Download Induction of CDPK (ZmCPK11)- and MAPK

Maize calcium-dependent protein kinase (ZmCPK11): local and systemic
response to wounding, regulation by touch and components of jasmonate
signaling
Jadwiga Szczegielniak*, Lidia Borkiewicz, Blanka Szurmak, Elżbieta LewandowskaGnatowska, Małgorzata Statkiewicz, Maria Klimecka, Jarosław Cieśla, Grażyna Muszyńska
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02106 Warsaw, Poland
*Corresponding author, e-mail: [email protected]
Tel. 48-22-5925717
Fax: 48-22-6584636
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Abstract
Expression of ZmCPK11, a member of the maize (Zea mays L.) Calcium-Dependent Protein Kinases
(CDPKs) family, is induced by mechanical wounding. A rapid increase of the activity of a 56-kDa
CDPK has been observed in damaged leaves. In the present work, it is shown that the 56-kDa CDPK,
identified as ZmCPK11, is also activated in non-wounded leaves as an element of systemic wound
response. Moreover, an increase of the enzyme’s activity and induction of ZmCPK11 expression was
observed after touching the leaves. To study the role of ZmCPK11 in wound and touch signaling,
transgenic Arabidopsis thaliana plants in which c-Myc-ZmCPK11 was expressed under control of the
CaMV 35S promoter were generated. Analysis of the transgenic plants showed that c-Myc-ZmCPK11
was activated upon wounding and touching. Furthermore, pretreatment with acetylsalicylic acid
(acSA), an inhibitor of jasmonic acid (JA)-dependent wound signaling, abolished the wound-induced
activation of ZmCPK11 in maize and the transgenic A. thaliana plants. Methyl jasmonate (MeJA) and
linolenic acid (LA) stimulated the activity of ZmCPK11 as well as induced the expression of
ZmCPK11 and other wound-responsive genes, lipoxygenase 1 (ZmLOX1) and proteinase inhibitor 1
(ZmWIP1). These results indicate that ZmCPK11, regulated at the enzymatic and transcriptional level
by LA and MeJA, is a component of touch- and wound-induced pathway(s), participating in early
stages of local and systemic responses.
Abbreviations: ABA, abscisic acid; acSA, acetylsalicylic acid; BSA, bovine serum albumin; CDPK,
calcium dependent protein kinase; DTT, dithiothreitol; JA, jasmonic acid; LA, linolenic acid; MAPK,
mitogen activated protein kinase; MeJA, methyl jasmonate; OPDA, 12-oxo-phytodienoic acid; PA,
phosphatidic acid; SA, salicylic acid; VOC, volatile organic compound.
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Introduction
In nature, plants are continuously exposed to numerous stresses, including mechanical wounding
caused by wind, rain, hail or herbivore attack. Wounding not only physically damages tissues affecting
the growth and reproduction of plants, but also provides gates for pathogen invasion. Plants respond to
wounding in the tissues directly damaged (local response) as well as in undamaged leaves (systemic
response) located distal to the wounded sites. An essential role in these defense responses play
hormones such as jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), ethylene (ET), and
other signaling molecules such as oligosaccharins and volatile organic compounds (VOCs) emitted by
plants (Schilmiller and Howe 2005, Koornneef and Pieterse 2008). JA, its precursors (e.g., 12-oxophytodienoic acid OPDA) and derivatives (e.g., methyl jasmonate MeJA) collectively referred to as
jasmonates, belong to the oxylipins (oxygenated polyunsaturated fatty acids). Jasmonates, synthesized
from -linolenic acid (LA, 18:3) via the octadecanoid pathway in response to mechanical wounding,
herbivore attack and necrotrophic pathogens (Wasternack 2007, Koo and Howe 2009), induce
expression of a wide range of defense genes (Reymond et al. 2000, Memelink 2009). For example,
insect feeding affects hundreds of transcripts in Arabidopsis; 67 - 84% of them are under the control of
the JA pathway (Reymond et al. 2004). Mutants deficient in JA biosynthesis and JA signaling show an
increased susceptibility to certain fungal pathogens and insects (Browse 2009). It has been
demonstrated that JA is responsible for the systemic response, functioning as a long-distance signal
molecule transmitting the information about wounding to distant, non-wounded tissues where a
defense response is invoked (Schilmiller and Howe 2005).
In response to a variety of stimuli, including wounding and JA treatment, spatial and temporal
changes in the concentration of cytosolic and nuclear free calcium (Ca2+), referred to as calcium
signature, are observed (Kudla et al. 2010). It has been shown that inhibitors of calcium channels
affect negatively the expression of JA-induced genes (León et al. 1998, Sun et al. 2006). These results
indicate an essential role of the changes in cytosolic Ca2+ concentration in JA signaling. The
information encoded by the Ca2+ signatures is decoded by Ca2+-binding sensor proteins. Among them,
Calcium Dependent Protein Kinases (CDPKs) are particularly important because they act as both
calcium sensors due to their ability to bind calcium by their calmodulin-like domain, and as effectors
exhibiting protein kinase activity (Harper et al. 2004). Upon Ca2+ binding to the calmodulin-like
domain, conformational changes occur in the protein and cause the release of the pseudosubstrate
autoinhibitor from the active site and activation of the CDPKs (Klimecka and Muszyńska 2007).
CDPKs are involved in many functions including responses to different stresses such as cold, drought,
high salinity and wounding (Klimecka and Muszyńska 2007, Xu et al. 2010, Asano et al. 2011).
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Mechanical wounding of Z. mays leaves stimulates the activity of a 56-kDa CDPK. Using protein
sequence data obtained from purified Z. mays 56-kDa CDPK the corresponding cDNA was cloned.
The clone was designated as ZmCPK11. Expression of ZmCPK11 was found to be induced by
wounding not only in injured but also in non-injured neighboring leaves (Szczegielniak et al. 2005).
The physiological role of an individual protein is usually studied using transgenic plants with
altered expression of its gene. Because of the difficulties of genetic manipulations in maize,
heterologous transformation of Z. mays genes in A. thaliana is frequently used. For example,
expression of the Z. mays calcineurin B-like protein (ZmCBL4) in A. thaliana confers salt tolerance
(Wang et al. 2007), whereas expression of a Z. mays transcription factor (ZmDREB2A) enhances stress
tolerance to drought and heat shock (Qin et al. 2007). Expression of another Z. mays transcription
factor ZmMYB42 in A. thaliana affected the cell wall structure and composition (Sonbol et al. 2009).
Here, the transgenic A. thaliana plants expressing c-Myc-ZmCPK11 were generated to monitor the
kinase activity. The effect of stresses and signaling molecules on the transcription and enzymatic
activity of ZmCPK11 was analyzed for elucidation of the involvement of this kinase in touch and JAdependent wound signaling.
Materials and methods
Generation of transgenic Arabidopsis thaliana plants expressing 35S:c-Myc-ZmCPK11
To construct 35S:c-Myc-ZmCPK11, PCR was performed on the previously cloned ZmCPK11 cDNA
(Szczegielniak et al. 2005) using Pfu DNA polymerase and the following primers: forward 5’CACCATGCAGCCGGACCCGAGCGGGAA-3’
and
reverse
5’-
GCGCTAGGTTTTGCTGGGATTCAAG-3’. The PCR product was cloned into pENTR/D-TOPO
vector (Invitrogen, www.invitrogen.com) and verified by DNA sequencing. Then the ZmCPK11
construct was recombined using Gateway® cloning system (Invitrogen) into the binary vector
pEarleyGate203 (N-c-Myc) (accession no. 1007964471) downstream of the 35S promoter of
Cauliflower mosaic virus. A. thaliana (Col-0) was transformed with the 35S:c-Myc-ZmCPK11
construct by floral dip method using Agrobacterium tumefaciens (strain GV3101) (Clough and Bent
1998). Transformants were selected based on their resistance to BASTA and the presence of the
transgene detected by PCR.
Plant material and treatments
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For stress treatments maize plants (Zea mays cv Mona) were cultivated in soil for two weeks in a
growth chamber with a daily cycle of 14 h light (70-80 watt/m2) at 24-25oC and 10 h dark at 20oC.
Transgenic A. thaliana plants expressing maize ZmCPK11 were grown in soil for four weeks in a
growth chamber under 16 h photoperiod at 22oC (day)/20oC (night). Two-week-old fully expanded
second and third leaves of maize or 4-week-old leaves of rosette stage transgenic A. thaliana plants
were subjected to one of the following treatments. Mechanical wounding was performed by rubbing
the bottom side of the lamina of Z. mays leaves with 50-grit sandpaper or by puncturing A. thaliana
leaves with forceps. Z. mays and A. thaliana plants were wounded in a different way because of the
differences in the size of the leaves. Both the wounded and upper non-wounded leaves were harvested
at the indicated time points, immediately frozen in liquid nitrogen and stored at -80oC. For analysis of
the touch response, plants were gently stroked back and forth with a gloved hand for 1 min. For LA or
acetylsalicylic acid (acSA) treatment detached Z. mays or A. thaliana leaves were stabilized for 3 to 4
h in water and then incubated with 250 μM LA or 100 μM acSA. Before use, LA was dissolved in
0.1% Triton X-100 and sonicated for 5 min with a Branson Ultrasonics Sonifier 250
(www.bransonultrasonics.com) and diluted with water to a final concentration of 250 μM LA and
0.001% Triton X-100, whereas acSA was dissolved in 96% ethanol and diluted with water to a final
concentration of 100 μM acSA and 0.001% ethanol. Control plants were incubated with 0.001% Triton
X-100 or 0.001% ethanol. At the indicated intervals, leaves were removed, immediately frozen in
liquid nitrogen and stored at -80oC. For MeJA treatment intact plants were placed in 11 L plexiglas
boxes for 12 to 15 hours, then 1 L of MeJA dissolved in 96% ethanol was applied to a paper wick
inside the box. At the indicated intervals, three or four leaves were excised, removed from the box,
and stored at -80oC. Triton-X-100 (Cat. No. T9284), α-LA (Cat. No. L2376), MeJA (Cat. No. 39,2707) and acSA (Cat. No. A5376) were obtained from Sigma-Aldrich (www.sigmaaldrich.com).
Preparation of clarified maize homogenates
Two Z. mays leaves (~ 200 mg) were ground in a mortar cooled with liquid nitrogen. The resulting
powder was suspended in 200 l of buffer containing 200 mM Tris, pH 7.5, 250 mM sucrose, 2 mM
EGTA, 2 mM EDTA, 50 mM -glycerophosphate, 100 M Na2VO4, 5 mM dithiothreitol (DTT), 1
mM phenylmethylsulfonyl fluoride (PMSF), and 16 l of Complete Protease Inhibitor Cocktail
(Roche, www.roche.com). The suspension was centrifuged for 30 min at 20000 x g and the
supernatant used for further analysis. Protein concentration was determined by the method of Bradford
(1979), using BSA as the standard.
Expression of GST:ZmCPK11 in Escherichia coli
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The expression in Escherichia coli and purification of Glutathione S-transferase (GST):ZmCPK11 was
described previously (Szczegielniak et al. 2005). The recombinant protein immobilized on glutathioneagarose beads (Amersham Biosciences, www.amershambiosciences.com) was cut off from GST with
enterokinase (Novagen, www.emdchemicals.com) which recognized the amino acid sequence “DDDK”
introduced to the construct between GST and ZmCPK11. The cleavage was carried out overnight at 4oC
on a rocker, using 3 U of enterokinase in a buffer containing 20 mM Tris, pH 7.5 and 50 mM NaCl.
ZmCPK11 protein was separated from agarose beads by centrifugation, glycerol was added to a final
concentration of 15% (v/v), then the protein was frozen in liquid nitrogen and stored at -80oC.
In–gel kinase activity assay
Procedure was performed as described previously (Szczegielniak et al. 2000, 2005) with some
modifications. Proteins (from 15 to 30 g) in clarified homogenates and protein size markers
(Fermentas, www.fermentas.com) were separated on 8% or 10% sodium dodecyl sulfate (SDS)polyacrylamide gels containing MBP (0.25 mg ml−1) as the immobilized substrate. After
electrophoresis, SDS was removed by washing the gel 3 times with 25 mM Tris, pH 7.5, containing 0.5
mM DTT, 0.1 mM Na2VO4, 5 mM NaF, 0.5 mg ml−1 BSA, and 0.1% (v/v) Triton X-100 for 30 min
each at room temperature. Next the gel was equilibrated in 25 mM Tris, pH 7.5, 5 mM NaF, 1 mM
DTT, and 0.1 mM Na2VO4 overnight with three changes of the buffer. Then, the gels were preincubated
for 30 min at 4oC in 10 ml of reaction buffer (20 mM Tris, pH 7.5, 15 mM MgCl2, 2 mM DTT,
containing 332 M Ca(CH3COOH)2 or 2 mM EGTA). Kinase activity was assayed for 1.5 h at 30oC in
the reaction buffer supplemented with 25 M ATP containing 50 Ci [32P] ATP. Unincorporated
[32P] ATP was removed by washing the gels in 5% TCA with 1% sodium phosphate. After washing,
the gels were stained with Coomassie Brilliant Blue R250 (Sigma-Aldrich), dried, and exposed to
Amersham Hyperfilm MP (Amersham Biosciences, www.amershambiosciences.com).
In-solution kinase activity assay
Kinase activity assay was performed as described previously using histone III-S (0.5 mg ml−1) as a
substrate (Szczegielniak et al. 2005) and [32P] ATP. For analysis of the effects of LA, MeJA and acSA
the compounds were added to the reaction mixture to final concentrations between 10 M and 2000
M, 1 and 200 M, and 10 and 1000 M, respectively.
Semi-quantitative RT-PCR
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Control (Col-0) and transgenic c-Myc-ZmCPK11 A. thaliana lines were analyzed by RT-PCR. RNA
isolation and RT-PCR analysis were performed as described previously (Szczegielniak et al. 2005).
Real-time PCR employing SYBR-Green chemistry
Isolation of total RNA from Z. mays leaves and synthesis of first-strand cDNA were carried out as
described previously (Szczegielniak et al. 2005). Quantitative PCR (QPCR) assays were carried out on
an Applied Biosystems 7500 apparatus. Each reaction was carried out in a mixture containing: 2x
concentrated commercial SYBR Green mix (MesaGreeen, Eurogentec, www.eurogentec.com),
forward and reverse primers 400 M each, cDNA template, and water to 25 µl of final volume. The
gene-specific primers, designed using Primer Express program (Applied Biosystems), for
amplification of ZmCPK11 (primers 1 and 2), ZmWIP1 (primers 3 and 4), ZmLOX1 (primers 5 and 6),
ZmEF-1 (primers 7 and 8), and Zm-tubulin (primers 9 and 10) are shown in Table 1 (see Appendix
S1, Supporting information). The primers were verified for equal efficiency of PCR reaction. The
conditions of QPCR were as follows: 95ºC for 3 min as initial denaturation step followed by 45 cycles
consisting of denaturation step at 95ºC for 15 s, and primer annealing and extension at 60ºC for 1 min.
Fluorescence was read during the annealing-extension step of each cycle. Melting-point temperature
analysis was performed in the range of 60ºC to 95ºC with temperature increments of 0.33ºC.
Background range and threshold for Ct evaluation in each experiment were adjusted manually. For
each cDNA sample four reactions were carried out using two template amounts, each in duplicate. For
each gene the amounts of cDNA were chosen individually (if possible the same for all genes) to obtain
Ct values in the range between 14 and 34 cycles. The quality of results was evaluated based on
expected Ct differences between two cDNA amounts as well as the product melting curves. Rare
outlying results were omitted in calculations. The resulting data were processed using files exported
from the QPCR cycler program and imported to Excel sheets and the ratios between the target and
reference gene expression levels were calculated. For each duration of treatment mean values (from 2
or 3 experiments) were calculated and normalized to time 0. These results were then normalized to the
expression of Zmα-tubulin (L27815) and ZmEF-1 (NM_001112117) mRNAs.
Antibodies and immunoblotting
Initial attempts to identify and monitor ZmCPK11 activation involved the use of specific antibodies.
Antibodies were produced against 16-meric peptides representing specific N-terminal and C-terminal
regions of ZmCPK11 (BioGenes, www.biogenes.de). These antibodies were able to recognize
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recombinant ZmCPK11. However, trials to identify this kinase in Z. mays extracts failed, most probably
due to a very low concentration of this protein. To circumvent this obstacle, the recombinant c-MycZmCPK11 was expressed in A. thaliana and immunoblot analysis of the expressed tagged kinase was
performed as described previously (Szczegielniak et al. 2005) using anti-c-Myc antibodies 9E10 diluted
1:10000 (Santa Cruz Biotechnology, www.scbt.com).
Immunoprecipitation
Leaves of Z. mays or transgenic A. thaliana were ground in a mortar cooled with liquid nitrogen. The
resulting powder was suspended in immunoprecipitation buffer (IP) containing 20 mM Tris, pH 7.5,
150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 100 μM Na3VO4, 50 mM βglycerophosphate, 2 μM DTT, 0.5 mM PMSF, and 16 l of Complete Protease Inhibitor Cocktail/200
l IP buffer (Roche). Z. mays protein extract (100 μg) was incubated with 2 μg anti-phospho-tyrosine
[anti-(P)Tyr] antibodies 4G10 (Millipore, www.millipore.com) at 4°C for 4 h on a rocking platform.
Protein A/G-agarose suspension 25 μl, (Santa Cruz Biotechnology) was added and the incubation
continued for 2 h. In the case of A. thaliana, anti-c-Myc mouse antibodies (~2 μg) were first bound to
protein A-agarose (Santa Cruz Biotechnology) and then this complex was incubated with the A.
thaliana protein extract (150 μg) at 4°C for 4 h. The agarose bead-protein complexes [with anti-(P)Tyr
or anti-c-Myc antibodies] were pelleted by brief centrifugation and washed four times with the IP
buffer and once with 20 mM Tris, pH 7.5. Next, sample buffer was added and the suspension heated at
95°C for 3 min. After a brief centrifugation, the supernatant was divided into two parts. One part was
analyzed by in-gel kinase assay, the second one by immunoblotting.
All experiments presented in this paper were repeated at least three times with similar results.
Results
Heterologous system study of ZmCPK11 in transgenic Arabidopsis thaliana plants
Our previous data indicate that the 56-kDa CDPK, whose molecular weight corresponds to the
ZmCPK11 calculated mass, is activated in response to wounding. Monitoring of the activity of
ZmCPK11 in response to stresses or treatment with LA or MeJA was not possible in Z. mays because
of a lack of specific antibodies against ZmCPK11 that could allow its identification. The antibodies
raised against specific peptides from the N- and C-termini of ZmCPK11 failed to detected this protein
kinase in Z. mays extracts, and generation of transgenic Z. mays plants is practically not possible at our
conditions. For the above reasons heterologous transformation was used and ZmCPK11 was expressed
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in A. thaliana. The full-length cDNA (1530 bp) for ZmCPK11 tagged with c-Myc was introduced into
A. thaliana in the sense orientation under the control of the CaMV 35S promoter. The c-Myc tag
allowed to follow the activity of ZmCPK11 and its protein amount during stress conditions using antic-Myc antibodies. Four independent A. thaliana transgenic lines were selected for further study.
Molecular analysis showed that ZmCPK11 was expressed in leaves of these transgenic lines at both
RNA and protein levels (see Appendix S2 in Supporting Information).
Maize 56-kDa CDPK is ZmCPK11
Identification of the 56-kDa protein kinase as ZmCPK11 was based on the facts: first, that observed
molecular mass of the 56-kDa kinase corresponds to the calculated mass of ZmCPK11; second, cMyc-ZmCPK11 expressed in A. thaliana showed the same responses to wounding, touch, and
signaling molecules (LA and MeJA) as the endogenous Z. mays enzyme; and third, the cDNA
encoding ZmCPK11 was isolated using protein sequence data obtained by mass spectrometry from
previously purified 56-kDa CDPK (Szczegielniak et al. 2005).
Wounding activates ZmCPK11 locally and systemically
Our previous study reported that ZmCPK11 was activated in wounded Z. mays leaves (Szczegielniak
et al. 2005). Here, it was examined whether the ZmCPK11 was also activated in maize in neighboring
non-wounded leaves. After wounding, a protein kinase was indeed activated within a few minutes in
the presence of Ca2+ in both the injured and neighboring Z. mays leaves. In the presence of a calcium
chelator (5 mM EGTA) the activity of this protein kinase was abolished confirming that it was a
CDPK. Coomassie Brilliant Blue staining of the gels has shown the equal amounts of the proteins in
the studied samples (Fig. 1A).
In addition to the above, both in the presence and in the absence of Ca2+ a 45-kDa kinase activity
was detected in wounded and neighboring Z. mays leaves (Fig. 1A). Because activation by stresses
such as wounding is a characteristic feature of MAPKs, it was checked whether the observed Ca2+independent, wound-induced 45-kDa kinase was a MAPK. To identify the phosphorylated active form
of this kinase immunoprecipitation with anti-(P)Tyr antibodies was performed (Ichimura et al. 2000).
The 45-kDa MAP kinase activity was detected in the anti-(P)Tyr immunoprecipitates from wounded
leaves, but not in those from the control ones (Fig. 1B). This result confirmed that maize leaves
responded properly to the applied stress.
In wounded leaves of A. thaliana plants expressing c-Myc-ZmCPK11 the recombinant kinase was also
activated within a few minutes. Immunoblot analysis showed that the level of c-Myc-ZmCPK11
9
protein was the same in the non-wounded and wounded leaves (Fig. 1C), indicating that the increase
of the kinase activity was a consequence of activation of the preexisting enzyme upon wounding.
LA and MeJA affect activity and expression of ZmCPK11
LA and MeJA play a role in the wound response not only as components of the octadecanoid pathway,
but also directly as effective inducers of defense responses (Farmer and Ryan 1992, Koch et al. 1999,
Halitschke et al. 2001, Memelink 2009). Therefore, the effects of wound-related signaling molecules
(LA and MeJA) on ZmCPK11 activity and expression were analyzed.
The protein kinase activity was determined in 2-week-old Z. mays leaves and 4-week-old
transgenic A. thaliana plants treated with 250 M LA for various times. Both the ZmCPK11 and the
45-kDa MAPK were activated by LA in Z. mays leaves (Fig. 2A). Activation of ZmCPK11 was
biphasic, peaking at 15 min and again at 1 h. As shown in Fig. 2B, c-Myc-ZmCPK11
immunoprecipitated from transgenic A. thaliana plants, like the ZmCPK11 in Z. mays leaves (Fig.
2B), was activated by LA. In both systems a transient activation of ZmCPK11 was detected 1 h after
the treatment. In contrast to the native system, there was no activation of c-Myc-ZmCPK11 after 15
min. It is possible that the first peak of ZmCPK11 activation with maximum 8 min after treatment with
18:3 (data not shown) is specific for the homologous Z. mays system, whereas the second peak of the
kinase activation with maximum 1 h after LA treatment is common for CDPKs from different plants.
Equal amounts of the protein were loaded per lane on the gel what is shown by staining the gel in
Coomassie Brilliant Blue (Fig. 2A) and by immunoblot analysis with anti-c-Myc antibodies (Fig. 2B),
what indicates that the increased activity was achieved without a change in the protein level.
Also, LA affects the in vitro activity of recombinant ZmCPK11. The activity of ZmCPK11 was
stimulated by LA in a dose-dependent manner (Fig. 2C).
The transcript levels of ZmCPK11 and two wound-responsive genes (ZmLOX1 and ZmWIP1) were
analyzed in 2-week-old Z. mays leaves treated with 250 M LA for various times. ZmLOX1 is a
member of the type 1-LOX subfamily with 9- and 13-LOX activities that is induced by MeJA and
wounding (Kim et al. 2003), whereas ZmWIP1 is serine proteinase inhibitor also induced by
wounding, which inhibits proteinase activity in pest midgut (Moeller and Tiffin 2008). Therefore, we
have selected ZmLOX1 and ZmWIP1 as a positive control of wounding. The levels of all three
transcripts were affected by exogenous LA in a similar manner. The levels of all transcripts increased
several-fold (4 to 10) with almost parallel biphasic kinetics with the maxima at 3-6 h and 24 h of
treatment (Fig. 2D).
The kinase activity of ZmCPK11 was analyzed in Z. mays and transgenic A. thaliana leaves
treated with MeJA for various times. In both plants the transient activation of ZmCPK11 was detected
10
after 1 h of MeJA treatment. In the Z. mays and A. thaliana system the increased activity of ZmCPK11
was achieved without a change in the protein level, as shown by staining the gel in Coomassie
Brilliant Blue (Fig. 3A) and by immunoblotting with a c-Myc-specific antibodies (Fig. 3B). The in
vitro effect of MeJA on ZmCPK11 activity was studied. The activity of recombinant ZmCPK11, using
histone III-S as a substrate, was not changed in the presence of MeJA at concentrations from 10 to
1000 M (data not shown).
It should be noted that treatment of Z. mays plants with MeJA have been performed in airtight
chambers as it was described in Material and methods and also using open flow system in which
detached Z. mays leaves were incubated in water and supplemented with 100 μM MeJA (data not
shown). Applying two different MeJA treatments of Z. mays leaves gave the same results in activation
of ZmCPK11. In both cases the ZmCPK11 was activated slowly with a maximum at 1 h.
The effect of MeJA on transcripts levels was unlike that of LA. Although all three genes were
induced by MeJA, the degree and kinetics of the observed induction were gene-specific and followed
different course. After MeJA treatment the ZmCPK11 transcript level increased 2.5-fold, peaking at 6
h, while that of ZmWIP1 increased ca. 60-fold, with a maximum at 36 h. The kinetics was biphasic for
ZmLOX1 with a 10-fold increase at 3 h and a 120-fold one at 36 h (Fig. 3C). Similar results for
ZmLOX1 were obtained by Kim et al. (2003).
Control experiments showed that 0.001% Triton X-100 and 0.001% ethanol did not affect the level
of ZmCPK11 activity and expression (data not shown).
acSA affects wound-activated ZmCPK11
It was previously shown that in wound signaling SA is antagonistic towards JA. SA and its acetylated
form (acSA) inhibit the expression of wound-responsive proteinase inhibitors probably by negatively
affecting the expression of JA biosynthesis enzymes (Leon-Reyes et al. 2010). To determine the
involvement of ZmCPK11 in the JA-dependent wound signaling pathway, the activity of protein
kinases was examined in Z. mays leaves and the transgenic A. thaliana plants treated with acSA prior
to wounding. The protein kinases were strongly activated within minutes after wounding. However,
when acSA was applied prior to wounding the ZmCPK11 activation in Z. mays leaves as well as the cMyc-ZmCPK11 activation in transgenic A. thaliana leaves was practically abolished (Fig. 4A and B).
Staining the gels in Coomassie Brilliant Blue and immunoblot analysis confirmed that equal amounts
of protein were loaded per lane on the gel (Fig. 4A and B).
The activity of recombinant ZmCPK11 was not changed in the presence of acSA at concentrations
from 10 to 1000 M (data not shown).
11
Touch affects activity and expression of ZmCPK11
Touch affects plant growth and development and also stimulates defense response against herbivores
(Braam 2005). Therefore, the question raised whether touch, a non-injuring stress weaker than
wounding also influenced the activity and transcript level of ZmCPK11.
ZmCPK11 activity was analyzed in protein extracts from touched leaves of Z. mays (Fig. 5A) and
transgenic A. thaliana plants expressing c-Myc-ZmCPK11 (Fig. 5B). Transient activation of
ZmCPK11 and c-Myc-ZmCPK11 was detected a few minutes after touching (Fig. 5A and B). Equal
amounts of the protein were loaded per lane on the gel what is shown on Fig. 5A (staining the gel in
Coomassie Brilliant Blue) and Fig. 5B (immunoblot analysis with anti-c-Myc antibodies).
Additionally, activation of the 45-kDa MAPK was observed (Fig. 5A). It was previously shown that in
A. thaliana two multifunctional MAPKs, AtMPK4 and AtMPK6, were rapidly activated by touch
(Ichimura et al. 2000).
The transcript level of ZmCPK11 increased after touch, reaching the highest level at 6 h (4-fold
increase), and then decreased to the basal level. The transcript level of ZmLOX1 increased quickly,
reaching the maximum 3 h after stress. In contrast to ZmCPK11 and ZmLOX1, the ZmWIP1 transcript
level increased in a biphasic manner with maxima 10 and 30 h after touch (Fig. 5C).
Discussion
Plants have evolved varied defense responses that allow them to survive mechanical damage caused
by herbivores or other agents such as adverse weather conditions. These responses are observed not
only in the tissues directly damaged but also in non-wounded areas.
In wounded Z. mays leaves, activity of a 56-kDa CDPK and expression of ZmCPK11 was
stimulated. Moreover, the transcript level of ZmCPK11 increased in non-injured neighboring leaves
(Szczegielniak et al. 2005). Presently, we have shown that wounding activates within minutes the 56kDa CDPK, identified as ZmCPK11, also in neighboring leaves. This is the first report of a rapid
activation of a CDPK as an element of systemic wound response. Activation of CDPK can be caused
by: (i) elevation of calcium level; (ii) phosphorylation of CDPK by an upstream kinase, and/or its
autophosphorylation; and (iii) change of subcellular localization and binding of membrane lipids
(Witte et al. 2010). It is possible that a rapid increase of cytoplasmic Ca2+ caused by wounding
activates ZmCPK11. ZmCPK11 present in Z. mays leaves seems to be a specific sensor and decoder of
calcium signaling in stress conditions.
The presence of activated ZmCPK11 in leaves of nominally non-stressed plants was sometimes
observed, probably due to handling of the plants during the experiment. The kinase activity was
12
usually about 10-30% of that in leaves of plants subjected to wounding (data not shown), Therefore,
we checked whether touch, a weaker stress not leading to plant injury, could also elicit the expression
and activity of ZmCPK11. Indeed, touch rapidly and transiently activated ZmCPK11 in Z. mays and
transgenic plants expressing c-Myc-ZmCPK11.
Touch, like wounding, stimulates the kinase activity and expression of ZmCPK11. Another CDPK
transcriptionally regulated by touch is AtCPK32 identified in ethylene-insensitive etr1-3 mutant plants
(Chotikacharoensuk et al. 2006). The positively regulated transcription of the wound- and JAinducible genes (ZmLOX1 and ZmWIP1) by touch reported by us is consistent with the results reported
by Tretner et al. (2008), showing a link between touch, accumulation of JA and gene expression
induced by JA.
The crucial role of the JA signaling pathway in wound response led us to investigate whether
ZmCPK11 is involved in JA-dependent wound signaling. Therefore, the effect of known wound
signaling molecules (MeJA and LA) on this kinase activity was determined. Treatment of Z. mays and
transgenic A. thaliana plants (expressing c-Myc-ZmCPK11) with LA activates ZmCPK11.
Additionally, LA activates recombinant ZmCPK11 in vitro in a dose-dependent manner suggesting a
possible direct interaction between ZmCPK11 and LA. It has previously been shown that the activity
of recombinant ZmCPK11 as well as native kinase isolated from Z. mays seedlings is stimulated in
vitro by phosphatidic acid (PA), phosphatidylserine (PS) and phosphatidylinositol (PI) (Szczegielniak
et al. 2000, 2005). Recently, we have shown that wound-induced activation of ZmCPK11 is dependent
on PA produced in the phospholipase D (PLD) pathway and that direct interaction of PA with
ZmCPK11 is possible (Klimecka et al. 2011). Those results suggest that the activity of ZmCPK11
might be regulated by lipids produced during stress conditions. Treatment of Z. mays plants with LA
activates also the 45-kDa MAPK. Early studies indicated that LA interacts with and inhibits the
activity of PP2C-type protein phosphatases (Baudouin et al. 1999, Schweighőfer et al. 2004), which
are negative regulators of MAPKs (Schweighőfer et al. 2007, Seo et al. 2007). Because unsaturated
fatty acids are liberated from chloroplast membrane lipids upon wounding (Ryu and Wang 1998),
inhibition of PP2C by LA might contribute to maintaining phosphorylated MAPK, and possibly
CDPKs (ZmCPK11), in an active state.
JA and its volatile methyl ester (MeJA) are elicitors of almost the same defense responses, which
applied exogenously regulate the expression of wound-responsive genes and induce emission of VOCs
(Koch et al. 1999, Pauw and Memelink 2005). The expression and activity of StCDPK2 from potato is
down-regulated by JA (Ulloa et al. 2002). Conversely, MeJA is known to induce the expression of
other CDPKs: NtCDPK1 in Nicotiana tabacum (Yoon et al. 1999), wound-responsive LeCDPK1 in
tomato leaves (Chico et al. 2002), and two CDPKs in rice (Akimoto-Tomiyama et al. 2003). The
transcript level of ZmCPK11 and marker wound-responsive genes (ZmLOX1 and ZmWIP1) increased
after LA and MeJA treatment with different kinetics and magnitudes. Exogenously applied LA and
13
MeJA were able to activate ZmCPK11, but in our experimental conditions the activation by MeJA was
much slower and weaker than that by LA. One can speculate that these differences are due to the
different uptake of these compounds, however, it is not the case because different modes of treatment
gave the same results (see Material and methods and Results). Tamogami et al. (2008) have
demonstrated that airborne MeJA activates defense responses in plants by converting itself into JA and
bioactive JA-Ile, which initiates defense response by emissions of VOCs and induction of endogenous
JA-Ile and JA-Leu biosynthesis. Thines et al. (2007) showed that JA-Ile, but not JA, MeJA or their
precursor OPDA are bioactive signals for certain COI1-dependent defense responses. Taking into
consideration the fact that JA and MeJA are not endogenously bioactive molecules it is possible that
the postponed effect of MeJA on ZmCPK11 activity is due to its conversion to bioactive jasmonates. It
may also explain why MeJA did not directly activate the recombinant ZmCPK11.
LA released from the chloroplast membrane lipids can be converted enzymatically into many
different oxylipins by various branches of LOX pathways and also by non-enzymatic pathways
(Mueller and Berger, 2009). The various oxylipins derived from LA are known to accumulate and
induce expression of numerous genes (Ribot et al. 2008, Wang et al. 2008). Moreover, in lima bean
two signaling molecules of the octadecanoid pathway, OPDA and JA, induced the synthesis of
different volatiles, and LA and its conjugates with Gln or Ile mimicked the action of OPDA (Koch et
al. 1999). The above results suggest that exogenously applied LA and MeJA induce a similar, but not
identical, signaling pathways in maize leaves and therefore lead to different responses.
To confirm the participation of ZmCPK11 in JA-dependent wound signaling, the effect of acSA on
the wound-activation of ZmCPK11 was studied. acSA, a negative regulator of JA-dependent wound
signaling (Leon-Reyes et al. 2010) does not influence the activity of ZmCPK11 in vitro. However,
acSA treatment of Z. mays or transgenic A. thaliana plants inhibited the wound-activated ZmCPK11
indicating that this kinase is involved in JA-dependent wound signal transduction pathway and it may
function down-stream of the octadecanoid pathway. The time course of ZmCPK11 activation in Z.
mays and A. thaliana (within minutes) is correlated with the increase in jasmonates level in A. thaliana
leaves (within 2 to 5 minutes) in response to wounding (Glauser et al. 2008).
Although it is now becoming evident that CDPKs are components of the wound-induced responses
in plants, their function is not elucidated yet. Interestingly, Sugiyama et al. (2000) have demonstrated
that a 52-kDa CDPK from Ventricaria ventricosa (Chlorophyta) participates in the cytoskeleton
rearrangements leading to the healing of the wounded alga. In this alga, the cytoplasm at the site of
injury forms an aggregation ring whose contraction leads to wound healing. It has been shown that the
contraction of the aggregation ring and formation of an f-actin bundle are suppressed by application of
antibodies against CDPK or by CDPK inhibitors. In this context it is interesting to speculate that the
rapid and transient activation of ZmCPK11, located in the cytoplasm (Szczegielniak et at. 2000), may
be involved in early stages of stress signal transduction in which changes in cytoskeleton organization
14
are possible. AtCPK4 and AtCPK11, the closest homologues of ZmCPK11, which both have
cytoplasmic and nuclear localization (Dammann et al. 2003, Rodriguez Milla et al. 2006), are able to
phosphorylate in vitro two ABA-responsive transcription factors, ABF1 and ABF4 (AREB2) and
positively regulate ABA signaling (Zhu et al. 2007). AtCPK11 also interacts with and phosphorylates
in vitro another transcription factor, AtDi19 (Rodriguez Milla et al. 2006). A recent comprehensive
study of A. thaliana CPKs showed that AtCPK4 and AtCPK11 are also specific regulators of MAMP
(microbe-associated molecular patterns)-mediated innate immune responses (Boudsocq et al. 2010).
Also OsCPK24, the closest homologue of ZmCPK11 in rice, is induced by rice blast pathogen (Wan
et al. 2007). Recently, two A. thaliana CPKs, AtCPK3 and AtCPK13, have been identified as
compounds of herbivore response signaling (Kanchiswamy et al. 2010).
Mechanical damage of leaves can mimic the wounding caused by herbivores. It has been shown
that wounding caused by a mechanical caterpillar (MecWarm) is sufficient to induce synthesis of
volatiles with a pattern similar to that caused by true herbivore attack (Mithöfer et al. 2005).
Moreover, microarray studies in A. thaliana showed that the majority of herbivore-inducible
transcripts were also up-regulated by mechanical wounding (Reymond et al. 2000). Because
ZmCPK11 is regulated by MeJA, LA and wounding we can speculate that ZmCPK11 may be
primarily involved in the early stages of the defense response to a herbivore attack.
In the present work we have demonstrated that ZmCPK11 participates in very early events of local
and systemic response to mechanical wounding and in response to touch. The regulation of expression
and enzymatic activity of ZmCPK11 by signaling molecules of the octadecanoid pathway suggests
that this CDPK is an important component of JA-dependent wound response leading to plant defense
against stresses. It is also worth emphasizing that the insertion of maize CPK11 into A. thaliana will
allow utilization of this heterologous model for analysis of the functions of biologically active
components such as jasmonates/oxylipins.
Acknowledgements
This work was funded by the Ministry of Science and Higher Education (grants no N N303 340835 to
JSz and N N303 306737 to GM). We are grateful to Professor Jan Fronk (Fuculty of Biology,
University of Warsaw) for critical reading of the manuscript, and Dr. Arkadiusz Ciesielski
(Department of Plant Biochemistry, Institute of Biochemistry and Biophysics PAS, Warsaw) for
everyday help.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Appendix S1. Primers for QPCR.
Appendix S2. Molecular analysis of transgenic Arabidopsis lines expressing c-Myc-ZmCPK11.
Figure legends
Fig. 1. Calcium-dependent and -independent protein kinase activities in leaves of maize and transgenic
A. thaliana after wounding. (A) Activity of protein kinases in wounded and non-wounded Z. mays
leaves. Protein extracts from leaves were analyzed by in-gel kinase assay in the presence of Ca2+ or
EGTA. The level of analyzed protein was monitored by staining with Coomassie Brilliant Blue. (B)
Identification of phosphorylated form of MAPK. Protein extracts from non-wounded (time 0) and
wounded (10 min after wounding) Z. mays leaves were immunoprecipitated with anti-(P)Tyr
antibodies and the resulting immunocomplexes were analyzed by in-gel kinase assay. (C) Activity of
c-Myc-ZmCPK11 in transgenic A. thaliana leaves non-wounded and 10 min after wounding. c-MycZmCPK11 was immunoprecipitated with anti-c-Myc antibodies and the immunocomplexes were
analyzed by in-gel kinase assay in the presence of Ca2+ and by immunoblotting with anti-c-Myc
antibodies. For details see Materials and methods section.
Fig. 2. Effect of LA on protein kinase activities and transcript level of wound-responsive genes in
leaves of Zea mays and transgenic Arabidopsis thaliana plants. (A) Protein kinase activities in protein
extracts from Z. mays leaves treated with 250 M LA. (B) Activity of c-Myc-ZmCPK11
immunoprecipitated from protein extracts of transgenic A. thaliana leaves treated with LA and
immunoblot analysis with anti-c-Myc antibodies. (C) Effect of LA on activity of recombinant
20
ZmCPK11. Activity of ZmCPK11 was measured in solution using histone III-S as a substrate at
different concentrations of LA (from 10 to 2000 M). Controls without LA (0), without LA but with
0.001% Triton X-100 (C). (D) Time-course of ZmCPK11, ZmLOX1 and ZmWIP1 expression in Z.
mays leaves after treatment with LA. Transcript level of genes was analyzed by QPCR. For details see
Materials and methods section and legend to Fig. 1.
Fig. 3. Effect of MeJA on protein kinase activities and transcript level of wound-responsive genes in
leaves of Zea mays and transgenic Arabidopsis thaliana plants. (A) Activity of ZmCPK11 in protein
extracts from leaves of Z. mays plants treated with MeJA. (B) Activity of c-Myc-ZmCPK11
immunoprecipitated from protein extracts of transgenic A. thaliana leaves treated with MeJA and
immunoblot analysis with anti-c-Myc antibodies. (C) Time-course of ZmCPK11, ZmLOX1 and
ZmWIP1 expression in Z. mays leaves after treatment with MeJA. For details see Materials and
methods section and legend to Figs. 1 and 2.
Fig. 4. Effect of acSA on wound-induced protein kinase activities in leaves of Zea. mays and
transgenic Arabidopsis thaliana plants. (A) Influence of acSA on wound-induced ZmCPK11 activity
in Z. mays leaves. Leaves of 2-week-old Z. mays plants were treated with 100 M acSA 3 h prior to
wounding. (B) Influence of acSA on wound-induced c-Myc-ZmCPK11 activity in transgenic A.
thaliana leaves and immunoblot analysis with anti-c-Myc antibodies. Leaves of 4-week-old transgenic
A. thaliana plants were treated with 100 M acSA 3 h prior to wounding. For details see Materials and
methods section and legend to Fig. 1.
Fig. 5. Effect of touch on protein kinase activities and transcript level of wound-responsive genes in
leaves of Zea mays and transgenic Arabidopsis thaliana plants. (A) Kinase activities in protein
extracts from Z. mays leaves after touching. (B) Activity of c-Myc-ZmCPK11 immunoprecipitated
from protein extracts of transgenic A. thaliana leaves after touching and immunoblot analysis with
anti-c-Myc antibodies. (C) Time-course of ZmCPK11, ZmLOX1 and ZmWIP1 expression in Z. mays
leaves after touching. For details see Materials and methods section and legend to Figs. 1 and 2.
21