Comparative phospho-proteomics analysis of salt-responsive phosphoproteins regulated by the MKK9-MPK6 cascade in Arabidopsis

Plant Science 241 (2015) 138–150

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

Comparative phospho-proteomics analysis of salt-responsive phosphoproteins regulated by the MKK9-MPK6 cascade in Arabidopsis Zhenbin Liu, Yuan Li, Hanwei Cao, Dongtao Ren ∗ State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China

a r t i c l e

i n f o

Article history: Received 10 July 2015 Received in revised form 9 October 2015 Accepted 10 October 2015 Available online 22 October 2015 Keywords: Salt stress Comparative phospho-proteomics Pro-Q Diamond staining MAPK cascade Rubisco activase

a b s t r a c t Mitogen-activated protein kinase (MAPK) cascades are involved in the salt stress response in plants. However, the identities of specific proteins operating downstream of MAPKs in the salt stress response remain unclear. Our studies showed that mkk9 and mpk6 null mutant seedlings are hyposensitive to salt stress. Moreover, we showed that MPK6 was activated by salt stress, indicating that the MKK9-MPK6 cascade mediated the salt stress response in Arabidopsis. To identify phosphoproteins downstream of the MKK9-MPK6 cascade in the salt stress response pathway, we performed two-dimensional electrophoresis (2-DE) with Pro-Q phosphoprotein staining and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to identify phosphoproteins induced by salt treatment in mkk9, mpk6, and wild-type seedlings. Phosphorylation of 4 proteins, including Rubisco activase (RCA), plastid ribosomal protein S 1 (PRPS1), plastid division protein (FtsZ2-2), and tortifolia2 (TOR2), was found to be regulated by activation of MKK9-MPK6 cascade. Further Phospho-proteomics analysis of MKK9DD mutant seedlings revealed that RCA phosphorylation was up-regulated as a result of MKK9 activation. The finding that the MKK9-MPK6 cascade functions in the salt stress response by regulating phosphorylation of RCA, PRPS1, FtsZ2-2, and TOR2, provides a novel insight into the MAPK-related mechanisms underlying the salt stress response in plants. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction High soil salinity is a major obstacle to agricultural development [1,2]. High soil salinity induces osmotic stress, ionic stress, and secondary oxidative stress in plant cells, eventually affecting plant growth and development [3]. Photosynthesis is a major factor in the determination of plant growth. Salt stress results in the decreased stomatal opening, and thereby restricts the diffusion of CO2 into chloroplast and reduces the leaf photosynthetic rate [4,5]. Salt stress can also inhibit photosynthesis by affecting the activity of photosynthesis-related proteins. For example, salt treat-

Abbreviations: 2-DE, two-dimensional electrophoresis; AAA+ , ATPase associated with various cellular activities; BAM1, beta-amylase 1; DEX, dexamethasone; FtsZ2-2, plastid division protein; IPGAM, 2, 3-biphosphoglycerate-independent phosphoglycerate mutase; LC, liquid culture; MALDI-TOF MS, matrix-assisted laser desorption ionization time of flight mass spectrometry; MAPK, mitogen activated protein kinase; MBP, myelin basic protein; PRPS1, plastid ribosomal protein S1; RCA, Rubisco activase; ROC4, cyclophilin 20-3; TCA, trichloroacetic acid; TOR2, tortifolia2. ∗ Corresponding author at: 2 West Yuanming yuan Rd., Haidian District, Beijing 100193, China. Fax: +86 010 62733794. E-mail address: [email protected] (D. Ren). http://dx.doi.org/10.1016/j.plantsci.2015.10.005 0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.

ments inhibit the activity of and reduce the content of Rubisco, a crucial enzyme in CO2 fixation process [6,7]. Plants are generally immobile and unable to select their environments. Therefore, in order to adapt to the environment, plants have evolved molecular mechanisms to perceive and respond to various biotic and abiotic stresses. Protein phosphorylation by kinases is an important post-translational modification that regulates cellular signal transduction in response to changes in the environment. In eukaryotes, activation of mitogen-activated protein kinase (MAPK) signaling cascades is a general mechanism through which external stimuli, including biotic and abiotic stresses, are translated into cellular responses. MAPK cascades are highly conserved signaling processes in eukaryotes that function downstream of sensors and receptors by converting signals generated at the sensors/receptors into cellular responses [8–10]. MAPK cascades are minimally composed of 3 types of kinase, MAP kinase kinase kinases (MAPKKK), MAP kinase kinases (MAPKK), and MAPK (denoted MKKK, MKK, and MPK in Arabidopsis, according to accepted systematic nomenclature), which are linked in various ways to upstream receptors and downstream targets [8,10]. Numerous MAPK pathways that respond to a variety

Z. Liu et al. / Plant Science 241 (2015) 138–150

of external stimuli have been characterized in yeast, animals, and plants [11]. Plant MAPK cascades have previously showed to be important for the regulation of salt stress responses. SIMK (salt stress-induced MAPK) and SIMKK (SIMK kinase) in alfalfa cells are activated by salt stress [12,13]. Salt and osmotic stress can enhance the activity of SIPK (salicylic acid-induced protein kinase) in tobacco protoplasts [14]. Three salt stress-induced MAPKs have been identified in Zea mays: ZmMPK3, ZmMPK5, and ZmSIMK1 [12,15,16]. In Arabidopsis, the most completely characterized MAPK cascade functioning in response to abiotic stress is the MEKK1-MKK2-MPK4/MPK6 cascade, which is activated by salt or cold stress [17]. Previous studies demonstrated that activation of MKK9 enhanced the sensitivity of transgenic seedlings to salt stress [18,19]. However, it is unclear how MKK9, MPK6, and other kinases regulate the response to salt stress. To understand how kinases mediate the salt stress response, it is vital to identify their downstream components. Phospho-proteomics is a powerful tool that can be used to identify MAPK substrates and downstream proteins, because it allows unbiased localization and site-specific quantification of many phosphorylated proteins in a single in vivo experiment. We used Pro-Q Diamond phosphoprotein gel stain [20–23] to study protein phosphorylation in Arabidopsis under salt stress [24]. The intensity of the Pro-Q Diamond stain was proportional to the degree of phosphorylation of each phosphoprotein, but not to the protein concentration [25]. In this study, we found that mkk9 and mpk6 mutants were hyposensitive to salt stress in comparison with wild-type plants. The fresh shoot weight of mkk9 and mpk6 seedlings was significantly heavier than that of the wild-type seedlings. Comparative analysis of the two-dimensional electrophoresis (2-DE) patterns in wild-type seedlings, mkk9 and mpk6 mutants under salt treatment was used to identify proteins with different phosphorylation statuses among the groups. Twenty salt- responsive phosphoproteins were identified with a high level of confidence in Arabidopsis wildtype seedlings. Four of these proteins was found to be regulated by activation of MKK9-MPK6 cascade.

2. Material and methods 2.1. Plant materials and treatments Wild-type (Col-0) and mutant Arabidopsis thaliana (ecotype Columbia) seeds were surface-sterilized. After cold treatment in the dark for 2 d at 4 ◦ C, the seeds were germinated and grown on Murashige and Skoog medium (containing 2.5% sucrose) with 0.5% phytagel agar (Sigma–Aldrich, St. Louis, MO, USA) plates. For the investigation of the mutant phenotype, wild-type and mutant seedlings were transferred to new plates containing 100 mM NaCl and grown at 22 ◦ C in a growth room with a 12-h photoperiod (photon flux density, 100 ␮E/m−2 /s−1 ). Three biological replicates were done for each treatment. For kinase assays and phospho-proteomics studies, seedlings were transferred to liquid culture medium (0.5 × Murashige and Skoog medium containing 0.025% 2-(N-morpholino) ethanesulfonic acid (MES) and 0.25% sucrose, pH 5.7) [18] and grown at 22 ◦ C under continuous light (photon flux density, 70 ␮E/m−2 /s−1 ). Two-week-old seedlings were treated with liquid culture medium containing NaCl to obtain final concentration of 100 mM. The control group was treated with liquid culture medium (LCM) only. The MKK9 active mutant (MKK9DD ), MKK9 inactive mutant (MKK9KR ), and MKK9DD /mpk6 crossed seedlings were treated with 2 ␮M dexamethasone (DEX) for 4 and 8 h [26,27]. Plant samples were collected at different time points, flash-cooled in liquid nitrogen, and stored at −80 ◦ C.

139

T-DNA insert mutants (mkk9, Salk 060 H06; mpk3, Salk 100651; mpk6, Salk 062471) were obtained from the Arabidopsis Biological Resource Center. MKK9KR , MKK9DD , and MKK9DD /mpk6 mutant plants were generated as previously described [18]. 2.2. Protein extraction Samples were ground to a fine powder in liquid nitrogen and transferred to a tube with precooled 10% TCA/acetone and 1% ␤-mercaptoethanol, vortexed briefly, and incubated overnight at −20 ◦ C. After centrifugation at 10,000 × g for 20 min at 4 ◦ C, the supernatants were discarded and the pellets were washed twice with precooled acetone containing 0.1% ␤-mercaptoethanol. After incubation for 30 min at −20 ◦ C, the samples were centrifuged at 10,000 × g for 20 min at 4 ◦ C. The final pellets were freeze–dried under vacuum and solubilized in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS detergent, 60 mM dithiothreitol (DTT), and 0.8% IPG buffer) to extract proteins. The solution was centrifuged (13,000 × g for 10 min at 25 ◦ C) and the supernatants were collected for 2-DE. Total protein concentrations were estimated using 2D Quant kits (GE Healthcare Life Sciences). 2.3. 2-DE and Pro-Q staining Total protein samples (1 mg) in rehydration buffer (8 M urea, 2% CHAPS detergent, 60 mM DTT, 0.8% IPG buffer, and a trace amount of bromophenol blue) were loaded onto 18-cm IEF linear strips (pH 4–7, GE Healthcare Life Sciences). Rehydration was performed at room temperature for 18 h. The IEF voltage was 100 V for 3 h, 300 V for 3 h, 1000 V for 1.5 h, 3000 V for 1.5 h, and finally 8000 V for a total of 75,000 Vh. When IEF was completed, the strips were equilibrated as previously described [28]. Proteins in the strips were separated on 11% polyacrylamide gels, fixed overnight in 500 mL of a solution of 50% methanol and 10% acetic acid, washed in 500 mL deionized water for 30 min, and stained with 3-fold-diluted Pro-Q stain for 2 h. After staining, the gels were destained with 250 mL of a solution of 50 mM sodium acetate (pH 4.0) in 20% acetonitrile (ACN) 4 times for 30 min. All steps from staining to washing were performed in a dark room. Gels were scanned with a Typhoon 9410 fluorescence scanner, stained with a blue-silver staining method as previously described [29], scanned with a UMAX 2000 scanner, and analyzed with PD-Quest 8.0 software. Three biological replicates were done for each condition. Protein spots were considered credible when they were detected in at least three biological replicates. 2.4. Protein identification by MALDI-TOF MS In-gel protein digestion was performed using trypsin (Roche) as previously described with minor modifications [28]. First, excess trypsin solution was removed, after which the gel pieces were submerged in working solution (1 mM CaCl2 and 25 mM NH4 HCO3 ). Supernatants were collected after incubation in the working solution for 12 h at 37 ◦ C. The gel pieces were extracted twice with 70% acetonitrile and 0.1% trifluoroacetic acid for 15 min with sonication. Supernatants were pooled and concentrated by freeze–drying. Samples were loaded onto an Anchor Chip target plate (Bruker Daltonics). Matrix solution (1 mg/mL of a-cyano-4-hydroxycinnamic acid dissolved in 70% acetonitrile and 0.1% trifluoroacetic acid) was added to the dried peptide samples. After fast evaporation, the peptides were washed with 0.1% trifluoroacetic acid and analyzed using an AutoFlexII TOF/TOF mass spectrometer. Spectrum masses of 700 to 4000 D were acquired. All mass spectra were externally calibrated with a peptide calibration standard. Monoisotopic peaks were collected and used for peptide fingerprinting identification with Flex Analysis 3.0 software (Bruker Daltonics). Proteins were identified by searching the National Center for Biotechnology Infor-

140

Z. Liu et al. / Plant Science 241 (2015) 138–150

mation non-redundant database with the MASCOT search engine using the following search parameters: cleaving enzyme, trypsin; peptide mass tolerance, 30 ppm; and one missed cleavage allowed. Carbamido methylation of Cys and oxidation of Met were set as fixed modifications. 2.5. In-gel kinase assays The in-gel kinase assay was performed as described previously [27]. Total protein samples (10 ␮g per lane) were separated on 10% SDS-poly acrylamide gels with 0.1 mg/mL myelin basic protein (Sigma) as a kinase substrate. After electrophoresis, the gels were washed 3 times at room temperature with washing buffer (25 mM Tris, 0.5 mM DTT, 0.1 mM Na3 VO4 , 5 mM NaF, 0.5 mg/mL bovine serum albumin, and 0.1% TritonX-100 (pH 7.5)) to remove SDS. The gels were incubated in a solution of 25 mM Tris, 1 mM DTT, 0.1 mM Na3 VO4 , and 5 mM NaF (pH 7.5) at 4 ◦ C overnight, with 3 changes of the buffer to re-nature the kinases. After incubation in reaction buffer (25 mM Tris, 2 mM EGTA, 12 mM MgCl2 , 1 mM DTT, 0.1 mM Na3 VO4 (pH 7.5)) with 200 nM ATP and 50 ␮Ci ␥-32 PATP (3000Ci/mmol) at room temperature for 60 min, the gels were washed several times in wash buffer (5% trichloroacetic acid and 1% sodium pyrophosphate) for at least 5 h to remove unincorporated ␥-32 P-ATP. Finally, the gels were dried and exposed to Kodak X-Ray film. 3. Results 3.1. mkk9 and mpk6 null mutants were hyposensitive to salt stress in comparison with wild-type plants The MKK9-MPK3/MPK6 cascade participates in regulation of the biosynthesis of ethylene and camalexin [18]. In order to further understand whether MPK3 and MPK6 are downstream of MKK9 in the salt stress response, we designed an experiment to test the tolerance phenotypes of mkk9, mpk3, and mpk6 null mutant plants under treatment with 100 mM NaCl. As shown in Fig. 1A, mkk9 and mpk6 seedlings were hyposensitive to NaCl treatment in comparison with wild-type seedlings (Col-0). Shoots of mkk9 and mpk6 seedlings were bigger than those of wild-type seedlings after salt stress. The mpk3 seedlings were slightly more sensitive to salt than the mkk9 and mpk6 seedlings. The weight of shoot of mkk9 and mpk6 seedlings was significantly heavier than that of the wild-type seedlings (Fig. 1B). The weight of the roots of the seedlings was not significantly different after salt treatment. These results suggest that MKK9 and MPK6 negatively regulate shoot growth under conditions of salt stress. To further understand the functions of MKK9 and MPK6 in the cellular response to salt stress, we used an in-gel kinase assay to detect changes of MAPK activity in seedling after salt treatment. The results showed that MPK6 was strongly activated in wildtype, mpk3, and mkk9 seedlings by salt treatment, while MPK3 was slightly activated in mpk6 seedlings (Fig. 1C). These results suggest that MPK3 and MPK6 participate together in the salt stress response in plants with at least some degree of functional redundancy. Salt stress also activated another MAPK (Fig. 1C), which was tentatively identified as MPK4 based on its size. A previous study demonstrated that MPK4 in Arabidopsis was activated by salt stress. [17]. Activation of MPK6 was more significant than that of MPK3 and MPK4, suggesting a major role for MPK6 in the salt stress response. MPK6 activity in mkk9 seedlings was reduced but not abolished in comparison with that of wild-type control seedlings. These results suggest that MKK9 is an up-stream MAPKK of MPK6 in the salt stress response pathway and other MKKs also may be involved in this pathway. Based on these results, we conclude that

the MKK9-MPK6 cascade participates in the salt response pathway and negatively regulates shoot growth under salt stress conditions. 3.2. Identification of differentially phosphorylated proteins in wild-type and mutant seedlings without NaCl treatment Before analyzing the differentially phosphorylated proteins induced by salt treatment, we compared Pro-Q stained 2-DE images of untreated wild-type, mkk9, and mpk6 seedlings to allow us to filter out non-salt-responsive proteins from the phospho-proteomic results collected after salt exposure. After TCA/acetone extraction and Pro-Q phosphoprotein staining for proteomic analysis, approximately 200 distinct spots were detected on the 2-DE image. Some protein spots in the image showed different intensities in the mkk9 and mpk6 mutant seedlings and the wild-type seedlings, indicating different degrees of phosphorylation. In untreated seedlings (0 h), spots 83, 84, 85, 89, and 109 were composed of proteins with increased phosphorylation in the mkk9 and mpk6 seedlings in comparison with those of the wild-type seedlings, indicating that MKK9 and MPK6 might involve some processes which regulate dephosphorylation of these proteins (Fig. 2). The protein spot 42 was down-regulated in the mpk6 seedlings. The expression levels of the proteins comprising spots 42, 83, 84, 85, 89, and 109 did not differ significantly in the null mutant and wild-type seedlings (Fig. S1). The identities of the protein spots are shown in Table 1. The enzyme 2, 3-biphosphoglycerate-independent phosphoglycerate mutase (IPGAM) (spots 83, 85, and 86) is involved in glycolysis and catalyzes the reversible interconversion of 3-phosphoglycerate to 2-phosphoglycerate. In addition, IPGAM plays a critical role in stomatal movement [30]. IPGAM phosphorylation was up-regulated in the mkk9 and mpk6 seedlings, suggesting that the MKK9-MPK6 cascade might be related to de-phosphorylation of IPGAM, and indicating that this process was not induced by salt stress. The control group was treated with LCM only. The proteins comprising some spots in the stained 2-DE images showed differential phosphorylation among the groups. As shown in Fig. 2, levels of the phosphorylated forms of proteins comprising 23 spots were upregulated 8 h after treatment with liquid culture medium (protein identities are shown in Table 2). This result suggested that phosphorylation of these proteins were induced by changes of some substances in liquid culture medium. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015.10.005. 3.3. Identification of phosphoproteins regulated by salt treatment in wild-type seedlings Because protein phosphorylation levels in wild-type seedlings are the basis of the analysis of protein phosphorylation levels in mkk9 and mpk6 seedlings, we identified proteins with phosphorylation induced by salt treatment in wild-type seedlings treated with 100 mM NaCl (dissolved in liquid culture medium) for 4 and 8 h. The control seedlings were treated with liquid culture medium only (Fig. 3). The Pro-Q and blue silver staining demonstrated that phosphorylation of several proteins was up-regulated by salt exposure, while their expression levels were unchanged (Fig. 3 and Fig. S2). The number of proteins showing increased phosphorylation increased gradually as the duration of salt stress was lengthened. After salt treatment for 8 h, the proteins comprising 26 spots were up-regulated, while the proteins comprising 9 spots were down-regulated. Proteins regulated by salt treatment were identified with a high level of confidence by MALDI-TOF MS (Table 3). Some proteins comprised several different spots, including Rubisco activase (spots 53, 60, 61, 62, and 63), ATP synthase CF 1 beta subunit (spots 74 and 75), and magnesium-chelatase subunit chlD (spots 100 and 101), perhaps due to post-translational mod-

Z. Liu et al. / Plant Science 241 (2015) 138–150

141

Fig. 1. Mutants growth and MPK6 activity in response to salt stress. (A) Analysis of the salt sensitivity of wild-type (Col-0), mkk9, mpk3, and mpk6 null mutants. Five-day-old seedlings were transferred to Murashige and Skoog medium (MS) with 100 mM (+NaCl) or without NaCl (−NaCl). Photographs were taken 15 d after transfer. (B) Analysis of the fresh weights of shoots and roots. Error bars represent SD (n > 10). Statistical significance was determined by Student’s t-test; * p < 0.05, ** p < 0.01. (C) Activation of MPK6 and MPK3 was measured in wild-type (Col-0) and mkk9, mpk3, and mpk6 null mutant liquid culture seedlings with (100 mM) or without (0 mM) NaCl. MAPK activity was determined by in-gel kinase assays using MBP as a substrate. Expression levels of MPK3 and MPK6 were detected by immunoblotting (IB). As an additional loading control, the large subunit (LSU) of Rubisco was subject to western blotting.

Table 1 Proteins differentially phosphorylated in mkk9 and mpk6 mutants in comparison with wild-type seedlings without exposure to any treatment. Spot no.

Protein name

Accession noa .

42 83 84 85 86 93 94 109 112

COP9 signalosome complex subunit 5a 2,3-Biphosphoglycerate-independent phosphoglycerate mutase 1 Beta-amylase 1 2,3-Biphosphoglycerate-independent phosphoglycerate mutase 2 2,3-Biphosphoglycerate-independent phosphoglycerate mutase 2 Luminal-binding protein 2 Luminal-binding protein 2 Tudor-SN protein 2 Cyclophilin 20-3

AT1G22920 AT1G09780 AT3G23920 AT3G08590 AT3G08590 AT5G42020 AT5G42020 AT5G61780 AT3G62030

Protein scoreb

105 83 96 204 186 97 108 209 76

Coverage (%)c

43 29 27 60 53 28 31 40 34

Matched peptidesd

11 9 6 11 12 8 10 13 6

Up-regulation (+)/ downregulation(−)e mkk9

mpk6

ns + + + ns + + + ns

− + + + + + + + +

ns: Not significant. a Accession number in the TAIR database. b Mascot score. c Sequence coverage by PMF using MALDI-TOF MS. d Number of peptides matched. e Up- or down-regulation of the protein in the identified spot.

ifications or alternative splicing or multiple sites phosphorylation [31]. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015.10.005. We found that phosphorylation of some proteins, e.g., ATPase synthase CF 1, xylose isomerase and luminal-binding protein 2, were induced in seedlings treated with liquid cultured. The levels of these phosphorylated proteins in seedlings treated with NaCl were further increased. The results suggest that phosphorylation of these proteins can be enhanced by salt treatment. To determine whether specific proteins in particular functional categories were affected disproportionately by salt stress,

we analyzed the functional categories of proteins regulated by salt treatment (Fig. 4A). Most proteins regulated by salt stress are involved in energy and metabolism (42%), followed by stress and defense (24%), whereas the remainder of the proteins made up 4 functional classes: signaling (10%), transport (10%), protein processing (7%), and RNA processing (6%). Analysis of the subcellular localization of proteins with salt-induced phosphorylation revealed that most were localized in the chloroplast, cell membrane, and nucleus, whereas others were localized in the cytosol, cytoplasm, mitochondria, cell wall, and Golgi apparatus.

142

Z. Liu et al. / Plant Science 241 (2015) 138–150

Fig. 2. 2-D gel electrophoresis (2-DE) analysis of phosphoproteins from seedlings not treated with NaCl. Proteins were extracted from wild-type (Col-0), mkk9, and mpk6 mutant seedlings that were not treated (0 h) or treated only with liquid culture medium (LCM). The seedlings were collected after 4 and 8 h. Proteins (1 mg) were focused on IPG strips (18 cm) with a linear gradient of pH 4–7. SDS-PAGE was performed using 11% acrylamide gels in the second dimension. The gels were stained with Pro-Q Diamond dye. Circles indicate spots changed in wild-type seedlings by adding liquid culture medium or spots different in the mutants and wild-type seedlings.

3.4. Comparative analysis of differentially phosphorylated proteins in mkk9, mpk6, and wild-type seedlings under salt treatment To identify phosphoproteins regulated by the MKK9-MPK6 cascade during the response to salt stress in Arabidopsis, we compared Pro-Q stained 2-DE gel images of proteins from mutants and wild-type seedlings after salt treatment. Several protein spots showed differences in the 2-DE gel images generated using samples from the mkk9, mpk6, and wild-type seedlings (Fig. 5). In comparison with wild-type seedlings, mkk9 seedlings had 9 down-regulated phosphoproteins and 3 up-regulated phosphoproteins, whereas mpk6 seedlings had 12 down-regulated phosphoproteins and 3 up-regulated phosphoproteins. However, the expression levels of the differentially regulated phosphoproteins were not changed significantly (Fig. S3). The phosphoproteins comprising spots 53, 57, 58, 63, 72, 100, and 101 were down-regulated in both of the mkk9 and mpk6 mutant seedlings, suggesting that MKK9 and MPK6 have some of the same downstream phosphoproteins. These results were consistent with

phenotypic observations, further indicating that MKK9 is upstream of MPK6 in the salt stress response pathway. Spots 53, 60, 61, 62 were all comprised of RCA. RCA phosphorylation was significantly up-regulated after salt treatment for 8 h in wild-type seedlings. However, the phosphorylation levels of the proteins comprising spots 53 and 63 in the mkk9 and mpk6 mutants were reduced in comparison with those of the same proteins in wild-type seedlings after salt treatment, with a greater effect in the mpk6 mutants (Fig. 7B and C). These results suggest that RCA is phosphorylated as a result of activation of the MKK9-MPK6 cascade in response to salt stress. The phosphoproteins comprising spot 57 (plastid ribosomal protein S 1 (PRPS1)), spot 58 (plastidial division protein (FtsZ2-2)), and spot 72 (tortifolia 2 (TOR2)) were also up-regulated by salt treatment in the wild-type seedlings, but the degree of phosphorylation of these proteins was reduced in the mkk9 and mpk6 null mutant seedlings. These results suggest that RCA, PRPS1, FtsZ2-2, and TOR2 are downstream of the MKK9-MPK6 cascade in the salt stress response pathway. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015.10.005.

Z. Liu et al. / Plant Science 241 (2015) 138–150

143

Table 2 Altered phosphoproteins in mkk9, mpk6 mutants and wild-type seedlings following treatment with liquid culture. Spot no.

14 15 17 31 32 67 74 75 76 93 94 112

Protein name

20S proteasome alpha subunit G1 Aluminium induced protein with YGL and LRDR motifs UDP-4-keto-6-deoxy-d-glucose-3,5-epimerase-4-reductase 1 ATPHOS34 Universal stress protein PHOS32 ERBB-3 Binding protein 1 ATP synthase CF1 beta subunit ATP synthase CF1 beta subunit Xylose isomerase Luminal-binding protein 2 Luminal-binding protein 2 Cyclophilin 20-3

Accession noa .

AT2G27020 AT5G43830 AT1G63000 AT4G27320 AT5G54430 AT3G51800 ATCG00480 ATCG00480 AT5G57655 AT5G42020 AT5G42020 AT3G62030

Protein scoreb

107 98 140 115 75 109 97 103 84 97 108 76

Coverage (%)c

42 49 54 52 50 48 37 40 30 28 31 34

Matched peptidesd

7 6 8 9 10 9 10 10 7 8 10 6

Up-regulation (+)/ down-regulation (−)e WT

mkk9

mpk6

+ + + + + + + + + + + +

+ + + + + ns + + + + + +

+ + + + + ns ns ns + ns ns +

ns: Not significant. a Accession number in the TAIR database. b Mascot score. c Sequence coverage by PMF using MALDI-TOF MS. d Number of peptides matched. e Up- or down-regulation of the protein in the identified spot.

Fig. 3. 2-D gel electrophoresis (2-DE) analysis of phosphoproteins induced by salt stress in wild-type seedlings. Total proteins were extracted from wild-type seedlings treated with liquid culture medium as a control (LCM) or with liquid culture medium containing NaCl (LCM + NaCl). The final concentration of NaCl was 100 mM. Proteins (1 mg) were focused on IPG strips (18 cm) with a linear gradient of pH 4–7. SDS-PAGE was performed using 11% acrylamide gels in the second dimension. Circles indicate spots changed after salt treatment.

144

Z. Liu et al. / Plant Science 241 (2015) 138–150

Table 3 Altered phosphoproteins in mkk9, mpk6 mutants and wild-type seedlings following treatment with NaCl. Spot no.

42 53 61 62 63 57 58 72 74 75 76 83 84 85 86 90 93 94 100 101 109 14 15 17 31 32 112

Protein name

COP9 signalosome complex subunit 5a Rubisco activase Rubisco activase Rubisco activase Rubisco activase Small subunit ribosomal protein S1 Plastidial division protein FtsZ2-2 Tubulin alpha-2/alpha-4 chain ATP synthase CF1 beta subunit ATP synthase CF1 beta subunit Xylose isomerase 2,3-Biphosphoglycerate-independent phosphoglycerate mutase 1 Beta-amylase 1 2,3-Biphosphoglycerate-independent phosphoglycerate mutase 2 2,3-Biphosphoglycerate-independent phosphoglycerate mutase 2 Chloroplast heat shock protein 70-1 Luminal-binding protein 2 Luminal-binding protein 2 Magnesium-chelatase subunit chlD Magnesium-chelatase subunit chlD Tudor-SN protein 2 20S proteasome alpha subunit G1 Aluminium induced protein with YGL and LRDR motifs UDP-4-keto-6-deoxy-d-glucose-3,5-epimerase-4-reductase 1 ATPHOS34 Universal stress protein PHOS32 Cyclophilin 20-3

Accession no.a

AT1G22920 AT2G39730 AT2G39730 AT2G39730 AT2G39730 AT5G30510 AT3G52750 AT1G04820 ATCG00480 ATCG00480 AT5G57655 AT1G09780 AT3G23920 AT3G08590 AT3G08590 AT4G24280 AT5G42020 AT5G42020 AT1G08520 AT1G08520 AT5G61780 AT2G27020 AT5G43830 AT1G63000 AT4G27320 AT5G54430 AT3G62030

Protein scoreb

105 144 71 75 121 92 91 94 97 103 84 83 96 204 186 156 97 108 122 138 209 107 98 140 115 75 76

Coverage (%)c

43 56 35 40 46 24 47 31 37 40 30 29 27 60 53 30 28 31 28 33 40 42 49 54 52 50 34

Matched peptidesd

11 10 10 8 9 7 12 11 10 10 7 9 6 11 12 11 8 10 11 10 13 7 6 8 9 10 6

Up-regulation (+)/ down-regulation (-)e WT

mkk9

mpk6

+ + + + + + + + + + + + + + + + + + + + + -

+ ns + ns + ns ns ns + + + + + + + + + + + + + − − − − − ns

+ ns + + + ns ns ns + + + + + + + + + + + + ns − − − − − ns

ns: Not significant. a Accession number in the TAIR database. b Mascot score. c Sequence coverage by PMF using MALDI-TOF MS. d Number of peptides matched. e Up- or down-regulation of the protein in the identified spot.

3.5. Identification of phosphoproteins induced by MKK9 activation To further explore proteins downstream of the MKK9-MPK6 cascade in vivo, we investigated changes of phosphoprotein after MKK9 expression in transgenic seedlings with MKK9DD (active MKK9), MKK9KR (inactive MKK9), and MKK9DD /mpk6 [18]. Previously, we showed that expression of MKK9DD phosphorylates and activates MPK6 [18]. The expressions of MKK9DD and MKK9KR can be induced by DEX treatment. In comparison with MKK9KR seedlings, we found 34 up-regulated protein spots and 8 downregulated protein spots in MKK9DD seedlings after DEX treatment (Fig. 6). Twenty-four of the proteins induced by MKK9 activation were identified using MALDI-TOF MS (Table 4). The expression levels of these changed proteins were not significantly different in blue-silver stained images (Fig. S4). In previous studies, the universal stress protein PHOS32 (spot 32) has been shown to be a substrate of MPK3 and MPK6 [32,33]. Theoretically speaking, MKK9 activation should enhance phosphorylation of downstream proteins. However, we found that IPGAM2 phosphorylation (spots 85 and 86) was down-regulated in MKK9DD seedlings treated with DEX, but not in MKK9DD /mpk6 seedlings treated with DEX. We speculated that activation of the MKK9-MPK6 cascade might involve the processes which regulate dephosphorylation of IPGAM2. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015.10.005. To identify phosphoproteins related to both salt stress and the MKK9-MPK6 cascade, we used a Venn diagram to visualize protein spots regulated in multiple groups of mutants treated with NaCl or DEX (Fig. 7A), resulting in the identification of 5 groups of differentially regulated protein spots that were regulated in more than

one mutant group. The 5 groups of differentially regulated protein spots, including phosphoprotein spots regulated in wild-type seedlings under NaCl treatment (WT + NaCl), protein spots differentially regulated in mkk9 or mpk6 mutants and wild-type seedlings under NaCl treatment (mkk9 + NaCl, mpk6 + NaCl), phosphoprotein spots induced by MKK9 activation in MKK9DD seedlings (MKK9DD + DEX), and phosphoprotein spots differentially regulated in MKK9DD /mpk6 and MKK9DD seedlings under DEX treatment (MKK9DD /mpk6 + NaCl). The protein comprising spot 63 (RCA) was the protein identified in all 5 groups. Phosphorylation of RCA (spots 63 and 53) was up-regulated in wild-type seedlings after salt treatment, suggesting that RCA is a salt-responsive phosphoprotein. RCA phosphorylation was down-regulated in mkk9 and mpk6 seedlings (Fig. 7B and C). RCA phosphorylation was also up-regulated in MKK9DD mutant seedlings after DEX treatment, while up-regulated to a much lesser degree in MKK9DD /mpk6 seedlings (Fig. 7D). These results indicate that RCA is a downstream phosphoprotein of the MKK9-MPK6 cascade in salt stress response. 4. Discussion 4.1. The advantages of 2-DE-based methods Although MS-based methods provide more protein information than gel-based method such as 2-DE, 2-DE analysis provides useful information that complements gel-free methods [34,35]. Moreover, 2-DE can provide information related to post-translational modifications and protein isoforms [36]. In particular, the use of 2-DE with stains designed to detect specific protein modifications, such as Pro-Q phosphoprotein stain, allows targeted research that can identify molecular signaling mechanisms.

Z. Liu et al. / Plant Science 241 (2015) 138–150

145

Fig. 4. Classification of phosphoproteins induced by salt treatment in wild-type seedlings. (A) Functional classification of differential phosphoproteins identified in the wild-type seedlings under salt stress. (B) Subcellular locations of differential phosphoproteins in wild-type seedlings under salt stress.

4.2. Phosphoproteins induced by salt treatment Using a 2-DE Pro-Q stain approach coupled with MALDI-TOF MS, we identified 20 salt stress-responsive phosphoproteins in wild-type Arabidopsis seedlings. Our functional analysis revealed that the salt-responsive phosphoproteins identified in wild-type Arabidopsis seedlings comprised 4 main functional groups: energy generation and metabolism, stress response, protein processing, and signal transduction-related function. Chloroplast ATP synthase CF1 is involved in energy generation and carbon fixation [37]. It is reported that cold stress induces phosphorylation of the beta subunit of ATP synthase CF1 in Arabidopsis [38]. In the present study, phosphorylation of the beta subunit of chloroplast ATP synthase CF1 was up-regulated in wild-type Arabidopsis seedlings after salt stress, suggesting that exposure to salt stress affected energy generation (Table 3). RCA, citrate synthase 4, and beta-amylase 1 are involved in metabolic processes. Phosphorylation of RCA, citrate synthase 4, and beta-amylase 1 was significantly up-regulated by salt exposure. activates ribulose-1,5-bisphosphate carboxyRCA lase/oxygenase (Rubisco) by relieving the dead-end inhibition caused by non-productive binding of the substrate or catalytic misfire products [39] and plays an important role in jasmonic acid-induced leaf senescence [40]. Citrate synthase is the first

enzyme of the tricarboxylic acid cycle and catalyzes condensation of acetyl-CoA and oxaloacetate, yielding citrate and CoA [41,42]. Beta-amylase (BAM) is a major enzyme involved in starch breakdown in leaves. Beta-amylase 4 (BAM4) facilitates/regulates starch breakdown and operates independently of BAM1 and BAM3 [43,44]. These results indicate that many critical metabolic processes in plants may be affected by salt stress. The primary salt stress-responsive phosphoproteins involved in protein processing in wild-type Arabidopsis seedlings were luminal-binding protein 2 (protein folding) and cyclophilin 20-3, also known in rice as outermost cell-specific 4 (ROC4). ROC4 is a unique member of the cyclophilin family that assists in protein folding and assembly. Previous studies demonstrated modulation of ROC4 phosphorylation in Arabidopsis seeds in response to ABA [45]. Moreover, our functional analysis demonstrates that phosphoprotein regulation in response to salt treatment affects a wide range of biological processes, indicating that the signaling network involved in the salt stress response in Arabidopsis is complex. High salinity induces osmotic stress, ionic stress, and secondary oxidative stress in plant cells. Salt stress is mainly considered as ionic stress. Secondary stresses, such as osmotic stress and oxidative stress, can adversely affect plant growth and development, as well as induce changes of phosphoproteins. Previous studies showed that osmotic stress can induce rapid activation of MAPK

146

Z. Liu et al. / Plant Science 241 (2015) 138–150

Fig. 5. Comparative analysis of the Pro-Q-stained 2-D gel electrophoresis (2-DE) patterns of mutants and wild-type seedlings after salt stress. Proteins (1 mg) were focused on IPG strips (18 cm) with a linear gradient of pH 4–7. SDS-PAGE was performed using 11% acrylamide gels in the second dimension. Circles indicate spots different in the mkk9 and mpk6 null mutant seedlings in comparison with the wild-type seedlings 4 and 8 h after salt treatment.

cascades (like HOG MAPK pathway in yeast) [46] and other kinase, (such as ASK1, Arabidopsis serine/threonine kinase1) [14]. Oxidative stress can also activate MAPK cascades in plant (e.g., MPK6 and MEKK1 − MKK1/2 − MPK4 pathway in Arabidopsis)[47,48]. Therefore, secondary stress caused by salt treatment may contribute to the observed changes in protein phosphorylation. Comprehensive future studies will be aimed at explaining the particular contributions of various types of stresses to protein phosphorylation and plant growth associated with salt treatment.

4.3. Phosphoproteins regulated by the MKK9-MPK6 cascade under salt stress RCA is essential for photosynthesis in plants and modulates CO2 fixation by Rubisco [40,49]. RCA is a member of the ATPase associated with various cellular activities (AAA+ ) superfamily and its activity requires ATP hydrolysis [40,50]. RCA phosphorylation associated with RCA dark inactivation has been reported in Arabidopsis leaves [51]. In the present study, phosphorylation of RCA was significantly up-regulated in wild-type Arabidopsis seedlings after

Z. Liu et al. / Plant Science 241 (2015) 138–150

147

Fig. 6. Comparative analysis of Pro-Q-stained 2-D gel electrophoresis (2-DE) patterns in seedlings with constitutively activated MKK9. Total protein extracted from the mutant seedlings with constitutively active MKK9 (MKK9DD ), MKK9DD /mpk6 seedlings, and inactive mutant MKK9KR seedlings treated with 2 ␮M DEX. Proteins (1 mg) were focused on IPG strips (18 cm) with a linear gradient of pH 4–7. SDS-PAGE was performed using 11% acrylamide gels in the second dimension. Circles indicate spots changed in MKK9DD seedlings with constitutively active MKK9 and spots different in MKK9DD /mpk6 seedlings compared with MKK9DD seedlings.

salt treatment; however, RCA phosphorylation was up-regulated to a lesser degree in the mkk9 and mpk6 null mutants (Fig. 7B and C). Phospho-proteomics analysis of MKK9DD mutant seedlings revealed that RCA phosphorylation was up-regulated as a result of MKK9 activation (Fig. 7D). These results demonstrate that RCA is a salt-responsive phosphoprotein and show that phosphorylation of RCA is regulated by the MKK9-MPK6 cascade during the response to salt stress in Arabidopsis. The kinases upstream of RCA and the effects of phosphorylation on RCA activity have not been reported in plant. In animals, the activity of some AAA+ proteins, including valosin-containing protein (p97) [52] and mitotic factor p60 [53], is inhibited by phosphorylation. Therefore, we speculate that the MKK9-MPK6 cascade negatively regulates RCA activity by

phosphorylating RCA when seedlings are under salt stress. The expectation that inhibition of RCA activity would inhibit plant growth was confirmed by the phenotypes of the mkk9 and mpk6 null mutant seedlings under salt treatment. Future studies should verify the effects of phosphorylation on RCA activity and determine whether RCA is a direct substrate of MAPKs. PRPS1, FtsZ2-2, and TOR2 were also identified in this study as salt stress-responsive phosphoproteins regulated by the MKK9MPK6 cascade; however, these three proteins were not identified in MKK9DD mutant seedlings. MKK9DD seedlings are artificial mutant seedlings with activation and overexpression of MKK9. Activation of MAPK cascade in MKK9DD seedlings may be different from that in wild-type seedlings treated with salt stress. It is likely

148

Z. Liu et al. / Plant Science 241 (2015) 138–150

Fig. 7. Phosphorylation of RCA can be induced by salt treatment and MKK9 activation. (A) Venn diagram depicting overlaps of spots changed in every experimental group after salt treatment or MKK9 activation. (B, C) Enlarged detail of the change in RCA (spots 53 and 63) in Pro-Q-stained 2-D gel electrophoresis (2-DE) images of seedlings under salt treatment. The seedlings were not treated (0 h) or treated with liquid culture medium (LCM). The final concentration of NaCl was 100 mM. (D) Enlarged detail of the change in RCA (spot 63) after MKK9 activation.

that PRPS1, FtsZ2-2, and TOR2 were not identified in MKK9DD seedlings because phosphorylation of these proteins requires specific regulation by some factor associated with salt stress, such as altered subcellular localization of proteins. As a result, MKK9 activation might be insufficient to induce phosphorylation of certain proteins. PRPS1 is involved in photosynthesis and chlorophyll biosynthesis; as a result, mutations in prps1 affect growth and photosynthesis [54]. FtsZ2-2 is a self-activating GTPase that plays an important role in plastid division and shares structural similarity with tubulin [55,56]. The phosphorylation status of FtsZ2-2 affects Z-ring formation, suggesting that FtsZ2-2 phosphorylation regulates chloroplast division. [55]. However, the identities of the kinases upstream of FtsZ2-2 have not been reported in plants. Previous research shows that AtFtsz2-2 is phosphorylated on Thr282 (L274 LAAVSQSTpPVTEAFNLADDILR296 ) in vivo [55]. The Thr282 phosphorylation site conforms to the specific phosphorylation site (Ser/Thr-Pro) of MAPK substrates; moreover, Ftsz2-2 has a docking domain for MAPKs [57]. Our results suggest that the MKK9MPK6 cascade can regulate phosphorylation of Ftsz2-2 under salt stress; therefore, we speculate that Ftsz2-2 may be a substrate of MAPK6 under salt stress. TOR2 is involved in cytoskeleton organization and microtubulebased processes. A. thaliana tor2 mutants carry a point mutation

in a-tubulin 4 and show aberrant cortical microtubule dynamics. Microtubule defects caused by disturbed TOR2 function lead to over-branching and right-handed helical growth in single-celled leaf trichomes [58,59]. Phosphorylation levels of some proteins, including IPGAM1, IPGAM2, luminal-binding protein (BIP2), and Tudor-SN protein 2 (TUDOR2), were increased in the mkk9 and mpk6 seedlings in comparison with those of the wild-type seedlings before salt treatment (Table 1). In MKK9DD seedlings, phosphorylation levels of IPGAM2 and BIP2 were down-regulated by MKK9 activation (Table 4). These results suggest that the MKK9-MPK6 cascade might involve the processes which regulate dephosphorylation of IPGAM2 and BIP2. We speculate that MAPKs participate in the processes by activating an unknown phosphatase responsible for dephosphorylating these proteins [60]; in addition, these proteins could be phosphorylated by other kinases, which might be up-regulated in mkk9 and mpk6 mutants. Such kinase up-regulation might have been responsible for the enhanced phosphorylation of IPGAM2 and BIP2 observed in mkk9 and mpk6 mutants. In this study, we report 4 newly identified salt-responsive phosphoproteins, PRPS1, FtsZ2-2, TOR2, and RCA, which are regulated by the MKK9-MPK6 cascade in Arabidopsis seedlings and play important roles in several biological processes. These findings provide a

Z. Liu et al. / Plant Science 241 (2015) 138–150

149

Table 4 MKK9 activation-induced phosphoproteins. Spot no. Protein name

Accession no.a Protein scoreb Coverage (%)c Matched peptidesd Up-regulation (+)/downregulation (−)e MKK9DD MKK9DD /mpk6

32 38 39 44 47 63 74 75 66 67 129 130 131 132 133 134 136 139 140 145 85 86 93 94

Universal stress protein PHOS32 Cell division protein FtsZ-like protein 1 Ran-binding protein 1-a Clathrin light chain protein Fructose-1,6-bisphosphatase, cytosolic Rubisco activase ATP synthase CF1 beta subunit ATP synthase CF1 beta subunit Citrate synthase 4 ERBB-3 Binding protein 1 TCP-1/cpn60 chaperonin family protein TCP-1/cpn60 chaperonin family protein Dihydroxy-acid dehydratase mutase 1 Actin 7 Phospholipase A 2A Rubisco activase Pyruvate dehydrogenase E1 component subunit alpha Cysteine synthase NAD(P)-binding Rossmann-fold superfamily protein Class I glutamine amidotransferase domain-containing protein 2,3-Biphosphoglycerate-independent phosphoglycerate mutase 2 2,3-Biphosphoglycerate-independent phosphoglycerate mutase 2 Luminal-binding protein 2 Luminal-binding protein 2

AT5G54430 AT5G55280 AT1G07140 AT3G51890 AT1G43670 AT2G39730 ATCG00480 ATCG00480 AT2G44350 AT3G51800 AT3G13470 AT3G13470 AT3G23940 AT5G09810 AT2G26560 AT2G39730 AT1G01090 AT2G43750 AT2G37660 AT1G15040 AT3G08590 AT3G08590 AT5G42020 AT5G42020

75 89 79 181 133 121 97 103 136 109 138 179 68 97 109 91 76 133 75 101 204 186 97 108

38 42 48 60 48 46 37 40 47 48 30 37 23 50 45 42 25 38 32 25 60 53 28 31

7 9 7 11 12 10 8 10 11 10 13 13 7 9 10 7 7 8 7 10 12 11 7 10

+ + + + + + + + + + + + + + + + + + + + -

ns − − ns ns − − − ns ns ns ns − ns ns ns ns ns ns ns − + ns ns

ns. Not significant. a Accession number in the TAIR database. b Mascot score. c Sequence coverage by PMF using MALDI-TOF MS. d Number of peptides matched. e Up- or down-regulation of proteins in MKK9DD seedlings after activation of MKK9. MKK9DD /mpk6 seedlings were compared with MKK9DD seedlings.

new foundation for research into the manner in which the MAPK cascade participates in the response to salt stress in plants. Future studies should explore whether the proteins identified in this study are phosphorylated directly by MAPKs and determine the effects of phosphorylation on their function. Acknowledgements This work was supported by grants from the State Basic Research Program (2012CB114200 and 2014CB138205) and the National Natural Science Foundation of China (31125006 and 31030010) to D. Ren. References [1] W. Wang, B. Vinocur, A. Altman, Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance, Planta 218 (2003) 1–14. [2] N.J. Atkinson, P.E. Urwin, The interaction of plant biotic and abiotic stresses: from genes to the field, J. Exp. Bot. 63 (2012) 3523–3543. [3] J.K. Zhu, Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol. 53 (2002) 247–273. [4] P. Stepien, G.N. Johnson, Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte thellungiella: role of the plastid terminal oxidase as an alternative electron sink, Plant Physiol. 149 (2009) 1154–1165. [5] M.M. Chaves, J. Flexas, C. Pinheiro, Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell, Ann. Bot. 103 (2009) 551–560. [6] S.Y. Lee, P.N. Damodaran, K.S. Roh, Influence of salicylic acid on rubisco and rubisco activase in tobacco plant grown under sodium chloride in vitro, Saudi J. Biol. Sci. 21 (2014) 417–426. [7] Y. He, C. Yu, L. Zhou, Y. Chen, A. Liu, J. Jin, J. Hong, Y. Qi, D. Jiang, Rubisco decrease is involved in chloroplast protrusion and Rubisco-containing body formation in soybean (Glycine max.) under salt stress, Plant Physiol. Biochem.: PPB/Societe francaise de physiologie vegetale 74 (2014) 118–124.

[8] G. Tena, T. Asai, W.L. Chiu, J. Sheen, Plant mitogen-activated protein kinase signaling cascades, Curr. Opin. Plant Biol. 4 (2001) 392–400. [9] D. Ren, Y. Liu, K.Y. Yang, L. Han, G. Mao, J. Glazebrook, S. Zhang, A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 5638–5643. [10] S. Zhang, D.F. Klessig, MAPK cascades in plant defense signaling, Trends Plant Sci. 6 (2001) 520–527. [11] R.J. Davis, Signal transduction by the JNK group of MAP kinases, Cell 103 (2000) 239–252. [12] L. Gu, Y. Liu, X. Zong, L. Liu, D.P. Li, D.Q. Li, Overexpression of maize mitogen-activated protein kinase gene, ZmSIMK1 in Arabidopsis increases tolerance to salt stress, Mol. Biol. Rep. 37 (2010) 4067–4073. [13] S. Kiegerl, F. Cardinale, C. Siligan, A. Gross, E. Baudouin, A. Liwosz, S. Eklof, S. Till, L. Bogre, H. Hirt, I. Meskiene, SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK, Plant Cell 12 (2000) 2247–2258. [14] M. Mikolajczyk, O.S. Awotunde, G. Muszynska, D.F. Klessig, G. Dobrowolska, Osmotic stress induces rapid activation of a salicylic acid-induced protein kinase and a homolog of protein kinase ASK1 in tobacco cells, Plant Cell 12 (2000) 165–178. [15] H.D. Ding, X.H. Zhang, S.C. Xu, L.L. Sun, M.Y. Jiang, A.Y. Zhang, Y.G. Jin, Induction of protection against paraquat-induced oxidative damage by abscisic acid in maize leaves is mediated through mitogen-activated protein kinase, J. Integr. Plant Biol. 51 (2009) 961–972. [16] J. Wang, H. Ding, A. Zhang, F. Ma, J. Cao, M. Jiang, A novel mitogen-activated protein kinase gene in maize (Zea mays), ZmMPK3, is involved in response to diverse environmental cues, J. Integr. Plant Biol. 52 (2010) 442–452. [17] M. Teige, E. Scheikl, T. Eulgem, R. Doczi, K. Ichimura, K. Shinozaki, J.L. Dangl, H. Hirt, The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis, Mol. Cell 15 (2004) 141–152. [18] J. Xu, Y. Li, Y. Wang, H. Liu, L. Lei, H. Yang, G. Liu, D. Ren, Activation of MAPK kinase 9 induces ethylene and camalexin biosynthesis and enhances sensitivity to salt stress in Arabidopsis, J. Biol. Chem. 283 (2008) 26996–27006. [19] I.A. Alzwiy, P.C. Morris, A mutation in the Arabidopsis MAP kinase kinase 9 gene results in enhanced seedling stress tolerance, Plant Sci. 173 (2007) 302–308. [20] R.T. El-Khatib, A.G. Good, D.G. Muench, Analysis of the Arabidopsis cell suspension phosphoproteome in response to short-term low temperature and abscisic acid treatment, Physiol. Plant. 129 (2007) 687–697. [21] G.K. Agrawal, J.J. Thelen, Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins, Proteomics 5 (2005) 4684–4688.

150

Z. Liu et al. / Plant Science 241 (2015) 138–150

[22] T.H. Steinberg, B.J. Agnew, K.R. Gee, W.Y. Leung, T. Goodman, B. Schulenberg, J. Hendrickson, J.M. Beechem, R.P. Haugland, W.F. Patton, Global quantitative phosphoprotein analysis using multiplexed proteomics technology, Proteomics 3 (2003) 1128–1144. [23] B. Schulenberg, R. Aggeler, J.M. Beechem, R.A. Capaldi, W.F. Patton, Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye, J. Biol. Chem. 278 (2003) 27251–27255. [24] B.R. Chitteti, Z. Peng, Proteome and phosphoproteome differential expression under salinity stress in rice (Oryza sativa) roots, J. Proteome Res. 6 (2007) 1718–1727. [25] C. Rampitsch, N.V. Bykova, The beginnings of crop phosphoproteomics: exploring early warning systems of stress, Front. Plant Sci. 3 (144) (2012). [26] K.Y. Yang, Y. Liu, S. Zhang, Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 741–746. [27] D. Ren, H. Yang, S. Zhang, Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis, J. Biol. Chem. 277 (2002) 559–565. [28] M. Giribaldi, I. Perugini, F.X. Sauvage, A. Schubert, Analysis of protein changes during grape berry ripening by 2-DE and MALDI-TOF, Proteomics 7 (2007) 3154–3170. [29] G. Candiano, M. Bruschi, L. Musante, L. Santucci, G.M. Ghiggeri, B. Carnemolla, P. Orecchia, L. Zardi, P.G. Righetti, Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis, Electrophoresis 25 (2004) 1327–1333. [30] Z. Zhao, S.M. Assmann, The glycolytic enzyme, phosphoglycerate mutase, has critical roles in stomatal movement, vegetative growth, and pollen production in Arabidopsis thaliana, J. Exp. Bot. 62 (2011) 5179–5189. [31] L. Wu, Z. Han, S. Wang, X. Wang, A. Sun, X. Zu, Y. Chen, Comparative proteomic analysis of the plant-virus interaction in resistant and susceptible ecotypes of maize infected with sugarcane mosaic virus, J. Proteomics 89 (2013) 124–140. [32] G. Merkouropoulos, E. Andreasson, D. Hess, T. Boller, S.C. Peck, An Arabidopsis protein phosphorylated in response to microbial elicitation AtPHOS32, is a substrate of MAP kinases 3 and 6, J. Biol. Chem. 283 (2008) 10493–10499. [33] W. Hoehenwarter, M. Thomas, E. Nukarinen, V. Egelhofer, H. Rohrig, W. Weckwerth, U. Conrath, G.J. Beckers, Identification of novel in vivo MAP kinase substrates in Arabidopsis thaliana through use of tandem metal oxide affinity chromatography, Mol. Cell. Proteomics: MCP 12 (2013) 369–380. [34] T. Kleffmann, A. von Zychlinski, D. Russenberger, M. Hirsch-Hoffmann, P. Gehrig, W. Gruissem, S. Baginsky, Proteome dynamics during plastid differentiation in rice, Plant Physiol. 143 (2007) 912–923. [35] Y. Nanjo, L. Skultety, Y. Ashraf, S. Komatsu, Comparative proteomic analysis of early-stage soybean seedlings responses to flooding by using gel and gel-free techniques, J. Proteome Res. 9 (2010) 3989–4002. [36] Z. Zhao, W. Zhang, B.A. Stanley, S.M. Assmann, Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways, Plant Cell 20 (2008) 3210–3226. [37] M. Kanekatsu, H. Saito, K. Motohashi, T. Hisabori, The beta subunit of chloroplast ATP synthase (CF0CF1-ATPase) is phosphorylated by casein kinase II, Biochem. Mol. Biol. Int. 46 (1998) 99–105. [38] E. Goulas, M. Schubert, T. Kieselbach, L.A. Kleczkowski, P. Gardestrom, W. Schroder, V. Hurry, The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature, Plant J. 47 (2006) 720–734. [39] X. Shan, J. Wang, L. Chua, D. Jiang, W. Peng, D. Xie, The role of Arabidopsis Rubisco activase in jasmonate-induced leaf senescence, Plant Physiol. 155 (2011) 751–764. [40] A.R. Portis Jr., Rubisco activase—Rubisco’s catalytic chaperone, Photosynthesis Res. 75 (2003) 11–27. [41] U. La Cognata, V. Landschutze, L. Willmitzer, B. Muller-Rober, Structure and expression of mitochondrial citrate synthases from higher plants, Plant & Cell Physiol. 37 (1996) 1022–1029.

[42] I. Pracharoenwattana, J.E. Cornah, S.M. Smith, Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination, Plant Cell 17 (2005) 2037–2048. [43] D.C. Fulton, M. Stettler, T. Mettler, C.K. Vaughan, J. Li, P. Francisco, M. Gil, H. Reinhold, S. Eicke, G. Messerli, G. Dorken, K. Halliday, A.M. Smith, S.M. Smith, S.C. Zeeman, Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts, Plant Cell 20 (2008) 1040–1058. [44] J.D. Monroe, A.R. Storm, E.M. Badley, M.D. Lehman, S.M. Platt, L.K. Saunders, J.M. Schmitz, C.E. Torres, beta-Amylase1 and beta-amylase3 are plastidic starch hydrolases in Arabidopsis that seem to be adapted for different thermal, pH, and stress conditions, Plant Physiol. 166 (2014) 1748–1763. [45] T. Ghelis, G. Bolbach, G. Clodic, Y. Habricot, E. Miginiac, B. Sotta, E. Jeannette, Protein tyrosine kinases and protein tyrosine phosphatases are involved in abscisic acid-dependent processes in Arabidopsis seeds and suspension cells, Plant Physiol. 148 (2008) 1668–1680. [46] F. Posas, H. Saito, Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK, Science 276 (1997) 1702–1705. [47] T. Yuasa, K. Ichimura, T. Mizoguchi, K. Shinozaki, Oxidative stress activates ATMPK6, an Arabidopsis homologue of MAP kinase, Plant & Cell Physiol. 42 (2001) 1012–1016. [48] A. Pitzschke, A. Djamei, F. Bitton, H. Hirt, A major role of the MEKK1-MKK1/2-MPK4 pathway in ROS signalling, Mol. Plant 2 (2009) 120–137. [49] R.J. Spreitzer, M.E. Salvucci, Rubisco: structure, regulatory interactions, and possibilities for a better enzyme, Annu. Rev. Plant Biol. 53 (2002) 449–475. [50] A.R. Portis Jr., C. Li, D. Wang, M.E. Salvucci, Regulation of Rubisco activase and its interaction with Rubisco, J. Exp. Bot. 59 (2008) 1597–1604. [51] E. Boex-Fontvieille, M. Daventure, M. Jossier, M. Hodges, M. Zivy, G. Tcherkez, Phosphorylation pattern of Rubisco activase in Arabidopsis leaves, Plant Biol. 16 (2014) 550–557. [52] C. Lavoie, E. Chevet, L. Roy, N.K. Tonks, A. Fazel, B.I. Posner, J. Paiement, J.J. Bergeron, Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13637–13642. [53] E. Whitehead, R. Heald, J.D. Wilbur, N-terminal phosphorylation of p60 katanin directly regulates microtubule severing, J. Mol. Biol. 425 (2013) 214–221. [54] I. Romani, L. Tadini, F. Rossi, S. Masiero, M. Pribil, P. Jahns, M. Kater, D. Leister, P. Pesaresi, Versatile roles of Arabidopsis plastid ribosomal proteins in plant growth and development, Plant J. Cell Mol. Biol. 72 (2012) 922–934. [55] D. Gargano, J. Maple-Grodem, S.G. Moller, In vivo phosphorylation of FtsZ2 in Arabidopsis thaliana, Biochem. J. 446 (2012) 517–521. [56] D. Gargano, J. Maple-Grodem, V. Reisinger, L.A. Eichacker, S.G. Moller, Analysis of the chloroplast proteome in arc mutants and identification of novel protein components associated with FtsZ2, Plant Mol. Biol. 81 (2013) 235–244. [57] A.D. Sharrocks, S.H. Yang, A. Galanis, Docking domains and substrate-specificity determination for MAP kinases, Trends Biochem. Sci. 25 (2000) 448–453. [58] T. Ishida, Y. Kaneko, M. Iwano, T. Hashimoto, Helical microtubule arrays in a collection of twisting tubulin mutants of Arabidopsis thaliana, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 8544–8549. [59] H. Buschmann, M. Hauptmann, D. Niessing, C.W. Lloyd, A.R. Schaffner, Helical growth of the Arabidopsis mutant tortifolia2 does not depend on cell division patterns but involves handed twisting of isolated cells, Plant Cell 21 (2009) 2090–2106. [60] H.C. Park, E.H. Song, X.C. Nguyen, K. Lee, K.E. Kim, H.S. Kim, S.M. Lee, S.H. Kim, D.W. Bae, D.-J. Yun, W.S. Chung, Arabidopsis MAP kinase phosphatase 1 is phosphorylated and activated by its substrate AtMPK6, Plant Cell Rep. 30 (2011) 1523–1531.