CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin FilamentStability and Stomatal Closure via Phosphorylation of ActinDepolymerizing Factor

ShuangshuangZhao,a,b Yuxiang Jiang,c YangZhao,b ShanjinHuang,c,dMingYuan,b Yanxiu Zhao,a,1 andYanGuob,1

a Key Laboratory of Plant Stress, Life Science College, Shandong Normal University, Jinan 250014, Chinab State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing100193, Chinac Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Science, Beijing 100093, ChinadCenter for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China

The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamicreorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largelyunclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), whichresponds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actinfilaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actindepolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure,deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA,whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive toABA. Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover,Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filamentreorganization and stomatal closure mainly through phosphorylation of ADF.

INTRODUCTION

Stomata regulate theuptakeofCO2 forphotosynthesis,water lossthrough transpiration, and defense responses during pathogenattack (Kim et al., 2010; Du et al., 2014). To cope with changes inenvironmental conditions, such as light, temperature, humidity,CO2, and salt in soil, plants must tightly regulate the opening andclosing of stomata (Roelfsema andHedrich, 2005; Vavasseur andRaghavendra, 2005; Israelsson et al., 2006). Many cellular signals(e.g., abscisic acid [ABA], H2O2, Ca

2+, CO2, and NO) regulatestomata by influencing the activities of H+, K+, Ca2+, and aniontransporters and channels (Pei et al., 2000; Schroeder et al., 2001;Hosy et al., 2003; Desikan et al., 2004; Hirayama and Shinozaki,2007; Wang and Song, 2008; Gayatri et al., 2013; Kollist et al.,2014). Actin filament reorganization occurs during stomatal clo-sure. The actin cytoskeleton in the guard cells changes fromwell-organized cortical filaments in the guard cells of open stomata, torandomly distributed filaments, and then finally reorganizes intohighlybundled longcables in the longitudinaldirection in theguardcells of closed stomata (Hwang and Lee, 2001; Zhao et al., 2011).This regulatory process involves actin binding proteins such asSCAB1 and the Arp2/3 complex (Zhao et al., 2011; Jiang et al.,

2012; Li et al., 2014). SCAB1 stabilizes actin filaments, and loss ofSCAB1 in plants causes defects in stomatal closure (Zhao et al.,2011). TheArp2/3complexmediatesstomatal closure in responseto external stimuli and regulates actin reorganization in guard cells(Jiang et al., 2012; Li et al., 2014). However, how such actin fil-ament reorganization in guard cells is regulated remains an openquestion.Actin filaments are highly dynamic, undergoing rapid reorgani-

zation and turnover regulated by actin binding proteins such asADF/cofilin, villin, profilin, fimbrin, and capping protein (Wasteneysand Galway, 2003; Hussey et al., 2006; Staiger and Blanchoin,2006; Higaki et al., 2007; Thomas et al., 2009; Li et al., 2010; Suet al., 2012;Quet al., 2013;Wanget al., 2015). ADF/cofilin proteinsfunction as key regulators of actin filament dynamics and re-organization through binding to both globular and filamentousactin. ADF/cofilin proteins promote actin filament severing anddepolymerization and inhibit nucleotide exchange on actinmonomers (Hotulainen et al., 2005; Andrianantoandro and Pollard,2006; Henty et al., 2011). The Arabidopsis thaliana genome enc-odes 11 ADF proteins, which play important roles in various bio-logical processes. ADF4 is involved in innate immune signaling(Tian et al., 2009;Henty-Ridilla et al., 2014); ADF7promotes pollentube growth (Zheng et al., 2013); and ADF2 is required for cellgrowth, development, and root-knot nematode infection (Clémentet al., 2009). In addition, the 14-3-3 l protein interacts withphosphorylated ADF1 to regulate actin dynamics during hypo-cotyl elongation (Zhao et al., 2015). Overexpression of ADF1causes disruption of F-actin cables in guard cells and results in

1 Address correspondence to guoyan@cau.edu.cn or zhaoyx@sdnu.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Yan Guo (guoyan@cau.edu.cn).www.plantcell.org/cgi/doi/10.1105/tpc.16.00078

The Plant Cell, Vol. 28: 1422–1439, June 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

a stomatal closure-defect phenotype following ABA treatment,suggesting that ADFproteinsmight function in this process (Donget al., 2001).

In animals and plants, many factors regulate the F-actin dis-assembling activity of ADF/cofilin. Two proteins, actin-interactingprotein-1 and cyclase-associated protein, enhance the F-actindisassembling activity of ADF/cofilin (Moriyama and Yahara,2002;Ono, 2003;Ketelaar et al., 2004;Shi et al., 2013). TheF-actindisassembling activity of ADF/cofilin can also be enhanced byincreased intracellular pH (Bernstein et al., 2000; Allwood et al.,2002). The F-actin disassembling activity of ADF/cofilin is de-creased by phosphoinositide and cortactin binding (Yonezawaet al., 1990; Allwood et al., 2002; Maciver and Hussey, 2002) aswell as by phosphorylation at the Ser-3 residue of animal cofilin(Agnew et al., 1995). Changes in the Ser-3 phosphorylation levelare tightly associated with extracellular stimuli, actin rearrange-ment, and cell activities, which implies a critical role of suchphosphorylation for modulation of cofilin activity (Mizuno, 2013).Ser-6 of plant ADFs has been considered to function analogouslyto Ser-3 of animal cofilin based on amino acid sequence similaritysearches and tertiary structure prediction (Chen et al., 2002).Similar to Ser-3 phosphorylation in cofilin, the phosphorylation ofthe Ser-6 in ADFs results in a reduction of their F-actin dis-assembling activity (Agnew et al., 1995; Moriyama et al., 1996;Nagaoka et al., 1996; Smertenko et al., 1998). In yeast, caseinkinase 1 (CK1) Hrr25 phosphorylates a number of actin bindingproteins, including the cofilin Cof1 (Peng et al., 2015). Two kinasefamilies, LIM (Lin-11/Isl-1/Mec-3) and TES (testicular protein),specifically phosphorylate and deactivate ADF/cofilin (Toshimaet al., 2001). However, mammalian LIMK does not phosphorylateplant ADFs, and the plant LIMK homologs do not phosphorylateADFs (Bernard, 2007). Rapid dephosphorylation of ADF/cofilinoccurs in response to various stimuli in animal cells and results inchanges of F-actin organization and assembly (Davidson andHaslam, 1994; Samstag et al., 1994; Kanamori et al., 1995). Inplants, the phosphorylation of the wheat (Triticum aestivum) ADFis regulated by low temperature (Ouellet et al., 2001). A proteinfraction purified from cell suspension cultures of French bean(Phaseolus vulgaris cv Immuna 1.1) and enriched in calmodulin-like domain protein kinase(s) (CDPKs) can phosphorylate maize(Zeamays) ADF3 at Ser-6, and this phosphorylation is inhibited bythe addition of anti-CDPK antibodies (Smertenko et al., 1998;Allwood et al., 2001). Recently, Arabidopsis CDPK6 protein hasbeen shown to phosphorylate ADF1 in vitro (Dong and Hong,2013). However, it remains to be determined whether these plantCDPKsassociatewith F-actin andwhat thephysiological functionof the phosphorylation is.

CK1 is a family of serine/threonine protein kinases highlyconserved in eukaryotic organisms. CK1 is involved in biologicalprocesses including circadian rhythm establishment, vesiculartrafficking, DNA repair, the cell cycle, and morphogenesis (Grosset al., 1995; Akashi et al., 2002; Cheong and Virshup, 2011). Anumber of cytoskeleton-related proteins have been identified astargets of CK1 kinases; these targets include cofilin, twinfilin,myosin, tropin, spectrin3, dynein, a-/b-tubulin, microtubule-associated protein, and kinesin-like protein (Boesger et al., 2014;Knippschild et al., 2014; Peng et al., 2015). In addition to the othercytoskeleton-related proteins, CK1 directly phosphorylates actin

protein in vitro (Shibayama et al., 1986; Knippschild et al., 2005).Arabidopsis casein kinase 1-like protein 6 (CKL6) phosphorylatestubulin and regulatesmicrotubule organization (Ben-Nissan et al.,2008).In this study, we show that CKL2 and actin depolymerizing

factor 4 (ADF4) are involved in stomatal closure in response todrought stress and ABA treatment. Although CKL2 does not bindto and stabilize actin filaments in vitro, it decorates and stabilizesactin filaments in guard cells. Interestingly, we found that CKL2physically interacts with ADF4 and that phosphorylation of ADF4decreases its F-actin disassembling activity in vitro. In terms ofABA-induced stomatal closure,CKL2 deletion andADF4 deletionhave opposite effects. Importantly, deletion of ADF4 in the ckl2mutant background partially rescues its ABA-insensitive stomatalclosure phenotype. Considering that CKL2 nonselectivelyphosphorylates Arabidopsis ADFs from subclass I in vitro, wepropose that CKL2 stabilizes actin filaments by phosphorylatingADFs and inhibiting their F-actin disassembling activity in guardcells to promote the reassembly of actin filaments duringdrought/ABA-induced stomatal closure.

RESULTS

The ckl2 Mutant Exhibits Rapid Water Loss and ImpairedABA-Induced Stomatal Closure

To obtain mutants that lost water faster and wilted earlier than thewild type, we performed a genetic screen of T-DNA insertion linesordered from the ABRC (Ohio State University). Rosette leaves of3-week-old plants were detached and the rate of water loss wasmonitored.After identificationofmutantswithbothmore rapidandless rapid water-loss phenotypes, we further performed a sto-matal aperture assay with an ABA stimulus as a standard duringthescreening.We identifiedamutant (SALK_104209) that showedfaster water loss and was less sensitive to ABA-induced stomatalclosure than the wild type (Figures 1A to 1C). The T-DNA insertionin the Arabidopsis gene At1g72710 was confirmed by PCR usingthe T-DNA left border and the gene-specific primers. BecauseAt1g72710 encodes CKL2, SALK_104209 was designated ckl2.RT-PCR analysis revealed that transcript accumulation of CKL2was significantly decreased in the ckl2 homozygous mutant(Supplemental Figures 1A and 1B). To determine the transpirationrates of shoots, we used an infrared camera to measure the leaftemperature of 4-week-old Col-0 and ckl2 mutant plants (Merlotet al., 2002). The leaf temperature of the ckl2 mutant was signif-icantly lower than that of Col-0, suggesting that themutant leavesdisplay a higher transpiration rate and lose more water than thewild type (Figures 1D and 1E).To rescue the water-loss and stomatal closure phenotypes of

the mutant, we transformed the ckl2 mutant with a constructharboring a 5.09-kb CKL2 genomic DNA fragment including1.06-kb promoter and 0.5-kb 39 untranslated region. The ex-pression level ofCKL2 in the transgenic lines was similar to that inthe wild type (Supplemental Figure 1B). The water-loss and sto-matal closure phenotypes were fully rescued in the transgeniclines (Figures 1B and 1C), suggesting the phenotype is indeedcaused by the loss of function of CKL2.

Phosphorylation of ADF Proteins by CKL2 1423

CKL2 Expression Is Induced by Water Loss andABA Treatment

Todeterminewhether theexpressionofCKL2 is regulatedbydroughtstress and ABA, total RNA was extracted from 7-d-old wild-typeseedlings treated with 20 mM ABA for 0, 0.5, and 1 h or water losstreatment until the seedlings lost 20%of their freshweight.Real-timeRT-PCRanalysisshowedthatCKL2expressionwas inducedbybothwater-loss and ABA treatments (Figures 1F and 1G). As a positivecontrol, the expression of RESPONSIVE TO DESICCATION 29A(RD29A) was confirmed to be induced by both treatments;RD29A isan abiotic stress/ABA-responsive gene (Ishitani et al., 1997). For thenegative control, we monitored the expression of a salt-responsivegene, SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCaBP8) (Quanetal.,2007;Linetal.,2009),whichshowednoobviouschangesunderthese conditions. The induction of CKL2 under water-loss and ABAtreatments is consistent with microarray data from AtGeneExpress

(http://jsp.weigelworld.org/expviz/expviz.jsp; Supplemental Figure1C) and a previous study (Cui et al., 2012).CKL2 is widely expressed in a variety of tissues throughout

Arabidopsis development (Gene ExpressMap of Arabidopsis,http://jsp.weigelworld.org/expviz/expviz.jsp; SupplementalFigure2A). To determine the tissue-specific expression patternof CKL2, real-time RT-PCR was performed using total RNAextracted from various tissues of 4-week-old Col-0 plants.Consistent with results obtained in the TAIRwebsite, expression ofCKL2was detected in roots, stems, cauline leaves, rosette leaves,flowers, and siliques (Supplemental Figure 2B).

CKL2 Colocalizes with Actin Filaments in Cells but Does NotDirectly Interact with Actin Filaments in Vitro

To determine the subcellular localization of CKL2, theGFP-CKL2construct driven by theCKL2 native promoter thatwas used in the

Figure 1. The ckl2 Mutant Shows Impaired Stomatal Closure.

(A) Leaves detached from wild-type and ckl2 plants for 0 (left) and 3 h (right) of water loss treatment.(B)Cumulative leaf transpirational water loss in Col-0, ckl2, and two rescued lines (com1 and com2) at the indicated times after detachment (means6 SD,n = 3).(C) Stomatal bioassays for ABA-induced closure in Col-0, ckl2, and two rescued lines (com1 and com2). The data represent the means 6 SD of threeindependent experiments; 50 stomata were analyzed per line. The data sets were tested as normal distribution by the Shapiro-Wilk test. Statisticalsignificance was determined by Student’s t test; significant differences are indicated by different lowercase letters. The t test analysis of the data indicatesthe levels of significance to be P = 0.0034, 0.9986, and 0.9347 for ckl2 and the two rescued lines, respectively, compared with Col-0 after ABA treatment.Before ABA treatment, the levels of significancewere P = 0.8075, 0.2705, and 0.6197 for ckl2 and the two rescued lines, respectively, comparedwithCol-0.(D) Representative pseudocolored infrared images of leaf temperature of Col-0 and ckl2 mutant plants.(E)Leaf (surface) temperatureofCol-0andckl2mutant plantsmeasured from imagesobtainedby infrared thermography, as in (D), andanalyzedby InfraTecreporter software. Twenty leaveswere analyzed per line. Data aremeans6 SD (n= 3). The data setswere tested for normal distribution by Shapiro-Wilk test.Statistical significance (**P < 0.01) was determined by Student’s t test.(F) and (G) Real-time PCR analysis revealed the induced expression of CKL2 by ABA treatment or water loss treatment. Seven-day-old seedlings weretreatedwith 20mMABA for 0.5 or 1 h (F) or were treated for water loss until leaves had lost 20%of their fresh weight (G).RD29A andSCaBP8were used ascontrols. Expression levels ofCKL2,RD29A, andSCaBP8without ABA/water loss treatment were set as 1.0, respectively. The experiments were repeatedthree times.Data aremeans6 SD. Statistical significance (**P < 0.01 and *P<0.05)wasdeterminedbyStudent’s t test. t test analysis of the data shown in (F)indicates the level of significance to be P = 0.0042 and 0.0023 for the data of CKL2 relative mRNA level at 0.5 and 1 h after ABA treatment, respec-tively, comparedwith the control. The positive controlRD29A also had a higher relativemRNA level when treatedwith ABA for 0.5 h (P = 0.0033) and 1 h (P=0.0012) comparedwith thecontrol. ThenegativecontrolSCaBP8 showednoobviousdifferencewhen treatedwithABA for0.5h (P=0.6890) and1h (0.7891)compared with the control. As shown in (G),CKL2was induced by water loss treatment (P = 0.0066). The positive control RD29A also had a higher relativemRNA levelwhen treatedwithwater loss until seedlings lost 20%of their freshweight (P= 0.0225). As anegative control, the relativemRNA level ofSCaBP8was not significantly different when treated with water loss until seedlings lost 20% of their fresh weight (P = 0.5851) compared with the control.

1424 The Plant Cell

genetic rescue experiment was transformed into Col-0 andthe ckl2 mutant. The stomatal closure phenotype of the ckl2mutant was rescued by the CKL2pro:GFP-CKL2 transgene(Figure 2A), and the expression level ofCKL2 in the transgeniclines was similar to that in the wild type (Supplemental Figure2C). Although the GFP signal formed filamentous structures inthe cells of these transgenic plants, it was veryweak, and it wasdifficult to obtain high-quality images. The GFP-labeled CKL2expressed from the CKL2 native promoter was observed infilamentous structures in the cytoplasm of hypocotyl epider-mal cells, leaf epidermal cells, guard cells, root epidermal cells,and root hairs of the Col-0 transgenic line (Figure 2B). Theseresults suggest that GFP-CKL2 accurately indicates the in-tracellular localization of CKL2. To further investigate thesubcellular localization of CKL2, we fused GFP to the N ter-minus of CKL2 under the control of the CaMV 35S promoter.This construct was used to transform Col-0 and the ckl2 mu-tant. TheGFP-CKL2 fusion gene rescued the stomatal-closuredefect and drought-sensitive phenotypes of the ckl2 mutant.GFP-labeled CKL2 in the transgenic plants formed fine fi-brous networks in the cytoplasm of hypocotyl epidermal cells(Supplemental Figure 3A), leaf epidermal cells (SupplementalFigure 3B), guard cells (Supplemental Figure 3C), root epi-dermal cells (Supplemental Figure 3D), and root hairs(Supplemental Figure 3E).

To determine if CKL2 was associated with actin filaments ormicrotubules, a suspension cell line was generated from therosette leaves of the GFP-CKL2 transgenic plants. The GFP-labeled CKL2 colocalized with rhodamine-phalloidin-stainedactin filaments in the suspension cells (Supplemental Figure3F). Colocalization was analyzed by plotting GFP-CKL2 andactin filament signal intensities using ImageJ software. ThePearson’s correlation coefficient value was 0.83 in the in-dicated regions of interest, suggesting a strong correlationbetween the spatial localizations of GFP-CKL2 and the actinfilaments (Supplemental Figure 3G). To further confirm theassociation of CKL2 with actin filaments, the GFP-CKL2transgenic plants were treated with latrunculin A (LatA), aninhibitor of actin polymerization that disrupts actin filamentsby binding actin monomers, or oryzalin, a microtubule-disruptingreagent. After 0.5 h treatment with 200 nM LatA, the filamentousnetwork of GFP-CKL2 was disrupted in most of the hypocotylcells. As a control, GFP-fABD2-GFP-labeled actin filaments werealso disrupted (Supplemental Figure 3H). However, after 1 htreatment with 10 mM oryzalin, the filamentous structure of GFP-CKL2 remained intact in most of the hypocotyl cells, whereasMBD-GFP-labeled microtubules were disrupted (SupplementalFigure 3I). Disruption of filamentous structures by LatA was alsoobserved in guard cells of CKL2pro:GFP-CKL2;ckl2 (Figure 2C).These results indicate that CKL2 colocalizes with actin filamentsbut not microtubules in cells.

To testwhetherCKL2binds toactinfilamentsdirectly invitro,weperformed an actin cosedimentation assay by high-speed cen-trifugation. We found that most of the CKL2 remained in the su-pernatant in both the absence and presence of F-actin; however,a significant amount of the actin binding protein Fim1 (Su et al.,2012) was pulled down by F-actin (Supplemental Figure 4A).

These results indicate that CKL2 does not directly bind to actinfilaments.To test if CKL2 has another effect on actin filaments in vitro, we

directly visualized actin filaments stained with Alexa-488-phal-loidin by epifluorescence light microscopy. The length of F-actinshowed no significant differences in the presence or absence ofHis-CKL2, indicating that CKL2 has no effect on the length dis-tribution of actin filaments in vitro (Supplemental Figures 4B and4C). To determinewhether CKL2 is involved in actin assembly, wedetermined the effect of CKL2 on spontaneous actin assemblyand found that CKL2 had no such activity in vitro (SupplementalFigure4D). Thus, these results suggest thatCKL2neither interactswith actin filaments nor affects actin assembly and disassemblyin vitro.

CKL2 Stabilizes Actin Filaments and RegulatesStomatal Closure

To test if CKL2 affects actin filaments in cells, wemeasured theF-actin stability in ckl2 and wild-type cells. To visualize theactin filaments, we generated transgenic plants harboring35S:GFP-fABD2-GFP (containing the second actin bindingdomain of At-Fim1) in the Col-0 and ckl2 background andexamined the stability of actin filaments in the guard cells of theckl2 mutant and Col-0. After the transgenic lines were treatedwith 200 nM LatA for 30 min, the actin filaments became morefragmented and less abundant in both Col-0 and ckl2 guardcells compared with the untreated guard cells (Figure 3A).However, the actin filaments were disrupted more rapidly inckl2 guard cells than in wild-type guard cells. In the ckl2mutant, more GFP-labeled dot-like structures were detectedafter LatA treatment (Figure 3A). When the GFP-fABD2-GFPsignal was monitored and quantified by measuring averageGFP fluorescence pixel intensity, the GFP signal was similarbetween ckl2 and Col-0 in widely opened stomata. However,the GFP signal in the ckl2 guard cells decreased to a greaterextent than in wild-type guard cells after the LatA treatment(Figure 3B). These results indicate that CKL2 plays a role instabilizing actin filaments in guard cells.We next determined the physiological relevance of the actin-

filament-stabilizing activity of CKL2 during stomatal closure.Since it has been shown that the actin cytoskeleton in guard cellsis reorganized during ABA-induced stomatal closure (Lemichezet al., 2001; Zhao et al., 2011) and the ckl2 mutant displayedimpaired ABA-induced stomatal closure, we examined whetherthe reorganizationof actinfilamentsduringABA-inducedstomatalclosure was altered in ckl2 by the exogenous application of ABA.When the stomata were in an opened state, the actin filamentswere present as well-organized cortical filaments in both Col-0andckl2guard cells. After 2mMABA treatment for 0.5 h,;73%of stomata were closed and the actin filaments were randomlyrearrangedand thenbundledpreferentially as longcables inCol-0.However, the stomatal closure was less sensitive to ABA treat-ment in the ckl2mutant and 71% of stomata were not closed; theactin filaments failed to form bundled cables and were trapped ina randomly distributed state (Figure 3C). To quantify the actinarchitecture in theguard cells, skewness anddensity (Higaki et al.,2010) parameters were measured to determine the extent of actin

Phosphorylation of ADF Proteins by CKL2 1425

filament bundling and the percentage of occupancy of actin fil-aments. In widely opened guard cells, the ckl2mutant had a lowerdensity value than wild type (Figure 3D); however, the skewnessvalue showed no significant difference in the ckl2 mutant com-pared with wild type (Figure 3E). After the plants were treated with2mMABA for 0.5h, 73%of stomatawereclosed inCol-0 andactinfilaments became bundled with higher skewness in these guardcells; however, 71%of stomatawere not closed in the ckl2mutantandactindisplayednoobviouschange inskewness in theseguardcells. The density value decreased in Col-0 and increased in theckl2mutant after the treatment (Figure 3D). These results suggestthat the actin-filament-stabilizing activity of CKL2 is required forthe subsequent construction of longitudinal actin cables andtherefore facilitates actin filament reorganization during stomatalclosure.

CKL2 Regulates the Activity of Actin Filament Severing inGuard Cells

Observation of a single actin filament lifetime, includingelongation rate and depolymerizing rate, in living cells hasbeen employed to study actin dynamics in vivo (Smertenko

et al., 2010; Henty-Ridilla et al., 2013; Qin et al., 2014; Li et al.,2014; Li et al., 2015). To examine the actin filament dynamicsin the guard cells of Col-0 and the ckl2 mutant, we performedtime-lapse imaging using spinning disk confocal microscopyto determine single actin filament dynamics (Figure 4A;Supplemental Movies 1 and 2). Several parameters of actinfilament dynamics in guard cells were significantly differentbetween wild type and the ckl2 mutant (Figure 4B). The av-erage lifetime of single actin filaments in Col-0 was ;2-foldlonger than that in ckl2 guard cells. Most of the single actinfilaments in Col-0 guard cells kept growing for more than 10 sbefore the first severing event occurred. However, most of thesingle actin filaments kept growing <8 s in ckl2 guard cells. InCol-0 guard cells, the severing rate was 0.0276 0.002 breaksmm21 s21 and a single actin filament elongated to an averagemaximum filament length of 4.3 6 0.2 mm. In the ckl2 mutant,however, the severing rate was increased to 0.039 6 0.05breaks mm21 s21, and the average maximum actin filamentwas 3.4 6 0.2 mm. There was about a 1.5-fold increase insevering frequency in the ckl2mutant compared with the wildtype. These results demonstrate that loss of CKL2 led to moresevering events in the ckl2 guard cells and suggest that CKL2

Figure 2. Localization of GFP-CKL2 Expressed from the Native CKL2 Promoter.

(A) Stomatal bioassays for ABA-induced closure in Col-0, ckl2, and two ProCKL2:GFP-CKL2 transgenic lines. The data represent themeans6 SD of threeindependent experiments; 50 stomata were analyzed per line. The data sets were tested for normal distribution by the Shapiro-Wilk test. Statisticalsignificancewas determined by Student’s t test; significant differences are indicated by different lowercase letters. The t test analysis of the data indicatedthe levels of significance to be P = 0.0024, 0.8673, and 0.8358 for ckl2 and two rescued lines data, respectively, compared with Col-0 after ABA treatment.Before ABA treatment, the levels of significance were P = 0.9023, 0.3251, and 0.6601 for ckl2 and two rescued lines, respectively, compared with Col-0.(B) Confocal images were taken of epidermal cells of hypocotyls (A), leaves (B), guard cells (C), roots (D), and root hairs (E) of ProCKL2:GFP-CKL2transgenic seedlings in the Col-0 background. Bars = 10 mm.(C) GFP-CKL2 driven by the CKL2 native promoter and GFP-fABD2-GFP transgenic seedlings were treated with 200 nM LatA for 0.5 h. Confocal imageswere taken of guard cells. Bars = 10 mm.

1426 The Plant Cell

plays an important role in controlling actin filament severing inguard cells.

CKL2 Interacts with and Phosphorylates ADF4

Our invitroanalyses indicated thatCKL2didnothaveanyeffectonthe actin cytoskeleton on its own; these results suggest that anactin binding protein (ABP) might mediate the regulatory effect ofCKL2 on the actin cytoskeleton in vivo. Furthermore, our findingsregarding actin filament dynamics and severing in Col-0 and ckl2guard cells suggest that an ABP that could sever actin filamentsandbe regulated by phosphorylationmight be relevant, especiallyconsidering that CKL2 is a protein kinase. ADF family membersfunction as important regulators of actin dynamics via severingactin filaments and promoting monomer dissociation from thepointed end of actin filaments, and phosphorylation of ADFs is

critical for regulating their activity. We reasoned that ADFs withsimilar expression patterns to CKL2 might act as substrates forCKL2 and mediate the regulation of actin dynamics by CKL2.ADF4 belongs to subclass I, members of which are expressedabundantly in all vegetative tissues and reproductive tissues, andits expression pattern is similar to that of CKL2 (SupplementalFigures 2A to 2C; Ruzicka et al., 2007; Henty et al., 2011). BothCKL2 and ADF4 are expressed in guard cells. However, the ex-pression ofCKL2was induced by ABA treatment, whereas that ofADF4was not (Supplemental Figure 5). The adf4mutant displaysdifferent F-actin dynamic behavior from that of the wild type,including decreased severing frequency and increasedmaximumfilament length and filament lifetime (Henty et al., 2011). Wetherefore selected ADF4 for further analysis as a potential targetof CKL2 kinase to mediate its regulatory effects on the actincytoskeleton.

Figure 3. CKL2 Stabilizes Actin Filaments in Guard Cells.

(A)Actin filament organization in guard cells of Col-0 and ckl2 35Sp:GFP-fABD2-GFP transgenic plants before (right) and after (left) 200 nMLatA treatmentfor 30 min.(B)Quantification of the relative average fluorescence pixel density of GFP-fABD2 signal in guard cells as shown in (A). After LatA treatment, Col-0 and ckl2mutant had lower relative average fluorescence pixel density of GFP-fABD2 signal compared with control (P = 0.0204, 0.0037).(C)Actin filament organization in guard cells of Col-0 and ckl2 35Sp:GFP-fABD2-GFP transgenic plants before (right) and after (left) 2mMABA treatment for0.5 h.(D) The extent of filament bundling (skewness) of guard cells shown in (C). The ckl2 mutant had significantly increased average actin filament densitycompared with Col-0 after ABA treatment (P = 0.0029). The ckl2mutant had significantly decreased average actin filament density than Col-0 before ABAtreatment (P = 0.0056).(E) Average filament density of Col-0 and ckl2 guard cells before and after 2 mM ABA treatment as shown in (C). ckl2mutant had significantly decreasedaverage actin filament skewness values comparedwithCol-0 after ABA treatment (P= 0.0068). No significant differenceof average actin filament skewnessvalues between Col-0 and ckl2 mutant was observed before ABA treatment (P = 0.0993).Values of (B), (D), and (E) represent the means6 SD of three independent experiments; 50 stomata were analyzed per line. The data sets were tested fornormal distribution by the Shapiro-Wilk test. Statistical significance was determined by Student’s t test. Significant differences are denoted with asterisks(**P < 0.01 and *P < 0.05) in (B). Significant differences (P < 0.01) are indicated by different lowercase letters in (D) and (E).

Phosphorylation of ADF Proteins by CKL2 1427

First, we determined the interaction between ADF4 and CKL2in vitro. His-CKL2 and GST-ADF4 recombinant proteins weretherefore purified. Pull-down assays showed that GST-ADF4coprecipitated with His-CKL2 (Figure 5A), suggesting that CKL2interactswithADF4.TodeterminewhichdomainofCKL2 interactswith ADF4, we fused the CKL2 N-terminal domain (catalytic do-main, from 1 to 295 amino acids) and CKL2 C-terminal domain(regulatory domain, including 296 to 464 amino acids) to His tag,respectively, purified both recombinant proteins and performedpull-down assays with ADF4. GST-ADF4 coprecipitated with theHis-CKL2 N terminus but not the C terminus. Immunoblottinganalyses further confirmed that the full-length CKL2 and the Nterminus of CKL2 interacted with ADF4 (Figure 5A).

To explore the interaction between CKL2 and ADF4 in vivo,we performed coimmunoprecipitation assays in vivo. Anti-ADF4antibodies were generated. They recognized endogenous ADF1and ADF4 but were not specific to ADF4 (Supplemental Figure6A). For coimmunoprecipitation assays in vivo, total protein wasisolated from the transgenic plants expressing the constructPro35S:Flag-HA-CKL2. CKL2was immunoprecipitatedwith anti-Flag antibodyconjugated agarose, andADFsweredetected in thepull-down products by anti-ADF antibodies. PKS5, a cytoplasmic

localized protein kinase (Fuglsang et al., 2007), was used asa control and showed no interaction with ADFs (SupplementalFigure 6B). We further used split-luciferase (split-LUC) comple-mentation assays in Nicotiana benthamiana to determine the in-teraction of ADF4 and CKL2 (Figure 5B). ADF4 was fused to the Cterminus of LUCIFERASE (ADF4-cLUC), and CKL2 was fused tothe N terminus of LUCIFERASE (nLUC-CKL2). The split-LUCassays showed that transient coexpression of nLUC-CKL2 andADF4-cLUC in N. benthamiana yielded strong fluorescence sig-nals, but no fluorescence signalwasdetected in thecontrol leavescoexpressing nLUC-CKL2 and cLUC or nLUC and ADF4-cLUC,which further confirmed that CKL2 interacts with ADF4 in vivo(Figure 5B). In addition, this interaction was induced by ABAtreatment (Figure 5B).To detect whether ADF4 is a substrate of CKL2, we conducted

in vitro kinase assays using the His-CKL2 andHis-ADF4 proteins.A phosphorylated ADF4 signal was detected (Figure 5C). How-ever,CKL2didnotphosphorylate theactinbindingproteinSCAB1in vitro (Zhao et al., 2011), suggesting that phosphorylation ofADF4 by CKL2 is relatively specific.To determine if CKL2 phosphorylates ADF4 in vivo, the

constructPro35S:3Flag-ADF4wasused to transformCol-0 and

Figure 4. The ckl2 Mutant Shows Different Actin Dynamics from the Wild Type.

(A) Time-lapse images of single actin filaments in guard cells of Col-0 and ckl2 35Sp:GFP-fABD2-GFP transgenic plants. Green dots highlight a repre-sentative single actin filament. Yellow arrows indicate actin filament-severing events. White arrows indicate the position at which growth of the single actinfilament began. Bars = 5 mm.(B) Actin filament dynamic parameters in Col-0 and ckl2. The parameters associated with single actin filament dynamics in Col-0 and ckl2 guard cells werequantified from spinning disk confocal micrographs. Values representmeans6 SD, n= 30. The data sets were tested for normal distribution by the Shapiro-Wilk test. Statistical significance was determined by Student’s t test. Quantification of *P < 0.05 and **P < 0.01.

1428 The Plant Cell

the ckl2mutant. Flag-ADF4 was immunoprecipitated with anti-Flag antibody-conjugated agarose and eluted using a Flagpeptide. Equal amounts of the Flag-ADF4 protein were used for2D immunoblotting assayswith anti-Flag antibody to detect thephosphorylation level of ADF4. TwoADF4 spotswith different pIvalues were detected in the 2D gel from both the wild type andthe ckl2 mutant. Spot A, close to the low pI region, was sig-nificantly decreased, and spotB, close to the high pI region,was

increased in the ckl2 mutant. When the wild-type sample wastreated with Lambda Protein Phosphatase, spot A almostdisappeared (Figures 5D and 5E), suggesting that spot A rep-resented phosphorylated ADF4. These results suggest thatADF4 is phosphorylated in plant cells and that CKL2 is requiredfor this phosphorylation.ArabidopsisADFsubclass I family proteins contain a conserved

amino acid Ser-6 (Supplemental Figure 6C). Phosphorylation at

Figure 5. CKL2 Interacts with and Phosphorylates ADF4.

(A)CKL2 interacts with ADF4 in pull-down assays. Equal amounts of affinity-purified His-CKL2 (left first panel), His-CKL2N (second panel), or His-CKL2Cfusionprotein (thirdpanel)were incubatedwithGST-ADF4.The inputandoutput proteinswerestainedwithCoomassieblueonaSDS-PAGEgel. Theoutputproteins were also subject to immunoblot assay with anti-ADF antibodies (right panel). The asterisks indicate the GST-ADF4 bands.(B) Split-luciferase complementation imaging assays in N. benthamiana. Quantitative analysis of luminescence intensity was determined. Relative valuesare mean 6 SD, n = 3. Higher luminescence intensity was observed after ABA treatment compared with control (denoted by asterisk, P = 0.0022, t test).(C) CKL2 phosphorylates ADF4 in vitro. The input proteins His-CKL2 and His-ADF4 were detected by Coomassie blue staining (left). Phosphorylationactivity was detected by [g-32P]ATP autoradiography (right).(D) Two-dimensional immunoblotting. The Flag-ADF4 protein was immunoprecipitated with anti-Flag agarose from Col-0 or ckl2 mutant plants. Equalamounts of Flag-ADF4 protein immunoprecipitated fromCol-0 were treated with l phosphatase as a control. Anti-Flag antibody was used for immunoblotassays. Arrowheads point to the location of the more acidic ADF4 spot, representing phosphorylated ADF4. Experiments were repeated three times.(E)Quantificationof relativeADF4phosphorylation level in (D). Values representmean6 SD,n=3.Statistical significancewasdeterminedbyStudent’s t test;significant differences (P < 0.05) are indicated with asterisks. The ckl2mutant had a lower relative ADF4 phosphorylation level compared with control (P =0.0258).(F) ADF4 Ser-6 is important for the phosphorylation of CKL2. Purified His-ADF4 and His-ADF4S6A as substrates were phosphorylated in the in vitro kinaseassays.(G)Quantification of relative ADF4 phosphorylation level in (F). Values represent mean6 SD, n = 3. The data sets were tested for normal distribution by theShapiro-Wilk test. Statistical significance was determined by Student’s t test; significant differences (P < 0.01) are indicated with asterisks. ADF4 Ser-6mutation led to a lower relative phosphorylation level compared with the wild type (P = 0.0091).

Phosphorylation of ADF Proteins by CKL2 1429

Ser-6 has been observed in all tested plant ADF subclass I familyproteins (Allwood et al., 2001, 2002; Chen et al., 2002). To de-termine whether Ser-6 is required for the phosphorylation byCKL2, we generated a mutated form of ADF4 with Ser-6 ex-changed for Ala (ADF4S6A). Recombinant proteins His-ADF4 andHis-ADF4S6A were purified and used for the kinase assay. Thephosphorylation level of ADF4 by CKL2was significantly reducedby the mutation at Ser-6, although this point mutation did notabolish phosphorylation (Figures 5F and 5G). Taken together, ourdata suggest that CKL2 phosphorylates ADF4 and that Ser-6 isone of the phosphorylation sites.

Given that ADF proteins are highly conserved and the ADFfamily in Arabidopsis consists of 11 members that are phyloge-netically divided into four subclasses (Mun et al., 2000; Ruzicka

et al., 2007), we wondered whether CKL2 could phosphorylateother ADFs besides ADF4 in Arabidopsis. To explore this, weselected other ADF proteins from subclass I (ADF1-4) and foundthat all 4 ADFs in the subclass I were phosphorylated by CKL2(Supplemental Figure 6D), suggesting that at least the subclass IADFs are potential targets of CKL2 in Arabidopsis.

Phosphorylation of ADF4 by CKL2 Inhibits the Activitiesof ADF4

Phosphorylation ofADFsat theN-terminal serine (Ser-3 in animalsandSer-6 inplants) is conserved inboth animal andplant cells andplays an important role in regulating ADF actin binding and dis-assembly activity (Ressad et al., 1998; Chen et al., 2002).

Figure 6. Phosphorylation of ADF4 by CKL2 Affects ADF4 Activity.

(A) The effect of ADF4 on F-actin disassembly was determined by a pyrene-actin assay. ADF4, after being phosphorylated by CKL2, was able to de-polymerize actin filamentsbutwas lesspotent thannonphosphorylatedADF4.Preassembled actinfilaments (from2mMG-actin, 10%pyrene-labeled)wereincubated with 250 nM ADF4 or 250 nM CKL2-phosphorylated ADF4 to induce actin disassembly at pH 7.0. Black closed circles, 0.5 mM F-actin; greenclosed squares, 0.5 mM F-actin + 4 mM ADF4; red closed diamonds, 0.5 mM F-actin + 4 mM ADF4 after phosphorylation by CKL2 for 0.5 h; blue closedtriangles, 0.5 mM F-actin + 4 mM ADF4 after phosphorylation by CKL2 for 1.5 h. a.u., arbitrary units.(B) Time-lapse TIRFmicroscopy analysis of actin filament severing by ADF4 after being phosphorylated byCKL2. Time-lapse imageswere recorded at 3-sintervals with TIRFmicroscopy. Actin filaments (from 1 mMG-actin, 50%Oregon-green labeled) weremonitored for 300 swithout ADF4 (A), in the presenceof 500 nM nonphosphorylated ADF4 (B) or 500 nMCKL2-phosphorylated ADF4 (C). The red pairs of scissors indicate severing events. See SupplementalMovie 3 for the entire series.(C)Quantification of ADF4-mediated actin-filament-severing frequency. Averages for each condition are fromat least 30 individual filaments obtained fromthree independent trials. Errorbars representmeans6 SD (n=3).Statistical significance (*P<0.5)wasdeterminedbyStudent’s t test. The t test analysisof thedata indicated the level of significance to be P = 0.0186 and 0.1380 for ADF4 and ADF4 + CKL2 data relative to the control data, respectively. The level ofsignificance between ADF4 and ADF4 + CKL2 data was P = 0.0219.

1430 The Plant Cell

Figure 7. ADF4 Is Required for CKL2-Mediated Stomatal Closure.

(A)Stomatal bioassays forABA-inducedclosure inCol-0,ckl2, adf4-1, adf4-2, and adf4ckl2plants. Thedata represent themeans6 SDof three independentexperiments; at least 50 stomata were analyzed per genetic background. The data sets were tested for normal distribution by the Shapiro-Wilk test.Statistical significancewasdeterminedbyStudent’s t test; significantdifferences (P<0.01) are indicatedbydifferent lowercase letters. Theckl2mutantshadwiderstomatalapertures thanCol-0 (P=0.0052).Bothadf4-1andadf4-2mutantshadsmaller stomatalapertures thanCol-0 (P=0.0017andP=0.0043). Theadf4 ckl2mutants hadsmaller apertures than theckl2mutants (P=0.0019) andwider apertures thanCol-0 (P=0.0012). BeforeABA treatment, therewerenosignificant differences in the data for ckl2, adf4-1, adf4-2, and adf4 ckl2 compared with Col-0 (P = 0.8216, 0.4420, 0.5870, and 0.2290, respectively).(B)Representative pseudocolored infrared images of leaf temperature of Col-0, ckl2, adf4-1, adf4-2, and adf4 ckl2plantswere obtained by infrared thermography.(C) Leaf (surface) temperatures of Col-0, ckl2, adf4-1, adf4-2, and adf4 ckl2 plants were analyzed by InfraTec reporter software from images in (B). Twentyleaveswere analyzed per line. Data aremeans6 SD (n=3). The data setswere tested for normal distribution by the Shapiro-Wilk test. Statistical significancewasdeterminedbyStudent’s t test; significantdifferences (P<0.01) are indicatedbydifferent lowercase letters. Theckl2mutanthad lower leaf temperaturesthanCol-0 (P = 0.0053). Both adf4-1 and adf4-2 had higher leaf temperatures thanCol-0 (P = 0.0012 andP=0.0091). adf4 ckl2 had higher leaf temperaturesthan ckl2 (P = 0.0036) and lower leaf temperatures than Col-0 (P = 0.0065).(D) Actin filament organization in guard cells fromCol-0, ckl2, adf4-1, adf4-2, and adf4 ckl2 transgenic plants harboring 35Sp:GFP-fABD2-GFP before andafter 2 mM ABA treatment for 0.5 h.(E)Quantitative analysis of actin filament density inguardcells asshown in (D). BeforeABA treatment, the levelsof significancewereP=0.0059, 0.0063, and0.0052 for actin filamentdensity inckl2,adf4-1, and adf4ckl2mutants, respectively, relative toCol-0. After ABA treatment, the levels of significancewereP=0.0041, 0.0033, and 0.0045 for ckl2, adf4-1, and adf4 ckl2, respectively, relative to Col-0.(F)Bundling (skewness) quantitative analysis inguardcells shown in (D). BeforeABA treatment, the levelsof significancewereP=0.0845, 0.0048, and0.137forckl2, adf4-1, and adf4 ckl2, respectively, relative to theCol-0 skewness value. After ABA treatment the levels of significancewereP=0.0072, 0.0013, and0.0025 for ckl2, adf4-1, and adf4 ckl2, respectively, relative to Col-0.(G)Stomatal bioassays for ABA-induced closure in leavesofCol-0, ckl2, and plants overexpressing ADF4 inCol-0 and ckl2mutant. BeforeABA treatment, nosignificant differences were observed when comparing the stomatal apertures of ckl2 and plants overexpressing ADF4 in Col-0 and ckl2mutant with Col-0,respectively (P = 0.66, 0.5830, 0.3932, 0.4523, and 0.6488). After ABA treatment, the levels of significance changed significantly (P = 0.0029, 0.0046, 0.0065,0.0035, and 0.0073, respectively).Values of (E) to (G) represent the means6 SD of three independent experiments; 50 stomata were analyzed per line. The data sets were tested for normaldistribution by Shapiro-Wilk test. Statistical significance was determined by Student’s t test; significant differences (P < 0.01) are indicated by differentlowercase letters.

Phosphorylation of ADF Proteins by CKL2 1431

To furtherdetect theeffectsofCKL2-mediatedphosphorylationon the actin disassembling activity of ADF4, we performed bulkactin disassembly assays and found that CKL2-mediated phos-phorylation decreased ADF4-induced actin disassembly (Figure6A).Wenextmonitored theeffects of thephosphorylationofADF4by CKL2 on the actin filament-severing activity in real time usingtotal internal reflection fluorescence (TIRF) microscopy. In theabsence of ADF proteins, few breaks were observed along actinfilaments (Figures 6Ba and 6C; Supplemental Movie 3). However,the addition of ADF4 resulted in increased filament breakage(Figures 6Bb and 6C; Supplemental Movie 4). By contrast, theadditionofADF4phosphorylatedbyCKL2 resulted in lessfilamentbreakage (Figures 6Bc and 6C; Supplemental Movie 5), sug-gesting that CKL2-mediated phosphorylation inhibited ADF4-induced actin filament severing. In summary, our results suggestthat the CKL2-mediated phosphorylation on ADF4 reduces theF-actin disassembling activity of ADF4.

Regulation of Stomatal Closure by CKL2 Is PartiallyDependent on ADF4

Todetermine thegenetic interaction betweenCKL2andADF4,wegenerated adf4 ckl2 doublemutants by crossing ckl2with an adf4T-DNA insertion line,adf4-1 (Tianetal., 2009). Thewild type,adf4-1,adf4-2 (Salk_121647, obtained from the ABRC), ckl2, and adf4-1ckl2mutantswereused tomonitor stomatal closure in response toABA treatment. RT-PCR results showed that the two adf4 ho-mozygous lines are knockout mutants (Supplemental Figure 7A).Stomatal closure in both adf4-1 and adf4-2wasmore sensitive toABA than was the wild type (Figure 7A). Consistent with ourprevious results (Figure 1C), the stomatal closure in ckl2 wasinsensitive to ABA compared with the wild type (Figure 7A). Thestomatal closure in the adf4-1 ckl2 double mutant in response toABA was intermediate between that in the respective singlemutants (Figure 7A). Consistent with the results of stomatal clo-sure measurement, leaf temperature in the adf4 ckl2 doublemutant was higher than that in the ckl2 single mutant but lowerthan that in the wild type (Figures 7B and 7C). Both adf4-1 andadf4-2mutant plants displayed higher leaf temperatures than didthe wild type (Figures 7B and 7C). These results suggest that lossof function of ADF4 partially rescues the stomatal closure phe-notype in ckl2.

To determine how CKL2 and ADF4 coordinately regulate theABA-induced actin dynamics in guard cells, we transformedthe 35S:GFP-fABD2-GFP construct into the adf4 and adf4 ckl2doublemutant. In widely opened guard cells, although the F-actinin the adf4mutant also displayed radially arranged filaments, theskewness value increased and actin filament density value de-creased significantly compared with these in Col-0, suggestingthat the extent of actin filament bundling increased in adf4 guardcells (Figures 7D to 7F). However, in the adf4 ckl2 double mutant,theeffect ofADF4deletiononactinfilamentswaspartially rescuedby CKL2 deletion. The double mutant showed an intermediatedensity between that of the adf4 and ckl2mutants, which was stilllower than that in Col-0. The skewness in the adf4 ckl2 doublemutant showedno significant difference fromCol-0 (Figures 7D to7F). After treatment with 2mMABA for 0.5 h, the skewness furtherincreased and the density further decreased in closed stomata

(69% of cell populations) of the adf4mutant compared with thesebefore the treatment (Figures7D to7F).However, in stomataof theadf4 ckl2 doublemutant (75%of cell populations), both skewnessand density displayed intermediate values between those of theadf4 and ckl2mutants after ABA treatment (Figures 7D to 7F). Theskewnessvalueof thedoublemutantwaslowerthanthatofCol-0andthe density value was higher than that of Col-0. These resultsshowed that deletion of ADF4 partially rescued the ckl2 mutantF-actin reorganization phenotype and suggest that loss of functionof ADF4 at least partially accounts for the F-actin reorganizationphenotype in ckl2.To further investigate the function of ADF4 in CKL2-mediated

stomatal closure, we generated ADF4 overexpression lines (35S:ADF4) in the Col-0 and ckl2 mutant background. There was nosignificant difference in ABA-induced stomatal closure betweenthe Col-0 and ckl2mutant upon overexpressing ADF4. Moreover,the plants overexpressing ADF4 in both the Col-0 and the ckl2mutantbackgroundswereeven lesssensitive than theckl2mutantplants in terms of ABA-induced stomatal closure (Figure 7G).These results further suggest that ADF4 interacts with CKL2 inregulating stomatal closure in response to ABA.

DISCUSSION

In this study, we found that an ABA-responsive actin filament-associated protein kinase, CKL2, regulates actin dynamics duringstomatal closure, presumably via phosphorylating ADF proteins.CKL2 therefore emerges as an important player in regulatingABA-mediated stomatal closure.CKL2andother factors, includingreceptor-like kinase, protein kinases (CDPK, SnRK2.2, 2.3, and2.6), transcription factors (ABI3, 4, and 5), and ion channel SLACs(Desikan et al., 2004; Israelsson et al., 2006; Kim et al., 2010; Huaet al., 2012), constitute a sophisticated regulatory network for ABA-mediated stomatal closure. Our study adds another importantcomponent to the ABA signaling network in guard cells and en-riches our understanding of the regulation of stomatal closure.In guard cells, actin filaments undergo dynamic reorganization,

which is important for proper stomatal closure (Kim et al., 1995;Eun andLee, 1997;HwangandLee, 2001;MacRobbie andKurup,2007; Zhao et al., 2011; Jiang et al., 2012). However, how thereorganization of the actin cytoskeleton is achieved duringstomatal closure remains poorly understood. Two stages are

Figure 8. A Simplified Working Model for the Role of CKL2 and ADF4 inRegulating Actin Reorganization during ABA-Induced Stomatal Closure.

During stomatal closure, microfilaments reorganize, first disassemblingand then reassembling. ABA/drought-induced CKL2 represses ADF ac-tivity to stabilize microfilaments and keep stomata closed.

1432 The Plant Cell

involved in this reorganization, with actin filament disassemblyfollowed by filament reassembly when stomata close (Eun andLee, 1997, 2000). Therefore, actin filament destabilization (fordisassembly) and stabilization (for reassembly) are both neces-sary aspects of this process. This work shows that a lack ofCKL2results in the blockage of actin filament reassembly and the actinfilaments remain in a disrupted state. These results suggest thatCKL2 has an effect on stabilization of actin filaments and, thus,that CKL2 functions in the reassembly of actin filaments duringstomatal closure.

ADFs bind to and sever actin filaments, thereby acting asmajorregulators of F-actin dynamics (Henty et al., 2011; Zheng et al.,2013) implicated in regulating F-actin reorganization during sto-matal closure and opening (Dong et al., 2001). Precise regulationof ADF activity is required for the correct balance between F-actinassembly and disassembly (Ressad et al., 1998; Bamburg, 1999).Exactly how ADFs regulate F-actin reorganization and how theactivity of ADFs is fine-tuned to meet the cellular demands duringstomatal closure andopening are interesting topics. Based onourexperimental results, we propose a brief model to explain howCKL2stabilizesactinfilaments topromoteactin reassemblyduringABA-induced stomatal closure (Figure 8). When ABA/drought-induced stomatal closure initiates, actin filaments begin to dis-assemble, which requires higher ADF activity. During this stage,CKL2 is accumulated due to upregulation by drought and ABA.The increased CKL2 level results in more phosphorylated ADFmolecules, which in turn reduces their actin filament binding andsevering activity, leading to actin arrays being reorganized intohighly bundled longcables. The resultingchanges inactinfilamentarchitecture promote stomatal closure. Furthermore, the stabili-zation of actin filaments would be maintained in the continuedpresence of ABA so that the stomata would remain closed.

Given that ADFs play an important role in actin filament dis-assemblyduringstomatal closure, how is their activity regulatedatthe early stage of ABA/drought response and reactivated after theactionofCKL2? It ispossible thataphosphatase(s) iscritical at thisstage by either activating ADFs or deactivating CKL2. PP2C isthought to be involved in actin reorganization in guard cells duringABA-induced stomatal closure. TheABA-insensitivemutant abi1-1 (aPP2Cmutant) displaysdisturbedactinfilament reorganizationin guard cells when treatedwith ABA (Eun et al., 2001; Hwang andLee, 2001; Lemichez et al., 2001). How PP2C cooperates withCKL2/ADFs to regulate actin dynamics in guard cells remains aninteresting question.

CKL2 associates with actin filaments but does not directly bindto theactinfilaments.We found thatABA induces the interactionofCKL2 with ADF4. Perhaps the interaction between CKL2 andADFs not only protects the actin filaments from severing by ADFs,but also plays a role in recruiting CKL2 to the actin filaments. Ourstudy suggests that Ser-6 in ADF4 is one of the residues phos-phorylated by CKL2; the other phosphorylated residues may playroles in regulation at different levels and stages for the full functionof ADF in modulating Arabidopsis stomatal closure.

The essential role of actin dynamics in regulating stomatalclosure and opening is well appreciated (Kim et al., 1995; Eun andLee, 1997; Hwang and Lee, 2001; MacRobbie and Kurup, 2007;Zhao et al., 2011; Jiang et al., 2012; Li et al., 2014), but how actindynamics are functionally coupled to the underlying cellular

processes in guard cells remains poorly understood. For instance,actin dynamics have been implicated in mediating Ca2+ concen-tration changes in the cytosol, K+ channel activity, and vacuoleshape determination (Hwang et al., 1997; Gao et al., 2009; Zhaoet al., 2013), but the related molecular mechanisms remain to beestablished. Onepossibility is regulation ofmembrane recycling byactin reorganization (Cheung et al., 2002; Lee et al., 2008; Zhaoetal., 2010;BouDaher andGeitmann, 2011;Zhuet al., 2013),whichwould change the localization of ion channels/transporters andthe area of vacuolar membrane. Actin dynamics may also act asan important component in signal transduction to mediate suchchanges (Gaoetal., 2008;Zhaoetal., 2011,2013;Jiangetal., 2012).

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana Col-0 was used as the wild type. The ckl2 mutant(Salk_104209) was obtained from the ABRC.

Primers used to confirm the homozygous mutant lines are listed inSupplemental Table 1. Arabidopsis seedlings were grown in soil under 8 hlight (light intensity of30mmolm–2 s–1)/16hdarkness (short-dayconditions)at 23°C and 60% relative humidity (RH) for 3 to 4 weeks. Nicotiana ben-thamiana plants were grown in soil under 16 h light/8 h darkness (long-dayconditions) at 23°C and 60% relative humidity for 3 to 4 weeks.

RNA Extraction and Real-Time Quantitative RT-PCR Analysis

Total RNAwas extracted from 7-d-old seedlings grown onMurashige andSkoog (MS) medium or the roots, stems, cauline leaves, rosettes, flowers,or siliques of 4-week-old plants grown in soil with Trizol reagent (Invi-trogen). Total RNA treated with RNase-free DNase I (Invitrogen) was usedfor reverse transcription with M-MLV reverse transcriptase (Promega).Real-time quantitative PCR was conducted using SYBR Premix Ex Taq(Takara). Elongation factor 1a (EF ) was used as an internal control. Theprimers used for RT-PCR or Real-time quantitative PCR are listed inSupplemental Table 1.

Water-Loss and Stomatal Aperture Assays

Todetect thestomatal closure in response toABAtreatment, rosette leavesof 5-week-old plants were detached and incubated in stomata-openingbuffer (containing 50 mM KCl, 10 mMCaCl2, and 10 mMMES, pH 6.15) ina growth chamber at 23°C under constant illumination. Stomatal apertureswere measured after adding 2 mM ABA for 0.5 h. The apertures of50 stomata were measured in three independent experiments.

For water-loss measurement, rosette leaves were detached from5-week-old plants. Theweight of the detached leaveswasmeasured every0.5 h.

Infrared Thermograph Imaging

To monitor the leaf temperature, thermal imaging was performed as de-scribed previously with slight modification (Xie et al., 2006). Well-watered3-week-old plants were transferred from a high humidity growth condition(RH 70%) to a low humidity condition (RH 40%). The leaf temperature wasrecorded by a thermal imaging camera after 3 d.

Subcellular Localization Assays

For subcellular localization analysis of GFP-CKL2, theCKL2 cDNA codingregion was amplified and cloned into the pCambia1205 vector (Zhao et al.,

Phosphorylation of ADF Proteins by CKL2 1433

2011) between the BamHI and EcoRI sites to generate Pro35S:GFP-CKL2. For the ProCKL2:GFP-CKL2 construct, the 1.06-kb CKL2 pro-moter fragment was amplified from BAC plasmid F18A22 (ABRC) andcloned into the pCAMBIA1305 vector (Zhao et al., 2011) between theHindIII and PstI sites, and then the CKL2 cDNA coding region wasamplified and cloned between the SalI and BamHI sites. The resultingPro35S:GFP-CKL2 and ProCKL2:GFP-CKL2 vectors were used totransform Col-0 and the ckl2mutant, respectively. The confocal imageswere taken from 6-d-old seedlings of the transgenic plants with a ZeissLSM 510Meta confocal microscope using a Plan-Apochromat 633/1.4oil immersion differential interference contrast lens. GFP was excited at488 nm.

Preparation of Suspension Cells

For suspension cell preparation, seedlings were grown on MS medium(4.43g/LMSsalt, 30 g/L sucrose, and0.3%agar) for 7 d. The leaveswerethen cut from the seedlings and cultured on MS medium with 1 mg/L2,4-D and 0.1 mg/L 6-benzylaminopurine to induce calli. The calli werecultured in the MS liquid medium with 1 mg/L 2,4-D on a rotor set to120 rpm in the dark at 23°C to induce suspension cells. The suspensioncells were subcultured in darkness for 3 weeks and then used for actinstaining assays.

Colocalization Analysis

For colocalization analysis, suspension cells generated from the 35Sp:GFP-CKL2 transgenic plants were incubated in actin staining buffer(0.18 mM rhodamine-phalloidin, 100 mM PIPES, 10 mM EGTA, 5 mMMgSO4, 5%DMSO, and 0.05%Nonidet P-40, pH 6.8) for 15min at roomtemperature. The samples were observed by confocal microscopy. Theimages were collected through visualizing the red/green fluorescencesignals from the GFP-CKL2 and the rhodamine-phalloidin-labeled actinfilaments. The colocalization between GFP-CKL2 and the actin cyto-skeleton was analyzed by calculating Pearson’s correlation coefficient(Dunn et al., 2011; Wu et al., 2012; McDonald and Dunn, 2013). Relativepixel intensity in the indicated regions of interest was measured usingImage J software. Thresholds were set manually to account for back-ground, and Pearson’s correlation coefficient was calculated to de-termine the correlation degree.

Confocal Laser Scanning Microscopy to Visualize Actin Filamentsin Vivo

To visualize actin filaments in plant cells, the Pro35S:GFP-fABD2-GFP construct was transformed into the wild type, the ckl2 mutant,the adf4-1 mutant, and the adf4 ckl2 double mutant. Six-day-oldseedlings of the transgenic lines grown on MS medium were used foractin filament visualization in hypocotyl cells and roots. Three-week-old plants were used for actin filament visualization in guard cells. Todetect the effect of LatA or ABA on actin filaments, the transgenicplants were treated with 200 nm LatA or 20 mM ABA for 0.5 h or 1 h.Then the actin cytoskeleton was visualized by detecting the GFPfluorescence signal in hypocotyl cells or guard cells with a Zeiss LSM510META confocalmicroscope using aPlan-Apochromat 633/1.4 oilimmersion differential interference contrast lens. GFP was excited at488 nm.

To observe actin filament dynamics in guard cells, time-lapse imageswerecapturedevery2swithanAndor iXoncharge-coupleddevicecamera.To observe actin filament dynamic behavior, parameters including maxi-mum filament lifetime, maximum filament length, elongation rate, severingfrequency, and depolymerization rate with single actin filament turnoverwere calculated using Image J software as described previously (Hentyet al., 2011; Qin et al., 2014).

Antibody Preparation and Immunoblotting

Anti-ADF4 antibodies were generated by immunizing mice with Es-cherichia coli-expressed ADF4. To determine the specificity of theantibodies in plants, total protein was isolated from the 7-d-old Col-0,the adf4 mutant (SALK_121647), the adf1 mutant (SALK_144459), andthe transgenic plants harboring the construct Pro35S:33Flag-ADF4 orPro35S:33Flag-ADF1 and subjected to immunoblot analysis with anti-ADF4 antibodies. The blots were probed with primary mouse anti-ADF4(diluted 1:1000).

Protein Purification and Pull-Down Assays

The coding regions of CKL2 and ADF4 were amplified and cloned into thevectors of PET28a and pGEX-6p-1, respectively. The N terminus of CKL2(295 amino acids, CKL2N) and the C terminus of CKL2 (170 amino acids,CKL2C) were cloned into the PET28a vector. The resulting vectors weretransformed into E. coli (strain BL21). The recombinant proteins werepurified with Ni-NTA agarose or glutathione sepharose.

For the pull-down assay, 5 mg His-CKL2, -CKL2N, or -CKL2C onNi-NTA agarose was incubated with 1 mg GST-ADF4 for 30 min at 4°C in100 mL binding buffer (20 mM Tris-HCl, pH 7.2, 10 mM MgCl2, and 2 mMDTT). After washing three times with the binding buffer, protein on theagarose was separated on 15% SDS-PAGE, followed by CoomassieBrilliant Blue R 250 staining. One microliter of agarose was analyzed byimmunoblot using anti-ADF antibodies.

For in vivo coimmunoprecipitation assays, theCKL2 coding region wasamplified and cloned into the pCM1307-N-Flag-HA vector between theXbaI and SalI sites. The resulting plasmid was transformed into wild-typeplants. Total protein was extracted from 7-d-old transgenic plants using2mL immunoprecipitation buffer (10 mMTris, pH 7.5, 0.5%Nonidet P-40,2mMEDTA,150mMNaCl, 1mMPMSF,and1%protease inhibitor cocktail[Sigma-Aldrich]). Flag-HA-CKL2 was purified with 30 mL anti-FLAG aga-rose (Sigma-Aldrich) from the total protein. After washing three times with2 mL immunoprecipitation buffer, the agarose was used for immunoblotassays with anti-ADFs antibodies.

Split-Luciferase Complementation Assays

For the split luciferase assays, the CKL2 and ADF4 cDNA coding regionswere amplified and cloned into the KpnI and SalI sites of the pCM1307-nLUC and pCM1307-cLUC vector, respectively. The plasmids were in-troduced into Agrobacterium tumefaciens GV3101 and coinfiltrated intothe leaves of N. benthamiana. After a 3-d incubation, the LUC activity wasmeasured using a cooled CCD imaging camera (1300B; Roper). Then,1 mM luciferin was sprayed onto the leaves. Relative LUC activity per cm2

infiltrated leaf area was calculated using Winview32 software. Each datacolumncontains at least 10 replicates, and three independent experimentswere performed.

Kinase Activity Assays

In vitro kinase activity assays were performed as described previously(Quan et al., 2007). Recombinant protein His-CKL2, His-ADF4, and His-ADF4S6A were purified with Ni-NTA agarose. One microgram of His-ADF4or His-ADF4S6A was incubated with 1 mg His-CKL2 in a kinase reactionbuffer (20 mM Tris-HCl, pH 8.0, 5 mMMgCl2, 10 mM ATP, 1 mM DTT, and2mCi [g-32P]ATP) in 15mL total volumeat 30°C for 30min. The reactionwasterminatedwith 23SDS loading buffer. After incubation at 100°C for 5min,the reaction products were separated on 15% SDS-PAGE and stainedusing Coomassie Brilliant Blue R 250. Then the SDS-PAGE gels wereexposed to a phosphor screen. The phosphor signals were detected bya Typhoon 9410 phosphor imager (Amersham Biosciences). Phosphory-lation signals were quantified using Image Quant 5.0 software.

1434 The Plant Cell

Two-dimensional SDS-PAGE was performed as described (Minamideet al., 1997; Dong et al., 2001). The Pro35S:33 Flag-ADF4 construct wastransformed into the wild type and ckl2mutant, respectively. Total proteinwas isolated from the resulting transgenic seedlings in extraction buffer(10mMTris, pH 7.5, 0.5%Nonidet P-40, 2mMEDTA, 150mMNaCl, 1mMPMSF, and 1% protease inhibitor cocktail [Sigma-Aldrich]). ADF4 proteinwas immunoprecipitated from the total proteins by incubation with anti-Flag agarose. For phosphatase treatment, theADF4proteinwas incubatedwith phosphatase (l phosphatase; New England Biolabs) at 30°C for15min. ImmunoblottingofADF4wasperformedwithananti-Flagantibody.

Quantification of GFP-fABD2-GFP Fluorescence Pixel Intensity,Density, and Skewness of the Actin Filaments in Guard Cells

Measurement of relative fluorescence pixel intensity levels of GFP-fABD2-GFP-labeled actin filaments in guard cells and hypocotyl epidermal cellswas performed as described previously (Huang et al., 2005). The averagefluorescence pixel intensity associated with GFP-fABD2-GFP was mea-sured using ImageJ software (http://rsb.info.nih.gov/ij/) to determine therelative amount of actin filaments. The skewness and density of actin fil-aments were quantified by ImageJ according to Higaki et al. (2010) withslight modification. Individual cells were segmented manually and actinfilaments at the cell border were eliminated. More than 50 guard cells wereused for the analysis.

Biochemical Assays to Determine the Effect of CKL2 onActin Polymerization

High-speed cosedimentation assays were performed to examine thebinding of CKL2 to actin filaments as previously described (Kovar et al.,2000; Huang et al., 2005). Direct visualization of actin filaments by epi-fluorescence light microscopy was performed as described previously(Okada et al., 2002). A spontaneous actin nucleation assay was performedaccording to previously published methods (Amann and Pollard, 2001;Huang et al., 2003; Andrianantoandro and Pollard, 2006). Various con-centrations of CKL2 were incubated with G-actin (2 mM 10% pyrene-labeled) for 5 min at room temperature. Actin polymerization was initiatedafter the addition of 1/10 volumeof 103KMEI (500mMKCl, 10mMMgCl2,10 mM EGTA, and 100 mM imidazole-HCl, pH 7.0). Pyrene fluorescencewas monitored using a QuantaMaster Luminescence QM 3 PH Fluo-rometer (Photo Technology International) with the excitation and emissionwavelength set at 365 and 407 nm, respectively.

Determination of the Effect of CKL2 Phosphorylation on ADF4-Mediated Actin Disassembly in Vitro

All proteins and buffers were preclarified by centrifugation at 200,000g for1 h at 4°C. Equal molar amounts of ADF4 and CKL2 were used for thephosphorylation reaction as described above. G-actin (2 mM, 10%pyrenelabeled)waspolymerizedat25°C for 2h inpolymerizationbuffer (describedabove). The resulting assembled actin filaments from500 nMG-actin werethen incubated with 500 nM nonphosphorylated ADF4 or 500 nM CKL2-phosphorylated ADF4 in 13 KMEI to induce actin filament disassembly.Actin disassembly was traced by monitoring the decrease in pyrenefluorescence for 30 min using a QuantaMaster Luminescence QM 3 PHFluorometer (Photon Technology International) with the excitation andemission wavelengths set at 365 and 407 nm, respectively.

Direct Visualization of Actin Filament Severing in Vitro byTIRF Microscopy

Single actin filament severing was directly visualized by TIRF microscopyas described previously (Amann and Pollard, 2001; Andrianantoandro andPollard, 2006; Jansen et al., 2014). The flow chamber was prepared as

described previously (Amann and Pollard, 2001; Jansen et al., 2014). Foractin-filament-severing assays, the assembled flow chamber was in-cubated with 25 nM N-ethylmaleimide-myosin for 5 min followed bywashing with 1% BSA for 3 min. Next, TIRF microscopy buffer (10 mMimidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 50 mM DTT,0.2 mM ATP, 50 mM CaCl2, 15 mM glucose, 20 mg/mL catalase,100 mg/mL glucose oxidase, and 0.5%methylcellulose) was injected intothe prepared flow chamber. Actin filaments (from 1 mM G-actin, 50%Oregon-Green labeled) in TIRF buffer were injected into the chamber andincubated in darkness for 5 min. Finally, TIRF buffer was injected into thechamber to remove attached actin filaments. After injection of ADF4 orCKL2-phosphorylated ADF4 into the flow chamber, single actin filamentsevering events were observed by time-lapse TIRF microscopy using anOlympus IX81 microscope equipped with a 3100 oil objective (1.49 nu-merical aperture). Time-lapse images were recorded every 3 s for 5 min.Actin filament severing frequencywas calculated as themaximum filamentlength divided by the number of breaks per filament length over time(breaks/mm/s). More than 30 actin filaments with length >10 mm wereselected for quantification under each condition.

Statistical Analysis

To test the data normality of continuous variables, statistical analysis wasperformed using the SPSS for Windows software package (version 11.5;IBM), and the Shapiro-Wilk test was applied. Relative values are means6SD or means 6 SE. All statistical analysis was performed using two-tailedStudent’s t test to determine group differences in means using GraphPadPrism 6.0 software or Kaleida Graph 4.1 (Synergy Software). Significantdifferences were indicated by different lowercase letters and the thresholdwas set at 0.01.

Accession Numbers

Sequence data in this study can be found in the Arabidopsis GenomeInitiative database under the following accession numbers: CKL2,At1g72710; ADF1, At3g46010; ADF4, At5g59890; EF1a, At5g60390;RD29A, At5g52310; and SCaBP8, At4g33000.

Supplemental Data

Supplemental Figure 1. Identification of the T-DNA Insertion in theckl2 Mutant.

Supplemental Figure 2. Expression of CKL2.

Supplemental Figure 3. CKL2 Colocalizes with Actin Filaments.

Supplemental Figure 4. CKL2 Does Not Bind to F-Actin Directly andHas No Effect on Actin Polymerization in Vitro.

Supplemental Figure 5. CKL2 and ADF4 Expression in Guard Cells.

Supplemental Figure 6. CKL2 Interacts with ADF4.

Supplemental Figure 7. Analysis of ADF Protein Level in TransgenicSeedlings.

Supplemental Table 1. Primers Used in This Study.

Supplemental Movie 1. Time-Series Movie Displaying the SeveringEvents of Single Filaments in Col-0 Guard Cell.

Supplemental Movie 2. Time-Series Movie Displaying the SeveringEvents of Single Filaments in the ckl2 Mutant Guard Cell.

Supplemental Movie 3. Time-Series Movie of Actin Filament Severingin the Absence of ADF4.

Supplemental Movie 4. Time-Series Movie of Actin Filament Severingin the Presence of Nonphosphorylated ADF4.

Phosphorylation of ADF Proteins by CKL2 1435

Supplemental Movie 5. Time-Series Movie of Actin Filament Severingin the Presence of CKL2-Phosphorylated ADF4.

Supplemental Movie Legends.

ACKNOWLEDGMENTS

We thank Christopher J. Staiger (Department of Biological Sciences,Purdue University) for kindly providing the Arabidopsis seeds of adf4-1(GARLIC_823_A11.b.1b.Lb3Fa), 35Sp:ADF4; adf4, and ABRC for ckl2(Salk_104209) and adf4-2 seeds. We thank Nancy Hofmann at PlantEditors for English editing. This work was supported by the National BasicResearch Program of China (Grant 2015CB910202 to Y.G.), the NationalNatural Science Foundation of China (Grant 31430012 to Y.G.), andFoundation for Innovative ResearchGroup of the National Natural ScienceFoundation of China (Grant 31121002).

AUTHOR CONTRIBUTIONS

S.Z., Y.J., andY.Z. performed the research. S.Z., Y.X.Z., andY.G. designedthe research and analyzed the data. Y.Z. contributed to the purification ofrecombinant proteins. Y.J. and S.H. performed the research on analysis ofsingle actin filament severing. S.Z., Y.G., S.H.,M.Y., andY.X.Z. contributedto the discussion and wrote the article.

Received February 3, 2016; revisedMay 12, 2016; accepted June 6, 2016;published June 7, 2016.

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Phosphorylation of ADF Proteins by CKL2 1437

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Phosphorylation of ADF Proteins by CKL2 1439

DOI 10.1105/tpc.16.00078; originally published online June 7, 2016; 2016;28;1422-1439Plant Cell

GuoShuangshuang Zhao, Yuxiang Jiang, Yang Zhao, Shanjin Huang, Ming Yuan, Yanxiu Zhao and Yan

via Phosphorylation of Actin Depolymerizing FactorCASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure

 This information is current as of September 21, 2020

 

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References /content/28/6/1422.full.html#ref-list-1

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