The Plant Journal (2015) 82, 232–244
doi: 10.1111/tpj.12808
Identification and characterization of an ABA-activated MAP
kinase cascade in Arabidopsis thaliana
Agyemang Danquah1,†, Axel de Zelicourt1,2,†, Marie Boudsocq1,†, Jorinde Neubauer1, Nicolas Frei dit Frey1, Nathalie
Leonhardt3, Stephanie Pateyron1, Frederik Gwinner4, Jean-Philippe Tamby1, Dolores Ortiz-Masia5, Maria J. Marcote5,
Heribert Hirt1,2,* and Jean Colcombet1
1
Institute of Plant Sciences Paris-Saclay, Institut National de Recherche Agronomique/Centre National de la Recherche Scientifique/Universite Paris Sud/Universite Paris Diderot/Universite d’Evry Val d’Essonne, Saclay Plant Sciences, 91405 Orsay, France,
2
Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900 Saudi Arabia,
3
Institut de Biologie Environnementale et Biotechnologie, Centre National de la Recherche Scientifique/Commissariat a
l’Energie Atomique/Universite Aix-Marseille II, 13108 Saint Paul les Durance, France,
4
Institut Pasteur, 75015 Paris, France, and
5
Biochemistry and Molecular Biology Department, Facultad de Farmacia, Universidad de Valencia, Avda. Vicente Andre s
Estelle s s/n, 46100 Burjassot, Spain
Received 25 October 2014; revised 6 February 2015; accepted 18 February 2015; published online 27 February 2015.
*For correspondence (e-mail [email protected]).
†
These authors contributed equally to the work.
Accession numbers Sequence data from this article can be found in Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession
numbers: At1g10210 (MPK1), At1g59580 (MPK2), At2g43790 (MPK6), At2g18170 (MPK7), At1g18150 (MPK8), At4g36450 (MPK14), At5g40440 (MKK3), At2g32510
(MAP3K17) and At1g05100 (MAP3K18).
SUMMARY
Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant
processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as
putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in
ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK
cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/
14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants.
Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent
genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK
pathway in plant stress signalling.
Keywords: abscisic acid, MAPK module, MKK3, MAP3K18, signalling pathway, Arabidopsis thaliana.
INTRODUCTION
Abiotic stresses such as drought, high salinity, temperature
or hypoxia are severe environmental stresses that impair
productivity in crop systems (Wang et al., 2003; Tuteja, 2007;
Qin et al., 2011). They trigger many biochemical, molecular
and physiological changes and the phytohormone abscisic
acid (ABA) plays a major role in the adaptation to these challenges (Seki et al., 2002; Yamaguchi-Shinozaki and Shinozaki, 2006; Shinozaki and Yamaguchi-Shinozaki, 2007).
Recently, the core signalling complex that perceives ABA
and transmits cues to downstream events has been deciphered (Fujii et al., 2009; Ma et al., 2009; Park et al., 2009):
Pyrabactin Resistance/Pyrabactin resistance-like/Regulatory
232
Component of ABA Receptor (PYR/PYL/RCARs) function as
the ABA receptors, Protein Phosphatases 2C (PP2Cs) act as
negative regulators, and SNF1-related protein kinases 2
(SnRK2s) are positive regulators (Mustilli et al., 2002; Park
et al., 2009; Umezawa et al., 2009). In the presence of ABA,
PYR/PYL/RCAR-PP2C complex formation leads to inhibition
of PP2C activity (Fujii et al., 2009; Ma et al., 2009; Park
et al., 2009; Santiago et al., 2009), thus allowing activation
of SnRK2s, which target membrane proteins, ion channels
and transcription factors, and facilitate transcription of
ABA-responsive genes (Sheard and Zheng, 2009; Umezawa
et al., 2010; Soon et al., 2012).
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd
ABA-activated MAPK cascade in Arabidopsis 233
Other signalling pathways have also been implicated in
ABA signal transduction. Among them, mitogen-activated
protein kinase (MAPK) modules are of particular interest as
they are known to be involved in stress signalling. A MAPK
module is minimally constituted of three kinases, a MAP3K
(or MAP2K Kinase), a MAP2K (or MAPK Kinase) and a
MAPK, which are able to phosphorylate and thereby activate
each other sequentially. These kinases belong to large families, for which we have incomplete functional information. A
number of the MAPK components have been clearly
involved in biotic and abiotic signal transduction (for review,
Rodriguez et al., 2010). But for a large number of these kinases, only sparse evidence is available on their physiological functions. MKK3, which displays an atypical MAP2K
structure, has been reported to act on MPK7 (Doczi et al.,
2007) and MPK8 (Takahashi et al., 2011) to mediate ROS signalling and to regulate MPK6 in response to jasmonic acid,
Salmonella typhimurium infection and blue light (Takahashi
et al., 2007; Schikora et al., 2008; Sethi et al., 2014).
The direct activation of MAPKs by ABA has been
reported for several plant species (for review, Danquah
et al., 2014). In Arabidopsis, studies showed that MPK1
and MPK2 activity increased after ABA treatment (OrtizMasia et al., 2007; Hwa and Yang, 2008; Umezawa et al.,
2013). Xing et al. (2008) demonstrated the ABA-dependent
MKK1-mediated activation of MPK6 to regulate CATALASE1 (CAT1) expression in ROS homeostasis. MPK9 and
MPK12, two MAPKs that are preferentially expressed in
guard cells, were shown to mediate ABA-induced guard
cell closure (Jammes et al., 2009). The role of these kinases, and particularly the connection between MAPK modules and the ABA core signalling pathway, is of primary
interest but still a matter of debate. Recently, phosphoproteomic analyses revealed a significant fraction of
ABA-induced phosphosites as potential MAPK targets
(Umezawa et al., 2013; Wang et al., 2013).
(a)
(b)
In the present work, we report the identification of an
entire ABA-dependent MAPK pathway. We show that ABA
induces two functionally redundant MAP3Ks, MAP3K17
and MAP3K18, to activate the MAPKK MKK3 which subsequently stimulates the activity of the four C group MAPKs:
MPK1, MPK2, MPK7 and MPK14. Our genetic analysis confirms that the ABA-dependent MAPK pathway plays a role
in ABA stress signalling.
RESULTS
mkk3-1 plants are hypersensitive to ABA in germination
and root elongation
Our previous work showed that MKK3 functions during
pathogen perception and ROS signalling (Doczi et al.,
2007). In an attempt to identify other MKK3 functions,
the mkk3-1 mutant and a line expressing a constitutively
active version of MKK3 (named MKK3-EE) were subjected
to ABA treatments. The ABA-insensitive line hab1G246D
(Robert et al., 2006) was used as control. In germination
assays, mkk3-1 knock-out seeds were hypersensitive to
increasing ABA concentrations when compared with WT
whereas MKK3-EE seeds were less sensitive (Figure 1a,b).
As ABA is involved in osmotic stress signalling, we also
performed germination assays on high salt (150 mM
NaCl) and mannitol (300 mM) conditions. Similarly, we
observed hypersensitivity of mkk3-1 seeds and insensitivity of MKK3-EE seeds (Figure S1). We also evaluated the
ABA root growth sensitivity of these lines. mkk3-1 plantlets grown in the presence of 1 lM ABA showed significantly stronger inhibition of root elongation than WT
plantlets while in this assay, MKK3-EE roots did not
show significant differences compared with WT (Figure 1c). These data suggest that the MKK3-dependent
MAPK pathway could mediate ABA signalling during germination and root elongation.
(c)
Figure 1. MKK3 mediates ABA signalling during germination and early development.
(a) Germination assays of WT (Col-0), mkk3-1 and MKK3-EE on ½MS medium containing different concentrations of ABA. Results are the mean of three biological experiments (n > 50 for each biological experiment).
(b) Pictures of 7-day-old germination of indicated genotypes on 1 and 3 lM ABA.
(c) Root growth inhibition of WT (Col-0), mkk3-1 and MKK3-EE seedlings on ½MS plates containing 1 lM ABA. Results are the mean of four biological experiments (32 > n > 12 for each biological experiment).
Asterisks indicate statistical differences compared with WT under the same conditions based on t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
234 Agyemang Danquah et al.
MKK3 acts upstream of the C group MAPKs in protoplasts
Previous reports showed that MKK3 acts upstream of the C
group MAPK MPK7 and interacts with the 4 MAPKs of this
clade (Doczi et al., 2007; Lee et al., 2008). MKK3 was also
published to act upstream of MPK6 and MPK8 (Takahashi
et al., 2007, 2011; Sethi et al., 2014). To identify which of
the six MAPKs were activated by MKK3, we expressed
these MAPKs as HA-tagged proteins in mesophyll protoplasts prepared from mkk3-1 leaves in the presence or
absence of a Myc-tagged constitutively active MKK3
(MKK3-EE). After MAPK immunoprecipitation with an antibody against the HA epitope, the activity of the MAPKs
was assayed by their ability to phosphorylate the heterologous substrate Myelin Basic Protein (MBP) (Figure 2a). The
activity of MPK2, MPK7, MPK14 and in a lesser extend
MPK1 was observed only when co-expressed with MKK3EE. In contrast, MPK6 and MPK8 showed a detectable basal
activity in mkk3-1 protoplasts that was not significantly
affected by MKK3-EE. These results confirm that MKK3 is
an activator of the C group MAPKs.
C group MAPKs are activated by ABA in an MKK3dependent manner
MPK1 and MPK2 were shown to be activated by ABA in
planta (Ortiz-Masia et al., 2007; Umezawa et al., 2013). To
test whether other C group MAPKs could be activated by
ABA, we expressed MPK1, MPK2, MPK7 and MPK14 as HAtagged proteins in WT Col-0 mesophyll protoplasts and
subjected them to ABA treatment before performing kinase
assays with the HA-immunoprecipitated MAPKs (Figure 2b). The four kinases were activated by ABA after 1 h.
In order to evaluate whether the ABA-dependent activation
of the MAPKs was mediated by MKK3, we used mkk3-1
mesophyll protoplasts to express HA-tagged MPK7 with
(or without) upstream WT Myc-tagged MKK3 and performed an ABA treatment. When MPK7 was co-expressed
(c)
(a)
(e)
(b)
(d)
Figure 2. ABA-dependent activation of C group MAPKs by MKK3.
(a) Kinase activity of HA-immunoprecipitated MAPKs expressed in mkk3-1 mesophyll protoplasts in the presence or absence of a constitutively active MKK3
(MKK3-EE). Western blots show protein expression levels.
(b) Kinase activity of HA-immunoprecipitated MAPKs expressed in Col-0 mesophyll protoplasts with ABA (30 lM, +) or mock ethanol (–) treatment for 1 h. Western blots show MAPK protein expression levels.
(c) Kinase activity of HA-immunoprecipitated MPK7 expressed with or without MKK3 in mkk3-1 mesophyll protoplasts before (T0) and after ABA (30 lM) or mock
(ethanol) treatment for 1 h. Western blots show protein expression levels.
(d) Kinase activity of MPK7 after immunoprecipitation using an anti-MPK7 specific antibody from Col-0 and mkk3-1 seedlings treated with ABA (50 lM) and mock
(ethanol) for indicated times.
(e) qRT-PCR analysis of MPK7 transcript accumulation in response to 50 lM ABA for 6 h in indicated genetic backgrounds. Transcript accumulation is expressed
relatively to ACTIN 2 transcript levels used as a reference. Data are the mean standard error (SE) of three biological replicates.
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
ABA-activated MAPK cascade in Arabidopsis 235
with MKK3, its activity was stimulated by ABA (Figure 2c).
Importantly, in the absence of MKK3, no MPK7 kinase
activity was detectable even in the presence of ABA,
although MPK7 was equally expressed in all samples. Similarly, the other MAPK members of the C clade (MPK1,
MPK2 and MPK14) were also activated by ABA in an
MKK3-dependent manner (Figure S2a). This ABA-dependent MAPK activation correlated with the increased phosphorylation of the TEY motif in the MAPK activation loop,
suggesting that MKK3 activated the MAPKs through TEY
phosphorylation, as expected in a functional MAPK module (Figure S2b).
In order to test whether MPK7 is activated by ABA in
planta, we used an anti-MPK7 specific antibody (Doczi
et al., 2007) to immunoprecipitate the endogenous MPK7
kinase and measure its activity on MBP. In vitro grown
10-day-old Col-0 and mkk3-1 plantlets were treated with
50 lM ABA for 2, 4 or 6 h. In WT, we observed an
ABA-dependent increase of MPK7 activity that was totally
abolished in mkk3-1 mutant (Figure 2d). As this slow ABA
activation of MPK7 could result from an ABA-dependent
transcriptional induction of MPK7, we performed qRT-PCR
analysis in WT and mkk3-1 backgrounds upon ABA treatment. MPK7 gene expression was indeed up-regulated by
ABA, reaching a maximum of expression at 4 h (Figures 2e
and S3). Interestingly, this transcriptional regulation was
independent of MKK3 as it still occurs in the mkk3-1
mutant background (Figure 2e). This suggests that MPK7
activation by ABA can be uncoupled from the transcriptional regulation of the MPK7 gene. All together, these
results identify MKK3 as the critical MAP2K that activates
MPK7 in response to ABA.
pairs of clade III MAP3Ks suggested that these genes might
function in a redundant manner. To confirm the ABA-triggered expression of MAP3K17 and 18, we performed qRTPCR analysis on 2-week-old in vitro grown Col-0 plantlets
after treatment with 50 lM ABA (Figure 3a,b). ABA induced
a strong accumulation of MAP3K17 and MAP3K18 transcripts peaking at 4 h. Similar results but with lower intensity were obtained when plantlets were subjected to
300 mM mannitol or 150 mM NaCl (Figure S6a,b).
The main ABA signalling cascade, referred to as the core
signalling module, consists of the ABA receptor, an SnRK2
kinase and a PP2C phosphatase (Fujii et al., 2009; Ma et al.,
2009; Park et al., 2009). To test whether the ABA-dependent
expression of MAP3K17 and MAP3K18 is regulated
(a)
(b)
The ABA core signalling module is required for MAP3K17
and MAP3K18 expression
We then attempted to identify the MAP3K(s) which could
act upstream of MKK3 in the ABA signalling pathway. We
hypothesised that such kinases could be transcriptionally
regulated by ABA and performed a bioinformatic analysis
using publicly available transcriptomic databases. Among
the 20 Arabidopsis MEKK-like MAP3K genes, MAP3K17
(At2g32510) and MAP3K18 (At1g05100) showed induction
by ABA and osmotic stresses (Figure S4), suggesting that
they could act as ABA signal transducers in the context of
abiotic stresses. A phylogenetic analysis of the Arabidopsis
MEKK-like genes showed that the MEKK family is organised in three distinct subclades (Figure S5). MAP3K17 and
MAP3K18 belong to a poorly characterized atypical subset
of eight members (referred to as clade III): this clade
encodes rather short kinases with small carboxy-termini.
Interestingly, clade III genes do not have any introns
whereas MEKK-like kinases from clades I and II always
have more than eight introns and mostly code for larger
proteins (Figure S5). Moreover, the sequence similarity of
(c)
(d)
Figure 3. MAP3K17 and MAP3K18 genes are transcriptionally regulated by
ABA and abiotic stresses.
(a, b) qRT-PCR analysis of MAP3K17 (a) and MAP3K18 (b) transcript accumulation in response to 50 lM ABA, in WT Col-0.
(c, d) qRT-PCR analysis of MAP3K17 (c) and MAP3K18 (d) transcript accumulation after 1.5 h treatment with 100 lM ABA in 10-day-old seedlings of WT
and mutants of the ABA core signalling.
Transcript accumulation is expressed relatively to ACTIN 2 transcript levels
used as a reference. Data are the mean standard error (SE) of three biological replicates.
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
236 Agyemang Danquah et al.
through this pathway, we performed ABA treatments on
the quadruple mutant of the ABA receptor, pyr1pyl1pyl2pyl4, and the hab1G246D line expressing a dominant ABI11-like mutation of the HAB1 phosphatase (Robert et al.,
2006; Park et al., 2009) (Figure 3c,d). The ABA-triggered
accumulation of MAP3K17 and MAP3K18 transcripts
observed in Col-0 plantlets was almost abolished in the
two ABA-insensitive genotypes. Similar results were
observed when plants were treated with 150 mM NaCl (Figure S6c,d). These results show that ABA- and salt-induced
MAP3K17 and MAP3K18 expression depends on the ABA
core signalling module.
MAP3K17 and MAP3K18 physically interact with MKK3 in
yeast and in planta
To investigate whether MAP3K17 and MAP3K18 could be
upstream activators of MKK3, we first tested if these kinases could interact with MKK3 in yeast. We performed yeast
two-hybrid experiments using the MAP3Ks fused to the
GAL4 Activation Domain (AD) and the 10 MAP2Ks fused to
the GAL4 Binding Domain (BD). Yeast co-transformed with
MKK3 and the two MAP3Ks were the only combinations
able to grow on selective media (Figure S7a). To confirm
that MAP3K17/18-MKK3 interactions also occur in planta,
we used Bimolecular Fluorescence Complementation
(BiFC) assays. MKK3 was fused to the C-terminal half of
YFP (YFPC) and MAP3K17 and MAP3K18 to the N-terminal
half of YFP (YFPN) before expression in pair-wise combinations in agro-infiltrated leaves of Nicotiana benthamiana.
Using confocal microscopy, we observed a functional cytoplasmic YFP signal in epidermal cells (Figure S7b). As positive and negative controls, we took advantage of the
(a)
specific interaction between MKK2 and MPK4 which occurs
largely in the same compartment (Gao et al., 2008). In our
expression system, MKK2 interacted with MPK4 but not
with MAP3K17 or MAP3K18 and MPK4 did not interact with
MKK3 (Figure S7b). All together, these results suggest that
the two MAP3Ks specifically interact with MKK3 in planta.
MAP3K17/18-MKK3-MPK7 defines a functional MAPK
cascade in vivo
To test whether the interaction detected between
MAP3K17, MAP3K18 and MKK3 in yeast and in N. benthamiana is biologically functional for the activation of
MKK3, we co-expressed tagged versions of MAP3K17,
MAP3K18 and MKK3 with MPK7-HA in mkk3-1 mesophyll
protoplasts. MPK7-HA was immunoprecipitated and its
activity was assayed in vitro on MBP. In contrast with
MKK3-EE, WT MKK3 alone was barely able to activate
MPK7 (Figure 4a), suggesting that MKK3 needs to be first
activated by an upstream component in the MAPK cascade. When MKK3 was expressed with either MAP3K17 or
MAP3K18, MPK7 became highly active, indicating that
MAP3K17 and MAP3K18 are indeed functional upstream
activators of the MKK3 pathway. As MPK7 activation did
not occur when MKK3 was omitted or replaced by another
MAP2K, MKK1 (Figure S8), the MAP3K-dependent activation of MPK7 is specifically mediated by MKK3. Taken
together, these results indicate that MAP3K17/18-MKK3MPK7 constitutes a functional MAPK signalling module in
Arabidopsis mesophyll protoplasts.
To test whether the MAP3K17/18-MKK3 module could
act upstream of other MAPKs, we co-expressed MAP3K18
and MKK3 with six HA-tagged MAPKs in mkk3-1 mesophyll
Figure 4. MAP3K17 and MAP3K18 functionally regulate MKK3 and C group MAPKs.
(a) Kinase activity of HA-immunoprecipitated MPK7
expressed in mkk3-1 mesophyll protoplasts in the
presence or absence of MKK3 (WT, +, or constitutively active, EE), MAP3K17 and MAP3K18.
(b) Kinase activity of HA-immunoprecipitated
MAPKs expressed in mkk3-1 mesophyll protoplasts
in the presence or absence of MKK3 and MAP3K18.
Western blots show protein expression levels.
(b)
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
ABA-activated MAPK cascade in Arabidopsis 237
protoplasts. The MAPKs were immunoprecipitated and
assayed for kinase activity on MBP (Figure 4b). As with
MPK7, a stronger activity was observed for MPK1, MPK2
and MPK14 when co-expressed with both MKK3 and
MAP3K18, whereas these MAPKs expressed alone or with
only one of the two upstream kinases displayed barely
detectable activities. MPK6 was also more active in the
presence of MAP3K18 but independently of MKK3
co-expression, suggesting that MAP3K18 activates MPK6
via other MAP2Ks. In contrast, MPK8 activity was not
affected by MAP3K18 or MKK3 expression (Figure 4b).
These results indicate that the MAP3K17/18-MKK3 module
can activate all members of the C group MAPKs MPK1,
MPK2, MPK7 and MPK14.
ABA-induced activation of the MKK3-MPK7 module
depends on MAP3K18
As ABA induces the expression of MAP3K17 and MAP3K18
(Figure 3) and constitutive expression of the MAP3Ks activates MKK3-MPK7 in protoplasts (Figure 4), we hypothesised that the ABA induction of endogenous MAP3Ks could
be sufficient to activate the MKK3-MPK7 module. To test
this hypothesis, we generated double mutant plants with
T-DNA insertions in both MAP3K17 and MAP3K18 genes
(Figure S9a). The characterization of the T-DNA insertion
sites in these lines suggested that the insertion in the
MAP3K18 gene results in a loss-of-function allele whereas
the insertion at the very end of the MAP3K17 coding region
might correspond to a knock-down as the truncated protein still possessed residual functionality in yeast (Figure S9b–d). We also complemented the map3k17map3k18
mutant with MAP3K18 fused to YFP under the control of its
own promoter (Figure S9c,e). We performed mesophyll
protoplast assays with these two genotypes to analyse the
requirement of the MAP3Ks on ABA-dependent MPK7 activation. In the map3k17map3k18 background, ABA was
unable to activate MPK7 even when co-expressed with
MKK3 (Figure 5a). Importantly, the ABA activation of MPK7
was restored in the MAP3K18–YFP complemented line, and
correlated with the accumulation of MAP3K18–YFP protein
upon ABA treatment, as observed in the immunoblot with
anti-GFP antibody (Figure 5a). These results demonstrate
that MAP3K17 and MAP3K18 mediate the ABA-dependent
activation of the MKK3-MPK7 module through ABAinduced de novo transcription and protein synthesis of the
two MAP3Ks.
ABA-triggered MPK7 activation is impaired in
map3k17map3k18 plants and blocked by the inhibition of
translation
We then wanted to confirm that the MAP3Ks function
upstream of the ABA-dependent MKK3-MPK7 pathway in
planta. In vitro grown map3k17map3k18 plantlets were
subjected to ABA treatment before immunoprecipitation of
endogenous MPK7 with the anti-MPK7 specific antibody.
As previously observed in mkk3-1 (Figure 2d), MPK7 activation was compromised, although not abolished, in
map3K17map3K18 mutant plantlets (Figure 5b). The
expression of a YFP-tagged MAP3K18 in the map3K17mapk3K18 background restored the ABA-dependent activation of MPK7 in plantlets (Figure 5c). Consistently with the
impaired ABA induction of MAP3K17/18 in the mutants of
the ABA core signalling module (Figure 3), the ABA-dependent MPK7 activation was blocked in hab1G246D plants
(Figure 5d). In addition, MPK7 activation correlates with
MAP3K18 protein accumulation induced by ABA with
respect to both its timing (Figure 5e) and its dose dependency (Figure 5f). These results confirm that the MAP3Ks
are critical upstream components for the ABA-dependent
activation of MPK7 and validate the conclusion that the
MAPK module MAP3K17/18-MKK3-MPK7 is activated by
ABA.
Whereas MPK3, MPK4 and MPK6 are activated by stresses within minutes (Ranf et al., 2011), corresponding to a
fast post-translational activation mechanism, the ABAdependent activation of MPK7 takes considerably longer
(Figure 5b,e). The slow ABA-dependent MPK7 activation is
compatible with the requirement of the de novo transcription and protein synthesis of the upstream MAP3Ks. To
test this hypothesis, we used the protein synthesis inhibitor cycloheximide on the MAP3K18–YFP complemented
line. Incubation with 100 lM cycloheximide 1 h before ABA
treatment completely abolished MPK7 activation by ABA
(Figure 5g) and was correlated with the absence of
MAP3K18 protein accumulation. Despite the fact that cycloheximide might also affect other pathways, these data support the hypothesis that MAP3K18 de novo production is a
critical step for the activation of the C group MAPKs in
response to ABA.
The MKK3 module is controlling genes involved in
response to abiotic stresses
Next, we wanted to test whether the MAPK module was
involved in ABA-mediated gene regulation. We thus performed a transcriptome analysis of Col-0 and mkk3-1
plants in mock conditions or upon treatment with 50 lM
ABA for 4 h (Figure S10a). When compared with wild-type
in resting conditions, 58 genes were shown to be up-regulated and 89 genes to be down-regulated in mkk3-1 mutant
plants, considering a two-fold change as cut-off (Figure 6a
and Table S1). Based on Gene Ontology (GO) annotation
analysis of these genes, we found enrichment in stress
response genes (Figure S10b). Conversely, among the upregulated genes, we also found an enrichment for ‘seed
development’ and ‘embryonic development ending in seed
dormancy’, suggesting that these genes might be connected to the observed germination phenotype of the
mkk3-1 mutant (Figure 1).
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
238 Agyemang Danquah et al.
(a)
(b)
(e)
(c)
(f)
(d)
(g)
Figure 5. ABA-dependent MPK7 activation requires MAP3K18.
(a) Kinase activity of HA-immunoprecipitated MPK7 expressed in mesophyll protoplasts from Col-0, map3k17map3k18 and the map3k17map3k18-MAP3K18locus-YFP
complemented line in the presence or absence of MKK3 and upon 3 h treatment with 30 lM ABA (+) or mock (ethanol, –). Western blots show protein expression
levels.
(b) Kinase activity of MPK7 after immunoprecipitation using an anti-MPK7 specific antibody from Col-0 and map3k17map3k18 seedlings treated with 50 lM ABA or
mock (ethanol) for 4 h.
(c) Kinase activity of MPK7 after immunoprecipitation with an anti-MPK7 specific antibody from seedlings of Col-0, mkk3-1, map3k17map3k18 and the map3k17map3k18-MAP3K18locus-YFP complemented line treated with 50 lM ABA (+) or mock (ethanol, –) for 4 h.
(d) Kinase activity of MPK7 after immunoprecipitation with an anti-MPK7 specific antibody from seedlings of Col-0 and hab1G246D treated with 50 lM ABA (+) or
mock (ethanol, –) for 4 h.
(e) Kinase activity of MPK7 after immunoprecipitation with an anti-MPK7 specific antibody from seedlings of the map3k17map3k18-MAP3K18locus-YFP complemented line treated with 50 lM ABA for indicated times. Western blot shows the MAP3K18–YFP accumulation in response to ABA in the complemented line.
(f) Kinase activity of MPK7 after immunoprecipitation with an anti-MPK7 specific antibody from seedlings of the map3k17map3k18-MAP3K18locus-YFP complemented line treated for 4 h with ABA at indicated concentrations. Western blot shows the MAP3K18–YFP accumulation in response to ABA in the complemented
line.
(g) Kinase activity of MPK7 after immunoprecipitation with an anti-MPK7 specific antibody from seedlings of the map3k17map3k18-MAP3K18locus-YFP complemented line treated for 4 h with 50 lM ABA (+) or mock (ethanol, –) with and without pretreatment with cycloheximide (100 lM, 1 h). Western blot shows the
MAP3K18–YFP accumulation in response to ABA in the complemented line.
© 2015 The Authors
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ABA-activated MAPK cascade in Arabidopsis 239
(a)
(b)
(c)
Figure 6. Transcriptome analysis of mkk3-1 and map3k17map3k18 after ABA treatment.
(a) Number of differentially expressed genes (two-fold change cut-off) between Col-0 and mkk3-1 in mock conditions and in response to ABA.
(b) Gene Ontology (GO) analysis of mkk3-1-dependent ABA-regulated genes. GO data of the gene list (377 genes highlighted in Figure S10) were extracted with
Agrigo GO Analysis Toolkit. Histograms of the values were produced to highlight GO enrichment of representative GO classes. P-values for each enriched class
are indicated (p-v).
(c) qRT-PCR analysis upon 6 h treatment with 50 lM ABA in the mutants of the MAPK module of six genes identified from transcriptomic experiment. Transcript
accumulation is expressed relatively to ACTIN 2 transcript levels used as a reference. Data are the mean standard error (SE) of three biological replicates.
In response to ABA, the transcriptome analysis revealed
that 377 genes displayed mkk3-1-dependent ABA induction
(Figure S10c). The GO analysis of these genes revealed
enrichment for processes mainly involved in abiotic stress
responses (Figure 6b). Using qRT-PCR of six selected
genes, we confirmed that this set of genes is regulated by
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
240 Agyemang Danquah et al.
ABA in an MKK3-dependent manner (Figure S10d). To test
whether the whole MAPK module was involved in this ABA
gene regulation, we compared the expression of the six
selected target genes in Col-0, mkk3-1, map3k17map3k18
and map3K17map3k18-MAP3K18locus-YFP plantlets upon
treatment with 50 lM ABA for 6 h (Figure 6c). We confirmed
the impaired ABA-dependent expression in mkk3-1 and
showed the same tendency, although to a lesser extent, in
map3K17map3K18 mutants, while the expression of
MAP3K18locus–YFP largely complemented the misregulation. Altogether, these results show that a set of ABA-regulated abiotic stress genes depends on the MAPK module.
DISCUSSION
The ABA core signalling pathway, composed of PYR/PYL
receptors, PP2Cs and SnRK2s (Fujii et al., 2009; Ma et al.,
2009; Park et al., 2009), controls a first and rapid wave of cellular responses, such as the stomatal pore closure through
the modulation of plasma-membrane channel activities (Kim
et al., 2010). ABA also modulates the expression of a large
set of nuclear genes, including MAP3K17 and MAP3K18
(Leonhardt et al., 2004). In this work, we report the identification of an entire MAPK cascade, composed of MAP3K17/
18, MKK3 and the C group MAPKs MPK1/2/7/14, which is
activated by ABA via the ABA core signalling module.
A MAPK module with an atypical mode of activation by
ABA
MAP3K17 and MAP3K18 belong to a monophyletic clade of
MEKK-like MAP3Ks whose functions are still unknown. We
confirmed that MAP3K17 and MAP3K18 are highly induced
by ABA (Leonhardt et al., 2004), which was compromised
in mutants of the ABA core signalling module. By yeast
two-hybrid analysis against all MAP2Ks, we identified
MKK3 as the only MAP2K interacting with MAP3K17 and
MAP3K18. MKK3 harbours a C-terminal Nuclear Transport
Factor 2 (NTF2) domain whose function is still unclear. All
currently known plant genomes contain only one gene
coding for such an MKK3-like MAP2K, indicating that this
fusion occurred early and is conserved during evolution.
MKK3 has been previously reported to regulate JA, Salmonella, ROS and blue light signalling (Doczi et al., 2007; Takahashi et al., 2007, 2011; Schikora et al., 2008; Sethi et al.,
2014) but its involvement in ABA responses has not been
reported so far, highlighting the importance of this MAP2K
in plant stress signalling. Our results show that a loss-offunction of MKK3 totally abolished ABA-dependent MPK7
activation, indicating that this MAP2K is an important actor
of the ABA-triggered MAPK pathway. MPK1 and MPK2,
which belong to the C group MAPKs, were previously
reported to be activated by ABA but the activation mechanism remained unclear (Ortiz-Masia et al., 2007; Umezawa
et al., 2013). MPK1 and MPK2 are members of the group C
MAPKs, and we show that all members of this group,
namely MPK1, MPK2, MPK7 and MPK14, are activated by
ABA in an MKK3-dependent manner. This redundant activation of four MAPKs by ABA is intriguing. One possibility
is that they have similar roles in different cell types. Alternatively, despite being activated by the same MAP2K, the
four MAPKs could phosphorylate distinct targets. This last
hypothesis is supported by the PAMP-activated MAPK
model in which MPK3, MPK4 and MPK6 were shown to
have both specific and shared targets (Frei dit Frey et al.,
2014). Identifying the targets of each MAPK and studying
the single mapk mutant phenotypes will help to solve
these questions. Previous studies have shown that MKK3
also regulates MPK6 and MPK8 activities (Takahashi et al.,
2007; Schikora et al., 2008). Although we could not confirm
these data in our assays, it is likely that MKK3 can activate
MPK6 or MPK8 under other culture conditions or developmental stages.
Interestingly, we found that activation of the ABA-triggered MAPK pathway depends on the transcription and protein synthesis of the MAP3Ks MAP3K17 and MAP3K18. As
far as we know, such an activation mechanism has not been
reported for a MAPK module neither in plants nor in animals, adding a level of complexity to MAPK signalling. Classical activation of MAPKs occurs within minutes upon
stress stimulation, as observed for MPK3/4/6 in response to
flg22 (Ranf et al., 2011), and involves post-translational
modifications (PTMs) such as phosphorylation. Although
we observed a slow and long-lasting activation of MPK7 by
ABA, this does not exclude the possibility that MAP3K17
and MAP3K18 are additionally activated through PTMs.
Importantly, mutations in the ABA core signalling SnRK2s
abolished ABA activation of MPK1 and MPK2 (Umezawa
et al., 2013), confirming our data that the ABA core machinery is required for the activation of the MAP3K17/18-MKK3MPK1/2/7/14. Interestingly, previous studies reported faster
kinetics for ABA-dependent MPK1 and MPK2 activation,
which could be more compatible with a protein synthesisindependent mechanism (Ortiz-Masia et al., 2007; Umezawa et al., 2013). A possible explanation for this apparent
discrepancy could be that different developmental and
experimental conditions might precondition ABA-triggered
MKK3-dependent MAPK activation. Future experiments will
have to show whether the transcriptional upregulation of
the MAP3Ks might also be part of a feed forward loop to
produce a long-term signal that is required for a persistent
adaptation response to the prevailing stress conditions.
The MAP3K17/18-MKK3-MPK7 cascade modulates ABA
responses
We showed that mkk3-1 knock-out plants are hypersensitive to ABA during germination and root elongation, while
the constitutively active 35S-MKK3-EE line was insensitive. By contrast, Hwa and Yang (2008) reported ABA
hypersensitivity of estradiol-inducible MKK3-EE lines.
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
ABA-activated MAPK cascade in Arabidopsis 241
These opposite results may come from differences in the
biological material or the technical setup of the experiments. Importantly, map3k17map3k18 displayed enhanced
ABA inhibition of root growth, although not as strong as
mkk3-1 mutant (Figure S11). This result is consistent with
the observation that ABA-triggered MPK7 activation is
totally abolished in the mkk3-1 background but only
reduced in map3k17map3k18 mutants. However, the
map3k17-1 T-DNA insertion line does not correspond to a
total loss-of-function allele and can thereby explain the
weaker phenotype. An alternative possibility that we cannot exclude is that other MAP3Ks, such as MAP3K19, could
redundantly act upstream of MKK3.
In an attempt to decipher what the downstream targets
of the ABA-triggered MAPK pathway are, we performed a
transcriptome analysis of mkk3 mutant plants in the
absence or presence of ABA. In mock conditions, we
observed a general enrichment for genes involved in stress
responses (Figure 6). However, the down-regulated genes
in mkk3-1 showed a specific role in defense responses,
which is consistent with previous results demonstrating
that mkk3-1 is more susceptible to bacterial infection (Doczi et al., 2007). On the other hand, the set of up-regulated
genes in mkk3-1 plants was enriched for genes involved in
seed development and more specifically in ‘embryonic
development ending in seed dormancy’ (Figure 6), in correlation with the germination phenotype observed for
mkk3-1 and MKK3-EE in response to ABA and abiotic stresses. In response to ABA, our transcriptome analysis
revealed a set of 377 ABA-regulated MKK3-dependent
genes with functions enriched in response to different
abiotic stimuli such as osmotic stress or temperature
variation (Figure 6). The ABA induction of those genes
was abolished or reduced in mkk3-1 and map3K17-
map3K18 mutants, while the complementation with the
MAP3K18locus–YFP in the double mutant background
restored the ABA responsiveness.
ABA is also known to be the major hormone involved in
abiotic stress signalling. Coherently, mkk3-1 knock-out
plants were also hypersensitive to germination under salt
and osmotic stress conditions (Figure S1). Interestingly,
dehydration was shown to trigger MPK7 activation and
MAP3K17/18 expression in an ABA-dependent manner,
which was correlated with higher water loss in mkk3-1 and
map3k17map3k18 mutants when subjected to a 10-day
drought treatment (Figure S12). Taken together, these
results suggest that the MAPK module functions in ABA
signalling in response to multiple abiotic stresses.
CONCLUSIONS
The discovery of an ABA-activated MAPK pathway completes our understanding of the ABA signalling in plants.
Our results support the concept (Figure 7) that the ABA core
signalling pathway controls a first and rapid wave of cellular responses, including a large transcriptional reprogramming (Sirichandra et al., 2010; Yoshida et al., 2010), that
precedes a second wave of late responses involved in stress
adaptation. Our data indicate that MAP3K17 and MAP3K18
belong to the set of genes that are regulated by the ABA
core signalling module, resulting in long-term activation of
C group MAPKs by MKK3. This MAPK module might
thereby contribute to generate a robust signal to induce
ABA-dependent responses in persisting stress conditions.
In support of this hypothesis, recent phosphoproteomic
studies revealed a number of ABA-induced phosphosites in
transcription factors and protein kinases among the potential MAPK targets (Umezawa et al., 2013; Wang et al., 2013),
including various receptor-like kinases, the protein kinases
AKIN10, PHOT1, PHOT2, SnRK2.10 and the transcription
factors ZIGA4, GT2, GTL1, GRF4, RAP2.7 and WRKY18.
Clearly, the phosphorylation of these protein kinases and
transcription factors could contribute to regulate changes in
the physiology and metabolism under long-lasting stress
conditions. Analysis of the role of this ABA-activated MAPK
module and its target proteins should provide valuable
information on the complex mechanism of plant stress
adaptation and should help to enhance crop stress tolerance that will be crucial for a future sustainable agriculture.
EXPERIMENTAL PROCEDURES
Table S2 provides the sequences of all primers used during this
work.
Plant material
Figure 7. Working model of ABA signalling network integrating the ABA
core module and the MAP3K17/18-MKK3-MPK1/2/7/14 pathway.
Details of the pyr1pyl1pyl2pyl4, hab1G246D, mkk3-1 and MKK3-EE
lines have been published previously (Nambara and Marion-Poll,
2005; Robert et al., 2006; Doczi et al., 2007; Park et al., 2009).
map3k17-1 (Salk_137069) and map3K18-1 (Gabi_244G02) were
identified from a publicly available T-DNA collection.
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
242 Agyemang Danquah et al.
The MAP3K18 locus upstream (3017 bp) or downstream (683 bp)
of the STOP codon was amplified using iProof (Bio-Rad, http://
www.bio-rad.com/) from Col-0 genomic DNA using a specific
3K18Locus5F/3K18Locus5R and 3K18Locus3F/3K18Locus3R pair of
primers. the YFP ORF was amplified with the primer set (YFP_F/
YFP_R) from pEXCS-GW-mYFP-nls (kindly provided by Dr Jane Parker). PCR fragments were cloned into the pGEMTeasy vector (Promega, http://worldwide.promega.com/) and, using KpnI, PstI and
NotI restriction enzymes, the MAP3K18 locus was reassembled in
the pGREEN0229 vector (Hellens et al., 2000) to generate the
pGREEN0229-MAP3K18locus. Then, the YFP DNA fragment was
cloned into the unique PstI site to build the pGREEN0229MAP3K18locus–YFP. The vector was transformed into Agrobacterium tumefaciens strain C58C1 containing pSOUP helper plasmid
(Hellens et al., 2000). Kanamycin-resistant Agrobacteria were then
used to transform map3k17-1map3k18-1 Arabidopsis plants using
the floral dip method (Clough and Bent, 1998). Segregation analysis
was performed to identify an homozygous line for both insertions.
Germination assay and root elongation assay
For in vitro culture, seeds were sterilized in 70% ethanol, 0.05%
sodium dodecyl sulphate (SDS) (10 min) washed twice in 96%
ethanol and dried before use. Seeds were sown on half-strength
Murashige and Skoog medium (½MS) plates and stratified for
3 days at 4°C before being transfered to a growth chamber (16 h
light, 8 h night, 22°C).
For germination assays, the ½MS plates contained either ethanol (ABA mock), ABA (Sigma, http://www.sigmaaldrich.com/),
NaCl (Sigma) or mannitol (Sigma). Germination was considered
as occurring when a radicle was detectable under the stereomicroscope.
For root growth assays, the ½MS plates contained either 0.002%
ethanol (mock) or 1 lM ABA. Growth was performed vertically for
7 days to facilitate the measurement of root length using IMAGEJ
software after scanning the plates (http://imagej.nih.gov/ij/).
Gene expression analysis
For ABA treatment, plantlets were grown in vitro, in 6-well plates
containing 4 ml ½MS 0.9% agar for 9 days. Then, 4 ml liquid ½MS
were added to the plants that were equilibrated for 24 h before
treatment with 0.2% ethanol, 50 lM ABA (Sigma), 150 mM NaCl or
300 mM mannitol. Plants were collected at indicated times and frozen in liquid nitrogen. RNA extraction with DNase treatment was
performed using the RNeasy plant mini kit and RNase-Free DNase
Set following manufacturer’s recommendations (Qiagen, https://
www.qiagen.com). RNA quantification and quality was measured
in a NanoDrop spectrophotometer (Thermo Scientific, http://
www.thermoscientific.com/). cDNAs were synthesized from 1 lg
total RNA using oligo(dT) and SuperScript II reverse transcriptase
(Invitrogen, http://www.lifetechnologies.com/) according to the
manufacturer’s instructions. qPCR reactions were carried out in
10 ll final volume with 1 ll of 10 times diluted reverse transcription (RT) reaction, 100 nM final concentration of each primer and
SYBR FAST Universal qPCR kit (Kapa Biosystems, http://
www.kapabiosystems.com/). ACTIN2 was used as a reference
gene. All reactions were done in a CFX384 TouchTM Real-Time PCR
Detection System (BIO-RAD) as follows: 95°C for 30 sec; 409 (95°C
for 5 sec and 60°C for 20 sec); and a dissociation step to validate
the PCR products. All reactions were performed with three technical replicates.
For transcriptomic analysis, the F2 progeny from an mkk3-1
backcrossed in Col-0 were used to minimize the effect of putative
side mutations. Col-0 (MKK3/MKK3) and mkk3-1 seeds (>50 per
condition) were grown in 25 ml ½MS, 1% sucrose (16 h light, 8 h
night, 70% humidity, 22°C) for 12 days. Plantlets were then treated
with ABA (50 lM) or mock (ethanol) for 4 h. Samples were collected in liquid nitrogen and grinded. Three independent biological replicates were produced. RNAs were extracted using Qiagen
kits as above. Microarray analysis was carried out using the CATMAv7 array based on AGILENT technology (Methods S4).
Gateway cloning to generate pDONR vectors
PCR reactions were performed using iProof (Bio-Rad). ORFs were
amplified from cDNA (if introns) or genomic DNA and recombined
in pDONR207 using the Gateway technology (Invitrogen),
following the protocol kindly provided by Lurin and co-workers
(http://www-urgv.versailles.inra.fr/atome/protocols.htm). Chimeric
ORFs as predicted from map3k17-1 and map3k18-1 loci were
amplified with 3K17_ORF_F/3K17_ORFtr_R and 3K18_ORF_F/
3K18_ORFtr_R couples of primers, respectively, and map3k17-1
map3k18-1 DNA as amplification matrix. Clones with and without
a stop codon were identified allowing N- and C-terminal protein
fusions and carefully sequenced.
Kinase assays using mesophyll protoplasts
Protoplasts from the different backgrounds were isolated and
transfected as previously reported (Yoo et al., 2007), using three
types of effector constructs: MAP3K–YFP, MKK-Myc and MPK-HA.
Constructs for MKK3-Myc and MKK3-EE-Myc were described previously (Doczi et al., 2007). To generate MPK-HA constructs,
MPK1, MPK2, MPK6, MPK7, MPK8 and MPK14 ORFs from
pDONR207 vector were recombined in pHaGWF7 using LR
enzyme mix (Invitrogen). For MAP3K–YFP, MAP3K17 and
MAP3K18 ORFs from pDONR207 vector were recombined in
p2FGW7. Typically, 0.4 ml protoplasts at a dwww.gehealthcare.com/ensity of 2 9 105/ml were transfected with 50 lg total DNA
including the three effectors constructs (MAP3K–YFP, MKK-Myc
and MPK-HA) in equal amount. Protoplasts were collected 14 h
after transfection. For ABA treatment, transfected protoplasts were
incubated for 14 h for protein expression before treatment with
30 lM ABA or mock ethanol for 3 h. For kinase assays, proteins
were extracted in immunoprecipitation buffer [50 mM Tris/HCl pH
7.5, 5 mM EDTA, 5 mM EGTA, 50 mM b–glycerophosphate, 10 mM
NaF, 1 mM orthovanadate, cocktail anti-protease 19 (Roche, http://
www.roche.com/)], 2 mM DTT, 1% Triton X100, 150 mM NaCl). The
protein extract was recovered after centrifugation at 20 000 g at
4°C for 5 min. The MPK-HA was immunoprecipitated with 1 ll
anti-HA antibody (Sigma) at 4°C for 2 h, and an additional 1 h
after addition of 20 ll of 50% slurry protein A-sepharose (GE
Healthcare, www.gehealthcare.com/). The immunoprecipitates were
washed three times in immunoprecipitation (IP) buffer and twice
in 20 mM Tris/HCl pH 7.5, 15 mM MgCl2, 5 mM EGTA, 1 mM DTT.
The kinase assay was performed for 30 min at RT in 15 ll reaction
buffer (20 mM Tris/HCl pH 7.5, 15 mM MgCl2, 5 mM EGTA, 1 mM
DTT, 50 lM cold ATP) supplemented with 2 lCi [c-33P]ATP and
1 lg MBP per reaction. The reaction was stopped with SDS loading buffer, heated at 95°C for 5 min and separated on 15% SDSPAGE. The radioactive MBP was detected on dried gels using a
Phosphor imaging system (GE Healthcare). The expression level
of epitope-tagged proteins was monitored by immunoblots with
anti-HA, anti-Myc and anti-GFP antibodies on the same transfected
protoplasts directly extracted with SDS loading buffer.
Kinase assays using plantlets
For ABA-induced MAPK activation, plantlets were grown vertically
on ½ MS plates containing 1% sucrose (16 h light, 8 h night, 22°C)
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244
ABA-activated MAPK cascade in Arabidopsis 243
for 12 days. Plantlets were transferred in liquid medium for
equilibration. Then plantlets were treated with 0.1% ethanol
(mock) or 50 lM ABA (Sigma). To stop treatments, plantlets were
rapidly dried and frozen in liquid nitrogen.
For drought experiment, plantlets were grown in ½ MS plates
containing 1% sucrose, for 15 days (16 h light, 8 h night, 22°C).
Then, the plates were unsealed and equilibrated under a fume
hood overnight. Drought stress was performed by removing the
lid. In these conditions, the medium loses 50% of its water in
5 h. Kinase assays were performed as previously described
(Ortiz-Masia et al., 2007). Briefly, samples were homogenized
using a TissueLyser II (Qiagen) and proteins were extracted in
extraction buffer (25 mM Tris/HCl pH 7.5, 75 mM NaCl, 5 mM
EDTA, 5 mM EGTA, 5 mM DTT, 20 mM b-glycerophosphate,
20 mM NaF, 0.05% Triton, cocktail anti-protease 19 [Roche]). The
protein extracts were recovered after centrifugation at 20 000 g
at 4°C for 5 min and normalized to a concentration of 4 lg ll 1
using Bradford. For immunoprecipitation, 300 lg of proteins
were incubated with 7 ll anti-MPK7 antibody (Doczi et al., 2007)
and 20 ll of 50% slurry protein A-sepharose (GE Healthcare) for
2 h at 4°C. The immunoprecipitates were washed twice with the
extraction buffer and once with kinase buffer (30 mM Tris/HCl pH
7.5, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 1 mM DTT, 20 mM b-glycerophosphate). The kinase assay was performed for 30 min at
RT in 15 ll reaction buffer (kinase buffer supplemented with
100 lM cold ATP, 1 lg ll 1 MBP and 2 lCi [c-33P]ATP per reaction). The reaction was stopped with SDS loading buffer, heated
at 95°C for 5 min and separated on 15% SDS-PAGE. The radioactive MBP was detected on dried gels using a Phosphor imaging system (GE Healthcare). When necessary, the expression
level of MAP3K18–YFP was monitored by immunoblots with
anti-GFP antibody.
ACKNOWLEDGEMENTS
We thank Sean Cutler, Sylvain Merlot and Jane Parker for kindly
providing hab1G246D line, pyr1pyl1pyl2pyl4 mutant and pEXCSGW-mYFP-nls plasmid, respectively. This work was supported by
the Institut National de Recherche Agronomique and the Agence
Nationale de la Recherche. AD was supported by the Ghanaian
Ministry of Education.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article.
Figure S1. MKK3 regulates germination on salt and mannitol.
Figure S2. C-group MAPKs are activated by ABA in an MKK3dependent manner.
Figure S3. Expression level of MPK1, MPK2, MPK7, MPK14 and
MKK3 in response to ABA.
Figure S4. Expression level of 20 MEKK-like MAP3K genes upon
treatment with ABA (a), drought (b) or salt (c).
Figure S5. Phylogenic analysis of the family of Arabidopsis MEKKlike MAP3Ks.
Figure S6. MAP3K17 and MAP3K18 genes are transcriptionally
regulated by abiotic stresses.
Figure S7. MAP3K17 and MAP3K18 interact with MKK3 in yeast
and in planta.
Figure S8. MKK3, but not MKK1, mediates MAP3K18-triggered
activation of MPK7.
Figure S9. Characterization of map3k17-1 and map3k18-1 mutations.
Figure S10. Transcriptomic analysis of mkk3 mutant upon ABA
treatment.
Figure S11. ABA hypersensitivity of map3k17map3k18 mutant.
Figure S12. Drought activation of MAPK module and droughtrelated phenotypes of mkk3-1 and map3k17map3k18 mutants.
Table S1. Lists of deregulated genes studied in the manuscript.
Table S2. Sequence of the primers used in the work.
Methods S1. Yeast two-hybrid.
Methods S2. BiFC experiment.
Methods S3. Relative Water Content measurements.
Methods S4. CATMAv7 microarray design, hybridization and Statistical Analysis.
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© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 232–244