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Proteome analysis of cell nuclei enriched subcellular fraction of apple (Malus × domestica Borkh.) Sidona Sikorskaite1, Perttu Haimi1, Minna Rajamäki2, Jari P.T. Valkonen2, Dalia Gelvonauskiene1, Grazina Staniene1, Vidmantas Stanys1, Danas Baniulis1 1Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai, Kaunas, LT; 2Department of Agricultural Sciences, University of Helsinki, Helsinki, FI. Abstract. Proteome analysis has been an important source of information for system biology analysis of complex molecular mechanisms involved in plant development, productivity and response to environmental stimuli. However, proteome of entire plant cell presents high demands on dynamic range and sensitivity of protein and analysis procedures. The problems encountered due to the complexity of sample could be overcome by application of subcellular fractionation in preparation of samples for proteomics analysis. Information on nuclear proteome of plants of the Rosaceae family remains vague. Apple, the most economically significant plant of the Rosaceae family, is also an ideal model for genomic studies on woody plants of the Rosaceae family due to availability of genomic information. In this study, we developed a procedure for apple cell nuclear protein enriched fraction preparation and 2D gel electrophoresis based analysis. Apple cell nuclei isolation conditions were established and optimised based on previously published results of the studies on plant nuclei preparation (Fig. 1-2). Efficient cell breakage of apple leave tissue was ensured by grinding in liquid nitrogen, and filtration followed by differential centrifugation led to separation of organellar fractions. For further enrichment of nuclei, a differential lysis of organelles was employed and concentration of detergent was optimized. The nuclei were separated from other organelles by equilibrium centrifugation on combined sucrose and Percoll gradient. The enrichment of nuclear proteins and contamination with non-nuclear proteins was analysed using specific antibodies (Fig. 3). Isolated protein samples were subjected to 2D gel electrophoresis (Fig. 4). Specificity and sensitivity of the method was assessed by comparing the results obtained using nuclear and total cell protein fractions. Fig. 1. Procedure for nuclear protein extraction and protein preparation from apple tissue. Fig. 3. Western blot analysis of fractionated proteins. Protein composition of protein fractions isolated from optimized combined Percoll/sucrose gradient was analyzed using Western blot analysis. Enrichment of nuclear proteins and contamination with non-nuclear proteins was analyzed using specific antibodies for nuclear protein, Histone H3 (H3), chloroplast protein, plastocyanin (PC), and endoplasmic reticulum protein, lumenalbinding protein 2 (BiP2). M, molecular marker, kDa; cell extract – cell protein extract after homogenization; cell lysate - a cell lysate after Triton X-100 treatment, from which organelles had been depleted by centrifugation at a low speed (1800g); 60% Percoll layer – Percoll layer of at the density gradient, from which nuclei were extracted; 60% P/2.5M S interphase – interphase between 60% Percoll and 2.5M sucrose layers of the density gradient; pellet – organelles depleted at the bottom of the gradient. Protein fraction, indicated in red brackets, represents nuclear proteins, isolated from 60% Percoll layer of the density gradient. Nuclear protein H3 was highly abundant in this fraction, whereas relative amount of chloroplast protein, plastocyanin, was highly reduced and endoplasdmic reticulum protein was absent from the nuclei, suggesting that the preparation was highly enriched in nuclear proteins. H3, detected in protein fractions in 60%P/2.5M S interphase and 2.5 M sucrose layer, represents proteins from unbroken cells, that sedimented at the interphase or penetrate to 2.5 M sucrose layer. Fig. 4. Separation of apple cell nuclear proteins by twodimensional gel electrophoresis. Nuclear and total protein fractions and reference sample were labeled with cystein specific fluorescent dye Em 560 (Dyomics), as described [Volke and Hoffmann, 2008]. Ten micrograms of proteins were used to rehydrate immobilized pH gradient strips (24 cm, pH 4-7). Proteins were loaded by in-gel rehydration method onto IEF strips and electrofocusing was performed using the IPGphor system (GE Healthcare) at 20ºC for 60700Vh. The focused strips were subjected to reduction followed by SDS-PAGE on 10-16% gradient gel. Typhoon FLA 9000 (GE Healthcare) imaging system was used for gel documentation. Gels demonstrate specific protein spots characteristic to the nuclear (upper panel) and total (lower panel) protein fractions. Plant material was homogenised in ten volumes of nuclei isolation buffer (NIB: 10 mM MES-KOH, pH 5.4, 10 mM NaCl, 10 mM KCl, 2.5 mM K-EDTA, 250 mM sucrose, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT, 1% PVP, 0.1% Protease Inhibitor Coctail) (modified according to J.C.Cushman (1995)). The homogenate was filtered through two layers of cheesecloth. The whole filtrate was passed through one layer of nylon mesh. Triton X-100 solution was added to a final concentration of 1%. The lysate was agitated for 20-30 min followed by centrifugation at 1800×g for 10 min. The supernatant was decanted and pellet resuspended in 5 ml of NIB. The mixture was fractionated on 60% Percoll / 2.5M sucrose gradient (1200×g for 30 min). Nuclei fraction collected from Percoll layer was washed with NIB buffer and sedimented in 35% Percoll solution. Nuclear proteins were extracted with Trizol (Invitrogen, Ltd.) according to manufacturer instructions. Fig. 2. Microscopy analysis of isolated apple nuclei. DAPI-stained nuclei isolated from the 60% Percoll layer of 60% Percoll/2.5 M sucrose density gradient. Fluorescent staining of nuclei with DAPI revealed non-uniform sphere shaped cell nuclei with an average diameter of approx. 15 µm. The concentration of nuclei, counted with hemocytometer, was approx. 7.5 x 106/ml. Micrographs displaying a composition of fractions of the density gradient. Components (unbroken cells, debris, chloroplasts, starch grains) pelleted at the interphase between 60% Percoll and 2.5 M sucrose layers (left panel). DAPI stained nuclei are mostly inside the unbroken cells. Nuclei sedimented in 60% Percoll layer (right panel). Total cell protein from plant tissue was prepared using method based on phenol extraction coupled with ammonium acetate precipitation, as described by Isaacson et al. [2006]. Concluding remarks. Subcellular fractionation techniques combined with high-resolution two-dimensional electrophoresis analysis is a method commonly used to analyze differential gene expression. In this study, we established a method for enrichment of apple cell nuclear fraction with minimal cellular contamination, protein preparation and protein separation procedures required for proteomics analysis. • A procedure of differential lysis and density gradient sedimentation was established for enrichment of cell nuclei from apple leaf tissue. An optimal concentration of detergent Triton X100 for lysis of contaminating organelles was established at 1%. Density centrifugation on Percoll/sucrose gradient was found effective for removal of contaminating cell components insoluble in Triton X-100. •Purity of isolated nuclear fractions was evaluated by Western blot analysis. Enrichment of nuclear proteins was established using antibody specific to Histone H3. Analysis using anti-PC and anti-BiP2 antibodies revealed that relative amount of chloroplast protein was highly reduced, whereas endoplasmic reticulum protein was not detected in nuclear protein fraction. •Isolated nuclear proteins were labeled with fluorescent dyes and subjected to 2D electrophoretic analysis. References. Cushman J.C., 1995. Methods Cell Biol. 50:113-127; Isaacson T., et al. 2006. Nat. Prot. 1(2): 769-774; Volke D. and Hoffmann R. 2008. Electrophoresis 29: 4516-4526. Acknowledgment. The European Social Fund under the Global Grant measure grant No. VP1-3.1ŠMM-07-K-01-041.