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First published online June 9, 2006; 10.1104/pp.106.079327 Plant Physiology 141:1316-1327 (2006) © 2006 American Society of Plant Biologists
Identification of Features Regulating OST1 Kinase Activity and OST1 Function in Guard Cells1,[W]Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette cedex, France (C. Belin, P.-O.F., C. Bourbousse, J.G., H.B.-B., S.T.); Institut Européen de Chimie et Biologie, 33607 Pessac, France (S.C., J.-M.S.); and Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Laboratoire des Echanges Membranaires et Signalisation, 13108 St. Paul lez Durance cedex, France (A.V.)
The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction. A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction. Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase. Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.
Drought is a major environmental constraint and plants have developed several protective strategies to survive this stress. Most plants first respond to drought by closing their stomata to prevent water loss via transpiration. Abscisic acid (ABA) is a major phytohormone that mediates drought responses in plants and, particularly, reduction of stomatal conductance through its action on guard cells (Tardieu et al., 1992  ). Regulation of potassium and anion channels in response to ABA leads to a rapid decrease in osmotic pressure and, consequently, a massive efflux of water from guard cells. This decrease in turgor results in stomatal pore closure. If the mechanism of the stomatal closure is now quite well described (Roelfsema and Hedrich, 2005), a thorough understanding of the signal transduction pathway of ABA in guard cells clearly requires further research. Many second messengers and genes have been shown to be involved in ABA signal transduction, but little evidence exists concerning molecular interactions between these components (Hetherington, 2001; Schroeder et al., 2001; Himmelbach et al., 2003). Moreover, although an ABA receptor was recently characterized (Razem et al., 2006), it is not active in guard cells and the molecular mechanisms of ABA perception in these cells remain unknown.
Previous studies reported the involvement of Snf1-related kinase 2 (SnRK2) proteins in ABA signal transduction pathways. First, PKABA1 was implicated in ABA signal transduction in wheat (Triticum aestivum; TaPKABA1) and barley (Hordeum vulgare; HvPKABA1) seeds (Anderberg and Walker-Simmons, 1992
Several studies have reported the phosphorylation of substrates by ABA-activated SnRK2 in different plant species. They include bZIP transcription factors, such as TaABF from wheat (Johnson et al., 2002
Many kinases are themselves regulated by phosphorylation and dephosphorylation events. Recent studies reported the importance of phosphorylation in rice SnRK2 activation in response to osmotic stresses (Kobayashi et al., 2004
Many previous studies using SnRK2 proteins from different plant species, including soybean (Glycine max), fava bean, tobacco (Nicotiana tabacum), wheat, or Arabidopsis, report that expression of recombinant proteins in Escherichia coli results in inactive proteins (Yoon et al., 1997 Here we demonstrate that the recombinant fusion protein 10xHis-OST1 produced in E. coli and purified under native conditions is active and is able to autophosphorylate. This allows the identification of phosphorylated Ser residues representing putative targets of upstream kinases or phosphatases. The importance of these residues is investigated both in vitro to study biochemical activity and in Arabidopsis guard cells to analyze their impact on OST1 function in signal transduction. Truncated versions also allow us to investigate the role of the C terminus. We report here the identification of conserved features critical for OST1 kinase activity and function in guard cells.
Recombinant OST1 Protein Is an Active Kinase and Autophosphorylates
To study the kinase activity of recombinant OST1, a vector was constructed that allowed expression of a 10xHis N-terminal-tagged OST1 protein in E. coli. Figure 1A
shows the different steps of 10xHis-OST1 production and purification under native conditions. Large amounts (>20 mg) of highly purified (>95%) recombinant OST1 were obtained. Assays for kinase activity show that this recombinant kinase is able to efficiently phosphorylate generic substrates such as histone (Fig. 1B) and myelin basic protein (data not shown). Moreover, the occurrence of a band corresponding to the size of OST1 (about 50 kD) suggests that it is able to autophosphorylate. This is confirmed by kinase assays performed in the absence of a substrate (Fig. 1C). In addition, a modified kinase assay, using either [
We tested whether OST1 activity is modulated by second messengers involved in ABA signal transduction or ABA itself. Changes of external pH (from 7–8.5) or calcium concentrations (from 0–500 µM CaCl2) did not significantly alter OST1 activity in vitro (data not shown). Finally, we demonstrated that ABA does not directly regulate OST1 in vitro activity (data not shown). We are thus able to purify under nondenaturing conditions a recombinant OST1 protein kinase and we demonstrate that it is active alone without any additional factor. This provides us with a powerful tool to investigate structure-function relationships of OST1 protein.
Active OST1 kinase is slightly shifted to higher apparent molecular mass compared to the inactive OST1G33R mutant (Fig. 1B). In rice, such shifts have been correlated with phosphorylations of SnRK2 kinases (Kobayashi et al., 2004
Ser-7, Ser-18, Ser-29, Ser-43, and T Loops Are Targets of Autophosphorylation of OST1 in Vitro Mass spectrometry (MS) studies were performed to detect phosphorylated residues in the recombinant OST1 protein. The average mass of 10xHis-OST1 measured by matrix-assisted laser-desorption ionization (MALDI)-MS analysis was about 400 D above the theoretical value calculated from the sequence. However, the pseudomolecular ion cluster was broad and unresolved. More convincing indications of phosphorylation were obtained by means of electrospray ionization (ESI)-MS analysis of recombinant proteins 10xHis-OST1 and 10xHis-OST1G33R. Seven different protein species were separated for active OST1 with masses ranging between 46,658 and 47,151 D (Table I ). A comparison between the theoretical mass of the recombinant protein (46,342 D, taking into account excision of the N-terminal Met) and experimental masses indicates that these different species correspond to isoforms of the protein carrying from four to 10 phosphate groups. Indeed, the average masses of the seven detected protein species differ by 80 D (i.e. the mass of one added phosphate group). The analysis of OST1G33R led to a major peak at 46,436 D that matches the theoretical mass of unphosphorylated OST1G33R and a minor one that probably corresponds to a degradation product. We may thus conclude that 10 residues of 10xHis-OST1 recombinant protein are targets of autophosphorylation.
In a second stage, tandem MS (MS/MS) analysis of a tryptic digest of recombinant OST1 was used to map target residues. A combination of MALDI-quadrupole time-of-flight (Q-TOF) and nano-liquid chromatography (LC)-ESI-Q-TOF analysis led to 68% sequence coverage of the fusion protein; this low score is explained by the fact that the [280–369] tryptic peptide accounting alone for 22% of the sequence was not detected. Nine potential phosphorylation sites were detected by a combination of exact mass measurement of precursor ions and observation of neutral loss (H3PO4) from these ions (Supplemental Table I). Four phosphorylated sites belong to the N-terminal tag (residues -25, -24, -10, and -9) and are not relevant for OST1 function. Ser-7, Ser-18, Ser-29, and Ser-43 belonging to the catalytic domain are modified by phosphate groups (Fig. 3 ). Peptide 175STVGTPAYIAPEVLLK190 corresponds to the activation loop (also called activation segment or T loop) that is conserved in most kinases. The MS/MS spectrum of this peptide ruled out the modification of Thr-179, but did not allow the assignment of the phosphate group to Ser-175 or Thr-176. No trace of a Ser-175 and Thr-176 doubly modified peptide was found, and incomplete coverage of the protein sequence presumably hindered identification of the tenth phosphorylated residue.
Thus, MS pointed out six residues that are putatively important for OST1 activity regulation by phosphorylation, namely, Ser-7, Ser-18, Ser-29, Ser-43, Ser-175, and Thr-176 (Fig. 3).
In a first step, the requirement of each of the candidate phosphorylated residues identified for kinase activity of the recombinant fusion protein was tested. Each was substituted, either for an Ala to prevent phosphorylation or for an Asp to mimic constitutive phosphorylation. All point mutants (OST1S7A, OST1S7D, OST1S18A, OST1S18D, OST1S29A, OST1S29D, OST1S43A, OST1S43D, OST1S175A, OST1S175D, OST1T176A, and OST1T176D) were produced as recombinant 10xHis N-terminal-tagged proteins in E. coli. Circular dichroism measurements in far UV (200–250 nm) revealed that none of the mutations drastically modified OST1 secondary structure (mostly
In contrast, both mutations of Ser-175 greatly affect ability of the kinase to phosphorylate a substrate (Fig. 4B). In this case, it would appear that substitution of Ser-175 for an Asp does not mimic constitutive phosphorylation of the residue because both mutants are equally impaired in kinase activity. The mutation Thr-176 to Asp is also critical for OST1 activity because we can only detect a slight band of autophosphorylation. However, the mutation of the same Thr to Ala has no effect on the kinase activity, demonstrating that phosphorylation of Thr-176 is not required for OST1 activity. Two hypotheses may account for these results: Either the Thr to Asp mutation does, in this case, mimic constitutive phosphorylation and phosphorylation of this residue inactivates the kinase, or the Thr-176 D mutation results in a misfolded protein when expressed in E. coli, which could explain the very low activity. At this stage, we cannot discriminate between the two hypotheses. We conclude that Ser-175 is critical for OST1 biochemical activity in contrast to Ser-7, Ser-18, Ser-29, and Ser-43. However, these residues might still be targets of regulatory phosphorylation events in planta.
To test the functionality of OST1 point mutants in plants, we generated a binary vector driving the expression of the protein of interest in fusion with a triple hemagglutinin (3xHA) N-terminal tag under the control of the OST1 promoter. The use of the gateway cloning system and of seed-specific green fluorescent protein expression as the selection marker (Bensmihen et al., 2004
The uidA gene was first inserted in this vector to test OST1 promoter specificity. Histochemical
An in-gel kinase assay (Fig. 5B) was performed on total protein extracts from Columbia-0 (Col-0) and srk2e guard cells. This experiment confirmed that OST1 is strongly activated by ABA in wild-type guard cells, whereas OST1 kinase activity is totally absent in the srk2e mutant background. In agreement with these results, the srk2e mutant shows increased water loss due to defects in stomatal closure. This phenotype can be monitored by infrared thermography (Merlot et al., 2002 ) as shown in Figure 5C. Guard cell-specific expression in the srk2e null mutant allows us to investigate OST1 function in the signaling pathways leading to stomatal closure, while avoiding possible side effects caused by ectopic expression from a 35S promoter or the presence of an inactive OST1 protein. OST1 function was investigated by testing mutant complementation using the infrared thermography approach on detached leaves to monitor stomatal aperture.
To investigate in planta the function of the Ser and Thr identified as putative phosphorylation targets, the point-mutated versions of OST1 were introduced into the srk2e null background and several independent lines were selected for all constructs. GUS and OST1 lines were used as negative and positive controls for mutant complementation, respectively. Infrared images of detached leaves with wild-type (Col-0) and mutant (srk2e) control leaves on each image were used to quantify the leaf temperature. Results in Figure 6B show that GUS is unable to complement the srk2e mutant, whereas the wild-type warm phenotype is restored when OST1 is expressed in srk2e guard cells. This figure shows one representative line for each of the residues targeted by site-directed mutagenesis. Similar results were obtained when the residues were mutated to Ala or Asp and in several lines for each substitution (Supplemental Fig. 2).
Both mutations affecting Ser-175 totally abolish OST1 ability to complement the srk2e mutant in agreement with the decreased kinase activity of the corresponding recombinant proteins (Fig. 6, A and B; Supplemental Fig. 2). In the case of Thr-176, both mutants (Thr to Ala and Thr to Asp) fully restore the wild-type phenotype when expressed in the mutant background (Supplemental Fig. 2). This Thr is thus not necessary for OST1 function. The lack of in vitro activity reported for the OST1T176D recombinant protein is most likely due to incorrect protein folding in E. coli. Ser-7, Ser-18, Ser-29, and Ser-43 are not required for OST1 kinase activity in vitro. Complementation assays show that mutations of Ser-7, Ser-18, or Ser-29 greatly impair the ability of OST1 to complement the srk2e mutant phenotype (Fig. 6B; Supplemental Fig. 2). Direct stomatal aperture measurements confirm that guard cells expressing point-mutated versions of Ser-7, Ser-18, or Ser-29 in the srk2e background do not respond to ABA (data not shown). To determine whether Ser-7, Ser-18, and Ser-29 mutations impair OST1 activation by ABA in planta, we tested the activity in response to ABA of the corresponding versions. Fusion proteins were immunoprecipitated from plants treated or not with ABA, using an antibody directed against the 3xHA tag. Kinase assays show that mutations on Ser-7, Ser-18, and Ser-29 do not affect activation of the kinase in response to ABA (Fig. 6C). In contrast, mutations of Ser-43 do not prevent srk2e complementation by OST1. Interestingly, the OST1S43A version immunoprecipitated from plants is not activated by ABA, but rather is constitutively active (Fig. 6C). We conclude that Ser-7, Ser-18, and Ser-29 are critical for OST1 function in the signaling pathway leading to stomatal closure, whereas Ser-43 is involved in negative regulation of OST1 activity in the absence of ABA.
To better understand how the carboxy-terminal domain affects OST1 function, we investigated the structure-function relationship of this domain. Alignment of 10 Arabidopsis SnRK2s, shown in Figure 7
, reveals a highly conserved feature among the 10 proteins between the Gln-303 and the Pro-318. We named it the SnRK2-specific box. This box is also conserved in all rice SAPKs (Supplemental Fig. 1). A second region after Ser-332, which we named the ABA-specific box, is conserved specifically among the three strongly ABA-activated kinases: OST1, OSKL2, and OSKL3 (Fig. 7), as well as in ABA-activated rice SAPK (Supplemental Fig. 1). Based on these observations, we decided to dissect the function of these two domains by generating five truncated versions of the OST1 protein: OST1
In vitro kinase assays of recombinant proteins (Fig. 8 ) show that OST1  280 and OST1302 truncated proteins have no detectable kinase activity, whereas OST1320 activity is similar to the full-length OST1 protein. Hence, the SnRK2-specific domain is necessary for OST1 kinase activity and its conservation suggests that it is responsible for the activation of all SnRK2 proteins. Longer truncated versions are all active when produced in E. coli. The second half of the C-terminal region, after Ala-320, is thus not necessary for OST1 kinase activity. The efficiency of substrate phosphorylation, however, differs between versions, suggesting a role of this domain in modulating OST1 kinase activity.
Results of in planta complementation assays are shown in Figure 9B and Supplemental Figure 3. As expected, OST1 280 and OST1302, which do not display kinase activity in vitro, do not complement the srk2e phenotype. More surprisingly, truncation after Ala-320, which results in an active protein when expressed in E. coli, totally abolishes activation of the kinase by ABA in planta and the ability of OST1 to complement the srk2e mutant (Fig. 9, B and C). Likewise, OST1331, which retains substantial kinase activity in vitro, is not activated by ABA in planta and is not able to complement the mutant. Accordingly, the stomata of srk2e plants expressing OST1331 do not respond to ABA (Fig. 10
). This indicates that the ABA-specific domain is very important for OST1 activation by ABA and function in plants. In contrast, OST1348 fully complements the srk2e transpiration phenotype and restores ABA responsiveness in stomatal bioassays (Fig. 10). OST1348 displays stronger activation by ABA than OST1 in plants (Fig. 9), although it shows a lower kinase activity in vitro than the full-length protein. The Asp-348 to Met-362 stretch may be involved in a mechanism that negatively regulates OST1 activity. We conclude that the Leu-333 to Asp-348 stretch within the ABA-specific box is critical for the activation and function of OST1 in response to ABA in guard cells. This led us to check whether ABA could directly regulate OST1 activity. However, in vitro treatment of OST1 protein immunoprecipitated from plants by 10 µM ABA did not induce kinase activity (data not shown).
OST1 protein kinase is a positive regulator of stomatal closure in guard cells (Mustilli et al., 2002 ; Yoshida et al., 2002). Here, we present results leading to a better understanding of how the structure of OST1 protein affects its function in guard cell-specific signal transduction.
Several previous works reported that recombinant SnRK2s were inactive (Yoon et al., 1997
The expression of SnRK2 recombinant proteins in E. coli allowed us to identify target residues of autophosphorylation in vitro. Using MS, we found four unequivocal target residues: Ser-7, Ser-18, Ser-29, and Ser-43, as well as a fifth peptide carrying one phosphate group for which we could not distinguish whether Ser-175 or Thr-176 was the target residue. This peptide corresponds to the OST1 activation loop (or T loop). Activation loops of many kinases have been extensively studied and their phosphorylation is often necessary for kinase activity of the protein (Johnson et al., 1996
Production of active recombinant proteins in E. coli allowed us to investigate the importance of this activation loop and other residue targets of autophosphorylation by testing in vitro the activity of versions of the protein in which each has been mutated. Our results are in agreement with reports on immunoprecipitated SAPKs (Kobayashi et al., 2004
Production of recombinant proteins also allowed us to investigate the structure-function relationships of the regulatory C terminus of the OST1 protein. Former studies on rice have demonstrated that the SnRK2 family may be divided into three subclasses and only rice kinases from subclass III (SAPK8, SAPK9, and SAPK10) were strongly activated in response to ABA (Kobayashi et al., 2004 We also identify four phosphorylated Ser residues (Ser-7, Ser-18, Ser-29, and Ser-43) and another box (ABA specific) between Leu-333 and the end of the protein (Met-362), which are not required for recombinant OST1 kinase activity. These features represent good candidates as regulatory sites for OST1 function in plants. Mechanisms of activation of OST1 in plants probably involve some of these features through binding of upstream partners or phosphorylations.
OST1 is expressed in vascular tissues of all plant organs, but its function in this context is unknown. In contrast, OST1 is a key regulator of stomatal movements. Because stomata are very specialized structures, it is likely that the mechanisms of OST1 regulation in guard cells differ with respect to other cell types. We use the OST1 promoter to focus our assay for in planta OST1 function on guard cell-signaling pathways regulating stomatal aperture. GUS histochemical staining (Fig. 5A) confirmed that the OST1 promoter principally drives expression in guard cells (Mustilli et al., 2002
Expression in the srk2e mutant of truncated OST1 versions that are inactive when produced in E. coli (OST1
Among the mutations that do not impair recombinant OST1 protein kinase activity, mutated versions on Ser-43 are able to complement the srk2e mutant for guard cell phenotype. Interestingly, the OST1S43A mutant kinase is constitutively active in plants. We propose that phosphorylation of this Ser, which is perfectly conserved in SnRK2 and SnRK3 families, represses OST1 activity in the absence of ABA. Mutations on the three other identified Ser residues affect the ability of OST1 to complement the mutant, but not its activation by ABA in plants. We suggest that Ser-7, Ser-18, and Ser-29 may be targets of phosphorylation and dephosphorylation events in the guard cell ABA-specific signaling pathway, and participate in the regulation of OST1 protein stability, target recognition, or docking of OST1 to a specific transduction complex. Ser-7, in the N-terminal extension peptide specific to OST1, and Ser-18, which links this N-terminal peptide with the catalytic domain (Fig. 3), are specific of the subclass III SnRK2 proteins as defined previously (Kobayashi et al., 2004
Domain-swapping experiments between ABA-responsive and non-ABA-responsive members of the rice SnRK2 family have shown that the carboxy-terminal domain confers ABA responsiveness (Kobayashi et al., 2004 Combining studies of OST1 kinase activity in vitro and OST1 function in guard cells, we could identify two classes of regulatory features. Ser-175 and the SnRK2-specific box are critical for kinase activity and the mechanisms involved are probably shared by all kinases of the SnRK2 family, whatever the signaling pathway in which they act. In contrast, Ser-7, Ser-18, and Ser-29 and the ABA-specific box are required for OST1 function in ABA responses of guard cells. In addition, our results suggest that Ser-43 is involved in repression of OST1 activity in the absence of ABA. These Ser residues may be targets of upstream phosphorylation or dephosphorylation events, and the ABA-specific motif may bind specific regulatory components of the pathway. These phosphorylations and interactions with upstream partners may regulate the function of OST1 kinase in guard cells by modulating its activity, stability, or recruitment of specific substrates.
Plant Material and Culture Conditions
The srk2e (Yoshida et al., 2002
Guard cell protoplasts (GCPs) were prepared essentially as reported (Pandey et al., 2002
Leaves from 4- to 5-week-old plants (grown in 8 h of light at 22°C and 16 h of darkness at 20°C; 70% relative humidity) were harvested in darkness at the end of the night. Abaxial epidermis was incubated for 30 min in darkness in 30 mM KCl and 10 mM MES-KOH, pH 6, at 20°C. Samples were then transferred to light for 3 h with or without 10 µM ABA in the bath solution. Stomatal apertures were measured with an optical microscope (Optiphot-2; Nikon) fitted with a camera lucida and a digitizing table (TG 1017; Houston Instruments) linked to a personal computer.
The OST1 cDNA was amplified by PCR from a pBS-OST1 (Mustilli et al., 2002
OST1 variants were introduced using LR recombination in a pET16b (Novagen) modified to a Gateway destination vector (Invitrogen). This modification was made by insertion of an RfC cassette (Invitrogen) into a blunt-ended BamH1 site. The recombined expression vectors were introduced into the Escherichia coli Rosetta (DE3) pLysS strain (Novagen). Bacteria were grown to OD 0.6 at 600 nm and 0.4 mM isopropylthio-
To detect and immunoprecipitate OST1 from wild-type plants, we produced an OST1-specific antibody (OST1N) against a synthetic peptide corresponding to the N terminus of the protein (MDRPAVSGPMDLC). The peptide was coupled to the keyhole limpet hemocyanin through cystein 13 and polyclonal antiserum was raised in rabbit (Eurogentec).
Whole plants were ground using a Mixer Mill MM 301 (Retsch). Proteins were extracted in an immunoprecipitation buffer: 20 mM HEPES, pH 7.5, 2 mM EDTA, 2 mM EGTA, 4 mM dithiothreitol, 10 mM NaF, 50 mM For phosphatase treatment of immunoprecipitated proteins, 30 µL of the protein A-Sepharose slurry were incubated at 37°C during 16 h with 10 units of calf intestinal alkaline phosphatase (New England Biolabs).
Full-length recombinant proteins purified from E. coli were subjected to MS analyses before any kinase assay. They were analyzed by MALDI-MS (reflex III; Bruker) in the linear mode using sinapinic acid as a matrix and bovine serum albumin for external calibration (singly and doubly charged species). ESI-MS (Q-TOF micro; Waters) of OST1 isoforms was achieved after online fast desalting on a C18 column (300-µm i.d.). Multiply charged species of horse heart myoglobin were used for external mass calibration. Nano-LC-ESI-MS/MS spectra of tryptic peptides were acquired with a Q-TOF micro (Waters) instrument interfaced to a CapLC chromatographic system using a C18 column, 75-µm i.d. (Waters). Accurate mass analysis was obtained by means of singly and doubly charged ion species of reference peptides as lock masses (lockspray system; Waters).
MALDI-MS and MALDI-MS/MS data were acquired with a Q-TOF Ultima mass spectrometer (Waters). The matrix was
Phosphorylation assays on recombinant proteins were performed by incubation for 45 min at room temperature of 100 ng kinase and 200 ng histone III-S substrate (Sigma) in 12.5 µL of 20 mM HEPES, pH 7.5, 0.5% (v/v) Triton X-100, 2 mM MnCl2, 1x protease inhibitor cocktail (Roche), 10 mM NaF, and 5 mM Proteins were separated by SDS-PAGE using a 10% (w/v) acrylamide gel and transferred to a nitrocellulose membrane. Radioactivity was detected on the dried membranes using a Storm imaging system (Molecular Dynamics). The same membrane was used, after scanning, for immunodetection of recombinant proteins (see below). Quantification of the activity was performed using the public domain image analysis program ImageJ, version 1.32j (http://rsb.info.nih.gov/ij). Radioactive bands were quantified using the plot lanes function on the image of the scan and data were normalized using the level of recombinant kinases quantified by the same method on immunoblot images.
Protein extraction and in-gel protein kinase assay were performed as described (Droillard et al., 2000
The OST1 promoter excised from the pOST1::GUS vector (Mustilli et al., 2002
A transcriptional fusion between the OST1 promoter and uidA was generated by recombination of the pENTR-gus vector (Invitrogen) with our p$POHA vector. We performed GUS histochemical staining on five srk2e/ProOST1:3xHA-gus lines as described (Jefferson et al., 1987
Thermal imaging was performed on leaves 5 to 10 min after they were detached from 3-week-old plants grown in the greenhouse. The Col-0 and srk2e controls were present on each image with transgenic lines (three to four leaves from independent plants for each line). Images were obtained using a Thermacam PM250 infrared camera (Inframetrics), saved on a PCMCIA memory card, and analyzed using the public domain image analysis program ImageJ, version 1.32j (http://rsb.info.nih.gov/ij) as described previously (Merlot et al., 2002
3xHA-tagged proteins extracted from plants were separated by SDS-PAGE using a 10% (w/v) acrylamide gel, transferred on nitrocellulose membranes, and detected by monoclonal anti-HA antibody (1:10,000; Sigma) and 10xHis-tagged recombinant proteins used for in vitro kinase assays were detected by monoclonal anti-His antibody (1:10,000; Sigma). Then they were revealed by a horseradish peroxidase-conjugated anti-mouse IgG (1:10,000; Sigma) or anti-rabbit IgG (1:10,000) using the chemiluminescence ECL plus kit (Amersham Biosciences). All western-blot analyses were performed as described (Sambrook et al., 1989
We thank Dr. J. Coffey, Dr. D. Uria, and Dr. N. Tomczyk for MS experiments performed with OST1 isoforms; Marie-Ange Badet-Denisot and Nathalie Leonhardt for their help with the circular dichroism technique and stomatal bioassays, respectively; Helen North, Thierry Gaude, Thierry Meinnel, and Sylvain Merlot for helpful discussions; and Daniel Couch for his critical reading of the manuscript. Received February 24, 2006; returned for revision May 31, 2006; accepted June 1, 2006.
1 This work was supported by the Centre National de la Recherche Scientifique and by a fellowship from the Ministère de l'Education Nationale de la Recherche et des Nouvelles Technologies (to C. Belin).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christophe Belin (belin{at}isv.cnrs-gif.fr).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.079327. * Corresponding author; e-mail belin{at}isv.cnrs-gif.fr; fax 33–1–69823768.
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TOP
ABSTRACT
RESULTS
-33P]ATP as a substrate or with the addition of unlabeled ATP 1 min prior to stopping the reaction, shows that the signal at OST1 size is due to autophosphorylation rather than binding of radioactive ATP (
-galactoside induction; S, soluble fraction; E, elution. B, In vitro kinase activity (top) of the native recombinant 10xHis-OST1 (OST1) and the mutated version 10xHis-OST1G33R (G33R). C, In vitro kinase activity (top) of the native recombinant 10xHis-OST1 without (–) or with (+) histone III-S substrate and with usual [
-33P]ATP (
