When a plant cell is challenged by a well-defined stimulus, complex signal transduction pathways are activated to promote the modulation of specific sets of genes and eventually to develop adaptive responses. In this context, protein phosphorylation plays a fundamental role through the activation of multiple protein kinase families. Although the involvement of protein kinases at the plasma membrane and cytosolic levels are now well-documented, their nuclear counterparts are still poorly investigated. In the field of plant defence reactions, no known study has yet reported the activation of a nuclear protein kinase and/or its nuclear activity in plant cells, although some protein kinases, e.g. MAPK (mitogen-activated protein kinase), are known to be translocated into the nucleus. In the present study, we investigated the ability of cryptogein, a proteinaceous elicitor of tobacco defence reactions, to induce different nuclear protein kinase activities. We found that at least four nuclear protein kinases are activated in response to cryptogein treatment in a time-dependent manner, some of them exhibiting Ca2+-dependent activity. The present study focused on one 47 kDa protein kinase with a Ca2+-independent activity, closely related to the MAPK family. After purification and microsequencing, this protein kinase was formally identified as SIPK (salicyclic acid-induced protein kinase), a biotic and abiotic stress-activated MAPK of tobacco. We also showed that cytosolic activation of SIPK is not sufficient to promote a nuclear SIPK activity, the latter being correlated with cell death. In that way, the present study provides evidence of a functional nuclear MAPK activity involved in response to an elicitor treatment.

INTRODUCTION

Plant cells challenged by stimuli develop adaptive responses based on the modulation of the expression of specific sets of genes whose activities are tightly controlled by signalling cascades. However, with the exception of transcription factors, little information is available regarding the signalling events operating in the nucleus [1]. Indeed, the possibility that the nucleus displays specific transduction processes has been poorly investigated. One of the most demonstrative studies highlighting the ability of the nucleus to perceive and to manage a specific response according to the applied stimulus came from Pauly et al. [2]. Using isolated nuclei from Nicotiana tabacum BY2 cells expressing the nuclear Ca2+ reporter aequorin, these authors showed that plant cell nuclei are able to generate their own Ca2+ signals independently of the changes in Ca2+ concentration occurring in the cytosol. These results point out the ability of nuclei to perceive extranuclear stimuli and to convert them into an appropriate nuclear response. A related and still poorly understood process concerns the molecular mechanisms linking cytosolic events and the modulation of nuclear gene expression. In other words, the question of how a specific signal is transmitted from the cytosol to the nucleus remains largely unanswered.

Protein phosphorylation/dephosphorylation is a major prototypic post-translational modification regulating protein function [3]. Based on animal studies, it is commonly assumed that once activated, plant nuclear PKs (protein kinases) could modulate the activity of target proteins, including transcription factors, leading, eventually, to the up- or down-regulation of gene expression. However, whereas various studies predict a nuclear localization for several PKs based on primary sequence analysis (that is the presence of a nuclear localization site [4,5]) and/or the nuclear localization of overexpressed chimaeric GFP (green fluorescent protein)–PK [6], the possibility that the activity of these PKs is modulated within the nucleus, as well as their putative translocation from the cytosol to the nucleus in physiological contexts, have been examined in only a few cases [7,8].

Cryptogein is a 10 kDa proteinaceous elicitor secreted by the oomycete Phytophthora cryptogea, an avirulent pathogen of tobacco. Upon application to tobacco leaves, cryptogein triggers the expression of defence-related genes, a HR (hypersensitive response) and a SAR (systemic acquired resistance) against various pathogenic micro-organisms [9]. The molecular mechanisms underlying the effects of cryptogein have been mainly investigated using tobacco cell suspensions (for a review see [10]). These studies highlight a key role for PKs in transducing the cryptogein signal. Notably, Lecourieux-Ouaked et al. [11] demonstrated that the elicitor induced, within 5 min, the phosphorylation of approx. 20 polypeptides, this process being partly controlled by a primary Ca2+ influx across the plasma membrane. The identity of these polypeptides has not been reported. Cryptogein was also shown to trigger the activation of cytosolic MAPKs (mitogen-activated PKs), including WIPK (wound-induced PK), SIPK [SA (salicylic acid)-induced PK] and Ntf4 [1215], as well as the activation of a 42 kDa PK operating downstream of anion effluxes [16]. Accordingly, PK inhibitors efficiently suppressed cryptogein-induced events including ion fluxes, ROS (reactive oxygen species) and RNS (reactive nitrogen species) production and gene expression (reviewed in [10,13]). A role for SIPK and WIPK in the elicitor-induced defence responses has been tentatively assigned. Zhang et al. [15] reported that staurosporine and K-252a, two serine/threonine kinase inhibitors that blocked WIPK activation, suppressed cell death, suggesting a role for WIPK in HR development. A similar role for SIPK was also postulated on the basis that the overexpression in tobacco plants of NtMEK2 {Nicotiana tabacum MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]}, a MKK (MAPK kinase) catalysing the phosphorylation and subsequent activation of different MAPKs including SIPK, led to the development of spontaneous necrosis [17]. Complementary results were obtained by Liu et al. [18] and Menke et al. [18a], who showed that WRKY1, a transcription factor involved in the control of cell death, was in vitro phosphorylated by SIPK; however, the conclusions drawn from these experiments require attention. First, a main problem facing these studies is the current lack of drugs capable of selectively acting on one specific PK. Secondly, the hypothesis that NtMEK2 activates PKs distinct from SIPK also involved in HR should not be ruled out. Finally, it should be mentioned that a recent study showed that NbSIPK, the Nicotiana benthamiana homologue of SIPK, is located in the nucleus [19]. In vitro analysis indicated that NbSIPK might be up-regulated by the upstream nuclear MKK NbMKK1 (N. benthamiana MKK1) which mediates Phytophthora infestans INF1 elicitor-mediated HR and non-host resistance to Pseudomonas cichorii. The demonstration that NbSIPK is indeed one of the downstream targets of NbMKK1 and is activated at the nuclear level in this biological model remains to be shown.

In the present study, we analysed the ability of cryptogein to trigger the activation of nuclear PKs and partly addressed the question of the identity of these PKs. We demonstrated that cryptogein activates a set of different nuclear PKs showing distinct kinetics of activation and biochemical properties in term of Ca2+-dependence. One of these PKs was firmly identified as SIPK. Finally we provided evidence that the activation of SIPK at the nuclear level is correlated with cell death.

MATERIALS AND METHODS

Cell culture and treatment

N. tabacum cv. Xanthi cell suspensions were cultivated as previously described [20]. Briefly, cell suspensions were maintained in Chandler's medium [21] on a rotary shaker (150 rev./min at 25 °C) under continuous light (photon flux rate 30–40 μmol·m−2·s−1). Cells were maintained in the exponential phase and subcultured 1 day prior to use. Treatment of cell suspensions was carried out directly in the culture medium.

Cryptogein was purified as previously described [22], and prepared as a 100 μM stock solution in water. Freeze-dried OGs (oligogalacturonates; with degrees of polymerization from 25 to 30) were provided by S.A. Goëmar and resuspended in water. All chemicals were purchased from Sigma–Aldrich. Sorbitol, SA and BA (butyric acid) were prepared in water. Gadolinium chloride (Gd3+) and cPTIO [carboxy-PTIO (carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide)] were prepared as stock solutions in water, except that cPTIO was dissolved immediately prior to use. DPI (diphenylene iodonium), glibenclamide and CHX (cycloheximide) were dissolved in DMSO. Chemicals used as inhibitors were added 10 min before elicitor treatment. When needed, equivalent volumes of DMSO or water were added to control cells to ensure that they did not interfere with the experiments. The final DMSO concentration did not exceed 0.25%. At various times, 30–40 ml of cells (typically 8–10 g of fresh weight) were harvested by filtration, quickly frozen in liquid nitrogen and stored at −80 °C until use.

Preparation of nuclear extracts

All steps were performed on ice and centrifugation steps were performed at 4 °C. Cell samples were ground in liquid nitrogen and thawed in 2 vol. of nucleus extraction buffer [NB1; 25 mM Tris/Mes (pH 7.5), 0.5 M hexylene glycol, 5 mM EDTA, 5 mM EGTA, 10 mM DTT (dithiothreitol), 10 mM NaF, 1 mM Na3VO4, 50 mM β-glycerophosphate and 1 mM PMSF]. The suspensions were filtered through a nylon mesh with a porosity of 31 μm (Sefar), and the filtrate was centrifuged at 500 g for 10 min. An aliquot (500 μl) of the supernatant was centrifuged at 21000 g for clarification and kept as the ‘cytosolic fraction’. The initial pellet was resuspended in 10 ml of NB1 and this suspension was layered on top of a 3 ml layer of 25% iodixanol (Optiprep) in NB1, and centrifuged at 3000 g for 30 min. The pellet was then washed by centrifugation at 300 g for 10 min in 15 ml of NB2 buffer (NB1 without hexylene glycol). After discarding the supernatant, the pelleted nuclei were frozen in liquid nitrogen and stored at −80 °C until use. Nucleus integrity was checked by fluorescence microscopy using a double coloration with propidium iodide and DIOC6(3) (3,3′-dihexyloxacarbocyanine iodide) as an ER (endoplasmic reticulum) dye. The nuclear-enriched fractions contamination rate was estimated by measuring the activity of marker enzymes [2325].

In-gel kinase activity assays

Nuclear proteins analysed by electrophoresis were extracted using TriReagent (Molecular Research Center), following the manufacturer's protocol. The nuclear proteins were resuspended in an urea buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS and 10 mM DTT] and diluted with at least 2 vol. of Laemmli buffer for in-gel kinase assays or Western blot experiments. In-gel kinase assays were performed as described previously [14] using 20 μg of nuclear proteins. Separating SDS/polyacrylamide gels were embedded with a PK substrate, i.e. HIIIS (histone IIIS; 0.14 mg·ml−1) or MBP (myelin basic protein; 0.12 mg·ml−1). Once the phosphorylation reaction was performed, the gels were dried on to Whatman 3MM paper and exposed to Kodak XAR-5 films. Pre-stained size markers (Fermentas) were used to estimate the apparent molecular mass of the PK.

Immunoblot analysis

Nuclear and cytosolic proteins from control and cryptogein-treated cells were submitted to SDS/PAGE (10% gels) and transferred on to nitrocellulose membranes. The membrane was blocked for 2 h at room temperature (25 °C) in TBST buffer [10 mM Tris (pH 7.5) and 100 mM NaCl containing 0.1% Tween 20] containing 1% (w/v) BSA, and then incubated overnight at 4 °C in the same buffer containing the anti-SIPK primary antibody raised against the specific SIPK N-terminal peptide [14]. After incubation and several washings with TBST, the membrane was then incubated with the appropriate HRP (horseradish peroxidase)-conjugated secondary antibodies (Bio-Rad). Detection was carried out using an ECL (enhanced chemiluminescence) Western blot detection kit (Cell Signaling Technology).

Immunoprecipitation

Nuclear proteins from untreated and cryptogein-treated cells were extracted by adding 2 vol. of lysis buffer (NB1 supplemented with 0.5 M NaCl and 0.5 MgCl2) to the nuclei. After ultracentrifugation [40000 rev./min for 1 h (70Ti rotor; Beckman)], immunoprecipitation using the nuclear proteins contained in the supernatant was carried out as previously described [26]. Immunoprecipitated proteins were analysed by in-gel kinase assays using HIIIS as a substrate, in the presence of Ca2+.

Purification of PK47 and MS sequencing

Nuclei obtained from 8 kg of tobacco cells were resuspended in 1 litre of lysis buffer (as above). After 60 min on ice, the suspension was ultracentrifuged at 40000 rev./min (70Ti rotor; Beckman) for 1 h at 4 °C. Using a 30 kDa molecular-mass cut-off centricon, the extracted nuclear proteins were concentrated in Q buffer [25 mM Tris/HCl (pH 9), 2 mM EDTA, 2 mM EGTA, 50 mM β-glycerophosphate, 25 mM MgCl2, 2 mM DTT, 5% (v/v) glycerol and 1 mM PMSF] and were loaded on to a 5 ml Q-Sepharose anion-exchange column (HiTrap Q HP column, Amersham BioSciences) equilibrated with the same Q buffer. After washing with 25 ml of buffer Q, the proteins were eluted with a 40 ml linear gradient of 0–700 mM NaCl in buffer Q. The PK47 activity was eluted with approx. 300 mM NaCl.

The fractions containing the highest PK47 activity, as determined by in-gel kinase assay using HIIIS as a substrate, were pooled and concentrated in buffer Q with 500 mM NaCl and loaded on to a 1 ml hydrophobic interaction column (HiTrap phenyl-Sepharose HP column, Amersham BioSciences) equilibrated in the same buffer. Bound proteins were eluted with a 20 ml linear gradient of 0–60% ethylene glycol and 500–0 mM NaCl in buffer Q. The PK47-active fractions (still determined using an in-gel kinase assay with HIIIS as a substrate) were then pooled and concentrated in buffer IP [50 mM Hepes/KOH (pH 7.5), 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 50 mM β-glycerophosphate, 5% (v/v) glycerol and 1 mM PMSF]. The proteins were then incubated with 20 μg of antibodies directed against SIPK and 85 μl of packed volume of Protein A–agarose beads (Sigma–Aldrich), at 4 °C for 12 h on a wheel. After washes, immunocomplexes were recovered in 30 μl of buffer IP added with 10 μl of a 5-fold-concentrated Laemmli sample buffer, and were analysed by one-dimensional electrophoresis. The gel was stained using a MS-compatible silver staining kit (Dodeca Silver Stain Kit, Bio-Rad). After one-dimensional electrophoresis, the band corresponding to PK47 was excised and protein was characterized after trypsin in-gel digestion as previously described [27]. The trypsin digest was separated and analysed by nanoLC–MS/MS (nano liquid chromatography–tandem MS) using an Ultimate 3000 system (Dionex) coupled to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific).

Cell death

Cell death was estimated as previously described [28] by using Neutral Red as a vital dye that accumulates in the acidic vacuole. Cells that did not accumulate Neutral Red were considered dead. For each treatment 500 cells were counted.

RESULTS

Cryptogein induces the activation of a set of nuclear PKs

In order to investigate cryptogein-activated nuclear PKs, we first set up an optimized protocol for the purification of nuclei from cultured tobacco cells. The extent of contamination by other cellular compartments including the cytosol, the ER, the Golgi apparatus and peroxisomes was followed by marker enzyme analysis. All of the marker enzymes assayed were poorly detectable in the nuclear preparation, the contamination rate being below 1% for each enzyme (see Supplementary Table S1 at http://www.BiochemJ.org/bj/418/bj4180191add.htm). The integrity of the nuclei was ensured by the use of a detergent-free protocol [29] and controlled by the staining of enriched nuclei with DIOC6(3), an ER dye (results not shown). However, it should be specified that a significant contamination by cell-wall fragments was observed (Supplementary Figure S1 at http://www.BiochemJ.org/bj/418/bj4180191add.htm), confirmed by Ruthenium Red and Phloroglucinol staining of the nuclear preparations, but changes in buffer conditions, ionic strength or pH value did not significantly modify this. The use of tobacco protoplasts as the starting material was not conceivable, since cryptogein does not induce any known signal transduction events in the protoplast (S. Bourque and A. Pugin, unpublished work).

To examine the putative involvement of nuclear PKs in cryptogein signalling, tobacco cell suspensions were treated for various times with 100 nM cryptogein and the corresponding nuclear protein extracts were analysed by in-gel kinase assay using HIIIS as a general phosphorylation substrate in the presence of 0.5 mM Ca2+. As shown in Figure 1(A), several nuclear PKs with molecular masses of 36, 55 and 60 kDa (named PK36, PK55 and PK60 respectively) were constitutively activated in control cells. In nuclear protein extracts prepared from cryptogein-treated tobacco cells, three additional PKs exhibiting molecular masses of 42, 47 and 80 kDa (named PK42, PK47 and PK80) were activated. Furthermore, the activity of PK36, or of another PK with the same apparent molecular mass, was significantly increased following cryptogein treatment. A fifth PK with a molecular mass of 75 kDa also showed a rise in activity in response to the elicitor; however, in contrast with PK36, PK42, PK47 and PK80, its activation was not systematically observed, as well as the enhanced activity of PK with the same molecular mass as the PK55 and PK60, detected in cryptogein-elicited cells.

Cryptogein activates several nuclear PK activities

Figure 1
Cryptogein activates several nuclear PK activities

Tobacco cells (N. tabacum cv. Xanthi) were treated with 100 nM cryptogein for various times (as indicated). The corresponding nuclear extracts were prepared and PK activities were analysed by in-gel kinase assay with HIIIS as a substrate. The phosphorylation reaction was performed in the presence of 0.5 mM CaCl2 (A) or without Ca2+ in the medium (2 mM EGTA in the medium) (B). The arrowheads on the right-hand side indicate the different PK activities. (C) PK patterns from nuclear proteins extracted using the TriReagent (lane 1; T) or the high-salinity buffer (lane 2; S) protocols. These experiments were repeated five times with similar results. The molecular mass (in kDa) is indicated on the left-hand side of the gels.

Figure 1
Cryptogein activates several nuclear PK activities

Tobacco cells (N. tabacum cv. Xanthi) were treated with 100 nM cryptogein for various times (as indicated). The corresponding nuclear extracts were prepared and PK activities were analysed by in-gel kinase assay with HIIIS as a substrate. The phosphorylation reaction was performed in the presence of 0.5 mM CaCl2 (A) or without Ca2+ in the medium (2 mM EGTA in the medium) (B). The arrowheads on the right-hand side indicate the different PK activities. (C) PK patterns from nuclear proteins extracted using the TriReagent (lane 1; T) or the high-salinity buffer (lane 2; S) protocols. These experiments were repeated five times with similar results. The molecular mass (in kDa) is indicated on the left-hand side of the gels.

In response to cryptogein, PK36, PK42, PK47 and PK80 displayed distinct kinetics of activation. PK36 activation was detected after 30 min of cryptogein treatment and lasted for 12 h; PK42 was slightly activated after 3–6 h, depending on the experiments, peaked at 9 h and remained detectable up to 12 h of treatment; PK47 was activated within 5–10 min following cryptogein application, and showed a high level of activity for at least 12 h; PK80 was activated after 30 min of cryptogein treatment and peaked at 3 h before returning to a basal level within 9 h (Figure 1A).

To further characterize the biochemical features of the cryptogein-activated nuclear PKs, their Ca2+-dependence was assessed. For this purpose, nuclear proteins were analysed by in-gel kinase assay using HIIIS as a substrate [a preferred substrate for CDPK (Ca2+-dependent PK)], but without Ca2+ in the phosphorylation buffer (Figure 1B). These conditions allow the detection of strictly Ca2+-independent PK activities. In the absence of Ca2+, only the activities of PK36, PK47 and PK60 were still detected. In contrast, the elicitor-triggered activation of PK42 and PK80, as well as the constitutive activity of PK55, were completely suppressed, highlighting the requirement of Ca2+ for their full activity. To confirm that these PK activities were not due in part to cell-wall contaminations, nuclear proteins were prepared after lysis of the nuclei by a high-salinity buffer and ultracentrifugation to remove DNA, nuclear envelopes and cell-wall fragments. In these conditions we obtained the same PK pattern (Figure 1C), indicating that all PK activities described previously correspond to nuclear proteins.

Taken together, these results highlight the ability of cryptogein to promote the activation of several nuclear PKs, displaying distinct kinetics of activation and Ca2+-dependence.

Cryptogein activates a nuclear PK related to MAPKs

Among Ca2+-independent PKs, MAPKs are the most studied PKs involved in signalling pathways leading to defence responses. Based on the findings that cryptogein induces the activation of cytosolic MAPKs, including WIPK, SIPK and Ntf4 [1214], and that MAPKs are translocated into the nucleus upon activation in various mammalian transduction processes [30], we investigated whether some of the cryptogein-induced nuclear PKs could correspond to MAPKs. Therefore nuclear proteins were analysed by in-gel kinase assay using MBP, a preferential phosphorylation substrate compared with HIIIS, in the absence of Ca2+ to favour the detection of Ca2+-independent MAPK activities [31]. As shown in Figure 2, only the PK36, PK47 and PK60 were able to phosphorylate MBP, the PK47 and PK60 activities being more intense than those observed using HIIIS as a substrate.

Cryptogein activates a nuclear PK related to MAPKs

Figure 2
Cryptogein activates a nuclear PK related to MAPKs

Tobacco cells (N. tabacum cv. Xanthi) were treated with 100 nM cryptogein for various times (as indicated). The corresponding nuclear extracts were prepared and PK activities were analysed by in-gel kinase assay with MBP as a substrate, a preferred substrate for MAPK. The phosphorylation reaction was performed without Ca2+ in the medium (2 mM EGTA in the medium). This experiment is representative of three independent experiments. The arrowheads on the right-hand side indicate the different PK activities. The molecular mass (in kDa) is indicated on the left-hand side of the gel.

Figure 2
Cryptogein activates a nuclear PK related to MAPKs

Tobacco cells (N. tabacum cv. Xanthi) were treated with 100 nM cryptogein for various times (as indicated). The corresponding nuclear extracts were prepared and PK activities were analysed by in-gel kinase assay with MBP as a substrate, a preferred substrate for MAPK. The phosphorylation reaction was performed without Ca2+ in the medium (2 mM EGTA in the medium). This experiment is representative of three independent experiments. The arrowheads on the right-hand side indicate the different PK activities. The molecular mass (in kDa) is indicated on the left-hand side of the gel.

PK47 displayed the same apparent molecular mass as SIPK [14]. We therefore investigated whether the PK47 activity could correspond to SIPK, or to a closely related PK. For this purpose, nuclear protein extracts from untreated and cryptogein-treated tobacco cell suspensions were immunodetected using a polyclonal antibody raised against SIPK [14]. As a control, the same experiment was carried out using cytosolic extracts. As expected, immunostaining revealed a reactive polypeptide of 47 kDa in the cytosolic protein fractions, corresponding to SIPK (Figure 3A and Supplementary Figure S2 at http://www.BiochemJ.org/bj/418/bj4180191add.htm). The immunoreactive band was observed in both the control and cryptogein-treated cytosolic extracts, indicating that SIPK is constitutively produced as previously reported [13]. In the nuclear extracts, the antibodies detected a polypeptide with an apparent molecular mass of 47 kDa, suggesting that SIPK could also be present in the nuclei from both control and cryptogein-treated cells; however, we were unable to detect any significant accumulation of SIPK in the nucleus that could have suggested a translocation in response to cryptogein treatment. Intriguingly, in the nuclear fractions, besides the 47 kDa polypeptide, a 45 kDa polypeptide was also detected, this last one showing the most reactive signal, but the immunodetection of P45 was not reproducible.

Nuclear PK47 is a PK closely related to SIPK

Figure 3
Nuclear PK47 is a PK closely related to SIPK

(A) Tobacco cells were treated with 100 nM cryptogein (Cry) for the times indicated. Cytosolic and nuclear extracts were prepared from harvested cells and 20 μg of protein per lane was separated on the same 10% polyacrylamide gel. After transfer on to nitrocellulose membrane, the immunoblot was performed using antibodies directed against the N-terminal part of SIPK. The black arrowhead indicates the band corresponding to SIPK. Cont., control. (B) Nuclear extracts containing 100 μg of proteins from untreated control (Cont.) or cells treated for 30 min with 100 nM cryptogein (Cry) were immunoprecipitated with 5 μg of antibodies directed against SIPK. Kinase activity of the immunocomplexes was then determined by in-gel kinase assay using HIIIS as a substrate, in the presence of 0.5 mM CaCl2 in the reaction buffer. The PK47-corresponding activity is indicated on the right-hand side (arrowhead). The molecular mass (in kDa) is indicated on the left-hand side of the gels.

Figure 3
Nuclear PK47 is a PK closely related to SIPK

(A) Tobacco cells were treated with 100 nM cryptogein (Cry) for the times indicated. Cytosolic and nuclear extracts were prepared from harvested cells and 20 μg of protein per lane was separated on the same 10% polyacrylamide gel. After transfer on to nitrocellulose membrane, the immunoblot was performed using antibodies directed against the N-terminal part of SIPK. The black arrowhead indicates the band corresponding to SIPK. Cont., control. (B) Nuclear extracts containing 100 μg of proteins from untreated control (Cont.) or cells treated for 30 min with 100 nM cryptogein (Cry) were immunoprecipitated with 5 μg of antibodies directed against SIPK. Kinase activity of the immunocomplexes was then determined by in-gel kinase assay using HIIIS as a substrate, in the presence of 0.5 mM CaCl2 in the reaction buffer. The PK47-corresponding activity is indicated on the right-hand side (arrowhead). The molecular mass (in kDa) is indicated on the left-hand side of the gels.

To determine whether the 47 kDa polypeptide carries PK47 activity, nuclear extracts from untreated and cryptogein-treated cells were immunoprecipitated with specific anti-SIPK polyclonal antibodies, and the resulting immunocomplexes were analysed with the in-gel kinase assay technique using HIIIS as a substrate and in the presence of Ca2+. These conditions were used because they allowed the detection of a higher number of nuclear PK activities as compared with MBP in the absence of Ca2+ (Figure 1 compared with Figure 2). As shown in Figure 3(B), only the PK47 activity was clearly immunoprecipitated from the nuclear extracts of cryptogein-treated cells. No signal could be detected at 45 kDa, leading us to conclude that we could not be certain of the putative activity of P45, especially we could not determine whether it could correspond to an inactive form of SIPK. Work is in progress to identify the 45 kDa polypeptide. Therefore the present results strongly suggest that the nuclear PK47 corresponds to SIPK and, although it is present in both control and cryptogein-treated cells, its activity is only observed in response to cryptogein treatment.

Purification and identification of SIPK from nuclear protein-enriched extracts

To confirm the assumption that the PK47 corresponds to SIPK, we undertook its purification from 70 mg of nuclear proteins corresponding to approx. 8 kg of cryptogein-treated tobacco cells. The nuclear proteins were first loaded on to a HiTrap Q anion-exchange column and each collected fraction was analysed by an in-gel kinase assay. As shown in Figure 4(A), several nuclear PKs showing an induced activity in response to cryptogein, mainly PK50–PK60, were eluted within the flowthrough. Other PKs of interest, including PK47, were retained by the column and subsequently eluted by increasing NaCl concentrations. The fractions containing the PK47 activity were pooled, loaded on to a HiTrap phenyl hydrophobic column and subsequently eluted with increasing concentrations of ethylene glycol and decreasing concentrations of NaCl. The hydrophobic chromatography did not permit an efficient fractionation of the nuclear PK activities as most of them were collected in both the flow-through and the eluted fractions (Figure 4B). In contrast, activity of PK47 was specifically detected in eluted fractions. The corresponding fractions were pooled, desalted, concentrated and finally immunoprecipitated using the specific anti-SIPK polyclonal antibodies. The immunoreactive polypeptides were loaded on to a preparative SDS/PAGE gel and the band corresponding to PK47 (Supplementary Figure S3 at http://www.BiochemJ.org/bj/418/bj4180191add.htm) was excised for microsequencing.

Enrichment of nuclear PK47

Figure 4
Enrichment of nuclear PK47

Nuclear proteins (70 mg) extracted from tobacco cells treated with 100 nM cryptogein for 3 h were used to enrich the PK47 activity through two chromatography steps. (A) Each fraction obtained after anion-exchange chromatography was analysed by in-gel kinase assay using HIIIS as a substrate, in the presence of 0.5 mM CaCl2. The arrowhead indicates the activity corresponding to PK47. (B) Each fraction after the hydrophobic chromatography was analysed by in-gel kinase assay using HIIIS as a substrate, in the presence of 0.5 mM CaCl2. The arrow indicates the activity corresponding to PK47. The molecular mass (in kDa) is indicated on the left-hand side of the gels.

Figure 4
Enrichment of nuclear PK47

Nuclear proteins (70 mg) extracted from tobacco cells treated with 100 nM cryptogein for 3 h were used to enrich the PK47 activity through two chromatography steps. (A) Each fraction obtained after anion-exchange chromatography was analysed by in-gel kinase assay using HIIIS as a substrate, in the presence of 0.5 mM CaCl2. The arrowhead indicates the activity corresponding to PK47. (B) Each fraction after the hydrophobic chromatography was analysed by in-gel kinase assay using HIIIS as a substrate, in the presence of 0.5 mM CaCl2. The arrow indicates the activity corresponding to PK47. The molecular mass (in kDa) is indicated on the left-hand side of the gels.

LC–MS/MS analysis of the tryptic digest from the immunopurified protein allowed the sequencing of four internal tryptic peptides, which unambiguously identified the PK47 protein as SIPK (Figure 5). The predicted molecular mass of SIPK is 45.1 kDa and it has a calculated pI of 5.53. Three out of the four sequenced peptides are shared with Ntf4, but the fourth ruled out the possibility that the purified PK47 could correspond to Ntf4. The MS/MS identification of SIPK from nuclear extracts confirmed the results obtained in immunoblot and immunoprecipitation experiments (Figure 3), and demonstrated that SIPK is present and active in response to cryptogein in the nuclei of treated tobacco cells.

Identification of PK47 as SIPK

Figure 5
Identification of PK47 as SIPK

The amino acid sequences of SIPK and Ntf4 were aligned. For Ntf4, only divergent amino acids are indicated. The sequences of the four tryptic peptides obtained by MS are indicated by the black lines over the SIPK sequence. The amino acid of the fourth microsequenced peptide highlighted in grey discriminates SIPK from Ntf4. Roman numerals indicate the 11 major conserved subdomains of serine/threonine protein kinases. The dotted line in subdomain VI indicates the catalytic site; the conserved residues essential for catalysis are marked with a black dot. The phosphorylated residues in the MAPK activation motif TEY are indicated with asterisks.

Figure 5
Identification of PK47 as SIPK

The amino acid sequences of SIPK and Ntf4 were aligned. For Ntf4, only divergent amino acids are indicated. The sequences of the four tryptic peptides obtained by MS are indicated by the black lines over the SIPK sequence. The amino acid of the fourth microsequenced peptide highlighted in grey discriminates SIPK from Ntf4. Roman numerals indicate the 11 major conserved subdomains of serine/threonine protein kinases. The dotted line in subdomain VI indicates the catalytic site; the conserved residues essential for catalysis are marked with a black dot. The phosphorylated residues in the MAPK activation motif TEY are indicated with asterisks.

Characterization of the nuclear SIPK activation

The cryptogein-induced signalling events acting upstream of cytosolic SIPK activation have been investigated in several studies. It was reported that SIPK activation is strictly dependent on the elicitor-triggered influx of extracellular Ca2+ [14]. In contrast, anion effluxes, NADPH oxidase-derived ROS and NO did not appear as essential components of the cryptogein-induced signalling pathway leading to the activation of SIPK [32]. To assess the involvement of signalling events in transmitting the cryptogein signal contributing to the activation of the nuclear SIPK, a pharmacological approach was performed.

The possible influence of the cryptogein-induced Ca2+ influx was investigated by blocking plasma membrane Ca2+-permeable channels using Gd3+. This compound has been shown to block the entrance of Ca2+ and subsequent Ca2+-dependent events in cryptogein-treated cells [33]. As shown in Figure 6, Gd3+ completely suppressed nuclear SIPK activation. To determine a putative link between nuclear SIPK activation and cryptogein-induced anion effluxes, ROS and NO production, we next examined the effects of the plasma membrane anion channel inhibitors glibenclamide and niflumic acid (results not shown) [16], the NADPH oxidase inhibitor DPI [20] and the NO scavenger cPTIO [33]. These compounds had no inhibitory effects on the elicitor-triggered nuclear SIPK activation. Finally, the effect of CHX, a widely used inhibitor of protein synthesis, was also tested. Here too, CHX did not impair the activation of the nuclear SIPK induced by cryptogein in cultured tobacco cells, suggesting that nuclear SIPK activation did not require prior de novo synthesis of SIPK itself or of other proteins acting upstream of its activation. These results fit well with those reported in the immunoblot (Figure 3A) in which the amount of nuclear SIPK was comparable in both control and cryptogein-treated cells.

Integration of SIPK nuclear activity in the cryptogein signalling pathway

Figure 6
Integration of SIPK nuclear activity in the cryptogein signalling pathway

(A) SIPK activity was first estimated in response to cryptogein alone. Control (Cont.) and treated (Cry) cells were harvested at the times indicated and SIPK activity was analysed in nuclear extracts by in-gel kinase assay using MBP as a substrate in the absence of Ca2+ (2 mM EGTA in the reaction buffer). (B) Control or cryptogein-treated (Cry) cells were pre-treated for 10 min with different inhibitors: 2 mM Gd3+, 10 μM DPI, 200 μM glibenclamide (Gli), 500 μM cPTIO and 10 μM CHX. Cells were harvested at the times indicated, and SIPK activity was analysed by in-gel kinase assay using MBP as a substrate in the absence of Ca2+ (2 mM EGTA in the reaction buffer).

Figure 6
Integration of SIPK nuclear activity in the cryptogein signalling pathway

(A) SIPK activity was first estimated in response to cryptogein alone. Control (Cont.) and treated (Cry) cells were harvested at the times indicated and SIPK activity was analysed in nuclear extracts by in-gel kinase assay using MBP as a substrate in the absence of Ca2+ (2 mM EGTA in the reaction buffer). (B) Control or cryptogein-treated (Cry) cells were pre-treated for 10 min with different inhibitors: 2 mM Gd3+, 10 μM DPI, 200 μM glibenclamide (Gli), 500 μM cPTIO and 10 μM CHX. Cells were harvested at the times indicated, and SIPK activity was analysed by in-gel kinase assay using MBP as a substrate in the absence of Ca2+ (2 mM EGTA in the reaction buffer).

Taken together, these results indicate that SIPK is first regulated at the post-translational level in the cytosol and the nucleus and that, similar to its cytosolic counterpart, the activation of the nuclear SIPK is strictly dependent on the elicitor-triggered Ca2+ influx, but independent of anion effluxes, ROS and NO production.

Activation of SIPK by various stimuli

Various stimuli, including biotic and abiotic stresses, activate cytosolic MAPKs, including SIPK [34]; however, to our knowledge, no study has analysed whether a cytosolic MAPK activation is paralleled by its activated form in the nucleus. We therefore investigated the ability of stimuli known to trigger the activation of SIPK in the cytosol to promote an active SIPK in the nucleus. As stimuli we tested: (i) OGs, non-necrotic elicitors of tobacco defence reactions [14]; (ii) SA [35]; (iii) BA [26]; and (iv) hyperosmotic shock triggered by sorbitol [36]. As previously reported [14,26,35,36], all of these compounds provoked SIPK activation in the cytosolic extracts, but with different kinetics (Figure 7). OGs triggered a transient activation of SIPK, peaking at 10 min and decreasing to basal levels within 60 min. Sorbitol also produced a very transient activation of SIPK, which peaked at 5 min and disappeared within 30 min. Similar results were obtained in response to SA, but with more extended kinetics. When used at 1 mM, BA used to acidify the cytosol [26] induced a slower activation of SIPK, occurring after 30 min of treatment and lasting for at least 2 h. Higher concentrations of BA (5 mM) induced a faster, stronger and long-lasting activation which occurred within 5 min and was maintained for at least 2 h.

Activity of SIPK in the cytosol and nucleus in response to different stimuli

Figure 7
Activity of SIPK in the cytosol and nucleus in response to different stimuli

Different treatments known to activate SIPK in the cytosol, were applied to cells: 100 μg·ml−1 OGs, 250 μM SA, 1 and 5 mM BA and 250 mM sorbitol. Control and treated cells were harvested at various times, and cytosolic (Cyt) and corresponding nuclear (Nuc) extracts were prepared. In-gel kinase assays were performed to analyse SIPK activity in these extracts, with MBP as a substrate in the absence of Ca2+ (2 mM EGTA in the reaction buffer).

Figure 7
Activity of SIPK in the cytosol and nucleus in response to different stimuli

Different treatments known to activate SIPK in the cytosol, were applied to cells: 100 μg·ml−1 OGs, 250 μM SA, 1 and 5 mM BA and 250 mM sorbitol. Control and treated cells were harvested at various times, and cytosolic (Cyt) and corresponding nuclear (Nuc) extracts were prepared. In-gel kinase assays were performed to analyse SIPK activity in these extracts, with MBP as a substrate in the absence of Ca2+ (2 mM EGTA in the reaction buffer).

When the nuclear activity of SIPK was assessed, its activation was detected only in response to 5 mM BA (Figure 7). In this condition, a slight activation occurred within 10 min. The activation became stronger after 60 min where it reached a level of activation similar to those observed in response to cryptogein. Interestingly, in contrast with 250 mM sorbitol, 1 mM BA, SA and OGs, only cryptogein and 5 mM BA triggered significant cell death within 24 h, reaching 35% and 100% respectively.

Taken together, these results indicate that the presence of the activated SIPK in the nucleus depends on the applied stimuli and is promoted by cell-death-triggering signals. Furthermore, they suggest that the cytosolic activation of SIPK is not systematically paralleled by the presence of its activity in the nucleus, confirming that detection of active SIPK in the nucleus is not due to cytosolic contamination or mechanical translocation when cells are harvested.

DISCUSSION

The possibility that nuclear PKs could be specifically activated in plant cells challenged by biotic stresses has been suggested, but not firmly established [8,18a,37]. The results of the present study support, for the first time, the conclusion that biotic stresses promote the activation of nuclear PK. Indeed, compared with control cells, at least four PKs, that is PK36, PK42, PK47 and PK80, showed increased PK activities in nuclear protein extracts prepared from cryptogein-treated tobacco cells. Each PK was activated in a specific time-dependent manner, varying from minutes in the case of PK47 to several hours in the case of PK42. Several lines of evidence indicate that these activities did not reflect a contamination by cytosolic PKs. G6PDH (glucose 6-phosphate dehydrogenase) activity, a marker of the cytosolic and plastidic compartments [39], was extremely low in the nuclear fraction. An ambiguous situation came from the analysis of the activity of SIPK, which was similarly detected in response to cryptogein treatment in both the cytosol and the nucleus. However, the observation that activated SIPK was specifically observed in cytosolic, but not in nuclear, extracts prepared from tobacco cells exposed to OGs, hyperosmotic stress, SA or 1 mM BA provided further arguments ruling out the hypothesis that SIPK activity in the nuclear extracts could result from a cytosolic contamination.

The nuclear PKs activated in response to cryptogein showed distinct biochemical features. PK activities of PK55 and PK80 were strictly Ca2+-dependent, suggesting that those were probably due to CDPK or CCaMK (Ca2+/calmodulin-dependent PK) [40]. Several representatives of these families have already been found to be located in the nuclear compartment. A study investigating the subcellular localization of several Arabidopsis thaliana CDPK isoforms fused to GFP showed that at least two of them were constitutively nuclear [4]. A well-known CCaMK, DMI3, involved in nodulation in Medicago truncatula, also exhibits a nuclear localization when fused to GFP [41]. With the exception of nuclear CCaMK-like activities in response to temperature stress [42], the activities of such PKs in the nuclear compartment have not yet been reported.

PK47 displayed features of MAPK, including a comparable molecular mass and an efficient phosphorylation activity toward MBP in a Ca2+-independent manner. Supporting these results, through combining immunological and biochemical purification approaches, we identified PK47 as the MAPK SIPK, providing the first evidence that MAPKs are active within the nucleus of plant cells challenged with a biotic stress. Experiments with plasma membrane Ca2+-channel inhibitors indicate that Ca2+ influx from the extracellular space is required for nuclear SIPK activation in cryptogein-treated cells. In contrast, neither anion effluxes nor ROS and NO production appeared to be necessary for nuclear SIPK activation. Interestingly, such regulation resembles those already described for cytosolic SIPK [14,16,26]. By using various stimuli known to activate SIPK, our results also clearly demonstrated that the cytosolic activation of SIPK is a prerequisite, but is not sufficient to induce, the activity of the nuclear SIPK.

Taken together, these findings raise the question of whether SIPK nuclear activity results from the translocation of the activated cytosolic enzyme from the cytosol to the nucleus or whether it results from the direct activation of the nucleus enzyme. Results from Western blot analysis performed using antibodies raised against SIPK indicated that this PK is expressed both in the cytosol and in the nucleus of control and cryptogein-treated tobacco cells. Accordingly, using a chimaeric SIPK–GFP construct under the control of the cauliflower mosaic virus 35S promoter, Menke et al. [18a] showed that SIPK is present both in the cytosol and the nucleus of tobacco BY2 cells. Similar results were obtained in N. benthamiana cells overexpressing a NbSIPK–GFP construct [19], but it was also shown in this case that a constitutively nuclear MKK, NbMKK1, interacts with and could be responsible for, the nuclear activation of NbSIPK. However, both studies did not provide evidence that SIPK could be active at the nuclear level and/or translocated from the cytosol. Two hypotheses are conceivable to explain the activation of a nuclear-localized SIPK: (i) SIPK is phosphorylated and activated in the cytosol by a MKK, presumably NtMEK2 [43], and then translocated into the nucleus; (ii) SIPK could also be phosphorylated at the nuclear level by a nuclear MKK, as suggested for NbMKK1, a nuclear MKK able to phosphorylate in vitro NbSIPK [19]. In this case, how this MKK could be activated in the nucleus remains undetermined. More generally, few studies have underlined the possibility that cytosolic MAPK could be translocated to the nucleus. For instance, in parsley cell suspensions, it was shown by immunolabelling that two MAPKs, PcMPK3 and PcMPK6, are translocated to the nucleus upon stimulation by the elicitor Pep13 [8]. Similarly, two A. thaliana MAPKs, AtMPK3 and AtMPK6, were shown to be translocated from the cytosol to the nucleus upon ozone treatment [37]. Here too, the demonstration that these MAPKs are active in the nucleus has not been provided.

Although several studies point to the presence of plant MAPKs in the nucleus, their roles in this compartment have been poorly investigated to date. Indeed, few substrates of these PKs have been identified. Among those, WRKY1, the only known substrate for SIPK to date, was shown to be implicated in HR-like cell death in association with SIPK [18a]. Similarly, Cheong et al. [44] found that BWMK1, a rice nuclear MAPK, could activate, by phosphorylation, the transcription factor OsEREBP1 which could lead to PR (pathogenesis related) gene activation. As several transcription factors were demonstrated to act downstream of MAPKs [4549], this supports the hypothesis that the nuclear activity of SIPK documented in the present study may be related to the control of transcription. It is then of major importance to identify the putative nuclear targets of SIPK and the associated plant genes.

In summary, this work greatly extends previous studies describing the involvement of PKs in plant defence by providing the first evidence of a functional nuclear MAPK activity involved in response to an elicitor treatment. Work in progress aims at investigating whether SIPK is directly activated within the nucleus or whether it is translocated from the cytosol to the nucleus in its activated form.

We thank Annick Chiltz, Agnès Klinguer and Delphine Desqué for technical assistance.

Abbreviations

     
  • AtMPK

    Arabidopsis thaliana MAPK

  •  
  • BA

    butyric acid

  •  
  • CCaMK

    Ca2+/calmodulin-dependent protein kinase

  •  
  • CDPK

    Ca2+-dependent protein kinase

  •  
  • CHX

    cycloheximide

  •  
  • cPTIO

    carboxy-PTIO (carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide)

  •  
  • DPI

    diphenylene iodonium

  •  
  • DTT

    dithiothreitol

  •  
  • ER

    endoplasmic reticulum

  •  
  • GFP

    green fluorescent protein

  •  
  • HIIIS

    histone IIIS

  •  
  • HR

    hypersensitive response

  •  
  • LC–MS/MS

    liquid chromatography–tandem MS

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MBP

    myelin basic protein

  •  
  • MKK

    MAPK kinase

  •  
  • NbMKK1

    Nicotiana benthamiana MKK1

  •  
  • NtMEK2

    Nicotiana tabacum MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]

  •  
  • OG

    oligogalacturonate

  •  
  • PcMPK

    Pseudomonas cichorii MAPK

  •  
  • PK

    protein kinase

  •  
  • ROS

    reactive oxygen species

  •  
  • SA

    salicylic acid

  •  
  • SIPK

    SA-induced PK

  •  
  • NbSIPK

    Nicotiana benthamiana SIPK

  •  
  • WIPK

    wound-induced PK

FUNDING

This work was supported by the Ministère de l'Education Nationale, de la Recherche et de la Technologie, and the Conseil Régional de Bourgogne.

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Supplementary data