The Cytosolic Kinases STY8, STY17, and STY46 ... - Plant Physiology

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dopsis (Arabidopsis thaliana; Martin et al., 2006). STY8 belongs to a ...... cotyledon1 and snowy cotyledon2 (Albrecht et al., 2006,. 2008), white cotyledon ...
The Cytosolic Kinases STY8, STY17, and STY46 Are Involved in Chloroplast Differentiation in Arabidopsis1[W] Giorgia Lamberti, Irene L. Gu¨gel, Jo¨rg Meurer, Ju¨rgen Soll, and Serena Schwenkert* Department of Biology I, Botany, Ludwig-Maximilians-Universita¨t Mu¨nchen, D–82152 Planegg-Martinsried, Germany (G.L., I.L.G., J.M., J.S., S.S.); and Munich Center for Integrated Protein Science, LudwigMaximilians-Universita¨t Mu¨nchen, D–81377 Munich, Germany

In Arabidopsis (Arabidopsis thaliana), transit peptides for chloroplast-destined preproteins can be phosphorylated by the protein kinases STY8, STY17, and STY46. In this study, we have investigated the in vitro properties of these plant-specific kinases. Characterization of the mechanistic functioning of STY8 led to the identification of an essential threonine in the activation segment, which is phosphorylated by an intramolecular mechanism. STY8 is inhibited by specific tyrosine kinase inhibitors, although it lacked the ability to phosphorylate tyrosine residues in vitro. In vivo analysis of sty8, sty17, and sty46 Arabidopsis knockout/knockdown mutants revealed a distinct function of the three kinases in the greening process and in the efficient differentiation of chloroplasts. Mutant plants displayed not only a delayed accumulation of chlorophyll but also a reduction of nucleus-encoded chloroplast proteins and a retarded establishment of photosynthetic capacity during the first 6 h of deetiolation, supporting a role of cytosolic STY kinases in chloroplast differentiation.

The majority of proteins localized to chloroplasts are encoded by the nuclear genome, synthesized in the cytosol of the plant cell, and posttranslationally imported into the organelle (Jarvis, 2008). In most cases, a cleavable transit peptide directs the preproteins to the chloroplast and facilitates recognition at the outer membrane TOC (for translocon at the outer envelope of chloroplasts) complex, which subsequently transports the preprotein across the membrane, handing it over to the TIC (for translocon at the inner envelope of chloroplasts) complex (Balsera et al., 2009). However, little is known about the stages of preprotein passage after translation in the cytosol and before their interaction with the TOC complex. Despite the diversity of transit peptides in their amino acid composition and the absence of any specific secondary structure, an overall positive charge and the predominant presence of Ser and Thr are two of the unifying features of chloroplast transit peptides (Bruce, 2000, 2001). In recent years, it has been shown that these Ser and Thr residues often lie within 14-3-3binding motifs and can be reversibly phosphorylated (Waegemann and Soll, 1996; May and Soll, 2000). 1

This work was supported by the Deutsche Forschungsgemeinschaft. * Corresponding author; e-mail [email protected]. 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: Serena Schwenkert ([email protected]). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.111.182774 70

Phosphorylation on Ser or Thr residues can regulate the affinity for 14-3-3 proteins with their substrates dynamically (Muslin et al., 1996). The 14-3-3 proteins are eukaryotic, small (approximately 30 kD) acidic proteins that readily dimerize and interact with a large number of different substrates involved in various cellular processes in plants and animals (Dougherty and Morrison, 2004; Bridges and Moorhead, 2005). Together with the molecular heat shock chaperone HSP70, they bind to chloroplast preproteins, most likely very soon after their translation, possibly preventing their aggregation and enhancing the import rate of the preproteins (May and Soll, 2000). Although lack of phosphorylation does not prevent protein import or lead to mistargeting (Nakrieko et al., 2004), it elevates transport rates mediated by a higher affinity to the receptor protein Toc34 (May and Soll, 2000). Analysis of the binding of 14-3-3 to preproteins revealed that this is not restricted to a few exceptions: approximately 25% out of a population of 41 preproteins were found to associate with 14-3-3 (Fellerer et al., 2011). Additionally, dephosphorylation of chloroplast preproteins has likewise been shown to influence protein import, since it is indispensable for efficient transport of preproteins (Waegemann and Soll, 1996). However, it is so far unclear at what stages of plant development or under which environmental conditions transit peptide phosphorylation is physiologically relevant in chloroplast biogenesis. In a recent attempt to isolate the kinase(s) responsible for transit peptide phosphorylation, the protein kinase STY8 was purified from a leaf extract of Arabidopsis (Arabidopsis thaliana; Martin et al., 2006). STY8 belongs to a plant-specific family of dual-specificity

Plant PhysiologyÒ, September 2011, Vol. 157, pp. 70–85, www.plantphysiol.org Ó 2011 American Society of Plant Biologists. All Rights Reserved.

Chloroplast Differentiation Influenced by STY Kinases

STY (for Ser/Thr/Tyr) kinases, which possess catalytic domains for both Ser/Thr and Tyr phosphorylation and comprise 57 representatives in Arabidopsis (Rudrabhatla et al., 2006). Two homolog isoforms of STY8, STY46 and STY17, are found in the Arabidopsis genome and share 89.3% amino acid sequence similarity. The kinase family is found exclusively in plants (i.e. monocots and dicots, green algae, mosses, and ferns) but is absent from animals, fungi, and yeast. All three kinases were shown to phosphorylate several chloroplast preproteins on Ser and Thr residues in vitro (Martin et al., 2006). In this study, we have thoroughly characterized the enzymatic properties of STY8, STY17, and STY46 in vitro. Our data revealed that STY8 activity is dependent upon the intramolecular phosphorylation of a conserved Thr residue in the activation segment and represents an unusual subclass of the STY kinases, since it shows conserved sequence motifs of Tyr kinases and biochemical characteristics of Ser/Thr kinases. In order to elucidate the function of the three STY kinases in vivo, we have analyzed single, double, and triple mutants of STY8, STY17, and STY46 in Arabidopsis, showing that chloroplast biogenesis in cotyledons is affected during the greening process in mutant plants, thus implying a possible role of preprotein phosphorylation in the differentiation process.

RESULTS A Conserved Autophosphorylated Thr Is Essential for the Activity of STY8, STY17, and STY46

To characterize the enzymatic properties of the three chloroplast transit peptide-phosphorylating kinases STY in vitro, STY8 (At2g17700), STY17 (At4g35708), and STY46 (At4g38470) full-length cDNAs were cloned into a pET21d vector, expressed in Escherichia coli, and purified via a C-terminal His tag on Ni2+-Sepharose. To investigate the dependence of the autophosphorylation of STY8 on the presence or absence of cations, 10 mM Mg2+, 10 mM Mn2+, and 10 mM Ca2+ were added during the phosphorylation reaction (Fig. 1A). Full autophosphorylation activity was only achieved in the presence of Mn2+. Mg2+only had a slight activating effect, whereas Ca2+ alone was not able to promote kinase activity at all but did not inhibit it either upon simultaneous incubation with Mn2+. To further investigate the necessity of phosphorylation for kinase activity and substrate phosphorylation, the His-tagged kinase was treated with l-phosphatase to dephosphorylate the kinase completely, repurified with Ni2+-Sepharose to remove the l-phosphatase, and subjected to an in vitro kinase assay. Efficient dephosphorylation was monitored with ProQ diamond stain (Invitrogen). The preprotein of the small subunit of the chloroplast ribulose-1,5bisphosphatase (pSSU), which is phosphorylated (Waegemann and Soll, 1996) in the transit peptide in Plant Physiol. Vol. 157, 2011

vitro, was used as a model substrate and subjected to a kinase assay with phosphorylated kinase as purified from E. coli or dephosphorylated kinase (Fig. 1B). Kinase phosphorylation is observed in the purified sample by radioactive labeling, which suggests that autophosphorylation takes place. Phosphorylation of pSSU was already visible after incubation for 1.5 min with STY8, whereas phosphorylation of the dephosphorylated kinase was clearly slower and no phosphorylation of the substrate could be observed, even after 3 min of reaction time (Fig. 1B). These results suggest that kinase phosphorylation or possibly even autophosphorylation is important for full activity of STY8. As a next step, therefore, we attempted to determine possible autophosphorylation site(s) and their roles in kinase activation. The primary sequences of all three kinases can be divided into 11 kinase-typical subdomains (Fig. 1C; Hanks et al., 1988) harboring the activation segment flanked by the highly conserved peptide motifs DFG (in subdomain VII) and APE (in subdomain VIII). Mass spectrometric analysis identified a phosphorylated Thr in all three kinases that is conserved among STY8, STY17, and ST46 and lies within the activation segment as the major phosphorylation site (for data from www.phosphat.mpimpgolm.mpg.de, see Supplemental Table S1; Heazlewood et al., 2008; Durek et al., 2009; Ito et al., 2009). Supplemental Table S1 includes information on the validated phosphorylated sites and the conditions under which the experiments were performed. The Thr was substituted to Ala by site-directed mutagenesis in all three kinases, resulting in the constructs STY8-T439A, STY17-T445A, and STY46-T443A (Fig. 1D). Phosphorylation of the purified kinase was abolished completely upon incubation with radiolabeled ATP, indicating that autophosphorylation occurs. Moreover, phosphorylation of the substrate pSSU was abolished completely (Fig. 1D). Additionally, activity was not restored when the Thr was substituted by a Ser or Tyr, which could potentially be phosphorylated (data not shown). Therefore, we conclude that the conserved Thr in the activation segment is indispensable for kinase activity. Autophosphorylation of the Kinase Occurs Intramolecularly

To investigate the molecular mechanism of Thr autophosphorylation, we chose two different approaches to distinguish between an intramolecular (in cis) and an intermolecular (in trans) autophosphorylation event. First, we generated a mutated kinase that lacked any activity but could still be phosphorylated on its Thr in the activation segment. An exchange of a conserved Lys residue at position 409 (located in subdomain VI) in STY8 (Fig. 1C) to an Arg resulted in complete loss of autophosphorylation and substrate phosphorylation (Supplemental Fig. S1). Such an invariant Lys, which is directly involved in the phospho71

Lamberti et al. Figure 1. STY8, STY17, and STY46 are autophosphorylated on a conserved Thr residue in the activation segment. A, An in vitro kinase assay was performed with 3 mg of purified STY8 in the presence of Mg2+, Mn2+, and/or Ca2+ for 10 min, and autophosphorylation was detected by autoradiography, showing a strong dependence on Mn2+. Coomassie blue staining (CBB) shows equal amounts of STY8 (bottom panel). B, STY8 was treated with l-phosphatase, and phosphorylation of STY8 and pSSU was monitored subsequently for the indicated time points. STY8 at 0.5 mg and 2 mg of pSSU were used. C, Protein alignment of STY8, STY17, and STY46. The ACT domain and the activation segment are boxed. Conserved motifs (1–3) mediating substrate specificity are shown in blue, and the conserved APE and DFG motifs flanking the activation segment are shown in yellow. The conserved Lys (position 409 in STY8, 415 in STY17, and 413 in STY46) as well as the Thr, which are essential for kinase activity, are shown in pink. D, A conserved Thr in the activation segment (position 439 in STY8, 445 in STY17, and 443 in STY46; highlighted in C) was substituted by an Ala in all three kinases and leads to complete loss of activity, as shown in an in vitro kinase assay with the substrate pSSU. STY8 at 0.5 mg and 2 mg of pSSU were used for the reactions. Autoradiographs (top two panels) and Coomassie blue staining showing the purified proteins (bottom two panels) are shown.

transfer reaction, is found in almost all kinases (Kamps and Sefton, 1986). Increasing amounts of STY8-K409R were used as a substrate for invariable amounts of wild-type STY8 (Fig. 2A). In the case of in trans phosphorylation of the inactive kinase by the active kinase, an increasing phosphorylation signal would be expected. However, since the amount of phosphorylated kinase remained unchanged, phosphorylation is likely to occur via an intramolecular mechanism. In a second approach addressing this question, we performed an in vitro kinase assay in a smaller (25 mL) and a larger (250 mL) reaction volume with equal amounts of kinase. Figure 2B shows that phosphorylation is not decreasing with a larger reaction volume, as we would expect in the case of an intermolecular process due to the dilution, thus also favoring an intramolecular mechanism for the autophosphorylation. 72

Kinase Activity Is Regulated by an ACT Domain

A BLAST search for conserved protein domains revealed that all three kinases contain an additional conserved functional domain upstream of the Ser/Thr and Tyr kinase domains, a so-called ACT domain, which has been described as a small molecule-binding regulative domain. The domain was named after the first proteins in which it was identified, Asp kinase, chorismate mutase, and tyrA (prephenate dehydrogenase; Grant, 2006). In STY8, the motif comprises amino acids 185 to 253. To investigate the function of the ACT domain in STY8, we deleted the domain and subjected the mutated kinase to an in vitro phosphorylation assay with pSSU and pOE23 as substrates (Fig. 2C). Surprisingly, the mutated protein exhibited higher activity than the wild-type protein. The yield of phosphorylation of pSSU and pOE23 Plant Physiol. Vol. 157, 2011

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autophosphorylation and substrate phosphorylation, even when applied at low concentrations ranging from 0.01 to 10 mM (Fig. 3A). Inhibitory effects were observed with respect to autophosphorylation (Fig. 3B) as well as substrate phosphorylation (Fig. 3C). Dimethyl sulfoxide, which was used as a solvent for the inhibitors, did not have an impact on the reaction. Strikingly, all three effective inhibitors, JNJ-10198409, tyrphostin, and Janex 1, are typical small-molecule Tyr kinase inhibitors. A summary of the tested inhibitors, their specificities, and their effect on STY8 is provided in Supplemental Table S2. To verify whether these inhibitors also have an effect on a typical Ser/Thr kinase, we tested the autophosphorylation or substrate phosphorylation of CPK4, a well-characterized plant Ser/Thr kinase (Hrabak et al., 2003; Fig. 3D). None of the inhibitors affected the phosphorylation of CPK4, indicating a resemblance between the dual-specificity STY kinases and classical Tyr kinases. In our experiments, we used an ATP concentration of 2.5 mM to allow the usage of an equal concentration of labeled and unlabeled ATP, which is below the determined Km value of 21.6 mM (Martin et al., 2006). To exclude the possibility that the low amount of ATP elevates the inhibitor sensitivity, we performed the experiment with 21.6 mM ATP (Supplemental Fig. S2). No change of the inhibitory effects was observed in comparison with Figure 3A. Substrate Phosphorylation Occurs Posttranslationally

Figure 2. Autophosphorylation of STY8 occurs by an intramolecular mechanism. A, Increasing amounts of mutated STY8-K409R were added to constant amounts of wild-type STY8 in an in vitro kinase assay. Phosphorylation of STY8-K409R did not increase with higher concentrations. B, An in vitro kinase assay was performed in a sample volume of 25 or 250 mL, and samples were taken at three time points as indicated. Equal amounts of STY8 are shown by Coomassie blue staining (CBB; bottom panel). C, The ACT domain regulates the activity of STY8. Phosphorylation of pSSU and pOE23 is shown with wild-type STY8 and STY8-DACT, which lacks the ACT domain. mSSU (the mature part of pSSU) is used as a nonphosphorylatable control (top panel). A Coomassie blue gel shows equal loading of proteins (bottom panel), and the purified proteins are indicated with arrows.

(Waegemann and Soll, 1996; Martin et al., 2006) was double than with wild-type kinase, suggesting that the domain could be involved in regulating kinase activity in planta. STY8 Is Inhibited by Tyr Kinase Inhibitors

To investigate the effect of potential inhibitors on STY8, a kinase inhibitor library comprising 64 different kinase inhibitors (Cayman; Supplemental Table S2), including 44 Ser/Thr and 20 Tyr kinase inhibitors, was screened for substances inhibiting STY8. Only three of those inhibitors proved to have a strong effect on STY8 Plant Physiol. Vol. 157, 2011

To gain insight into whether phosphorylation of preproteins occurs cotranslationally or posttranslationally, we followed the phosphorylation status of pSSU during in vitro translation in a wheat germ lysate, which contains endogenous kinase. The nonphosphorylatable pSSU S31/34A mutant was used as a control. The 35S-labeled translated proteins were purified after the translation reaction via a C-terminal His tag and separated on two-dimensional gels (Fig. 4A, top panel). Three distinct spots were visible in the wild-type pSSU sample representing nonphosphorylated (isoelectric point [IEP]: 7.71), single phosphorylated (IEP: 6.89) and double phosphorylated (IEP: 6.54) pSSU. In the pSSU S31/34A sample, only the nonphosphorylated form (IEP: 7.71) was detected, as expected (Fig. 4A, middle panel). When wild-type pSSU was treated with phosphatase prior to twodimensional gel electrophoresis, only the nonphosphorylated form of pSSU was detected, comparable to the mutant (Fig. 4A, bottom panel). A possible cotranslational mechanism of substrate phosphorylation was investigated by stalling the ribosomes to the nascent peptide chain during translation. Wild-type pSSU and pSSU S31/34A, both equipped with a C-terminal stalling sequence (Bhushan et al., 2010), were translated in vitro, purified with a N-terminal His tag, and the phosphorylation status was visualized on two-dimensional gels (Fig. 4B). In this case, only the nonphosphorylated form was detected (IEP: 8.21), 73

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Figure 3. STY8 is inhibited by typical Tyr kinase inhibitors. A, The kinase inhibitors JNJ-10198409, tyrphostin, and Janex 1 were added to an in vitro kinase assay in increasing amounts. Constant amounts of 1 mg of STY8 and 2 mg of pSSU were used. A control reaction with the highest used concentration of dimethyl sulfoxide (DMSO; corresponding to 0.1% of the reaction volume) is shown. B and C, Autophosphorylation of STY8 (B) as well as substrate phosphorylation (pSSU; C) decrease in the presence of kinase inhibitors in a concentration of 0 to 10 mM. D, The Ser/Thr kinase CPK4 is not inhibited by 10 mM JNJ-10198409, tyrphostin, or Janex 1. CBB, Coomassie blue.

indicating that the phosphorylation sites are not accessible to the kinase during the translation process but that phosphorylation of the preproteins rather occurs posttranslationally.

Substrate Phosphorylation and Autophosphorylation Occur on Ser and Thr Residues

Since the kinases belong to a family of dual-specificity kinases that have been shown to phosphorylate hydroxyl groups of Ser, Thr, as well as Tyr, phosphoamino acid analysis was performed with autophosphorylated kinase as well as the substrate protein MBP (for myelin basic protein). MBP was used as a substrate protein, since Tyr phosphorylation on MBP has been observed previously (Reddy and Rajasekharan, 2006) and the transit peptide of pSSU does not contain any Tyr residues that could potentially be phosphorylated. The proteins were subjected to an in vitro kinase assay in the presence of 32P, and phosphorylated proteins were excised from a SDS gel and hydrolyzed in 6 M HCl. The hydrolyzed phospho-amino acids were then separated by thin-layer chromatography (TLC), and phosphorylated amino acids were visualized by autoradiography. The phosphorylated amino acids were compared with ninhydrin-stained amino acids run in parallel. Interestingly, phosphorylation was restricted to Ser and Thr residues not only in the kinase but also in the model substrate MBP (Fig. 4C). To confirm the lack of Tyr phosphorylation by STY8, we performed a kinase assay with STY8 and Yes, 74

a typical Tyr kinase of the Src protein kinase family (Fig. 4D). Phosphorylated amino acids were detected by specific antisera. The substrate protein MBP was efficiently phosphorylated by Yes on Tyr residues but not by STY8, even though almost 10-fold STY8 excess over Yes was used. Thr phosphorylation, however, was observed only with STY8, not with Yes. Inactivation of STY8, STY17, and STY46 in Arabidopsis Results in Retarded Growth

STY8 was previously shown to localize to the cytosol (Martin et al., 2006). The two homolog kinases STY17 and STY46 likewise do not constitute any predicted signaling sequences (TargetP; Emanuelsson et al., 2007) and were localized to the cytosol when expressed as N-terminal GFP fusion proteins in isolated Arabidopsis protoplasts (Supplemental Fig. S3). To study the effect of transit peptide phosphorylation in planta, we isolated loss-of-function mutants for STY8 and STY46. Homozygous lines with the T-DNA insertion located in exon 3 (sty8) and in exon 9 (sty46) were obtained (Fig. 5A) and crossed to generate double mutants. No residual RNA was detected in either of the single or double mutant lines, as shown by reverse transcription (RT)-PCR (Fig. 5B). Since no T-DNA insertion lines were available for STY17, an RNA interference (RNAi) approach was applied to generate sty17 knockdown lines in the wild type as well as the sty8 sty46 double mutant background. A 400-bp fragment corresponding to the N-terminal part of STY17, Plant Physiol. Vol. 157, 2011

Chloroplast Differentiation Influenced by STY Kinases

Figure 4. Substrate phosphorylation occurs posttranslationally on Ser and Thr residues. A, pSSU was in vitro translated in wheat germ lysate and subjected to isoelectric focusing, resulting in three distinct spots corresponding to nonphosphorylated pSSU (0P) and pSSU phosphorylated on one residue (1P) or on both residues (2P; top panel). pSSU S31/34A and pSSU dephosphorylated after the translation reaction were used as controls, only showing nonphosphorylated protein (middle and bottom panels). TL shows equal translation efficiencies for pSSU and pSSU S31/34A. B, pSSU fused to a C-terminal ribosomal stalling sequence was subjected to in vitro translation and again separated by isoelectric focusing. In wild-type pSSU (top panel) and pSSU S31/34A (bottom panel), only a spot corresponding to the nonphosphorylated pSSU (0P) was detectable. TL shows equal translation efficiencies for pSSU and pSSU S31/34A. C, STY8 and MBP were phosphorylated, and the corresponding bands were cut from a SDS gel and hydrolyzed. Phosphorylated amino acids were subsequently analyzed by TLC and identified by comparison with marker proteins. D, MBP was phosphorylated with STY8 and Yes, and phosphorylated amino acids were detected with phospho-Thr or phospho-Tyr antibodies. Yes at 2 mg and 15 mg of STY8 were used. Coomassie blue (CBB) staining of MPB is shown as a loading control.

which does not contain any of the conserved protein domains, was cloned in the sense and antisense orientations into the Gateway vector pB7GWIWG2(II), and wild-type as well as sty8 sty46 plants were transformed with the construct. Transformants were selected by BASTA resistance, and the F2 generation was analyzed at the RNA and protein levels to verify the extent of STY17 reduction. RNA levels of STY17 in the Plant Physiol. Vol. 157, 2011

double mutant background were significantly reduced to 10% to 30% in lines 14, 16, and 21, as demonstrated by quantitative RT-PCR (Fig. 5C). Analyses of the protein level with specific STY17 antisera confirmed these results, showing a reduction to below 25% of wild-type protein levels (Fig. 5D) in the respective lines, which were consequently used for further analyses. Growth phenotypes of all mutant plants were analyzed under several conditions, including long day (16 h of light), short day (8 h of light), constant light (24 h of light), low light (10 mmol photons m22 s21), high light (500 mmol photons m22 s21), and cold stress (+10°C). sty8 single mutants did not show any visible phenotype under all conditions tested, whereas sty46 single and sty8 sty46 double mutants were clearly retarded in growth, especially under long-day conditions (Fig. 5E, top panel). sty17 knockdown mutants in the wild-type background were indistinguishable from wild-type plants, even if the protein levels of STY17 were severely reduced to less than 10% of wildtype levels (Supplemental Fig. S4). RNAi knockdown in the background of sty8 sty46 resulted in an even more pronounced retardation of growth (Fig. 5E, middle panel). Five-week-old mutant plants displayed a delay of bolting by over 1 week in comparison with the wild type (Fig. 5F), although the principal inflorescence stem reached the same length after 6 weeks. The delayed development was solely due to retardation in growth, since germination was not found to be affected in the mutants (data not shown). To ensure that the observed phenotype resulted from inactivation of the kinases and was not due to background mutations in any of the lines, complementation analyses were performed with the double mutant sty8 sty46. The full-length cDNA of STY8 and STY46 was cloned under the control of the 35S cauliflower mosaic virus promoter, and wild-type as well as mutant plants were transformed with the construct. STY8 cDNA was not able to rescue the sty8 sty46 phenotype (data not shown). The STY46 cDNA, however, was sufficient to completely restore the wild-type phenotype (Fig. 5, E, bottom right panel, and F), which is consistent with the observation of a growth phenotype in the sty46 single mutant. Successful expression of STY46 in three independent complemented lines is demonstrated by RT-PCR (Fig. 5G). Chloroplast Ultrastructure Is Changed in Cotyledons of Kinase Mutant Plants

To verify whether retardation in growth was accompanied by ultrastructural changes, we analyzed wildtype and mutant plants by transmission electron microscopy. Overviews of the mutant tissues frequently showed cells with slightly smaller vacuoles and plastids that were more round bodied. Plastids of 7- and 14-d-old cotyledons of sty8 sty46 double mutants were analyzed in more detail. They showed abnormal shape, and thylakoid formation was overall affected (Figs. 6 and 7; Supplemental Fig. S3). The 75

Lamberti et al. Figure 5. Isolation of sty8 and sty46 T-DNA insertion lines, generation of sty17 RNAi knockdown lines, and complementation of the mutants. A, Gene model showing the positions and orientations of T-DNA insertions in SALK 072890 (sty8) and SALK 116340 (sty46). Oligonucleotides used in B are indicated. LB, Left border; RB, right border. B, RT-PCR was performed with single and double mutants of sty8 and sty46 and the wild type (WT). HCF136 was amplified as a control. C, Independent sty17 RNAi lines (14, 16, and 21) in the background of sty8 sty46 were analyzed by quantitative RT-PCR. Values were calculated relative to 103 molecules of ACTIN2, and expression levels relative to the wild type are given. D, Immunoblot analysis of sty17 RNAi lines (14, 16, and 21) in the background of sty8 sty46. For 100%, 20 mg of protein was loaded on a 12% SDS gel, transferred to a polyvinylidene difluoride membrane, and immunodecorated with specific STY17 antisera. Ponceau staining of the large subunit of Rubisco is shown as a control. E, Phenotype analysis of wild-type, mutant, and complemented lines. Plants are shown 4 weeks after germination on soil. Bars = 1 cm. F, Flowering phenotype of wild-type and mutant plants 5 weeks after germination. G, RT-PCR analysis of the wild type, sty8 sty46, and sty8 sty46/35SSTY46, demonstrating the expression of STY46 in the complemented line.

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results of ultrastructural analysis of the organelles revealed a time gradient of developmental retardation. In 7-d-old cotyledons of wild-type plants (Fig. 6A; Supplemental Fig. S5A), chloroplasts in mesophyll cells showed a typically ellipsoidal shape with grana stacks and stroma thylakoids (Pyke, 2007). In 7-d-old cotyledons of mutant plants (Fig. 6B; Supplemental

Figure 6. Ultrastructure of chloroplasts from 7-d-old cotyledons of Arabidopsis plants. A, The wild type. Defined poles are indicated by arrows. B, sty8 sty46. sty8 sty46 affects chloroplast morphology and thylakoid ultrastructure in different and extensive stages. Blown-up thylakoids, grana stacks loosely appressed with blown-up lumen (left panels), membrane-bound vesicles mainly in the vicinity of grana stacks indicated by the arrow (right panels), and often missing membrane connections between closely juxtaposed grana stacks are shown. Bars = 1 mm (top panels) and 200 nm (middle and bottom panels). The inset magnification is 110,0003. Plant Physiol. Vol. 157, 2011

Figure 7. Ultrastructure of chloroplasts from 14-d-old cotyledons of Arabidopsis plants. A, The wild type. B, sty8 sty46. sty8 sty46 chloroplasts with curved structured grana stacks and disoriented arrangement (top panels), grana stacks lacking connection to stroma thylakoids (left middle panel, inset), curved structure of grana stacks (right middle panel), star-shaped fragments of vesicular and tubular membranes (left bottom panel), and nearly hexagonal vesicular or tubular membranes (right bottom panel and insets) are shown; described structures are indicated by arrows. Bars = 1 mm (top panels) and 200 nm (middle and bottom panels). Magnification of the insets in the left panels is 110,0003. 77

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Fig. S6B), discrepancies from wild-type chloroplasts could be observed in every chloroplast of the mesophyll cells. We observed different and extensive stages of disordered thylakoids. Grana stacks of thylakoids were only loosely appressed and the lumen was blown up, mainly at both of the chloroplast-defined poles, where we found converging thylakoids in the wild type (Pyke, 2007; Fig. 6A, top panel) but also at the top and bottom parts of the plastids (Fig. 6B, left panel). Plastids from mutant plants contained more and larger membrane-bound vesicles, frequently in the vicinity of developing grana stacks (Fig. 6B, right panel). Membrane connections between closely juxtaposed grana stacks were often missing. In addition, the network of stroma thylakoids was only partially developed, and the thylakoids appeared more like isolated membrane arrays in many cases. Altogether, the chloroplasts were less elongated and more disordered, and the thylakoids seemed to be improperly arranged in comparison with the wild type (Fig. 6; Supplemental Fig. S5, A and B). In 14-d-old cotyledons of mutant plants (Fig. 7B; Supplemental Fig. S5, C–E), developing chloroplasts frequently showed grana stacks with a curved structure and disoriented arrangement. In many cases, the grana stacks lacked a connection to stroma thylakoids and ended in a curved conformation (Fig. 7B, top and middle panels). The thylakoid lumen appeared normal, in contrast to 7-d-old plants. Sometimes, we observed star-shaped fragments of vesicular and tubular membranes, also in combination with small osmiophilic structures between nonconnected grana stacks (Fig. 7B, bottom panel, left). These structures are most likely distinct from a typical prolamellar body and have been observed before in electron microscopic studies on plastid differentiation (Menke, 1960). Furthermore, in mutant plants, stacks of nearly hexagonally arranged vesicular or tubular membranes were regularly found at all anatomical areas of plastids, meaning the defined poles and at the top and bottom parts of the plastids (Fig. 7B, bottom panel right). In the wild type, these were almost never observed (this was also the case in 21-d-old cotyledons; data not shown). These membrane stacks resemble those described as Heitz-Leyonsche crystals (Menke, 1960). The ultrastructure of the plastids in cotyledons of complemented plantlets was fully restored to the wildtype phenotype (Supplemental Fig. S5, A and E). Kinase Mutant Cotyledons Are Affected in Chlorophyll Accumulation and Show Impaired Photosynthesis during Greening

Chloroplast differentiation from etioplasts to chloroplasts requires the massive influx of preproteins into the organelle to form thylakoids and the photosynthetic machinery. Therefore, seedlings of the wild type and mutants were grown in darkness for 6 d and subsequently transferred to light for several hours to study possible differences during chloroplast differ78

entiation. Interestingly, the greening process was significantly delayed in sty8 sty46 and sty8 sty46 sty17, as observed by coloring of the cotyledons (Fig. 8A) as well as by quantitative measurements of the chlorophyll a and b contents 2, 4, and 6 h after the transition to light (Fig. 8, B and C). The measurements demonstrate that greening is hampered in the mutants and

Figure 8. Greening is delayed in kinase mutant plants. A, Five representative seedlings of the wild type (WT) and mutants are shown 2, 4, and 6 h after illumination. B, Chlorophyll concentration was measured 2, 4, and 6 h after exposure of etiolated seedlings to light (50 mE m22 s21). Chlorophyll concentration was reduced to 50% to 60% of wildtype levels during the entire greening process in double and triple mutants. C, Chlorophyll concentration was measured in mg mg21 fresh weight and increases in the wild type and mutants during the first 6 h of illumination (n = 3). D, Fv/Fm was measured at the indicated time points after illumination in wild-type and mutant plants (n = 3). Plant Physiol. Vol. 157, 2011

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that chlorophyll accumulation only reaches about 50% of the wild-type level after 2, 4, and 6 h (Fig. 8B). Nevertheless, chlorophyll accumulation is not completely impaired, as the total chlorophyll concentration increases continuously during the first 6 h after illumination in mutants and the wild type (Fig. 8C). Chlorophyll a fluorescence measurements performed during the greening process clearly demonstrated a delay in the photosynthetic performance of PSII in mutant plants. The maximum quantum yield of PSII, measured as a ratio Fv/Fm, was severely decreased to 0.22 6 0.034 in sty8 sty46 sty17-14 compared with wild type, with 0.36 6 0.011 after 4 h of illumination (Fig. 8D). Measurements after 2 h of illumination were not feasible, since the signal obtained by the saturating light pulse was hardly above background noise. However, a significant difference in Fv/Fm was observed in the early time period from 4 to 6 h. Mutant plants only reached the same photosynthetic efficiency as the wild type after 22 h of illumination (wild type, 0.74 6 0.016; sty8 sty46 sty17-14, 0.72 6 0.01). Nucleus-Encoded Chloroplast Proteins Accumulate Slower during the Transition from Etioplasts to Chloroplasts

To elucidate defects of chloroplast development on the protein level, total proteins were isolated 2 h after illumination and immunodecorated with several antisera against chloroplast-localized proteins (Fig. 9A). All immunoblots were repeated at least three times, and the bands were quantified (Fig. 9B). Several membrane as well as soluble nucleus-encoded chloroplast proteins with diverse functions were found to be reduced to 50% to 80%, mostly showing a stronger effect in the triple mutant. LHCb2 (for light-harvesting complex of PSII) displayed the strongest reduction, to about 50% of the wild-type level in the triple mutant, and subunits CF0-II and CFI-g of the ATP synthase only reached about 60% of wild-type levels. Furthermore, PAC (for pale cress; Meurer et al., 1998a), which plays a role in plastid RNA processing, HCF101 (for high chlorophyll fluorescence 101; Schwenkert et al., 2010), a PSI assembly factor, and HCF136 (Meurer et al., 1998b), a membrane protein involved in early PSII biogenesis, were reduced to 70% to 80% compared with wild-type levels. Strikingly, the plastid-encoded CF1 a- and b-subunits of the ATP synthase were not down-regulated, underlining a deficiency in the accumulation of nucleus-encoded chloroplast proteins only. However, a number of other nucleus-encoded proteins tested were found to be present in comparable amounts to the wild type, such as PsaD, PsaG, PsbO, OE23, LHCa1, and SSU (data not shown). The wild type and mutants did not show any differences in protein levels when immunoblotting was performed with leaves from 3-week-old plants (data not shown). Analysis of the RNA levels of STY8, STY17, and STY46 in Arabidopsis seedlings by quantitative RTPCR 2 h after illumination revealed a comparable Plant Physiol. Vol. 157, 2011

relative expression level of all three kinases in wildtype plants (data not shown). This indicates a potential function of all three isoforms in the greening process, a fact that is also supported by the additive effect of the sty17 knockdown in the double mutant background. To rule out any regulatory effects on the transcriptional level of the down-regulated chloroplast proteins, we analyzed expression levels of LHCb2, CF0-II, CFI-g, HCF101, PAC, and HCF136 by quantitative RTPCR with RNA isolated from etiolated seedlings 2 h after illumination (Fig. 9C). No down-regulation of these genes was observed in comparison with the wild type, indicating that RNA metabolism is not affected in the mutants. Some of the genes were slightly upregulated, although not more than 1.3-fold of the wild type. Our next aim was to verify whether LHCb2, CF0-II, HCF101, and PAC preproteins can be phosphorylated by our kinase, as has already been shown for two of the down-regulated proteins, CFI-g and HCF136 (Martin et al., 2006). Chimeric proteins of the transit peptide of LHCb2/CF0-II/HCF101 and mSSU, as well as full-length HCF101 and PAC and the mature part of SSU, were purified via a C-terminal His tag and subjected to an in vitro kinase assay. All proteins but mSSU, which was used as a nonphosphorylatable control, were found to be phosphorylated (Fig. 9D). A control reaction without kinase is provided in Supplemental Figure S6A.

DISCUSSION STY8 Is Autophosphorylated on an Essential Thr by an Intramolecular Mechanism

The kinases (STY8, STY17, and STY46) responsible for transit peptide phosphorylation were recently isolated from Arabidopsis (Martin et al., 2006). Since little is known about the mechanistic functioning of dual-specificity kinases in plants, we have analyzed characteristics of STY8 with respect to autophosphorylation and substrate phosphorylation in more detail. Sequence analysis of STY8, STY17, and STY46 demonstrates that the kinases contain typical Ser/Thr motifs as well as Tyr motifs, as have been described before (Rudrabhatla et al., 2006; see below). Comparison with other Tyr kinases allowed dissection of the kinase domain into 11 typical subdomains (Fig. 1C; Hanks et al., 1988). Subdomains VII and VIII harbor the activation segment flanked by the highly conserved DFG and APE amino acids. Within this activation segment, we identified the conserved autophosphorylated Thr, which was shown to be indispensable for kinase activity. Autophosphorylation usually leads to a conformational change, thus stabilizing the kinase in its active conformation (Nolen et al., 2004). The activation loop can be phosphorylated by several mechanisms, either involving an upstream kinase or by in-trans phosphorylation of an inactive 79

Lamberti et al. Figure 9. Protein levels of nucleus-encoded chloroplast proteins are reduced in kinase mutants. A, Immunoblot analysis of wild-type (WT) and mutant seedlings isolated 2 h after illumination. Nucleus-encoded proteins (LHCb2, CF0-II, CF1g, PAC, HCF101, and HCF136) as well as the plastid-encoded CF1-b subunit of the ATP synthase were analyzed. The CF1-b antibody recognizes CF1-b (bottom band) and cross-reacts with CF1-a (top band). A total of 15 mg of protein corresponds to 100%, and the bottom panel shows a Coomassie blue-stained gel (CBB) as a loading control. B, Quantification of immunoblotting was performed with ImageQuant software (GE Healthcare). All immunoblots were repeated at least three times, and the bands corresponding to 50% were quantified. C, Quantitative RT-PCR was performed with LHCb2, CF0II, CF1-g, PAC, HCF101, and HCF136 to analyze the expression level (n = 3). D, Two micrograms of mSSU, pLHCb2-mSSU, pCF0-II-mSSU, pPAC, pHCF101, and pHCF101-mSSU was subjected to an in vitro kinase assay with 1 mg of STY8. The positions of the kinase and the purified substrate proteins are marked with arrows in the autoradiograph and the corresponding Coomassie bluestained gel. A second, smaller band is detected in the pPAC sample (marked with an asterisk), which is also phosphorylated and probably contains a degradation product of pPAC.

kinase molecule by an active molecule or via an intramolecular mechanism in cis. Autophosphorylation in cis, as demonstrated here for the STY kinases, is a rather poorly characterized activation mechanism either depending on the help of molecular chaperones, such as HSP90, or involving translational intermediates to overcome conformational hindrances during autophosphorylation (Lochhead, 2009). Further analyses are required to verify whether other binding partners are required for STY8 maturation. We additionally investigated the function of a conserved ACT domain located upstream of the kinase domains in STY8, STY17, and STY46. The ACT domain is mainly found in enzymes involved in amino acid and purine metabolism and is thought to act as a regulatory element by the binding of small molecules. Its conservation becomes most evident on the structural level, since it comprises four b-strands and two a-helices arranged in a babbab fold (Grant, 2006). We found that deletion of the ACT domain promotes 80

activation of the kinase, doubling its phosphorylation activity, suggesting a regulatory function, possibly upon binding metabolites or other small molecules. Changes in the metabolite composition of the cell upon stress conditions or environmental changes, therefore, might regulate kinase activity in vivo. STY8 Is Distinguished from Other Plant STY Kinases

Inhibition of kinases by specific inhibitors provides a possibility to study their biochemical properties in vitro and is widely used as a therapeutic strategy. Therefore, we have tested a set of 64 kinase inhibitors and found that STY8 is strongly inhibited by the typical Tyr inhibitors JNJ-10198409, tyrphostin, and Janex 1. Tyrphostin is a rather broad inhibitor of Tyr kinases, and an inhibitory effect of tyrphostin has likewise been reported for a phylogenetically related peanut (Arachis hypogaea) STY kinase (Rudrabhatla and Rajasekharan, 2004), whereas Janex 1 is known to Plant Physiol. Vol. 157, 2011

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act specifically on Janus kinase 3, which is a nonreceptor Tyr kinase functioning in the JAK-STAT pathway (Sudbeck et al., 1999; Lv et al., 2009). Thus, STY8 seems to bear a certain structural relationship to typical Tyr kinases that allows blocking by these inhibitors, although it only phosphorylates Ser and Thr in vitro. Three conserved motifs, which are thought to mediate substrate specificity, are found in subdomains VI, VIII, and XI. Motif 1 (DLKTAN) differs from both a typical Ser/Thr-specific motif, which is usually DLKPEN, and the DLR/AAR/AN motif, which is a strong indicator of Tyr kinase activity (Hanks et al., 1988). Strikingly, the Lys within this motif has been found to be essential for activity in our study, emphasizing a certain importance of the conservation of this motif. The second motif conferring substrate specificity lies within the activation segment and is typical for plant dual-specificity kinases (Rudrabhatla et al., 2006). Subdomain XI harbors the conserved motif CW(X)6RPXF, which is often found in Tyr kinases. Nevertheless, we were unable to detect any Tyr phosphorylation activity, although Tyr autophosphorylation has been reported previously for a closely related STY kinase (Rudrabhatla and Rajasekharan, 2002; Reddy and Rajasekharan, 2007). Therefore, it seems that STY8 is clearly distinguished from this closely related peanut STY kinase. However, considering that Tyr is one of the most uncommon amino acids in chloroplast transit peptides (Zhang and Glaser, 2002), an ability to phosphorylate Tyr is dispensable and might consequently have been lost in the STY8, STY17, and STY46 kinase family. STY8, STY17, and STY46 Are Plant Specific and Play a Role in the Transition of Etioplasts to Chloroplasts in Cotyledons

To emphasize the presence of the STY kinases in green plants, we have conducted a phylogenetic analysis of STY8, STY17, and STY46 homologs in plants. Homologs are found in all green plants (i.e. green algae, mosses, ferns, monocots, and dicots), but not in species containing rhodoplasts or complex plastids. A phylogenetic tree and a sequence alignment with representatives of the respective species are provided in Supplemental Figures S7 and S8. Algae, nonvascular plants, monocots, and dicots each form a distinct group. Dicots can be subdivided into three clades, among which two or three isoforms of each species are unevenly distributed (Dereeper et al., 2008, 2010). STY8 and STY17 are very closely related and are located in a larger context of duplicated genes, as verified with the Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication; Tang et al., 2008). To further elucidate the role of STY8, STY17, and STY46 in vivo, we analyzed Arabidopsis knockout/ knockdown mutants of STY8, STY17, and STY46. The data obtained revealed that the transition of etioplasts Plant Physiol. Vol. 157, 2011

to chloroplasts in cotyledons is affected during the initial phase of illumination. Protein levels of several nucleus-encoded proteins only accumulated to 50% to 80% of wild-type levels in the mutants, whereas mRNA levels corresponding to the reduced proteins as well as chloroplast-encoded proteins were unchanged, suggesting that a posttranslational defect may be in protein import due to a lack of transit peptide phosphorylation. As shown by chlorophyll a fluorescence measurements, the phenotype is most prominent early during the greening process, since Fv/Fm is reduced to 50% at 4 h after light exposure, whereas the wild type and mutants reach the same efficiency after 22 h. Reduction of Fv/Fm as a measure of active PSII centers correlates with reduced amounts of HCF136, as determined from immunoblots, which is an indispensable factor for PSII assembly (Meurer et al., 1998b). Interestingly, HCF136, CF0-g, and LHCII, which were found to be reduced in mutants during greening, have been described to be phosphorylated in their transit peptides (Waegemann and Soll, 1996; Martin et al., 2006). Additionally, we show here that LHCb2, CF0-II, PAC, and HCF101 could likewise be phosphorylated by STY8. Sequence analysis of HCF136, CF0-g, and CF1-II reveals at least one common 14-3-3-binding motif in their N-terminal sequences at positions 33 to 38 for HCF136, 42 to 47 for CF0-g, and 37 to 42/48 to 53 for CF1 and CFII (Supplemental Fig. S6B). In contrast, only one of the tested proteins, OE23, which was not found to be reduced, has a predicted 14-3-3-binding site. Tobacco (Nicotiana tabacum) pSSU also has been shown to bind 14-3-3 (May and Soll, 2000); however, the deduced motif differs slightly from the common 14-3-3-binding motifs and from the motifs found in Arabidopsis pSSU isoforms and therefore could account for comparable protein levels in the wild type and mutants. The influence of phosphorylation may also act individually on different preproteins, since their import competence also could be regulated by abundance or solubility. During the first days of germination and almost immediately upon illumination, etioplasts in cotyledons are converted into chloroplasts to facilitate the transition from heterotrophic to autotrophic growth and enable the seedlings to photosynthesize. Several mutants have been identified, with distinct phenotypes in chloroplast biogenesis restricted to either cotyledons or true leaves. In a mutant of the FtsH complex, var2, and in var3, pigment deficiencies are confined to true leaves only (Chen et al., 2000; Naested et al., 2004), whereas in other mutants, such as snowy cotyledon1 and snowy cotyledon2 (Albrecht et al., 2006, 2008), white cotyledon (Yamamoto et al., 2000), and cyo1 (Shimada et al., 2007), chloroplast formation is solely affected in cotyledons. All of these genes encode for chloroplast-localized proteins, which exert their function within the organelle and are involved in the reorganization of the plastid on the transcriptional or protein level. However, chloroplast development is 81

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severely dependent upon the import of more than 3,000 nucleus-encoded proteins synthesized in the cytosol (Richly and Leister, 2004), which could likewise function as a regulatory step. The STY kinases play an important role during this transition phase, maybe by facilitating preprotein import into chloroplasts by transit peptide phosphorylation. In general, chloroplasts in primordia directly emerge from proplastids. This transition is difficult to approach experimentally due to the scarcity of material, but we are currently trying to analyze this tissue to see if we can observe chloroplast retardation. Cell differentiation and expansion in leaves is accompanied by a massive increase in the number and size of chloroplasts, which requires the massive influx of preproteins from the cytosol, comparable to the deetiolation process. Therefore, the physiological relevance of STY kinases is most pronounced during differentiation phases and less important or even dispensable during phases of maintenance, as in fully differentiated adult leaf tissue. Analysis of 7-d-old cotyledons on the ultrastructural level revealed distinct changes between the wild type and mutants concerning the formation and shape of thylakoids. The defined poles appear unstructured, thylakoids are less appressed, and the thylakoid lumen is swollen. Possibly, this is due to the lack of several photosynthetic membrane and lumenal proteins or proteins involved in lipid trafficking and thylakoid formation, as has been observed in photosynthesis mutants, such as mutants deficient in the ATP synthase assembly (Bosco et al., 2004; Benz et al., 2009). The massive accumulation of vesicles may be due to the retarded development, since vesicle trafficking is involved in the formation of thylakoids (Joyard et al., 1998; Westphal et al., 2003). In 14-d-old mutant plants, the phenotype is less severe, although thylakoids still often appear disordered and unstructured. Similar structures to the star-shaped fragments of vesicular and tubular membranes were described previously as an aggregation of tubuli, but no physical presence of osmiophilic structures was observed or mentioned in that study (Menke, 1960). Heitz-Leyonsche crystals, to which we found resembling structures, were observed only in plastids of meristematic cells with highly developed endoplasmic reticulum in the cytoplasm, termed the squamulae intravaginales. Similar “rows of vesicles” were found in Avena coleoptiles (Schnepf, 1964). Direct connections between the inner membrane of plastids and the vesicular, tubular, or diagonally cut tubules were often observed (Fig. 7B, bottom right panel). Structural relationships between those tubules and the thylakoid layers exist, but they have to be analyzed in more detail. More frequent observation of these special structures in mutant plants might be due to the retardation in growth, which allows the observation of different developmental stages from the wild type. Likewise, ultrastructural analyses of tobacco and pea (Pisum sativum) leaves incubated at 12°C revealed an accumulation of membranes and membrane vesicles (Morre´ et al., 1991). 82

Apart from the observed defects in cotyledons, a general retardation in growth was observed in the mutants, indicating that any disturbance at the cotyledon stage has a more severe impact in general plant development. This has also been observed in other mutants affected in chloroplast formation in cotyledons (Albrecht et al., 2006) and might also hint at a function of transit peptide phosphorylation during chloroplast development or under other environmental conditions in true leaves.

MATERIALS AND METHODS Plant Materials and Growth Conditions Arabidopsis (Arabidopsis thaliana) wild-type Columbia ecotype and the respective mutants were grown either on soil or on half-strength Murashige and Skoog (MS) medium supplied with 1% Suc under controlled conditions in a growth chamber. For phenotyping analysis, plants were grown on soil in long-day conditions (16 h/8 h of light/dark, 22°C, 120 mE m22 s21). For greening experiments, dry seeds were surface sterilized and vernalized at 4°C for 2 d. Petri dishes were exposed to light (120 mE m22 s21) for 6 h and then placed in the dark. After 6 d in the dark, petri dishes were exposed to light for the indicated periods of time. Seedlings for transmission electron microscopy recordings were either grown on half-strength MS medium without Suc (7 d) or on soil (14 d) in long-day conditions and were harvested in darkness at the end of night. T-DNA insertion lines SALK 072890 (sty8) and SALK 116340 (sty46) were obtained from the SALK collection (http://signal.salk.edu), and homozygous lines were isolated by PCR using the oligonucleotides STY85#UTR for, STY8Ex4 rev, STY46Ex7 for, STY46Ex10 rev, and LBa1 (Supplemental Table S3). Homozygous sty8 and sty46 lines were crossed, and double homozygous lines were isolated in the T2 progeny. For the generation of independent sty17 RNAi lines, a 400-bp fragment of STY17 was amplified using the oligonucleotides STY17RNAi for and STY17RNAi rev (Supplemental Table S3), cloned into the binary vector pB7GWIWG2(II) (Plant Systems Biology) with the Gateway system (Invitrogen), and introduced into Agrobacterium tumefaciens strain GV3101. Wild type and homozygous sty8 sty46 mutant plants were transformed using the floral dip method (Clough and Bent, 1998), and seeds from the progeny of three independent lines were used for molecular and biochemical analysis. For complementation analysis, the full length STY46 and STY8 cDNA was cloned into the pH2GW7 vector (Plant Systems Biology), and wild type and homozygous sty8 sty46 mutant plants were transformed. Segregating lines from the sty17 RNAi or complemented T2 progeny were either grown on MS medium supplemented with the appropriate antibiotic or checked by PCR for the presence of the respective construct if grown on soil.

Protein Sequence Analysis Protein alignments were performed with AlignX/ClustalW (Invitrogen). The 14-3-3-binding sites were analyzed using the Eukaryotic Linear Motif server (http://elm.eu.org). BLAST searches were performed with the National Center for Biotechnology Information database (Marchler-Bauer et al., 2011).

Spectroscopic Analysis Chlorophyll a fluorescence of wild-type and mutant leaves was measured using a pulse-modulated fluorimeter (PAM 101; Walz) as described (Meurer et al., 1996).

Quantitative RT-PCR Analysis of Transcripts Plantlets grown on MS medium were ground in liquid nitrogen, and total RNA was isolated from several seedlings using the Plant RNeasy extraction kit (Qiagen). cDNA was synthesized from 1 mg of RNA (DNase treated) by RT (Moloney murine leukemia virus reverse transcriptase; Promega). For quantitative RT-PCR, the FastStart DNA Master SYBR-Green Plus kit was used, and Plant Physiol. Vol. 157, 2011

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the reaction was performed in a LightCycler (Roche). Forty-five cycles were performed as follows: 1 s at 95°C, 7 s at 49°C, 19 s at 72°C, and 5 s at 79°C. The relative abundance of all transcripts amplified was normalized to the expression level of 18S rRNA or actin. All oligonucleotides are given in Supplemental Table S3.

Protein Blot-Gel Analysis of Protein Extracts Fifty to 150 mg (fresh weight) of Arabidopsis was harvested and homogenized in homogenization buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 2 mM EGTA, and 1 mM dithiothreitol [DTT]) using a micropestle. The extract was solubilized in SDS sample buffer, and proteins were separated on an SDSpolyacrylamide gel, transferred on a polyvinylidene difluoride membrane, incubated with the appropriate primary antibody, and developed with enhanced chemiluminescence as described (Schwenkert et al., 2006). For STY17 antisera production, full-length purified STY17 was injected into rabbits (Biogenes). Lhcb2 antiserum was purchased from Agrisera. Production of HCF101 and PAC antisera is described elsewhere (Schwenkert et al., 2010; Stoppel et al., 2011). Phosphospecific antisera were purchased from Cell Signaling.

Chlorophyll Extraction Chlorophyll determination of Arabidopsis leaves was performed following the method described by Porra et al. (1989). A total of 200 mg of leaf tissue was harvested and incubated in 2 mL of dimethylformamide for 2 h in the dark. Absorbance was measured at 663, 750, and 645 nm. Chlorophyll concentration was calculated as described (Arnon, 1949).

Overexpression and Purification of Recombinant Proteins The coding regions for STY8, STY46, STY17, LHCb2-mSSU, CF0-II-mSSU, HCF101, and PAC were cloned in the expression vector pET21d with a Cterminal His tag (Novagen) using oligonucleotides introducing appropriate restriction sites (Supplemental Table S3). The chimeric constructs (LHCb2mSSU, CF0-II-mSSU, and HCF101-mSSU) were generated by overlap PCR using the oligonucleotides tpLHCb2-rev, tpCF0-II-rev, and tpHCF101-rev to fuse the N-terminal 30, 74, and 120 respective amino acids to the mature part (amino acids 58–180) of mSSU (mSSU-for). Point mutations leading to single amino acid substitutions were introduced by site-directed mutagenesis with appropriate oligonucleotides (Supplemental Table S3; Kunkel et al., 1987). Clones for the substrate proteins pSSU and pOE23 are described elsewhere (Waegemann and Soll, 1996). All constructs were expressed in E. coli BL21 (pLysS) cells by induction with 1 mM isopropyl-1-thio-b-D-galactopyranoside for 12 h at 16°C. Cells were lysed in lysis buffer (200 mM NaCl, 20 mM Tris-HCl, pH 7.5, and 20 mM imidazol), centrifuged for 30 min at 18,000 rpm, and the supernatant was incubated with 350 mL of nickel-Sepharose fast flow (GE Healthcare) for 1 h at 4°C. The Sepharose was washed twice with washing buffer (200 mM NaCl, 20 mM Tris-HCl, pH 7.5, and 40 mM imidazol), and recombinant proteins were eluted by increasing the imidazol concentration up to 200 mM.

In Vitro Kinase Assays Recombinant STY kinase was incubated with recombinant substrate in the presence of 3 mCi of [32P]ATP and 2.5 mM ATP in a total volume of 50 mL of kinase buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 0.5 mM MnCl2). Exact amounts of kinase and substrate proteins used for the individual experiments are given in the figure legends. The reaction was carried out for 10 min at 23°C and stopped by adding 12 mL of SDS sample buffer. Kinase reactions with CPK4 were accomplished in kinase buffer containing 7 mM CaCl2 and 1 mM DTT instead of MnCl2, and Yes kinase reactions were performed in 20 mM MOPS, pH 7.9, and 5 mM MgCl2. Purified Yes kinase (Summy et al., 2003) was obtained from Proteinkinase.de. The proteins were separated on a 12% SDSpolyacrylamide gel followed by autoradiography. For the inhibition studies, the STY kinase was preincubated for 10 min with the inhibitors JNJ-10198409, tyrphostin, and Janex 1 (Cayman) at the indicated concentrations. The reaction was then carried out as described before. The reaction product was separated on a 12% SDS-polyacrylamide gel, the Coomassie blue-stained protein bands Plant Physiol. Vol. 157, 2011

were cut out from the gel, and the radioactivity was determined by liquid scintillation counting.

Phospho-Amino Acid Analysis Phosphorylated proteins were separated by SDS-PAGE and extracted from gels by incubation in 10 mM CHAPS and 20 mM Tris-HCl, pH 7.5, for 12 h at 20°C. Proteins were precipitated with TCA and resuspended in 50 mL of water. For acid hydrolysis, proteins were heated at 110°C for 2 h in 6 M HCl. After hydrolysis, residues were lyophilized and dissolved in distilled water. TLC was performed on cellulose plates (TLC Cellulose Plastic Sheets, 20 3 20 cm; Merck) for 4 h at 1,000 V in a mixture of acetic acid:formic acid:water (78:25:897, v/v). 32P-labeled amino acids were detected by autoradiography. Standards of Ser(P), Thr(P), and Tyr(P) were run in parallel and detected by staining with 0.25% ninhydrin.x

In Vitro Transcription, Translation, and Affinity Pull Down Linear plasmid DNA was transcribed using SP6 RNA polymerase (Fermentas). Protein synthesis was performed in the presence of 50% homemade wheat germ lysate for 45 min at 25°C as described (Fellerer et al., 2011). Samples were centrifuged for 10 min at 20,000g at 4°C, and supernatant was added to 15 mL of TALON magnetic beads (Clontech). After incubation for 1 h at 4°C, beads were washed two times with washing buffer (50 mM NaPi, pH 7.3, 300 mM NaCl, 13 complete protease inhibitor cocktail [Roche], and 13 PhosSTOP phosphatase inhibitor cocktail [Roche]), and proteins were eluted from the beads in 15 mL of elution buffer (50 mM NaPi, pH 7.3, 300 mM NaCl, 250 mM imidazol, 13 complete protease inhibitor cocktail [Roche], and 13 PhosSTOP phosphatase inhibitor cocktail [Roche]). For protein dephosphorylation, eluted proteins were incubated with l phosphatase (Sigma) for 30 min at 30°C.

Isoelectric Focusing Rehydration buffer (7 M urea, 2 M thiourea, 0.2% Biolytes 3–10 [Bio-Rad], 2% CHAPS, 100 mM DTT, bromphenol blue, 13 complete protease inhibitor cocktail [Roche], and 13 PhosSTOP phosphatase inhibitor cocktail [Roche]) was added to protein samples and incubated for 1 h at 20°C. Samples were centrifuged for 10 min at 20,000g, and supernatant was added to immobilized pH gradient strips (ReadyStrip immobilized pH gradient strips, pH range 3–10; Bio-Rad) and incubated 1 h at 20°C. Isoelectric focusing was performed using a Protean IEF Cell (Bio-Rad) at 8,000 V. After isoelectric focusing, the immobilized pH gradient strips were incubated for 20 min in equilibration buffer I (6 M urea, 2% SDS, 50 mM Tris-HCl, pH 8, 20% glycerol, and 2% DTT) and for 10 min in equilibration buffer II (6 M urea, 2% SDS, 50 mM Tris-HCl, pH 8, 20% glycerol, and 2.5% iodoacetamide). The second dimension was performed on a 12% SDS-polyacrylamide gel, and the 35S-labeled proteins were detected by autoradiography.

Protoplast Transformation and Transient Expression of GFP Fusion Proteins Full-length constructs of STY8, STY17, and STY46 were cloned into the vector p2GWF7 (Plant Systems Biology) with the Gateway system (Invitrogen) and transiently expressed in Arabidopsis mesophyll protoplasts. Protoplasts were isolated and transformed as described (Duy et al., 2007), and GFP fluorescence was observed with a confocal laser scanning microscope (Zeiss LSM 510 Meta) 18 h after transfection.

Electron Microscopy Distal pieces (approximately 1-mm2 segments) of cotyledons were prefixed in 2.5% (w/v) glutaraldehyde in 75 mM cacodylate buffer (pH 7.0). Cotyledon segments were rinsed in cacodylate buffer and fixed in 1% (w/v) osmium tetroxide in the same buffer for 2.5 h at room temperature. The specimens were stained en block with 1% (w/v) uranyl acetate in 20% acetone, dehydrated in a graded acetone series, and embedded in Spurr’s low-viscosity epoxy resin (Spurr, 1969). Ultrathin sections (50–75 nm thick) were cut with a diamond knife on an Ultramicrotome Leica EM UC6 and poststained with lead citrate 83

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(Reynolds, 1963). Micrographs were taken at 1,8003 (overviews) and 11,0003, 44,0003, and 110,0003 (details) at 80 kV on a Fei Morgagni 268 electron microscope. All sequences are available from GenBank (http://www.ncbi.nlm.nih.gov). Accession numbers are as follows: STY8 (At2g17700), STY17 (At4g35708) STY46 (At4g38470), pSSU (AAA34116), LHCb2 (At2g05100), CF0-II (At4g32260), CFI-g (At4g04640), HCF101 (At3g24430), PAC (At2g48120), HCF136 (At5g23120), and CPK4 (At4g09570).

Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Exchange of Lys-409 to Arg in STY8 results in a completely inactive kinase. Supplemental Figure S2. Inhibition of STY8 by Tyr kinase inhibitors. Supplemental Figure S3. Localization of STY8, STY17, and STY46. Supplemental Figure S4. Analysis of sty17 RNAi lines in the wild-type background. Supplemental Figure S5. Chloroplast ultrastructure of 7- and 14-d-old cotyledons. Supplemental Figure S6. Phosphorylation of chloroplast transit peptides. Supplemental Figure S7. Cladogram of the Arabidopsis isoforms of STY8, STY17, and STY46 with isoforms found in Chlamydomonas reinhardtii, Physcomitrella patens, Selaginella moellendorffii, Oryza sativa, Medicago truncatula, Populus trichocarpa, and Vitis vinifera. Supplemental Figure S8. Sequence alignment of sequences used for the generation of the cladogram. Supplemental Table S1. Mass spectrometric data on phosphorylation sites of STY8, STY17, and STY46 obtained from PhosPhAt (www.phosphat. mpimp-golm.mpg.de). Supplemental Table S2. Kinase inhibitor library tested with STY8. Supplemental Table S3. Oligonucleotides used for the experiments as indicated.

ACKNOWLEDGMENTS Katharina Scho¨ngruber is acknowledged for excellent technical assistance. HCF136 antisera were a kind gift from Peter Westhoff. ATP synthase antisera were generated in the laboratory of Richard Berzborn and kindly provided by Stephan Greiner. We appreciate the generous gift of purified CPK4 from Norbert Mehlmer. We thank Joshua Heazlewood for providing mass spectrometric phosphorylation data prior to publication. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. Received July 1, 2011; accepted July 27, 2011; published July 28, 2011.

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