Proteomics Analysis of Rice Lesion Mimic Mutant ... - ACS Publications

For this, we focused our attention on a rice lesion mimic mutant (LMM), spotted leaf 1 (spl1), which has been used to study the programmed cell death ...
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Proteomics Analysis of Rice Lesion Mimic Mutant (spl1) Reveals Tightly Localized Probenazole-Induced Protein (PBZ1) in Cells Undergoing Programmed Cell Death Sun Tae Kim,† Sang Gon Kim,† Young Hyun Kang,‡ Yiming Wang,‡ Jae-Yean Kim,†,‡,§ Nari Yi,| Ju-Kon Kim,| Randeep Rakwal,⊥ Hee-Jong Koh,# and Kyu Young Kang*,†,‡,§ Environmental Biotechnology National Core Research Center, Division of Applied Life Science (BK21 program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, Korea, Division of Bioscience and Bioinformatics, Myongji University, Yongin 449–728, Korea, Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba West, 16-1 Ongogawa, Tsukuba 305-8569, Japan, and School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea Received December 23, 2007

Numerous reports have predicted/hypothesized a role for probenazole-induced protein (PBZ1) as a molecular marker in rice self-defense mechanism. However, the precise function of PBZ1 remains unknown. In the present study, we examined PBZ1 as a putative cell death marker in rice. For this, we focused our attention on a rice lesion mimic mutant (LMM), spotted leaf 1 (spl1), which has been used to study the programmed cell death (PCD) phenomenon during lesion development in leaf. Using twodimensional gel electrophoresis (2-DGE), 18 colloidal Coomassie brilliant blue stained protein spots were found to be differentially expressed in the leaves of spl1 mutant. After analysis of these spots by MALDI-TOF-MS, we identified the PBZ1 protein to be highly inducible in spl1. On the basis of these results, we proceeded to verify whether PBZ1 is highly expressed in the tissues undergoing PCD in rice. To do so, we performed immunoblot analysis and immunolocalization and used transgenic lines carrying the PBZ1 promoter fused with GFP. Results demonstrated that the expression levels and localizations of PBZ1 dramatically coincided with tissues undergoing PCD, namely, during leaf senescence, root aerenchyma formation, coleoptiles senescence, root cap, and seed aleurone layer. Furthermore, localization of the PBZ1 protein was also tightly correlated with TUNEL signal in the seed aleurone layer. As DNA fragmentation is a hallmark of PCD, this result clearly indicates a role for PBZ1 in rice tissues undergoing PCD. In conclusion, our results provide strong support for the hypothesis that PBZ1 is a molecular marker in rice defense response, and can serve as a novel potential marker for cell death/PCD in rice. Keywords: Oryza sativa L. • PBZ1 • two-dimensional gel electrophoresis • mass spectrometry • immunochemistry • PCD

1. Introduction Programmed cell death (PCD), a genetically determined process that occurs in response to environmental signals (including abiotic and biotic stresses) and development, plays an essential role in plant growth and survival in multicellular organisms by removing undesirable cells.1 PCD has been wellcharacterized in animals by genetically controlled processess.2 * To whom correspondence should be addressed. Dr. Kyu Young Kang: e-mail, [email protected]; fax, +82-55-757-0178. † Environmental Biotechnology National Core Research Center, Gyeongsang National University. ‡ Division of Applied Life Science, Gyeongsang National University. | Myongji University. ⊥ National Institute of Advanced Industrial Science and Technology (AIST). # Seoul National University. § Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University.

1750 Journal of Proteome Research 2008, 7, 1750–1760 Published on Web 03/14/2008

In plants, PCD has been observed during developmental events, such as tracheary element development in pea, leaf senescence in rice, cell senescence in Arabidopsis, formation of aerenchyma in water logged roots, seed alurone layer, and during the hypersensitive response (HR) to pathogen attack.3–9 Senescence, which is the final stage of plant vegetative and reproductive cell and tissues, has certain features in common with PCD.10 In the rice plant, the progression of senescence and PCD associated with aerenchyma formation develops in the coleoptiles as well as the leaf.4,5,11 In particular, lesion mimic mutants (LMMs) exhibiting HRlike lesions in the absence of pathogen attack have been usually used as tools for genetic analysis of PCD during lesion development.12 Therefore, many LMMs have been isolated and characterized in numerous plant species including maize, barley, soybean, and rice.13–17 In addition, LMMs have revealed the induction of a series of defense responses such as callose 10.1021/pr700878t CCC: $40.75

 2008 American Chemical Society

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Localization of PBZ1 in Cells Undergoing PCD deposition, inducible expression of defense-related genes, production of reactive oxygen species (ROS), and accumulation of phytoalexins, conferring enhanced resistance to infection by pathogens.18 In rice, the LMMs, spotted leaf (spl1 and spl11), lesion mimic cell death and resistance (cdr), and blast lesion mimic (blm) not only have been reported to show enhanced resistance to rice blast fungus, but also bacterial pathogen have been characterized.16,19,20 So far, three genes of the spl7, ttm1, and spl11 for controlling lesion mimic phenotypes encoding a heat stress transcription factor, protein kinase, and a U-box/ ARM protein, respectively, have been isolated.21–23 Since proteomics is a powerful tool for identifying proteins that are up- or down-regulated under specific physiological conditions, proteomic approaches have been applied to monitor global changes in protein levels to understand an important aspect of biotic and abiotic stresses in plants.24 Therefore, comparative proteomics has been used to elucidate differences in protein levels between wild-type and rice LMMs, cdr2, blm, and spl6.25–27 For example, defense-related proteins and metabolic enzymes were found to be altered during lesion formation in cdr2.25 Many pathogen-related (PR) proteins (OsPR5 and OsPR10), a phytoalexin biosynthesis-related protein (naringenin 7-O-methyltransferase), and three oxidative-stress-related proteins [catalase, ascorbate peroxidae (APX), and superoxide dismutase] were also found to be differentially expressed in the blm mutant.26 Most recently, protein disulfide isomerase (PDI), transketolase, and thioredoxin peroxidase (TPX) proteins were found to be down-regulated in the leaves of the spl6 mutant.27 It has been well-documented that production of PR proteins is induced when plant recognizes the invading pathogen(s) through activation of a host resistance gene, which is accompanied by HR resulting in the triggering of rapid and effective defense responses.2,28 Moreover, the induction of PR proteins is also a ubiquitous and common plant response to PCD and pathogen attack.29–31 The probenazole (3-allyloxy-1, 2-benzisothiazole-1, 1-dioxide; PBZ)-induced protein (PBZ1), which is a PR 10 family member, was found to be expressed both in rice suspension-cultured cells and leaf blades inoculated with blast fungus.32–34 The PBZ1 protein was also induced upon treatment with jasmonic acid (JA), and in the rice LMMs.19,32,33 To date, the correlation between the accumulation of PBZ1 and enhanced resistance of LMMs to rice blast fungus has been only associated with three mutants, spl1, spl5, and spl11.19 However, Takahashi et al.17 correlated PBZ1 gene expression with lesion formation in cdr1, cdr2, and cdr3 mutants.17 Furthermore, the in planta localization of the PBZ1 protein in rice leaves was found to be correlated with cell death caused by rice blast fungus infection.32 In spite of the above research linking PBZ1 with defense/ stress response and LMMs, its biological function remains to be clarified. To address this question, we analyzed the spl1 mutant showing severe lesion mimic phenotype using twodimensional gel electrophoresis (2-DGE) in conjunction with MALDI-TOF-MS. It was reasoned that a differential proteome analysis of LMMs might broaden our understanding on signaling networks involved in PCD and defense signaling in plants. Among the numerous proteins induced in spl1, for example, one significantly and highly induced protein spot in the leaves was identified as rice PBZ1. Using detailed immunohistochemistry and PBZ1 gene promoter experiments in PCD tissues such as LMMs, senescence tissues, seed aleurone layer, and root

aerenchyma cells, we attempt to shed new light on PBZ1 function/role.

2. Materials and Methods 2.1. Plant Material and Treatments. Fourth- and fifth-leafstage rice (Oryza sativa L.) seedlings grown under natural light in a greenhouse (20–30 °C) were used for inoculation of whole plants with the blast fungus (Magnaporthe grisea KJ401 and KJ101). The LMMs (spl1 and spl2) used in this study were also grown in the greenhouse. For light-induced senescence, leaves detached from fourth- and fifth-leaf stage plants were placed onto Petri dishes containing 20 mL of distilled water and harvested in accordance with the extent of leaf senescence, which was judged by observing changes in total chlorophyll content. Chlorophyll was analyzed in 80% acetone extracts as described by Bruinsma.35 For submersion studies to induce the coleoptiles senescence, rice seedlings were completely submerged (8 cm below the water surface) in distilled water in a glass bottle as previously reported.11 To initiate cell death, seedlings that had been submerged for 5 days were exposed to air for 1, 2, 3, 4, 5, and 6 days. Root aerenchyma formation was performed using the method described by Kawai et al.36 Dehulled seeds were germinated at 28 °C in dark conditions. Endosperm from germinated seeds was excised with a razor blade and then used as samples for this study. 2.2. Protein Extraction and 2-DGE. Protein was extracted with Mg/NP-40 buffer (0.5 M Tris-HCl (pH 8.3), 2% (v/v) NP40, 20 mM MgCl2, 5% β-mercaptoethanol, 1 mM phenyl methyl sulfonyl fluoride, and 1% (w/v) polyvinyl polypyrrolidone) and fractionated with polyethylene glycol (PEG) 4000. The 15% PEG supernatant fractions were used for 2-DGE analysis, essentially following the method described in Kim et al.37 Isoelectric focusing (IEF) for the first dimension was carried out using 18 cm (length) glass tube gels. The IEF gel mixture consisted of a 4.5% (w/v) acrylamide solution, 9.5 M urea, 2% (v/v) NP-40, and 2.5% (v/v) pharmalytes (pH 3–10/pH 5–8/pH 4–6.5 ) 1:3.5: 2.5; Amersham Pharmacia Biotech, San Francisco, CA). Each sample (250 µg) was mixed in the IEF sample buffer and then loaded onto 18 cm IEF gels.37,38 SDS-PAGE in the second dimension was carried out as described by Laemmli using 12.5% polyacrylamide gels.39 The 2-D gels were stained by colloidal Coommassie brilliant blue G-250 (hereafter called colloidal CBB). Three replicated gels were used in this experiment. 2.3. Image and Data Analysis. Gels were scanned (300 dpi, 16 bit grayscale pixel depth, TIFF file) for image/data analysis performed using the ImageMaster 2D Platinum imaging software ver. 5.0 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The 2-DGE patterns of protein spots between the spl1 and the wild-type were compared, and the abundance of spots was normalized as relative intensity according to the normalization method provided by the software. The changed (p < 0.05) protein spots were marked and selected for further identification by MS if they were confirmed in three independent sample sets. 2.4. In-Gel Digestion and MALDI-TOF-MS. The excised colloidal CBB-stained protein spot was cut into small pieces, washed with 50% acetonitrile in 0.1 M NH4HCO3, and vaccuum-dried. Protein digestion and all procedures for MALDITOF-MS (Voyager STR, PerSeptive Biosystems, Framingham, MA) analysis and searching were followed as described.40 The gel pieces were reduced for 45 min at 55 °C in 10 mM DTT in 0.1 M NH4HCO3. After that, the DTT solution was immediately Journal of Proteome Research • Vol. 7, No. 4, 2008 1751

research articles replaced with 55 mM iodoacetamide in 0.1 M NH4HCO3 and incubated for 30 min at room temperature in the dark. Gel pieces were washed with 50% acetonitrile in 0.1 M NH4HCO3 and dried in a SpeedVac evaporator. The dried gel pieces were swollen in a minimum volume of 10 µL of digestion buffer containing 25 mM NH4HCO3 and 12.5 ng/µL trypsin (Promega, sequencing grade) and incubated at 37 °C overnight. Digestion mixture was redissolved using a solution of distilled water/ acetonitrile/trifluoroacetic acid (93:5:2). The samples were sonicated for 5 min and centrifuged for 2 min. The matrix solution [dissolved R-cyano-4-hydroxycinnamic acid (Sigma) in acetone (40 mg/mL) and nitrocellulose in acetone (20 mg/ mL)], the nitrocellulose solution, and isopropanol were mixed 100:50:50. A two-point internal standard for calibration was used with a des-Arg1-Bradykinin peak (m/z 904.4681) and an angiotensin 1 peak (m/z 1296.6853). The samples were analyzed using a Voyager-DE STR MALDI-TOF mass spectrometer with the following parameters. Parent ion masses were measured in the reflection/delayed extraction mode with an accelerating voltage of 20 kV, a grid voltage of 76%, a guide wire voltage of 0.010%, and a delay time of 150 ns. Peptides were selected in the mass range of 600–2500 Da. For data processing, the MoverZ (http://bioinformatics.genomicsolutions.com) software was used. The acquired peak lists were analyzed by database (National Center for Biotechnology Information nonredundant, NCBInr) searches for identification by peptide mass fingerprint (PMF) using ProFound (http://www.unb.br/cbsp/paginiciais/ profound.htm) and MASCOT (www.matrixscience.com). The searching parameters were followed by which O. sativa was chosen for the taxonomic category. A peptide mass accuracy of below 50 ppm was applied. The results with MOWSE score higher than 65 (p < 0.05) were consider valuable. Only the best matches with high MOWSE score were selected. Spectra matching proteins are provided (Supplementary Figure 1 in Supporting Information). 2.5. Western Blot Analysis. The PEG-fractionated samples were analyzed by SDS-PAGE and transferred onto a polyvinyldiene difluoride (PVDF) membrane with a semidry electrophoretic apparatus (Hoefer, San Francisco, CA). The blotted membrane was blocked for 2 h in TTBS (50 mM Tris-HCl, pH 8.2, 0.1% (v/v) Tween 20, and 150 mM NaCl) containing 5% (w/v) nonfat dry milk. After blocking, the membrane was incubated with the purified PBZ1 polyclonal antibody (1:1000) in TTBS, The antigen–antibody interaction was carried out for 2 h. The membrane was washed (3 × 20 min) in TTBS, and a secondary goat anti-rabbit IgG conjugated with horseradish peroxidase diluted 1:5000 in TTBS was used for immunodetection. After the blots were washed with TTBS, the immunoblot signals were detected using enhanced chemiluminescent, ECL (PerkinElmer Life Sciences, Boston, MA). 2.6. Immunolocalization. Preparation of tissue sections and immunolocalization was followed as published previously.32 Tissue samples were fixed in phosphate buffer saline (PBS)buffered paraformaldehyde [2.5% paraformaldehyde (w/v)] at 4 °C overnight, dehydrated in a graded ethanol series, and embedded with Paraplast Plus (Sigma). Specimen (8 µm) was transferred to poly L-lysine-coated microscope slides that were deparaffinized by washing with xylene for 15 min and rehydrated in an ethanol series (100, 90, 80, 70, 60, 50, and 35%). For immunohistochemistry, thin sections were incubated with the blocking buffer comprising 2% BSA in PBS containing 0.02% sodium azide and 0.05% Tween-20 for 1 h at room temperature, and incubated with the purified PBZ1 primary antibody (1:50) 1752

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Kim et al. in a dilution buffer (PBS containing 0.1% [v/v] Tween 20 [PBST] and blocking buffer [1:1, v/v]) for 2 h. The specimens were rinsed three times with PBS-T (10 min each) and incubated with the alkaline phosphatase (Bio-Rad, Hercules, CA) or Rhodamine-conjugated anti-rabbit IgG antibody (Bio-Rad, 1:300) for 1 h, followed by three rinses in PBS-T (10 min each). Sample using alkaline phosphatase as a secondary antibody was incubated with NBT/BCIP solution for 20 min prior to visualization. 2.7. Construction of Ti Plasmid Vector Harboring PBZ1 Promoter and Generation of Transgenic Rice Plants. We searched the rice genome (O. sativa L.) database using the PBZ1 cDNA sequence (GenBank accession number, D38170). To isolate the PBZ1 promoter from rice genomic DNA, a forward primer (5′- GGTGCATGGTTGCGACCATT-3′) and a reverse primer (5′- AGCTAGTTGCAACTGATCAC-3)′ were designed using the PRIMER DESIGNER 4 software program (Scientific and Educational Software) and amplified by PCR. The resulting PCR product showing a 1403 bp genomic DNA fragment was cloned into the pGEM-T Easy vector (Promega, WI) and sequenced. The PBZ1 promoter was cloned into the pFANTA Gateway vector including GFP as a reporter protein using forward primer (attB1) 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCGGTGCATGGTTGCGACCATT-3′ and reverse (attB2) 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCTAGTTGCAACTGATCAC-3′. The resulting vector for PBZ1 promoter fused with GFP was introduced into the rice callus to generate transgenic rice plant by the use of Agrobacteriummediated transformation as followed by Jang et al.41 Regenerated plants were selected with 4 mg/mL phosphinotricin (Duchefa) and 0.5% Basta solution.42 Senescent leaves and leaves infected by the rice blast fungus were observed for GFP fluorescence under a LAS3000 (Fujifilm). GFP signal in germinated endosperms was observed under Laser-Scanning Confocal Microscopy (Olympus Co. Ltd., Tokyo, Japan). 2.8. Cell Death Assay. Cell death in senescent coleoptiles and seed aleurone cells were evaluated by histochemical analysis using Evans blue staining, which is a nonpermeating dye with low toxicity in plant cells, and propiodium iodide (3 µg/mL), which is a membrane-impermeable dye that binds to nucleotides and that is generally stained with dead cells, respectively. 43 2.9. TUNEL for Nuclear DNA Fragmentation. Seed endosperms harvested at 3 days after imbibition were longitudinally sectioned with a razor blade and fixed in 2.5% paraformaldehyde in PBS. TUNEL for nuclear DNA fragmentation was performed using DIG DNA labeling and detection kit according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). Stained sections were observed under a bright-field light microscope and immediately photographed. For nuclear staining, the fluorescent dye DAPI (100 µg/mL) was used. The samples were examined with light and LaserScanning Confocal Microscopy (Olympus Co. Ltd., Tokyo, Japan).

3. Results and Discussion 3.1. Proteome Analysis of the Rice LMM, spl1. To determine which proteins were specifically induced in LMM, proteins extracted from the leaves of the spl1 mutant were analyzed by 2-DGE. We initially examined both the phenotypes of spl1 and spl2 mutants, and observed that the spl1 mutant exhibited more severe lesion formation with large hypersensitive spots compared to the spl2 mutant. So, we choose the spl1

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Figure 1. 2-DGE analysis of PEG-fractionated proteins induced in the leaves of the spl1 mutant. Protein spots on gels are the enlarged images of differentially induced proteins in rectangles. Protein samples (250 µg) in 15% PEG supernatant fractions were separated on 2-D gels (pI 4–7), followed by colloidal CBB staining. 3-D images were generated by ImageMaster 2D Platinum, Version 5.01 (GE Healthcare Amersham Biosciences). 2D, two-dimensional; 3D, three-dimensional.

Figure 2. Quantitative analysis of differentially induced proteins on the 2-D gels. The mean relative expression level of three replicate samples is shown in the histograms based on relative protein intensities compared with background levels. Quantification of 17 protein spots from samples was made with ImageMaster 2D Platinum, Version 5.01 (GE Healthcare Amersham Biosciences). Error bars indicate the standard deviation. Wild-type, black bars; spl1 mutant, white bars.

mutant for proteome analysis in this study. The total protein extracts from spl1 mutant leaves were fractionated with PEG, and then the supernatant fraction was precipitated with acetone. The resolubilized protein precipitates were analyzed by 2-DGE with the isoelectric focusing (pH 4–7) for the first dimension and SDS-PAGE (12.5%) for the second dimension.37 Numerous colloidal CBB protein spots were detected on the 2-D gel (Supplementary Figure 2 in Supporting Information). Representative proteins that were differentially expressed in the leaves of the spl1 mutant are shown in Figure 1 (2D, twodimensional; 3D, three-dimensional). Eleven spots were found to be increased and seven showed decrease in protein amounts in spl1. Proteome analyses with spl1 mutant resulted in differential changes in the abundance of these proteins. The intensity of these differentially expressed protein spots on the 2-D gels obtained in three independent experiments were

quantitatively measured to obtain statistical information on variations in the protein levels using the ImageMaster program (Figure 2) 3.2. Characterization of Differentially Expressed Protein Spots on 2-D Gels. We excised the protein spots indicated in Figures 1 and 2 from the 2-D gels, which were digested with trypsin. After extraction of peptides, the proteins were identified by PMF using MALDI-TOF-MS. The resultant mass spectra were searched against the NCBInr protein database for identification using two different PMF search programs, ProFound and MASCOT (Table 1). Proteins differentially expressed in spl1 were identified as thaumatin-like protein (spot 1), two fructosebisphosphate aldolases (FBA, spots 4 and 10), three glyceraldehydes-3-phosphate dehydrogense (G3PDH, spots 9, 14, and 17), peroxidase (spot 5), APX (spot 11), 5-methyltetrahydropteroyltriglutamate homocysteine S-methyltransferase (spot Journal of Proteome Research • Vol. 7, No. 4, 2008 1753

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Table 1. Identification of Differentially Induced Proteins in spl1 Mutant spot no

protein name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Thaumatin-like protein 2-Cys peroxiredoxin Coproporphyrinogen III oxidase Fuctose-bisphosphate aldolase Peroxidase Glycosyl hydrolase family 3 Rubisco large subunit Glutathione reductase Glyceraldehyde-3-phosphate dehydrogenase Fuctose-bisphosphate aldolase Ascorbate peroxidase Sulfilte reductase, alpha subunit NAD-specific isocitrate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase 5-methyltetrahydropteroyltrigultamate-homocystein S-methyltransferase Serine hydroxymethyltransferase Glyceraldehyde-3-phosphate dehydrogenase Probenazole induced proteinb

16 17 18

AC

MS

database

401190 0.67 149390911 94 115460466 89 108864048 65 20286 82 32488698 74 115468792 98 115449517 72 115458768 75 115468886 69 125559715 111 115446541 69 41052915 66 115458768 75 115489654 75

ProFound MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT MASCOT

115455221 120 115476618 64 115489022 80

MASCOT MASCOT MASCOT

MP SC

4 4 6 6 4 5 7 8 7 6 6 4 5 7 5

21 33 25 13 19 9 18 21 19 19 36 22 12 19 9

ExMr

ThMr

ExpI ThpI expression

18.43 19.2 38.46 37.16 34.6 85.26 55.27 56.16 44.59 44.58 25.49 43.27 45.19 44.29 86.34

18. 28.09 36.47 41.58 32.86 89.24 52.8 53.5 42.69 37.73 27.11 40.67 46.04 42.49 84.67

5.23 5.2 5.6 5.6 5.9 6.4 6.3 6.5 6.79 6.81 4.9 6.2 6.5 6.79 6.4

10 25 57.29 56.38 6.8 5 9 57.5 53.37 6.5 6 41 17 16.69 4.7

5.1 5.7 6 6.07 5.8 6.5 6.2 6.2 7.62 7.57 5.2 8 6.3 7.62 5.9

up down down up up down down down up up up down down up up

8.4 6.6 4.9

up up up

a The proteins induced were identified by peptide mass fingerprinting using MALDI-TOF; SC, sequence coverage; MS, MOWSE score; AC, accession number; MP, matched peptides; b Probenazol induced protein was confirmed by Western blot using purified PBZ1 antibody.

15), serine hydroxymethyltransferase (spot 16), and PBZ1 (spot 18). All these proteins were induced in the leaves of the spl1 mutant. The other proteins including 2-Cys peroxiredoxin (spot 2), coproporphyrinogen III oxidase (spot 3), glycosyl hydrolase family 3 (spot 6), rubisco large subunit (spot 7), glutathione reductase (spot 8), sulfilte reductase (spot 12), and alpha subunit NAD-specific isocitrate dehydrogenase (spot 13) were reduced in the leaves of the spl1 mutant and are also shown in Table 1. The induction of many metabolic enzymes, such as two FBAs and three G3PDHs, was also found in the leaves of the spl1 mutant, whereas two proteins were reduced in the previously characterized blm mutant.26 G3PDH which is a glycolytic enzyme has been reported in plants in response to biotic and abiotic stresses as well as LMMs.25,44,45 This protein is not only a glycolytic enzyme, but also functions as a protein kinase, an mRNA-binding protein, and has uracil DNA glycosylate activity for DNA repair.46 Thus, the accumulation of some proteins involved in energy metabolism such as glycolytic (FBA and G3PDH) and tricarboxylic cycles (NAD-specific isocitrate dehydrogenase) may be necessary to increase the production of ATP, NAD(P)H, and carbon skeletons which are needed to sustain the demands of cells for increased protein synthesis in the LMMs. Serine hydroxymethyltransferase and 5-methyltetrahydropteroyltrigultamate-homocystein S-methyltransferase were also identified in spl1. These proteins are involved in the biosynthesis of glutamate, cysteine, and O-acetyl-serine, a precursor in the cysteine biosynthesis pathway, which are required for reduced glutathione (GSH). A large family of antioxidant enzymes, including 2-Cys peroxiredoxin (spot 2), peroxidase (spot 5), and glutathione reductase (spot 8), which are down-regulated in spl1 were also identified. A similar observation was reported in the spl6 mutant.27 However, APX, which is an important component of the ascorbate-GSH cycle for H2O2detoxification in chloroplasts and cytosol, was found to be induced in spl1 mutant. In contrast, the expression of APX was found to be reduced in blm.26 Therefore, these results imply that biosynthesis of GSH and other ROS enzymes may be necessary to protect the cells against oxidative damage by 1754

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reducing ROS in spl1. Furthermore, when the antioxidant enzymes show down-regulations in the LMMs, it may cause a failure to protect cells against oxidative burst, eventually leading to cell death. Interestingly, coproporphyrinogen III oxidase (spot 2) was reduced in spl1, suggesting that the down-regulation of this protein in spl1 is likely to be a one of the key responses in causing cell death, which occurs during lesion formation. It has been previously reported that lesion mimic phenotypes were produced by the inhibition of protoporphyrinogen oxidase expression.47 Many proteins including PR proteins (PR5 and PBZ1), oxidative-stress-related proteins, and metabolic enzymes which are either up- or down-regulated in LMMs were identified, implying that disruption of cellular homeostasis by increasing or decreasing of related proteins leads to a wide range of metabolic perturbations and subsequently causes cell death in plants. From the spl1 proteome analysis, however, we realized that proteins (FBA, G3PDH, and APX) induced in spl1 were reduced in the two recently characterized LMMs, blm and spl6. These results suggest that expression patterns of proteins could be differentially regulated since genes causing mutations might display a diverse signaling cross-talk according to genetic background of mutants. 3.3. The PBZ1 Protein Accumulates in Tissues Undergoing PCD Induced by Abiotic Stresses. Interestingly, PBZ1 was highly induced in the spl1 mutant (Figure 3A). To verify whether PBZ1 was indeed induced in spl1, the PEG supernatant protein fraction of the spll mutant leaves was examined by SDS-PAGE. Western blot analysis using 2-DGE and SDS-PAGE revealed that the PBZ1 protein was also accumulated in spl2 as well as spl1 (Figure 3B,C) over no such accumulation in the wild-type. The leaves infected with the incompatible blast fungus (M. grisea strain, KJ401) was used as a positive control.23As shown in Figure 3B, the PBZ1 protein was more markedly accumulated in the leaves of the spl1 mutant that exhibited more severe lesion formation than in the spl2 mutant. Expression of PBZ1 in tissues undergoing cell death was observed in HR induced by the rice blast fungus pathogen and

Localization of PBZ1 in Cells Undergoing PCD

research articles cumulation of PBZ1 and senescence was first investigated by monitoring its level during senescence of detached rice leaves (Figure 4). The extent of leaf senescence was measured as the change in total chlorophyll content (Figure 4A). As shown in Figure 4, the accumulation of PBZ1 protein was gradually increased during leaf senescence of detached leaves (Figure 4B) and natural leaf senescence (Figure 4C) of whole plants. In addition, the PBZ1 protein was also highly accumulated at early stages of leaf senescence in which leaves still had more than 80% chlorophyll. After that, leaf senescence was significantly enhanced. We performed immunolocalization to determine the precise localization of PBZ1 in the spl1 mutant and senescent leaf. As shown in Figure 5, accumulation of the PBZ1 protein in spl1 was localized around lesion in the leaves (Figure 5A,B) and senescent leaf (Figure 5 C,D). These results are in agreement with previous PBZ1 localization results for which the PBZ1 protein did accumulate in cells surrounding dead cells resulting from strong HR.32

Figure 3. Accumulation of PBZ1 protein in the spl1 mutant. (A) Enlarged image of PBZ1 protein induced in spl1mutant; 3D, threedimensional. (B) Western blot on 2-DGE in spl1. (C) Western blot on SDS-PAGE. Western blot analyses using the purified PBZ1 antibody in whole leaves of two LMMs (spl1 and spl2) and wildtype plants were used to monitor the accumulation of PBZ1 proteins. Leaf inoculated with rice blast fungus (KJ401) was used as a positive control. Protein samples were harvested and separated by SDS-PAGE. The total protein (20 µg) was loaded for SDS-PAGE.

in LMMs (blm, cdr2, and spl1).25,26,32 This prompted us to further investigate whether this phenomenon extended to other systems in which PCD or cell death occurred. Senescence is a unique developmental process that is characterized by massive PCD and nutrient recycling. The correlation between ac-

Recently, it was suggested that coleoptiles senescence occurs when elongated coleoptiles grown under submergence for 5 days were transferred to the aerated condition. Such coleoptiles exhibited cell death events that were associated with senescence.11 To check whether PBZ1 protein was associated with coleoptiles senescence, Western blot analysis was performed (Figure 6A). We devised a condition to induce synchronous PCD in coleoptiles from rice seeds by submerging them in water for 4 days followed by transfer to aerobic conditions, in which coleoptiles senescence starts rapidly. Similar to the observations in senescent leaves, the level of PBZ1 protein was significantly increased as soon as coleoptiles were transferred from the submerged condition to the aerobic environment (Figure 6A), accompanied by the PCD when stained with Evans blue (Figure 6E). Because the PBZ1 protein highly accumulated during coleoptiles senescence, we also examined their cellular localization in senescent coleoptiles by immunohistochemistry. Immunohistochemistry of PBZ1 revealed that it is expressed

Figure 4. Accumulation of PBZ1 protein during leaf senescence. (A) Chlorophyll content at various time points as an indicator of senescence of detached leaves; (B) Western blot analysis of PBZ1 protein with proteins extracted from samples collected at the same time points; (C) Chlorophyll content and accumulation of PBZ1 protein during natural senescence of rice whole plants. The 100 refers to fully expanded green leaf (100% Chl); 80 refers to senescent leaves (70–95% Chl); 50 refers to senescent leaves (50–70% Chl). CBB, Coomassie blue; IB, immuno blot. Each protein sample (20 µg) was loaded onto a 12.5% SDS-PAGE gel. Equal loading of protein was confirmed by Ponceau S staining of the membrane. Journal of Proteome Research • Vol. 7, No. 4, 2008 1755

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Figure 5. Immunolocalization of PBZ1 protein in spl1 and senescent leaves. Immunolocalization of PBZ1 proteins in spl1 (A and B) and senescent leaves (C). (D) Inserted image indicates a negative control used with free PBZ1 antibody. The sections were incubated with alkaline phosphatase-conjugated anti-rabbit IgG antibody (dilution 1:300) for 1 h and visualized after the addition of NBT/BCIP solution for 20 min.

in cells adjacent to expanding aerenchyma cells (Figure 6F,G), where cell death occurs, compared to the negative controls (Figure 6B-D). We also investigated PBZ1 accumulation in roots during aerenchyma formation obtained from seedlings grown under aerobic or submerged conditions. Root aerenchyma cells in the cortex also undergo PCD during development.7,10 Formation of root aerenchyma cells occurred in seedlings that had been transferred from aerobic conditions (5 days) to anaerobic ones (48 h) as described by Kawai et al.36 Aerenchyma in cortex started to form in the roots of plants following 24 h in submerged conditions and was fully developed by 48 h (Figure 7A). For Western blot analysis, proteins were extracted at 0, 3, 6, 12, 24, and 48 h after submergence. Western blot analysis revealed that the PBZ1 expression level gradually increased in accordance with incubation time of anaerobic growth as shown in Figure 7B. In addition, to further investigate whether PBZ1 is specifically expressed in root aerenchyma cell, immunohistochemstry was performed with roots grown under submergence condition (Figure 7C). The PBZ1 signal was dramatically localized in the cortical region where aerenchyma cells develop in the root cortex (Figure 7 C). No signal was detected in negative control performed using the secondary antibody alone. 3.4. Promoter Analysis of the PBZ1 Gene. To further characterize the PBZ1 expression and localization pattern, we generated transgenic plants harboring the GFP gene driven by the PBZ1 promoter using Agrobacterium-mediated transformation.41 We cloned a DNA fragment (about 1405 bp) between the rice genomic DNA and used it as the promoter region of PBZ1 to drive GFP expression. We carefully examined the GFP signals in the leaves of the transgenic plants inoculated with rice blast fungus. As shown in Figure 8A, overall, GFP flores1756

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Figure 6. Accumulation and immunolocalization of PBZ1 protein in coleoptiles obtained from seedling grown under aerobic or submerged conditions. (A) Induction of PBZ1 protein in the rice coleoptiles obtained from seedlings that had been transferred from submergence (5 days) to aerobic condition. Proteins were extracted at 0, 1, 2, 3, 4, and 5 days after exposure to air. Crosssections of and senescent coleoptiles of 4 days old seedlings grown under aerobic conditions (B-D, F, and G). Each crosssection was treated with purified specific anti-PBZ1 for immunohistochemical analysis and detected by NBT/BCIP (F and G). Blue-colored signals indicate accumulation of PBZ1. Most of PBZ1 was accumulated in aerenchyma cell; ae, aerenchyma. (E) Cell death in coleoptiles was stained with Evans blue. (D) Tissue treated with free antibodies: negative control.

cence signals showed strong PBZ1 promoter activity in or around neighboring cells invaded by rice blast fungus as revealed by immunolocalization.23 Furthermore, we found that the PBZ1 promoter was activated during leaf senescence (Figure 8B), whereas no such signal was detected in senescent nontransgenic plant. This result is consistent with the Western blot and immunolocalization data described above. 3.5. The PBZ1 Protein Accumulates with Developmental PCD Tissues. So far, we investigated the PBZ1 expression in cell death tissues caused by biotic and abiotic stresses. To test whether PBZ1 is induced in development tissues involved in natural PCD as well as biotic and abiotic stresses, we used immunolocalization with the PBZ antibody in seed aleurone cells, which is an example of a typical developmental PCD tissue in plants.10 The accumulation of PBZ1 protein in germinated seed endosperm was examined by Western blot analysis in dry, imbibed, and germinating seeds. Results revealed highly accumulated PBZ1 protein in the seed endosperm at 3 days during seed germination (data not shown),

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Figure 7. Accumulation and immnolocalization of PBZ1 protein in root during aerenchyma formation obtained from seedling grown under aerobic and submerged conditions. (A) Induction of PBZ1 protein in root aerenchyma cell obtained from seedlings that had been transferred from aerobic conditions (5 days) to submergence (48 h). Root sections grown under aerobic (left panel in A); root sections grown for 24 h under submerged conditions (middle panel in A); root sections grown for 48 h under submerged conditions (right panel in A). (B) Western blot analysis of induced PBZ1 protein. Proteins were extracted at 0, 3, 6, 12, 24, and 48 h after submergence. For Western blot analysis, total proteins (20 µg/mL) were electrophoresed using SDS-PAGE and transferred onto a PVDF membrane. (C) Immunolocalization of root submerged for 48 h. Each root cross-section was treated with purified specific anti-PBZ1 for immunohistochemical analysis and detected by NBT/BCIP. Most of PBZ1 was accumulated in aerenchyma cell in the root cortex; ae, aerenchyma.

but not imbibed seeds. In addition, immunohistochemical analysis revealed that PBZ1 accumulated in aleurone cells during seed germination (Figure 9A). Furthermore, intense GFP signal of PBZ1 promoter transgenic line was also activated in germinated seeds but not in imbibed seeds (Figure 9B), which is consistent with the Western blot and immunolocalization data. To dissect whether the PBZ1 signal coincides with PCD cells, we used propiodium iodide which is an indicator of cell death using 3-day-old germinated seeds. Germinated seeds were cut in half with a razor blade and observed under confocal microscopy. As shown in Figure 9 C, the PBZ1 signal dramatically coincided with propiodium iodide signal in the seed aleurone layer. The PBZ1 signal and propiodium iodide signal significantly co-accumulated around root cells where lateral root is emerging, indicating that it undergoes PCD during root growth (Supplementary Figure 3 in Supporting Information). Moreover, PBZ1 protein using immunolocalization was dramatically accumulated in root caps as well as root aerenchyma cells (Supplementary Figure 4 in Supporting Information).

Figure 8. Fluorescence image of PBZ1 promoter after M. grisea treatment and leaf senescence. (A) Transgenic leaves harboring PBZ1 promoter::sGFP (PBZ1 pro::sGFP) chimeric gene were inoculated with compatible race, KJ101 for 3 days. GFP signal specifically appeared (green) in the lesion invaded by rice blast fungus. (B) GFP signal in transgenic leaves after induction of senescence by dark condition; NS, nonsenescent leaf; S, senescent leaf.

3.6. In Situ Detection of DNA Fragmentation by TUNEL Analysis. TUNEL which typically hallmarks PCD was employed to examine whether nuclear DNA fragmentation occurs in the seed aleurone layer. DNA fragmentation was detected in paraformaldehyde-fixed seed aleurone layer after germination at 3 days. As shown in Figure 9D, the spots visualized in the TUNEL analysis were observed in seed aleulone layer only after staining with DAPI. To verify that the TUNEL signals were from nuclei, we stained the nuclei with DAPI. We did not observe digoxigenin incorporation in the aleurone layer of imbibed seeds (data not shown). To investigate whether accumulation of the PBZ1 protein coincided with seed aleurone layer showing TUNEL signal, we coanalyzed TUNEL and immunolocalization in the seed aleurone layer (Figure 9E). The TUNEL signals Journal of Proteome Research • Vol. 7, No. 4, 2008 1757

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Figure 9. Expression of PBZ1 protein in seed aleurone layer associated with PCD. (A) Immunolocalization of PBZ1 in seed aleurone layer. For immunohistochemical analysis, seeds at 72 h after germination were used, cross-sectioned by hand, and analyzed with antibody (dilution; 1/100) against PBZ1 protein. Tissue treated with pre-immune serum was used for negative control (pre-immune). Sections of seed endosperm were reacted with anti-PBZ1. The specimens were incubated with the alkaline phosphatase-conjugated anti-rabbit IgG antibody (1:300) for 1 h and detected with NBT/BCIP solution for 20 min for visualization. al, aleurone layer; se, seed endosperm. (B and C) Activation of PBZ1 promoter in seed (B) and hand-cut seed germinated for 3 days. (C) Observation of sGFP fluorescence in transgenic rice plants expressing PBZ1 promoter::sGFP (PBZ1 pro::sGFP) chimeric gene using confocal microscopy. Propiodium iodide was used as an indicator of cell death. (D) Detection of DNA fragmentation using TUNEL in seed aleurone cell during germination. DNA fragmentation in seed aleurone cell after germination for 48 h was detected with an in situ cell death fluorescent detection kit (Boehringer Mannheim GmbH, Germany) and DNA fragmentation was detected by adding a fluorescent (FITC)-labeled group to the 3′-ends of broken DNA strands. For nuclear staining, the fluorescent dye DAPI which stains DNA was used. (E) Colocalization both of DNA fragmentation and PBZ1 protein in seed aleurone cell during germination. Rhodamin-conjugated secondary antibody (dilution 1:200) was used to detect PBZ1 protein.

tightly coincided with localization of the PBZ1 protein, indicating that PBZ1 is indeed colocalized with tissues undergoing PCD in development tissues as well as biotic and abiotic stresses. Several members of the PR protein class show induced expression levels in LMMs and during senescence as well as in pathogen attack.17,26,31,32 Similarly, the senescence-associated genes are expressed only in chlorotic tissue surrounding the sites of HR during infection of tobacco with tobacco mosaic virus.44 In many studies, transgenic plants that constitutively express rice PR genes showed enhanced disease resistance accompanied by HR.48 Recently, Sasaki et al. studied the blast fungus-induced peroxidase (R2329) in great detail by the precise characteristics of its promoter::GUS-fusion genes such as tissue specific expression profiles and expression patterns in response to rice blast fungus.49 In blast fungus inoculated R2329 promoter::GUS leaves, GUS staining analysis revealed that signal was accumulated just around fungus-induced local lesions. This result was in agreement with immunolocalization of PBZ1 protein and activation of PBZ1 promoter::sGFP. However, the R2329 promoter did not coincide with PCD tissues like PBZ1.32 Instead, the GUS signal was commonly found in vascular bundle and exodermises in leaves and roots, which are all not PCD cells.49 1758

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3.7. Putative Function of the PBZ1 Protein. In this study, induction and localization of PBZ1 were observed in rice PCD cells. However, the exact roles and biochemical function of the PBZ1 protein in plant cytoplasm are still not clear. Most of the previous publications on the PR-10 family proteins point to a putative RNase function based on amino acid sequence similarity to the ginseng callus RNases.50 This characteristic feature has also been reported with the birch pollen Betv1 and white lupin PR-10 -like proteins, both in their native and recombinant forms.51,52 Recently, it was reported that RNase LX is specifically expressed during endosperm mobilization and leaf/flower senescence.53 With the use of immunofluorescence, RNase LX protein was detected in immature tracheary elements, suggesting a function in xylem differentiation. Walter et al. have also proposed that cytosolic ribonucleases could be involved in selective and/or highly regulated degradation of existing mRNAs during stress or pathogen attack.54 These results support a physiological function of RNase in selective cell death processes that are also thought to involve PCD. Recently, it was reported that the rice JIOsPR10 protein exhibits RNase activity.55 However, there is no evidence for RNase activity for PBZ1. In general, the PCD process causes hydrolytic enzymes, such as RNases, DNases, and proteases enriched in lytic compartments, to invade the soluble cell content for

Localization of PBZ1 in Cells Undergoing PCD 56

degradation. These proteins are usually considered as biochemical and molecular marker for cell death in plants. However, little is known about cell death marker in rice. Recently, Vacca et al. found that cytochrome c, which was a typical cell death marker protein in animal system, was induced in a ROS-dependent manner during heat-induced PCD in tobacco BY-2 cells.57 However, it is likely that cytochrome c release might not be a common or essential PCD signal in plants.58

4. Conclusions We carried out proteomics analysis to understand cell death and identify which proteins were activated in LMMs. Eleven up-regulated and 7 down-regulated proteins in spl1 mutant compared to wild-type plant were identified by the use of 2-DGE coupled with MADLI-TOF MS analysis of tryptically digested proteins. The proteins differentially expressed in spl1 mutant were mainly involved in ROS scavenging, metabolism, and stress responses. This was in agreement of results of proteome analysis of other LMMs (blm, cdr2, spl6), suggesting that these proteins are common features occurring in LMMs when cell death is caused. In addition, the correlation between accumulation of PBZ1 and PCD tissues was first investigated by monitoring its expression level using Western blot, immunohistochemical analysis, and its promoter analysis, resulting in a clear demonstration that the PBZ1 protein is tightly associated with plant cell death tissues. In rice, precise localization of protein as well as its promoter in PCD tissues specifically has been poorly studied so far. We suggest for the first time that PBZ1 could serve as a cell death marker protein as well as defense protein in rice. Thus, the precise PBZ1 promoter analysis described here showing induction locally at the pathogen target regions could be useful for generation of transgenic plants to prevent crops from attacking pathogens including rice blast fungus and leaf blight bacteria. However, further investigations will be required to elucidate the evidence for the role of PBZ1 as RNase activities and its functional relevance for plant cell death or defense responses remain unknown.

Acknowledgment. This work was supported by ARPC, by a grant from MOST/KOSEF to the Environmental Biotechnology National Core Research Center (R15-2003-012-010020), and by scholarships from the Brain Korea 21 Program (Y.H.K.). Supporting Information Available: Figures showing spectra matching proteins, 2-DGE analysis of PEG-fractionated proteins in spl1 mutant, fluorescence image of PBZ1 promoter around root cells where lateral root is emerging, and immunolocalization of PBZ1 protein in root cap. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) (2) (3) (4)

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