Plant Physiology Preview. Published on May 1, 2014, as DOI:10.1104/pp.113.230268
1
Running head:
2
A rice CPK gene involved abiotic stress tolerance
3
4
5
6
Corresponding author:
7
Blanca San Segundo
8
Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB.
9
Edifici CRAG, Campus UAB. Bellaterra (Cerdanyola del Vallés).
10
08193 Barcelona, Spain.
11
Tlf: 34 935636600 Ext. 3131.
12
FAX: 34 935636601.
13
E-mail: [email protected]
14
15
16
17
Journal Research Area:
18
Signaling and Response
19
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Copyright 2014 by the American Society of Plant Biologists
1
20
Overexpression of a Calcium-dependent protein kinase confers salt and drought
21
tolerance in rice by preventing membrane lipid peroxidation
22
23
24
Sonia Campo1, Patricia Baldrich1, Joaquima Messeguer1, Eric Lalanne2,
25
María Coca1 and Blanca San Segundo1*
26
1 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB.
27
Edifici CRAG, Campus UAB. Bellaterra (Cerdanyola del Vallés). 08193 Barcelona,
28
Spain
29
2
Oryzon Genomics, St Ferran 74, 08940 Cornellà de Llobregat, Barcelona, Spain.
30
31
32
33
34
35
One-sentence summary:
36
A plasma membrane Calcium-dependent protein kinase prevents membrane lipid
37
peroxidation and confers tolerance to salt and drought stress in rice plants
38
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
2
39
Financial source:
40
This work was supported by the European Research Area-Plant Genomics Network
41
project TRIESTER (Trilateral Initiative for Enhancing Salt Tolerance in rice, Grant
42
GEN2006-27794-C4-1-E from MICINN), and grants BIO2009-08719/BIO2012-32838
43
from MINECO. We also thank the Consolider-Ingenio CSD2007-00036 to CRAG and
44
the Generalitat de Catalunya (Xarxa de Referencia en Biotecnología and SGR 09626)
45
for substantial support.
46
47
48
Present address (S.C): Donald Danforth Plant Science Center (DDSPC), St Louis,
49
Missouri, USA.
50
51
*Corresponding author:
52
Blanca San Segundo
53
e-mail, [email protected]
54
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
3
55
Abstract
56
The OsCPK4 gene is a member of the complex gene family of Calcium-dependent
57
protein kinases (CPKs or CDPK) in rice. Here, we report that OsCPK4 expression is
58
induced by high salinity, drought and the phytohormone abscisic acid. Moreover, a
59
plasma membrane localization of OsCPK4 was observed by transient expression assays
60
of green fluorescent protein-tagged OsCPK4 in onion epidermal cells. Overexpression
61
of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress.
62
Knock-down rice plants, however, were severely impaired in growth and development.
63
Compared to control plants, OsCPK4 overexpressor plants exhibit stronger water-
64
holding capability and reduced levels of membrane lipid peroxidation and electrolyte
65
leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings
66
accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice
67
overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on
68
the rice transcriptome. Moreover, no genes were found to be commonly regulated by
69
OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in
70
lipid metabolism and protection against oxidative stress appear to be up-regulated by
71
OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no
72
effect on the expression of well-characterized abiotic stress-associated transcriptional
73
regulatory networks (i.e. OsDREB and OsNAC6 genes) and LEA genes in their roots.
74
Taken together, our data show that OsCPK4 functions as a positive regulator of the salt
75
and drought stress response in rice via protection of cellular membranes from stress-
76
induced oxidative damage.
77
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
4
78
INTRODUCTION
79
80
Plants have evolved diverse mechanisms for sensing and responding to adverse
81
environmental conditions. If the acclimation potential is surpassed by environmental
82
stress, plants encounter metabolic imbalances and disturbance of developmental
83
processes, which might result in reduced productivity and eventually plant death.
84
Therefore improvement of abiotic stress tolerance might increase actual yields in most
85
crops.
86
Plants acquire tolerance to abiotic stress by reprogramming metabolism and gene
87
expression (Rabbani et al., 2003; Matsui et al., 2008). Early events in most abiotic stress
88
responses involve changes in the cellular redox status and uncontrolled redox reactions,
89
these processes occurring in different phases depending on the stress intensity: redox-
90
dependent deregulation in metabolism, development of oxidative damage, and cell
91
death. Thus, the primary goal of cellular regulation during adaptation to abiotic stresses
92
is the readjustment of metabolic processes under conditions of increasing redox
93
imbalances.
94
Abiotic-stress responsive genes can be categorized in three groups in terms of their
95
protein products. One group comprises genes encoding proteins involved in protection
96
of macromolecules and cellular structures (i.e. late embryogenesis abundant proteins, or
97
LEA) and enzymes responsible for the synthesis of osmolites. This group also includes
98
genes playing a role in protection from damage produced by reactive oxygen species
99
(ROS), such as peroxidase, superoxide dismutase and glutathione-S-transferase (GST),
100
among others. A second group of abiotic-stress responsive genes comprises regulatory
101
genes involved in stress signal transduction (mitogen-activated protein kinases and
102
protein phosphatases) and transcriptional control (transcription factors). Finally, the
103
third group comprises genes that are involved in ion transport (aquoporins, ion
104
transporters). It is, however, difficult to distinguish critical genes that determine the
105
phenotype of tolerance to the plant from housekeeping genes that are activated as part of
106
the stress-induced perturbation. The emerging picture also defines that, in addition to
107
transcriptional changes, post-transcriptional and post-translational mechanisms (i.e.
108
protein phosphorylation) might represent additional layers of regulatory systems
109
governing plant adaptation to abiotic stresses. It is also well established that the
110
phytohormone abscisic acid (ABA) is an important signal that mediates plant responses
111
to abiotic stresses (Hirayama and Shinozaki, 2007), although salt stress inducible genes
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
5
112
which are ABA-independent have been described (Nakashima et al 2009; Kumar et al.
113
2013).
114
Global transcript profiling reveals that many genes are coordinately regulated by salt
115
and drought stress, supporting a high degree of crosstalk between these types of stresses
116
(Rabbani et al., 2003; Nakashima et al., 2009). In this respect, it is well known that salt
117
stress disrupts homeostasis of water potential (osmotic homeostasis) and ion distribution
118
(ionic homeostasis). Then, the initial response to salt stress (the osmotic stress
119
component) shares similarities with drought stress although long-term exposure to high
120
salinity introduces the ion toxicity component. Homeostasis of sodium (Na+) and
121
potassium (K+) ions is of particular importance for plant salt tolerance.
122
Increasing evidence supports that calcium levels are altered in plant cells in response
123
to abiotic stress (Boudsocq and Sheen, 2010). The stress-induced perturbation in
124
cytosolic Ca2+ levels is sensed by calcium binding proteins (Ca2+ sensors). In plants, the
125
Ca2+-dependent protein kinase (CPKs, or CDPKs) family represents a unique group of
126
calcium sensors (Harper et al., 2004). Within a single polypeptide, CPKs contain a
127
catalytic serine/threonine kinase domain fused to a calmodulin domain. This unique
128
structure allows the CPKs to recognize specific calcium signatures and transduce the
129
signal into phosphorylation cascades. The existence of salt stress-induced Ca2+-
130
dependent phosphorylation events controlling cellular Na+ homeostasis and salt
131
tolerance is well illustrated by the salt-overlay-sensitive (SOS) signalling pathway
132
(Quintero et al., 2002).
133
CPKs have been identified throughout the plant kingdom and in some protozoans, but
134
not in animals (Harper et al., 2004). The available information on plant CPKs indicates
135
that CPKs are encoded by multigene families consisting of 31 genes in Oryza sativa and
136
34 genes in Arabidopsis (Hrabak et al., 2003; Asano et al., 2005; Ray et al., 2007;
137
Schulz et al., 2013). Whereas some of the CPK genes are ubiquitously expressed, others
138
show a tissue-specific pattern of expression or are regulated by stress (wounding,
139
salinity, cold, drought, pathogen infection) (Klimecka and Muszynska, 2007; Coca and
140
San Segundo, 2010). Microarray analysis of rice seedlings subjected to cold, drought or
141
salinity stress revealed up-regulation of six CPK genes (OsCPK4, 10, 12, 13, 15 and 21)
142
and down regulation of one gene (OsCPK1) (Ray et al., 2007; Das and Pandey, 2010).
143
Even though the expression of several rice CPK genes has been reported to be regulated
144
by abiotic stresses, only a few members of this multigene family have been functionally
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
6
145
characterized in rice (Saijo et al., 2000; Komatsu et al., 2007; Asano et al., 2011; Asano
146
et al., 2012).
147
In this study, we report the functional characterization of OsCPK4, a member of the
148
CPK gene family from rice. OsCPK4 gene expression is up-regulated in response to salt
149
and drought stress. Transient expression of the OsCPK4-gfp (green fluorescent protein)
150
gene in onion epidermal cells showed that OsCPK4 localizes to the plasma membrane.
151
Overexpression of OsCPK4 leads to increased tolerance to salt and drought stress in rice
152
plants. Even though OsCPK4 overexpression has a low impact in the rice transcriptome,
153
transcript profiling revealed up-regulation of a significant number of genes involved in
154
lipid metabolism and protection against oxidative stress in roots of OsCPK4
155
overexpressor plants. Contrary to this, alterations in the expression of genes typically
156
associated to the salt stress response did not occur in roots of OsCPK4-rice plants.
157
Compared with control plants, OsCPK4 overexpressor plants showed reduced levels of
158
membrane lipid peroxidation and electrolyte leakage under salt stress conditions.
159
OsCPK4 rice seedlings also accumulated less Na+ in their roots. Collectively, these
160
results suggest that the protective effect conferred by OsCPK4 overexpression in rice
161
might be mediated by an increased capacity of the transgenic plants to prevent oxidative
162
163
164
165
166
damage and membrane lipid peroxidation under salt stress conditions.
RESULTS
167
168
OsCPK4 expression is induced by salt and drought stresses
169
The OsCPK4 gene (Os02g03410) encodes a Calcium-dependent protein kinase
170
containing the four-domain structure typical of CDPKs: an amino terminal variable
171
domain, a serine/threonine kinase domain, a junction autoinhibitory domain, and a C-
172
terminal calmodulin domain (Fig. 1A). When examining OsCPK4 expression in rice
173
plants at different developmental stages (14d-, 28d- and 50d-old plants), higher levels of
174
expression were found in roots compared to leaves at all the developmental stages here
175
analysed (Fig. 1B).
176
We examined OsCPK4 expression in response to salt and drought stress, and at
177
different times of stress treatment. OsCPK4 was rapidly and transiently activated upon
178
exposure to salt stress in rice roots (aprox. 5-fold increase after 1h of salt-treatment)
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
7
179
(Fig. 1C, left panel). Under drought stress imposed by either air-drying treatment or
180
polyethylene glycol (PEG) treatment, induction of OsCPK4 expression was also
181
observed, its activation being maintained along the entire period of stress treatment
182
(Fig. 1C, middle and right panels). To assess the effectiveness of treatment we analysed
183
the expression of the marker gene OsLEA23, a dehydrin gene also known as OsDip1
184
(Dehydration inducible protein 1) (Fig. 1D). The expression of OsLEA23/OsDip1 is
185
known to be up-regulated by salt and drought stress (early-responsive gene) (Rabbani et
186
al., 2003). As expected, OsLEA23/Dip1 transcripts increased in response to high salt
187
conditions, with the highest levels recorded at 1h of salt treatment and then decreasing
188
at subsequent times of salt treatment (Fig.1D, left panel). Air-drying and PEG treatment
189
also induced OsLEA23/Dip1 expression (Fig.1D, middle and right panels). When
190
examining salt responsiveness of OsCPK4 expression in leaves of rice plants, a less
191
intense and delayed activation of OsCPK4 was observed compared with root tissues
192
(Supplemental Fig S1).
193
Together, this study indicated that OsCPK4 gene expression is rapidly activated by
194
salt and drought stress in the rice root. Moreover, OsCPK4 and OsLEA23/OsDip1 share
195
similar expression patterns in response to salt stress, these two genes representing early
196
salt-stress responsive genes.
197
198
199
OsCPK4 expression is induced in response to treatment with ABA
200
ABA plays a major role in plant adaptation to high salinity and drought, and many
201
genes that are induced by these stresses contain specific cis-regulatory elements in their
202
promoters (Hirayama and Shinozaki, 2007). A detailed analysis of the OsCPK4
203
promoter (2000 bp upstream of the transcription start site) revealed the presence of
204
multiple ABA-responsive cis-elements, including several ABRE (ABA Responsive
205
Element; PyACGTGGC) elements, MYB recognition sites (TGGTTAG), and MYC
206
recognition sites (CACATG) (Fig. 2A, Supplemental Table SI). In particular, ABRE is
207
considered a major cis-element in the promoter activity of genes regulated by ABA and
208
osmotic stress (Yamaguchi-Shinozaki and Shinozaki, 2006). However, a single ABRE
209
is not sufficient for ABA response, and either additional ABRE elements or coupling
210
elements are required. In line with this, several ABRE motifs were identified in the
211
OsCPK4 promoter region here examined (Fig. 2A). Consistent with the observation that
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
8
212
OsCPK4 promoter contains cis-acting elements for ABA-dependent gene expression, a
213
marked increase in OsCPK4 transcripts occurred in rice plants in response to ABA (Fig.
214
2B, left panel). OsLEA26/RAB16B was used as a marker gene to show the effectiveness
215
of the ABA treatment (Fig. 2B, right panel).
216
In this work, we also examined OsCPK4 protein accumulation in rice roots in
217
response to treatment with ABA, high salt and drought stress. Towards this end,
218
polyclonal antibodies were generated against the N-terminal domain of OsCPK4 (Met1–
219
Arg58), a region that is known to be highly variable among CPK proteins (Supplemental
220
Fig. S2A,B). In agreement with real-time PCR data, western blotting with the anti-
221
OsCPK4 protein showed an important increase in the accumulation of this protein after
222
ABA, NaCl and drought treatment (Fig 2C). No serological reaction occurred when the
223
preimmune serum was used to probe protein extracts from roots of either control or
224
stressed rice plants (results not shown). Together, results here presented indicate that the
225
transcriptional activation of OsCPK4 expression in response to salt and drought stress
226
or ABA treatment is accompanied by the accumulation of the OsCPK4 protein in rice
227
roots.
228
229
230
OsCPK4 localizes to the plasma membrane
231
CPK proteins have been shown to localize in many different cellular compartments
232
including the nucleus, cytosol, peroxisome, plasma membrane, oil bodies, mitochondria
233
and endoplasmic reticulum, as well as in association with actin filaments (Dammann et
234
al., 2003; Klimecka and Muszynska, 2007; Campos-Soriano et al., 2011). To localize
235
OsCPK4 in the plant cell, we transiently expressed an OsCPK4-GFP fusion gene in
236
onion epidermal cells. Confocal laser scanning microscopy revealed an OsCPK4-
237
specific labeling in the cell periphery, likely the plasma membrane (Fig. 3A). As
238
expected, onion cells expressing the GFP gene alone showed distribution of green
239
fluorescence throughout the cell (Fig. 3B). Next, OsCPK4-GFP-transformed onion cells
240
were also incubated in a hypertonic solution (15min in 750mM mannitol) to induce
241
plasmolysis. In plasmolysed onion cells, the OsCPK4-GFP displayed a pattern
242
consistent with its location in the plasma membrane of the shrunken protoplasm (Fig.
243
3C). Under these conditions, protoplast pull away from the cell wall, leaving large
244
numbers of thin plasma membrane bridges, known as Hechtian strands, firmly anchored
245
to the cell wall (Fig. 3D), thus, confirming a plasma membrane localization for the
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
9
246
OsCPK4 protein. Consistent with its subcellular localization site, a myristoylation site
247
and a potential site for palmitoylation (Cys4) are present at the N-terminal end of
248
OsCPK4 (Fig. 1A).
249
250
251
Overexpression of OsCPK4 improves tolerance to salt and drought stress.
252
Transgenic rice lines overexpressing OsCPK4 were generated. Northern blot and
253
western blot analyses confirmed transgene expression and OsCPK4 protein
254
accumulation (Supplemental Fig. S3). Under normal growth conditions, there were no
255
obvious phenotypical differences between homozygous OsCPK4 plants (T2 to T4
256
generation) and wild type plants (results not shown).
257
The transgenic rice plants overexpressing OsCPK4 and transgenic rice lines
258
expressing the empty vector (5 independent homozygous OsCPK4 lines, and 3
259
independent homozygous empty vector lines), as well as wild-type (Nipponbare) plants
260
were tested for salt tolerance. The rice plants were grown hydroponically until they
261
reached the three-leaf stage (Fig.4 A and B, D0). Then, plants were exposed to salt
262
stress (60 mM NaCl). Phenotypical differences between OsCPK4 and control plants
263
were evident by 11 days of salt treatment (results not shown). By 18 days of salt
264
treatment, OsCPK4 plants showed a clear phenotype of tolerance to salt stress compared
265
to both vector control, and wild-type plants (Fig. 4B, D18). Survival of OsCPK4
266
transgenic lines after 25 days in the presence of salt was much higher than in control
267
plants, the highest OsCPK4-expressing line consistently exhibiting the highest level of
268
tolerance (line 1, 60% survival) (Fig. 4C and D). Under the same experimental
269
conditions, only 2-12% of the control plants survived. From these results, it is
270
concluded that overexpression of OsCPK4 in transgenic rice results in improved
271
tolerance to salt stress.
272
Next, we examined whether overexpression of OsCPK4 affects tolerance to drought
273
stress in rice. Transgenic plants and vector control plants were grown for 15 days under
274
a fully watered regime (Fig. 5A and B, D0). Plants were deprived of irrigation for 17
275
days and then returned to the regular watering conditions to allow their recovery. By 14
276
days without water, vector control plants showed visual symptoms of drought-induced
277
damage, such as leaf rolling and wilting, whereas transgenic OsCPK4 plants remained
278
green (results not shown). On day 17 of drought treatment, all the control plants were
279
severely affected, while OsCPK4 plants remained healthy and vigorous (Fig. 5B, D17).
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
10
280
After 9 days of rewatering, only 18% of the control plants still contained green tissues
281
(Fig. 5B, RW and Fig.5C). Under the same experimental conditions, aprox. 90% of the
282
OsCPK4 rice plants recovered from the stress. None of the control plants survived
283
drought treatment.
284
It is generally assumed that plants with higher water retention ability can better
285
survive under drought stress conditions. Accordingly, we checked whether OsCPK4
286
overexpression has an effect on the water loss rate and water retention in the rice plant.
287
Under drought conditions, overexpression of OsCPK4 reduces water losses in leaves of
288
these plants (Fig. 5D). Leaves from OsCPK4 overexpressor plants also showed higher
289
water retention ability than control plants (Fig. 5E).
290
291
Altogether, results obtained on salt and drought tolerance assays demonstrated that
OsCPK4 overexpression confers tolerance to salt and drought stress in rice plants.
292
293
294
OsCPK4 Knockout Results in Severe Growth Inhibition
295
To further investigate into the function of OsCPK4, we searched for loss-of-function
296
mutants. Two T-DNA insertion mutants of OsCPK4 were identified in the POSTECH
297
collection: 2D-00040 (Dongjin background) and 1D-03351 (Hwayoung background).
298
Both of them contained the T-DNA insertion in the kinase domain of the OsCPK4 gene
299
(Fig. 6A). Results obtained for the 2D-00040 cpk4 mutant are presented in Fig. 6B-D
300
(similar results were obtained for the second cpk4 mutant, the 1D-03351 mutant; results
301
not shown). PCR-based genotyping with combinations of different gene-specific and the
302
T-DNA primers allowed the identification of homozygous and heterozygous individuals
303
for the cpk4 mutants and qRT-PCR confirmed OsCPK4 suppression in homozygous
304
mutant plants (Fig. 6 B, C). To note, the homozygous cpk4 mutant plants were severely
305
affected in their growth and development compared with the azygous plants (Fig. 6D).
306
These findings support that OsCPK4 disruption has a strong impact on plant growth,
307
this gene being required for normal growth and development of rice plants.
308
309
310
Transcript profiling of rice plants overexpressing OsCPK4.
311
Knowing that OsCPK4 overexpression enhances salt and drought tolerance, we
312
initially hypothesized an OsCPK4-mediated activation of genes with a known role in
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
11
313
conferring tolerance to abiotic stresses. We examined the expression level of rice LEA
314
genes in OsCPK4 plants, including Dehydrin genes (Group 2 of LEA proteins). Because
315
the expression of many dehydrins is induced by ABA, they are also referred as RAB
316
(Responsive to ABA) proteins (Wang et al., 2007). However, overexpression of
317
OsCPK4 does not result in a significant increase in transcript accumulation of either
318
LEA genes (OsLEA19a and OsLEA21) or dehydrin genes (OsLEA23, also named DIP1,
319
OsLEA24 and OsLEA27) (Supplemental Fig. S4A).
320
Several major regulons are known to be active in the plant response to abiotic stress,
321
such as the DREB1/CBF (Dehydration-Responsive Element Binding protein1/C-repeat
322
Binding Factor) and DREB2 regulons, the AREB/ABF (ABA Responsive Element
323
Binding protein/ABRE Binding Factor) regulon, and the NAC regulon (Todaka et al.,
324
2012). In particular, overexpression of OsDREB1A, OsNAC6 and ONAC045 in rice or
325
Arabidopsis plants has been shown to confer drought and salt tolerance, whereas
326
overexpression of DREB2B enhances drought and termotolerance (Dubouzet et al.,
327
2003; Ito et al., 2006; Sakuma et al., 2006; Nakashima et al., 2007; Zheng et al., 2009;
328
Matsukura et al., 2010). In this work, we examined the expression of transcription
329
factors belonging to one or another of the salt/drought-associated regulons in roots of
330
OsCPK4 rice plants (in all the cases, plants were grown under non stress conditions).
331
They were: OsDREB1A and OsDREB2B (DREB1/CBF and DREB2 regulons,
332
respectively), OsNAC6 (NAC regulon), as well as ONAC045. Although the expression
333
of these genes varied among the individual transgenic lines, none of the transcription
334
factor genes here assayed was found to be significantly misregulated (over- or under-
335
expressed) in any of the OsCPK4 lines compared to vector control plants (Supplemental
336
Fig. S4B). There was, however, the possibility that the salt-associated genes here
337
examined (LEA, transcription factors) might show stronger differential regulation in
338
transgenic plants only under salt stress conditions (as opposed to being constitutively
339
activated in the transgenic plants). However, when examining the expression of these
340
genes in salt-stressed OsCPK4 and vector control transgenic plants (1h of treatment, 60
341
mMNaCl), no differences in the intensity of the response to salt stress could be
342
observed for any of them (results not shown).
343
To obtain further insights into molecular mechanisms by which OsCPK4 mediates
344
tolerance to salt stress in rice plants, gene expression profiles in roots and leaves of
345
OsCPK4 overexpressor plants and vector control plants were compared. Plants were
346
grown under non-stressed conditions (two independent OsCPK4 lines and two
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
12
347
independent control plants were analysed). Differentially expressed genes were selected
348
based on fold change (FC ≥ 2.0) and p-value (P ≤ 0.05). Only 62 genes were found to be
349
up-regulated in roots of the transgenic lines relative to vector control plants, supporting
350
that OsCPK4 overexpresion has a low impact on the root transcriptome of rice plants.
351
Using the same criteria, genes showing down-regulation in roots of OsCPK4 plants
352
were not identified (FC ≤ -2.0 and P ≤ 0.05).
353
Examination of the functional distribution of genes showing up-regulation in roots of
354
OsCPK4 plants revealed that the majority of these genes grouped into the category of
355
“Metabolism” (46.77% of the total number of differentially expressed genes), most of
356
them being involved in “Lipid Metabolism” and “Protein Metabolism” (16.13% and
357
14.52%, respectively) (Fig. 7A and Table I). Based on the molecular function, genes
358
encoding proteins with oxido-reductase activity were represented in different
359
subcategories of Metabolism (Table I). We also noticed that genes involved in oxidative
360
stress and redox regulation, such as peroxidase, thioredoxin, GST and laccase were
361
present in the set of OsCPK4-regulated genes (6.45% of the total number of OsCPK4
362
regulated genes) (Fig. 7A; Table I). In this respect, it is well recognized that salt and
363
drought stress induce ROS generation and that antioxidant enzymes play an important
364
role in tolerance to abiotic stress in plants, including rice plants (Dionisio-Sese and
365
Tobita, 1998; Miller et al., 2010). Consistent with data obtained by microarray analysis,
366
qRT-PCR analysis confirmed up-regulation of thioredoxin and laccase in roots of
367
OsCPK4 overexpressor plants (Fig. 7B). In the category of Lipid Metabolism, genes
368
encoding proteins with lipid binding activities and Lipid Transfer Proteins, as well as
369
lipases were found to be up-regulated in roots of OsCPK4 plants (Table I).
370
The category of “Signal transduction and intracellular trafficking” was also highly
371
represented in the set of genes that are up-regulated in OsCPK4 plants (12.90%; Fig.
372
7A) comprising several protein kinases, receptor-like protein kinases, and calmodulin
373
binding proteins. To note, our comparative transcript profiling analysis of root tissues
374
confirmed that OsCPK4 overexpression does not result in up-regulation of the major
375
transcription factors that function under salt and drought stress, such as DREB or NAC
376
transcription factors (as previously noticed by qRT-PCR analysis of transgenic plants).
377
Only the cold-inducible OsMyb4 gene was identified among the differentially expressed
378
transcription factor genes in OsCPK4 rice plants (Table I). OsMyb4 overexpression in
379
rice has been shown to reduce membrane injury under stress conditions (Laura et al.,
380
2010). Finally, the expression of genes encoding ion transporters, namely a cation
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
13
381
antiporter and a potassium transporter, was found to be up-regulated in OsCPK4 plants
382
(Table I).
383
We also investigated whether OsCPK4 overexpression has an impact on the leaf
384
transcriptome. Microarray analysis showed that only 94 genes were differentially
385
expressed in leaves of OsCPK4 plants relative to control plants, of which 22 and 72
386
genes were up-regulated (FC ≤ 2.0 and P ≤ 0.05) and down regulated (FC ≤ -2.0 and P ≤
387
0.05), respectively (Supplemental Table SII). The functional categories of the
388
differentially expressed genes in leaves of OsCPK4 plants are presented in Fig 7C. The
389
majority of the misregulated genes grouped into the categories of Metabolism (31.91%)
390
and Transcription and RNA binding (22,34%). Validation of microarray gene
391
expression for selected genes, namely the OsWRKY71 and OsWRKY76 transcription
392
factors (up-regulated and down-regulated, respectively) and aspartic proteinase is
393
presented in Fig 7D. Intriguingly, overexpression of OsCPK4 in rice causes a reduction
394
in the expression of the salt-associated DREB1A, DREB1B and LEA21 genes in leaves,
395
whereas the expression of these genes was not affected in root tissues. A trehalose-6-P-
396
phosphatase (OsTPP) gene was down-regulated in leaves of OsCPK4 plants, this gene
397
being involved in the biosynthesis of trehalose. Although trehalose has been proposed to
398
act as an osmoprotectant during desiccation, it is also true that trehalose does not
399
accumulate in plants in quantities high enough to directly protect against abiotic stress
400
as a compatible solute. Similarly, overexpression of OsTPP1 in rice rendered the plant
401
more tolerant to salinity and cold even though no increase in the trehalose content could
402
be observed (Ge et al., 2008).
403
Collectively, these findings indicated that overexpression of OsCPK4 results in few
404
transcriptional changes in either root or leaf tissues. No genes were found to be
405
commonly regulated by OsCPK4 in leaves and roots of rice plants. In root tissues,
406
OsCPK4 positively regulates the expression of genes involved in metabolic processes,
407
mainly lipid metabolism, as well as genes involved in protection against oxidative
408
stress. Because roots are the first organs that encounter excess salinity, they are also the
409
first sites of potential damage. Presumably, up-regulation of genes involved in lipid
410
metabolism and protection against oxidative stress in root tissues might be relevant in
411
conferring salt-tolerance to the rice plant. Noticeably, no significant alterations in the
412
expression of salt-associated genes appear to occur in roots of OsCPK4 rice plants,
413
whereas a reduction in the expression of certain salt-associated genes occurs in leaves of
414
OsCPK4 plants.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
14
415
416
417
Effect of salt stress on membrane lipid peroxidation and electrolyte leakage in
418
OsCPK4-overexpressing rice plants
419
The control of membrane integrity and membrane-associated functions is crucial for
420
salt tolerance (Lopez-Perez et al., 2009). As previously mentioned, salt stress induces
421
ROS production, which in turn, might cause oxidative damage on membrane lipids and
422
perturbation of cell membrane functioning. Salt stress-induced membrane damage also
423
results in electrolyte leakage (Verslues et al., 2006). As lipid peroxidation is the
424
symptom mostly ascribed to oxidative damage, it is often used as an indicator of
425
damage to cell membranes in plants under salt stress condition.
426
To further investigate into the mechanism underlying salt tolerance in OsCPK4 rice
427
plants, we examined lipid peroxidation levels in OsCPK4 overexpressor plants, with or
428
without salt treatment. Lipid peroxidation was measured as malondialdehyde (MDA)
429
content, MDA being a typical breakdown product of peroxidized polyunsaturated fatty
430
acids in plant membranes (Weber et al., 2004). Under normal conditions (no NaCl
431
treatment), the MDA levels were lower in OsCPK4 rice plants than in vector control
432
plants (Fig. 8A). Most importantly, under salt stress conditions, the MDA levels were
433
significantly higher in vector control plants than in OsCPK4 plants, indicating a lower
434
degree of lipid peroxidation due to salt stress in OsCPK4 plants.
435
Next, we measured electrolyte leakage in unstressed and salt-stressed OsCPK4 plants
436
and control plants. Both, the OsCPK4 transgenic plants and vector control plants
437
showed similar levels of relative electrolyte leakage under non-stress conditions.
438
However, the presence of NaCl induces an increase in electrolyte leackage in vector
439
control plants, but not in OsCPK4-rice plants (Fig. 8B). Altogether, these observations
440
suggest that OsCPK4 overexpression prevents salt-stress induced lipid peroxidation and
441
electrolyte leakage in cellular membranes under salt stress conditions.
442
443
OsCPK4 regulates Na+ accumulation
444
Growth under salt stress conditions results in increased levels of cytoplasmic Na+
445
accumulation (Munns and Tester, 2008) which, in turn, causes membrane injury and
446
affects MDA accumulation in the cell. Fluorescent indicators of Na+ such as CoroNa-
447
Green, are valuable tools for non-destructive monitoring of the spatial and temporal
448
distribution of Na+ in plant tissues. In order to measure the relative concentration of Na+
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
15
449
in OsCPK4 overexpressor and control plants, roots were incubated with NaCl, stained
450
with CoroNa-Green and analysed by confocal fluorescence microscopy. Root cells were
451
visualized by propidium iodide staining. This study revealed that roots from vector
452
control plants had much stronger fluorescence than roots from the OsCPK4 plants (Fig.
453
8C). Difference in Na+ accumulation was quantified from confocal CoroNaGreen data,
454
this analysis confirming that Na+ levels were substantially lower in the OsCPK4
455
overexpressor roots (p ≤ 0.001) (Fig. 8D). These findings further support that, under salt
456
stress conditions, the OsCPK4 transgenic plants accumulate less Na+ in their roots than
457
control plants.
458
459
460
DISCUSSION
461
In this study, we show that OsCPK4 is induced by high salinity and drought stress as
462
well as by ABA treatment. Transcriptional activation of OsCPK4 is consistent with the
463
presence of various abiotic stress-related cis-elements in the OsCPK4 promoter,
464
including the ABRE, MYB and MYC motifs. Overexpression of OsCPK4 enhances salt
465
and drought tolerance in transgenic rice. In addition to its involvement in abiotic stress
466
tolerance, OsCPK4 plays a key role in controlling plant development, as inferred from
467
the severe growth inhibition observed in cpk4 mutants. At the present time, however,
468
the function of OsCPK4 in controlling developmental processes remains to be solved.
469
Transcript profiling indicated that OsCPK4 overexpression has a low impact on the
470
transcriptome of rice plants growing under normal conditions. Moreover, no significant
471
differences in the expression of the typical salt/drought associated genes was observed
472
in roots of OsCPK4-rice plants. Interestingly, an important number of genes involved in
473
lipid metabolism and protection against oxidative stress were found to be up-regulated
474
in roots of OsCPK4 rice plants compared to control plants (i.e. peroxidase, thioredoxin,
475
laccase and GST genes). In this respect, it has long been recognized that the expression
476
of genes encoding antioxidant enzymes is activated by salt and drought stress, and that
477
overexpression of these genes is an effective way of protecting plants from abiotic
478
stress-induced oxidative damage (Edwards et al., 2000; Roxas et al., 2000; Liang et al.,
479
2006; Csiszar et al., 2012). ROS induce oxidative stress by reacting with several
480
macromolecules. When ROS target lipids, they initiate a lipid peroxidation process,
481
resulting in cell damage. An interesting observation arising from this study was that the
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
16
482
OsCPK4-rice plants exhibited a lower rate of membrane lipid peroxidation than control
483
plants under salt stress conditions. It is known that salt-tolerant rice genotypes have a
484
lower lipid peroxidation rate compared to salt-sensitive cultivars (Demiral and Turkan,
485
2005; Khan and Panda, 2008). Moreover, a relationship between expression of
486
antioxidant genes, lipid peroxidation and abiotic stress tolerance is also documented in
487
different plant species (Oberschall et al., 2000; Roxas et al., 2000; Katsuhara et al.,
488
2005). Thus, up-regulation of genes involved in protection against oxidative stress in
489
roots of OsCPK4-rice plants might prevent salt-stress induced lipid peroxidation and
490
oxidative damage in cellular membranes. Protection from oxidative damage of
491
membrane lipids will also positively influence membrane permeability to ions, neutral
492
solutes and water. Along with this, OsCPK4 overexpressor plants accumulate less Na+
493
in their roots compared to control plants. The up-regulation of ion transporter genes in
494
roots of OsCPK4 rice plants could be, at least in part, responsible for the observed lower
495
level of Na+ accumulation in roots of OsCPK4 rice plants. Furthermore, OsCPK4
496
overexpressor plants exhibited stronger water-holding capability and lower level of
497
electrolyte leackage in their leaves under stress conditions. Presumably, OsCPK4
498
overexpression promotes salt tolerance by reducing membrane lipid peroxidation which,
499
in turn, reduces electrolyte leakage and Na+ content under salt stress conditions.
500
Genes involved in lipid metabolism, including lipid binding genes and LTP genes,
501
were highly represented among the set of genes that are up-regulated in roots of
502
OsCPK4 rice plants. As for LTPs, these proteins have been implicated in the regulation
503
of cell functions where movement of lipids is thought to be important, including
504
maintenance of the cell membrane stability under abiotic stress conditions (Wu et al.,
505
2004). The expression of LTP genes has also been shown to respond to salt and drought
506
stress in different plant species (Hughes et al., 1992; Guo et al., 2013). We also noticed
507
up-regulation of several lipases and GDSL-like lipases in OsCPK4 overexpressor plants,
508
the expression of these genes being regulated by abiotic stress conditions in different
509
plant species (Hong et al., 2008). Presumably, this set of OsCPK4-regulated genes
510
might also contribute to the phenotype of salt tolerance that is observed in OsCPK4 rice.
511
Recently, a role of lipid metabolism in the adquisition of dessication-tolerance in the
512
resurrection plant Craterostigma plantagineum has been proposed (Gasulla et al., 2013).
513
It is also accepted that changes in membrane integrity and modulation of lipid
514
composition are key factors in the primary sensing of salt and drought (Lopez-Perez et
515
al., 2009). Clearly, the maintenance of membrane integrity in root tissues, the first site
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
17
516
at which salt stress is perceived, is important to protect the cell from abiotic stress
517
injuries, and accordingly, to confer tolerance in rice plants to abiotic stress.
518
Collectively, results here presented suggest that the protective effects of OsCPK4
519
overexpression in conferring salt and drought tolerance might rely more on protection
520
against oxidative damage of membranes and activation of genes involved in lipid
521
metabolism, rather than on the activation of the typical salt/drought-stress associated
522
transcriptional networks in root tissues.
523
The finding that the expression of OsDREB1A and OsDREB1B genes are down-
524
regulated in leaves of OsCPK4 rice plants is paradoxical because these genes are
525
positive regulators of abiotic stress tolerance. Here, it should be mentioned that whereas
526
the involvement of DREB genes in conferring tolerance to abiotic stress is well
527
documented in Arabidopsis, the contribution of these genes in abiotic stress tolerance in
528
rice is not fully understood. In Arabidopsis, DREB1 and DREB2 distinguish two
529
different signal transduction pathways, in which DREB1 preferentially functions in cold
530
stress tolerance and DREB2 is mainly involved in drought and salt stress, but not in cold
531
stress (Nakashima et al., 2009; Akhtar et al., 2012). Different results are, however,
532
found in the literature concerning transgenic rice overexpressing OsDREB1 genes.
533
Whereas, Ito et al. (2006) described tolerance to cold, drought and high-salt stresses in
534
DREB1-rice plants, Lee et al. (2004) did not observe any protective effect on tolerance
535
to either cold or drought stress in rice constitutively expressing DREB1. Different rice
536
cultivars with different level of tolerance to abiotic stresses (Dongjin and Kita-ake) were
537
used for transgenic expression of DREB1 genes, this fact being proposed as a possible
538
explanation for the different responses to abiotic stress in DREB1-overexpressor plants
539
(Lee et al., 2004; Ito et al., 2006). Although the exact mechanism underlying the
540
connection between up-regulation of OsCPK4 expression and down-regulation of
541
OsDREB1A, OsDREB1B (and OsLEA21) expression is unclear, the dramatic effect of
542
OsCPK4 on the expression of these genes in leaves of rice plants reveals such
543
connection. On the other hand, OsCPK4 overexpressor plants showed up-regulation of
544
OsWRKY76 in their leaves. Very recently, it was reported that overexpression of
545
OsWRKY76 improves tolerance to cold stress in rice plants, a protective effect that
546
appears to be mediated by protection of membrane damage from oxidative stress
547
(Yokotani et al., 2013). Results here presented on OsCPK4 rice plants are reminiscent
548
of those reported by Yokotani et al. (2013) on OsWRKY76 overexpressor plants.
549
Furthermore, examination of the OsWRKY76-regulated genes (Yokotani et al., 2013),
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
18
550
revealed down-regulation of OsDREB1B in leaves of OsWRKY76 overexpressor plants
551
(as it is also the case in OsCPK4 overexpressor plants). These observations might be
552
indicative of a functional connection between OsCPK4 and OsWRKY76 in conferring
553
stress tolerance in rice.
554
It is also intriguing the finding that there is no overlap between OsCPK4-regulated
555
genes in roots and leaves. Based on the results here presented, it is tempting to speculate
556
that different OsCPK4-mediated protective mechanisms might operate in roots and
557
leaves of rice plants in relation to abiotic stress tolerance. In favor of this hypothesis,
558
Minh-Thu et al. (2013) recently reported that although leaf and root tissues shared some
559
common gene expression during drought stress, the two tissues appeared to act
560
differently in response to dehydration. Other studies in maize indicated that under salt
561
stress conditions maize leaves and roots employed distinct mechanisms to cope with salt
562
stress (Qing et al., 2009). Then, tolerance mechanisms to abiotic stress may be
563
differentially regulated based on plant organs. Another possibility is that OsCPK4 might
564
act on different protein substrates which might, in turn, activate different responses (i.e.
565
phosphorylation cascades) in one or another tissue. Further studies are needed to
566
decipher whether OsCPK4 exerts its protective effect in abiotic stress tolerance by
567
modulating specific mechanisms in either root or leaf tissues.
568
Results here presented demonstrate that OsCPK4 localizes to the plasma membrane.
569
Clearly, the plasma membrane contains proteins that are essential for receiving signals
570
from the environment and transducing them for activation of downstream responses. As
571
previously mentioned, OsCPK4 harbours potential myristoylation and palmitoylation
572
sites at its N-terminal end. The N-terminal myristoylation appears to mediate membrane
573
localization of distinct CPKs whereas other CPKs require both myristoylation and
574
palmitoylation for its membrane association (Martín and Busconi, 2000; Benetka et al.,
575
2008; Coca and San Segundo, 2010; Mehlmer et al., 2010; Lu and Hrabak, 2013). In
576
other studies, it was shown that the variable domain of a plant CPK dictates not only its
577
proper subcellular localization but also confers substrate specificity (Asai et al., 2013).
578
If demonstrated, myristoylation and/or palmitoylation of OsCPK4 would explain that
579
OsCPK4 is exclusively localized at the plasma membrane.
580
It is also well recognized that the level of Ca2+ rapidly increases in plant cells in
581
response to abiotic stress (Reddy et al., 2011). Ca2+ homeostasis is achieved by the
582
combined action of channels, pumps and antiporters. Very recently, Choi et al. (2014)
583
proposed the existence of a plant-wide signaling system based on the rapid, long-
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
19
584
distance transmission of Ca2+ waves. This Ca2+-dependent signalling system elicits
585
molecular responses in distant parts of the plant upon perception of localized salt stress
586
(e.g. application of salt stress to the root tip triggers systemic molecular responses in
587
shoot tissue). The peculiar structural features of CPKs, make these proteins ideally
588
suited to sense the stress-induced alterations in cytoplasmic Ca2+ levels by the binding
589
Ca2+ to its intrinsic calmodulin-like domain for activation of downstream
590
phosphorylation processes. Then, in addition to its transcriptional activation, a direct
591
regulation by Ca2+ of the OsCPK4 enzyme activity might induce changes in the
592
phosphorylation status of a variety of protein targets in response to salt stress. In this
593
regard, alterations in the phosphoproteome of rice plants in response to salt stress are
594
documented, some of these events occurring at the plasma membrane (Chitteti and
595
Peng, 2007). Multiple phosphorylation of plasma membrane aquaporins in response to
596
salt stress has also been described in Arabidopsis (Prak et al. (2008). Furthermore, the
597
guard cell anion channel SLAC1 (SLOW ANION CHANNEL-ASSOCIATED1) is
598
phosphorylated by AtCPK21 and AtCPK23 (Geiger et al., 2010), and another vacuolar
599
channel, TPK1 (two-pore K+ channel 1), is phosphorylated by AtCPK3 (and other
600
CDPKs) in response to salt stress (Latz et al., 2013). The important role of Ca2+-
601
dependent phosphorylation processes during the salt stress response in plants is also
602
illustrated by the SOS pathway in which SOS3 (a Ca2+ sensor) activates the protein
603
kinase SOS2 which then regulates the plasma membrane Na+/H+antiporter SOS1
604
(Quintero et al., 2002). These findings raise the question of whether OsCPK4 could play
605
a role at the plasma membrane, for example by regulating the function of ion channels
606
or transporters through phosphorylation. Indeed, the observation that OsCPK4
607
overexpression has a low impact in the rice transcriptome favours that OsCPK4 might
608
act at the post-translational level for regulation of target proteins, perhaps in a tissue-
609
specific manner, during adaptation of rice plants to abiotic stress conditions.
610
In Arabidopsis, certain CPKs are known to be involved in abiotic stress tolerance and
611
ABA signalling (Sheen, 1996; Zhu et al., 2007; Boudsocq and Sheen, 2010; Xu et al.,
612
2010; Franz et al., 2011). In rice, however, the function of most CPK genes in relation
613
to abiotic stress is less characterized. Overexpression of OsCPK12, OsCPK13
614
(encoding OsCDPK7) and OsCPK21 was reported to confer salt tolerance (Saijo et al.,
615
2000; Asano et al., 2011; Asano et al., 2012), whereas OsCPK23 overexpression
616
confers cold tolerance (Komatsu et al., 2007). As it is the case in OsCPK4
617
overexpressor plants, no significant difference in the expression levels of LEA and
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
20
618
OsNAC6 genes was observed between plants overexpressing one or another of the
619
above mentioned CPK genes (OsCPK12, OsCPK21 and OsCPK13) under unstressed
620
conditions (Saijo et al., 2000; Asano et al., 2011; Asano et al., 2012). The knowledge
621
gained in this study, not only reveals an important regulatory function of OsCPK4 in
622
abiotic stress tolerance, but also provides a foundation for further investigation of stress-
623
induced signalling pathways in which rice CPK genes participate. For instance, it will
624
then be of interest to determine whether OsCPK4 acts in a cooperative manner with one
625
or more of other CPK genes during adaptation of rice plants to salt and/or drought
626
conditions. Knowing that rice has been adopted as the model system for cereal
627
genomics (Goff, 1999), a better understanding of abiotic stress mechanisms in rice
628
might also benefit stress tolerance in other monocot cereal crops.
629
Finally, in this study we determined salt tolerance of OsCPK4-rice plants at the
630
seedling stage. Rice is considered to be a salt sensitive crop, its tolerance to high salinity
631
varying through the life cycle of the plant (Maas and Hoffman, 1977). Thus, the rice
632
plant is very sensitive to high salinity at the seedling and reproductive stages (Zeng and
633
Shannon, 2000). Further studies will determine whether OsCPK4 overexpression
634
confers tolerance to salt/drought stress at any other developmental stage (i.e.
635
reproductive stage). Equally, the practical question is whether the benefits observed by
636
transgenic expression of OsCPK4 in rice in terms of salt and drought tolerance could be
637
used to engineer tolerant rice plants under field conditions. The results described in this
638
study point to the need to further dissect the pathways in which OsCPK4 is involved in
639
tolerance to abiotic stresses in rice plants
640
641
642
MATERIALS AND METHODS
643
644
Plant materials, growth condition and stress treatments
645
Rice plants (O.sativa L. cv. Nipponbare) were grown at 26 – 28ºC with a 18h light/8h
646
dark cycle. For expression studies of OsCPK4 in response to salt treatment, 7 day-old
647
rice plants were transferred to nutrient solution containing 100mM NaCl. To examine
648
the expression pattern of OsCPK4 in relation to drought stress, 7 day-old rice plants
649
were left to air dry, or stressed in a 20% (w/v) PEG-8000 solution, for the required
650
period of time. In all the cases, roots of at least 10 individual plants were collected and
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
21
651
used for total RNA isolation. Control seedlings were treated with water. For treatment
652
with ABA, 2 week-old plants were treated with a solution of 100μM ABA (100 mM
653
stock solution in ethanol) for 1h. T-DNA insertional mutant lines for OsCPK4 were
654
grown as described above and genotyped by PCR (Supplemental Methods).
655
656
Rice transformation
657
The full-length complementary DNA sequence of OsCPK4 (Os02g03410) was cloned
658
into the pCAMBIA1300 vector under the control of the maize ubiquitin 1 (ubi)
659
promoter and the nopaline synthase (nos) terminator. Transgenic rice (O. sativa cv.
660
Nipponbare) plants were produced by Agrobacterium-mediated transformation (A.
661
tumefaciens EHA105 strain). As control, transgenic rice lines expressing the empty
662
vector (pCAMBIA1300) were also produced. Details on construct preparation and rice
663
transformation are presented in Supplemental Methods.
664
665
qRT-PCR
666
Total RNA was extracted from plant tissues using the Trizol reagent (Invitrogen) and
667
used for reverse transcription reactions. The first cDNA was synthesized from DNase-
668
treated total RNA (1 μg) with the Transcriptor Reverse Transcriptase (Roche) and oligo
669
(dT)18 following the manufacturer’s instructions. qRT-PCR was performed in optical
670
96-well plates in the Roche Light Cycler® 480 instrument using SYBR Green I dye.
671
Primers were designed using Primer Express software (Applied Biosystems) (primers
672
are listed in Supplemental Tables SIV). Routinely, three replicate reactions were used
673
for each sample. Data were normalized using the geometric mean of the OsCYP
674
cyclophylin (Os02g02890) and OsUbi Ubiquitin (Os06g46770) genes as internal
675
controls. Three independent biological replicates were analysed. Controls of the qRT-
676
PCR reactions without adding the reverse transcriptase enzyme were systematically
677
included in our experiments.
678
679
Microarray analysis.
680
Total RNA was isolated from tissues, both roots and leaves, of OsCPK4 and vector
681
control plants that had been grown hydroponically for 15 days using the Plant total
682
RNA extraction kit (Qiagen). Two independent homozygous OsCPK4 lines (lines 1 and
683
13) and two vector control lines (lines 3 and 11) were examined. The quality and
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
22
684
concentration of the RNA was analysed using the Agilent 2100 bioanalyzer and
685
NanoDrop™ ND-1000 (Thermo Scientific). Samples with RNA integrity number (RIN)
686
< 6.0 were discarded. Three biological samples and three replicates per biological
687
sample were analysed. Details of microarray construction, probe labelling and
688
hybridization conditions are presented in Supplemental Methods. Bootstrap analysis
689
with Significance Analysis of Microarrays (SAM) was used to identify differentially
690
expressed genes using a cut-off of 2.0 (Tusher et al., 2001). SAM calculates the fold
691
change and significance of differences in expression. The delta value, false significant
692
number (FSN) and false discovery rate (FDR) were 0.749, 8.88 and 4.99, respectively.
693
We considered genes with P values ≤ 0.05. Data sets for microarray analyses (roots and
694
leaves) have been deposited in the National Center for Biotechnology Information
695
(NCBI) Gene Expression Omnibus (GEO) (GSE52353)
696
697
Tolerance to salt and drought stress
698
For salt stress treatment, plants were grown hydroponically in Yoshida medium for 15
699
days and transferred to nutrient solution containing 60 mM NaCl, or fresh nutrient
700
solution (control plants). Five independent homozygous OsCPK4 lines, three
701
independent vector control plants, and at least 30 plants per line were assayed. Salt
702
tolerance was evaluated by determining the percentage of surviving plants after 25 days
703
of salt treatment.
704
For drought stress treatment, plants were grown in soil for 15 days under a normal
705
watering regime. Drought stress was induced by withholding water for 17 days. Plants
706
were then irrigated normally for 9 days. Drought tolerance was evaluated by
707
determining the percentage of surviving plants after the period of recovery. To measure
708
the water loss under dehydration conditions, leaves of wild-type and homozygous
709
transgenic plants at the three-leaf stage were cut and exposed to air under controlled
710
conditions (27º +/- 2ºC). The leaves were weighed four hours after being cut down.
711
Water loss was calculated from the equation:
712
dessicated weight)/fresh weight X100. The Water Retention Ability was measured
713
according to the formula: WRA (%)=(desiccated weight–dry weight)/(fresh weight–dry
714
weight)X100. Statistical analysis of data obtained in these experiments was performed
715
using the ANOVA test at a 5% confidence level.
Water loss (%) = (fresh weight–
716
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
23
717
Subcellular localization.
718
An OsCPK4-GFP fusion gene was prepared and used for particle bombardment
719
experiments in onion epidermal cells as previously described (Murillo et al., 2003) (for
720
details see Supplemental Methods). Confocal microscopy was used to visualize GFP
721
fluorescence. To confirm plasma membrane localization, cells were plasmolysed with
722
0.75 M mannitol for 15min.
723
724
Preparation of anti-OsCPK4 N-terminal antiserum and immunoblotting
725
The N-terminal domain of OsCPK4 (Met1-Arg58) was produced in Escherichia coli
726
and the purified polypeptide was used for antiserum production in rabbits. Protein
727
extracts were prepared from roots of control and OsCPK4 rice plants, separated by
728
SDS-PAGE, or 2D-PAGE, and probed with the anti-OsCPK4 antiserum produced in
729
this work (1:1000 dilution). Blots were then incubated with protein A–peroxidase
730
(Sigma) at a dilution of 1:10000. Peroxidase activity was made visible by incubating the
731
blot with ECL Western Blotting Substrate (Pierce) for 5min on an LAS-4000 Image
732
analyzer (Fujifilm). Details on bacterial expression, preparation of the antiserum and
733
immunoblotting are presented in Supplemental Methods.
734
735
Lipid peroxidation, electrolyte leakage and detection of Na+
736
The level of lipid peroxidation was determined by measuring the amount of
737
malondialdehyde (MDA) produced by the thiobarbituric acid (TBA) reaction as
738
described by Hodges et al. (1999). Leaf samples 0.05 g were homogenized in 1 mL of
739
80% (v/v) ethanol on ice, and then centrifuged at 16,000g for 20 min at 4ºC. The
740
supernatant (0.6 mL) was mixed with 0.6 mL of 20% (w/v) trichloroacetic acid
741
containing 0.65% (w/v) thiobarbituric acid. The mixture was heated at 95°C for 30 min
742
and then quickly cooled in an ice bath. After centrifugation at 10,000g for 10 min, the
743
absorbance of the supernatant was measured at 532 nm. The value for non-specific
744
absorption at 600 nm was subtracted from the 532 nm reading. The MDA content was
745
calculated using its molar extinction coefficient (155 mM-1 cm-1) and the results
746
expressed as μmol MDA mg-1fresh weight. For measurement of relative electrolyte
747
leakage, three OsCPK4 and 2 vector control lines were treated with 150mM NaCl for
748
2h. Leakage ratio values were determined following the method described by Cao et al.
749
(2007). Briefly, the leaf segments from at least 3 plants each line were placed in
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
24
750
deionized water and the conductivities of the obtained solutions were determined. Then
751
the leaf segments in deionized water were boiled for 15 min. After being thoroughly
752
cooled to room temperature, the conductivities of the resulting solutions were
753
determined. For each line, the relative electrolyte leakage after salt treatment was
754
calculated as the ratio of the conductivity before boiling the rice leaves to that after
755
boiling. Each data point represents average from two independent experiments.
756
Intracellular Na+ was detected in 5 day-old rice roots, control and salt-treated roots (150
757
mM NaCl, 16h) by staining with 10 μM CoroNa-Green dye (Molecular Probes), and
758
images were obtained by confocal laser scanning microscopy using an Olympus
759
FV1000 microscope.
760
performed using the FV10-ASW4.1 software for image analysis. The data were
761
subjected to statistical analysis using ANOVA.
Quantification of the fluorescent signals from the cells was
762
763
764
ACKNOWLEDGEMENTS
765
We thank Dr. Mauricio Soto for assistance in microarray data analysis and Dra.
766
Montserrat Pagés for critical reading of this manuscript. We are also grateful to Gisela
767
Peñas, Vanessa Alfaro and Vanessa Vega for technical assistance.
768
769
770
771
LITERATURE CITED
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
Akhtar M, Jaiswal A, Taj G, Jaiswal JP, Qureshi MI, Singh NK (2012)
DREB1/CBF transcription factors: their structure, function and role in abiotic
stress tolerance in plants. J Genet 91: 385-395
Asai S, Ichikawa T, Nomura H, Kobayashi M, Kamiyoshihara Y, Mori H, Kadota
Y, Zipfel C, Jones JDG, Yoshioka H (2013) The Variable Domain of a Plant
Calcium-dependent Protein Kinase (CDPK) Confers Subcellular Localization
and Substrate Recognition for NADPH Oxidase. J Biol Chem 288: 14332-14340
Asano T, Hakata M, Nakamura H, Aoki N, Komatsu S, Ichikawa H, Hirochika H,
Ohsugi R (2011) Functional characterisation of OsCPK21, a calcium-dependent
protein kinase that confers salt tolerance in rice. Plant Mol Biol 75: 179-191
Asano T, Hayashi N, Kobayashi M, Aoki N, Miyao A, Mitsuhara I, Ichikawa H,
Komatsu S, Hirochika H, Kikuchi S, Ohsugi R (2012) A rice calciumdependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance
and blast disease resistance. Plant J 69: 26-36
Asano T, Tanaka N, Yang G, Hayashi N, Komatsu S (2005) Genome-wide
Identification of the Rice Calcium-dependent Protein Kinase and its Closely
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
25
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
Related Kinase Gene Families: Comprehensive Analysis of the CDPKs Gene
Family in Rice. Plant Cell Physiol 46: 356-366
Benetka W, Mehlmer N, Maurer-Stroh S, Sammer M, Koranda M, Neumüller R,
Betschinger J, Knoblich JA, Teige M, Eisenhaber F (2008) Experimental
testing of predicted myristoylation targets involved in asymmetric cell division
and calcium-dependent signalling. Cell Cycle 7: 3709-3719
Boudsocq M, Sheen J (2010) Calcium Sensing and Signaling. In A Pareek, S Sopory,
H Bohnert, Govindjee, eds, Abiotic Stress Adaptation in Plants: Physiological,
Molecular and Genomic Foundation Springer, Dordrecht, The Netherlands, pp
75-90
Campos-Soriano L, Gomez-Ariza J, Bonfante P, San Segundo B (2011) A rice
calcium-dependent protein kinase is expressed in cortical root cells during the
presymbiotic phase of the arbuscular mycorrhizal symbiosis. BMC Plant Biol
11: 90
Cao W-H, Liu J, He X-J, Mu R-L, Zhou H-L, Chen S-Y, Zhang J-S (2007)
Modulation of Ethylene Responses Affects Plant Salt-Stress Responses. Plant
Physiol 143: 707-719
Chitteti BR, Peng Z (2007) Proteome and Phosphoproteome Differential Expression
under Salinity Stress in Rice (Oryza sativa) Roots. J Proteome Res 6: 1718-1727
Choi W-G, Toyota M, Kim S-H, Hilleary R, Gilroy S (2014) Salt stress-induced
Ca2+ waves are associated with rapid, long-distance root-to-shoot signaling in
plants. Proc Natl Acad Sci USA: doi:10.1073/pnas.1319955111
Coca M, San Segundo B (2010) AtCPK1 calcium-dependent protein kinase mediates
pathogen resistance in Arabidopsis. Plant J 63: 526-540
Csiszar J, Galle Å, Horvath E, Dancso P, Gombos M, Vary Z, Erdei L, Gyorgyey
J, Tari I (2012) Different peroxidase activities and expression of abiotic stressrelated peroxidases in apical root segments of wheat genotypes with different
drought stress tolerance under osmotic stress. Plant Physiol Biochem 52: 119129
Dammann C, Ichida A, Hong B, Romanowsky SM, Hrabak EM, Harmon AC,
Pickard BG, Harper JF (2003) Subcellular Targeting of Nine CalciumDependent Protein Kinase Isoforms from Arabidopsis. Plant Physiol 132: 18401848
Das R, Pandey GK (2010) Expressional Analysis and Role of Calcium Regulated
Kinases in Abiotic Stress Signaling. Curr Genomics 11: 2-13
Demiral T, Turkan I (2005) Comparative lipid peroxidation, antioxidant defense
systems and proline content in roots of two rice cultivars differing in salt
tolerance. Environ Exp Bot 53: 247-257
Dionisio-Sese ML, Tobita S (1998) Antioxidant responses of rice seedlings to salinity
stress. Plant Sci 135: 1-9
Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M,
Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB genes in rice, Oryza
sativa L., encode transcription activators that function in drought-, high-salt- and
cold-responsive gene expression. Plant J 33: 751-763
Edwards R, Dixon DP, Walbot V (2000) Plant glutathione S-transferases: enzymes
with multiple functions in sickness and in health. Trends Plant Sci 5: 193-198
Franz S, Ehlert B, Liese A, Kurth J, Cazale A-C, Romeis T (2011) CalciumDependent Protein Kinase CPK21 Functions in Abiotic Stress Response in
Arabidopsis thaliana. Mol Plant 4: 83-96
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
26
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
Gasulla F, vom Dorp K, Dombrink I, Zähringer U, Gisch N, Dörmann P, Bartels D
(2013) The role of lipid metabolism in the acquisition of desiccation tolerance in
Craterostigma plantagineum: a comparative approach. Plant J 75: 726-741
Ge L-F, Chao D-Y, Shi M, Zhu M-Z, Gao J-P, Lin H-X (2008) Overexpression of
the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in
rice and results in the activation of stress responsive genes. Planta 228: 191-201
Geiger D, Scherzer S, Mumm P, Marten I, Ache P, Matschi S, Liese A, Wellmann
C, Al-Rasheid KAS, Grill E, Romeis T, Hedrich R (2010) Guard cell anion
channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+
affinities. Proc Natl Acad Sci USA 107: 8023-8028
Goff SA (1999) Rice as a model for cereal genomics. Curr Opin Plant Biol 2: 86-89
Guo L, Yang H, Zhang X, Yang S (2013) Lipid transfer protein 3 as a target of
MYB96 mediates freezing and drought stress in Arabidopsis. J Exp Bot 64:
1755-1767
Harper JE, Breton G, Harmon A (2004) Decoding Ca2+ signals through plant protein
kinases. Annu Rev Plant Biol 55: 263-288
Hirayama T, 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
Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the
thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in
plant tissues containing anthocyanin and other interfering compounds. Planta
207: 604-611
Hong J, Choi H, Hwang I, Kim D, Kim N, Choi D, Kim Y, Hwang B (2008)
Function of a novel GDSL-type pepper lipase gene, CaGLIP1, in disease
susceptibility and abiotic stress tolerance. Planta 227: 539-558
Hrabak EM, Chan CWM, Gribskov M, Harper JF, Choi JH, Halford N, Kudla Jr,
Luan S, Nimmo HG, Sussman MR, Thomas M, Walker-Simmons K, Zhu JK, Harmon AC (2003) The Arabidopsis CDPK-SnRK Superfamily of Protein
Kinases. Plant Physiol 132: 666-680
Hughes MA, Dunn MA, Pearce RS, White AJ, Zhang L (1992) An abscisic-acidresponsive, low temperature barley gene has homology with a maize
phospholipid transfer protein. Plant Cell Environ 15: 861-865
Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K,
Yamaguchi-Shinozaki K (2006) Functional Analysis of Rice DREB1/CBFtype Transcription Factors Involved in Cold-responsive Gene Expression in
Transgenic Rice. Plant Cell Physiol 47: 141-153
Katsuhara M, Otsuka T, Ezaki B (2005) Salt stress-induced lipid peroxidation is
reduced by glutathione S-transferase, but this reduction of lipid peroxides is not
enough for a recovery of root growth in Arabidopsis. Plant Sci 169: 369-373
Khan MH, Panda SK (2008) Alterations in root lipid peroxidation and antioxidative
responses in two rice cultivars under NaCl-salinity stress. Acta Physiol Plant 30:
81-89
Klimecka M, Muszynska G (2007) Structure and functions of plant calcium-dependent
protein kinases. Acta Biochim Pol 54: 219-233
Komatsu S, Yang G, Khan M, Onodera H, Toki S, Yamaguchi M (2007) Overexpression of calcium-dependent protein kinase 13 and calreticulin interacting
protein 1 confers cold tolerance on rice plants. Mol Genet Genomics 277: 713723
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
27
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
Latz A, Mehlmer N, Zapf S, Mueller TD, Wurzinger B, Pfister B, Csaszar E,
Hedrich R, Teige M, Becker D (2013) Salt Stress Triggers Phosphorylation of
the Arabidopsis Vacuolar K+ Channel TPK1 by Calcium-Dependent Protein
Kinases (CDPKs). Mol Plant 6: 1274-1289
Laura M, Consonni R, Locatelli F, Fumagalli E, Allavena A, Coraggio I, Mattana
M (2010) Metabolic response to cold and freezing of Osteospermum ecklonis
overexpressing Osmyb4. Plant Physiol Biochem 48: 764-771
Lee S, Huh K, An K, An G, Kim S (2004) Ectopic expression of a cold-inducible
transcription factor, CBF1/DREB1b, in transgenic rice (Oryza sativa L.).
Molecules and Cells 18: 107-114
Liang M, Haroldsen V, Cai X, Wu Y (2006) Expression of a putative laccase gene,
ZmLAC1, in maize primary roots under stress. Plant Cell Environ 29: 746-753
Lopez-Perez L, Martinez-Ballesta MdC, Maurel C, Carvajal M (2009) Changes in
plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an
adaptation mechanism to salinity. Phytochemistry 70: 492-500
Lu S, Hrabak E (2013) The myristoylated amino-terminus of an Arabidopsis calciumdependent protein kinase mediates plasma membrane localization. Plant Mol
Biol 82: 267-278
Maas EV, Hoffman NC (1977) Crop salt tolerance - current assessment. J Irrig Drain
E- ASCE 103: 115-134
Martín ML, Busconi L (2000) Membrane localization of a rice calcium-dependent
protein kinase (CDPK) is mediated by myristoylation and palmitoylation. Plant J
24: 429-435
Matsui A, Ishida J, Morosawa T, Mochizuki Y, Kaminuma E, Endo TA, Okamoto
M, Nambara E, Nakajima M, Kawashima M, Satou M, Kim J-M,
Kobayashi N, Toyoda T, Shinozaki K, Seki M (2008) Arabidopsis
Transcriptome Analysis under Drought, Cold, High-Salinity and ABA
Treatment Conditions using a Tiling Array. Plant Cell Physiol 49: 1135-1149
Matsukura S, Mizoi J, Yoshida T, Todaka D, Ito Y, Maruyama K, Shinozaki K,
Yamaguchi-Shinozaki K (2010) Comprehensive analysis of rice DREB2-type
genes that encode transcription factors involved in the expression of abiotic
stress-responsive genes. Mol Genet Genomics 283: 185-196
Mehlmer N, Wurzinger B, Stael S, Hofmann-Rodrigues D, Csaszar E, Pfister B,
Bayer R, Teige M (2010) The Ca2+-dependent protein kinase CPK3 is required
for MAPK-independent salt-stress acclimation in Arabidopsis. Plant J 63: 484498
Miller GAD, Suzuki N, Ciftci-Yilmaz S, Mittler RON (2010) Reactive oxygen
species homeostasis and signalling during drought and salinity stresses. Plant
Cell Environ 33: 453-467
Minh-Thu P-T, Hwang D-J, Jeon J-S, Nahm B, Kim Y-K (2013) Transcriptome
analysis of leaf and root of rice seedling to acute dehydration. Rice 6: 38
Munns R, Tester M (2008) Mechanisms of Salinity Tolerance. Annu Rev Plant Biol
59: 651-681
Murillo I, Roca R, Bortolotti C, Segundo BS (2003) Engineering photoassimilate
partitioning in tobacco plants improves growth and productivity and provides
pathogen resistance. Plant J 36: 330-341
Nakashima K, Ito Y, Yamaguchi-Shinozaki K (2009) Transcriptional Regulatory
Networks in Response to Abiotic Stresses in Arabidopsis and Grasses. Plant
Physiol 149: 88-95
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
28
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
Nakashima K, Tran L-SP, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Ito
Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K (2007) Functional
analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and
biotic stress-responsive gene expression in rice. Plant J 51: 617-630
Oberschall A, Deák M, Török K, Sass L, Vass I, Kovács I, Fehér A, Dudits D,
Horváth GV (2000) A novel aldose/aldehyde reductase protects transgenic
plants against lipid peroxidation under chemical and drought stresses. Plant J 24:
437-446
Prak S, Hem S, Boudet J, Viennois G, Sommerer N, Rossignol M, Maurel C,
Santoni V (2008) Multiple Phosphorylations in the C-terminal Tail of Plant
Plasma Membrane Aquaporins: Role in Subcellular Trafficking of AtPIP2;1 in
Response to Salt Stress. Mol Cell Proteomics 7: 1019-1030
Qing D-J, Lu H-F, Li N, Dong H-T, Dong D-F, Li Y-Z (2009) Comparative Profiles
of Gene Expression in Leaves and Roots of Maize Seedlings under Conditions
of Salt Stress and the Removal of Salt Stress. Plant Cell Physiol 50: 889-903
Quintero FJ, Ohta M, Shi H, Zhu J-K, Pardo JM (2002) Reconstitution in yeast of
the Arabidopsis SOS signaling pathway for Na+ homeostasis. Proc Natl Acad
Sci USA 99: 9061-9066
Rabbani MA, Maruyama K, Abe H, Khan MA, Katsura K, Ito Y, Yoshiwara K,
Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Monitoring Expression
Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and
Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot
Analyses. Plant Physiol 133: 1755-1767
Ray S, Agarwal P, Arora R, Kapoor S, Tyagi A (2007) Expression analysis of
calcium-dependent protein kinase gene family during reproductive development
and abiotic stress conditions in rice (Oryza sativa L. ssp. indica). Molecular
Genetics & Genomics
Reddy ASN, Ali GS, Celesnik H, Day IS (2011) Coping with Stresses: Roles of
Calcium- and Calcium/Calmodulin-Regulated Gene Expression. Plant Cell 23:
2010-2032
Roxas VP, Lodhi SA, Garrett DK, Mahan JR, Allen RD (2000) Stress Tolerance in
Transgenic Tobacco Seedlings that Overexpress Glutathione STransferase/Glutathione Peroxidase. Plant Cell Physiol 41: 1229-1234
Saijo Y, Hata S, Kyozuka J, Shimamoto K, Izui K (2000) Over-expression of a
single Ca2+-dependent protein kinase confers both cold and salt/drought
tolerance on rice plants. Plant J 23: 319-327
Sakuma Y, Maruyama K, Qin F, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki
K (2006) Dual function of an Arabidopsis transcription factor DREB2A in
water-stress-responsive and heat-stress-responsive gene expression. Proc Natl
Acad Sci USA 103: 18822-18827
Schulz P, Herde M, Romeis T (2013) Calcium-Dependent Protein Kinases: Hubs in
Plant Stress Signaling and Development. Plant Physiol 163: 523-530
Sheen J (1996) Ca2+-dependent protein kinases and stress signal transduction in plants.
Science 274: 1900-1902
Todaka D, Nakashima K, Shinozaki K, Yamaguchi-Shinozaki K (2012) Toward
understanding transcriptional regulatory networks in abiotic stress responses and
tolerance in rice. Rice 5: 6
Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied
to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116-5121
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
29
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu J-K (2006) Methods and
concepts in quantifying resistance to drought, salt and freezing, abiotic stresses
that affect plant water status. Plant J 45: 523-539
Wang X-S, Zhu H-B, Jin G-L, Liu H-L, Wu W-R, Zhu J (2007) Genome-scale
identification and analysis of LEA genes in rice (Oryza sativa L.). Plant Sci 172:
414-420
Weber H, Chételat A, Reymond P, Farmer EE (2004) Selective and powerful stress
gene expression in Arabidopsis in response to malondialdehyde. Plant J 37: 877888
Wu G, Robertson AJ, Liu X, Zheng P, Wilen RW, Nesbitt NT, Gusta LV (2004) A
lipid transfer protein gene BG-14 is differentially regulated by abiotic stress,
ABA, anisomycin, and sphingosine in bromegrass (Bromus inermis). J Plant
Physiol 161: 449-458
Xu J, Tian Y-S, Peng R-H, Xiong A-S, Zhu B, Jin X-F, Gao F, Fu X-Y, Hou X-L,
Yao Q-H (2010) AtCPK6, a functionally redundant and positive regulator
involved in salt/drought stress tolerance in Arabidopsis. Planta 231: 1251-1260
Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in
cellular responses and tolerance to dehydration and cold stresses. Annu Rev
Plant Biol 57: 781-803
Yokotani N, Sato Y, Tanabe S, Chujo T, Shimizu T, Okada K, Yamane H,
Shimono M, Sugano S, Takatsuji H, Kaku H, Minami E, Nishizawa Y
(2013) WRKY76 is a rice transcriptional repressor playing opposite roles in
blast disease resistance and cold stress tolerance. J Exp Bot 64: 5085-5097
Zeng L, Shannon MC (2000) Salinity effects on seedling growth and yield components
of rice. Crop Sci 40: 996-1003
Zheng X, Chen B, Lu G, Han B (2009) Overexpression of a NAC transcription factor
enhances rice drought and salt tolerance. Biochem Biophys Res Commun 379:
985-989
Zhu S-Y, Yu X-C, Wang X-J, Zhao R, Li Y, Fan R-C, Shang Y, Du S-Y, Wang XF, Wu F-Q, Xu Y-H, Zhang X-Y, Zhang D-P (2007) Two Calcium-Dependent
Protein Kinases, CPK4 and CPK11, Regulate Abscisic Acid Signal Transduction
in Arabidopsis. Plant Cell 19: 3019-3036
1019
1020
FIGURE LEGENDS
1021
1022
Figure 1. Expression of OsCPK4 in response to salt and drought stress. Transcript
1023
levels were determined by qRT-PCR and normalized to OsCYP and OsUbi mRNAs. A,
1024
Structure of OsCPK4 showing the typical domains of plant CPKs: a kinase catalytic
1025
domain joined via a junction (J) domain sequence to a calmodulin-like domain (CaM)
1026
with four calcium-binding EF hands (rhombus in the calmodulin domain). The N-
1027
terminal amino acid sequence of OsCPK4 is indicated in the upper part. The bar
1028
indicates the DNA fragment used as probe in Northern blot analyses of OsCPK4 in
1029
Supplemental Fig. S3. Arrows indicate the position of primers used for qRT-PCR. B,
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
30
1030
Expression of OsCPK4 in leaves and roots of rice plants at the indicated developmental
1031
stages. C, D, Expression of OsCPK4 (C) and OsLEA23 (D) in roots of rice plants in
1032
response to salt and drought stress. Seven-day old seedlings were exposed to 100mM
1033
NaCl, air-dried or treated with 20% PEG 8000 for the indicated periods of time. The
1034
same RNA samples were used in C and D. Values represent mean+/-SE of three
1035
replicates (*, P ≤ 0.05; **, P ≤ 0.001).
1036
1037
Figure 2. OsCPK4 expression in response to ABA and accumulation of OsCPK4 in
1038
response to ABA treatment, salt and drought stress. A, Diagram of the OsCPK4
1039
promoter showing the distribution of abiotic stress-related cis-elements. B, OsCPK4 and
1040
OsLEA26 expression in rice roots in response to ABA. The OsCYP and OsUBI mRNAs
1041
were used as internal controls for normalization. Significant differences are indicated
1042
(*P < 0.05, **P < 0.001). C, Accumulation of OsCPK4 in protein extracts of rice roots
1043
that have been subjected to salt stress, ABA treatment or drought stress (air drying) for
1044
1h. Protein extracts (20 µg each) were probed with an antiserum prepared against the N-
1045
terminal domain of OsCPK4. Upper panel, protein extracts from control (line C) and
1046
salt-stressed (line S) roots. Lower panel, protein extracts from control (lines C, controls
1047
for ABA treatment; C’, control for drought treatment), ABA-treated (line A) and
1048
drought-stressed rice roots (line D). Ponceau-S staining of Western blots was used as
1049
the loading control.
1050
1051
Figure 3. Plasma membrane localization of OsCPK4. Onion epidermal cells were
1052
transformed with the OsCPK4-GFP gene via particle bombardment. Confocal images
1053
were taken 5h post-bombardment. Projection (A, B) and individual (C, D) sections are
1054
shown. A, Localization of OsCPK4 fusion protein. Fluorescence and transmission
1055
images are shown in the left and middle panels, respectively. Merged pictures of the
1056
green fluorescence channel with the corresponding light micrographs are shown on the
1057
right panel. B, Onion epithelial cell expressing gfp. C, Onion cells transformed with the
1058
OsCPK4-GFP gene after plasmolysis with mannitol (15 min of treatment). Light
1059
micrographs show the shrinkage of the protoplast. D, Higher magnification of a
1060
plasmolysed onion cell showing the Hechtian strands (h). Treatment with mannitol
1061
renders the Hechtian strands attaching the plasma membrane to the cell wall. Scale bars
1062
= 20 µm. cw, cell wall; pm, plasma membrane.
1063
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
31
1064
Figure 4. Salt tolerance of rice plants overexpressing OsCPK4. A, Diagram showing
1065
the experimental design for salt tolerance assays. Salt stress was imposed to both
1066
transgenic and control plants grown in hydroponic culture until they reached the three-
1067
leaf stage (D0). Plants were then transferred to nutrient solution containing 60mM
1068
NaCl, or to fresh nutrient solution. In each experiment, wild-type (Nipponbare), vector
1069
control (pC, 3 independent lines) and OsCPK4 (OsCPK4-OX, 5 independent lines)
1070
transgenic lines were assayed. B, Phenotype of OsCPK4-OX rice plants under salt stress
1071
conditions. Whereas control plants (WT and pC) grew poorly and exhibited chlorosis,
1072
the OsCPK4-OX lines grew in the presence of NaCl. Photographs were taken after 18
1073
days of salt stress (D18). C, Survival rates of OsCPK4-OX and vector control plants rice
1074
plants at different times of salt treatment. D, OsCPK4 expression in roots of transgenic
1075
and control plants used in salt tolerance assays as determined by qRT-PCR analysis.
1076
Specific primers for analysis of OsCPK4 transgen expression were used (Supplemental
1077
Table SIV). Samples were taken at D0 time point (onset of salt treatment). Salt
1078
tolerance assays were carried out three times and at two successive generations (T2 and
1079
T3). In each experiment, at least 30 plants per line were assayed. Asterisks denote
1080
significant differences between the OsCPK4-OX and control (pC and WT) groups (*, P
1081
≤ 0.05; **, P ≤ 0.001).
1082
1083
Figure 5. Drought tolerance of rice plants overexpressing OsCPK4. A, Diagram
1084
showing the experimental design for drought tolerance assays. Transgenic and control
1085
(wild-type and transgenic lines expressing the empty vector) plants were grown for 2
1086
weeks (D0), subjected to 17 days of drought stress (D17), followed by 9 days of re-
1087
watering (RW) in the greenhouse. B, Phenotype of OsCPK4 overexpressor (OsCPK4-
1088
OX) and vector control (pC) plants during drought stress. Photographs were taken at day
1089
0 (onset of drought treatment), at 17 days of drought stress (D17) and after 9 days of re-
1090
watering (RW). C, Survival rates of OsCPK4-OX and pC plants exposed to drought
1091
stress. D, Water loss and E, water retention rates of detached leaves of OsCPK4-OX and
1092
pC plants at the three-leave stage (*, P ≤ 0.05). Drought tolerance assays were repeated
1093
3 times using five independent OsCPK4 transgenic lines and two independent vector
1094
control lines (at least 30 plants per line). Values represent the mean+/-SE of three
1095
replicates (*, P ≤ 0.05).
1096
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
32
1097
Figure 6. Analysis of cpk4 mutants. A, OsCPK4 gene structure showing the T-DNA
1098
insertion sites for the cpk4 mutants identified in the POSTECH collection (the 2D-
1099
00040 and 1D-03351 mutants). Exons and introns are indicated by black boxes and
1100
lines, respectively. Gene-specific primers used for the analysis of T-DNA integration
1101
are indicated by arrows. B, Analysis of T-DNA integration in the 2D-00040 cpk4
1102
mutant. “Ho”,“he” and “Az” indicates homozygous, hemizygous and azygous plants,
1103
respectively. C, qRT-PCR analysis of OsCPK4 expression in leaves of 2D-00040 cpk4
1104
mutant plants. D, Appearance of the homozygous and hemizygous 2D-00040 cpk4
1105
mutant plants. Similar results were obtained for the 1D-03351 cpk4 mutant. Asterisks
1106
denote significant differences (**, P ≤ 0.001).
1107
1108
Figure 7. Differentially expressed genes in OsCPK4 rice plants. A, Functional
1109
categories of up-regulated genes in roots of OsCPK4-OX plants as determined by
1110
microarray analysis. B, Expression of Thioredoxin and Laccase genes in roots of
1111
OsCPK4-OX plants. C, Functional categories of misregulated genes in leaves of
1112
OsCPK4-OX plants. D. Expression of WRKY71, WRKY76 and aspartic proteinase genes
1113
in leaves of OsCPK4-OX plants. Transcript levels were determined by qRT-PCR using
1114
total RNA. Representative results are shown for two OsCPK4 lines (lines 1 and 14) and
1115
two vector control lines (lines 3 and 11) plants from a total of 5 and 3 independent
1116
OsCPK4 and vector control lines, respectively, analysed in this work.
1117
1118
Figure 8. Effect of salt treatment on lipid peroxidation, electrolyte leackage and Na+
1119
content. A and B, Detection of lipid peroxidation (A) and electrolyte leackage (B) in
1120
leaves of 3 week-old plants, both vector control (pC, 2 independent lines) and OsCPK4-
1121
overexpressor (OsCPK4-OX, three independent lines) plants, treated with 100mM NaCl
1122
for 14 days. Data shown correspond to unstressed (grey bars) and salt-stressed (blue and
1123
orange bars) OsCPK4-OX and pC lines. FW, fresh weight. C, Imaging of Na+ content in
1124
salt-stressed vector control (upper planels) and OsCPK4-OX (lower panels) roots.
1125
Confocal images from roots of five-day-old seedlings that had been treated with
1126
150mM NaCl for 16h and stained with CoroNa-Green (green) and propidium iodide
1127
(red) are shown. Bars, 20 μm. D, Quantitative comparison of CoroNa-Green fluorescent
1128
intensities. Two independent lines and three individual plants per line were examined
1129
for each genotype. Values represent the mean +/- SE of average intensities of root
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
33
1130
sections at 700 μm distance to the tip. Asterisks indicate a significant differences (**, P
1131
≤ 0.001, ANOVA).
1132
1133
1134
SUPPLEMENTAL DATA
1135
1136
Supplemental Figure S1. Expression of OsCPK4 in rice leaves in response to salt
1137
stress.
1138
1139
Supplemental Figure S2. Amino acid sequence of OsCPK4 and alignment of the N-
1140
terminal variable domain of rice CPKs.
1141
1142
Supplemental Figure S3. Overexpression of OsCPK4 in transgenic rice.
1143
1144
Supplemental Figure S4. Expression of salt-associated genes in roots of rice plants
1145
overexpressing OsCPK4.
1146
1147
Supplemental Table SI. cis-related motifs identified in the 2000 bp upstream region of
1148
the OsCPK4 promoter.
1149
1150
Supplemental Table SII. Genes misregulated in leaves of plants overexpressing
1151
OsCPK4 relative to vector control plants, identified by microarray analysis.
1152
1153
Supplemental Table SIII. List of primers used for OsCPK4 cloning purposes and
1154
mutant analysis.
1155
1156
Supplemental Table SIV. List of primers used for expression analysis of rice genes by
1157
qRT-PCR.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
34
1158
Table I. Genes up-regulated in roots of plants overexpressing OsCPK4 relative to vector control
1159
1160
1161
plants, identified by microarray analysis. Asterisks denote genes involved in oxidation-reduction
processes. Indicated are the genes with a fold change ≥ 2.0-fold OsCPK4 plants. Genes with P values ≤
0.05 were considered.
Biological
function
1. Metabolism
Carbohydrates
Lipids
Aminoacids and Proteins
DNA/ RNA modification
Others
Fold
change
Gene ID
Description
LOC_Os01g62420
LOC_Os03g36530
LOC_Os10g32980
LOC_Os01g46250
LOC_Os03g64170
LOC_Os05g36000
LOC_Os05g46580
LOC_Os06g20430
LOC_Os06g29650
LOC_Os07g07930
LOC_Os10g25400
LOC_Os10g40530
LOC_Os12g16640
LOC_Os01g47410
LOC_Os01g48740
LOC_Os01g69040
LOC_Os04g27850
LOC_Os04g49160
LOC_Os05g41010
LOC_Os05g41630
LOC_Os01g60790
LOC_Os11g24240
LOC_Os01g01400
LOC_Os02g47560
LOC_Os02g30630
LOC_Os04g33670
None
LOC_Os07g28400
LOC_Os07g37420
Triose phosphate isomerase, cytosolic
FAD-binding and arabino-lactone oxidase domains containing protein*
Cellulose synthase
Lipase
GDSL-like lipase/acyl hydrolase
FabA-like domain containing protein
Polyprenyl synthetase (Similar to Farnesyl diphosphate synthase)
Lipid binding protein (BPI/LBP family protein At3g20270 precursor
CDP-diacylglycerol-inositol 3-phosphatidyltransferase 1
Lipid Transfer protein (LTPL78)- Protease inhibitor/seed storage/LTP family
GDSL-like lipase/acylhydrolase
Lipid Transfer protein (LTPL146)-Protease inhibitor/seed storage/LTP family
Similar to cyclopropane fatty acid synthase*
Aspartic proteinase oryzasin-1 precursor
Aspartyl protease family protein
zinc finger, C3HC4 type domain containing protein (ubiquitination)
Oxidoreductase*
zinc finger, C3HC4 type domain containing protein (ubiquitination)
Oxidoreductase*
Translation initiation factor eIF3 subunit
40S ribosomal protein S26
Sinapoyl glucose choline sinapoyl transferase
Polynucleotidyl transferase, Ribonuclease H fold domain containing protein
DNA helicase
thiamine pyrophosphate enzyme, C-terminal TPP binding domain
Carbonic anhydrase, eukaryotic family protein.
cytochrome P450* (AK106236)
CDGSH iron sulfur domain-containing protein 1
short-chain dehydrogenase/reductase*
2.20
2.03
2.23
2.00
2.06
2.36
2.21
2.01
2.03
2.65
2.20
2.00
2.10
2.00
2.19
2.04
2.09
2.51
2.00
2.14
2.03
2.05
2.14
2.14
2.18
2.07
2.48
2.01
2.16
LOC_Os09g29100
Cyclin
2.07
Peroxidase*
Thioredoxin*
Laccase*
glutathione S-transferase*
2.19
2.00
2.19
2.00
MYB family transcription factor (OsMYB4)
G-patch domain containing protein
FACT complex subunit SPT16
zinc finger C-x8-C-x5-C-x3-H type family protein
helix-loop-helix DNA-binding protein
2.02
2.00
2.32
2.33
2.11
2. Cellular processes
3. Oxidative stress and redox regulation
LOC_Os02g14160
LOC_Os02g42700
LOC_Os10g20610
LOC_Os07g07320
4. Transcription and RNA binding
LOC_Os01g50110
LOC_Os03g14860
LOC_Os04g25550
LOC_Os04g57010
LOC_Os11g06010
5. Signal transduction and Intracellular trafficking
LOC_Os03g23960
LOC_Os04g49530
LOC_Os04g53998
LOC_Os06g36400
LOC_Os07g44330
LOC_Os09g02410
LOC_Os11g44310
LOC_Os12g06630
IQ calmodulin-binding motif family protein
GTP1/OBG domain containing protein.
S-locus receptor-like kinase RLK13.
HAD superfamily phosphatase
Kinase
S-domain receptor-like protein kinase
Calmodulin binding protein
OsFBT14 - F-box and tubby domain
2.00
2.28
2.25
2.26
2.33
2.37
2.00
2.06
LOC_Os02g20330
LOC_Os08g39950
Cation antiporter
Potassium transporter
2.00
2.01
LOC_Os01g26370
LOC_Os01g39080
LOC_Os01g55160
LOC_Os01g74170
LOC_Os04g39660
LOC_Os05g01210
LOC_Os05g03410
LOC_Os05g39980
LOC_Os06g05470
LOC_Os06g22410
LOC_Os07g02360
LOC_Os11g31680
LOC_Os11g42970
Expressed protein
Expressed protein
Expressed protein
Expressed protein
OsFBDUF22 - F-box and DUF domain containing protein
Expressed protein
Expressed protein
Protein of unknown function DUF3615 domain containing protein.
Expressed protein
Expressed protein
Expressed protein
Retrotransposon protein
membrane associated DUF588 domain containing protein
2.17
2.07
2.04
2.09
2.03
2.24
2.00
2.34
2.03
2.30
2.34
2.01
2.13
7. Ion transport
8. Unknown
1162
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
35
A!
B!
MGACFSSHTATAAADGGSGKRQQR !
Relative expression level !
(x102)!
Fig. 1!
0.015
1.5!
TGA!
5’-UT!
J!CaM domain!
Kinase!
0.003
0.3!
Relative expression level !
(x10)!
0.0
0.000
C!
0.050
0.5!
OsCPK4
0.040
0.4!
0.030
0.3!
0.030
0.3!
**!
0.020
0.2!
0.010
0.1!
0.000
0.0!
30’!
Relative expression level!
1.000
1.0!
1h!
1h
4h!
3h
*!
*!
9h!
24h!
4h
OsLEA23
*!
CONTROL!
NaCl!
*!
0.200
0.2!
*
1h!
1h
4h!
3h
9h!
4h
24h!
9h
!
14d
14d!
**!
28d
28d!
30’!
1h!
1h
4h!
3h
50d
50d!
CONTROL!
PEG!
*!
0.030
0.3!
*
9h!
4h
0.400
0.4!
*!
24h!
0.020
0.2!
0.000
0.0!
9h
CONTROL!
Air-dried!
0.3!
0.300
0.000
0.0!
*!
*
**!
0.010
0.1!
30'
0.1!
0.100
*!
30'
*
30’!
30'
1h!
1h
4h!
3h
0.400
0.4!
9h!
4h
*!
24h!
9h
CONTROL!
PEG!
0.300
0.3!
**!
0.2!
0.200
0.2!
0.200
*!
30’!
0.000
0.0!
9h
0.600
0.6!
0.000
0.0!
**!
0.010
0.1!
0.800
0.8!
0.400
0.4!
0.050
0.5!
CONTROL!
Air-dried!
0.040
0.4!
0.020
0.2!
*!
30'
D!
0.050
0.5!
CONTROL!
NaCl!
0.040
0.4!
*!
0.009
0.9!
3’-UT!
0.006
0.6!
N-terminal!
ROOT!
ROOT
LEAF
LEAF!
*!
0.012
1.2!
*!
30’!
30'
**!
1h!
1h
*!
*!
*!
4h!
9h!
24h!
3h
4h
9h
*!
0.1!
0.100
0.000
0.0!
30’!
30'
1h!
1h
**!
4h!
3h
*!
9h!
4h
24h!
9h
Figure 1. Expression of OsCPK4 in response to salt and drought stress. Transcript levels were determined by qRT-PCR
and normalized to OsCYP and OsUbi mRNAs. A, Structure of OsCPK4 showing the typical domains of plant CPKs: a
kinase catalytic domain joined via a junction (J) domain sequence to a calmodulin-like domain (CaM) with four calciumbinding EF hands (rhombus in the calmodulin domain). The N-terminal amino acid sequence of OsCPK4 is indicated in the
upper part. The bar indicates the DNA fragment used as probe in Northern blot analyses of OsCPK4 in Supplemental Fig.
S3. Arrows indicate the position of primers used for qRT-PCR. B, Expression of OsCPK4 in leaves and roots of rice plants
at the indicated developmental stages. C, D , Expression of OsCPK4 (C) and OsLEA23 (D) in roots of rice plants in
response to salt and drought stress. Seven-day old seedlings were exposed to 100mM NaCl, air-dried or treated with 20%
PEG 8000 for the indicated periods of time. The same RNA samples were used in C and D. Values represent mean+/-SE of
three replicates (*, P ≤ 0.05; **, P ≤ 0.001).
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
+176!
+1!
-147!
-241!
-346!
-469!
-584!
-686!
-885!
-865!
-831!
-790!
-963!
-1121!
-1356!
-1310!
-1538!
A!
-1873!
Fig. 2!
UTR!
MYB!
ABRE!
MYC!
C!
B!
OsCPK4!
CONTROL!
ABA!
0.06
6!
*!
*!
0.04
4!
2!
0.02
0.00
0!
OsLEA26!
8.00
8!
Relative expression level!
Relative expression level!
(x102) !
0.08
8!
*!
1h!
1h
2.00
2!
2h!
2h
3h!
3h
0.00
0!
S!
**!
KDa!
C! C’! A! D!
75!
**!
1h!
1h
OsCPK4
50!
**!
4.00
4!
C!
75!
CONTROL!
ABA!
6.00
6!
KDa!
OsCPK4
50!
2h!
2h
3h!
3h
Figure 2. OsCPK4 expression in response to ABA and accumulation of OsCPK4 in response to ABA treatment, salt and drought
stress. A, Diagram of the OsCPK4 promoter showing the distribution of abiotic stress-related cis-elements. B, OsCPK4 and
OsLEA26 expression in rice roots in response to ABA. The OsCYP and OsUBI mRNAs were used as internal controls for
normalization. Significant differences are indicated (*P < 0.05, **P < 0.001). C, Accumulation of OsCPK4 in protein extracts of
rice roots that have been subjected to salt stress, ABA treatment or drought stress (air drying) for 1h. Protein extracts (20 µg
each) were probed with an antiserum prepared against the N-terminal domain of OsCPK4. Upper panel, protein extracts from
control (line C) and salt-stressed (line S) roots. Lower panel, protein extracts from control (lines C, controls for ABA treatment;
C’, control for drought treatment), ABA-treated (line A) and drought-stressed rice roots (line D). Ponceau-S staining of Western
blots was used as the loading control.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Fig. 3!
A
cw!
cw!
B!
C
cw
pm!
cw!
cw!
D
h!
Figure 3. Plasma membrane localization of OsCPK4. Onion epidermal cells were transformed with the OsCPK4GFP gene via particle bombardment. Confocal images were taken 5h post-bombardment. Projection (A, B) and
individual (C, D) sections are shown. A, Localization of OsCPK4 fusion protein. Fluorescence and transmission
images are shown in the left and middle panels, respectively. Merged pictures of the green fluorescence channel
with the corresponding light micrographs are shown on the right panel. B, Onion epithelial cell expressing gfp. C,
Onion cells transformed with the OsCPK4-GFP gene after plasmolysis with mannitol (15 min of treatment). Light
micrographs show the shrinkage of the protoplast. D, Higher magnification of a plasmolysed onion cell showing the
Hechtian strands (h). Treatment with mannitol renders the Hechtian strands attaching the plasma membrane to the
cell wall. Scale bars = 20 µm. cw, cell wall; pm, plasma membrane.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Fig. 4!
D0
A!
D11
Yoshida medium
D18
D25
Yoshida medium + 60mM NaCl
B!
D0
D18
14!
48!
OsCPK4-OX!
1!
10!
13!
pC!
10!
26!
11!
9!
WT!
1!
13!
14!
pC !
48!
100.0%$
C!
100.0%$
100!
*
80.0%$
26
**
60.0%$
60!
26
**
40!
40.0%$ 40.0%$
**
14
14
48
48
20!
20.0%$
20.0%$
14!
48!
pC !
OX !
OsCPK4!
*
0.18000#
0.18!
0.15000#
0.15!
*
0.12000#
0.12!
0.09000#
0.09!
*
0.06000#
0.06!
*
*
0.03000#
0.03!
0!
0.0%$
0.0%$
1
1
OsCPK4-OX!
Survival (%)!
9
*
13!
0.21000#
0.21!
pC!
WT
9
10!
26!
11!
9!
WT!
1!
D! 0.24000#
0.24!
WT
*
80.0%$
80!
60.0%$
WT!
OX !
Relative expression level!
WT!
0
0
0!
11
11
11!
18
18!
18
25
25!
25
Days (salt treatment)!
0.00!
0.00000#
WT!
3! 9! 11! 1# 1! 10! 13! 14! 48!
pC!
OsCPK4-OX!
Figure 4. Salt tolerance of rice plants overexpressing OsCPK4. A, Diagram showing the experimental design for salt
tolerance assays. Salt stress was imposed to both transgenic and control plants grown in hydroponic culture until they
reached the three-leaf stage (D0). Plants were then transferred to nutrient solution containing 60mM NaCl, or to fresh
nutrient solution. In each experiment, wild-type (Nipponbare), vector control (pC, 3 independent lines) and OsCPK4
(OsCPK4-OX, 5 independent lines) transgenic lines were assayed. B, Phenotype of OsCPK4-OX rice plants under salt
stress conditions. Whereas control plants (WT and pC) grew poorly and exhibited chlorosis, the OsCPK4-OX lines grew in
the presence of NaCl. Photographs were taken after 18 days of salt stress (D18). C, Survival rates of OsCPK4-OX and
vector control plants rice plants at different times of salt treatment. D, OsCPK4 expression in roots of transgenic and control
plants used in salt tolerance assays as determined by qRT-PCR analysis. Specific primers for analysis of OsCPK4 transgen
expression were used (Supplemental Table SIV). Samples were taken at D0 time point (onset of salt treatment). Salt
tolerance assays were carried out three times and at two successive generations (T2 and T3). In each experiment, at least
30 plants per line were assayed. Asterisks denote significant differences between the OsCPK4-OX and control (pC and WT)
groups (*, P ≤ 0.05; **, P ≤ 0.001).
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Fig. 5!
A!
D0!
Regular Watering!
D17!
Drought! (17d)!
(15d)!
RW!
Re-Watering! (9d)!
B!
D17!
D0!
13!
14!
OsCPK4-OX!
D!
120!
120%
Survival rate (%)!
100!
100%
*!
*!
*!
80!
80%
60!
60%
40!
40%
20!
20%
0%0!
11!
13!
13
14!
14
OsCPK4-OX!
3!
3
pC!
13!
14!
3!
OsCPK4-OX
50!
50
pC
40!
40
*!
30!
30
20!
20
*!
10!
10
0!
0
0.5!
0.5
1!
1
2!
2
Time (h)!
13!
14!
3!
pC!
OsCPK4-OX!
E!
60!
60
00!
1!
pC!
OsCPK4-OX!
pC!
Water Loss (%)!
C!
1!
3!
100!
100
Water Retention(%)!
1!
RW!
*!
90!
90
80!
80
*!
70!
70
60!
60
60
50!
50
R53
OsCPK4-OX
40!
50
40
30!
30
40
30
R56
pC
0!
0
0.5!
0.5
11!
2!
2
Time (h)!
20
Figure 5. Drought tolerance of rice plants overexpressing OsCPK4. A, Diagram showing the10
experimental design for drought
tolerance assays. Transgenic and control (wild-type and transgenic lines expressing the empty vector) plants were grown for
0 (RW) in the greenhouse. B,
2 weeks (D0), subjected to 17 days of drought stress (D17), followed by 9 days of re-watering
0.5 Photographs
1
2
Phenotype of OsCPK4 overexpressor (OsCPK4-OX) and vector control (pC) plants during drought0stress.
were
taken at day 0 (onset of drought treatment), at 17 days of drought stress (D17) and after 9 days of re-watering (RW). C,
Survival rates of OsCPK4-OX and pC plants exposed to drought stress. D, Water loss and E, water retention rates of
detached leaves of OsCPK4-OX and pC plants at the three-leaf stage (*, P ≤ 0.05). Drought tolerance assays were repeated
3 times using five independent OsCPK4 transgenic lines and two independent vector control lines (at least 30 plants per
line). Values represent the mean+/-SE of three replicates (*, P ≤ 0.05).
Fig. 6!
A!
1!
2!
3! 4! 5!
6!
p1! p2!
7! 8! 9!10!
11!
7! 8! 9!10!
11!
2D-00040!
p3!
1!
2!
3! 4! 5!
6!
1D-03351!
B!
Ho! he! Az!
_!
D!
p1+p2!
p1+p3!
oscpk4!
Relative expression!
level (x103)!
C! 0.0040#4! OsCPK4!
0.0030#3!
0.0020#2!
0.0010#1!
0.0000#0!
** Ho!
3
he!
4
oscpk4!
Az!
WT
Ho! Ho! he! Az!
oscpk4!
Figure 6. Analysis of cpk4 mutants. A, OsCPK4 gene structure showing the T-DNA insertion sites for the cpk4
mutants identified in the POSTECH collection (the 2D-00040 and 1D-03351 mutants). Exons and introns are
indicated by black boxes and lines, respectively. Gene-specific primers used for the analysis of T-DNA
integration are indicated by arrows. B, Analysis of T-DNA integration in the 2D-00040 cpk4 mutant. “Ho”,“he”
and “Az” indicates homozygous, hemizygous and azygous plants, respectively. C, qRT-PCR analysis of
OsCPK4 expression in leaves of 2D-00040 cpk4 mutant plants. D, Appearance of the homozygous and
hemizygous 2D-00040 cpk4 mutant plants. Similar results were obtained for the 1D-03351 cpk4 mutant.
Asterisks denote significant differences (**, P ≤ 0.001).
Fig. 7!
C!
0!
Carbohyd
rate
s
Unknown
(20.97%)
Unknown
(28.72%)
0!
Carbohydrates!
Carboh
ydrates!
A!
0!
Metabolism
0!
(46.77%)
Metabolism
(31.91%)
DNA/ RNA modifica
Others!
0!
Ion transport
(3.23%)
Aminoa
cids
Proteins &
Signal transduction &
Intracellular trafficking
(12.90%)
Cellular processes!
Cellular processes!
Others!
Disease Resistance!
Ion transport
Oxidative stress!
Transcription and RNA b
Cellular processes
Signal transduction and
(3.19%)
Signal transduction and
Intracellular trafficking!
Signal transduction &
Ion transport!
Unknown!
Transcription & RNA binding
(22.34%)
(x10)!
*
1.5
0.0015#
0.4
0.0400#
1.0
0.0010#
0.2
0.0200#
0.0
0.0000#
2.5
0.0025#
2.0
0.0020#
0.6
0.0600#
*
0.5
0.0005#
3 11 1# 1 14
pC
OsCPK4
0.0
0.0000#
3 11 1# 1 14
pC
OsCPK4
5.0
5.0#
4.0
4.0#
3.0
3.0#
2.0
2.0#
1.0
1.0#
0.0
0.0#
3 11 1# 1 14
pC
OsCPK4
0.6
0.6#
0.5
0.5#
0.4
0.4#
0.3
0.3#
0.2
0.2#
0.1
0.1#
0.0
0.0#
3 11 1# 1 14
pC
OsCPK4
Asp. proteinase
Nepenthin(
3.0 Os12g39360
3.0#
2.5
2.5#
(x102)!
0.8
0.0800#
3.0
0.0030#
WRKY76
WRKY76'
0.7 Os09g25060
0.7#
Relative expression level!
**
WRKY71
WRKY71'
6.0 Os02g08440
6.0#
(x102)!
1.0
0.1000#
**
(x103)!
1.2
0.1200#
3.5 Os10g20610
0.0035#
Relative expression level!
Relative expression level!
1.4 Os02g42700
0.1400#
Laccase
Laccase&
Relative expression level !
D!
Thioredoxin
Thioredoxin*
Relative expression level!
B!
Intracellular trafficking!
Ion transport!
Oxidative stress
(4.26%) Unknown!
Intracellular trafficking
(7.45%)
Cellular processes (1.61%)
Oxidative stress (6.45%)
Disease Resistance!
Oxidative stress!
(2.13%)
Transcription and RNA binding!
Transcription &
RNA binding (8.06%)
Lipids!
Amino acids and Pr
2.0
2.0#
1.5
1.5#
1.0
1.0#
0.5
0.5#
0.0
0.0#
3 11 1# 1 14
pC
OsCPK4
Figure 7. Differentially expressed genes in OsCPK4 rice plants. A, Functional categories of up-regulated genes in roots
of OsCPK4-OX plants as determined by microarray analysis. B, Expression of Thioredoxin and Laccase genes in roots
of OsCPK4-OX plants. C, Functional categories of misregulated genes in leaves of OsCPK4-OX plants. D. Expression of
WRKY71, WRKY76 and aspartic proteinase genes in leaves of OsCPK4-OX plants. Transcript levels were determined
by qRT-PCR using total RNA. Representative results are shown for two OsCPK4 lines (lines 1 and 14) and two vector
control lines (lines 3 and 11) plants from a total of 5 and 3 independent OsCPK4 and vector control lines, respectively,
analysed in this work.
Fig. 8!
A
B!
1.60#
1.6!
1.60#
1.20#
1.00#
0.80#
0.60#
0.40#
1.4!
1.40#
1.20#
1.2!
*!
1.00#
1.0!
30.00#
*!
*!
15.00#
0.60#
0.6!
10.00#
0.4!
0.40#
0.20#
0.2!
0.00#
0.0!
0.00#
1#
14#9!
9#
5.00#
11!
48#
11#
9#11# !
pC!
C! pC!
25.00#
20.00#
0.80#
0.8!
0.20#
NaCl!
10mM#NaCl#
10mM#NaC
H2O!
Mock#(H2O)#
Mock#(H2O
35.00#
30!
30.00#
H
Mock#(H2O)#
Mock#(H2O)#
2O!
14!
11#
14#
48!
48#
0.00#
25!
25.00#
20!
20.00#
pC!
*!
14!
11#14#
48!
48#
15!
15.00#
10!
10.00#
5!
5.00#
0.00#
0!
1#
14#9!
9#
OsCPK4-OX!
pC!
*!
*!
48#11!
11#
pC!
9# 11# !
OsCPK4-OX!
pC!
D!
OsCPK4-Ox!
OsCPK4-Ox!
OsCPK4-Ox!
OsCPK4-Ox!
Fluorescence intensity!
1.40#
40.00#
35!
35.00#
NaCl!
10mM#NaCl#10mM#NaCl#
Relative Electrolyte Leakage!
1.8!
1.80#
MDA(µmol/mgFW)!
1.80#
1200
1000
800
600
**
400
**
200
0
3
11
pC
1
13
OsCPK4
OX
Figure 8. Effect of salt treatment on lipid peroxidation, electrolyte leackage and Na+ content. A and B, Detection of lipid
peroxidation (A) and electrolyte leackage (B) in leaves of 3 week-old plants, both vector control (pC, 2 independent
lines) and OsCPK4-overexpressor (OsCPK4-OX, three independent lines) plants, treated with 100mM NaCl for 14 days.
Data shown correspond to unstressed (grey bars) and salt-stressed (blue and orange bars) OsCPK4-OX and pC lines.
FW, fresh weight. C, Imaging of Na+ content in salt-stressed vector control (upper planels) and OsCPK4-OX (lower
panels) roots. Confocal images from roots of five-day-old seedlings that had been treated with 150mM NaCl for 16h and
stained with CoroNa-Green (green) and propidium iodide (red) are shown. Bars, 20 µm. D, Quantitative comparison of
CoroNa-Green fluorescent intensities. Two independent lines and three individual plants per line were examined for
each genotype. Values represent the mean +/- SE of average intensities of root sections at 700 µm distance to the tip.
Asterisks indicate a significant differences (**, P ≤ 0.001, ANOVA).