Download Casein Kinase1-Like Protein2 Regulates Actin Filament

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. The data sets were tested for normal
1053
distribution by Shapiro-Wilk test. Statistical significance was determined by Student’s
1054
t-test; significant differences (P < 0.01) are indicated by different lowercase letters.
1055
1056
Figure 8. A Simplified Working Model for the Role of CKL2 and ADF4 in
1057
Regulating Actin Reorganization during ABA-Induced Stomatal Closure.
1058
During stomatal closure, microfilaments reorganize, first disassembling and then
1059
reassembling. ABA/drought-induced CKL2 represses ADF activity to stabilize
1060
microfilaments and keep stomata closed.
43
Parsed Citations
Agnew, B.J., Minamide, L.S., and Bamburg, J.R. (1995). Reactivation of phosphorylated actin depolymerizing factor and
identification of the regulatory site. J. Biol. Chem. 270, 17582-17587.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Akashi, M., Tsuchiya, Y., Yoshino, T., and Nishida, E. (2002). Control of intracellular dynamics of mammalian period proteins by
casein kinase I epsilon (CKIepsilon) and CKI delta in cultured cells. Mol Cell Biol. 22, 1693-1703.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Allwood, E.G., Anthony R.G., Smertenko A.P., Reichelt S., Drobak B.K., Doonan J.H., Weeds A.G., and Hussey P.J. (2002).
Regulation of the pollen-specific actin-depolymerizing factor LlADF1. Plant Cell 14, 2915-2927.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Allwood, E.G., Smertenko, A.P., and Hussey, P.J. (2001). Phosphorylation of plant actin-depolymerising factor by calmodulin-like
domain protein kinase. FEBS Lett 499, 97-100.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Amann, K.J., and Pollard, T.D. (2001). Direct real-time observation of actin filament branching mediated by Arp2/3 complex using
total internal reflection fluorescence microscopy. Proc. Natl. Acad. Sci. USA 98, 15009-15013.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Andrianantoandro, E., and Pollard, T.D. (2006). Mechanism of actin filament turnover by severing and nucleation at different
concentrations of ADF/cofilin. Mol cell 24, 13-23.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bamburg, J.R. (1999). Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu Rev Cell Dev Biol. 15, 185230.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ben-Nissan, G., Cui, W., Kim, D.J., Yang, Y., Yoo, B.C., and Lee, J.Y. (2008). Arabidopsis Casein Kinase 1-Like 6 contains a
microtubule-binding domain and affects the organization of cortical microtubules. Plant Physiol. 148, 1897-1907.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bernard, O. (2007). Lim kinases, regulators of actin dynamics. Int J. Biochem Cell Biol. 39, 1071-1076.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bernstein, B.W., Painter, W.B., Chen, H., Minamide, L.S., Abe, H., and Bamburg, J.R. (2000). Intracellular pH modulation of
ADF/cofilin proteins. Cell Motil. Cytoskeleton 47, 319-336.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Boesger, J., Wagner, V., Weisheit, W., and Mittag, M. (2014). Comparative phosphoproteomics to identify targets of the clockrelevant casein kinase 1 in C. reinhardtii Flagella. Methods Mol Biol 1158, 187-202.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bou Daher, F., and Geitmann, A. (2014). Actin is involved in pollen tube tropism through redefining the spatial targeting of
secretory vesicles. Traffic 12, 1537-1551.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Chen, C.Y., Wong E.I., Vidali, L., Estavillo, A., Hepler, P.K., Wu, H.M., and Cheung, A.Y. (2002). The regulation of actin organization
by actin-depolymerizing factor in elongating pollen tubes. Plant Cell 14, 2175-2190.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cheong, J.K., and Virshup, D.M. (2011). Casein kinase 1: Complexity in the family. Int J Biochem Cell Biol 43, 465-469.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cheung, A.Y., Chen, C.Y., Glaven, R.H., de Graaf, B.H., Vidali, L., Hepler, P.K., and Wu, H.M. (2002). Rab2 GTPase regulates vesicle
trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth. Plant Cell 14, 945-962.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Clement, M., Ketelaar, T., Rodiuc, N., Banora, M.Y., Smertenko, A., Engler, G., Abad, P., Hussey, P.J., and de Almeida Engler, J.
(2009). Actin-depolymerizing factor2-mediated actin dynamics are essential for root-knot nematode infection of Arabidopsis. Plant
cell 21, 2963-2979.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cui, Y., Ye, JZ., Guo, XH., Chang, HP., Yuan, CY., Wang, Y., Hu, S., Liu, XM., Li, XS.,. (2012). Arabidopsis casein kinase 1-like 2
involved in abscisic acid signal transduction pathways. J. Plant Inter 9, 19-25.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Davidson, M.M., Haslam, R.J. (1994). Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+.
Biochemical J. 301, 41-47.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Desikan, R., Cheung, M.K., Bright, J., Henson, D., Hancock, J.T., and Neill, S.J. (2004). ABA, hydrogen peroxide and nitric oxide
signalling in stomatal guard cells. J. Exp. Bot. 55, 205-212.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dong, C.H., Xia, G.X., Hong, Y., Ramachandran, S., Kost, B., and Chua, N.H. (2001). ADF proteins are involved in the control of
flowering and regulate F-actin organization, cell expansion, and organ growth in Arabidopsis. Plant Cell 13, 1333-1346.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dong, C.H., and Hong, Y. (2013). Arabidopsis CDPK6 phosphorylates ADF1 at N-terminal serine 6 predominantly. Plant Cell Rep 32,
1715-1728.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Du, M., Zhai, Q., Deng, L., Li, S., Li, H., Yan, L., Huang, Z., Wang, B., Jiang, H., Huang, T., Li, C. B., Wei, J., Kang, L., Li, J., Li, C.
(2014). Closely related NAC transcription factors of tomato differentially regulate stomatal closure and reopening during pathogen
attack. Plant cell 26, 3167-3184.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dunn, K.W., Kamocka, M.M., McDonald, J.H. (2011). A practical guide to evaluating colocalization in biological microscopy. Am J
Physiol Cell Physiol 300, C723-742.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Eun, S.O., and Lee, Y. (1997). Actin filaments of guard cells are reorganized in response to light and abscisic acid. Plant Physiol.
115, 1491-1498.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Eun, S.O., and Lee, Y. (2000). Stomatal opening by fusicoccin is accompanied by depolymerization of actin filaments in guard cells.
Planta 210, 1014-1017.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Eun, S.O., Bae, S.H., and Lee, Y. (2001). Cortical actin filaments in guard cells respond differently to abscisic acid in wild-type and
abi1-1 mutant Arabidopsis. Planta 212, 466-469.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Fuglsang, A.T., Guo, Y., Cuin, T.A., Qiu, Q., Song, C., Kristiansen, K.A., Bych, K., Schulz, A., Shabala, S., Schumaker, K.S., Palmgren,
M.G., and Zhu, J.K. (2007). Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction
with 14-3-3 protein. Plant cell 19, 1617-1634.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gao, X.Q., Chen, J., Wei, P.C., Ren, F., Chen, J., and Wang, X.C. (2008). Array and distribution of actin filaments in guard cells
contribute to the determination of stomatal aperture. Plant Cell Rep 27, 1655-1665.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gao, X.Q., Wang, X.L., Ren, F., Chen, J., Wang, X.C. (2009). Dynamics of vacuoles and actin filaments in guard cells and their roles
in stomatal movement. Plant Cell Environ. 32, 1108-1116.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gayatri, G., Agurla, S., and Raghavendra, A.S. (2013). Nitric oxide in guard cells as an important secondary messenger during
stomatal closure. Front Plant Sci 4, 425.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gross, S.D., Hoffman D.p., Fisette, P.L., Baas, P., and Anderson, R.A. (1995). A phosphatidylinositol 4,5-bisphosphate-sensitive
casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins. J. Cell Biol. 130, 711-724.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Henty-Ridilla, J.L., Li, J., Blanchoin, L., and Staiger, C.J. (2013). Actin dynamics in the cortical array of plant cells. Curr. Opin. Plant
Biol. 16, 678-687.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Henty-Ridilla, J.L., Li, J., Day, B., and Staiger, C.J. (2014). ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during
innate immune signaling in Arabidopsis. Plant cell 26, 340-352.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Henty, J.L., Bledsoe, S.W., Khurana, P., Meagher, R.B., Day, B., Blanchoin, L., and Staiger, C.J. (2011). Arabidopsis actin
depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells. Plant
cell 23, 3711-3726.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Higaki, T., Sano, T., and Hasezawa, S. (2007). Actin microfilament dynamics and actin side-binding proteins in plants. Curr. Opin.
Plant Biol. 10, 549-556.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Higaki, T., Kutsuna, N., Sano, T., Kondo, N., and Hasezawa, S. (2010). Quantification and cluster analysis of actin cytoskeletal
structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells. Plant J. 61,
156-165.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hirayama, T., and Shinozaki, K. (2007). Perception and transduction of abscisic acid signals: keys to the function of the versatile
plant hormone ABA. Trends Plant Sci. 12, 343-351.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hosy, E., Vavasseur, A., Mouline, K., Dreyer, I., Gaymard, F., Poree, F., Boucherez, J., Lebaudy, A., Bouchez, D., Very, A.A.,
Simonneau, T., Thibaud, J.B., and Sentenac, H. (2003). The Arabidopsis outward K+ channel GORK is involved in regulation of
stomatal movements and plant transpiration. Proc. Natl. Acad. Sci. USA 100, 5549-5554.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hotulainen, P., Paunola, E., Vartiainen, M.K., and Lappalainen, P. (2005). Actin-depolymerizing Factor and Cofilin-1 play overlapping
roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells. Mol. Biol. Cell 16, 649-664.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hua, D., Wang, C., He, J., Liao, H., Duan, Y., Zhu, Z., Guo, Y., Chen, Z., and Gong, Z. (2012). A plasma membrane receptor kinase,
GHR1, mediates abscisic acid- and hydrogen peroxide-regulated stomatal movement in Arabidopsis. Plant cell 24, 2546-2561.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Huang, S., Robinson, R.C., Gao, L.Y., Matsumoto, T., Brunet, A., Blanchoin, L., and Staiger, C.J. (2005). Arabidopsis VILLIN1
generates actin filament cables that are resistant to depolymerization. Plant cell 17, 486-501.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Huang, W., Anvari B., Torres, J.H., LeBaron, R.G., and Athanasiou, K.A. (2003). Temporal effects of cell adhesion on mechanical
characteristics of the single chondrocyte. J Orthop Res. 21, 88-95.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hussey, P.J., Ketelaar, T., and Deeks, M.J. (2006). Control of the actin cytokeleton in plant cell growth. Annu. Rev. Plant Biol. 57,
109-125.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hwang, J.U., and Lee, Y. (2001). Abscisic Acid-induced actin reorganization in guard cells of dayflower is mediated by cytosolic
calcium levels and by protein kinase and protein phosphatase activities. Plant Physiol. 125, 2120-2128.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hwang, J.U., Suh, S., Yi, H., Kim, J., and Lee, Y. (1997). Actin filaments modulate both stomatal opening and inward K+-channel
activities in guard cells of Vicia faba L. Plant Physiol. 115, 335-342.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ishitani, M., Xiong, L., Stevenson, B., and Zhu, J.K. (1997). Genetic analysis of osmotic and cold stress signal transduction in
Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant cell 9, 19351949.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Israelsson, M., Siegel, R.S., Young, J., Hashimoto, M., Iba, K., and Schroeder, J.I. (2006). Guard cell ABA and CO2 signaling network
updates and Ca2+ sensor priming hypothesis. Curr. Opin. Plant Biol. 9, 654-663.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lee, Y.J., Szumlanski, A., Nielsen, E., and Yang, Z. (2008). Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle
targeting and exocytosis during tip growth. J. Cell Biol. 181, 1155-1168.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jansen, S., Collins, A., Golden, L., Sokolova, O., Goode, B.L. (2014). Structure and mechanism of mouse cyclase-associated protein
(CAP1) in regulating actin dynamics. J. Biol. Chem. 289, 30732-30742.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jiang, K., Sorefan, K., Deeks, M.J., Bevan, M.W., Hussey, P.J., and Hetherington, A.M. (2012). The ARP2/3 complex mediates guard
cell actin reorganization and stomatal movement in Arabidopsis. Plant cell 24, 2031-2040.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kanamori, T., Hayakawa T., Suzuki, M., and Titani, K. (1995). Identification of two 17-kDa rat parotid gland phosphoproteins,
subjects for dephosphorylation upon beta-adrenergic stimulation, as destrin- and cofilin-like proteins. J. Biol. Chem. 270, 80618067.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ketelaar, T., Allwood, E.G., Anthony, R., Voigt, B., Menzel, D., and Hussey, P.J. (2004). The Actin-interacting protein AIP1 is
essential for actin organization and plant development. Curr. Biol. 14, 145-149.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kim, M., Hepler, P.K., Eun, S.O., Ha, K.S., and Lee, Y. (1995). Actin filaments in mature guard cells are radially distributed and
involved in stomatal movement. Plant Physiol. 109, 1077-1084.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kim, T.H., Bohmer, M., Hu, H., Nishimura, N., and Schroeder, J.I. (2010). Guard cell signal transduction network: advances in
understanding abscisic acid, CO2, and Ca2+ signaling. Annu. Rev. Plant Biol. 61, 561-591.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Knippschild, U., Gocht A., Wolff, S., Huber, N., Lohler, J., Lohler J., and Stoter, M. (2005). The casein kinase 1 family: participation in
multiple cellular processes in eukaryotes. Cell Signal. 17, 675-689.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Knippschild, U., Kruger, M., Richter, J., Xu, P., Garcia-Reyes, B., Peifer, C., Halekotte, J., Bakulev, V., and Bischof, J. (2014). The
CK1 Family: Contribution to cellular stress response and its role in carcinogenesis. Front Oncol 4, 1-17.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kollist, H., Nuhkat, M., and Roelfsema, M.R. (2014). Closing gaps: linking elements that control stomatal movement. New Phyto 203,
44-62.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kovar, D.R., Staiger, C.J., Weaver, E.A., and McCurdy, D.W. (2000). AtFim1 is an actin filament crosslinking protein from Arabidopsis
thaliana. Plant J. 24, 625-636.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lemichez, E., Wu, Y., Sanchez, J.P., Mettouchi, A., Mathur, J., and Chua, N.H. (2001). Inactivation of AtRac1 by abscisic acid is
essential for stomatal closure. Genes Dev. 15, 1808-1816.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li, J., Blanchoin, L., and Staiger, C.J. (2013). Signaling to Actin Stochastic Dynamics. Annu. Rev. Plant Biol. 66, 1411-1425.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li, X., Li, JH., Wang, W., Chen, NZ., Ma, TS., Xi, YN., Zhang, XL., Lin, HF., Bai Y., Huang, SJ., Chen, YL.,. (2014). ARP2/3 complexmediated actin dynamics is required for hydrogen peroxide-induced stomatal closure in Arabidopsis. Plant Cell Environ 37, 15481560.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li, Y., Shen, Y., Cai, C., Zhong, C., Zhu, L., Yuan, M., and Ren, H. (2010). The type II Arabidopsis formin14 interacts with
microtubules and microfilaments to regulate cell division. Plant cell 22, 2710-2726.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lin, H., Yang, Y., Quan, R., Mendoza, I., Wu, Y., Du, W., Zhao, S., Schumaker, K.S., Pardo, J.M., and Guo, Y. (2009). Phosphorylation
of SOS3-LIKE CALCIUM BINDING PROTEIN8 by SOS2 Protein Kinase Stabilizes Their Protein Complex and Regulates Salt
Tolerance in Arabidopsis. Plant Cell 21, 1607-1619.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Maciver, S.K., and Hussey, P.J. (2002). The ADF/cofilin family: actin-remodeling proteins. Genome Biol 3, 3001-3012.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
MacRobbie, E.A., and Kurup, S. (2007). Signalling mechanisms in the regulation of vacuolar ion release in guard cells. New Phyto.
175, 630-640.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mcdonald, J.H., Dunn, K.W. (2013). Statistical tests for measures of colocalization in biological microscopy. J. Microsc. 252, 295-302.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Merlot, S., Mustilli, A.-C., Genty, B., North, H., Lefebvre, V., Sotta, B., Vavasseur, A., and Giraudat, J. (2002). Use of infrared thermal
imaging to isolate Arabidopsis mutants defective in stomatal regulation. Plant J. 30, 601-609.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Minamide, L.S., Painter, W.B., Schevzov, G., Gunning, P., and Bamburg, J.R. (1997). Differential regulation of actin depolymerizing
factor and cofilin in response to alterations in the actin monomer pool. J. Biol. Chem. 272, 8303-8309.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mizuno, K. (2013). Signaling mechanisms and functional roles of cofilin phosphorylation and dephosphorylation. Cell. Signal. 25,
457-469.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Moriyama, K., and Yahara, I. (2002). The actin-severing activity of cofilin is exerted by the interplay of three distinct sites on cofilin
and essential for cell viability. J. Biochem. 365, 147-155.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Moriyama, K., Iida K., and Yahara, I. (1996). Phosphorylation of Ser-3 of cofilin regulates its essential function on actin. Genes Cells
1, 73-86.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mun, J.H., Yu H.J., Lee, H.S., Kwon, Y.M., Lee, J.S., Lee, I., and Kim, S.G. (2000). Two closely related cDNAs encoding actindepolymerizing factors of petunia are mainly expressed in vegetative tissues. Gene 257, 167-176.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Nagaoka, R., Abe H., and Obinata, T. (1996). Site-directed mutagenesis of the phosphorylation site of cofilin: its role in cofilin-actin
interaction and cytoplasmic localization. Cell Motil. Cytoskeleton 35, 200-209.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Okada K., B.L., Abe H, Chen H, Pollard TD, Bamburg JR. (2002). Xenopus Actin-interacting Protein 1 (XAip1) enhances cofilin
fragmentation of filaments by capping filament ends. J. Biol. Chem. 277, 43011-43016.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ono, S. (2003). Regulation of Actin Filament Dynamics by Actin Depolymerizing Factor/Cofilin and Actin-Interacting Protein 1:? New
Blades for Twisted Filaments†. Biochemistry 42, 13363-13370.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ouellet, F., Carpentier E., Cope, M.J., Monroy, A.F., and Sarhan, F. (2001). Regulation of a wheat actin-depolymerizing factor during
cold acclimation. Plant Physio. 125, 360-368.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Pei, Z.M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen, G.J., Grill, E., and Schroeder, J.I. (2000). Calcium channels
activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406, 731-734.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Peng, Y., Grassart, A., Lu, R., Wong, C.C.L., III, J.Y., Barnes, G., Drubin, D.G.,. (2015). Casein Kinase 1 promotes initiation of
clathrin-mediated endocytosis. Developmental Cell 32, 231-240.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Qin, T., Liu, X., Li, J., Sun, J., Song, L., and Mao, T. (2014). Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube
growth by severing actin filaments. Plant cell 26, 325-339.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Qu, X., Zhang, H., Xie, Y., Wang, J., Chen, N., and Huang, S. (2013). Arabidopsis villins promote actin turnover at pollen tube tips
and facilitate the construction of actin collars. Plant cell 25, 1803-1817.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Quan, R., Lin, H., Mendoza, I., Zhang, Y., Cao, W., Yang, Y., Shang, M., Chen, S., Pardo, J.M., and Guo, Y. (2007). SCABP8/CBL10, a
Putative Calcium Sensor, Interacts with the Protein Kinase SOS2 to Protect Arabidopsis Shoots from Salt Stress. Plant Cell 19,
1415-1431.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ressad, F.R., Didry, D., Xia, G.X., Hong, Y., Chua, N.H. (1998). Kinetic analysis of the interaction of actin-depolymerizing factor
(ADF)/Cofilin with G- and F-actin. J. Biol. Chem. 273, 20894-20902.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Roelfsema, M.R., and Hedrich, R. (2005). In the light of stomatal opening: new insights into 'the Watergate'. New phyto. 167, 665691.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ruzicka, D.R., Kandasamy, M.K., McKinney, E.C., Burgos-Rivera, B., and Meagher, R.B. (2007). The ancient subclasses of
Arabidopsis Actin Depolymerizing Factor genes exhibit novel and differential expression. Plant J. 52, 460-472.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Samstag, Y., Eckerskorn C., Wesselborg, S., Henning, S., Wallich R., and Meuer, S.C. (1994). Costimulatory signals for human T-cell
activation induce nuclear translocation of pp19/cofilin. Proc. Natl. Acad. Sci. USA 91, 4494-4498.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M., Scholkopf, B., Weigel, D., Lohmann, J.U. (2005). A gene
expression map of Arabidopsis thaliana development. Nature Gene. 37, 501-507.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schroeder, J.I., Kwak, J.M., and Allen, G.J. (2001). Guard cell abscisic acid signalling and engineering drought hardiness in plants.
Nature 410, 327-330.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shi, M., Xie, Y., Zheng, Y., Wang, J., Su, Y., Yang, Q., and Huang, S. (2013). Oryza sativaactin-interacting protein 1 is required for
rice growth by promoting actin turnover. Plant J. 73, 747-760.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shibayama T., Shinkawa, K., Nakajo, S., Nakaya, K., and Nakamura, Y. (1986). Phosphorylation of muscle and non-muscle actins by
casein kinase 1 in vitro. Biochem Int 13, 367-373.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Smertenko, A.P., Deeks, M.J., and Hussey, P.J. (2010). Strategies of actin reorganisation in plant cells. J Cell Sci 123, 3019-3028.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Smertenko, A.P., Jiang, C.J., Simmons, N.J., Weeds, A.G., Davies, D.R., and Hussey, P.J. (1998). Ser6 in the maize actindepolymerizing factor, ZmADF3, is phosphorylated by a calcium-stimulated protein kinase and is essential for the control of
functional activity. Plant J. 14, 187-193.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Spudich, J.A., and Watt, S. (1971). The Regulation of Rabbit Skeletal Muscle Contraction: I. Biochemical Studies of the interaction
of the tropomyosin-troponin complex wirh actin and the proteolytic fragments of myosin. J. Biol. Chem. 246, 4866-4871.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Staiger, C.J., and Blanchoin, L. (2006). Actin dynamics: old friends with new stories. Curr. Opin. Plant Biol. 9, 554-562.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Su, H., Zhu, J., Cai, C., Pei, W., Wang, J., Dong, H., and Ren, H. (2012). FIMBRIN1 is involved in lily pollen tube growth by stabilizing
the actin fringe. Plant cell 24, 4539-4554.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Thomas, C., Tholl, S., Moes, D., Dieterle, M., Papuga, J., Moreau, F., and Steinmetz, A. (2009). Actin bundling in plants. Cell Motil.
Cytoskeleton 66, 940-957.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tian, M., Chaudhry, F., Ruzicka, D.R., Meagher, R.B., Staiger, C.J., and Day, B. (2009). Arabidopsis actin-depolymerizing factor
AtADF4 mediates defense signal transduction triggered by the Pseudomonas syringae effector AvrPphB. Plant Physio. 150, 815824.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Toshima, J., Toshima, J.Y., Takeuchi, K., Mori, R., and Mizuno, K. (2001). Cofilin phosphorylation and actin reorganization activities
of testicular protein kinase 2 and its predominant expression in testicular Sertoli cells. J. Biol. Chem. 276, 31449-31458.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Vavasseur, A., and Raghavendra, A.S. (2005). Guard cell metabolism and CO2 sensing. New Phytol. 165, 665-682.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang, P., and Song, C.P. (2008). Guard-cell signalling for hydrogen peroxide and abscisic acid. New Phytol. 178, 703-718.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang, C., Zheng, Y., Zhao, Y., Zhao, Y., Li, J., Guo, Y. (2015). SCAB3 is required for reorganization of actin filaments during light
quality changes. J Genet Genomics. 42, 161-168.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wasteneys, G.O., and Galway, M.E. (2003). Remodeling the cytoskeleton for growth and form: an overview with some new views.
Annu. Rev. Plant Biol. 54, 691-722.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Winter, D., Vinegar, B., Nahal, H., Ammar, R., Wilson, G.V., Provart, N.J. (2007). An "Electronic Fluorescent Pictograph" browser for
exploring and analyzing large-scale biological data sets. PLoS ONE 2, e718.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wu, Y., Zinchuk, V., Grossenbacher-Zinchuk, O., and Stefani, E. (2012). Critical evaluation of quantitative colocalization analysis in
confocal fluorescence microscopy. Interdiscip Sci. 4, 27-37.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xie, X., Wang, Y., Williamson, L., Holroyd, G.H., Tagliavia, C., Murchie, E., Theobald, J., Knight, M.R., Davies, W.J., Leyser, H.M.O.,
and Hetherington, A.M. (2006). The identification of genes involved in the stomatal response to reduced atmospheric relative
humidity. Curr. Biol. 16, 882-887.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yonezawa, N., Nishida, E., Iida, K., Yahara, I., and Sakai, H. (1990). Inhibition of the interactions of cofilin, destrin, and
deoxyribonuclease I with actin by phosphoinositides. J. Biological Chem. 265, 8382-8386.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhao, Y., Yan, A., Feijo, J.A., Furutani, M., Takenawa, T., Hwang, I., Fu, Y., and Yang, Z. (2010). Phosphoinositides regulate clathrindependent endocytosis at the tip of pollen tubes in Arabidopsis and tabacco. Plant cell 22, 4031-4044.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhao, Y., Pan, Z., Zhang, Y., Qu, X., Zhang, Y., Yang, Y., Jiang, X., Huang, S., Yuan, M., Schumaker, K.S., and Guo, Y. (2013). The
actin-related Protein2/3 complex regulates mitochondrial-associated calcium signaling during salt stress in Arabidopsis. Plant cell
25, 4544-4559.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhao, Y., Zhao, S., Mao, T., Qu, X., Cao, W., Zhang, L., Zhang, W., He, L., Li, S., Ren, S., Zhao, J., Zhu, G., Huang, S., Ye, K., Yuan, M.,
and Guo, Y. (2011). The plant-specific actin binding protein SCAB1 stabilizes actin filaments and regulates stomatal movement in
Arabidopsis. Plant cell 23, 2314-2330.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhao, S., Zhao, Y., Guo, Y. (2015). 14-3-3 ? protein interacts with ADF1 to regulate actin cytoskeleton dyanmics in Arabidopsis. Sci
China Life Sci. 58, 1142-1150.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zheng, Y., Xie, Y., Jiang, Y., Qu, X., and Huang, S. (2013). Arabidopsis actin-depolymerizing factor7 severs actin filaments and
regualtes actin cable turnover to promote normal pollen tube growth. Plant Cell 25, 3405-3423.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhu, L., Zhang, Y., Kang, E., Xu, Q., Wang,M., Rui, Y., Liu, B., Yuan, M., and Fu, Y. (2013). MAP18 regulates the direction of pollen
tube growth in Arabidopsis by modulating F-actin organization. Plant Cell 25, 851-867.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Casein Kinase1-Like Protein2 Regulates Actin Filament Stability and Stomatal Closure via
Phosphorylation of Actin Depolymerizing Factor
Yan Guo, Shuangshuang Zhao, Yuxiang Jiang, Yang Zhao, Shanjin Huang, Ming Yuan and Yanxiu Zhao
Plant Cell; originally published online June 7, 2016;
DOI 10.1105/tpc.16.00078
This information is current as of August 3, 2017
Supplemental Data
/content/suppl/2016/06/07/tpc.16.00078.DC1.html
/content/suppl/2016/06/16/tpc.16.00078.DC2.html
/content/suppl/2016/06/30/tpc.16.00078.DC3.html
Permissions
https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X
eTOCs
Sign up for eTOCs at:
http://www.plantcell.org/cgi/alerts/ctmain
CiteTrack Alerts
Sign up for CiteTrack Alerts at:
http://www.plantcell.org/cgi/alerts/ctmain
Subscription Information
Subscription Information for The Plant Cell and Plant Physiology is available at:
http://www.aspb.org/publications/subscriptions.cfm
© American Society of Plant Biologists
ADVANCING THE SCIENCE OF PLANT BIOLOGY