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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.