Choi JS

Heo HY

Joo EJ

Kim AR

Kim KH

Nam JH

Park CS

Seo HS

Seo YW

Woo SH

English

Sydney eScholarship

 1Proteins identification of wheat-rye translocation lines by MALDI-TOF-TOF mass spectrometry and ESI-QTOF/MS  A-Reum Kim1,5, Ju-Hyun Nam3, Hyeong-Seok Seo1, Ki-Hyeon Kim1, Chul-Soo Park2, Hwa-Young Heo3, Yong-Weon Seo4, Jong-Soon Choi5 and Sun-Hee Woo1 1. Dept. of Crop Science, Chungbuk National University, Cheongju 361-763, KOREA 2 Honam Agricultural Research Institute, NICS, RDA, Iksan, 570-080, KOREA 3 Breeding Resource Development, National Institute of Crop Science, Suwon 441-857, KOREA 4. Division of Biotechnology, Korea University, Seoul 136-701, KOREA 5  Proteome Research Team, Korea Basic Science Institute, Daejeon 305-333, KOREA    ABSTRACT  The wheat-rye translocation lines have been agriculturally developed for the crop resistant to powdery mildew, leaf rust, Hessian fly, barley yellow dwarf virus, and drought stress. Chaupon rye contains 2RL chromatin to harbor resistance genes for powdery mildew and leaf rust. In order to identify 2RL chromosome-derived specific proteins, we compared the proteome of ‘Coker797’ (non-2RL) with those of ‘Hamlet’ (2RL) and near-isogenic line (NIL) carrying 2RL by 2D-gel electrophoresis and MALDI/ESI-MS. In the leaf proteome, 24 protein spots were clearly increased in 2RL-carrying lines compared to non-2RL line. The specific proteins of 2RL-lines included heat shock protein 70, chaperon protein DnaK, malate dehydrogenase I, and triosephosphate isomerase, which were confirmed in the EST database of NILs. In the root proteome, three protein spots were identified as putative peroxidase, cytoplasmic aldolase, and oxo-phytodienoic acid reductase. These results suggest that defense mechanism-related proteins and enhanced catabolic enzymes play roles of acquiring the resistance to biotic and abiotic stress in wheat-rye translocation lines.  INTRODUCTION  Cultivated ryes (Secale cereale) have been known to be more resistant to the pest than wheat (Triticum aetivum). Thus, several molecular breeding have been attempted to develop resistant wheat-rye translocation lines. At present, the most common translocation line is the 1BL/1RS1. Far from the merits of pest-resistant rye 1R short-chromosome, lines carrying this chromosome display several problems in quality such as reduced gluten strength, dough stickiness, and poor loaf volume of wheat2. Another line, Hamlet (PI549276) is derived from a 2BS/2RL wheat-rye translocation resistant to Hessian fly in which Chaupon rye was used as the donor of 2RL chromatin to harbor resistance genes for powdery mildew and leaf rust 3. The Hamlet-line was obtained by crossing between wheat cultivar ND7532 and rye cultivar Chaupon to translocate the chromosome 2RL (H21 resistant gene to biotype L of Hessian fly) from rye to wheat. In this study, we performed gel-based proteomics to identify the specifically expressed proteins of leaf and root of 2RL-carrying wheat-rye translocation Hamlet line compared to non-2RL ‘Coker797’ and near isogenic-line (NIL). MATERIALS AND METHODS  Plant Materials A near isogenic-line (NIL) carrying the H21 gene resistant to biotype L of Hessian fly was developed by backcross introgression (Coker797*4/Hamlet) and repeated selection by verifying the resistance to larvae of biotype L of Hessian fly4.  Total protein extraction and quantitation The proteins from leaf and root tissues of Coker797, Hamlet, and NIL lines were extracted by trichloroacetic acid/acetone precipitation method. The leaf and root proteins of the dried powder were solubilized in rehydration buffer containing 9 M urea/2 M thiourea/4% CHAPS. The solubilized proteins were quantitated by a modified Bradford method5. 2D-PAGE/Image Analysis Total proteins of 250 µg were electrofocused using 3-10 NL IPG strip gel (GE Healthcare) for 90,000 Vhr in leaf sample and for 12,000 Vhr in root sample. The IPG strips were equilibrated and the isoelectrofocused proteins were separated on 13% SDS-PAGE gel on Ettan Dalt system (GE Healthcare). After the electrophoresis, the 2D-gels were stained with silver staining kit (GE Healthcare). After silver staining, the gels were scanned using scanner (Powerlook III, UMAX) and the 2D-images were analyzed using Progenesis workstation version 2005 (Nonlinear Dynamics). In order assay consistent features of the 2D-gel patterns, we performed at least duplicate experiments per group. MS analysis/Bioinformatics Altered protein spots with more than 50% spot intensity were cut into fine slices with a razor blade, then transferred to Eppendorf tubes, and subjected to in-gel trypsin digestion according to the previous protocol6. All mass spectra were acquired at a reflection mode by MALDI-TOF MS (4700 Proteomics Analyzer, Applied Biosystems) and nano-LC Q-TOF MS (Premier II, Micromass). Since the genomic database of wheat cultivar is not sufficient, de novo internal sequencing were conducted using MS/MS fragmentation analysis and homology search against nr-BLAST database. The protein identification was performed by MASCOT (Matrixscience) and further searched by the expressed sequence tag (EST) database of wheat-rye translocation lines7.  2    Figure 1. The silver-stained 2D-gel images of leaf -Coker797 (A), Hamlet (B), and NIL (C).   Table 1. The catalogue of the leaf proteins expressed specifically in 2RL-carryling Hamlet and Near-isogenic Line  Spot  No. Accession No. identified protein Organism MW(kDa)/pI MS Sequence  Cover. (%) EST-DB 1 BAD34660 methionine synthase  Hordeum vulgare subsp. vulgare 84452/5.68 ESI 5 - 2 AAF23074 heat shock protein 70  Triticum aestivum 39680/4.56 ESI 18 Yes ABA97211 chaperone protein DnaK  Oryza sativa (japonica cultivar-group) 74041/5.11 ESI 15 Yes 3 NP114266 ATP synthase CF1 beta chain Triticum aestivum 53824/5.06 ESI 26 - 4 56 - 5 24 - 6 ABA92225 expressed protein  Oryza sativa (japonica cultivar-group) 8638/5.21 MALDI 47 - 7 NP114266  ATP synthase CF1 alpha subunit Triticum aestivum 53824/5.06 ESI 22 - 8 AAD41663 resistance protein  Oryza sativa  19146/8.57 MALDI 48 - 9 CAD54448 ribulose 1,5-bisphosphate carboxylase  large subunit  Haworthia vittata 49166/6.43 ESI 4 - 10 AAP55143 plastid-lipid associated protein, putative  Oryza sativa (japonica cultivar-group) 40016/4.42 ESI 2 - 11 AAU11110 ribulose-1,5-bisphosphate carboxylase  /oxygenase large subunit  Psathyrostachys fragilis subsp. Seca 53064/6.44 ESI 8 - CAA59228 NADPH dehydrogenase Hordeum vulgare 42122/9.25 ESI 12 - 12 CAA44032 rbcL  Aegilops crassa 46871/6.46 ESI 4 - CAB43994 malate dehydrogenase 1 Brassica napus 37708/7.59 ESI 10 Yes 13 CAA44027 rbcL  Triticum aestivum 46973/6.60 MALDI 39 - 14 41 15 CAD30025 ferredoxin-NADP(H) oxidoreductase  Triticum aestivum 40206/6.92 MALDI 38 - 16 CAC14917 triosephosphat-isomerase  Triticum aestivum 26786/5.38 MALDI 37 Yes 17 44 18 CAA44027 rbcL  Triticum aestivum 46973/6.60 MALDI 39 - 19 AAK72543  ribulose-1,5-biphosphate carboxylase  Wolffia arrhiza 49590/6.32 MALDI 37 - 20 CAA44027 rbcL  Triticum aestivum 46973/6.60 MALDI 37 - 21 CAC83406  ribulose-1,5-biphosphate-carboxylase  Moraea namaquamontana 49179/6.46 MALDI 34 - 22 NP114256 ATP synthase CF1 alpha subunit  Triticum aestivum 55261/6.11 MALDI 4 - 23 NP114267  ribulose-1,5-bisphosphate carboxylase /oxygenase large subunit  Triticum aestivum 52817/6.22 MALDI 35 - 24 NP114256  ATP synthase CF1 alpha subunit Triticum aestivum 55261/6.11 MALDI 4 -     3  Figure 2. The silver-stained 2D-gel images of root -Coker797 (A), Hamlet (B), and NIL (C).  Table 2. The catalogue of the root proteins expressed specifically in 2RL-carryling Hamlet and Near-isogenic Line. Spot No. Accession No. identified protein Organism MW(kDa)/pI Sequence  Coverage (%) EST-DB 1 CAB79456 putative peroxidase Arabidopsis thaliana 34846/10.05 4 - 2 BAA02729 cytoplasmic aldolase Oryza sativa  38695/6.56 3 - 3 CAD89604 oxo-phytodienoic acid reductase Oryza sativa(japonica cultivar-group)  42438/5.79 4 - BAD06519 hypothetical protein Pisum sativum 40922/5.52 5 -  RESULTS AND DISCUSSION In the leaf proteomic analysis, 24 protein spots were clearly increased in 2RL-carrying lines compared to non-2RL line (Figure 1). From the selected spots, 27 proteins in total were putatively identified by tandem mass spectrometry, which corresponded to 18 unique proteins. The spot number 2, 11, and 12 included two different proteins in single spot. Twelve protein spots were identified against non-wheat organisms due to the incomplete wheat genomic database. Interestingly, heat shock protein 70, chaperon protein DnaK, malate dehydrogenase I, and triosephosphate isomerase were confirmed in the EST database of NILs cDNA library. Thus, these suggest that up-regulation of heat shock proteins and metabolic enzymes are involved in the acquired resistance of wheat-rye translocation to the biotic and abiotic stress. In additions, methionine synthase, ATP synthase CF1 α/β chain, Rubisco large subunit, NADPH dehydrogenase, ferredoxin-NADP(H) oxidoreductase, resistance protein, and plastid lipid- associated proteins were exclusively identified in 2RL-lines by proteomic approach. Four protein spots out of 9 spots assigned as Rubisco large subunit were solely identified by T. aestivum and previous wheat leaf proteomic data8. Most of leaf proteins in this study did not match with previous leaf proteomic data. However, methionine synthase, HSP70, DnaK and triosephosphate isomerase, were commonly identified as wheat root proteome9. By the root proteome analysis of 2RL-lines, three known protein spots were identified as putative peroxidase, cytoplasmic aldolase, and oxo-phytodienoic acid reductase. In particular, the hydrogen peroxide and peroxidase are known to play a key role in the defense response of plants to the pathogen10. Interestingly, the spot number 3 was exclusively identified in Hamlet as oxo-phytodienoic acid reductase and hypothetical protein. In summary, this proteomic study of wheat-rye translocation lines will give helpful clues to solve the defense mechanism of plants against pathogens and provide useful information to select marker-based selection for crop development.  REFERENCES 1. Reynaldo, L. et al., 1998. Agronomic performance of chromosomes 1BS and T1BL1.1RS near-isogenic-lines in the spring bread wheat Seri M82. Euphytica 103, 195-202. 2. Fenn, D.O. et al., 1994. Milling and baking quality of 1BL/1RS translocation wheats. I. effects of genotype and environment. Cereal Chem. 71, 189-195. 3. Sears, R. G. et al., 1992. Registration of Hamlet, a Hessian fly resistant hard red winter wheat germplasm. Crop Sci. 302, 506. 4. Seo, Y. W. et al., 1997. A molecular marker associated with the H21 Hessian fly resistance gene in wheat. Mol. Breed 3, 177-181. 5. Ramagli, L. 1999. Quantifying protein in 2-D PAGE solubilization buffers. Methods Mol. Biol. 112, 99-103. 6. Shevchenko, A. et al., 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850-858. 7. Jang, C. S. et al., 2003. Expressed sequence tags from a wheat-rye translocation line (2BS/2RL) infested by larvae of Hessian fly [Mayetiola destructir (Say)]. Plant Cell Rep. 22, 150-158. 8. Able, A. J. et al., 2000. Hydrogen peroxidase yields during the incompatible interaction of tobacco suspension cells ionulated with Phytophthroa nicotianae. Plant Physiol. 124, 899-910. 9. Donnelly, B. E. et al., 2005. The wheat (Triticum aestivum L.) leaf proteome. Proteomics 5, 1624-1633. 10. Song, X. et al., 2007. Wheat (Triticum aestivum L.) root proteome and differentially expressed root proteins between hybrid and parents. Proteomics 7, 3538-3557. 

Proteins identification of wheat-rye translocation lines by MALDI-TOF-TOF mass spectrometry and ESI-QTOF/MS

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Proteins identification of wheat-rye translocation lines by MALDI-TOF-TOF mass spectrometry and ESI-QTOF/MS

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