breaks were observed along actin filaments (Figure 6Ba, Figure 6C and Supplemental 449 Movie 3). However, the addition of ADF4 resulted in increased filament breakage 450 (Figure 6Bb, Figure 6C and Supplemental Movie 4). By contrast, the addition of 451 ADF4 phosphorylated by CKL2 resulted in less filament breakage (Figure 6Bc, 452 Figure 6C and Supplemental Movie 5), suggesting that CKL2-mediated 453 phosphorylation inhibited ADF4-induced actin filament severing. In summary, our 454 results suggest that the CKL2-mediated phosphorylation on ADF4 reduces the F-actin 455 disassembling activity of ADF4. 456 457 Regulation of Stomatal Closure by CKL2 Is Partially Dependent on ADF4 458 To determine the genetic interaction between CKL2 and ADF4, we generated adf4 459 ckl2 double mutants by crossing ckl2 with an adf4 T-DNA insertion line, adf4-1 (Tian 460 et al., 2009). The wild type, adf4-1, adf4-2 (Salk_121647, obtained from the ABRC), 461 ckl2, and adf4-1 ckl2 mutants were used to monitor stomatal closure in response to 462 ABA treatment. RT-PCR results showed that the two adf4 homozygous lines are 463 knockout mutants (Supplemental Figure 7A). Stomatal closure in both adf4-1 and 464 adf4-2 was more sensitive to ABA than was the wild type (Figure 7A). Consistent 465 with our previous results (Figure 1C), the stomatal closure in ckl2 was insensitive to 466 ABA compared to the wild type (Figure 7A). The stomatal closure in the adf4-1 ckl2 467 double mutant in response to ABA was intermediate between that in the respective 20 468 single mutants (Figure 7A). Consistent with the results of stomatal closure 469 measurement, leaf temperature in the adf4 ckl2 double mutant was higher than that in 470 the ckl2 single mutant but lower than that in the wild type (Figure 7B and 7C). Both 471 adf4-1 and adf4-2 mutant plants displayed higher leaf temperatures than did the wild 472 type (Figures 7B and 7C). These results suggest that loss of function of ADF4 473 partially rescues the stomatal closure phenotype in ckl2. 474 To determine how CKL2 and ADF4 coordinately regulate the ABA-induced actin 475 dynamics in guard cells, we transformed the 35S:GFP-fABD2-GFP construct into the 21 476 adf4 and adf4 ckl2 double mutant. In widely opened guard cells, although the F-actin 477 in the adf4 mutant also displayed radially arranged filaments, the skewness value 478 increased and actin filament density value decreased significantly compared to these 479 in Col-0, suggesting that the extent of actin filament bundling increased in adf4 guard 22 480 cells (Figures 7D to 7F). However, in the adf4 ckl2 double mutant, the effect of ADF4 481 deletion on actin filaments was partially rescued by CKL2 deletion. The double 482 mutant showed an intermediate density between that of the adf4 and ckl2 mutants, 483 which was still lower than that in Col-0. The skewness in the adf4 ckl2 double mutant 484 showed no significant difference from Col-0 (Figures 7D to 7F). After treatment with 485 2 μM ABA for 0.5 h, the skewness further increased and the density further decreased 486 in closed stomata (69% of cell populations) of the adf4 mutant compared to these 487 before the treatment (Figures 7D to 7F). However, in stomata of the adf4 ckl2 double 488 mutant (75% of cell populations), both skewness and density displayed intermediate 489 values between those of the adf4 and ckl2 mutants after ABA treatment (Figure 7D to 490 7F). The skewness value of the double mutant was lower than that of Col-0 and the 491 density value was higher than that of Col-0. These results showed that deletion of 492 ADF4 partially rescued the ckl2 mutant F-actin reorganization phenotype and suggest 493 that loss of function of ADF4 at least partially accounts for the F-actin reorganization 494 phenotype in ckl2. 495 To further investigate the function of ADF4 in CKL2-mediated stomatal closure, 496 we generated ADF4 overexpression lines (35S:ADF4) in the Col-0 and ckl2 mutant 497 background. There was no significant difference in ABA-induced stomatal closure 498 between the Col-0 and ckl2 mutant upon overexpressing ADF4. Moreover, the plants 499 overexpressing ADF4 in both the Col-0 and the ckl2 mutant backgrounds were even 500 less sensitive than the ckl2 mutant plants in terms of ABA-induced stomatal closure 501 (Figure 7G). These results further suggest that ADF4 interacts with CKL2 in 502 regulating stomatal closure in response to ABA. 503 504 23 505 DISCUSSION 506 507 In this study, we found that an ABA-responsive actin filament-associated protein 508 kinase, CKL2, regulates actin dynamics during stomatal closure, presumably via 509 phosphorylating ADF proteins. CKL2 therefore emerges as an important player in 510 regulating ABA-mediated stomatal closure. CKL2 and other factors, including 511 receptor-like kinase (GHR), protein kinases (CDPK, SnRK2.2, 2.3 and 2.6), 512 transcription factors (ABI3, 4 and 5), and ion channel SLACs (Desikan et al., 2004; 513 Israelsson et al., 2006; Kim et al., 2010; Hua et al., 2012), constitute a sophisticated 514 regulatory network for ABA-mediated stomatal closure. Our study adds another 515 important component to the ABA signaling network in guard cells and enriches our 516 understanding of the regulation of stomatal closure. 517 In guard cells, actin filaments undergo dynamic reorganization, which is 518 important for proper stomatal closure (Kim et al., 1995; Eun and Lee, 1997; Hwang 519 and Lee, 2001; MacRobbie and Kurup, 2007; Zhao et al., 2011; Jiang et al., 2012). 520 However, how the reorganization of the actin cytoskeleton is achieved during stomatal 521 closure remains poorly understood. Two stages are involved in this reorganization, 522 with actin filament disassembly followed by filament reassembly when stomata close 523 (Eun and Lee, 1997, 2000). Therefore, actin filament destabilization (for disassembly) 524 and stabilization (for reassembly) are both necessary aspects of this process. Our 525 present work shows that a lack of CKL2 results in the blockage of actin filament 526 reassembly and the actin filaments remain in a disrupted state. These results suggest 527 that CKL2 has an effect on stabilization of actin filaments, and thus, that CKL2 528 functions in the reassembly of actin filaments during stomatal closure. 529 ADFs bind to and sever actin filaments, thereby acting as major regulators of 530 F-actin dynamics (Henty et al., 2011; Zheng et al., 2013) implicated in regulating 531 F-actin reorganization during stomatal closure and opening (Dong et al., 2001). 532 Precise regulation of ADF activity is required for the correct balance between F-actin 533 assembly and disassembly (Ressad et al., 1998; Bamburg, 1999). Exactly how ADFs 534 regulate F-actin reorganization and how the activity of ADFs is fine-tuned to meet the 24 535 cellular demands during stomatal closure and opening are interesting topics. Based on 536 our experimental results, we propose a brief model to explain how CKL2 stabilizes 537 actin filaments to promote actin reassembly during ABA-induced stomatal closure 538 (Figure 8). When ABA/drought-induced stomatal closure initiates, actin filaments 539 begin to disassemble, which requires higher ADF activity. During this stage, CKL2 is 540 accumulated due to up-regulation by drought and ABA. The increased CKL2 level 541 results in more phosphorylated ADF molecules, which in turn reduces their actin 542 filament-binding and -severing activity, leading to actin arrays being reorganized into 543 highly bundled long cables. The resulting changes in actin filament architecture 544 promote stomatal closure. Furthermore, the stabilization of actin filaments would be 545 maintained in the continued presence of ABA so that the stomata would remain 546 closed. 547 Given that ADFs play an important role in actin filament disassembly during 548 stomatal closure, how is their activity regulated at the early stage of ABA/drought 549 response and reactivated after the action of CKL2? It is possible that a phosphatase(s) 550 is critical at this stage by either activating ADFs or deactivating CKL2. PP2C is 551 thought to be involved in actin reorganization in guard cells during ABA-induced 552 stomatal closure. The ABA-insensitive mutant abi1-1 (a PP2C mutant) displays 553 disturbed actin filament reorganization in guard cells when treated with ABA (Eun et 554 al., 2001; Hwang and Lee, 2001; Lemichez et al., 2001). How PP2C cooperates with 555 CKL2/ADFs to regulate actin dynamics in guard cells remains an interesting question. 25 556 CKL2 associates with actin filaments, but does not directly bind to the actin 557 filaments. We found that ABA induces the interaction of CKL2 with ADF4. Perhaps 558 the interaction between CKL2 and ADFs not only protects the actin filaments from 559 severing by ADFs, but also plays a role in recruiting CKL2 to the actin filaments. Our 560 study suggests that Ser6 in ADF4 is one of the residues phosphorylated by CKL2; the 561 other phosphorylated residues may play roles in regulation at different levels and 562 stages for the full function of ADF in modulating Arabidopsis stomatal closure. 563 The essential role of actin dynamics in regulating stomatal closure and opening is 564 well appreciated (Kim et al., 1995; Eun and Lee, 1997; Hwang and Lee, 2001; 565 MacRobbie and Kurup, 2007; Zhao et al., 2011; Jiang et al., 2012; Li et al., 2014), but 566 how actin dynamics are functionally coupled to the underlying cellular processes in 567 guard cells remains poorly understood. For instance, actin dynamics have been 568 implicated in mediating Ca2+ concentration changes in the cytosol, K+ channel activity, 569 and vacuole shape determination (Hwang et al., 1997; Gao et al., 2009; Zhao et al., 570 2013), but the related molecular mechanisms remain to be established. One possibility 571 is regulation of membrane recycling by actin reorganization (Cheung et al., 2002; Lee 572 et al., 2008; Zhao et al., 2010; Bou et al., 2011; Zhu et al., 2013), which would change 573 the localization of ion channels/transporters and the area of vacuolar membrane. Actin 574 dynamics may also act as an important component in signal transduction to mediate 575 such changes (Gao et al., 2008; Zhao et al., 2011; Jiang et al., 2012; Zhao et al., 576 2013). 577 578 Materials and Methods 579 Plant Materials and Growth Conditions 580 Arabidopsis thaliana Col-0 was used as the wild type. The ckl2 mutant (Salk_104209) 581 was obtained from ABRC. 582 Primers used to confirm the homozygous mutant lines are listed in Supplemental 583 Table 1. Arabidopsis seedlings were grown in soil under 8 h light (light intensity of 30 584 µmol m–2s–1)/16 h darkness (short-day conditions) at 23°C and 60% relative humidity 585 (RH) for 3-4 weeks. Nicotiana benthamiana plants were grown in soil under 16 h 26 586 light/8 h darkness (long-day conditions) at 23°C and 60% relative humidity for 3-4 587 weeks. 588 589 RNA Extraction and Real-Time Quantitative RT-PCR Analysis 590 Total RNA was extracted from 7-d-old seedlings grown on MS medium or the roots, 591 stems, cauline leaves, rosettes, flowers, or siliques of 4-week-old plants grown in soil 592 with Trizol reagent (Invitrogen). Total RNA treated with RNase-free DNase I 593 (Invitrogen) was used for reverse transcription with M-MLV reverse transcriptase 594 (Promega). Real-time quantitative PCR was conducted using SYBR Premix Ex Taq 595 (Takara). Elongation factor 1α (EF) was used as an internal control. The primers used 596 for RT-PCR or Real-time quantitative PCR are listed in Supplemental Table 1. 597 598 Water-Loss and Stomatal Aperture Assays 599 To detect the stomatal closure in response to ABA treatment, rosette leaves of 600 5-week-old plants were detached and incubated in stomata-opening buffer (containing 601 50 mM KCl, 10 μM CaCl2, and 10 mM MES, pH 6.15) in a growth chamber at 23°C 602 under constant illumination. Stomatal apertures were measured after adding 2 μM 603 ABA for 0.5 h. The apertures of 50 stomata were measured in three independent 604 experiments. 605 606 For water-loss measurement, rosette leaves were detached from 5-week-old plants. The weight of the detached leaves was measured every 0.5 h. 607 608 Infrared Thermograph Imaging 609 To monitor the leaf temperature, thermal imaging was performed as described 610 previously with slight modification (Xie et al., 2006). Well-watered 3-week-old plants 611 were transferred from a high humidity growth condition (RH 70%) to a low humidity 612 condition (RH 40%). The leaf temperature was recorded by a thermal imaging camera 613 after 3 days. 614 615 Subcellular Localization Assays 27 616 For subcellular localization analysis of GFP-CKL2, the CKL2 cDNA coding region 617 was amplified and cloned into the pCambia1205 vector (Zhao et al., 2011) between 618 the BamHI and EcoRI sites to generate Pro35S:GFP-CKL2. For the 619 ProCKL2:GFP-CKL2 construct, the 1.06-kb CKL2 promoter fragment was amplified 620 from BAC plasmid F18A22 (ABRC) and cloned into the pCAMBIA1305 vector 621 (Zhao et al., 2011) between the HindⅢ and PstΙ sites, and then the CKL2 cDNA 622 coding region was amplified and cloned between the SalΙ and BamHΙ sites. The 623 resulting Pro35S:GFP-CKL2 and ProCKL2:GFP-CKL2 vectors were used to 624 transform Col-0 and the ckl2 mutant, respectively. The confocal images were taken 625 from 6-day-old seedlings of the transgenic plants with a Zeiss LSM 510 Meta 626 confocal microscope using a Plan-Apochromat 63 ×/ 1.4 oil immersion differential 627 interference contrast lens. GFP was excited at 488 nm. 628 629 Preparation of Suspension Cells 630 For suspension cell preparation, seedlings were grown on MS medium (4.43 g/L 631 Murashige and Skoog salt, 30 g/L sucrose, and 0.3% agar) for 7 days. The leaves were 632 then cut from the seedlings and cultured on MS medium with 1 mg/L 2,4-D and 0.1 633 mg/L 6-benzylaminopurine to induce calli. The calli were cultured in the MS liquid 634 medium with 1 mg/L 2,4-D on a rotor set to 120 rpm in the dark at 23°C to induce 635 suspension cells. The suspension cells were subcultured in darkness for 3 weeks and 636 then used for actin staining assays. 637 638 Co-localization Analysis 639 For co-localization analysis, suspension cells generated from the 35Sp:GFP-CKL2 640 transgenic plants were incubated in actin staining buffer (0.18 μM 641 rhodamine-phalloidin, 100 mM PIPES, 10 mM EGTA, 5 mM MgSO4, 5% DMSO, 642 and 0.05% Nonidet P-40, pH 6.8) for 15 min at room temperature. The samples were 643 observed by confocal microscopy. The images were collected through visualizing the 644 red/green fluorescence signals from the GFP-CKL2 and the 645 rhodamine-phalloidin-labeled actin filaments. The colocalization between GFP-CKL2 28 646 and the actin cytoskeleton was analyzed by calculating Pearson’s correlation 647 coefficient (Dunn et al., 2011; Wu et al., 2012; Mcdonald et al., 2013). Relative pixel 648 intensity in the indicated regions of interest was measured using Image J software. 649 Thresholds were set manually to account for background, and Pearson’s correlation 650 coefficient was calculated to determine the correlation degree. 651 652 Laser-Scanning Confocal Microscopy to Visualize Actin Filaments in Vivo 653 To visualize actin filaments in plant cells, the Pro35S:GFP-fABD2-GFP construct 654 was transformed into wild type, the ckl2 mutant, the adf4-1 mutant, and the adf4 ckl2 655 double mutant. Six-day-old seedlings of the transgenic lines grown on MS medium 656 were used for actin filament visualization in hypocotyl cells and roots. 657 Three-week-old plants were used for actin filament visualization in guard cells. To 658 detect the effect of Lat A or ABA on actin filaments, the transgenic plants were treated 659 with 200 nm Lat A or 20 μM ABA for 0.5 h or 1 h. Then the actin cytoskeleton was 660 visualized by detecting the GFP fluorescence signal in hypocotyl cells or guard cells 661 with a Zeiss LSM 510 META confocal microscope using a Plan-Apochromat 63 ×/ 662 1.4 oil immersion differential interference contrast lens. GFP was excited at 488 nm. 663 To observe actin filament dynamics in guard cells, time-lapse images were 664 captured every 2 s with an Andor iXon charge-coupled device camera. To observe 665 actin filament dynamic behavior, parameters including maximum filament lifetime, 666 maximum filament length, elongation rate, severing frequency, and depolymerization 667 rate with single actin filament turnover were calculated using Image J software as 668 described previously (Henty et al., 2011; Qin et al., 2014). 669 670 Antibody Preparation and Immunoblotting 671 Anti-ADF4 antibodies were generated by immunizing mice with E. coli-expressed 672 ADF4. To determine the specificity of the antibodies in plants, total protein was 673 isolated from the 7-d-old Col-0, the adf4 mutant (SALK_121647), the adf1 mutant 674 (SALK_144459), and the transgenic plants harboring the construct 675 Pro35S:3×Flag-ADF4 or Pro35S:3×Flag-ADF1, and subjected to immunoblot 29 676 analysis with anti-ADF4 antibodies. The blots were probed with primary mouse 677 anti-ADF4 (diluted 1:1000). 678 679 Protein Purification and Pull-Down assays 680 The coding regions of CKL2 and ADF4 were amplified and cloned into the vectors of 681 PET28a and pGEX-6p-1, respectively. The N-terminus of CKL2 (295 amino acids, 682 CKL2N) and the C-terminus of CKL2 (170 amino acids, CKL2C) were cloned into 683 the PET28a vector. The resulting vectors were transformed into E. coli (strain BL21). 684 The recombinant proteins were purified with Ni-NTA agarose or glutathione 685 sepharose. 686 For the pull-down assay, 5 μg His-CKL2, -CKL2N or -CKL2C on Ni-NTA 687 agarose was incubated with 1 μg GST-ADF4 for 30 min at 4°C in 100 μL binding 688 buffer (20 mM Tris-HCl, pH 7.2, 10 mM MgCl2, and 2 mM DTT). After washing 689 three times with the binding buffer, protein on the agarose was separated on 15% 690 SDS-PAGE, followed by Commassie Brilliant Blue R250 staining. One microliter of 691 agarose was analyzed by immunoblot using anti-ADF antibodies. 692 For in vivo coimmunoprecipitation assays, the CKL2 coding region was amplified 693 and cloned into the pCM1307-N-Flag-HA vector between the XbaI and SalI sites. The 694 resulting plasmid was transformed into wild-type plants. Total protein was extracted 695 from 7-d-old transgenic plants using 2 mL immunoprecipitation buffer (10 mM Tris, 696 pH 7.5, 0.5% Nonidet P-40, 2 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 1% 697 protease inhibitor cocktail [Sigma-Aldrich]). Flag-HA-CKL2 was purified with 30 μL 698 anti-FLAG agarose (Sigma-Aldrich) from the total protein. After washing three times 699 with 2 mL immunoprecipitation buffer, the agarose was used for immunoblot assays 700 with anti-ADFs antibodies. 701 702 Split-Luciferase Complementation Assays 703 For the split luciferase assays, the CKL2 and ADF4 cDNA coding regions were 704 amplified and cloned into the KpnI and SalI sites of the pCM1307-nLUC and 705 pCM1307-cLUC vector, respectively. The plasmids were introduced into 30 706 Agrobacterium tumefaciens GV3101 and co-infiltrated into the leaves of Nicotiana 707 benthamiana. After a 3-d incubation, the LUC activity was measured using a cooled 708 CCD imaging camera (1300B; Roper). Then, 1 mM luciferin was sprayed onto the 709 leaves. Relative LUC activity per cm2 infiltrated leaf area was calculated using 710 Winview32 software. Each data column contains at least ten replicates, and three 711 independent experiments were carried out. 712 713 Kinase Activity Assays 714 In vitro kinase activity assays were carried out as described previously (Quan et al., 715 2007). Recombinant protein His-CKL2, His-ADF4, and His-ADF4S6A were purified 716 with Ni-NTA agarose. One microgram His-ADF4 or His-ADF4S6A was incubated 717 with 1 μg His-CKL2 in a kinase reaction buffer (20 mM Tris-HCl, pH 8.0, 5 mM 718 MgCl2, 10 μM ATP, 1 mM DTT and 2 μCi [γ-32P]ATP) in 15 μL total volume at 30°C 719 for 30 min. The reaction was terminated with 2×SDS loading buffer. After incubation 720 at 100°C for 5 min, the reaction products were separated on 15% SDS-PAGE and 721 stained using Coomassie Brilliant Blue R250. Then the SDS-PAGE gels were exposed 722 to a phosphor screen. The phosphor-signals were detected by a Typhoon 9410 723 phosphor imager (Amersham Biosciences). Phosphorylation signals were quantified 724 using Image Quant 5.0 software. 725 Two-dimensional SDS-PAGE was performed as described (Minamide et al., 1997; 726 Dong et al., 2001). The Pro35S:3× Flag-ADF4 construct was transformed into the 727 wild type and ckl2 mutant, respectively. Total protein was isolated from the resulting 728 transgenic seedlings in extraction buffer (10 mM Tris, pH 7.5, 0.5% Nonidet P-40, 2 729 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 1% protease inhibitor cocktail 730 [Sigma-Aldrich]). ADF4 protein was immunoprecipitated from the total proteins by 731 incubation with anti-Flag agarose. For phosphatase treatment, the ADF4 protein was 732 incubated with phosphatase (λ phosphatase, New England BioLabs) at 30°C for 15 733 min. Immunoblotting of ADF4 was performed with an anti-Flag antibody. 734 735 Quantification of GFP-fABD2-GFP Fluorescence Pixel Intensity, Density and 31 736 Skewness of the Actin Filaments in Guard Cells 737 Measurement of relative fluorescence pixel intensity levels of 738 GFP-fABD2-GFP-labeled actin filaments in guard cells and hypocotyl epidermal cells 739 was carried out as described previously (Huang et al., 2005). The average 740 fluorescence pixel intensity associated with GFP-fABD2-GFP was measured using 741 ImageJ software (http://rsb.info.nih.gov/ij/) to determine the relative amount of actin 742 filaments. The skewness and density of actin filaments were quantified by ImageJ 743 according to Higaki et al. (2010) with slight modification. Individual cells were 744 segmented manually and actin filaments at the cell border were eliminated. More than 745 50 guard cells were used for the analysis. 746 747 Biochemical Assays to Determine the Effect of CKL2 on Actin Polymerization 748 High-speed co-sedimentation assays were performed to examine the binding of CKL2 749 to actin filaments as previously described (Kovar et al., 2000; Huang et al., 2005). 750 Direct visualization of actin filaments by epifluorescence light microscopy was 751 performed as described previously (Okada et al., 2002). A spontaneous actin 752 nucleation assay was performed according to previously published methods (Amann 753 and Pollard, 2001; Huang et al., 2003; Andrianantoandro et al., 2006). Various 754 concentrations of CKL2 were incubated with G-actin (2 μM 10% pyrene-labeled) for 755 5 min at room temperature. Actin polymerization was initiated after the addition of 756 1/10 volume of 10 × KMEI (500 mM KCl, 10 mM MgCl2, 10 mM EGTA, and 100 757 mM imidazole-HCl, pH 7.0). Pyrene fluorescence was monitored using a 758 QuantaMaster Luminescence QM 3 PH Fluorometer (Photo Technology International) 759 with the excitation and emission wavelength set at 365 nm and 407 nm, respectively. 760 761 Determination of the Effect of CKL2 Phosphorylation on ADF4-Mediated Actin 762 Disassembly in Vitro 763 All proteins and buffers were pre-clarified by centrifugation at 200,000 g for 1 h at 764 4°C. Equal molar amounts of ADF4 and CKL2 were used for the phosphorylation 765 reaction as described above. G-actin (2 μM, 10% pyrene labeled) was polymerized at 32 766 25°C for 2 h in polymerization buffer (described above). The resulting assembled 767 actin filaments from 500 nM G-actin were then incubated with 500 nM 768 non-phosphorylated ADF4 or 500 nM CKL2-phosphorylated ADF4 in 1 × KMEI to 769 induce actin filament disassembly. Actin disassembly was traced by monitoring the 770 decrease in pyrene fluorescence for 30 min using a QuantaMaster Luminescence QM 771 3 PH Fluorometer (Photon Technology International) with the excitation and emission 772 wavelengths set at 365 nm and 407 nm, respectively. 773 774 Direct Visualization of Actin Filament Severing in Vitro by Total Internal 775 Reflection Fluorescence (TIRF) Microscopy 776 Single actin filament severing was directly visualized by TIRF microscopy as 777 described previously (Amann and Pollard, 2001; Andrianantoandro et al., 2006; 778 Jansen et al., 2014). The flow chamber was prepared as described previously (Amann 779 and Pollard, 2001; Jansen et al., 2014). For actin-filament-severing assays, the 780 assembled flow chamber was incubated with 25 nM N-ethylmaleimide-myosin for 5 781 min followed by washing with 1% BSA for 3 min. Next, TIRF microscopy buffer (10 782 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 50 mM DTT, 0.2 783 mM ATP, 50 mM CaCl2, 15 mM glucose, 20 mg/mL catalase, 100 mg/mL glucose 784 oxidase, and 0.5% methylcellulose) was injected into the prepared flow chamber. 785 Actin filaments (from 1 μM G-actin, 50% Oregon-Green labeled) in TIRF buffer were 786 injected into the chamber and incubated in darkness for 5 min. Finally, TIRF buffer 787 was injected into the chamber to remove attached actin filaments. After injection of 788 ADF4 or CKL2-phosphorylated ADF4 into the flow chamber, single actin filament 789 severing events were observed by time-lapse TIRF microscopy using an Olympus 790 IX81 microscope equipped with a ×100 oil objective (1.49 numerical aperture). 791 Time-lapse images were recorded every 3 s for 5 min. Actin filament severing 792 frequency was calculated as the maximum filament length divided by the number of 793 breaks per filament length over time (breaks/μm/s). More than 30 actin filaments with 794 length > 10 μm were selected for quantification under each condition. 795 33 796 Statistical Analysis 797 To test the data normality of continuous variables, statistical analysis was performed 798 using the SPSS for Windows software package (ver. 11.5, IBM Corp, Armonk, NY, 799 USA), and the Shapiro-Wilk test was applied. Relative values are means ± SD or 800 means ± SE. All statistical analysis was performed using two-tailed Student’s t-test to 801 determine group differences in means using GRAPHPAD PRISM 6.0 Software (San 802 Diego, California, USA) or Kaleida Graph 4.1 (Synergy Software, Reading, PA, 803 USA). Significant differences were indicated by different lowercase letters and the 804 threshold was set at 0.01. 805 806 Accession Numbers 807 Sequence data in this study can be found in the Arabidopsis Genome Initiative 808 database under the following accession numbers: CKL2, At1g72710; ADF1, 809 At3g46010; ADF4, At5g59890; EF1α, At5g60390; RD29A, At5g52310; SCaBP8, 810 At4g33000. 811 812 Supplemental Data 813 Supplemental Figure 1. Identification of the T-DNA Insertion in the ckl2 Mutant. 814 Supplemental Figure 2. Expression of CKL2. 815 Supplemental Figure 3. CKL2 Co-localizes with Actin Filaments. 816 Supplemental Figure 4. CKL2 Does Not Bind to F-actin Directly and Has No Effect 817 on Actin Polymerization in Vitro. 818 Supplemental Figure 5. CKL2 and ADF4 Expression in Guard Cells. 819 Supplemental Figure 6. CKL2 Interacts with ADF4. 820 Supplemental Figure 7. Analysis of ADF Protein Level in Transgenic Seedlings. 821 Supplemental Table 1. Primers used in this study. 822 Supplemental Movie 1. Time-series movie displaying the severing events of single 823 filaments in Col-0 guard cell. 824 Supplemental Movie 2. Time-series movie displaying the severing events of single 825 filaments in the ckl2 mutant guard cell. 34 826 Supplemental Movie 3. Time-series movie of actin filament severing in the absence of 827 ADF4. 828 Supplemental Movie 4. Time-series movie of actin filament severing in the presence 829 of non-phosphorylated ADF4. 830 Supplemental Movie 5. Time-series movie of actin filament severing in the presence 831 of CKL2-phosphorylated ADF4. 832 833 Supplemental Movie Legends. 834 ACKNOWLEDGEMENTS 835 We thank Christopher J. Staiger (Department of Biological Sciences, Purdue 836 University) for kindly providing the Arabidopsis seeds of adf4-1 837 (GARLIC_823_A11.b.1b.Lb3Fa), 35Sp:ADF4; adf4, and ABRC for ckl2 838 (Salk_104209) and adf4-2 seeds. We thank Dr. Nancy Hofmann at Plant Editors for 839 English editing. This work was supported by the National Basic Research Program of 840 China (Grant 2015CB910202 to YG), National Natural Science Foundation of China 841 (Grant 31430012 to YG) and Foundation for Innovative Research Group of the 842 National Natural Science Foundation of China (Grant 31121002). 843 844 AUTHOR CONTRIBUTIONS 845 SS.Z., YX. J. and Y.Z. performed the research. SS.Z., YX.Z. and Y.G. designed the 846 research and analyzed the data. Y.Z. contributed to the purification of recombinant 847 proteins. YX. J. and SJ.H. performed the research on analysis of single actin filament 848 severing. SS.Z., Y.G., SJ.H., M.Y., and YX.Z. contributed to the discussion and wrote 849 the article. 35 850 36 851 Figure Legends 852 Figure 1. The ckl2 Mutant Shows Impaired Stomatal Closure. 853 (A) Leaves detached from wild-type and ckl2 plants for 0 (left) and 3 h (right) of 854 water loss treatment. 855 (B) Cumulative leaf transpirational water loss in Col-0, ckl2, and two rescued lines 856 (com1 and com2) at the indicated times after detachment (means ± SD, n = 3). 857 (C) Stomatal bioassays for ABA-induced closure in Col-0, ckl2, and two rescued lines 858 (com1 and com2). The data represent the means ± SD of three independent 859 experiments; 50 stomata were analyzed per line. The data sets were tested as normal 860 distribution by the Shapiro-Wilk test. Statistical significance was determined by 861 Student’s t-test; significant differences are indicated by different lowercase letters. 862 The t-test analysis of the data indicates the levels of significance to be P = 0.0034, 863 0.9986, and 0.9347 for ckl2 and the two rescued lines, respectively, compared with 864 Col-0 after ABA treatment. Before ABA treatment, the levels of significance were P = 865 0.8075, 0.2705, and 0.6197 for ckl2 and the two rescued lines, respectively, compared 866 with Col-0. 867 (D) Representative pseudocolored infrared images of leaf temperature of Col-0 and 868 ckl2 mutant plants. 869 (E) Leaf (surface) temperature of Col-0 and ckl2 mutant plants measured from images 870 obtained by infrared thermography, as in (D), and analyzed by InfraTec GmbH 871 reporter software. Twenty leaves were analyzed per line. Data are means ± SD (n = 3). 872 The data sets were tested for normal distribution by Shapiro-Wilk test. Statistical 873 significance (**P < 0.01) was determined by Student’s t-test. 874 (F) and (G) Real time PCR analysis revealed the induced expression of CKL2 by 875 ABA treatment or water loss treatment. Seven-day-old seedlings were treated with 20 876 μM ABA for 0.5 h or 1 h (F), or were treated for water loss until leaves had lost 20% 877 of their fresh weight (G). RD29A and SCaBP8 were used as controls. Expression 878 levels of CKL2, RD29A and SCaBP8 without ABA/water loss treatment were set as 879 1.0, respectively. The experiments were repeated three times. Data are means ± SD. 880 Statistical significance (**P < 0.01 and *P < 0.05) was determined by Student’s t-test. 37 881 t-test analysis of the data shown in (F) indicates the level of significance to be P = 882 0.0042, and 0.0023 for the data of CKL2 relative mRNA level at 0.5 h and 1 h after 883 ABA treatment, respectively, compared with the control. The positive control RD29A 884 also had a higher relative mRNA level when treated with ABA for 0.5 h (P = 0.0033) 885 and 1 h (P = 0.0012) compared with the control. The negative control SCaBP8 886 showed no obvious difference when treated with ABA for 0.5 h (P = 0.6890) and 1 h 887 (0.7891) compared with the control. As shown in (G), CKL2 was induced by water 888 loss treatment (P = 0.0066). The positive control RD29A also had a higher relative 889 mRNA level when treated with water loss until seedlings lost 20% of their fresh 890 weight (P = 0.0225). As a negative control, the relative mRNA level of SCaBP8 was 891 not significantly different when treated with water loss until seedlings lost 20% of 892 their fresh weight (P = 0.5851) compared with the control. 893 894 Figure 2. Localization of GFP-CKL2 Expressed from the Native CKL2 Promoter. 895 (A) Stomatal bioassays for ABA-induced closure in Col-0, ckl2, and two 896 ProCKL2:GFP-CKL2 transgenic lines. The data represent the means ± SD of three 897 independent experiments; 50 stomata were analyzed per line. The data sets were 898 tested for normal distribution by the Shapiro-Wilk test. Statistical significance was 899 determined by Student’s t-test; significant differences are indicated by different 900 lowercase letters. The t-test analysis of the data indicated the levels of significance to 901 be P = 0.0024, 0.8673, and 0.8358 for ckl2, and two rescued lines data, respectively 902 compared with Col-0 after ABA treatment. Before ABA treatment, the levels of 903 significance were P = 0.9023, 0.3251, and 0.6601 for ckl2, and two rescued lines, 904 respectively, compared with Col-0. 905 (B) Confocal images were taken of epidermal cells of hypocotyls (a), leaves (b), 906 guard cells (c), roots (d), and root hairs (e) of ProCKL2:GFP-CKL2 transgenic 907 seedlings in the Col-0 background. Bars = 10 μm. 908 (C) GFP-CKL2 driven by the CKL2 native promoter and GFP-fABD2-GFP 909 transgenic seedlings were treated with 200 nM LatA for 0.5 h. Confocal images were 910 taken of guard cells. Bars = 10 μm. 38 911 912 Figure 3. CKL2 Stabilizes Actin Filaments in Guard Cells. 913 (A) Actin filament organization in guard cells of Col-0 and ckl2 914 35Sp:GFP-fABD2-GFP transgenic plants before (right) and after (left) 200 nM LatA 915 treatment for 30 min. 916 (B) Quantification of the relative average fluorescence pixel density of GFP-fABD2 917 signal in guard cells as shown in (A). After LatA treatment, Col-0 and ckl2 mutant had 918 lower relative average fluorescence pixel density of GFP-fABD2 signal compared 919 with control (P = 0.0204, 0.0037). 920 (C) Actin filament organization in guard cells of Col-0 and ckl2 921 35Sp:GFP-fABD2-GFP transgenic plants before (right) and after (left) 2 μM ABA 922 treatment for 0.5 h. 923 (D) The extent of filament bundling (skewness) of guard cells shown in (C). The ckl2 924 mutant had significantly increased average actin filament density compared to Col-0 925 after ABA treatment (P = 0.0029). The ckl2 mutant had significantly decreased 926 average actin filament density than Col-0 before ABA treatment (P = 0.0056). 927 (E) Average filament density of Col-0 and ckl2 guard cells before and after 2 μM 928 ABA treatment as shown in (C). ckl2 mutant had significantly decreased average actin 929 filament skewness values compared to Col-0 after ABA treatment (P = 0.0068). No 930 significant difference of average actin filament skewness values between Col-0 and 931 ckl2 mutant was observed before ABA treatment (P = 0.0993). 932 Values of (B), (D) and (E) represent the means ± SD of three independent 933 experiments; 50 stomata were analyzed per line. The data sets were tested for normal 934 distribution by the Shapiro-Wilk test. Statistical significance was determined by 935 Student’s t-test. Significant differences are denoted with asterisks (**P < 0.01 and *P 936 < 0.05) in (B). Significant differences (P < 0.01) are indicated by different lowercase 937 letters in (D) and (E). 938 939 Figure 4. The ckl2 Mutant Shows Different Actin Dynamics from the Wild Type. 940 (A) Time-lapse images of single actin filaments in guard cells of Col-0 and ckl2 39 941 35Sp:GFP-fABD2-GFP transgenic plants. Green dots highlight a representative single 942 actin filament. Yellow arrows indicate actin filament-severing events. White arrows 943 indicate the position at which growth of the single actin filament began. Bars = 5 μm. 944 (B) Actin filament dynamic parameters in Col-0 and ckl2. The parameters associated 945 with single actin filament dynamics in Col-0 and ckl2 guard cells were quantified 946 from spinning disk confocal micrographs. Values represent means ± SD, n = 30. The 947 data sets were tested for normal distribution by the Shapiro-Wilk test. Statistical 948 significance was determined by Student’s t-test. Quantification of *P < 0.05 and **P < 949 0.01. 950 951 Figure 5. CKL2 Interacts with and Phosphorylates ADF4. 952 (A) CKL2 interacts with ADF4 in pull-down assays. Equal amounts of 953 affinity-purified His-CKL2 (left first panel), His-CKL2N (second panel), or 954 His-CKL2C fusion protein (third panel) were incubated with GST-ADF4. The input 955 and output proteins were stained with Coomassie Blue R250 on a SDS-PAGE gel. The 956 output proteins were also subject to immunoblot assay with anti-ADF antibodies 957 (right panel). The asterisks indicate the GST-ADF4 bands. 958 (B) Split-luciferase complementation imaging assays in Nicotiana benthamiana. 959 Quantitative analysis of luminescence intensity was determined. Relative values are 960 mean ± SD, n = 3. Higher luminescence intensity was observed after ABA treatment 961 compared with control (denoted by asterisk, P = 0.0022, t test). 962 (C) CKL2 phosphorylates ADF4 in vitro. The input proteins His-CKL2 and 963 His-ADF4 were detected by Coomassie blue staining (left). Phosphorylation activity 964 was detected by [γ-32P] ATP autoradiography (right). 965 (D) Two-dimensional immunoblotting. The Flag-ADF4 protein was 966 immunoprecipitated with anti-Flag agarose from Col-0 or ckl2 mutant plants. Equal 967 amounts of Flag-ADF4 protein immunoprecipitated from Col-0 were treated with λ 968 phosphatase as a control. Anti-Flag antibody was used for immunoblot assays. 969 Arrowheads point to the location of the more acidic ADF4 spot, representing 970 phosphorylated ADF4. Experiments were repeated three times. 40 971 (E) Quantification of relative ADF4 phosphorylation level in (D). Values represent 972 mean ± SD, n = 3. Statistical significance was determined by Student’s t-test; 973 significant differences (P < 0.05) are indicated with asterisks. The ckl2 mutant had a 974 lower relative ADF4 phosphorylation level compared with control (P = 0.0258). 975 (F) ADF4 Ser6 is important for the phosphorylation of CKL2. Purified His-ADF4 and 976 His-ADF4S6A as substrates were phosphorylated in the in vitro kinase assays. 977 (G) Quantification of relative ADF4 phosphorylation level in (F). Values represent 978 mean ± SD, n = 3. The data sets were tested for normal distribution by the 979 Shapiro-Wilk test. Statistical significance was determined by Student’s t-test; 980 significant differences (P < 0.01) are indicated with asterisks. ADF4 Ser6 mutation led 981 to a lower relative phosphorylation level compared with wild type (P = 0.0091). 982 983 Figure 6. Phosphorylation of ADF4 by CKL2 Affects ADF4 Activity. 984 (A) The effect of ADF4 on F-actin disassembly was determined by a pyrene-actin 985 assay. ADF4, after being phosphorylated by CKL2, was able to depolymerize actin 986 filaments but was less potent than non-phosphorylated ADF4. Preassembled actin 987 filaments (from 2 µM G-actin, 10% pyrene-labeled) were incubated with 250 nM 988 ADF4 or 250 nM CKL2-phosphorylated ADF4 to induce actin disassembly at pH 7.0. 989 Black closed circles, 0.5 µM F-actin; green closed squares, 0.5 µM F-actin + 4 µM 990 ADF4; red closed diamonds, 0.5 µM F-actin + 4 µM ADF4 after phosphorylation by 991 CKL2 for 0.5 h; blue closed triangles, 0.5 µM F-actin + 4 µM ADF4 after 992 phosphorylation by CKL2 for 1.5 h. a.u., arbitrary units. 993 (B) Time-lapse TIRF microscopy analysis of actin filament severing by ADF4 after 994 being phosphorylated by CKL2. Time-lapse images were recorded at 3-s intervals 995 with TIRF microscopy. Actin filaments (from 1 µM G-actin, 50% Oregon-green 996 labeled) were monitored for 300 s without ADF4 (a), in the presence of 500 nM 997 non-phosphorylated ADF4 (b) or 500 nM CKL2-phosphorylated ADF4 (c). The red 998 pairs of scissors indicate severing events. See Supplemental Movie 3 for the entire 999 series. 1000 (C) Quantification of ADF4-mediated actin-filament-severing frequency. Averages for 41 1001 each condition are from at least 30 individual filaments obtained from three 1002 independent trials. Error bars represent means ± SD (n = 3). Statistical significance (* 1003 P < 0.5) was determined by Student’s t-test. The t-test analysis of the data indicated 1004 the level of significance to be P = 0.0186 and 0.1380 for ADF4 and ADF4 + CKL2 1005 data relative to the control data, respectively. The level of significance between ADF4 1006 and ADF4 + CKL2 data was P = 0.0219. 1007 1008 Figure 7. ADF4 Is Required for CKL2-Mediated Stomatal Closure. 1009 (A) Stomatal bioassays for ABA-induced closure in Col-0, ckl2, adf4-1, adf4-2, and 1010 adf4 ckl2 plants. The data represent the means ± SD of three independent experiments; 1011 at least 50 stomata were analyzed per genetic background. The data sets were tested 1012 for normal distribution by the Shapiro-Wilk test. Statistical significance was 1013 determined by Student’s t-test; significant differences (P < 0.01) are indicated by 1014 different lowercase letters. The ckl2 mutants had wider stomatal apertures than Col-0 1015 (P = 0.0052). Both adf4-1 and adf4-2 mutants had smaller stomatal apertures than 1016 Col-0 (P = 0.0017 and P = 0.0043). The adf4 ckl2 mutants had smaller apertures than 1017 the ckl2 mutants (P = 0.0019) and wider apertures than Col-0 (P = 0.0012). Before 1018 ABA treatment, there were no significant differences in the data for ckl2, adf4-1, 1019 adf4-2, and adf4 ckl2 compared with Col-0 (P = 0.8216, 0.4420, 0.5870, and 0.2290, 1020 respectively). 1021 (B) Representative pseudocolored infrared images of leaf temperature of Col-0, ckl2, 1022 adf4-1, adf4-2, and adf4 ckl2 plants were obtained by infrared thermography. 1023 (C) Leaf (surface) temperatures of Col-0, ckl2, adf4-1, adf4-2, and adf4 ckl2 plants 1024 were analyzed by InfraTec GmbH reporter software from images in (B). Twenty 1025 leaves were analyzed per line. Data are means ± SD (n = 3). The data sets were tested 1026 for normal distribution by the Shapiro-Wilk test. Statistical significance was 1027 determined by Student’s t-test; significant differences (P < 0.01) are indicated by 1028 different lowercase letters. The ckl2 mutant had lower leaf temperatures than Col-0 (P 1029 = 0.0053). Both adf4-1 and adf4-2 had higher leaf temperatures than Col-0 (P = 1030 0.0012 and P = 0.0091). adf4 ckl2 had higher leaf temperatures than ckl2 (P = 0.0036) 42 1031 and lower leaf temperatures than Col-0 (P = 0.0065). 1032 (D) Actin filament organization in guard cells from Col-0, ckl2, adf4-1, adf4-2, and 1033 adf4 ckl2 transgenic plants harboring 35Sp:GFP-fABD2-GFP before and after 2 μM 1034 ABA treatment for 0.5 h. 1035 (E) Quantitative analysis of actin filament density in guard cells as shown in (D). 1036 Before ABA treatment, the levels of significance were P = 0.0059, 0.0063, and 0.0052 1037 for actin filament density in ckl2, adf4-1, and adf4 ckl2 mutants, respectively, relative 1038 to Col-0. After ABA treatment, the levels of significance were P = 0.0041, 0.0033 and 1039 0.0045 for ckl2, adf4-1 and adf4 ckl2 respectively relative to Col-0. 1040 (F) Bundling (skewness) quantitatively analysis in guard cells shown in (D). Before 1041 ABA treatment the levels of significance were P = 0.0845, 0.0048 and 0.137 for ckl2, 1042 adf4-1, and adf4 ckl2 respectively relative to the Col-0 skewness value. After ABA 1043 treatment the levels of significance were P = 0.0072, 0.0013 and 0.0025 for ckl2, 1044 adf4-1 and adf4 ckl2 respectively relative to Col-0. 1045 (G) Stomatal bioassays for ABA-induced closure in leaves of Col-0, ckl2, and plants 1046 overexpressing ADF4 in Col-0 and ckl2 mutant. Before ABA treatment, no significant 1047 differences were observed when comparing the stomatal apertures of ckl2 and plants 1048 overexpressing ADF4 in Col-0 and ckl2 mutant with Col-0, respectively (P = 0.66, 1049 0.5830, 0.3932, 0.4523, and 0.6488). After ABA treatment, the levels of significance 1050 changed significantly (P = 0.0029, 0.0046, 0.0065, 0.0035, and 0.0073, respectively). 1051 Values of (E), (F) and (G) represent the means ± SD of three independent 1052 experiments; 50 stomata were analyzed per line. 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