Systematic Secretome Analyses of Rice Leaf and Seed Callus

Systematic Secretome Analyses of Rice Leaf and Seed Callus Suspension-Cultured Cells: Workflow Development and Establishment of High-Density Two-Dimensional Gel Reference Maps Young-Ho Jung,† Seung-Hee Jeong,† So Hee Kim,† Raksha Singh,† Jae-eun Lee,† Yoon-Seong Cho,† Ganesh Kumar Agrawal,‡,§ Randeep Rakwal,‡,| and Nam-Soo Jwa*,† Department of Molecular Biology, Sejong University, Gunja-dong, Seoul 143-747, South Korea, Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal, Interdisciplinary Plant Group and Division of Biochemistry, 109 Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211, and Health Technology Research Center (HTRC), National Institute of Advanced Industrial Science and Technology (AIST) West, Tsukuba 305-8569, Ibaraki, Japan Received July 9, 2008

Secreted proteins control a multitude of biological and physiological processes in multicellular organisms such as plants. Identification of secreted proteins in reference plants like Arabidopsis and rice under normal growth conditions and adverse environmental conditions will help better understand the secretory pathways. Here, we have performed a systematic in planta and in vitro analyses of proteins secreted by rice leaves (in planta) and seed callus suspension-cultured cells (SCCs; in vitro), respectively, using a combination of biochemical and two-dimensional gel electrophoresis (2-DGE) coupled with liquid chromatography mass spectrometry analyses. Secreted proteins prepared from either leaves or SCCs medium were essentially free from contamination of intracellular proteins as judged by biochemical and Western blot analyses. 2-DGE analyses of secreted proteins collectively identified 222 protein spots with only 6 protein spots common to both in planta and in vitro derived data sets. Data were used to establish high-resolution and high-density 2-D gel reference maps for both in planta and in vitro secreted proteins. Identified proteins belonged to 11 (in planta) and 6 (in vitro) functional classes. Proteins involved in carbon metabolism (33%) and cell wall metabolism having plant defense mechanism (18%) were highly represented in the in planta secreted proteins accounting for 51% of total identified proteins, whereas proteins of cell wall metabolism having plant defense mechanism (64%) were predominant in the in vitro secreted proteins. Interestingly, secreted proteins possessing signal peptides were significantly lower in an in planta (27%) prepared secreted protein population than in vitro (76%) as predicted by SignalP prediction tool, implying the notion that plant might possess yet unidentified secretory pathway(s) in addition to the classical endoplasmic reticulum/Golgi pathway. Taken together, this systematic study provides evidence for (i) significant difference in protein population secreted in planta and in vitro suggesting both approaches are complementary, (ii) identification of many novel and previously known secreted proteins, and (iii) the presence of large number of functionally diverse proteins secreted in planta and in vitro. Keywords: endoplasmic reticulum/Golgi secretory pathway • rice secretome • gel-based approach • proteomics • in vivo/in vitro secretion

1. Introduction Secreted proteins constitute an important class of active molecules that control and regulate a multitude of biological * To whom correspondence should be addressed. Dr. Nam-Soo Jwa, Department of Molecular Biology, Sejong University, Gunja-dong, Seoul 143747, Korea. E-mail, [email protected]; fax, +82-2-3408-4336. † Sejong University. ‡ Research Laboratory for Biotechnology and Biochemistry (RLABB). § University of Missouri. | National Institute of Advanced Industrial Science and Technology (AIST) West. 10.1021/pr8005149 CCC: $40.75

 2008 American Chemical Society

and physiological processes in multicellular organisms, such as growth and development, cell division and differentiation, and defense- and stress-related responses. These proteins have been shown to act both locally and systemically.1-4 The global study of secreted proteins by a cell, tissue, organ or organism at any given time and conditions is called ‘secretome’.5,6 Secretome is also a term referring to both the machineries for protein transport and the secreted proteins themselves. Like other higher organisms, protein secretion in plants is a highly sophisticated and tightly regulated process. Despite decades of studies of the plant secretory pathways,7,8 our knowledge Journal of Proteome Research 2008, 7, 5187–5210 5187 Published on Web 11/06/2008

research articles on the secretory pathways and the mechanisms of protein secretion is highly limited. A list of proteins involved in the secretory pathways is still incomplete. Moreover, little is known about the secreted proteins in plants in response to chemical elicitors or pathogens upon plant-pathogen interaction. Proteomics approaches are particularly well-suited and preferred technologies for secretome analysis over gene expression approach, such as gene array-based transcriptomics study. This is because secreted proteins are usually expressed in low abundance, are expressed by specialized cell types and during specific stages of development, or have an induced expression during specific cellular responses, including those in the innate immune response. Such compartmentalized secreted proteins are possible to isolate and enrich using techniques like differential extraction. Use of proteomics in case of plant-pathogen interactions is the key, as there is more than one genome present in this situation. Literature survey indicates that largescale secretome studies have been conducted mostly in animals, yeast, and bacteria, but rarely in plants. Bacillus subtilis and rat liver homogenates are good examples of a large-scale secretome analysis,5,6,9,10 where a widely used proteomics approach two-dimensional gel electrophoresis (2-DGE) was employed. To date, no large-scale secretome analysis has been reported on establishment of 2-D gel reference map in plants. This is mainly due to technical difficulties in isolation and preparation of highly enriched secreted proteins suitable for gel- and gelfree based proteomic approaches. Preparation of secreted proteins from in vivo plant tissue has more problems. The most widely used system for secreted protein preparation is the suspension-cultured cells (SCCs), which is an in vitro system. In this in vitro system, secreted proteins are present in cultured medium and therefore very easy to separate medium from cells to proceed with preparing the secreted proteins. Moreover, a few methods already have been developed to isolate secreted proteins suitable for 2-DGE analysis, such as trichloroacetic acid (TCA) precipitation.11 However, these techniques largely for generating a highly confident secretome data have yet to be fully standardized to ensure reproducibility from laboratory to laboratory. Recently, in a first study, Arabidopsis SCCs were used to investigate the secreted proteins in response to salicylic acid (SA) with an aim for targeted study on selected proteins.12 A total of 13 proteins were identified by MALDI-TOF-MS that showed relative differential abundance between treated and nontreated SCCs with SA. No 2-D gel reference map was developed. One of these proteins was identified as GDSL motif lipase/hydrolase-like protein, called GLIP1, and selected for its functional study. Functional analysis suggested that GLIP1 might be a critical component in plant resistance to Alternaria brassicicola. Walton’s laboratory took 1-DGE- and shotgunbased proteomics approaches to systematically identify proteins secreted by Fusarium graminearum (Gibberella zeae) during growth on 13 media in vitro and in planta during infection of wheat heads.13 The culture filtrates and vacuum infiltration were used to collect in vitro and in planta secreted proteins. A total of 289 proteins secreted by F. graminearum were identified. Altogether, though one in vitro SCCs study in Arabidopsis using 2-DGE-based proteomics approach is available, the complete spectrum of in vitro secreted proteins is still lacking. Moreover, in planta secreted proteins have not yet been investigated at proteomics level. Identification of a complete set of secreted proteins in reference plants like Arabidopsis and rice is required to begin 5188

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Jung et al. deciphering the secretory pathways and the host-pathogen interactions on a global level. To this end, it is important to develop a suitable, reproducible isolation procedure for preparation of highly enriched secreted proteins free from cytosolic and other contaminants. This is specifically necessary for in vivo secretome analysis of plant tissues. Analysis in fully sequenced Arabidopsis and rice genome plants will be very useful for secretome study. In such reference plants, a comprehensive list of potentially secreted proteins can also be generated using computational programs, if necessary. Experimentally generated secretome data can then help in better designing of programs suitable for plants. In this study, we have developed a novel method, termed gravity extraction method (GEM), to prepare pure secreted proteins from rice leaves. The 2-DGE-based proteomics approach was utilized to develop high-resolution 2-D gel reference maps of rice leaf (in planta) and SCCs (in vitro) secreted proteins. A total of 192 and 30 abundant protein spots were identified by nanoelectrospray ionization liquid chromatography mass spectrometer (nESILC-MS/MS) and mapped to the reference gel. Several previously known and novel secreted proteins were identified. Both in planta and in vitro systems were found to be complementary approaches for in-depth secretome study.

2. Materials and Methods 2.1. Plant Materials. Rice (Oryza sativa L. japonica-type cv. Nipponbare) seeds were grown in soil (Punong, Kyung-Ju, Korea) in a growth chamber [white fluorescent light (wavelength 390-500 nm, 150 µmol · m-2 · s-1), light/dark photoperiods of 16 h (32 °C)/8 h (28 °C), and 70% relative humidity]. Three-week-old rice seedlings (4th leaf stage) were treated with 250 ppm Tween-20 (Sigma) using an air sprayer. Seedlings were wrapped with polyethylene film to protect evaporation of Tween-20 and kept in a humidity chamber at 28 °C for 24 h. Leaves were harvested from treated seedlings followed by immediate isolation of secreted proteins. 2.2. Suspension-Cultured Cells. Dehulled mature rice seeds were sterilized in 70% ethanol for 10 min and then in 3% sodium hypochlorite for 20 min followed by extensive washing in distilled water to remove the disinfectant. Sterilized seeds were placed on the N6 induction medium [3% (w/v) sucrose, 0.03% (w/v) casamino acid, proline (2.878 mg/L), CHU (3.981 g/L), 1 mL of 2,4-D (2 mg/mL ethanol), pH 5.8, 0.2% (w/v) gellan gum] for 3 weeks in the dark at 28 °C.14,15 Growing calli (0.5-1 g) were transferred into 50 mL induction liquid media14,15 to establish cell suspension culture. Suspension-cultured cells were maintained by subculturing (1 mL) into 50 mL of fresh induction medium once every week with gentle agitation (120 rpm) at 28 °C in the dark. 2.3. Isolation of Secreted and Total Proteins from Rice Leaves. The flowchart for secreted protein isolation from rice leaf is schematically depicted in Figure 1. Secreted proteins were isolated form rice leaves harvested at 24 h post-treatment with 250 ppm Tween-20. For this purpose, leaf was cut with scissor at the basal end and sap was collected in 50 mL Falcon tube containing a plastic sheet laid with holes (2 mm diameter) followed by centrifugation at 1000g for 20 min at 15 °C. Collected saps containing secreted proteins were again centrifuged at 18 500g for 10 min at 4 °C to remove any impurities. The clear saps were immediately freeze-dried and dried pellet was resuspended in 300 µL of LB-TT [7 M (w/v) Urea, 2 M (w/v) Thiourea, 4% (w/v) CHAPS, 18 mM (w/v) Tris-HCl (pH 8.0), 14 mM (w/v) Trizma base, 0.2% (v/v) Triton X-100, 50 mM

Systematic Secretome Analyses of Rice Leaf and Seed SCCs

research articles in LB-TT for 30 min with occasional vortexing and sonication, followed by centrifugation at 18 500g for 15 min at 20 °C.

Figure 1. Flowchart for isolation of in planta secreted proteins from rice leaves. Three-week old rice seedlings were treated with 250 ppm Tween-20 and sap was collected using GEM (gravity extraction method) followed by CTAB precipitation for 8 times before subjecting the isolated secreted proteins for 2-DGE analyses.

(w/v) DTT, and one EDTA-free proteinase inhibitor (Roche) tablet per 50 mL buffer] for further purification of secreted proteins. Equal volume of CTAB buffer [100 mM (w/v) TrisHCl (pH 8.0), 10 mM (w/v) EDTA (pH8.0), 4% (w/v) CTAB, and one EDTA-free proteinase inhibitor tablet in a final volume of 50 mL buffer] was added, mixed, and centrifuged at 18 500g for 10 min at 4 °C. Supernatant was transferred to a new 1.5 mL tube and 1 vol of supernatant was mixed with 1 vol of 100% methanol and 0.5 vol of chloroform [i.e., supernatant/methanol/ chloroform 1:1:1/2 (v/v)] followed by centrifugation again at 18 500g for 10 min at 4 °C. Aqueous phase was carefully removed and protein was precipitated with 3 vol of 100% methanol. Pellet was air-dried for 5 min and resuspended in 300 µL of isoelectric focusing (IEF) LB-TT. Steps from CTAB precipitation were repeated 8 times. Total protein was extracted using two-step trichloroacetic acid/acetone protein extraction protocol (TCAAEB) with some modifications.11 Leaves (300 mg) were placed in liquid nitrogen and ground thoroughly to a fine powder with a mortar and pestle (precooled). Proteins were precipitated with TCAAEB [acetone containing 10% (w/v) TCA, and 0.07% (v/v) 2-mercaptoethanol (2-ME)] for 1 h at -20 °C, and centrifuged at 18 500g for 15 min at 4 °C. The pellet was washed thrice with wash buffer [acetone containing 0.07% (v/v) 2-ME, 2 mM (w/v) EDTA, and two EDTA-free proteinase inhibitor tablets in a final volume of 100 mL buffer] followed by removal of acetone by air drying the pellet. Protein pellet was resuspended

2.4. Isolation of Secreted and Total Proteins from Suspension-Cultured Cells. Five-day-old suspension-cultured cells, the day after replacing the culture medium with the fresh culture medium, were used for isolation of secreted and total proteins. Callus secreted proteins were prepared by culture medium passing through a membrane filter (0.45 µm) to remove the callus. The filtered medium was freeze-dried and dialyzed against Tris buffer (10 mM Tris-HCl, pH 8.0). The prepared secreted proteins were freeze-dried and stored at -80 °C. Collected cells after membrane filtration were used for total protein isolation. Total protein was extracted using TCAAEB with some modifications. Callus (300 mg) was placed in liquid nitrogen and ground thoroughly to a fine powder with a mortar and pestle (precooled). Proteins were precipitated with TCAAEB as described above. 2.5. Bradford Assay for Protein Quantification. Secreted or total protein pellets were resuspended in LB-TT and briefly centrifuged to obtain clear supernatant. Supernatant was then used for protein quantification using a modified Bradford assay.16 Briefly, bovine serum albumin (BSA) was prepared with LB-TT to use as protein standard. Each protein sample (2 µL) and BSA standards (0, 2, 4, and 6 µg) were mixed with 20% Bradford reagent (Bio-Rad, Hercules, CA) containing 0.1 mM hydrochloric acid in a final volume of 1 mL. The absorbance (at 595 nm) of each sample mixture was measured using Pharmacia LKB Ultrospec III (Amersham Biosciences, Uppsala, Sweden). 2.6. Enzyme Activity Assay of Malate Dehydrogenase. Enzymatic activity of malate dehydrogenase (MDH) was determined in secreted and total protein fractions as described previously.17 Briefly, 100 mg of rice leaves and callus powder was mixed with 1 mL of extraction buffer [30 mM sorbitol, 1% (w/v) BSA, and 1% (w/v) polyvinylpyrrolidone (PVP) in 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]KOH, pH 8.0]. The mixture was centrifuged at 18 500g for 15min at 4 °C and the supernatant was collected and analyzed immediately. Secreted proteins as a sap removed cell debris and other impurities was added by final concentration containing 30 mM sorbitol, 1% (w/v) bovine serum albumin, and 1% (w/v) PVP in 10 mM HEPES-KOH, pH 8.0, and measured at once (see Figure 2). Total and secreted protein for measuring the same amount of proteins was freeze-dried and resolublized into the extraction buffer. Briefly, total proteins were homogenized in 100 mg of rice leaves and callus powder was mixed with 1 mL of distilled water containing 250 ppm Tween-20 and centrifuged at 18 500g for 15 min at 4 °C. Both total (centrifuged supernatant) and secreted (sap removed cell debris and other impurities) proteins were freeze-dried and resolublized into the distilled water and assayed for protein quantification (see section 2.5). For measuring the enzyme activity, both protein samples were added by final concentration containing 30 mM sorbitol, 1% (w/v) bovine serum albumin, and 1% (w/v) PVP in 10 mM HEPES-KOH, pH 8.0. The MDH activity was analyzed at 25 °C by measuring the rate of decrease at A340 nm in 1 mL (final volume) of the following mixture: 0.1 mM NADH, 46.5 mM Tris-HCl, pH 9.5. The enzyme reaction was carried out with 10 µL of protein samples and started by the addition of 0.4 mM oxalacetate (see Supplementary Figure 1 in Supporting Information). 2.7. Western Blot Analysis. Proteins (20 µg) were separated on 12% SDS-PAGE and electrotransferred onto a PVDF memJournal of Proteome Research • Vol. 7, No. 12, 2008 5189

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Figure 2. Secreted proteins fraction is essentially free from intracellular malate dehydrogenase (MDH) activity. (A) Biochemical analyses of an intracellular MDH activities was perfored in protein fractions of total and secreted proteins isolated from rice leaves and suspension-culture medium. (B) The MDH activity is also mentioned that was recorded at 1 min after reaction in different protein fractions.

brane (NT-31, 0.45 µm pore size; Nihon Eido) as described previously.18,19 The blotted membranes were incubated with anti-OsRacB, anti-R-tubulin, and antiglucanase antibodies. Membranes were then incubated with secondary antibody conjugated with horseradish peroxidase. Immunoblot signals were detected using the ECL detection system as per manufacturer’s protocol (WEST-ZOLTM plus Western blotting detection system, iNtRON, Korea). All procedures were carried out at ambient room temperature. 2.8. 2-DGE Analysis. 2-DGE was carried out using precast IPG strip gels on an IPGphor unit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) followed by the second dimension using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) on a Nihon Eido horizontal electrophoresis unit (Nihon Eido, Tokyo, Japan). The volume carrying 150 µg total soluble protein was mixed with LB-TT containing 0.5% (v/v) pH 4-7 IPG buffer and 1% DeStreak Reagent (GE Healthcare) to bring to a final volume of 450 µL. IPG strips (pH 4-7; 24 cm, GE Healthcare) were carefully placed onto the protein samples avoiding air bubbles between the sample and the gel. The IPG strips were allowed to passively rehydrate with the protein samples for 1.5 h, followed by overlaying the IPG strips with cover fluid (mineral oil), and this was directly linked to a five-step active rehydration and focusing protocol as described. IEF parameter at 50 µÅ max, step 1 (active rehydration), step-n-hold at 50 V 5190


Jung et al. for 12 h; step 2, step-n-hold at 100 V for 1 h; step 3, gradient to 500 V for 4 h; step 4, gradient to 8000 V for 12 h; step 5, stepn-hold at 8000 V for 3 h. The whole procedure was controlled at 20 °C. Preceding the second-dimension separation, the strips were placed in a reduction buffer [Urea 6 M, SDS 4%, BPB 0.002%, Glycerol 30%, 50 mM Tris-HCl (pH8.8), 2% DTT] for 10 min twice with gentle shaking. The strips were then transferred to an alkylation buffer [Urea 6 M, SDS 4%, BPB 0.002%, Glycerol 30%, 50 mM Tris-HCl (pH8.8), 2.5% Iodoacetamide] and shaken for 10 min twice. IPG strips were then rinsed with cathode running buffer [0.025 M Tris, 0.192 M Glycine and 0.2% (w/v) SDS] and placed onto 12% PAG and overlaid with overlay agarose solution [60 mM Tris-HCl, pH 6.8, 60 mM SDS, 0.5% (w/v) Agarose, 0.01% (w/v) BPB]. The SDS-PAG was run in a lower anode buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS) at a current setting of 340 V at 20 °C. Molecular masses were determined by running standard protein markers (DualColor PrecisionPlus Protein Standard; Bio-Rad). For each sample, triplicate IPG strips and PAGs were processed for electrophoresis under the same conditions. 2.9. Protein Visualization and Image Analysis. To visualize the protein spots, the polyacrylamide gels were stained with silver nitrate (Plus One Silver Staining Kit Protein; GE Healthcare). Protein patterns in the gels were recorded as digitalized images using a digital scanner (EPSON perfection 4490 photo, resolution 400 dpi), and saved as TIFF files. The gels were quantitated in profile mode as instructed in the operating manual of the EasyPro-2D software (BioBud, Korea). High quality protein spots present in three biological replicate gels were excised using a gel picker (One Touch Spot Picker, P2D1.5 and 3.0, The Gel Company, San Francisco, CA), and stored at -80 °C. 2.10. In-Gel Digestion and Mass Spectrometry Analysis. Two stained protein spots were excised from the silver-stained 2-D gels and transferred to sterilized Eppendorf tubes (1.5 mL). The gel pieces were incubated until removal of the silver stain in 15 mM potassium ferricyanide and 50 mM sodium thiosulfate. The gel pieces were transferred to sterilized water and washed two times. The gel pieces were incubated in 0.2 M NH4HCO3 (pH 7.8) for 20 min. Gel pieces were shrunk by dehydration in acetonitrile, which was then removed, followed by washing with vortexing in the same volume of acetonitrile and 0.1 M NH4HCO3 (pH 7.8). After removing the solution, the gel pieces were dehydrated with vortexing by addition of acetonitrile, and swelled by rehydration in 0.1 M NH4HCO3 (pH 7.8). After repeating the above dehydration process twice, the gel pieces were completely dried in a vacuum centrifuge. The gel pieces were swollen in a digestion buffer containing 10 mg/ mL trypsin (Promega, sequencing grade) in ice. After a 45 min incubation, the digestion buffer was removed and replaced with 20 µL of 50 mM NH4HCO3 (pH 7.8), and the gel pieces were incubated at 37 °C for 12 h. The supernatant was desalted through a C18 ZipTip (Millipore, Bedford, MA) according to the instructions and a 2-5 µL solution was injected for analysis with LC-MS/MS (Agilent, Palo Alto, CA). The nLC was performed with an Agilent 1100 Nano LC-1100 system combined with a microwell-plate sampler and thermostatted column compartment for preconcentration (LC Packings, Agilent). The samples were loaded on the column (Zorbax 300SB-C18, 150 mm × 75 µm, 3.5 µm) using a preconcentration step in a microprecolumn cartridge (Zorbax 300SB-C18, 5 mm × 300 µm, 5 µm). Sample (2 to 5 µL) was loaded on the precolumn at a flow rate of 30 µL/min. After 5 min, the precolumn was


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connected with the separating column, and the gradient was started at 300 nL/min. The buffers used were 0.1% HCOOH in water (A) and 0.1% HCOOH in acetonitrile (B). A linear gradient from 15 to 45% B for 25 min was applied. A single run took 75 min, which included the regeneration step. An LC/MSD Trap XCT with a nanoelectrospary interface (nESI; Agilent) was used for MS. Ionization (2.0 kV ionization potential) was performed with a liquid junction and a noncoated capillary probe (New Objective, Cambridge). 2.11. Database Search and Protein Identification. Tandem MS spectra were searched against the rice protein database using the Agilent Spectrum Mill MS Proteomics Workbench (Spectrum Mill version; Agilent Technologies, Santa Clara, CA). This software includes a Data Extractor function that identifies good quality MS/MS spectrum for peptides by seeking CID fragment differences that correspond to known amino acids (sequence tag length >1) and thus functions as a filter to discard spectra that are unlikely to arise from peptides. MS/ MS spectra were created with the Spectrum Mill Data Extractor program with the following setting in modified Patrichorth method (with some modifications).20 Search parameters included unmodified and carbamidomethylation of cysteine. The extracted MS/MS spectra were searched against the rice protein database for tryptic peptides with a mass tolerance of ( 2.5 Da for the precursor ions and a tolerance of ( 0.7 Da for the fragment ions and 3 allowed maximum missed cleavages, in identity search mode. A Spectrum Mill autovalidation was followed by Partichorth method in the protein details mode. The mass spectra of low-abundance proteins/peptides are given in Supplementary Figure 2 in Supporting Information.

3. Results and Discussion The 2-DGE-based proteomics approach is schematically depicted in Figure 3 for development of high-density 2-D gel reference maps of in planta and in vitro secreted proteins. A novel procedure (Figure 1) for isolation of in planta secreted proteins has also been developed in this study. 3.1. Development of Isolation Procedure for Secreted Protein Particularly from in Planta Rice Leaves. Preparation of secreted proteins essentially free from contamination remains a daunting task for several reasons including their low concentration and contamination with abundant cytosolic proteins. Hence, there is a lack of optimized isolation procedure for plant tissues (in planta) and SCCs (in vitro). The vacuum infiltration method is widely used to extract apoplastic and secreted proteins in planta.21 It is worth mentioning that all apoplastic proteins are not secreted proteins, but all secreted proteins are the apoplastic proteins. In planta apoplastic proteins have been isolated using the vacuum infiltration method and analyzed by 2-DGE.22 Though the vacuum infiltration method is supposed to extract intercellular proteins after infiltration of intercellular washing fluid,23-27 it suffers from certain drawbacks. First, infiltration is always performed with buffer containing high concentration of ionic reagents such as KCl, NaCl, or LiCl, which dissolve apoplastic proteins but also membrane-bound proteins. Second, vacuum infiltration is usually carried out 2-3 times on the same tissue to obtain a satisfactory quantity of proteins in the intercellular washing fluids, and this process increases contaminants from cytoplasm and other subcellular compartments. And third, the prepared intercellular washing fluids possess interfering compounds like carbohydrates, which interfere with downstream proteomics analysis, especially for 2-DGE-based proteomics. Our aim is

Figure 3. Schematic depiction of experimental plan for establishment of high-resolution 2-D gel reference maps of secreted proteins by rice seedling leaves and rice seed suspensioncultured cells. Soluble and secreted proteins were isolated from 3-week-old rice seedling leaves treated with 250 ppm Tween 20 buffer for 24 h (called in planta experiment) and rice callus cultured in liquid medium (2N614,15) for 5 days after replacing the culture medium with the fresh culture medium (called in vitro experiment). The purity of secreted protein isolation was determined using biochemical assays for MDH activity and Western blot analyses for extracellular and intracellular protein markers. Isolated proteins (150 µg) from three independent biological replicates were then separately subjected to 2-DGE analyses using linear IPG strips (pH 4-7; 24 cm) in the first dimension followed by SDS-PAGE (12%) in the second dimension to generate four high-quality 2-D gels. Proteins were visualized with silver nitrate staining. Upon image analyses, 230 and 45 protein spots were excised from secreted proteins reference gels of rice leaf and seed callus-generated suspension-cultured cells, respectively, and digested with trypsin enzyme. Peptides were analyzed by nESI-LC-MS/MS on Agilent 1100 nanoLC-1100 system resulting in confident protein assignments of 192 (rice leaf) and 30 (seed callus suspension-cultured cells) protein spots. Acquired MS/MS data were searched against the rice database using the MASCOT search engine [the Agilent Spectrum Mill MS Proteomics Workbench (Spectrum Mill version; Agilent Technologies, Santa Clara, CA)] followed by further bioinformatics analyses of obtained results for data analysis, integration, and construction.

to develop a high-quality 2-D gel reference map of in planta rice leaf secretome. Keeping in mind the drawbacks of vacuum infiltration method, we developed a novel extraction method, called “gravity extraction method (GEM),” to prepare pure and Journal of Proteome Research • Vol. 7, No. 12, 2008 5191

research articles intact secreted proteins from in planta rice leaves suitable for 2-DGE analysis. The flowchart of GEM is schematically depicted in Figure 1. The GEM includes collection of secreted sap (i.e., liquid) on leaves in a Falcon tubes under low speed centrifugation (1000g). The approach of leaf sampling and low speed centrifugation largely avoids any excessive stress to tissue and therefore contaminants from other subcellular compartments. Collected sap is then CTAB precipitated. CTAB precipitation was added to GEM to further purify the secreted proteins from interfering compounds, most likely carbohydrates. CTAB precipitation step is necessary. Lack of this step showed very high background upon staining with silver nitrate, masking lowabundance proteins (Figure 1). The prepared secreted proteins using GEM were found to be highly enriched, pure, and suitable for 2-DGE analysis, as described in the following sections. In vitro SCCs have been the preferred system for secretome analysis in plants and other organisms including mammals, bacteria, and fungus.13,28 This is mainly due to availability of more mature secreted protein extraction method than in planta.12,29 Importantly, it is also easy to scale up the preparation of secreted proteins. The key in this in vitro system is to concentrate the secreted proteins from relatively large volume of culture medium. Two widely accepted methods for such purpose are precipitation of filtered supernatants with TCA30,31 and molecular weight cutoff filters.32,33 Though these two methods have been slightly modified to better serve the purpose of isolated highly pure and enrich secreted proteins, they also have a number of drawbacks. Standard TCA precipitation gave a poor yield, a high background, and difficulties in resolubilizing the precipitated pellet. Molecular cutoff column was found to suffer from the blocking of the column during centrifugation and loss of low molecular weight proteins. Recently, a third method was used to prepare secreted proteins of high quality from Arabidopsis SCCs, in which both abovementioned methods were bypassed and simply freeze-dried approach was used.12 In this method, the filtered supernatants were freeze-dried in a vacuum lyophilizer and the resuspended pellet in small volume of water was dialyzed against MES buffer (10 mM, pH 5.8). We basically adapted the same method to prepare secreted proteins from in vitro rice SCCs medium, which were found to be highly pure and suitable for 2-DGE analyses. 3.2. Biochemical and Western Blot Analyses Indicate That Secreted Protein Preparations are Largely Free of Nonsecreted Proteins Contamination. Both biochemical and Western blot analyses were performed to determine the purity of secreted protein preparations using intracellular enzyme activity of MDH and antibodies specific to intra- (OsRacB and R-tubulin) and extracellular (glucanase) known proteins, respectively. The MDH activities were measured up to 3-5 min after adding the MDH enzyme to total and secreted protein fractions (Figure 2). MDH is an intracellular enzyme and has beenusedasmarkerenzymeformanykeycellularfunctions.12,17,34 Almost no MDH activities were found in secreted protein fractions of an in planta and in vitro compared to total protein fractions (Figure 2) The MDH activities measured at 1 min after reaction are also mentioned in Figure 2B. On the other hand, Western blot analyses of total and secreted protein fractions with antibody protein markers detected intense bands of OsRacB (21 kDa) and R-tubulin (50 kDa) in total but not in secreted protein fractions (Figure 4). Antibody against glucanase detected a prominent 37 kDa band in an in planta secreted protein fraction, but was absent in total protein 5192


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Figure 4. Western blot analyses of prepared secreted protein fractions and corresponding controls with intracellular and extracellular protein markers. (A) Prepared protein fractions (20 µg) from in planta and in vitro experiments were resolved by 12% SDS-PAGE and separated proteins were visualized with silver nitrate staining. (B) Gels were subjected to Western blot analyses with OsRacB (accession number: AAF91343), R-tubulin (accession number: AAG16905), and glucanase (accession number: AAD10383) protein markers without prior silver nitrate staining. T and S stand for total and secreted proteins, respectively.

fractions (Figure 4B). No detection of glucanase in an in vitro secreted protein fraction suggests that in planta and in vitro may secrete different protein populations differing in there abundance. These results indicate that prepared secreted protein fractions from in planta and in vitro are highly enriched and essentially free of nonsecreted proteins such as intracellular proteins. 3.3. Establishment of High-Resolution and High-Density 2-D Gel Reference Maps of in Planta and in Vitro Secretomes. To establish high-resolution and high-density 2-D gel reference maps of in planta and in vitro secreted proteins, medium range IPG strips of pH 4-7 (24 cm) were exclusively used followed by 12% SDS-PAGE analyses (Figure 3). This selection is based on the fact that total protein separation on the wide range IPG strip of pH 3-10 (24 cm) populates protein spots between pH 4 and 7 causing spots overlaps and therefore problems in downstream analyses such as precise detection of spots and their identification by MS.35,36 2-DGE analyses of secreted proteins from in planta and in vitro detected 327 and 70 highquality silver-nitrate stained protein spots in reference gels, respectively. In parallel, 2-DGE analyses of their total protein were also performed. Comparison of 2-D gel images indicated dramatic differences in protein patterns of in planta and in vitro secreted and total proteins (Figure 5); gel sections presented are representative of three high-quality 2-D gels. Moreover, when in planta and in vitro 2-D reference gel images were merged together, none of the protein spots were unequivocally matched (data not shown). These results suggest that in planta and in vitro secreted proteins are of largely different physical and biochemical properties with no or very minimum overlap. Of 327 and 70 protein spots, 230 and 45 intensely stained protein spots were excised from in planta and in vitro reference


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Figure 5. The 2-D gel profiles of secreted protein spots along with corresponding control protein spots. Three different gel areas (Area I-III) are representative of high-resolution 2-D gels generated from four independent biological samples in an in planta and in vitro experiments. These selected areas are also marked in the 2-D gel reference maps of secreted proteins in (A) (in planta) and (B) (in vitro) and in the 2-D gels of corresponding control proteins. These gel areas display drastic differences in profiles of protein spots between control and secreted proteins but also between in planta and in vitro secreted proteins.

gels, respectively, covering the entire gel from the high to low molecular mass and acidic to basic pI range. Protein spots were digested with trypsin and identified by nESI-LC-MS/MS in combination with Spectrum Mill search engine. A total of 192 and 30 protein spots were confidently assigned for in planta and in vitro secreted proteins (Tables 1 and 2). The protein IDs of only 6 spots were found to be common between the data sets, indicating that in planta and in vitro are complementary systems for in-depth investigation of secretome in rice, and perhaps in plants. These common proteins were putative antifungal zeamatin-like protein, Chitinase, secretory protein, and alpha galactosidase precursor (Tables 1 and 2; highlighted with light yellow color). Chitinase, a pathogenesis-related protein (PR protein), was predominantly expressed and accounted for 50% of the commonly identified proteins. Acquired data were used to develop 2-D gel reference maps of in planta and in vitro secretomes (Figures 6 and 7). To date, there is no report on the secretome 2-D gel reference maps in rice, and perhaps yet in plants to the best of our knowledge. Therefore, the generated high-density landmarks can be very useful for comparative secretome analysis. One of the abilities of 2-DGE analysis is the identification of proteins with fractional pI and mass changes.35,36 In line with other large-scale 2-DGE studies, this study also identified large number of proteins that differ in their pI and/or mass, such as arabinoxylan arabinofuranohydrolase (AXAH) isoenzyme AXAH-II, beta 1,3-glucanase, Chitinase, and transketolase. These protein spots might be due to alternative splicing of the same gene and/or posttranslation modifications. 3.4. In Vitro Secreted Proteins Possessing Signal Peptides are Significantly Higher than in Planta. SignalP prediction software is the only program available to date and generally used to search for signal sequences and their cleavages sites in a secreted protein.37 Hence, the SignalP program (http:// www.cbs.dtu.dk/services/ SignalP/) was used in this study to determine signal peptide in the identified secreted proteins. A total of 27% (in planta) and 76% (77%) (in vitro) of the identified secreted proteins were found to possess signal peptides (Figure 8). Use of TargetP program (http://www.cbs. dtu.dk/services/TargetP/) on the rest of the secreted proteins without signal peptides predicted to have chloroplastic, mito-

chondria, and others localization. Secreted proteins of chloroplastic localization were only found in the in planta experiments. This is highly reasonable given the tissue materials, leaves versus SCCs in the in planta versus in vitro experiments, used for experiments and extraction of secreted proteins. Some of these chloroplastic proteins were the proteins involved in a parallel glycolytic pathway in plastid, HSP70, leucine aminopeptidase, glutamine synthetase, adenosine kinase, and superoxide dismutase. There were very little or no difference in secreted proteins with prediction of mitochondria localization between the in planta and in vitro experiments. Arabidopsis SCCs is the only secretome study conducted systematically in plants at the global level. Of 91 different proteins identified by MALDI-TOF-MS out of 107 protein spots, only 54% of these proteins possessed signal peptides.12 Results obtained in Arabidopsis and rice (this study) SCCs indicate that (i) not all secreted proteins contain signal peptides, (ii) secreted proteins with signal peptides perhaps varies between 50 and 75%, which might change as we collect more and more secretome data, and (iii) in vitro secreted proteins having signal peptides are dramatically higher than proteins secreted by in planta. A number of reasons could be attributed to the third finding. The most notable is the presence of many in planta secreted proteins having a known biochemical function in primary metabolism (Tables 1 and 2). One such example is the proteins involved in glycolysis and amino acid synthesis. Glycolytic enzymes show strong sequence identity across organisms from mammals to bacteria, and to plants. Enzymes of amino acid synthesis via methionine pathway are also strictly conserved in many organisms. None of the identified proteins of glycolysis and amino acid synthesis have signal peptides. Secretome analyses of the pathogenic fungus and cancer cell lines have identified secreted proteins involved in primary metabolism and almost all of them do not possess signal peptides.13,38 These results suggest that secretion of proteins without signal peptides may be due to presence of yet unknown ER/Golgi independent secretory pathway in rice and Arabidopsis, and perhaps in plants. However, the possibility that some of these proteins are released nonspecifically into the Journal of Proteome Research • Vol. 7, No. 12, 2008 5193

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Table 1. The List of Rice Secreted Proteins in Planta

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a At, Arabidopsis thaliana Os, Oryza sativa; Hv, Hordeum vulgare; St, Solanum tuberosum; CM, carbon metabolism; PD, plant defense; UC, uncharacterized; PF, protein folding; AM, amino acid synthesis or metabolism; CW, cell wall metabolism; PP, proteolysis and peptidolysis; SG, signaling and gene regulation; EM, energy metabolism; RR, redox regulation; LM, liquid metabolism; CHL, Chloroplast; SEC, Secretory pathway; MT, Mitochondrion, OTH, Others; Y, yes, and N, no.

extracellular microenvironment as a consequence of cell autolysis cannot be overruled. 3.5. In Planta and in Vitro Secreted Proteins Are of Diverse Biological Functions. In planta and in vitro identified 192 and 30 redundant proteins which belonged to 11 and 6 functional classes, respectively (Figure 8). Of in planta proteins, proteins involved in carbon metabolism (CM; 35%), plant defense (PD; 19%), uncharacterized (UC; 11%), amino acid synthesis and metabolism (AM; 7%), protein folding (PF; 6%), and cell wall (CW; 5%) were highly represented, collectively accounting for 83% of the total identified secreted proteins. On the other hand, proteins predominantly expressed in an in vitro system were involved in PD (56%), UC (17%), and proteolysis and peptidolysis (PP; 10%) and collectively accounted for 91% of the total identified proteins. Comparison of proteins with functional classes indicates that a high number of proteins found in planta were not found in the in vitro experiment. For example, identified proteins of the glycolytic pathway mainly belong to CM functional class, which is absent from in vitro system. One comparative study on in vitro and in planta extracellular proteins from the pathogenic fungus F. graminearum reported that more than 50% of the proteins (including most of those proteins involved in the glycolytic pathway) identified in planta were not identified in any of the 13 in vitro experiments.13 On the basis of previous single study and the present study, it is likely that differences in the in planta and in vitro secreted proteins and their biological function are largely due to experimental approach and associated source material for secretome study. 3.6. Identification of Novel and Previously Known Secreted Proteins. Identified in planta and in vitro secreted proteins are schematically presented in Figure 9, revealing distinct differences in secreted proteins. Many in planta secreted proteins were not identified in an in vitro experiment and vice versa to some extent (Tables 1 and 2). For example, cell growth and division proteins, expansin and amylase, were not identified in planta, whereas essential proteins of metabolism and several other were not identified in vitro. Unlike

plants, in vitro-grown cells undergo rapid cell growth and division and this might cause accumulation of expansin and amylase to control rapid cell wall synthesis and degradation through active cell differentiation. Hence, in planta and in vitro experiments are complementary, and could be used for indepth investigation of plant secretome. Although some in vitro identified secreted proteins in this study were previously reported in an in vitro SCCs studies,12,29 almost all in planta proteins are the novel secreted proteins identified from rice leaf. Since this study is the only in planta study conducted to date in plants, identified novel proteins have been discussed with secretome studies in other organisms such as mammals, bacteria, and fungus. 3.6.1. Pathogenesis-Related (PR) Proteins Are the Major Secreted Proteins of the Plant Defense Proteins. The PR proteins are differentially regulated by diverse environmental (biotic and abiotic) factors and during growth and development of plants.39,40 The characteristic features of most PR proteins are that they form a small protein family, are inducible, possess antimicrobial activities, and perhaps are involved in defense signaling.39,40 Today, there are 17 recognized families of PR proteins in plants.40 This study identified 28 and 16 protein spots as PR proteins in both in planta and in vitro experiments, respectively (Tables 1 and 2). In general, these identified PR proteins have been found in many plants including crop, weed, and woody plants, and to have antifungal activities against a wide number of fungi, including plant and human pathogens. Moreover, almost all these proteins had signal peptides and were predicted to be secreted. In planta secreted PR proteins were identified as glucanases, chitinases, thaumatin like-protein, germin, and osmotin forming more than 4 PR families. Of these proteins, glucanases were predominant and expressed with 17 protein spots. Glucanases belong to PR2 family, which are thought to be involved in hydrolyzing the structural 1,3-beta-glucan present in the fungal cell wall, particularly at the hyphal apex of filamentous molds.40 Chitinases (6 spots) were the second PR proteins that showed preponderance expression in the in planta secretome. CurJournal of Proteome Research • Vol. 7, No. 12, 2008 5203

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a Os, Oryza sativa PD, plant defense; UC, uncharacterized; CW, cell wall metabolism; PP, proteolysis and peptidolysis; SG, signaling and gene regulation; EM, energy metabolism; SEC, Secretory pathway; MT, Mitochondrion; Y, yes, and N, no.

rently, there are four PR protein families (PR3, PR4, PR8, and PR11) for chitinases.40 By analogy with beta-glucanases, chitinases are in general involved in cleaving the cell wall chitin polymers, resulting in a weakened cell wall and rendering fungal cells osmotically sensitive.40 Phylogenetic analysis of rice full-length chitinases41 with the chitinases detected in this study revealed that the secreted chitinases were classified primarily into class III, except for one member belonging to class II (Figure 10). Since the functions are not characterized according to each class, functions of class III chitinases cannot be predicted. However, these class III secreted chitinases can be phylogenetically regarded as a unique group, which has functions outside of cells where there are limited studies. Both betaglucanases and chitinases proteins act synergistically in inhibiting fungal growth.42 Thaumatin (2 spots) and osmotin (1 spot) proteins of PR5 family were also identified. Like glucanases and chitinases, PR5 proteins possess function to kill fungi; however the precise mechanism of action is not completely understood. Regarding cell wall, PR5 proteins are known to cause cell permeability changes in fungal cells with a cell wall.43 It was previously shown that the amino-terminal signal of the osmotin-like protein is essential for the transport of protein to the endoplasmic reticulum (ER) both in tobacco and yeasts.44 Two protein spots were identified as germin of PR15 protein family. In vitro identified PR proteins were chitinases, peroxidases, beta-glucosidases, osmotins, and chitin-binding protein (Table

2). Except chitinase and osmotins, other in vitro PR proteins were not identified in an in planta experiments. Chitinases (9 spots) and peroxidases (5 spots) were the most predominantly expressed PR proteins accounting for 14 protein spots out of the total 16 protein spots identified as PR proteins. Peroxidase protein belongs to PR9 family and was previously identified in an in vitro secretome analysis of Arabidopsis suspension-cultured medium,12 and tobacco cultured BY2 medium.29 Expression of peroxidases at detectable levels only in an in vitro experiment suggests their possible involvement in controlling rapid cell division and growth as well as suppressing oxidative stress induced by accumulated phenolic compounds in liquid medium. Endo-1,3-beta-glucosidase (1 spots) and osmotin (1 spot) proteins were also identified. These in planta and in vitro results indicate that relatively large fraction of PR protein families are most likely to be secreted with the primary function in altering cell wall properties. The presence of predicted N-terminal signal peptide in almost all the identified PR proteins suggests translocation into the ER, followed by secretion into the apoplast. Our results also indicate that these PR proteins are accumulated extracellularly and can also be collected easily in intercellular washing fluid.40 Secretion of these PR proteins to extracellular matrix might be required to actively and synergistically combat biotic and abiotic stresses including the microbial infection.39,40 A recent study in Nicotiana benthamiana has shown that syntaxins may Journal of Proteome Research • Vol. 7, No. 12, 2008 5205

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Figure 6. The 2-D gel reference map of rice leaves secreted in planta secretome. High-quality protein spots successfully assigned by nESI-LC-MS/MS were only circled. Protein assignment of some spots are also mentioned. Three different areas (Areas I-III; marked by dotted squares) are expanded in Figure 5. Molecular masses of protein markers are shown on the left.

Figure 7. The 2-D gel reference map of suspension-cultured cells secreted in vitro secretome. High-quality protein spots successfully assigned by nESI-LC-MS/MS were only circled. Protein assignment of some spots are also mentioned. Three different areas (Areas I-III; marked by dotted squares) are expanded in Figure 5. Molecular masses of protein markers are shown on the left.

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accepted marker genes widely used to dissect the disease- and stress-related pathways in plants.39 We propose that PR

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Figure 8. Localization and functional classification of secreted proteins. Pie charts show the distribution of in planta and in vitro derived secreted proteins to protein localization (upper raw) and to their functional classes (lower raw) by relative percentage. The localization of secreted proteins was performed using the Signal IP software (http://www.cbs.dtu.dk/services/TargetP). Functional classification was carried out according to rice KOME Web site (http://cdna01.dna.affrc.go.jp/cDNA/) with some modification as described in Materials and Methods. Abbreviations are CM, carbon metabolism; PD, plant defense; UC, uncharacterized; PF, protein folding; AM, amino acid synthesis or metabolism; CW, cell wall metabolism; PP, proteolysis and peptidolysis; SG, signaling and gene regulation; EM, energy metabolism; RR, redox regulation; LM, liquid metabolism; CHL, Chloroplast; SEC, Secretory pathway; and MT, Mitochondrion.

Figure 9. Schematic depiction of identified in planta and in vitro secreted proteins.

proteins can also be used to determine the purity of secreted protein preparation. For example, as all members of the PR10 protein seem to be cytoplasmic, these proteins can be an excellent marker. The high-density 2-D gel reference maps of in planta and in vitro did not identify PR10 proteins, indicating that the prepared secreted proteins are pure and free from cytoplasmic proteins. 3.6.2. Peptidase. Peptidases are the proteolytic enzymes that break up small proteins into amino acids by a process known as proteolysis. Likewise PR proteins, peptidases also possess a variety of functions during normal growth and stress conditions including pathogens.46-48 Extracellular peptidases are central components of the plant defense response during pathogen

attack and environmental stresses.48,49 The intercellular fluid is a rich source of peptidase. Peptidases are classified on the basis of their catalytic mechanisms into aspartic, cysteine, metallo, serine, and threonine peptidases.47,50,51 This study identified five unique peptidases/proteases (Tables 1 and 2): leucine aminopeptidase (1 spot), aspartyl protease (3 spots), oligopeptidase A-like protein (1 spot), cysteine proteinase (1 spot), and cathepsin B-like cysteine protease (1 spot). Except aspartyl proteases, all others were identified only in an in planta experiment (Table 1). Moreover, cysteine proteinase was the only peptidase/proteiase that did not possess the signal peptide. Recently, a secretome study in mammalian cells revealed that caspase-1, a cysteine protease, is a regulator of Journal of Proteome Research • Vol. 7, No. 12, 2008 5207

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Figure 10. Phylogenetic analysis of secreted chitinases. Known rice full-length chitinases and chitinases detected in in vivo and in vitro secretomes from this study were grouped using CLUSTAL W (http://www.ebi.ac.uk/Tools/clustalw2/). In case of other species, only one representing chitinase out of homologous closely related ones was used.41 Dicot class I chitinase was not included since it is not classified into the same group with chitinases which were detected in secretomes.

unconventional protein secretion and involved in mediating secretion of proteins lacking an N-terminal signal peptide to extracellular space.52 It remains unknown whether the identified cysteine proteinase in this study also plays a role as a carrier in an ER/Golgi-independent protein secretion pathway. The extracellular localization of a plant cysteine peptidase has also been shown.21 Beside cysteine peptidase, aspartic and 5208


serine peptidases have been identified in Nicotiana tabacum leaf intercellular fluid.21 Hence, this study demonstrates the presence of peptidases/proteiases in the extracellular matrix of rice leaf and seed culture medium. 3.6.3. Heat Shock and Chaperone Proteins. Heat shock and chaperone proteins were identified only in an in planta experiments (Tables 1 and 2). A total of four and five protein

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Systematic Secretome Analyses of Rice Leaf and Seed SCCs spots were identified as HSP70 and chaperonins (60 and 21 kDa), respectively. None of these proteins possessed signal peptide. A previous in vitro secretome study in Arabidopsis SCCs identified HSP17 and HSP100 but not HSP70 and chaperonins.12 These studies suggest that though HSPs and chaperonins lacks an identifiable N-terminal signal peptide, they are present in the extracellular environment. HSPs and chaperonins are usually present at very low level under the normal condition within the cytoplasm of almost all organisms, but can also be released into the extracellular environment.53 Emerging evidence, largely in mammals, suggests that HSPs and chaperonins are important mediators of intercellular signaling and transport.53,54 It has been proposed that such stress protein release occurs both through physiological secretion mechanisms and during cell death by necrosis.54 However, the exact mechanisms by which these proteins are dynamically released by living cells have not yet been elucidated. 3.6.4. Housekeeping Proteins. Housekeeping proteins are essential for proper growth and development, and for survival of living organisms. As glycolysis, citric acid cycle, and protein metabolism are the central and evolutionarily conserved pathways across organisms supplying carbohydrates, proteins, and energy (in terms of oils55,56), proteins involved in these pathways are considered as housekeeping proteins. In total, 20 protein spots corresponding to 9 proteins of the glycolytic pathway were identified (Tables 1 and 2). These proteins are UDP-glucose pyrophosphorylase (2 spots), phosphoglucomutase (4 spots), fructokinase (1 spot), fructose bisphosphate aldolase (1 spot), triosephosphate isomerase (3 spots), glyceraldehydes 3-phosphate dehydrogenase (2 spots), phosphoglycerate kinase (2 spots), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (3 spots), and enolase (2 spots). Proteins involved in calvin cycle of photosynthesis were also identified: transketolase (8 spots), phosphoribulokinase (3 spots) and RuBisCO LSU (6 spots). Surprisingly, except transketolase, all identified proteins of glycolysis and calvin cycle lack an identificable N-terminal signal peptide sequence. In addition to these proteins, methionine synthase and adenosylhomocysteinase proteins were also identified as secreted proteins; these proteins are involved in storage protein synthesis and stress responses, and again lack signal peptide sequence. Other proteins involved in primary metabolism were also identified (Table 1); none of them have predicted signal peptides. It is worth mentioning that all these proteins lacking predicted N-terminal signal peptide can not be assumed to arise from either cell lysis or the extraction method used.13,38 Such housekeeping proteins have been previously reported as secreted proteins in bacteria.,57 fungi,13 and cancer cell lines.38 These independent secretome studies in different organisms and cell lines provide strong evidence on the presence of housekeeping proteins involved in primary metabolism such as glycolysis in the extracellular environment. The question, whether or not such housekeeping proteins are actively secreted through the ER/Golgi-independent protein secretion pathway, remains to be investigated. These housekeeping proteins were not identified in our in vitro experiments. These proteins were also absent from an in vitro secretome studies in other organisms including Arabidopsis.12,13 One possible explanation could be the difference in source organism. In an in vitro experiment of SCCs, all essential elements such as carbon and vitamin are supplied externally, and therefore cells survival do not solely depend on

in vivo synthesis of carbohydrates, proteins, and energy. However, other possibilities cannot be ruled out. 3.6.5. Novel Proteins. Although almost all in planta identified secreted proteins are novel mainly due to lack of in vivo secretome study in plants, the identification of AXAH protein with signal peptide is unique to this study. All previous secretome studies have failed to identify this protein. The AXAH protein has been isolated from barley and form a multigene family.58 In higher plants, arabinoxylans constitute a major component of cell walls. The AXAH enzymes have a central role in cell wall metabolism during normal growth and development and might also be involved in the modification of the wall in response to biotic and abiotic stresses.58 Another potentially exciting finding was the detection of a secreted receptor-like protein kinase (RLK) in both in planta and in vivo secretomes (spots 158 and 159, and spot C9, respectively). The RLKs are known to have possible functions in biotic and abiotic stress signaling.59,60 It is already known that rice blast fungus as well as JA strongly induce RLK1, which is the same as the identified secreted protein in this study.59 Although there are numerous RLKs in plants their functions are not well characterized. It is speculated that RLK1 may have an early stress response activation function because they are being constantly secreted under normal conditions both in planta and in vivo.

4. Concluding Remarks This study conducts the first systematic in vivo and in vitro secretome analyses in rice using a combination of biochemical methods and 2-DGE-based proteomics approach. This study has (i) established a novel method for preparation of secreted proteins from rice leaf essentially free from intracellular contamination, (ii) developed high-resolution and high-density 2-D gel reference maps of in planta rice leaf and in vitro rice seed suspension-cultured medium, (iii) shown that in vivo and in vitro experiments are complementary approaches for indepth secretome investigation, and (iv) provided large inventory of novel secreted proteins. High percentage of identified in planta proteins lacks an identifiable N-terminal signal sequence, whose functional investigation might help in better understanding the plant secretory pathways other than the classical ER/Golgi secretory pathway. Large list of experimentally identified secreted proteins opens a door for improving the prediction algorithm for secreted proteins and in finding the regulatory components that mediate the secretion of proteins with known and unknown extracellular functions.

Acknowledgment. The study was in part funded by a grant from the Plant Signaling Network Research Center, Korea Science and Engineering Foundation. The study was also carried out with the support of “On-Site Cooperative Agriculture Research Project (No. 20070301080003), RDA, Republic of Korea (N.-S.J.), and by grant number CG2130 from the Crop Functional Genomics Center of the 21st Century Frontier R&D Program, which is funded by Ministry of Science and Technology of the Republic of Korea. The nESI-LC-MS/MS was carried out at the Carbohydrate Bioproduct Research Center in Sejong University, Korea. Young-Ho Jung expresses his thanks to the Seoul Science Fellowship. Financial support to Ganesh Kumar Agrawal at University of Missouri was provided by National Science Foundation grant DBI-0604439 awarded to Jay J. Thelen (University of Missouri). Journal of Proteome Research • Vol. 7, No. 12, 2008 5209

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