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Aug 17, 2005 - Furthermore, when the hormone/stress regulated phosphoproteins are compared in rice leaf sheath, leaf blade and root, only cytoplasmic ...
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Comprehensive Phosphoproteome Analysis in Rice and Identification of Phosphoproteins Responsive to Different Hormones/Stresses Monowar Khan, Hironori Takasaki, and Setsuko Komatsu* Department of Molecular Biology, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Received April 25, 2005

Phosphoproteins in rice were detected by in vitro protein phosphorylation followed by two-dimensional polyacrylamide gel electrophoresis. Forty-four phosphoproteins were detected on a 2D-gel after in vitro protein phosphorylation of the crude extract from rice leaf sheath. Among the phosphoproteins detected, 42 were identified through analysis by Q-TOF MS/MS and/or MALDI-TOF MS. The largest percentage of the identified phosphoproteins are involved in signaling (30%), while 18% are involved in metabolism. When rice seedlings were treated with various hormones and stresses, it was observed that the phosphorylation of 13 proteins was enhanced differentially by different hormone and stress treatments. Furthermore, when the hormone/stress regulated phosphoproteins are compared in rice leaf sheath, leaf blade and root, only cytoplasmic malate dehydrogenase was found to be phosphorylated in all the tissues. Results suggest that in the phosphorylation cascade of rice, glycolytic metabolism processes and Ca2+-signaling seem to be important targets in response to hormones and stresses. Furthermore, the direct visualization of phosphoproteins by 32P-labeling and their mass spectrometric identification provides an accurate and reliable method of analyzing the rice phosphoproteome. Keywords: phosphoproteome • rice • hormone • stress

Introduction Phosphorylation is the most important post-translational modifications of proteins that modulate protein activity and transmit signals within cellular pathways and networks.1,2 In signal transduction in eukaryotes, protein phosphorylation is a key event. A wide range of cellular process, particularly, protein kinase activation, cell cycle progression, cellular differentiation and transformation, developmental, and hormone response, are all regulated by changes in the state of protein phosphorylation. It is believed that as many as one-third of all cellular proteins derived from mammalian cells are phosphorylated. Protein phosphatases and kinases regulate a variety of cellular processes.3 Understanding the post-translational modifications of proteins is rather a daunting task. Owing to the importance of protein phosphorylation in regulating cellular signaling, a major goal of current proteomic efforts is to identify the phosphoproteins in higher organisms and understand their definite functions. In plants, most of the developmental events and environmental responses including the regulation of growth and differentiation are controlled by the phosphorylation of different proteins. The phosphorylation activity of two proteins in rice leaf was increased by cAMP.4 In celery (Apium graveolens), the calcium-binding properties of a calcium binding protein, VCaB45 are modulated by phosphorylation.5 In potato (Solanum * To whom correspondence should be addressed. Setsuko Komatsu, Department of Molecular Genetics, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan. Tel: 81-29-838-7446. Fax: 81-29-838-7408. E-mail: [email protected].

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tuberosum L.), StMBF1, a member of the MBF1 transcriptional co-activator family is phosphorylated by fungal elicitor.6 Further, in gid2, a gibberellin (GA)-insensitive dwarf rice mutant, SLR1 protein is strongly phosphorylated by exogenous application of GA.7 Similarly, many proteins in different plants have been reported to be phosphorylated under various stimuli and showed specific response. Recently, Nuhse et al. have identified more than 300 phosphorylation sites from Arabidopsis thaliana plasma membrane proteins, demonstrating a large number of proteins phosphorylated in plants.8 Now it is well-known that hundreds of proteins are phosphorylated during the normal growth of plants. Only a very small fraction of the thousands of protein kinases and phosphates in rice has been studied experimentally to date. To understand the molecular mechanism of signal transduction in plants, research on the analysis of phosphoproteins needs to be studied systematically. Rice proteomics research entered into the new era since the completion of genome sequences for few species in 2002 and a rapid progress in other species/varieties. During the past few years, efforts were being put to unveil the functionality of the genes following proteomic approach, and tremendous progress has been made in generating large-scale data sets for tissue and organelle composition, protein function and profiles in rice.9,10 Recent systematic protein analyses of various subcellular compartments/organelles from rice such as plasma membrane, vacuolar membrane, Golgi membrane, mitochondria and chloroplast,11 and nucleus12 provides enormous information on plant function. Further, a detailed mitrochondria13 proteome analysis from rice provides much information 10.1021/pr0501160 CCC: $30.25

 2005 American Chemical Society

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Phosphoproteome Analysis in Rice

on the function of rice michondria. Unlike straightforward protein analysis, phosphoproteins in rice have not yet been analyzed in detailed. Clearly, rice phosphoproteome research is still at its early stage. After the completion of genome sequencing in rice, the identification of various signaling components is currently feasible with phosphoproteomics. To address the challenging problem, the advantage of in vitro protein phosphorylation technique using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by mass spectrometric analysis has been taken. Labeling of the proteins with [32P] is generally used for phosphoproteomics as a highly selective and sensitive technique of detecting phosphoproteins.14,15 In vitro protein phosphorylation using [γ-32P]ATP followed by 2D-PAGE separation and expose to X-ray film allows direct visualization and quantification of phosphoproteins spots. Further, the most powerful method of analyzing the phosphoproteins is the use of mass spectrometry.16 Besides the analysis of individual phosphoproteins, it is very important to elucidating the phosphorylation state of a collection of proteins at an organelle wide or cell-wide level. Such global analysis promise to provide a new window into the inside changes in a cell. A comprehensive identification of phosphoproteins in different tissues of rice, the rice phosphoproteomics will provide a basis for understanding the signaling components of the plant under any stimuli. Signaling pathways have to be regarded as complex networks. These signal networks are characterized by multiple points of convergence and divergence that enable integration of signaling pathways at different levels and provide the molecular basis for appropriate downstream responses. For example, Nicotiana benthamiana plants with reduced levels of NtCDPK1 show severe abnormalities in cell morphology, spontaneous necrotic lesions, and increased expression of marker genes for the plant defense.17 Similarly, cold tolerance and GA-dependent elongation were promoted through distinct signaling pathways that crosstalk at the level of OsCDPK13.18 In this study, a detailed phosphoproteome analysis in different tissues of rice has been carried out using in vitro protein phosphorylation technique followed by mass analysis. Furthermore, the changes of protein phosphorylation by different hormones and stresses have been investigated.

Experimental Section Plant Material and Growth Condition. Rice (Oryza sativa L. cv. Nipponbare) seedlings were grown in pots under white fluorescent light (600 µmol/m2/s, 12 h light period/day) at 25 °C and 70% relative humidity in a growth chamber. At 2 weeks growth stage, the plants were treated with various hormones, 5.0 µM gibberellin (GA3, Wako, Osaka, Japan), 0.1 µM brassinolide (BL, Daiichi fine chemical, Toyama, Japan), 0.45 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D, Wako), 1.0 µM abscisic acid (ABA, Wako) and 50 mM NaCl for 24 h. For cold treatment, the plants were placed in a growth chamber at 5 °C for 24 h. Leaf sheath tissues were collected from the seedlings after treatments. Preparation of Crude Protein Extract. The following procedures were carried out at 4 °C. For protein extraction, leaf sheaths (200 mg), leaf blade (100 mg) and root (150 mg) were homogenized in a mortar with a pestle in a 300 µL extraction buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EGTA, 5 µM sodium vanadate and 1 mM phenylmethylsulfonyl fluoride. The

homogenate was centrifuged at 20 000 × g for 5 min and the supernatant was used as the crude protein extract. In Vitro Protein Phosphorylation and 2D-PAGE. Five micro liter protein extract was incubated in a 25 µL reaction mixture containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.2 mM CaCl2 and 39 µM [γ-32P]ATP (110 TBq/mmol, Amersham Bioscience, Piscataway, NJ). The reaction mixture was incubated for 10 min at 30 °C and terminated by the addition of a lysis buffer containing 8 M urea, 2% Triton X-100, 2% ampholine (pH 3.5-10) and 10% poly(vinylpyrrolidone)-40 19. Denatured proteins were subjected to isoelectric focusing (IEF) in tube gels and then 15% SDS-PAGE was carried out. The gels were stained with silver staining kit (Bio-Rad, Hercules, CA) or Coomassie brilliant blue (CBB) and exposed to X-ray film (Fuji, Tokyo, Japan) at -80 °C for 2-6 days. ESI-Q/TOF-MS/MS Analysis. Silver and CBB stained phosphoprotein spots were excised from gels, washed with 15 mM potassium ferricyanide and 50 mM sodium thiosulfate, and destained with 50 mM NH4HCO3 in 50% methanol at 40 °C for 15 min. Proteins were reduced with 10 mM dithiothreitol in 100 mM NH4HCO3 at 50 °C for 1 h and incubated with 40 mM iodoacetamide in 100 mM NH4HCO3 for 30 min. The gel pieces were minced and allowed to dry and then rehydrated in 10 mM Tris-HCl (pH 8.5) with 1 pM trypsin at 37 °C for 10 h. The digested peptides were extracted from the gel slices with 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile/water for 3 times. The peptide solutions were analyzed by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q/TOF-MS/ MS) (Q-TOF micro; Micromass, Manchester, UK). MS/MS data were processed with a maximum entropy data enhancement program MaxEnt 3 (Micromass). The resultant spectra were interpreted with SeqMS, a software aid for de novo sequencing by MS/MS. The sequence tags obtained were also used for the homology search in the database with Mascot software (Matrix Science Ltd., London, UK). MALDI-TOF MS Analysis. Silver and CBB stained phosphoprotein spots were excised from gels and prepared automatically by using DigestPro 96 (M & S Instruments Trading Inc., Tokyo, Japan) following the manufacturer’s instruction as follows. The gel pieces were digested with equal volume of 100% acetonitrile and 50 mM NH4HCO3 for 15 min followed by 10 min dehydration with 10% acetonitrile. Proteins were then reduced with 10 mM dithiothreitol for 10 min and incubated with 50 mM iodoacetamide in 50 mM NH4HCO3 for 15 min. The reduced proteins were digested with 1 pM trypsin or lysylendpeptidase at 37 °C for 4 h. The digested peptides were extracted with equal volume of 100% acetonitrile and 10% formic acid, dried up and reconstituted with 30 µL of 0.1% TFA in 5% acetonitrile/water, and then desalted by ZipTipC18 pipet tips (Millipore, Bedford, MA). The peptide solution was mixed with the matrix solution, R cyano-4- hydroxycinnamic acid, and air-dried on the flat surface of a carbon plate and analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) (Voyager RP, Applied Biosystems, Framingham, MA). Calibrations were carried out using a standard peptide mixture. The mass spectra were subjected to sequence database search with Mascot software (Matrix Science Ltd.).

Results and Discussion Detection of Phosphoproteins in Rice Leaf Sheath by In Vitro Labeling. The leaf sheath of rice seedlings, including the crown, is a functionally important region where many of the Journal of Proteome Research • Vol. 4, No. 5, 2005 1593

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Figure 1. Phosphoprotein pattern of rice leaf sheath proteins. Crude proteins were extracted from leaf sheath and incubated in a reaction mixture containing [γ-32P]ATP. After in vitro protein phosphorylation, proteins were subjected to 2D-PAGE. The 2D-gel was exposed to X-ray film for 2 (A), 4 (B), and 6 (C) days and phosphoproteins detected. After in vitro protein phosphorylation and separation by 2D-PAGE, the proteins were detected by silver staining (D). The pI and molecular weight of each phosphoprotein were determined using 2D-PAGE Marker. Arrows denote the positions of phosphoproteins.

critical metabolic and regulatory activities that eventually control the height and robustness of the plant take place. In a previous study, we analyzed the leaf sheath proteome, which includes proteins of the basal region.20, 21 No clear function could be predicted for 20% of the proteins in the leaf sheath proteome, but a majority of proteins identified are involved in central metabolic pathways and energy production. Although the leaf sheath containing basal region is important for rice plant growth, there are as yet no quantitative measures of proteome data. To identify phosphoproteins in rice leaf sheath, proteins extracted from the tissue were labeled by incubating with [γ-32P]ATP. After stopping the reaction, protein samples were separated by 2D-PAGE. The proteins were visualized by silver staining of 2D-gels and phosphoproteins were visualized by exposing the 2D-gels on X-ray film. Phosphoproteins visualized on X-ray film are shown in Figure 1. The gels were exposed for 2 (Figure 1A), 4 (Figure 1B), and 6 (Figure 1C) days respectively, in order to visualize the largest number of phosphorylated proteins in rice leaf sheath. Using this method a total of 44 phosphoproteins were visualized. The corresponding phosphoproteins visualized on X-ray film were observed on 2Dgel by silver staining (Figure 1D). The levels of phosphorylation as visualized on X-ray film do not necessarily correlate with levels of protein as detected by silver staining of the 2D gel. For example, spot 10 (Figure 1D) is strongly stained but is weakly labeled (Figure 1A) whereas spot 15 (Figure 1D) is weakly stained but strongly labeled (Figure 1C). The former protein may therefore have a low degree of phosphorylation under the experimental conditions. Alternatively, it is possible that it was already phosphorylated 1594

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before the incubation with [γ-32P]ATP so that unlabeled phosphates occupied most of the phosphorylation sites. A total of about 900 protein spots were detected on 2D-gel by silver staining (Figure 1D). Of these, only 44 are phosphoproteins as defined by their being detected on X-ray film (Figure 1C). This corresponds to 4.9% of the total number of proteins. Although it is believed that a large percentage of cellular proteins are phosphorylated,22,13 there have been very few phosphoproteome studies in rice. This lack of experimental work leaves unanswered key questions concerning the phosphorylation of proteins during the normal growth of the plant. Predictions of phosphorylation status of proteins from sequence patterns have limited sensitivity and lack specificity.24 It is therefore preferable that protein phosphorylation should be observed directly. The present findings provide the first, basic understanding of the rice phosphoproteome. Identification of Rice Leaf Sheath Phosphoproteins by Mass Spectrometry. Phosphoproteins in rice leaf sheath visualized on X-ray film were excised from 2D-gels and identified by ESIQ/TOF MS/MS and MALDI-TOF MS. Out of 44 phosphoprotein spots, 42 phosphoproteins could be identified by these methods and they are grouped based on their functions in Table 1. Furthermore, 6 proteins out of them were identified phosphorylation site of protein by MASCOT software search after LC Q-TOF MS/MS analysis (Table 2). Two phosphoproteins (spot number 01 and 33) could not be analyzed by either of the methods. These 2 phosphoproteins were very weakly visible on 2D-gels, indicating their low abundance. The probability

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Phosphoproteome Analysis in Rice Table 1. Identification of Phosphoproteins in Leaf Sheath of Rice Seedling by Mass Spectrometry spot MW no. 03 12 18 19 20 21 28 29 02 07 08 09 10 23 24 25 27 35 37 38 41 17 31 04 22 30 06 42 43 14 32 39 26 34 36 05 11 13 15 16 40 44 01 33 a

pI

homologous protein

Metabolism 64.3 5.5 Citrate synthase 43.3 6.2 OsGA2ox2 gene for putative gibberellin 2-oxidase 39.2 5.6 UDP-galactose 4-epimerase-like protein 38.3 5.9 Glyceraldehyde-3-phosphate dehydrogenase 37.5 5.7 Cytoplasmic malate dehydrogenase 36.4 5.1 Aldo/keto reductase family protein 33.2 5.6 Glyoxalase -I 31.5 5.8 5-aminolevulinate synthase Signaling 77.3 5.0 Serine/threonine protein kinase (YK21) 60.1 5.0 Signal recognition particle 54-kDa protein (spr54-2) 59.2 5.1 GTP-binding protein Rab36 57.3 4.7 Calcium binding protein 57.2 4.2 Zinc finger family protein 36.1 4.7 Putative zinc finger protein 35.4 5.7 Putative IAA1 protein (iaa1 gene) 35.3 4.0 Signal recognition particle 54-kDa protein (Srp54-1) 34.2 5.9 GTP binding protein 25.3 5.5 GTPase Ras 2p (ras2) 23.4 5.7 myb protein 23.1 5.2 Serine/threonine protein phosphatase 20.4 5.1 myb protein Cell growth and maintenance 39.3 4.0 Transcriptional factor B3 family protein contains Pfam profile PF023622 29.6 5.7 Calmodulin-related protein Protein folding and degradation 63.1 5.2 NAD ADP-ribosyltransferase 36.2 5.5 26S proteosome regulatory partial triple-A (OsRPT1b) 31.3 5.2 26S proteosome regulatory partial triple-A (OsRPT1b) 60.3 4.2 Chaperonin R subunit Energy 19.3 5.7 Chlorophyll a/b binding protein precursor 18.1 5.5 RuBisCO Transcription 41.2 5.9 Histone deacetylase Unclear function 28.4 4.6 PC-2 xylanase (xyn A) 22.3 5.0 Guichao2 RSSG8 35.2 4.2 Hypothetical protein 26.2 5.6 Reversibly glycosylated polypeptide-2 24.3 5.3 Hypothetical protein Unknown 61.5 5.4 Not significantly matched 48.7 4.7 Not significantly matched 42.4 5.8 FLJ32871 fis, clone TESTI2003914 41.0 6.3 Not significantly matched 39.5 5.7 Not significantly matched 21.2 5.5 At clone 4329 17.5 5.1 Hypothetical protein Not detected 80.1 4.8 Not detected 27.1 4.6 Not detected

accession IDa,b no.

AF302906 MALDI AB092484 MALDI

Table 2. Identification of Phosphorylation Modification of Protein by Q-TOF LC-MS/MS and MASCOT Software spot no.

MW

pI

accession no.

04

63.1 5.2 AK103479

06

60.3 4.2 AK100602

43

18.1 5.5 AK105600

26

35.2 4.2 AK100503

34

26.2 5.6 AK098933

36

24.3 5.3 AK058391

AB099292 MALDI U31676

Q-TOF

AF353203 Q-TOF AK100718 MALDI AB017042 Q-TOF AF073341 MALDI AF230510 MALDI AB115419 MALDI AB023061 AB021259 AK108060 AY224508 AJ251791 AB114831

MALDI Q-TOF MALDI MALDI MALDI MALDI

AK069539 AF321464 AF467733 AK101918

MALDI MALDI MALDI MALDI

AF467733 MALDI AK106332 MALDI AK062711 MALDI AK103479 Q-TOF AB070252 MALDI AB070252 MALDI AK100602 Q-TOF AY445626 MALDI AK105600 Q-TOF AK099909 MALDI U57819 AY034141 AK100503 AK098933

MALDI MALDI Q-TOF Q-TOF

AK058391 Q-TOF MALDI MALDI AK057433 MALDI MALDI MALDI AY088216 MALDI AK109550 MALDI Q-TOF QTOF

MALDI: MALDI-TOF MS. a Q-TOF: LC-MS/MS.

for each of the identified proteins to be phosphorylated was evaluated by NETPHOS 2.0 (www.cbs.dtu.dk/services/NetPhos).

start-end amino acid

amino acid sequences

15-21 104-110 178-188 29-39 154-168 235-243 127-143 144-162 327-340 341-370

SGRSSCK C(pT)IEVAK DAPSSGQTSSK LTYYTPEYETK TFQGPPHGIQVERDK SQAE(pT)GEIK AVLQDIAIVTGAEFLAK DLGLLVENATEEQLGTARK YENLIEAGVIDPAK VTRCALQNAA(pS)VAGMVL (pT)TQAIVVEKPKPK DLELKICLVK REK(pS)GHGTK SGHGTK(pS)K YVF(pT)IDDDCFVAK ASNPFVNLK GIFWQEDIIPFFQNATIPK IDPYFVK LGISSNNSK RRVETVA(pS)WDSLP(pT) SLIK

112-121 226-274 269-276 105-117 276-284 289-307 330-336 2-10 154-171

Analysis of the function of the identified phosphoproteins allows the proteins to be put into groups based on shared function. Proteins involved in cell signaling form the largest percentage of the phosphoproteins identified (13/44 ) 29.5%). Among these are serine/threonine protein kinase, signal recognition particle 54-kDa protein, GTP-binding protein, calcium binding protein etc. A considerable percentage (8/44 ) 18.2%) of phosphoproteins in rice leaf sheath were found to be involved in metabolism. The next largest set are involved in protein folding and degradation (4/44 ) 9.1%) and finally cell growth and maintenance proteins account for just 2 out of the 44 proteins identified (4.5%). Other phosphoproteins identified are involved in energy production and transcription. Of the phosphoproteins identified, the functions of 7 proteins are unknown, (this number also includes the proteins that did not give a significant match in the available database). Furthermore, 5 other proteins, namely PC-2 xylanase, guichao 2 RSSG8, reversibly glycosylated polypeptide-2, and 2 hypothetical proteins were detected for which the function is not clear to date and which are categorized into an unclear function group. As described above, the identified rice leaf sheath phosphoproteins can be grouped into different categories according to their predicted functions (Table 1). This is a very useful tool for the understanding of the overall function of the phosphoproteins in a given tissue. Phosphoproteins identified in leaf sheath are dominated by signaling molecules, indicating that phosphoproteins largely form the signaling component of a cell. Nevertheless, the second largest group consisted of proteins involved in metabolism. In rice proteome analysis, metabolic proteins form the largest percentage of proteins in leaf sheath20 while in phosphoproteome analysis, they formed the second largest percentage. Phosphoproteins involved in other cellular functions such as energy, cell growth, and maintenance and protein folding and degradation were also identified in considerable numbers (Table 1). The unique function of different proteins in rice leaf sheath is better understood through this comprehensive phosphoproteome analysis than by simple proteome analysis.20 Changes of Protein Phosphorylation Status in Rice Leaf Sheath by Treatment with Different Hormones. To demonstrate whether the exogenous application of hormones could change the phosphorylation status of rice leaf sheath proteins, Journal of Proteome Research • Vol. 4, No. 5, 2005 1595

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rice seedlings were treated with 5.0 µM GA3, 0.1 µM BL, and 0.45 µM 2, 4-D for 24 h. When rice leaf sheath segments were treated with plant hormones in Petri dishes, a 6 h treatment was enough to enable phopshoprotein detection (data not shown). However, in the case of intact rice plant treatment, only a few phosphoproteins were detected after gibberellin treatment for 6 h. Because a 6 h treatment was insufficient, in this study we treated rice plants with hormones for 24 h. As described above, crude proteins were extracted and phosphorylated in vitro using [γ-32P]ATP. After in vitro protein phosphorylation, the proteins were separated by 2D-PAGE and the phosphoproteins influenced by the different hormones visualized on X-ray film. The results show that a similar overall pattern of phosphorylation is observed whichever hormone is used for the treatment. However, there are some spots which do change differentially with the different hormones. With GA3 the phosphorylation status of 6 proteins varies. These are calcium binding protein (spot no. 09), glyceraldehyde-3phosphate dehydrogenase (spot no. 19), cytoplasmic malate dehydrogenase (spot no. 20), putative zinc finger protein (spot no. 23), glyoxalase-I (spot no. 28) and another unknown protein (spot no. 36). With BL there are 3 proteins whose phosphorylation status varies namely, glyceraldehyde-3-phosphate dehydrogenase (spot no. 19), cytoplasmic malate dehydrogenase (spot no. 20), and aldo/keto reductase family protein (spot no. 21). With 2,4-D there are 5 proteins which vary. These are glyceraldehyde-3-phosphate dehydrogenase (spot no. 19), cytoplasmic malate dehydrogenase (spot no. 20), putative zinc finger protein (spot no. 23), glyoxalase-I (spot no. 28), and calmodulin-related protein (spot no. 31) (Figure 2 and Table 1). From these results it can be seen that the phosphorylation of 4 proteins (glyceraldehyde-3-phosphate dehydrogenase, cytoplasmic malate dehydrogenase, putative zinc finger protein and glyoxalase-I) is increased by both GA3 and 2, 4-D treatment. In addition, the phosphorylation of one protein (aldo/keto reductase family protein) was increased by BL alone, while the phosphorylation of another protein (calmodulin-related protein) was increased by 2, 4-D alone. The phosphorylation of two of the proteins (calcium binding protein and an unknown protein) changed only with GA3 treatment. These changes thus demonstrate hormone specific phosphorylation. In contrast, the phosphorylation of 2 proteins, glyceraldehyde-3-phosphate dehydrogenase and cytoplasmic malate dehydrogenase was enhanced by all of the hormones used (Figure 2). Glyceraldehyde-3-phosphate dehydrogenase and cytoplasmic malate dehydrogenase play a critical role in the glycogen metabolism pathway in the cell. Glyceraldehyde-3-phosphate dehydrogenase is an NAD+ specific enzyme that reversibly catalyzes the oxidation and phosphorylation of D-glyceraldehyde-3-phosphate to D-1, 3-biphosphoglycerate. Malate dehydrogenase reversibly catalyzes the incorporation of malate to oxaloacetate utilizing the NAD+/NADH cofactor system25 and is universally present in all eukaryotic cells in 2 isoforms, cytoplasmic malate dehydrogenase and mitochondrial malate dehydrogenase. Glyceraldehyde-3-phosphate dehydrogenase and cytoplasmic malate dehydrogenase are involved in the synthesis of different metabolites and the subsequent production of energy. The enhanced phosphorylation of these 2 proteins by different hormones indicates that this is the mechanism through which the hormones activate metabolic pathways in rice leaf sheath and thus play a crucial role in plant growth. 1596

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Figure 2. Effect of various hormones on phosphorylation of rice leaf sheath proteins. Rice seedlings were treated with 5.0 µM GA3, 0.1 µM BL and 0.45 µM 2, 4-D for 24 h. Crude proteins from leaf sheath were extracted and incubated in a reaction mixture containing [γ-32P]ATP. After in vitro protein phosphorylation, proteins were subjected to 2D-PAGE. The 2D-gels were exposed to X-ray film for 6 days and phosphoproteins detected. The proteins phosphorylated as a result of treatment by various hormones as observed on X-ray film were compiled in one chart to show hormone dependent protein phosphorylation. Arrowheads denote the positions of proteins phosphorylated by different hormones. Venn diagram analysis shows phosphorylation of the proteins that overlapped between hormones. The pI and molecular weight of each phosphoprotein were determined using 2D-PAGE Marker. Spot numbers are corresponding to numbers in Figure 1 and Table 1.

Changes of Protein Phosphorylation Status in Rice Leaf Sheath by Various Stresses. To investigate the effect of various stresses on phosphorylation activities of rice leaf sheath proteins, rice seedlings were treated with 1.0 µM ABA, 50 mM NaCl or 5 °C for 24 h. Crude proteins from leaf sheath were extracted and the proteins were separated by 2D-PAGE after in vitro protein phosphorylation. Phosphorylation of the proteins enhanced by different stresses was detected on X-ray film and the results have been compiled in Figure 3. It was observed that the phosphorylation of 3 proteins, Zinc finger family protein (spot no. 10), cytoplasmic malate dehydrogenase (spot no. 20), and calreticulin (“spot a”) was increased by ABA treatment. The phosphorylation of 4 proteins, cytoplasmic malate dehydrogenase (spot no. 20), calreticulin (“spot a”), and 2 unknown proteins (spot no. 34 and “spot b”) was increased by NaCl. The phosphorylation of 4 proteins, glyoxalase-I (spot no. 28), calmodulin-related protein (spot no. 31), calreticulin (“spot a”) and an unknown protein (“spot c”) by cold treatment.

Phosphoproteome Analysis in Rice

Figure 3. Effect of various stresses on phosphorylation of rice leaf sheath proteins. Rice seedlings were treated with 1.0 µM ABA, 50 mM NaCl and 5 °C cold for 24 h. Crude proteins from leaf sheath were extracted and incubated in a reaction mixture containing [γ-32P]ATP. After in vitro protein phosphorylation, proteins were subjected to 2D-PAGE. The 2D-gels were exposed to X-ray film for 6 days and phosphoproteins detected. The proteins phosphorylated by various stresses as observed on X-ray film were compiled in one chart to show stress dependent protein phosphorylation. Arrowheads denote the positions of proteins phosphorylated by different stresses. Venn diagram analysis shows phosphorylation of the proteins that overlapped between stresses. The pI and molecular weight of each phosphoprotein were determined using 2D-PAGE Marker. Spot numbers correspond to numbers in Figure 1 and Table 1. “Spots a, b and c” are the phosphoproteins which could not be detected in the plants without stress.

The results show that one common phosphoprotein, calreticulin was regulated by ABA, NaCl and cold treatments but no other common phosphoprotein was regulated either by ABA and NaCl, ABA and cold, or NaCl and cold treatments. Phosphorylation of 3 proteins, calreticulin (“spot a”), and 2 unknown proteins (“spot b and c”) was not detected by in vitro protein phosphorylation during the normal growth of rice (Figure 1), but observed only under different stress conditions. This is because these proteins likely to be very weakly phosphorylated during normal plant growth. Considering the importance of the common signaling component of stresses, we analyzed phosphoprotein ‘a’, which was identified as calreticulin. The molecular weight and pI of calreticulin were 56 kDa and 4.3, respectively on the gel. Calreticulin, a Ca2+ binding protein, was shifted from basic site to scidic site by cold stress in rice leaf sheath 21, which might be due to phosphorylation. Calreticulin accomplishes the regeneration of rice cultured suspension cells 26. The enhanced phosphorylation of calreticulin by different stresses in rice indicates that it might be a very important signaling component whereby stresses invoke various cellular and developmental events in cells.

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Figure 4. Protein phosphorylation showing tissue specific expression under various hormones and stresses. Crude proteins from leaf blade (A), leaf sheath (B) and roots (C) were extracted and incubated in a reaction mixture containing [γ-32P]ATP. After in vitro protein phosphorylation, proteins were subjected to 2DPAGE. The 2D-gels were exposed to X-ray film for 2 (right) and 6 (left) days and phosphoproteins detected. The pI and molecular weight of each phosphoprotein were determined using 2D-PAGE Marker. Arrows denote the positions of phosphoproteins, which showed enhanced phosphorylation by various hormones and stresses in different tissues.

Tissue Specific Changes of Phosphorylation by Different Hormones and Stress. Attempts were made to identify phosphoproteins in leaf sheath whose phosphorylation was increased by different hormones and stresses and to ascertain whether similar changes were observed in leaf blade and root.In leaf sheath a total of 13 hormone and stress responsive phosphoproteins were found. Of these, only 2 proteins, namely cytoplasmic malate dehydrogenase (spot no. 20) and another unknown protein (“spot c”) were detected in leaf blade (Figure 4A). In root 3 phosphoproteins, glyceraldehyde-3-phosphate dehydrogenase (spot no. 19), 5-aminolevulinate synthase (spot no. 29) and an unknown protein (spot no. 36) were responsive (Figure 4C). One protein, cytoplasmic malate dehydrogenase was found to be phosphorylated in all the tissues tested. Different organs of a plant plays differential roles during the growth of a plant and respond against biotic/abiotic and/or environmental stimuli. Here, the phosphorylation of malate Journal of Proteome Research • Vol. 4, No. 5, 2005 1597

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sense and antisense plants compared to that of control plants (Figure 5). The results suggest that these 3 proteins might be an important signaling component in the response of rice to GA and cold stress.

Concluding Remarks An inclusive phosphoprotein analysis using phosphoproteomics provides a clear insight into the molecular function of various proteins in a particular cell/tissue under a set of environmental conditions. The phosphoproteome research conducted here indicates that rice seedlings respond differently to hormones and stresses, with alterations in the phosphorylation status of particular proteins or signaling molecules. Here, glycolytic metabolism and Ca2+-signaling processes were found to be important targets of the phosphorylation cascades in rice under hormone and stress conditions. Some cross talk or common signaling molecules for various hormones and different stresses in rice were observed. Furthermore, a common signaling molecule was identified in various parts of the rice plant, indicating a coordinated response of the plant to various stimuli. Direct visualization of phosphoprotein spots on X-ray film after in vitro phosphorylation of the proteins using [γ-32P]ATP followed by 2D-PAGE separation and mass spectrometric analysis provided a very useful tool for reliable and accurate analysis of phosphoproteins in rice. Chemical modifications of phosphoproteins by isotope-coded affinity tags (ICAT) and the use of immobilized metal affinity chromatography (IMAC) for purification and quantification of low abundance phosphopeptides from a complex mixture of proteins are also useful techniques for phosphoproteome analysis.27,28 Fluorescent labeling of phosphoproteins seems to be another promising approach in this area of research.29 It is hoped that more progress on phosphoproteome analysis in rice will be made soon to elucidate various signaling cascades in the plant. Figure 5. Phosphorylation of proteins in the OsCDPK13 transgenic rice plants. Crude proteins were extracted from leaf sheath of vector control (A) and OsCDPK13 transgenic rice seedlings containing sense (B) and antisense (C) constructs and incubated with the reaction mixture containing 0.2 mM CaCl2. After in vitro protein phosphorylation, phosphorylated proteins were separated by 2-PADE with IEF in the first dimension and SDS-PAGE in the second dimension. The proteins were detected by silver staining and exposed to X-ray film. A typical result is shown from 5 independent experiments. Arrows denote the positions of phosphoproteins changed in the sense and antisense transgenic plants. Spot numbers correspond to numbers in Figure 1 and Table 1.

dehydrogenase in all the tissues of rice indicates its ubiquitous role in plant growth and development. Phosphorylation of Proteins in OsCDPK13 Transgenic Plants. An alternative explanation for the apparent involvement of OsCDPK13 in both cold stress and GA signaling is crosstalk between the two pathways18. Antisense OsCDPK13 transgenic rice lines were shorter than the vector control lines, and sense OsCDPK13 transgenic rice lines had higher recovery rates after cold stress than vector control rice.18 To evaluate the changes in phosphorylation status of proteins in OsCDPK13 transgenic rice plants containing the sense and antisense constructs, in vitro phosphorylation of the proteins was performed. Results indicate that the phosphorylation status of 3 proteins, spot numbers 23, 28, and 36, (Figures 3 and 4) was changed in the 1598

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Abbreviations: GA, gibberellin; BL, brassinolide; 2, 4-D, 2, 4-dichlorophenoxyacetic acid; ABA, abscisic acid; CBB, Coomassie brilliant blue; ESI-Q/TOF-MS/MS, electrospray ionization quadrupole time-of-flight mass spectrometry; TFA, trifluoroacetic acid; MALDI-TOF MS, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

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