Plant Physiology Preview. Published on August 24, 2015, as DOI:10.1104/pp.15.01112
1
Running head:
2
SSPP negatively regulates leaf senescence
3
4
5
To whom all correspondence should be sent
6
Ning Ning Wang
7
Tel: +86 22 23504096
8
Email: [email protected].
9
10
11
12
13
14
15
16
17
Research areas:
18
Genes, Development and Evolution
19
20
21
1
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Copyright 2015 by the American Society of Plant Biologists
22
The Protein Phosphatase SSPP Directly Interacts with the Cytoplasmic Domain of
23
AtSARK and Negatively Regulates Leaf Senescence in Arabidopsis1
24
Dong Xiao , YanJiao Cui , Fan Xu, Xinxin Xu, GuanXiao Gao, YaXin Wang, Zhao Xia Guo,
25
Dan Wang, and Ning Ning Wang
†
†
*
26
27
Department of Plant Biology and Ecology, College of Life Sciences, Nankai
28
University, Tianjin 300071, China
29
†
Co-first authors
30
31
32
33
34
35
36
37
One-sentence Summary
38
A
39
dephosphorylating a senescence-promoting receptor-like kinase.
protein
phosphatase
negatively
regulates
Arabidopsis
leaf
senescence
2
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
through
40
1
41
31170261), the Key Grant Project of Chinese Ministry of Education (grant no. 313032), the
42
Specialized Research Fund for the Doctoral Program of Higher Education (grant no.
43
20130031130003) and the Key Project on the Breeding of New Genetically Modified Species
44
(grant nos. 2014ZX08004-005-004 and 2014ZX08009-030B-002).
45
* Corresponding author: [email protected].
This work was supported by the National Natural Science Foundation of China (grant no.
3
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
46
Abstract
47
Reversible protein phosphorylation mediated by protein kinases and phosphatases plays an
48
important role in the regulation of leaf senescence. We previously reported that the leucine-rich
49
repeat receptor-like kinase AtSARK positively regulates leaf senescence in Arabidopsis. Here, we
50
report the involvement of a PP2C-type protein phosphatase, SENESCENCE-SUPPRESSED
51
PROTEIN PHOSPHATASE (SSPP), in the negative regulation of Arabidopsis leaf senescence.
52
SSPP transcript levels decreased greatly during both natural senescence and SARK-induced
53
precocious senescence. Overexpression of SSPP significantly delayed leaf senescence in
54
Arabidopsis. Protein pull-down and bimolecular fluorescence complementation assays
55
demonstrated that the cytosol-localized SSPP could interact with the cytoplasmic domain of the
56
plasma membrane-localized AtSARK. In vitro assays showed that SSPP has protein phosphatase
57
function and can dephosphorylate the cytosolic domain of AtSARK. Consistent with these
58
observations, overexpression of SSPP effectively rescued AtSARK-induced precocious leaf
59
senescence and changes in hormonal responses. All our results suggested that SSPP functions in
60
sustaining proper leaf longevity and preventing early senescence by suppressing or perturbing
61
SARK-mediated senescence signal transduction.
62
4
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
63
Introduction
64
As the final stage of leaf development, senescence occurs in an age-dependent manner and in
65
response to interplays of multiple internal and external signals (Gan and Amasino, 1995; Miller et
66
al., 1999; Guo and Gan, 2005; Zhang and Zhou, 2012). Leaf senescence also plays important roles
67
in plant fitness by recycling nutrients to vigorously growing organs (Lohman et al., 1994).
68
Modifications of this process directly affect agricultural traits of crop plants (Zhang et al., 1987;
69
Rivero et al., 2007). Substantial progress has been made in addressing the underlying molecular
70
mechanisms of senescence (Lim et al., 2007; Thomas, 2013), but the distinct pathways that
71
transduce different signals to control the initiation and progression of leaf senescence remain
72
unclear.
73
Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, plays a
74
critical role in cellular signaling. The involvement of specific protein kinases in the regulation of
75
leaf senescence has also been suggested. For example, the membrane-bound receptor protein
76
kinase RPK1 affects Arabidopsis leaf senescence induced by abscisic acid (ABA); loss-of-function
77
mutants of RPK1 exhibit delayed symptoms in both age-dependent and ABA-induced senescence
78
(Lee et al., 2011). Arabidopsis AHK3 (ARABIDOPSIS HISTIDINE KINASE 3） functions as the
79
major cytokinin receptor kinase that mediates the anti-senescence effect of cytokinins through
80
specific phosphorylation of ARR2 (ARABIDOPSIS RESPONSE REGULATOR 2) (Kim et al.,
81
2006). EDR1 (ENHANCED DISEASE RESISTANCE 1), a CTR1-like kinase, functions as a
82
negative regulator of ethylene-induced senescence in an EIN2 (ETHYLENE INSENSITIVE2)
83
dependent manner (Frye et al., 2001; Tang et al., 2005). In addition, a member of the ABC1
84
(Activity of bc1 complex) protein kinase family, OsABC1-2, confers enhanced tolerance to
85
dark-induced senescence in rice (Gao et al., 2012). A MAPK cascade involving MKK9, and its
86
downstream target MPK6, plays a positive role in regulating leaf senescence (Zhou et al., 2009).
87
Also, SnRK1, an energy sensor kinase, plays a negative role in the regulation of leaf senescence
88
(Cho et al., 2012).
89
Dephosphorylation by protein phosphatases functions as a balancing switch to reverse the
90
effects of phosphorylation by protein kinases. Removal of phosphates often renders protein
5
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
91
kinases inactive, effectively halting their cellular functions. Such examples including KAPP
92
(Kinase-associated Protein Phosphatase) which functions as a negative regulator of the
93
CLAVATA1 signal transduction pathway, and group A PP2Cs which efficiently inactivates
94
SnRK2s (subclass III SnRK2) in ABA signaling (Stone et al., 1998; Schweighofer et al., 2004;
95
Umezawa et al., 2009). In recent years, evidence has emerged that protein phosphatases also have
96
pivotal functions in the regulation of leaf senescence. For example, the PP2C family protein
97
phosphatase SAG113, which serves as a negative regulator of ABA signal transduction, is
98
involved in the control of water loss during leaf senescence in Arabidopsis (Zhang et al., 2012).
99
Silencing of the protein phosphatase AtMKP2, which positively regulates oxidative stress
100
tolerance and inactivates the MPK3 and MPK6 MAPKs in Arabidopsis, promotes early
101
senescence (Lee and Ellis, 2007; Li et al., 2012). However, few key protein phosphatases that
102
interact with a known senescence-associated protein kinase and function in the regulation of leaf
103
senescence have been characterized.
104
We previously reported that a soybean dual-specificity kinase, GmSARK, and its Arabidopsis
105
homolog, AtSARK, regulate leaf senescence through synergistic actions of auxin and ethylene (Xu
106
et al., 2011). In this study, we cloned and identified a PP2C type protein phosphatase
107
SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), which negatively regulates
108
leaf senescence in Arabidopsis. The transcript level of SSPP was greatly reduced during both
109
natural senescence and SARK-induced precocious senescence in Arabidopsis. Overexpression of
110
SSPP significantly delayed leaf senescence in transgenic Arabidopsis. Protein pull-down and
111
bimolecular fluorescence complementation (BiFC) assay demonstrated that the cytosol-localized
112
SSPP could interact with the plasma membrane-localized AtSARK both in vitro and in vivo. SSPP
113
also dephosphorylated the cytoplasmic domain of AtSARK in vitro. Overexpression of SSPP
114
effectively restored SARK-induced precocious leaf senescence. All results suggested that SSPP
115
negatively regulates leaf senescence through suppressing or perturbing SARK-mediated
116
senescence signal transduction by directly dephosphorylating AtSARK.
6
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
117
Results
118
The expression of SSPP is suppressed during leaf senescence
119
We previously performed a microarray analysis to detect changes in the transcriptome of the
120
SARK-induced early-senescent Arabidopsis seedlings, and identified one gene (GenBank
121
accession No. At5g02760) with substantially decreased transcript levels (unpublished data). Here,
122
based on our subsequent observations, we called this gene SENESCENCE-SUPPRESSED
123
PROTEIN PHOSPHATASE (SSPP). To confirm the involvement of SSPP in leaf senescence, we
124
used quantitative RT-PCR to measure its transcript levels in both natural leaf senescence and
125
SARK-induced early senescence processes. The SSPP transcript level was high in young leaves,
126
and decreased gradually as the leaves developed from non-senescent to late senescence stages (Fig
127
1A). For SARK-induced senescence, we used the dexamethasone (DEX)-inducible construct
128
GVG:AtSARK; as shown in our previous report (Xu et al., 2011), DEX treatment resulted in a
129
continuous increase in the transcript level of AtSARK and an early senescence phenotype in the
130
vertically grown 4-d-old GVG:AtSARK transgenic seedlings (Fig. 1B). In these seedlings, a rapid
131
decrease in the SSPP transcript levels was found after 1 h of DEX treatment. Upon 2 h of DEX
132
treatment, the SSPP mRNA level dropped to 10% of its untreated-level. No accumulation of
133
AtSARK transcript was detected in the mock-treated GVG:AtSARK seedlings in which the
134
transcription level of SSPP was also not affected (Fig. 1B).
135
SSPP encodes a PP2C-type protein phosphatase
136
The complete sequence of SSPP consists of 1543 bp of cDNA and encodes a protein of 370
137
amino acids. The predicted SSPP protein shows high sequence similarity to a Thellungiella
138
halophila putative protein serine/threonine phosphatase 2C (PP2C) family protein (GenBank
139
Accession No.BAJ33929), with 91% amino acid sequence identity. Refseq Protein database
140
searches (http://blast.ncbi.nlm.nih.gov) revealed that SSPP exhibits 66% amino acid identity with
141
Vitis vinifera PP2C38-like, and Cicer arietinum PP2C38-like, respectively. In Arabidopsis, SSPP
142
is most similar to the predicted protein phosphatase 2C family protein AT3G12620, with 58%
143
identity on the amino acid level (Fig. 2A). SSPP contains the amino acids that form the catalytic
7
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
144
sites in the channel of the beta-sandwich of PP2C homologs (Das et al., 1996) (Fig. 2A).
145
Consistent with a previous report (Schweighofer et al., 2004), a phylogenetic tree also shows that
146
SSPP has high similarity to the PP2C group D subfamily proteins (Fig. 2B).
147
To determine if SSPP can function as a protein phosphatase, the recombinant SSPP protein
148
with a glutathione S-transferase (GST) tag was expressed in Escherichia coli and purified by
149
affinity chromatography using a Glutathione Sepharose 4B column. After treating GST-SSPP with
150
the site-specific PreScission protease to remove the GST tag, we tested the purified SSPP in a
151
phosphatase assay using p-nitrophenyl phosphate (pNPP) as a substrate (An and Carmichael,
152
1994). SSPP exhibited obvious phosphatase activity, successfully cleaving the phosphate from
153
pNPP and generating a yellow nitrophenol product that was quantitated by absorbance at 405 nm
154
(A405). As shown in Fig. 2, SSPP functions at an optimum pH between 6.5 and 8.5 (Fig. 2C), and
155
an optimum temperature between 42°C and 57°C (Fig. 2D). In addition, SSPP exhibited an
156
absolute requirement for Mg2+, indicating that SSPP is a Mg2+-dependent phosphatase enzyme
157
(Fig. 2E).
158
Overexpression of SSPP significantly delays leaf senescence
159
To further examine the biological functions of SSPP, we tested the effects of SSPP
160
overexpression. Multiple independent lines of 35S:SSPP transgenic Arabidopsis were generated
161
by the Agrobacterium-mediated floral-dip method (Clough and Bent, 1998). Except for line 35 in
162
which the expression of SSPP was silenced, the other 6 lines exhibited a significant delay in
163
senescence (Fig. 3A) and we selected line 38 as a typical line for further study. To gain a better
164
view of the function of SSPP in leaf senescence, the rosette leaves of 33-d-old 35S:SSPP plants
165
and the developing 5th leaves of 35S:SSPP plants were compared with their corresponding wild
166
type controls, respectively. As shown in Fig. 3B, the 35S:SSPP transgenic plants showed an
167
obvious delay in natural leaf senescence. We also tested dark-induced senescence using either
168
attached or detached 5th and 6th leaves at mature stage (from plants at the Boyes growth stage 5.10,
169
Fig. S1). It was found that both the attached 35S:SSPP leaves individually covered by aluminum
170
foil for 9 d (Fig. 3C (a)) and the detached 35S:SSPP leaves incubated in darkness for 4 d (Fig. 3C
171
(b)) exhibited a much delayed senescence when compared with their corresponding wild type
8
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
172
controls (Fig. 3C).
173
Based on the Boyes growth stage ontology (Boyes et al., 2001), the 6th leaves of the wild
174
type and 35S:SSPP transgenic plants at four different developmental stages, including stage 3.50,
175
stage 5.10, stage 6.00 and stage 6.10 (Fig. S1), were sampled respectively to assay the transcript
176
levels of known senescence-associated genes. The tested genes included: the age-related
177
senescence marker gene SAG12 (Gan and Amasino, 1995), the PP2C family protein phosphatase
178
gene SAG113 (Zhang et al., 2012), and four critical senescence-related transcription factors, NAC1
179
(Kim et al., 2009), NAC2 (Kim et al., 2009), WRKY6 (Robatzek and Somssich, 2002) and AtNAP
180
(Guo and Gan, 2006). Quantitative RT-PCR analysis revealed a remarkable increase in the
181
transcript levels of these genes when the wild type plants developed from stage 5.10 to stage 6.00
182
and stage 6.10, however, the increase in the expression of these senescence-associated genes was
183
significantly inhibited in the 35S:SSPP plants (Fig. 3D). Besides significantly delayed leaf
184
senescence, overexpression of SSPP in Arabidopsis also resulted in shorter roots, smaller rosette
185
leaves with a curved surface, and shorter plant height (Fig. S2A-F). The 35S:SSPP plants also
186
exhibited a delay of 5 d in bolting time.
187
To further reveal the function of SSPP in senescence, we next examined the SSPP
188
loss-of-function phenotype. We obtained a Salk T-DNA line, SALK_099356C, which has a T-DNA
189
insertion in the last (4th) exon and named it sspp-1. However, except for the weak advance in
190
dark-induced senescence, no significant difference in growth and development between the sspp-1
191
mutant and wild-type plants was observed (Fig. S3), indicating that redundant genes may regulate
192
leaf senescence in Arabidopsis.
193
Overexpression of SSPP sustains chloroplast structure and function
194
To investigate the cellular events caused by the overexpression of SSPP, mesophyll cells
195
from the 6th leaves of 35S:SSPP transgenic plants at 3.50 and 6.00 stages were examined by
196
electron microscopy. As shown in Fig. 4A, the color of the osmium-fixed chloroplasts of
197
35S:SSPP transgenic plants were darker than those of the wild type plants, suggesting that the
198
35S:SSPP chloroplasts were more active at both stages. In the younger 6th leaves from plants at
9
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
199
stage 3.50, both the SSPP-overexpressing and the wild-type chloroplasts exhibited the similar
200
inner membrane systems (Fig. 4A). However, when the plants developed from stage 3.50 to stage
201
6.00, the 6th leaves of wild type plant displayed early senescence symptom companied with many
202
huge starch grains accumulated in their chloroplasts; oppositely, the SSPP-overexpressing
203
chloroplasts sustained a much better-organized inner membrane system (Fig. 4A). The chlorophyll
204
contents in the 6th leaves at all the tested four developmental stages, including stage 3.50, 5.10,
205
6.00 and 6.10, were significantly increased in the 35S:SSPP transgenic plants (Fig. 4B).
206
Quantitative RT-PCR was used to measure the expression of genes encoding key enzymes
207
involved in chlorophyll metabolism and chloroplast functions in the 35S:SSPP transgenic plants at
208
the above-mentioned four developmental stages. It was found that when developing from stage
209
3.50 to stage 6.10, the transcript levels of GTR1, which encodes the chlorophyll biosynthesis
210
enzyme glutamyl tRNA reductase (McCormac et al., 2001), and two photosynthetic genes RbcL
211
and RbcS (Krebbers et al., 1988; Isono et al., 1997) were gradually decreased in the 6th leaves of
212
wild type plants, however, the decrease in the transcription of these genes was significantly
213
retarded in the corresponding 35S:SSPP transgenic leaves (Fig. 4C). On the contrary, the
214
age-induced increase in the transcript levels of ACD1, which encodes the chlorophyll breakdown
215
enzyme pheide α oxygenase (Pruzinska et al., 2003), and SIG5, which is induced under adverse
216
conditions to protect plants from stresses by enhancing repair of the PSⅡreaction center
217
(Nagashima et al., 2004), was greatly suppressed in the SSPP-overexpressing plants (Fig. 4C).
218
These results, consistent with the ultrastructural morphology analysis of the transgenic
219
chloroplasts (Fig. 4A), suggested that overexpression of SSPP sustains the structure and function
220
of chloroplasts.
221
Effects of SSPP overexpression on cytokinin, auxin and ethylene responses
222
Cytokinin is well known as a senescence-delaying hormone. Increases in cytokinin levels lead
223
to delayed senescence in many plant species, including rice (Kudo et al., 2012), maize (Pineda
224
Rodo et al., 2008), and iris (Pineda Rodo et al., 2008). To examine the effect of SSPP
225
overexpression on cytokinin pathways, quantitative RT-PCR was used to measure transcript levels
226
of several cytokinin-responsive marker genes, including IPT3, which encodes the key enzyme of
10
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
227
cytokinin biosynthesis and the type A ARRs ARR5 and ARR6, which have been commonly used as
228
cytokinin-inducible markers (Cui et al., 2010), GRXS13, a cytokinin up-regulated gene encoding
229
for two CC-type GRX (Glutaredoxin) isoforms (Nemhauser et al., 2006; Laporte et al., 2012) and
230
At2g18300, which encodes a bHLH cytokinin-responsive transcription factor (Brenner et al.,
231
2005). Quantitative RT-PCR analysis revealed that the expressions of these 5 genes in leaves at
232
mature stage were all significantly higher in the SSPP overexpression plants (Fig. 5A). These
233
results indicated that SSPP overexpression enhanced cytokinin responses in the transgenic
234
Arabidopsis plants.
235
DR5 is a synthetic promoter consisting of 7 tandem repeats of an auxin-responsive TGTCTC
236
element and a minimal 35S CaMV promoter (Ulmasov et al., 1997). DR5 promoter fused to a
237
reporter gene has been widely used as a good tool to monitor auxin response in planta (Sabatini et
238
al., 1999; Wang et al., 2005). To examine auxin pathways, we used DR5:GFP to detect auxin
239
accumulation and distribution in the SSPP-overexpressing seedlings. In the wild-type background,
240
fluorescence of DR5:GFP was mainly detected in the quiescent center and columella cells of roots
241
(Fig. 5B). In the SSPP-overexpressing background, besides in the quiescent center cells, the
242
DR5:GFP signals also occurred in the epidermis of the root apical meristem (Fig. 5B). We
243
observed no significant difference of the intensity of the DR5:GFP signal between the
244
DR5:GFP/35S:SSPP and DR5:GFP/WT roots (Fig. 5B).
245
Ethylene plays a critical role in the regulation of leaf senescence. To examine ethylene
246
pathways in the SSPP-overexpressing plants, the expression levels of another reporter construct,
247
5×EBS:GUS, were examined. 5×EBS is also a synthetic promoter which consists of five tandem
248
repeats of the EIN3 binding site (EBS) followed by the minimal 35S promoter (Stepanova et al.,
249
2007). EIN3 is a transcription factor that acts as a positive regulator of the ethylene signal
250
transduction pathway (Chao et al., 1997; Solano et al., 1998), thus 5×EBS:GUS has been
251
previously used to monitor the primary ethylene response in planta (Vandenbussche et al., 2010).
252
The 5×EBS:GUS/35S:SSPP plants were obtained by crossing 5×EBS:GUS with 35S:SSPP
253
transgenic plants. Histochemical GUS staining revealed a dramatic decrease in the activity of
254
5×EBS:GUS in the SSPP-overexpressing plants (Fig. 5C). We also used gas chromatography to
11
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
255
measure ethylene levels and found that 3-d-old etiolated seedlings of 35S:SSPP plants showed a
256
slight but significant reduction of ethylene emission compared with the wild type control (Fig.
257
S4A (a)). Moreover, when treated with 1 μM ACC, the ratio of exaggerated apical hook formation
258
in the 35S:SSPP etiolated seedlings was dramatically decreased (Fig. S4A (b and c)). Consistent
259
with the GUS staining results in 5×EBS:GUS/35S:SSPP plants, the transcript levels of several
260
ethylene response markers (PDF1.2, CHI-B, PR4 and ERF11) were all greatly decreased by the
261
overexpression of SSPP (Fig. S4B). These results indicated that the overexpression of SSPP not
262
only suppressed the biosynthesis of ethylene, but also reduced the responses to ethylene in
263
Arabidopsis.
264
Subcellular localization of AtSARK and SSPP
265
To determine the subcellular localization of the AtSARK and SSPP proteins, enhanced yellow
266
fluorescent protein (EYFP) fused C-terminally to AtSARK and SSPP were transiently expressed in
267
Arabidopsis mesophyll protoplasts under the control of the cauliflower mosaic virus 35S promoter
268
respectively. In the EYFP control, yellow fluorescence was observed in the cytoplasm. The yellow
269
fluorescence of AtSARK-EYFP was detected as a fine ring at the cell periphery, external to the
270
chloroplasts, indicating that AtSARK localizes to the plasma membrane (Fig. 6). SSPP-EYFP
271
expression was detected in the cytoplasm (Fig. 6), indicating that SSPP localizes in the cytoplasm.
272
SSPP partially dephosphorylates the auto-phosphorylated cytoplasmic domain of AtSARK
273
To determine whether AtSARK and SSPP can serve as substrates for each other, we
274
performed in vitro phosphorylation and de-phosphorylation assays. SSPP and the cytoplasmic
275
domain of AtSARK (AtSARK-CD) were fused with glutathione S-transferase (GST) and
276
expressed in Escherichia coli, respectively. The GST fusion proteins were purified by affinity
277
chromatography using a Glutathione Sepharose 4B column, and the GST tag was removed from
278
SSPP
279
autophosphorylated for 10 min before incubating with SSPP or GST for further analysis. The in
280
vitro protein phosphorylations were detected by immunoblot using anti-Phospho-Threonine
281
antibody (anti-pT). As shown in Figure 7, when the autophosphorylated GST-AtSARK-CD (lane 1)
using
the
site-specific
PreScission
protease.
Purified
GST-AtSARK-CD
12
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
was
282
or the autophosphorylated GST-AtSARK-CD together with purified GST (lane 5) were incubated
283
in the ATP-containing protein phosphorylation reaction mixture, only the GST-AtSARK-CD band
284
was revealed by immunoblot analysis, confirming that the active AtSARK-CD protein exhibited
285
autophosphorylation and the GST tag had no effect on the ability of AtSARK-CD to
286
auto-phosphorylate. Purified SSPP (line 2), GST (line 4) or both combined together (line 6)
287
displayed no signal of protein phosphorylation, indicating that neither SSPP nor GST protein has
288
autophosphorylation activity. When the auto-phosphorylated GST-AtSARK-CD was incubated
289
with purified SSPP in the reaction mixture, no matter whether GST was added, no band
290
corresponding to SSPP was detected by anti-pT antibody. However, the intensity of the
291
auto-phosphorylated GST-AtSARK-CD bands decreased significantly (Fig. 7 lanes 3, and 7).
292
These results indicated that AtSARK does not phosphorylate SSPP, but SSPP can
293
de-phosphorylate
294
dephosphorylation of AtSARK-CD was independent of the GST tag.
295
SSPP interacts with AtSARK in vitro and in vivo
the
auto-phosphorylated
AtSARK-CD.
Also,
the
SSPP-mediated
296
SSPP dephosphorylates the cytoplasmic domain of AtSARK (AtSARK-CD); therefore, we
297
tested whether SSPP and AtSARK physically interact. We first tested for this interaction using an
298
in vitro pull-down assay. We expressed AtSARK-CD fused to His and SSPP fused to GST in E.
299
coli and purified the fusion proteins. GST-SSPP or GST was bound to a glutathione Sepharose
300
column and incubated with His-AtSARK-CD, and then the eluted proteins were separated by
301
SDS-PAGE and subjected to immunoblot analysis with His antibody (Fig. 8A). Immunoblotting
302
detected a single band corresponding to His-AtSARK-CD in the proteins eluted from the
303
GST-SSPP column (Fig. 8A lane 5), but detected no signal in the proteins eluted from the GST
304
column (Fig. 8A lane 4), indicating that SSPP physically interacts with AtSARK-CD in vitro.
305
To confirm the interaction between AtSARK and SSPP in plant cells, we used a bimolecular
306
fluorescence complementation (BiFC) assay based on split yellow fluorescent protein (YFP)
307
(Bracha-Drori et al., 2004). The different combinations of the N- or C-terminal end of YFP fused
308
to AtSARK or SSPP were transiently co-expressed, together with a plasma membrane marker
309
pm-rk-CD3-1007 (Nelson et al., 2007), in Nicotiana benthamiana leaves. As shown in Figure 8B,
13
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
310
an obvious fluorescent signal was detected in the plasma membrane for the AtSARK-nYFP
311
+SSPP-cYFP combination, but no significant signals were detected in controls lacking either
312
AtSARK or SSPP. The fluorescent signal perfectly overlapped with the red fluorescence of the
313
co-expressed plasma membrane marker pm-rk. The BiFC assay thus confirmed that SSPP interacts
314
with AtSARK in the plasma membrane of cells.
315
Additionally, we found that the kinase domain of GmSARK (GmSARK-KD), a soybean
316
homolog of AtSARK, interacts with SSPP in our yeast two-hybrid system (Fig. S5), suggesting
317
that the interaction between SARK and SSPP may be conserved.
318
Overexpression of SSPP rescues SARK-induced premature leaf senescence in Arabidopsis
319
To investigate whether SSPP affects AtSARK function, we tested whether overexpression of
320
SSPP could alter the induction of senescence by AtSARK. To this end, we obtained the
321
35S:SSPP/GVG:AtSARK plants by transferring SSPP into the GVG:AtSARK transgenic plants.
322
When grown on DEX-containing plates, the 7-d-old transgenic seedlings of G28, a homozygous
323
GVG:GUS control line (Xu et al., 2011), displayed normal growth and development (Fig. 9A). By
324
contrast, the seedlings of AtS20, a typical GVG:AtSARK line, displayed an early senescence
325
phenotype (Fig. 9A). However, the 35S:SSPP/GVG:AtSARK seedlings showed no early
326
senescence (Fig. 9A). The DEX-induced AtSARK overexpression and the 35S promoter-derived
327
SSPP overexpression in the transgenic plants were confirmed by quantitative RT-PCR (Fig. 9B).
328
In addition to suppressing the visible senescence phenotype, overexpression of SSPP in the
329
35S:SSPP/GVG:AtSARK double transgenic seedlings also suppressed the AtSARK-induced
330
increases in expression of several marker genes for senescence, such as SAG12, NAC1, WRKY6
331
and SAG113 (Fig. 9C). All these results suggested that SSPP functions as a negative regulator of
332
AtSARK during leaf senescence.
333
AtSARK was previously suggested to regulate leaf senescence through auxin and ethylene
334
(Xu et al., 2011). Quantitative RT-PCR revealed that overexpression of SSPP also suppressed the
335
AtSARK-induced increases in transcript levels of CKX3, a gene encoding cytokinin oxidase, which
336
degrades cytokinin; TSA1, an auxin synthesis-related gene; GH3.5, an auxin-responsive gene; and
14
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
337
two ethylene-responsive marker genes, GST2 and PR4. The AtSARK-induced decreases in
338
expression of At2g18300, a cytokinin-responsive marker gene, was also recovered in the
339
35S:SSPP/GVG:AtSARK double transgenic seedlings (Fig. 10).
340
Comparison of the spatial and temporal expression of AtSARK and SSPP during leaf
341
development
342
To further reveal the molecular mechanism underlying interactions between SSPP and
343
AtSARK, the promoter-GUS reporter system was used to compare the spatial and temporal
344
expression of SSPP and AtSARK during Arabidopsis leaf development. 28-d-old SSPP:GUS and
345
AtSARK:GUS transgenic Arabidopsis plants with the 1st and 2nd leaves beginning to yellow were
346
sampled for histochemical GUS staining, respectively. The SSPP promoter showed strong activity
347
in juvenile leaves and this activity gradually decreased with increasing leaf age (Fig. 11). By
348
contrast, the AtSARK promoter showed weak activity in juvenile leaves, but this activity gradually
349
increased with increasing leaf age (Fig. 11). The expression of SSPP and AtSARK partially
350
overlapped from the 2nd leaf to the 8th leaf in 28-d-old plants, corresponding to the mature leaves
351
to early-senescent leaves stages, implying that during these stages SSPP might suppress the
352
functions of AtSARK.
353
15
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
354
Discussion
355
Protein kinase and phosphatase-mediated reversible protein phosphorylation plays a critical
356
role in cellular signaling. Many reports have revealed the involvement of protein kinases in the
357
regulation of leaf senescence (Zhou et al., 2009; Xu et al., 2011; Cho et al., 2012); however, the
358
reports on protein phosphatase are relatively few. In this study, we found that a
359
senescence-suppressed type 2C protein phosphatase (SSPP) functions in the regulation of leaf
360
senescence. Overexpression of SSPP effectively attenuated age-dependent natural leaf senescence
361
and SARK-induced early leaf senescence in Arabidopsis (Fig. 1). SSPP overexpression prevented a
362
multitude of changes induced by leaf senescence, including the increased expression of
363
senescence-associated marker genes, enhanced ethylene responses, and down-regulated cytokinin
364
functions (Fig. 9C and 10). All these results suggested that SSPP functions as a negative regulator
365
of leaf senescence.
366
We previously reported that AtSARK functions as a key positive regulator of Arabidopsis leaf
367
senescence (Xu et al., 2011). AtSARK localizes to the plasma membrane and SSPP localizes to the
368
cytoplasm (Fig. 6); thus, it was interesting to find that SSPP can directly interact with the
369
cytoplasmic domain of AtSARK (AtSARK-CD), as shown by our in vitro pull down assay (Fig.
370
8A) and bimolecular fluorescence complementation (Fig. 8B). Also, the auto-phosphorylated
371
cytoplasmic domain of AtSARK could be dephosphorylated by SSPP (Fig. 7), implying that SSPP
372
might exert its negative regulatory function on leaf senescence by keeping de-phosphorylation
373
status of AtSARK. Kinases often autocatalytically phosphorylate key amino acid residues to
374
relieve autoinhibition or enhance catalytic efficiency (Zenke et al., 1999; Canova et al., 2008). Our
375
preliminary results suggested that SARK function requires autophosphorylation of key residues
376
(data not shown). Thus it is not surprising to find that overexpression of SSPP effectively rescued
377
the AtSARK-induced premature leaf senescence in Arabidopsis (Fig. 9A). Overexpression of SSPP
378
also effectively suppressed the AtSARK-mediated induction of senescence marker genes SAG12,
379
NAC1, WRKY6 and SAG113 (Fig. 9C), and the AtSARK-induced changes in expression of the
380
phytohormone-related marker genes CKX3, At2g18300, TSA1, GH3.5, PR4 and GST2 (Fig. 10).
381
All these observations support our model that SSPP functions by dephosphorylating AtSARK to
16
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
382
attenuate SARK-mediated senescence signaling.
383
It is notable that SSPP could interact directly with the cytoplasmic domain of GmSARK, a
384
soybean homolog of AtSARK, in a yeast two-hybrid system (Fig. S5). This result suggested that
385
regulation of leaf senescence by SSPP and SARK might be a conserved mechanism (Fig. S5).
386
Leaves are the major organs for photosynthesis. The leaf longevity directly affects the
387
lifetime carbon fixation of plants, and accordingly affects plant growth and development
388
(Kikuzawa and Ackerly, 1999). Different plant species have different leaf longevities (Kikuzawa,
389
1991), and normal growth and development of each plant species requires proper leaf longevity.
390
(Jonasson, 1989). Comparison of the spatial and temporal expression patterns of SSPP and
391
AtSARK revealed that SSPP was highly expressed in young leaves and mature leaves where the
392
promoter activity of AtSARK was quite low (Fig. 1A and Fig. 11). Additionally, the accumulation
393
of SSPP protein would, as mentioned above, inhibit function of AtSARK by direct interaction.
394
Taken together, these results indicated that SSPP repressed the function of AtSARK to sustain leaf
395
function and prevent early leaf senescence. As the leaves developed from mature to early
396
senescence stage, SSPP promoter activity gradually decreased (Fig. 1A and Fig. 11). Because of
397
the reduction of accumulation of SSPP, the inhibitory effects on AtSARK were relieved, thereby
398
promoting leaf senescence.
399
Cytokinin is best known as a senescence-delaying hormone. Many plant species show a
400
decline in levels of foliar cytokinin during leaf senescence (Singh et al., 1992). Accordingly,
401
sustaining the proper cytokinin level in plants can effectively prolong leaf longevity and delay
402
senescence (Hwang et al., 2012). Although exogenous application of cytokinin had no obvious
403
effect on the activity of the SSPP promoter (data not shown), the transcription levels of genes
404
involved in cytokinin biosynthesis and responses all increased in 35S:SSPP plants (Fig. 5A).
405
These results implied that besides functioning as a negative regulator of AtSARK, SSPP also
406
played a role in sustaining or enhancing cytokinin responses in plants.
407
Ethylene functions as an endogenous modulator of plant ageing and as a strong promoter of
408
senescence (Jing et al., 2005; Kim et al., 2009; Li et al., 2013). SARK-induced leaf senescence
17
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
409
requires increases in ethylene biosynthesis and responses. Exogenous application of AVG, an
410
inhibitor of ethylene biosynthesis, or mutation in EIN2, a critical component of ethylene signal
411
transduction, could effectively suppress the SARK-induced early leaf senescence symptoms (Xu et
412
al., 2011). Overexpression of SSPP significantly reduced ethylene responses both in the
413
developing rosettes and in the SARK-overexpressing senescent leaves (Fig. 5C, Fig. 10C and Fig.
414
S4). Because of the low transcription level of AtSARK in young seedlings, the above results
415
suggested that SSPP might exert its negative effects on the onset and progression of leaf
416
senescence through two independent pathways: directly inhibiting the functions of AtSARK and
417
negatively regulating the biosynthesis and functions of ethylene. Characterization and
418
identification of more components involved in SSPP-mediated ethylene repression will help
419
elucidate the mechanisms controlling leaf longevity.
420
In conclusion, we postulate that SSPP functions in sustaining normal leaf development from
421
young leaf to mature leaf stages, maintaining functions of the mature leaf, and specifying proper
422
leaf longevity by preventing early senescence. The significant decrease in expression of SSPP
423
during natural leaf senescence (Fig. 1A) suggested that leaf age or other developmental signals
424
negatively regulate SSPP. Moreover, the observation that AtSARK also suppressed SSPP
425
expression (Fig. 1A) implied that once leaf senescence began, the increase in AtSARK expression
426
or the senescence process itself exerted a negative feedback effect on the functions of SSPP, to
427
further facilitate the progression of senescence. Further work on the mechanisms underlying the
428
regulation of SSPP expression and isolation of the upstream regulators of SSPP will help to
429
improve our understanding of the roles SSPP plays in leaf senescence.
430
The sspp-1 mutants, which have a T-DNA knockout allele of SSPP, developed similar to the
431
wild type plants (Fig. S3), suggesting the existence of other functionally redundant genes of SSPP.
432
Identification of more interaction partners for AtSARK and SSPP will help to elucidate the
433
molecular mechanisms underlying the SSPP and SARK-mediated regulation of leaf development
434
in higher plants.
435
Auxin triggers a plethora of developmental events (Lofke et al., 2013). Many if not all of
436
auxin’s actions, such as embryonic axis formation, postembryonic organ formation, tropic growth
18
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
437
responses, and vascular tissue development, rely on its differential distribution within plant tissues
438
manifested by local auxin maxima and minima, also referred to as auxin gradients (Benkova et al.,
439
2003; Friml et al., 2003; Scarpella et al., 2006; Tanaka et al., 2006; Kleine-Vehn et al., 2010). The
440
role of auxin in regulating senescence is not clear. Several studies support the idea that auxin
441
negatively regulates leaf senescence (Shoji et al., 1951; Lim et al., 2010; Kim et al., 2011), but
442
other lines of evidence indicate that auxin positively regulates leaf senescence. We have
443
previously reported that SARK-mediated leaf senescence requires an increase in the biosynthesis
444
of and responses to auxin (Xu et al., 2011). Most recently, a gene known to be strongly induced by
445
auxin, SAUR36, was shown to promote leaf senescence (Hou et al., 2013). In summary, the role of
446
auxin in leaf senescence appears to be very complex and may not be simply explained by variation
447
in the auxin level during leaf senescence. Although exerting opposite effects on leaf senescence,
448
both SSPP and AtSARK could alter the expression pattern of the auxin reporter DR5:GUS (Xu et
449
al., 2011). These results implied that the changes in auxin distribution, but not the variations in
450
auxin level, play a major role in the regulation of leaf senescence. Further studies are needed to
451
elucidate the detailed mechanisms of auxin function in senescence.
452
19
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
453
Materials and Methods
454
Amino acid sequence alignment and phylogenetic analyses
455
The full-length PP2C protein sequences were retrieved from GenBank. Amino acid sequences
456
were aligned using CLUSTALX2 (http://www.clustal.org/) (Thompson et al., 1994). The
457
phylogenetic tree was generated in CLUSTALX2 by the neighbour-joining method and a thousand
458
replicates and displayed using MEGA5 (http://www.megasoftware.net/) (Kumar et al., 1994).
459
Plant materials and growth conditions
460
Arabidopsis thaliana ecotype Columbia-0 was used in the study. Seeds were surface-sterilized in
461
10% (v/v) sodium hypochlorite for 2 min, washed 10 times with sterilized water, germinated and
462
grown on vertical plates (1/2 MS medium containing 0.8 % agar, pH 5.7, 1 % sucrose,
463
supplemented with or without antibiotics and chemical reagents) at 20 ±1°C with cycles of 16 h of
464
light and 8 h of darkness under 100-150 μ mol m-2 s-1 light intensity. The 10-d-old seedlings were
465
transferred to soil and grown under the same conditions for further experiments and seed
466
production.
467
For phenotypic analyses of the etiolated seedlings of 35S:SSPP transgenic Arabidopsis, seeds
468
were germinated and grown on vertical plates (1/2 MS medium containing 0.8 % agar, pH 5.7,
469
1 % sucrose, supplemented with 1 μM ACC). 3-d-old etiolated seedlings were imaged with a
470
scanner.
471
The dark-induced senescence analysis was performed using both attached and detached 5th
472
and 6th leaves at mature stage (from plants at the Boyes growth stage 5.10). The attached leaves
473
under normal growth conditions were individually covered by aluminum foil for 9d and the
474
detached leaves on wet filter paper were incubated in darkness for 4d before the effects of
475
dark-treatments were recorded using a camera (Canon PowerShot G10) or scanner (Epson 1260).
476
Measurements of ethylene emission
477
Ethylene emission of the wild type and 35S:SSPP was determined in 3-d-old etiolated
478
seedlings by gas chromatography (Agilent 6890N) as described by Li et al. (Li et al., 2009).
479
Constructs, Plant Transformation, and Crossing
480
To construct the 35S:SSPP fusion gene, the full-length cDNA of SSPP was amplified from
20
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
481
Arabidopsis cDNA by RT-PCR. The pair of primers used in the PCR was SSPP-35S-TA-1 and
482
SSPP-35S-TA-2. The DNA fragment was inserted into the binary vector pBI121 to create the
483
recombinant transcription unit 35S:SSPP.
484
To construct the SSPP:GUS fusion gene, a 1516-bp DNA fragment covering the 5’ flanking region
485
of the SSPP gene was amplified from Arabidopsis genomic DNA by PCR. The pair of primers
486
used in the PCR was cP-SSPP-T-1 and cP-SSPP-T-2. The DNA fragment was inserted into the
487
binary vector pCAMBIA1301 to create the recombinant transcription unit SSPP:GUS. The
488
transcript was terminated with a NOS terminator.
489
For constructing the GST-SSPP recombinant transcription unit, the SSPP coding region was
490
amplified with SSPP cDNA as the template using SSPP-EcoRI-F and SSPP-XhoI-R. The DNA
491
fragment was inserted into the vector pGEX-6P-1 to create the recombinant transcription unit
492
GST-SSPP.
493
For the construction of His-AtSARK-CD and GST-AtSARK-CD recombinant transcription unit,
494
DNA fragment covering the intracellular domain of AtSARK (residues 432-1515; 1137 bp) was
495
amplified by PCR from the corresponding full-length cDNA using AtSARK-CD-EcoRI-F and
496
AtSARK-CD-SalI-R. The fragment was inserted into the vector pET28a and pGEX-6P-1 to create
497
the fusion gene His-AtSARK-CD and GST-AtSARK-CD, respectively.
498
For the construction of AtSARK-EYFP, SSPP-EYFP, AtSARK-nYFP and SSPP-cYFP recombinant
499
transcription units, the SSPP and AtSARK coding regions were amplified from the corresponding
500
full-length cDNA using the following primer pairs: AtSARK-EcoRI-F and AtSARK-SalI-R for
501
AtSARK-EYFP, AtSARK-XbaI-F and AtSARK-BamHI-R for AtSARK-nYFP, SSPP-EcoRI-F and
502
SSPP-SalI-R for SSPP-EYFP, SSPP-XbaI-F and SSPP-SalI-R for SSPP-cYFP. The corresponding
503
fragment of AtSARK and SSPP was inserted into the vector pSAT6-EYFP-N1 to create the
504
constructs AtSARK-EYFP and SSPP-EYFP. The fragment of AtSARK was inserted into the vector
505
pSPYNE-35S to create the construct AtSARK-nYFP, and the fragment of SSPP was inserted into
506
the pSPYCE-35S to create the construct SSPP-cYFP.
507
For the construction of BD-GmSARK-KD recombinant transcription unit, DNA fragment covering
508
the kinase domain of GmSARK (residues 660-940; 840 bp) was amplified by PCR from Glycine
21
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
509
max cDNA by RT-PCR. The pair of primers used in the PCR was GmKD-1 and GmMKD-2. The
510
fragment was inserted into the vector pGBKT7 to create the fusion gene BD-GmSARK-KD.
511
For the construction of the AD-SSPP recombinant transcription unit, the full-length cDNA of
512
SSPP was amplified from Arabidopsis cDNA by RT-PCR. The pair of primers used in the PCR
513
was SSPP-EcoRI-F and SSPP-XhoI-R. The fragment was inserted into the vector pGBKT7 to
514
create the fusion gene AD-SSPP.
515
The recombinant plasmids were introduced into Agrobacterium tumefaciens strain GV3101 and
516
transformed into wild-type Columbia-0 Arabidopsis plants using the floral dip method (Clough
517
and Bent, 1998). Transformants were screened on 0.5×MS medium containing 30 mg L-1
518
hygromycin or 30 mg L-1 kanamycin, and the resistant seedlings were transferred to soil and
519
verified by semi-quantitative RT-PCR. The PCR primers used to confirm the recombinant
520
transgenes in transgenic plants are listed in Supplemental Table S1. Homozygous T3 plants were
521
used for all experiments.
522
The 5×EBS:GUS/35S:SSPP plants were obtained by crossing the 35S:SSPP transgenic line with
523
5×EBS:GUS plants. The Agrobacterium strain GV3101 carrying DR5:GFP construct was used to
524
transform the homozygous 35S:SSPP line to produce DR5:GFP/35S:SSPP plants. The
525
Agrobacterium strain GV3101 carrying 35S:SSPP construct was used to transform the
526
homozygous GVG:AtSARK line to produce 35S:SSPP/GVG:AtSARK plants. Homozygous plants
527
were identified by segregation analysis, comparison with the parental phenotypes and PCR-based
528
genotyping in the F3 progeny.
529
RNA Isolation and RT-PCR Analysis of Gene Expression
530
RNA extraction, cDNA synthesis and RT-PCR analysis were done as described previously (Liu et
531
al., 2010). Real-time RT-PCR analysis was performed using SYBR Green Perfect mix (TaKaRa,
532
Dalian, China) on an iQ5 (Bio-Rad, California, USA), following the manufacturer’s instructions.
533
Three independent repeats were done to give the typical results shown here. All primers used in
534
RT-PCR analysis are listed in Supplemental Table S1.
535
Histochemical GUS Staining, Chlorophyll Content Determination, and Transmission
536
Electron Microscopy
22
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
537
Histochemical GUS staining and transmission electron microscopy were done as described
538
previously (Liu et al., 2010). Chlorophyll content was spectrophotometrically measured as
539
described (Arnon, 1949). At least three independent samples were examined to give the typical
540
results shown in this paper.
541
Protoplast transfection, BIFC assay and Fluorescence microscopy analyses
542
Protoplasts were prepared and transformed according to the protocols of Yoo et al. (Yoo et al.,
543
2007). The plasmids of SSPP-EYFP, AtSARK-EYFP and EYFP control were prepared with the
544
Vigorous Plasmid Maxprep Kit (Vigorous Biotechnology, Beijing, China). The transformed
545
protoplasts were assayed for fluorescence 12-18 h after transfection.
546
For infiltration of N. benthamiana, the Agrobacterium tumefaciens strain GV3101 was infiltrated
547
into the abaxial air space of 2-4-week-old plants as described (Voinnet et al., 2003). The p19
548
protein of tomato bushy stunt virus was used to suppress gene silencing. Co-infiltration of
549
Agrobacterium strains containing the BiFC constructs and the p19 silencing plasmid was carried
550
out at OD600 of 0.6:0.6:0.3. Epidermal cell layers of tobacco leaves were assayed for fluorescence
551
2 days after infiltration.
552
The fluorescence signal was collected with a laser scanning confocal microscope (Leica TCS-SP5,
553
Germany). The image data were processed using Adobe Photoshop (www.adobe.com).
554
Protein Expression, Purification and Pull down assay
555
The GST-SSPP, GST-AtSARK-CD and HIS-AtSARK-CD fusion proteins were expressed in
556
Escherichia coli Rosetta 2 (DE3) plysS and purified by affinity chromatography using Glutathione
557
Sepharose 4B columns (GE Healthcare) and Ni2+NTA agarose columns (GE Healthcare) according
558
to the manufacturer’s recommendations. The GST tag of GST-SSPP was removed by PreScission
559
Protease (GE Healthcare) according to the manufacturer’s recommendations.
560
The in vitro pull down assay was performed as described previously (Zhang et al., 2013). Briefly,
561
the columns or beads bound with GST-SSPP and GST were washed with PBS (0.14 M NaCl, 2.7
562
mM KCl, 10.1 mM Na2HPO4, and 1.8 mM KH2PO4) for GST pull-down assays. Each reaction
563
contained approximately 30 μg His-tagged fusion protein. After being added to glutathione
23
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
564
Sepharose 4B columns carrying a GST fusion protein, reaction mixtures were incubated for at
565
least 1 h at 4°C under gentle rotation. After being washed five times with PBS, proteins were
566
eluted and boiled for 10 min. The proteins were separated by 12% SDS-PAGE for Coomassie
567
Brilliant Blue staining and immunodetection with anti-His antiserum at 1:2000 dilutions.
568
Phosphatase Activity Assay
569
The optimal activity conditions of SSPP were determined as described previously (Wu et al.,
570
2011) with some modifications. Briefly, to determine the optimal pH for SSPP activity,
571
phosphatase activity assays were performed in 100 mM buffers, including sodium acetate buffer
572
(pH 4.5 and 5.5), Tris/HCl buffer (pH 6.5 to 8.5), or glycine/ NaOH buffer (pH 9.5 and 10.5) with
573
1 μg purified SSPP protein and 7.5 mM pNPP (New England Biolabs), and the reactions were
574
initiated by adding purified SSPP and incubated for 30 min at 47°C. To determine the optimal
575
temperature for SSPP activity, phosphatase activity was assayed by incubating 1 μg of the protein
576
and 7.5 mM pNPP in 100 mM Tris-HCl buffer (pH 7.5). The Mg2+-dependent phosphatase activity
577
of SSPP was assayed by incubating 1 μg of SSPP and 7.5 mM pNPP in 100 mM Tris-HCl buffer
578
(pH 7.5) containing 1 mM EDTA with or without 50 mM Mg2+ and incubated for 30 min at 47°C.
579
In Vitro Kinase Assay
580
The in vitro protein phosphorylation experiments were performed essentially as reported
581
previously (Shalitin et al., 2003) with minor modifications. 0.2 μg GST-AtSARK-CD protein was
582
incubated in the phosphorylation buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM MnCl2,
583
1mM dithiothreitol, 1 μg/μl ATP) at 28°C for 10 min to auto-phophorylate before an equal amount
584
of SSPP was added in the mixture and incubated together with GST-AtSARK-CD for further 20
585
min. The phosphorylation reactions were stopped by adding 2 × SDS-PAGE sample buffers and
586
boiling for 10 min. The proteins were separated on 12% SDS-PAGE gels. Gels were stained with
587
Coomassie blue. Immunoblot analysis was performed to check the in vitro phosphorylation states
588
of the purified proteins by anti-Phospho-Threonine antibody (anti-pT) (CST). Protein bands were
589
visualized using a ECL Western Blotting Reagent Pack (GE Healthcare) following the
590
manufacturer’s instructions. The images were recorded by a chemiluminescence imaging system
591
(Tanon 5500). The PVDF membrane was stripped and re-probed with anti-GST antibody (CST) to
24
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
592
ensure equivalent protein loading. Data shown are representative of at least three independent
593
experiments.
594
Yeast two-hybrid assays
595
Yeast two-hybrid analysis was performed using the MATCHMAKER GAL4 system (Clontech,
596
Palo Alto, CA) as described in the yeast protocols handbook (Clontech). The bait plasmids
597
containing sequences for GmSARK-KD and prey plasmids containing SSPP were sequentially
598
transformed into the yeast strain, AH109 (Clontech). Confirmation of the presence of both vectors
599
was performed by growing the yeast on medium lacking Trp and Leu. Experimental
600
protein-protein interaction was determined by growth on SD/-Leu-Trp-His with 1mM 3AT
601
medium or SD/-Trp/ X-α-gal plates. For control experiments, yeast strains were generated with the
602
pGADT7-T plasmid and either the pGBKT7-53 or the pGBKT7-Lam vector for positive and
603
negative controls, respectively. Yeast was transformed with the BD-GmSARK-KD plasmid for
604
self-activation assay.
605
Accession Numbers
606
Sequence data from this article can be found in the Arabidopsis Information Resource (TAIR)
607
or GenBank/EMBL database under the following accession numbers: SSPP (At5g02760), AtSARK
608
(At4g30520), VvPP2C38-like (XM_002276595), RcPP2Cc (XM_002525162), CaPP2C38-like
609
(XM_004493866), SAG12 (At5g45890), SAG113 (AT5G59220), AtNAP (At1g69490), WRKY6
610
(At1g62300), NAC1 (At1g56010), NAC2 (At5g04410), GTR1 (AT1G58290), ACD1 (AT3G44880),
611
RbcL (AtCG00490.1), RbcS (At1G67090), SIG5 (At5g24120), IPT3 (At3g63110), ARR5
612
(AT3G48100), ARR6 (AT5G62920), GRXS13 (At1g03850), At2g18300, CKX3 (At5g56970),
613
TSA1 (At3g54640), GH3.5 (At4g27260), GST2 (AT4G02520), PR4 (At3g04720), PDF1.2
614
(At5g44420), CHI-B (AT3G12500), ERF11 (At1g28370), GmSARK (AY687391), and TIP41-like
615
(AT4G34270).
616
Supplemental Data
617
The following materials are available in the online version of this article.
25
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
618
Table S1. Gene-specific primers used in this study.
619
Figure S1. Photograph of the wild type (WT) and 35S:SSPP transgenic plants at four
620
developmental stages (stage 3.50, 5.10, 6.00 and 6.10).
621
Figure S2. Morphology of SSPP over-expressing plants.
622
Figure S3. Morphology of SSPP T-DNA insertion line sspp-1.
623
Figure S4. Effects of SSPP overexpression on the functions of and responses to ethylene in
624
Arabidopsis.
625
Figure S5. SSPP interacts with the kinase domain of GmSARK (GmSARK-KD) in the yeast
626
two-hybrid system.
627
ACKNOWLEDGMENTS
628
We gratefully acknowledge Dr. Nam-Hai Chua (The Rockefeller University) for providing the
629
pTA7002 vector. The homozygous 5×EBS:GUS seeds were kindly provided by Dr. Hongwei Guo
630
(Peking University, Beijing, China). We also deeply thank Dr. Shuhua Yang (China Agricultural
631
University, Beijing, China) for her guidance on the BiFC assay of the SSPP and AtSARK
632
interaction. The plasma membrane marker pm-rk was kindly provided by Dr. Qingqiu Gong
633
(College of Life Sciences, Nankai University). Thanks also go to Miss Yuanyuan Mei (College of
634
Life Sciences, Nankai university) for editing the final manuscript.
26
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
635
Literature Cited
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
An J, Carmichael WW (1994) Use of a colorimetric protein phosphatase inhibition assay and enzyme
linked immunosorbent assay for the study of microcystins and nodularins. Toxicon 32:
1495-1507
Arnon DI (1949) Copper Enzymes in Isolated Chloroplasts. Polyphenoloxidase in Beta Vulgaris. Plant
Physiol 24: 1-15
Benkova E, Michniewicz M, Sauer M, Teichmann T, Seifertova D, Jurgens G, Friml J (2003)
Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell
115: 591-602
Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Görlach J (2001)
Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput
functional genomics in plants. Plant Cell 13: 1499-1510
Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N (2004)
Detection of protein-protein interactions in plants using bimolecular fluorescence
complementation. Plant Journal 40: 419-427
Brenner WG, Romanov GA, Kollmer I, Burkle L, Schmulling T (2005) Immediate-early and
delayed cytokinin response genes of Arabidopsis thaliana identified by genome-wide
expression profiling reveal novel cytokinin-sensitive processes and suggest cytokinin action
through transcriptional cascades. Plant Journal 44: 314-333
Canova MJ, Veyron-Churlet R, Zanella-Cleon I, Cohen-Gonsaud M, Cozzone AJ, Becchi M,
Kremer L, Molle V (2008) The Mycobacterium tuberculosis serine/threonine kinase PknL
phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation
sites and their role in the recruitment of Rv2175c. Proteomics 8: 521-533
Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR (1997) Activation of the
ethylene
gas
response
pathway
in
Arabidopsis
by
nuclear
protein
ETHYLENE-INSENSITIVE3 and related proteins. Cell 89: 1133-1144
Cho YH, Hong JW, Kim EC, Yoo SD (2012) Regulatory functions of SnRK1 in stress-responsive
gene expression and in plant growth and development. Plant Physiol 158: 1955-1964
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana. Plant Journal 16: 735-743
Cui X, Ge C, Wang R, Wang H, Chen W, Fu Z, Jiang X, Li J, Wang Y (2010) The BUD2 mutation
affects plant architecture through altering cytokinin and auxin responses in Arabidopsis. Cell
Res 20: 576-586
Das AK, Helps NR, Cohen PT, Barford D (1996) Crystal structure of the protein serine/threonine
phosphatase 2C at 2.0 A resolution. EMBO J 15: 6798-6809
Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jurgens G (2003)
Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426:
147-153
Frye CA, Tang D, Innes RW (2001) Negative regulation of defense responses in plants by a
conserved MAPKK kinase. Proc Natl Acad Sci U S A 98: 373-378
Gan S, Amasino RM (1995) Inhibition of leaf senescence by autoregulated production of cytokinin.
Science 270: 1986-1988
Gao Q, Yang Z, Zhou Y, Yin Z, Qiu J, Liang G, Xu C (2012) Characterization of an Abc1 kinase
27
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
family gene OsABC1-2 conferring enhanced tolerance to dark-induced stress in rice. Gene
498: 155-163
Guo Y, Gan S (2005) Leaf senescence: signals, execution, and regulation. Curr Top Dev Biol 71:
83-112
Guo Y, Gan S (2006) AtNAP, a NAC family transcription factor, has an important role in leaf
senescence. Plant Journal 46: 601-612
Hou K, Wu W, Gan SS (2013) SAUR36, a small auxin up RNA gene, is involved in the promotion of
leaf senescence in Arabidopsis. Plant Physiol 161: 1002-1009
Hwang I, Sheen J, Muller B (2012) Cytokinin signaling networks. Annu Rev Plant Biol 63: 353-380
Isono K, Niwa Y, Satoh K, Kobayashi H (1997) Evidence for transcriptional regulation of plastid
photosynthesis genes in Arabidopsis thaliana roots. Plant Physiology 114: 623-630
Jing HC, Schippers JH, Hille J, Dijkwel PP (2005) Ethylene-induced leaf senescence depends on
age-related changes and OLD genes in Arabidopsis. Journal of Experimental Botany 56:
2915-2923
Jonasson S (1989) Implications of Leaf Longevity, Leaf Nutrient Re-Absorption and Translocation for
the Resource Economy of Five Evergreen Plant Species. Oikos 56: 121-131
Kikuzawa K (1991) A Cost-Benefit Analysis of Leaf Habit and Leaf Longevity of Trees and Their
Geographical Pattern. The American Naturalist 138: 1250-1263
Kikuzawa K, Ackerly D (1999) Significance of leaf longevity in plants. Plant Species Biology 14:
39-45
Kim HJ, Ryu H, Hong SH, Woo HR, Lim PO, Lee IC, Sheen J, Nam HG, Hwang I (2006)
Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in
Arabidopsis. Proc Natl Acad Sci U S A 103: 814-819
Kim JH, Woo HR, Kim J, Lim PO, Lee IC, Choi SH, Hwang D, Nam HG (2009) Trifurcate
feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis.
Science 323: 1053-1057
Kim JI, Murphy AS, Baek D, Lee SW, Yun DJ, Bressan RA, Narasimhan ML (2011) YUCCA6
over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis
thaliana. Journal of Experimental Botany 62: 3981-3992
Kleine-Vehn J, Ding Z, Jones AR, Tasaka M, Morita MT, Friml J (2010) Gravity-induced PIN
transcytosis for polarization of auxin fluxes in gravity-sensing root cells. Proc Natl Acad Sci U
S A 107: 22344-22349
Krebbers E, Seurinck J, Herdies L, Cashmore AR, P.Timko M (1988) Four genes in two diverged
subfamilies encode the ribulose-1,5-bisphosphate carboxylas small subunit polypeptides of
Arabidopsis thaliana. Plant Molecular Biology 11: 745-759
Kudo T, Makita N, Kojima M, Tokunaga H, Sakakibara H (2012) Cytokinin activity of cis-zeatin
and
phenotypic
alterations
induced
by
overexpression
of
putative
cis-Zeatin-O-glucosyltransferase in rice. Plant Physiol 160: 319-331
Kumar S, Tamura K, Nei M (1994) MEGA: Molecular Evolutionary Genetics Analysis software for
microcomputers. Comput Appl Biosci 10: 189-191
Laporte D, Olate E, Salinas P, Salazar M, Jordana X, Holuigue L (2012) Glutaredoxin GRXS13
plays a key role in protection against photooxidative stress in Arabidopsis. Journal of
Experimental Botany 63: 503-515
Lee IC, Hong SW, Whang SS, Lim PO, Nam HG, Koo JC (2011) Age-dependent action of an
28
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
ABA-inducible receptor kinase, RPK1, as a positive regulator of senescence in Arabidopsis
leaves. Plant and Cell Physiology 52: 651-662
Lee JS, Ellis BE (2007) Arabidopsis MAPK phosphatase 2 (MKP2) positively regulates oxidative
stress tolerance and inactivates the MPK3 and MPK6 MAPKs. J Biol Chem 282:
25020-25029
Li H, Wong WS, Zhu L, Guo HW, Ecker J, Li N (2009) Phosphoproteomic analysis of
ethylene-regulated protein phosphorylation in etiolated seedlings of Arabidopsis mutant ein2
using two-dimensional separations coupled with a hybrid quadrupole time-of-flight mass
spectrometer. Proteomics 9: 1646-1661
Li Z, Peng J, Wen X, Guo H (2012) Gene Network Analysis and Functional Studies of
Senescence-associated Genes Reveal Novel Regulators of Arabidopsis Leaf Senescence. J
Integr Plant Biol
Li Z, Peng J, Wen X, Guo H (2013) ETHYLENE-INSENSITIVE3 Is a Senescence-Associated Gene
That Accelerates Age-Dependent Leaf Senescence by Directly Repressing miR164
Transcription in Arabidopsis. Plant Cell
Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58: 115-136
Lim PO, Lee IC, Kim J, Kim HJ, Ryu JS, Woo HR, Nam HG (2010) Auxin response factor 2
(ARF2) plays a major role in regulating auxin-mediated leaf longevity. Journal of
Experimental Botany 61: 1419-1430
Liu D, Gong Q, Ma Y, Li P, Li J, Yang S, Yuan L, Yu Y, Pan D, Xu F, Wang NN (2010) cpSecA, a
thylakoid protein translocase subunit, is essential for photosynthetic development in
Arabidopsis. Journal of Experimental Botany 61: 1655-1669
Lofke C, Luschnig C, Kleine-Vehn J (2013) Posttranslational modification and trafficking of PIN
auxin efflux carriers. Mech Dev 130: 82-94
Lohman KN, Gan S, John MC, Amasino RM (1994) Molecular analysis of natural leaf senescence in
Arabidopsis thaliana. Physiologia Plantarum 92: 322-328
McCormac AC, Fischer A, Kumar AM, Söll D, Terry MJ (2001) Regulation of HEMA1 expression
by phytochrome and a plastid signal during de-etiolation in Arabidopsis thaliana. Plant Journal
25: 549-561
Miller JD, Arteca RN, Pell EJ (1999) Senescence-associated gene expression during ozone-induced
leaf senescence in Arabidopsis. Plant Physiol 120: 1015-1024
Nagashima A, Hanaok M, Shikanai T, Fujiwara M, Kanamaru K, Takahashi H, Tanaka K (2004)
The multiple-stress responsive plastid sigma factor, SIG5, directs activation of the psbD blue
light-responsive promoter (BLRP) in Arabidopsis thaliana. Plant Cell Physiol 45: 357-368
Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for
co-localization studies in Arabidopsis and other plants. Plant Journal 51: 1126-1136
Nemhauser JL, Hong F, Chory J (2006) Different plant hormones regulate similar processes through
largely nonoverlapping transcriptional responses. Cell 126: 467-475
Pineda Rodo A, Brugiere N, Vankova R, Malbeck J, Olson JM, Haines SC, Martin RC, Habben
JE, Mok DW, Mok MC (2008) Over-expression of a zeatin O-glucosylation gene in maize
leads to growth retardation and tasselseed formation. Journal of Experimental Botany 59:
2673-2686
Pruzinska A, Tanner G, Anders I, Roca M, Hortensteiner S (2003) Chlorophyll breakdown:
Pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell
29
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
death 1 gene. Proc Natl Acad Sci U S A 100: 15259-15264
Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E (2007)
Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl
Acad Sci U S A 104: 19631-19636
Robatzek S, Somssich IE (2002) Targets of AtWRKY6 regulation during plant senescence and
pathogen defense. Genes Dev 16: 1139–1149
Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold
N, Weisbeek P, Scheres B (1999) An auxin-dependent distal organizer of pattern and polarity
in the Arabidopsis root. Cell 99: 463-472
Scarpella E, Marcos D, Friml J, Berleth T (2006) Control of leaf vascular patterning by polar auxin
transport. Genes Dev 20: 1015-1027
Schweighofer A, Hirt H, Meskiene I (2004) Plant PP2C phosphatases: emerging functions in stress
signaling. Trends in Plant Science 9: 236-243
Shalitin D, Yu X, Maymon M, Mockler T, Lin C (2003) Blue light-dependent in vivo and in vitro
phosphorylation of Arabidopsis cryptochrome 1. Plant Cell 15: 2421-2429
Shoji K, Addicott FT, Swets WA (1951) Auxin in Relation to Leaf Blade Abscission. Plant Physiol
26: 189-191
Singh S, Letham DS, Palni LMS (1992) Cytokinin biochemistry in relation to leaf senescence. VII.
Endogenous cytokinin levels and exogenous applications of cytokinins in relation to
sequential leaf senescence of tobacco. Physiologia Plantarum 86: 388-397
Solano R, Stepanova A, Chao Q, Ecker JR (1998) Nuclear events in ethylene signaling: a
transcriptional
cascade
mediated
by
ETHYLENE-INSENSITIVE3
and
ETHYLENE-RESPONSE-FACTOR1. Genes and Development 12: 3703-3714
Stepanova AN, Yun J, Likhacheva AV, Alonso JM (2007) Multilevel interactions between ethylene
and auxin in Arabidopsis roots. Plant Cell 19: 2169-2185
Stone JM, Trotochaud AE, Walker JC, Clark SE (1998) Control of meristem development by
CLAVATA1 receptor kinase and kinase-associated protein phosphatase interactions. Plant
Physiol 117: 1217-1225
Tanaka H, Dhonukshe P, Brewer PB, Friml J (2006) Spatiotemporal asymmetric auxin distribution:
a means to coordinate plant development. Cell Mol Life Sci 63: 2738-2754
Tang D, Christiansen KM, Innes RW (2005) Regulation of plant disease resistance, stress responses,
cell death, and ethylene signaling in Arabidopsis by the EDR1 protein kinase. Plant Physiol
138: 1018-1026
Thomas H (2013) Senescence, ageing and death of the whole plant. New Phytologist 197: 696-711
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22: 4673-4680
Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ (1997) Aux/IAA proteins repress expression of
reporter genes containing natural and highly active synthetic auxin response elements. Plant
Cell 9: 1963-1971
Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F, Yamaguchi-Shinozaki K,
Ishihama Y, Hirayama T, Shinozaki K (2009) Type 2C protein phosphatases directly
regulate abscisic acid-activated protein kinases in Arabidopsis. Proc Natl Acad Sci U S A 106:
17588-17593
30
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
839
840
841
Vandenbussche F, Petrasek J, Zadnikova P, Hoyerova K, Pesek B, Raz V, Swarup R, Bennett M,
Zazimalova E, Benkova E, Van Der Straeten D (2010) The auxin influx carriers AUX1 and
LAX3 are involved in auxin-ethylene interactions during apical hook development in
Arabidopsis thaliana seedlings. Development 137: 597-606
Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in
plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.
Plant Journal 33: 949-956
Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY (2005) Control of root cap formation by
MicroRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17: 2204-2216
Wu HY, Liu MS, Lin TP, Cheng YS (2011) Structural and Functional Assays of AtTLP18.3 Identify
Its Novel Acid Phosphatase Activity in Thylakoid Lumen. Plant Physiol
Xu F, Meng T, Li P, Yu Y, Cui Y, Wang Y, Gong Q, Wang NN (2011) A soybean dual-specificity
kinase, GmSARK, and its Arabidopsis homolog, AtSARK, regulate leaf senescence through
synergistic actions of auxin and ethylene. Plant Physiol 157: 2131-2153
Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for
transient gene expression analysis. Nature Protocols 2: 1565-1572
Zenke FT, King CC, Bohl BP, Bokoch GM (1999) Identification of a central phosphorylation site in
p21-activated kinase regulating autoinhibition and kinase activity. J Biol Chem 274:
32565-32573
Zhang H, Zhou C (2012) Signal transduction in leaf senescence. Plant Mol Biol
Zhang K, Xia X, Zhang Y, Gan SS (2012) An ABA-regulated and Golgi-localized protein
phosphatase controls water loss during leaf senescence in Arabidopsis. Plant Journal 69:
667-678
Zhang R, Letham DS, Parker CW, Schroeder H, Higgins TJV (1987) Retardation of soybean leaf
senescence and associated effects on seed composition. Journal of Plant Growth Regulation 6:
15-21
Zhang Y, Li B, Xu Y, Li H, Li S, Zhang D, Mao Z, Guo S, Yang C, Weng Y, Chong K (2013) The
cyclophilin CYP20-2 modulates the conformation of BRASSINAZOLE-RESISTANT1, which
binds the promoter of FLOWERING LOCUS D to regulate flowering in Arabidopsis. Plant
Cell 25: 2504-2521
Zhou C, Cai Z, Guo Y, Gan S (2009) An arabidopsis mitogen-activated protein kinase cascade,
MKK9-MPK6, plays a role in leaf senescence. Plant Physiol 150: 167-177
31
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
842
FIGURE LEGENDS
843
Figure 1. The expression of SSPP is suppressed during leaf senescence. A. Quantitative RT-PCR
844
analysis of the expression level of SSPP in senescing leaves. YL, young leaves; ML, mature leaves;
845
ES, early senescence stage leaves; LS, late senescence stage leaves. The TIP41-like gene was used
846
as an internal control in RT-PCR and the data are displayed as relative expression to YL. Three
847
biological replicates with at least three technical repeats were done. Error bars represent SD. B.
848
Comparative analyses of the time-course expression profiles of AtSARK and SSPP in a typical
849
GVG:AtSARK line, AtS20, upon mock and DEX treatments. 4-day-old transgenic Arabidopsis
850
seedlings were incubated on a 10 μM DEX-containing plate for 0, 0.5, 1, 1.5, 2, 6, 24, or 48 h.
851
Data are normalized to the mock-treated control at time point 0 h. Three biological replicates with
852
at least three technical repeats were done. Error bars represent SD.
853
Figure 2. Amino acid sequence analysis of SSPP and biochemical characterization of SSPP
854
phosphatase activity. A. Alignment of the predicted amino acid sequence of SSPP with its
855
homologs from different plant species. Comparison of the protein sequences of SSPP and its
856
homologues from Arabidopsis thaliana (AT3G12620), Vitis vinifera (VvPP2C38-like,
857
XP_002276631.1), Ricinus communis (RcPP2C, XP_002525208.1) and Cicer arietinum
858
(CaPP2C38-like, XP_004493923.1) were performed by CLUSTALX and displayed by DNAMAN
859
software. Closed arrowhead indicates the conserved active sites that feature PP2C homologs. B. A
860
phylogenetic tree of PP2Cs related to SSPP. A neighbor-joining tree was built on the full-length
861
protein sequences of PP2C subfamily D members. The scale bar is an indicator of genetic distance
862
based on branch length. C and D. pH (C) and temperature (D) optima for SSPP activity with pNPP
863
as the substrate. E. The phosphatase activity of SSPP was determined in the presence or absence of
864
Mg2+. Reaction conditions are described in Materials and Methods. Three independent replicates
865
were done to give the average results shown here. Error bars represent SD.
866
Figure 3. Overexpression of SSPP delays leaf senescence. A. (a). Seven independent 35S:SSPP
867
transgenic lines (line 16, 18, 20, 26, 38, 35 and 39) and their wild type controls were cultivated
868
under long-day photoperiod conditions for up to 57 days. (b). Determination of SSPP transcript
32
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
869
levels in the above 35S:SSPP transgenic lines by semi-quantitative RT-PCR. The 6th leaves of
870
33-d-old plants were sampled. The TIP41-like gene was used as an internal control. Three
871
biological replicates were done to give the typical results shown here. B. (a). Leaves from
872
33-d-old wild-type (WT) and 35S:SSPP plants were laid out in order of emergence. (b).
873
Age-dependent senescence phenotype of the 5th rosette leaves of wild-type (WT) and 35S:SSPP
874
plants. DAE, Days after emergence. C. Dark-induced senescence was delayed in the 35S:SSPP
875
transgenic Arabidopsis. (a). The 5th and 6th leaves of wild-type (WT) and 35S:SSPP transgenic
876
plants at stage 5.10 under normal growth conditions were covered by aluminum foil for up to 9
877
days. (b). The 5th leaves of wild type (WT) and 35S:SSPP transgenic plants at stage 5.10 were
878
detached and incubated in darkness for 4 days. D. Overexpression of SSPP reduced the transcript
879
levels of several senescence-related marker genes in Arabidopsis plants. The 6th leaves of the wild
880
type (WT) and 35S:SSPP transgenic plants at four different developmental stages were sampled.
881
The transcript levels of the marker genes were determined by quantitative RT-PCR, with the
882
expression of TIP41-like as an internal control. Values are normalized relative to the expression in
883
the wild type control (WT) at stage 3.50. Three biological replicates with at least three technical
884
repeats were done for each gene. Error bars represent SD.
885
Figure 4. Overexpression of SSPP sustains the structure and function of chloroplasts. A.
886
Ultrastructural morphology of chloroplasts in mesophyll cells from the 6th leaves of the wild-type
887
(WT) and 35S:SSPP transgenic plants at the stage 3.50 and 6.00 respectively. p, plastoglobuli; s,
888
starch. Bars = 1.0 μm. B. Comparisons of chlorophyll contents of the 6th leaves of the wild type
889
(WT) and 35S:SSPP transgenic plants at four different developmental stages. Data are means (±
890
SD) of three experiments. Significant differences between wild type (WT) and 35S:SSPP
891
transgenic plants are indicated by asterisks on the error bars (Student’s t-test, P < 0.05). FW, fresh
892
weight. C. Overexpression of SSPP changes the expression levels of genes involved in chlorophyll
893
metabolism and chloroplast functions in Arabidopsis plants. The 6th leaves of the wild type (WT)
894
and 35S:SSPP transgenic plants at four developmental stages were sampled. The transcript levels
895
of the marker genes were determined by quantitative RT-PCR, with the expression of TIP41-like
896
as an internal control. Values are normalized relative to the expression in the wild type control
897
(WT) at stage 3.50. Three biological replicates with at least three technical repeats were done for
33
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
898
each gene. Error bars represent SD.
899
Figure 5. Overexpression of SSPP alters multiple hormone responses in Arabidopsis. A. Changes
900
in the expression of cytokinin-related marker genes in the 35S:SSPP plants. The 6th leaves of the
901
wild type (WT) and SSPP-overexpressing plants at stage 5.10 were sampled. The transcript levels
902
of the marker genes were determined by quantitative RT-PCR, with the expression of TIP41-like
903
as an internal control. The data are shown relative to the wild type control (WT). Three biological
904
replicates with at least three technical repeats were done. Error bars represent SD. B. Expression
905
of DR5:GFP in the roots of 7-d-old wild-type (DR5:GFP/WT) and SSPP-overexpressing
906
(DR5:GFP/35S:SSPP) seedlings. C. Expression of 5×EBS:GUS in 2-week-old (top panel) and
907
4-week-old
908
(5×EBS:GUS/35S:SSPP) plants.
909
Figure 6. Subcellular localizations of AtSARK and SSPP in Arabidopsis protoplasts. Confocal
910
laser scanning microscopy images of Arabidopsis protoplasts transiently expressing EYFP,
911
AtSARK-EYFP, SSPP-EYFP were shown. Scale bar = 20 μm.
912
Figure 7. SSPP partially dephosphorylates the auto-phosphorylated cytoplasmic domain of
913
AtSARK. Equal amounts (0.2 μg) of autophosphorylated GST-AtSARK-CD, SSPP or GST were
914
incubated in reaction buffer for 20 min. The reaction products were separated by 12% SDS-PAGE.
915
Autophosphorylation of AtSARK-CD was detected by anti-Phospho-Threonine antibody (anti-pT).
916
The PVDF membrane was stripped and re-probed with anti-GST antibody to ensure equivalent
917
protein loading. A solid arrow indicates the migration of each protein and the dotted arrows
918
indicate the predicted positions of SSPP and GST. Three independent replicates were done to give
919
the typical results shown here.
920
Figure 8. SSPP interacts with AtSARK in vitro and in vivo. A. A pull-down assay showing the
921
interaction between SSPP and AtSARK-CD. His-AtSARK-CD proteins were incubated with
922
GST-SSPP or GST bound columns, respectively. Proteins bound to GST or GST-SSPP columns
923
were pelleted, subjected to 12% SDS-PAGE, stained with Coomassie blue (bottom), or detected by
924
immunoblot analysis using anti-His antibody (top). A solid arrow indicates the migration of each
(bottom
panel)
wild-type
(5×EBS:GUS/WT)
and
SSPP-overexpressing
34
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
925
protein. Three independent replicates were done to give the typical results shown here. B.
926
Bimolecular Fluorescence Complementation (BiFC) analysis of the interaction between AtSARK
927
and SSPP in Nicotiana benthamiana leaves. YFP and red fluorescence of plasma membrane
928
marker pm-rk microscope images of N. benthamiana epidermal cells in leaves agro-infiltrated
929
with a mixture of Agrobacterium strains harboring constructs encoding the indicated fusion
930
proteins were shown. Each image is a representative picture of at least three experiments. Scale
931
bar = 20 μm.
932
Figure 9. Overexpression of SSPP rescues the SARK-induced precocious leaf senescence in
933
Arabidopsis. A. 4-d-old GVG:GUS (G28), GVG:AtSARK (AtS20), 35S:SSPP and 35S:SSPP/
934
GVG:AtSARK transgenic plants were grown on vertical plates containing either 10 μM DEX
935
(DEX) or its mock solution (mock) for an additional 7 days. Results from one out of three
936
biological replicates are shown. B. Determination of AtSARK and SSPP transcript levels by
937
quantitative
938
GVG:AtSARK/35S:SSPP were incubated with either 10 μM DEX (DEX) or its mock solution
939
(mock) for 24 h. The data are displayed as relative expression to the mock-treated control. Three
940
biological replicates with at least three technical repeats were done. Error bars represent SD. C.
941
Overexpression of SSPP effectively reduces the AtSARK-induced expression of senescence-related
942
marker genes in Arabidopsis. Quantitative RT-PCR analysis was used to determine the expression
943
levels of SAG12, NAC1, WRKY6 and SAG113 in the 4-d-old GVG:GUS (G28), GVG:AtSARK
944
(AtS20), 35S:SSPP and 35S:SSPP/GVG:AtSARK transgenic seedlings treated with either 10 μM
945
DEX (DEX) or its mock solution (mock) for 24 h. TIP41-like was used as an internal control. In
946
all cases, data are shown relative to the mock-treated control. Three biological replicates with at
947
least three technical repeats were done.
948
Figure 10. AtSARK-induced changes in the expression of cytokinin- (A), auxin- (B), and
949
ethylene-related (C) genes were suppressed by the overexpression of SSPP in Arabidopsis. The
950
transcript levels of the hormone-related genes were determined by quantitative RT-PCR. 4-d-old
951
GVG:GUS (G28), GVG:AtSARK (AtS20), 35S:SSPP and 35S:SSPP/GVG:AtSARK transgenic
952
seedlings were treated with either 10 μM DEX (DEX) or its mock solution (mock) for 24 h.
RT-PCR.
4-d-old
GVG:GUS
(G28),
GVG:AtSARK
35
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
(AtS20)
and
953
TIP41-like was used as an internal control. In all cases, data are shown relative to the mock-treated
954
control. Three biological replicates with at least three technical repeats were done.
955
Figure 11. Comparison of the spatial and temporal expression of SSPP and AtSARK during leaf
956
development. 28-d-old SSPP:GUS (top panel) and AtSARK:GUS (bottom panel) transgenic
957
Arabidopsis plants were sampled for histochemical GUS staining. Leaves from the transgenic
958
plants were laid out in order of leaf emergence as indicated by the numbers. Results from one out
959
of three biological replicates are shown.
36
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Parsed Citations
An J, Carmichael WW (1994) Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay
for the study of microcystins and nodularins. Toxicon 32: 1495-1507
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Arnon DI (1949) Copper Enzymes in Isolated Chloroplasts. Polyphenoloxidase in Beta Vulgaris. Plant Physiol 24: 1-15
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Benkova E, Michniewicz M, Sauer M, Teichmann T, Seifertova D, Jurgens G, Friml J (2003) Local, efflux-dependent auxin
gradients as a common module for plant organ formation. Cell 115: 591-602
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Görlach J (2001) Growth stage-based phenotypic analysis of
Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13: 1499-1510
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N (2004) Detection of protein-protein interactions in
plants using bimolecular fluorescence complementation. Plant Journal 40: 419-427
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Brenner WG, Romanov GA, Kollmer I, Burkle L, Schmulling T (2005) Immediate-early and delayed cytokinin response genes of
Arabidopsis thaliana identified by genome-wide expression profiling reveal novel cytokinin-sensitive processes and suggest
cytokinin action through transcriptional cascades. Plant Journal 44: 314-333
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Canova MJ, Veyron-Churlet R, Zanella-Cleon I, Cohen-Gonsaud M, Cozzone AJ, Becchi M, Kremer L, Molle V (2008) The
Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation
loop phosphorylation sites and their role in the recruitment of Rv2175c. Proteomics 8: 521-533
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR (1997) Activation of the ethylene gas response pathway in
Arabidopsis by nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell 89: 1133-1144
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cho YH, Hong JW, Kim EC, Yoo SD (2012) Regulatory functions of SnRK1 in stress-responsive gene expression and in plant
growth and development. Plant Physiol 158: 1955-1964
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
Journal 16: 735-743
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cui X, Ge C, Wang R, Wang H, Chen W, Fu Z, Jiang X, Li J, Wang Y (2010) The BUD2 mutation affects plant architecture through
altering cytokinin and auxin responses in Arabidopsis. Cell Res 20: 576-586
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Das AK, Helps NR, Cohen PT, Barford D (1996) Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A
resolution. EMBO J 15: 6798-6809
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jurgens G (2003) Efflux-dependent auxin gradients
establish the apical-basal axis of Arabidopsis. Nature 426: 147-153
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Google Scholar: Author Only Title Only Author and Title
Frye CA, Tang D, Innes RW (2001) Negative regulation of defense responses in plants by a conserved MAPKK kinase. Proc Natl
Acad Sci U S A 98: 373-378
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gan S, Amasino RM (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270: 1986-1988
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gao Q, Yang Z, Zhou Y, Yin Z, Qiu J, Liang G, Xu C (2012) Characterization of an Abc1 kinase family gene OsABC1-2 conferring
enhanced tolerance to dark-induced stress in rice. Gene 498: 155-163
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Guo Y, Gan S (2005) Leaf senescence: signals, execution, and regulation. Curr Top Dev Biol 71: 83-112
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Guo Y, Gan S (2006) AtNAP, a NAC family transcription factor, has an important role in leaf senescence. Plant Journal 46: 601-612
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hou K, Wu W, Gan SS (2013) SAUR36, a small auxin up RNA gene, is involved in the promotion of leaf senescence in Arabidopsis.
Plant Physiol 161: 1002-1009
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hwang I, Sheen J, Muller B (2012) Cytokinin signaling networks. Annu Rev Plant Biol 63: 353-380
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Isono K, Niwa Y, Satoh K, Kobayashi H (1997) Evidence for transcriptional regulation of plastid photosynthesis genes in
Arabidopsis thaliana roots. Plant Physiology 114: 623-630
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jing HC, Schippers JH, Hille J, Dijkwel PP (2005) Ethylene-induced leaf senescence depends on age-related changes and OLD
genes in Arabidopsis. Journal of Experimental Botany 56: 2915-2923
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jonasson S (1989) Implications of Leaf Longevity, Leaf Nutrient Re-Absorption and Translocation for the Resource Economy of
Five Evergreen Plant Species. Oikos 56: 121-131
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kikuzawa K (1991) A Cost-Benefit Analysis of Leaf Habit and Leaf Longevity of Trees and Their Geographical Pattern. The
American Naturalist 138: 1250-1263
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kikuzawa K, Ackerly D (1999) Significance of leaf longevity in plants. Plant Species Biology 14: 39-45
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kim HJ, Ryu H, Hong SH, Woo HR, Lim PO, Lee IC, Sheen J, Nam HG, Hwang I (2006) Cytokinin-mediated control of leaf longevity
by AHK3 through phosphorylation of ARR2 in Arabidopsis. Proc Natl Acad Sci U S A 103: 814-819
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kim JH, Woo HR, Kim J, Lim PO, Lee IC, Choi SH, Hwang D, Nam HG (2009) Trifurcate feed-forward regulation of age-dependent
cell death involving miR164 in Arabidopsis. Science 323: 1053-1057
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Kim JI, Murphy AS, Baek D, Lee SW, Yun DJ, Bressan RA, Narasimhan ML (2011) YUCCA6 over-expression demonstrates auxin
function in delaying leaf senescence in Arabidopsis thaliana. Journal of Experimental Botany 62: 3981-3992
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kleine-Vehn J, Ding Z, Jones AR, Tasaka M, Morita MT, Friml J (2010) Gravity-induced PIN transcytosis for polarization of auxin
fluxes in gravity-sensing root cells. Proc Natl Acad Sci U S A 107: 22344-22349
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Krebbers E, Seurinck J, Herdies L, Cashmore AR, P.Timko M (1988) Four genes in two diverged subfamilies encode the ribulose1,5-bisphosphate carboxylas small subunit polypeptides of Arabidopsis thaliana. Plant Molecular Biology 11: 745-759
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kudo T, Makita N, Kojima M, Tokunaga H, Sakakibara H (2012) Cytokinin activity of cis-zeatin and phenotypic alterations induced
by overexpression of putative cis-Zeatin-O-glucosyltransferase in rice. Plant Physiol 160: 319-331
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kumar S, Tamura K, Nei M (1994) MEGA: Molecular Evolutionary Genetics Analysis software for microcomputers. Comput Appl
Biosci 10: 189-191
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Laporte D, Olate E, Salinas P, Salazar M, Jordana X, Holuigue L (2012) Glutaredoxin GRXS13 plays a key role in protection against
photooxidative stress in Arabidopsis. Journal of Experimental Botany 63: 503-515
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lee IC, Hong SW, Whang SS, Lim PO, Nam HG, Koo JC (2011) Age-dependent action of an ABA-inducible receptor kinase, RPK1,
as a positive regulator of senescence in Arabidopsis leaves. Plant and Cell Physiology 52: 651-662
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lee JS, Ellis BE (2007) Arabidopsis MAPK phosphatase 2 (MKP2) positively regulates oxidative stress tolerance and inactivates
the MPK3 and MPK6 MAPKs. J Biol Chem 282: 25020-25029
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li H, Wong WS, Zhu L, Guo HW, Ecker J, Li N (2009) Phosphoproteomic analysis of ethylene-regulated protein phosphorylation in
etiolated seedlings of Arabidopsis mutant ein2 using two-dimensional separations coupled with a hybrid quadrupole time-of-flight
mass spectrometer. Proteomics 9: 1646-1661
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li Z, Peng J, Wen X, Guo H (2012) Gene Network Analysis and Functional Studies of Senescence-associated Genes Reveal Novel
Regulators of Arabidopsis Leaf Senescence. J Integr Plant Biol
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li Z, Peng J, Wen X, Guo H (2013) ETHYLENE-INSENSITIVE3 Is a Senescence-Associated Gene That Accelerates Age-Dependent
Leaf Senescence by Directly Repressing miR164 Transcription in Arabidopsis. Plant Cell
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58: 115-136
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lim PO, Lee IC, Kim J, Kim HJ, Ryu JS, Woo HR, Nam HG (2010) Auxin response factor 2 (ARF2) plays a major role in regulating
auxin-mediated leaf longevity. Journal of Experimental Botany 61: 1419-1430
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Liu D, Gong Q, Ma Y, Li P, Li J, Yang S, Yuan L, Yu Y, Pan D, Xu F, Wang NN (2010) cpSecA, a thylakoid protein translocase subunit,
is essential for photosynthetic development in Arabidopsis. Journal of Experimental Botany 61: 1655-1669
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lofke C, Luschnig C, Kleine-Vehn J (2013) Posttranslational modification and trafficking of PIN auxin efflux carriers. Mech Dev 130:
82-94
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lohman KN, Gan S, John MC, Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana.
Physiologia Plantarum 92: 322-328
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
McCormac AC, Fischer A, Kumar AM, Söll D, Terry MJ (2001) Regulation of HEMA1 expression by phytochrome and a plastid signal
during de-etiolation in Arabidopsis thaliana. Plant Journal 25: 549-561
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Miller JD, Arteca RN, Pell EJ (1999) Senescence-associated gene expression during ozone-induced leaf senescence in
Arabidopsis. Plant Physiol 120: 1015-1024
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Nagashima A, Hanaok M, Shikanai T, Fujiwara M, Kanamaru K, Takahashi H, Tanaka K (2004) The multiple-stress responsive
plastid sigma factor, SIG5, directs activation of the psbD blue light-responsive promoter (BLRP) in Arabidopsis thaliana. Plant Cell
Physiol 45: 357-368
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and
other plants. Plant Journal 51: 1126-1136
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Nemhauser JL, Hong F, Chory J (2006) Different plant hormones regulate similar processes through largely nonoverlapping
transcriptional responses. Cell 126: 467-475
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Pineda Rodo A, Brugiere N, Vankova R, Malbeck J, Olson JM, Haines SC, Martin RC, Habben JE, Mok DW, Mok MC (2008) Overexpression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation. Journal of
Experimental Botany 59: 2673-2686
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Pruzinska A, Tanner G, Anders I, Roca M, Hortensteiner S (2003) Chlorophyll breakdown: Pheophorbide a oxygenase is a Riesketype iron-sulfur protein, encoded by the accelerated cell death 1 gene. Proc Natl Acad Sci U S A 100: 15259-15264
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E (2007) Delayed leaf senescence induces
extreme drought tolerance in a flowering plant. Proc Natl Acad Sci U S A 104: 19631-19636
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Robatzek S, Somssich IE (2002) Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev 16:
1139-1149
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold N, Weisbeek P, Scheres B (1999)
An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 99: 463-472
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Scarpella E, Marcos D, Friml J, Berleth T (2006) Control of leaf vascular patterning by polar auxin transport. Genes Dev 20: 10151027
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schweighofer A, Hirt H, Meskiene I (2004) Plant PP2C phosphatases: emerging functions in stress signaling. Trends in Plant
Science 9: 236-243
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shalitin D, Yu X, Maymon M, Mockler T, Lin C (2003) Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis
cryptochrome 1. Plant Cell 15: 2421-2429
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shoji K, Addicott FT, Swets WA (1951) Auxin in Relation to Leaf Blade Abscission. Plant Physiol 26: 189-191
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Singh S, Letham DS, Palni LMS (1992) Cytokinin biochemistry in relation to leaf senescence. VII. Endogenous cytokinin levels and
exogenous applications of cytokinins in relation to sequential leaf senescence of tobacco. Physiologia Plantarum 86: 388-397
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Solano R, Stepanova A, Chao Q, Ecker JR (1998) Nuclear events in ethylene signaling: a transcriptional cascade mediated by
ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes and Development 12: 3703-3714
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Stepanova AN, Yun J, Likhacheva AV, Alonso JM (2007) Multilevel interactions between ethylene and auxin in Arabidopsis roots.
Plant Cell 19: 2169-2185
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Stone JM, Trotochaud AE, Walker JC, Clark SE (1998) Control of meristem development by CLAVATA1 receptor kinase and kinaseassociated protein phosphatase interactions. Plant Physiol 117: 1217-1225
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tanaka H, Dhonukshe P, Brewer PB, Friml J (2006) Spatiotemporal asymmetric auxin distribution: a means to coordinate plant
development. Cell Mol Life Sci 63: 2738-2754
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tang D, Christiansen KM, Innes RW (2005) Regulation of plant disease resistance, stress responses, cell death, and ethylene
signaling in Arabidopsis by the EDR1 protein kinase. Plant Physiol 138: 1018-1026
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Thomas H (2013) Senescence, ageing and death of the whole plant. New Phytologist 197: 696-711
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ (1997) Aux/IAA proteins repress expression of reporter genes containing natural and
highly active synthetic auxin response elements. Plant Cell 9: 1963-1971
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F, Yamaguchi-Shinozaki K, Ishihama Y, Hirayama T, Shinozaki K (2009)
Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proc Natl Acad Sci U S A
106: 17588-17593
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Vandenbussche F, Petrasek J, Zadnikova P, Hoyerova K, Pesek B, Raz V, Swarup R, Bennett M, Zazimalova E, Benkova E, Van Der
Straeten D (2010) The auxin influx carriers AUX1 and LAX3 are involved in auxin-ethylene interactions during apical hook
development in Arabidopsis thaliana seedlings. Development 137: 597-606
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of
gene silencing by the p19 protein of tomato bushy stunt virus. Plant Journal 33: 949-956
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY (2005) Control of root cap formation by MicroRNA-targeted auxin response
factors in Arabidopsis. Plant Cell 17: 2204-2216
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wu HY, Liu MS, Lin TP, Cheng YS (2011) Structural and Functional Assays of AtTLP18.3 Identify Its Novel Acid Phosphatase Activity
in Thylakoid Lumen. Plant Physiol
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xu F, Meng T, Li P, Yu Y, Cui Y, Wang Y, Gong Q, Wang NN (2011) A soybean dual-specificity kinase, GmSARK, and its Arabidopsis
homolog, AtSARK, regulate leaf senescence through synergistic actions of auxin and ethylene. Plant Physiol 157: 2131-2153
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression
analysis. Nature Protocols 2: 1565-1572
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zenke FT, King CC, Bohl BP, Bokoch GM (1999) Identification of a central phosphorylation site in p21-activated kinase regulating
autoinhibition and kinase activity. J Biol Chem 274: 32565-32573
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhang H, Zhou C (2012) Signal transduction in leaf senescence. Plant Mol Biol
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhang K, Xia X, Zhang Y, Gan SS (2012) An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf
senescence in Arabidopsis. Plant Journal 69: 667-678
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhang R, Letham DS, Parker CW, Schroeder H, Higgins TJV (1987) Retardation of soybean leaf senescence and associated effects
on seed composition. Journal of Plant Growth Regulation 6: 15-21
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhang Y, Li B, Xu Y, Li H, Li S, Zhang D, Mao Z, Guo S, Yang C, Weng Y, Chong K (2013) The cyclophilin CYP20-2 modulates the
conformation of BRASSINAZOLE-RESISTANT1, which binds the promoter of FLOWERING LOCUS D to regulate flowering in
Arabidopsis. Plant Cell 25: 2504-2521
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhou C, Cai Z, Guo Y, Gan S (2009) An arabidopsis mitogen-activated protein kinase cascade, MKK9-MPK6, plays a role in leaf
senescence. Plant Physiol 150: 167-177
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.