- Open Access
Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families
© Sikorskaite et al.; licensee BioMed Central Ltd. 2013
- Received: 3 May 2013
- Accepted: 24 July 2013
- Published: 26 July 2013
In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.
- Nuclei isolation
The nucleus is a highly specialized, complex and heterogeneous organelle of the cell. It contains most of the cell’s hereditary information and controls most of the cellular functions [1, 2]. The studies on transcription of genes , synthesis and processing of RNA , and posttranscriptional gene silencing  are enhanced by the availability of isolated nuclei. Isolated nuclei are also used to obtain high-quality chromosomal DNA with minimized chloroplast and mitochondrial DNA contamination , to determine ploidy level, and to measure nuclear DNA content using flow cytometry [7–11].
Nuclear proteins play a central role in regulating gene expression. Studies on nuclear proteome have been conducted in model species, such as Arabidopsis thaliana[12, 13], Oryza sativa, and Medicago truncatula, as well as in chickpea (Cicer arietinum) . The studies have estimated the size of the proteome, placed the identified proteins by functional classes, revealed targeting of the proteins and their posttranslational modifications. Pendle et al.  conducted the first proteomic analysis of nucleoli of A. thaliana, whereas Calikowski et al.  characterised nuclear matrix in Arabidopsis. Tan et al.  performed proteomic and phosphoproteomic analysis of the chromatin-associated proteins in rice, whereas Aki et al.  identified nuclear proteins involved in evolutionarily conserved mechanisms for sugar response in plants. Nuclear proteome analyses have also revealed major changes in nuclear protein composition during the influence of environmental stimuli [12, 21, 22]. Species-specific or tissue-specific comparative analyses of nuclear proteomes have provided new insights into understanding of the composition of the nucleus. Repetto et al.  compared nuclear proteomes of seeds of Medicago, leaves of Arabidopsis and seedlings of chickpea. On the other hand, Choudhary et al.  performed a comparative analysis of nuclear proteome at the organism level and assessed differences in protein composition of the samples isolated from rice, Arabidopsis, Medicago and chickpea.
Isolation of nuclei from protoplasts is commonly used and provides high yields of pure nuclei [23, 24]. However, the protocols for protoplast preparation are laborious and available for a limited number of species. Therefore, methods for isolation of nuclei from intact plant cells and tissues have been developed for a number of plant species using embryos [25, 26], seed coat , cultured plant cells , meristematic root tissues  or leaf tissues [30, 31]. Most of the isolation methods use the same steps of the procedure, including tissue disruption, filtration, centrifugation, solubilisation of membranes of contaminating organelles with non-ionic detergents, and separation of nuclei by density gradient centrifugation. Preparation of samples for flow cytometry usually requires only the first three steps . Non-ionic detergents, such as Triton X-100, facilitate the release of nuclei from cells and prevent nuclei from clumping . Moreover, application of detergent is crucial for isolation of nuclei from green tissue, in which nuclei need to be separated from chloroplasts by disrupting the latter with the detergent . However, high concentrations of the detergent or prolonged exposure to it may also disrupt the nuclear membrane [34, 35]. To facilitate analysis of nuclei from different types of cells, Deal and Henikoff  developed a method for isolation of nuclei tagged in specific cell types (INTACT), which allows isolation of biotin-labelled nuclei from specific types of cells of a tissue by streptavidin-coated magnetic beads. The method provides high purity of nuclei, but requires construction of transgenic plants for tissue-specific expression of affinity-labelled nuclear envelope protein, which is time consuming and not easily applicable for many plant species.
Different buffers for isolation of nuclei have been reported with variable compositions according to the plant material and the purpose of study for which the nuclei are isolated. During the release of nuclei from intact cells, nuclear isolation buffers must ensure the integrity of nuclear membrane and stability of nuclei throughout the experiment. Organic buffers (MES, HEPES, PIPES, TRIS, MOPS) are required to stabilize pH in the solution. Inorganic salts (KCl, NaCl) maintain ionic strength of the solution, and certain organic agents (sucrose, glycerol, hexylene glycol) stabilize membranes . Polyamines in the presence of metal chelators or divalent cations help to stabilize the nuclear chromatin and to avoid aggregation of nuclei . Reducing compounds, such as β-mercaptoethanol and dithiothreitol (DTT), are crucial for nuclear protein preparations, because they maintain cysteine residues in reduced form. The requirement for protease inhibitors depends on further application of the isolated nuclei. While protease inhibitors are used in some studies [34, 37], they are omitted from the buffers used for intact nuclei in other investigations [3, 12]. Many plant species contain large amounts of phenolic compounds and the use of reducing agents and resins absorbing phenolics (such as polyvinylpyrrolidone) is needed [10, 16].
Plant species and tissues vary in their physico-chemical composition. Therefore, adjustment and optimization of the protocols are needed to obtain pure preparations of intact nuclei that are free from contaminating non-nuclear proteins. The aim of this study was to develop a simple protocol applicable to isolation of nuclei and nuclear proteins from leaf tissues of three different plant species representing the families Solanaceae and Rosaceae. This was achieved by modification of the protocol of Cushman  by adjusting the concentration of detergents used for lysis of organelles and the parameters of density gradient and centrifugation, which were dependent on species-specific size and density of the nuclei. Furthermore, adjustments were made to remove phenolics and to stabilize protein samples for proteomics analyses. The nuclei-enriched fractions obtained with the optimised protocol showed negligible contamination with plastids, other cellular organelles and non-nuclear proteins. The isolation procedure could be concluded in 2 h.
2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) (Carl-Roth cat. No. 6335.1)
2-(N-morpholino)ethanesulfonic acid (MES) (Carl-Roth cat. No. 4256.2)
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Carl-Roth cat. No. 1479.2)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Carl-Roth cat. No. 9105.4)
Bradford Reagent (Sigma cat. No. B6916)
Diethyl ether (Carl-Roth cat. No. 5920.2)
Dithiothreitol (DTT) (Carl-Roth cat. No. 6908.2)
Ethylenediaminetetraacetic acid (EDTA) (Sigma cat. No. EDS)
Glycerol (Sigma cat. No. 49781)
MgCl2 hexahydrate (Carl-Roth cat. No. HN03.2)
Percoll (Sigma cat. No. P1644)
Polyvinylpyrrolidone (PVP) (Sigma cat. No. PVP40)
Potassium chloride (KCl) (Fluka cat. No. 60128)
Potassium phosphate monobasic (KH2PO4) (Sigma cat. No. P8416)
Protease inhibitor cocktail (Sigma cat. No. P9599)
Sodium chloride (NaCl) (Carl-Roth cat. No. 9265.1)
Sodium dodecyl sulfate (SDS) (Carl-Roth cat. No. 2326.2)
Sodium phosphate dibasic (Na2HPO4) (Sigma cat. No. S9763)
Spermidine (Sigma cat. No. S0266)
Spermine (Carl-Roth cat. No. 7162.1)
Sucrose (Sigma cat. No. S0389)
Thiourea (Sigma cat. No. T7875)
Triton X-100 (Carl-Roth cat. No. 3051.2)
TRizol reagent (Invitrogen cat. No. 15596–026)
Urea (Bio-Rad cat. No. 161–0730)
ECL anti-rabbit IgG, Horseradish Peroxidase-linked whole antibody (GE Healthcare cat. No. NA934V)
Rabbit anti-histone H3 (Agrisera cat. No. AS10 710)
Rabbit anti-lumenal-binding protein 2 (Agrisera cat. No. AS09 481)
Rabbit anti-plastocyanin (Agrisera cat. No. AS06 141)
SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific cat. No. 34080)
Falcon tubes (15 ml, 50 ml)
Homogenizer (UltraTurrax T25, IKA)
Hybond-P polyvinylidene fluoride membrane (GE Healthcare)
Leitz Laborlux S microscope with an epifluorescence extension (Leitz Ploemopak) and DAPI filter
Mortar and pestle
Orbital shaker incubator
Swinging rotor centrifuge (e.g., Eppendorf 5804R)
Nuclei isolation buffer (NIB)
1× NIB: 10 mM MES-KOH (pH 5.4), 10 mM NaCl, 10 mM KCl, 2.5 mM EDTA, 250 mM sucrose, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT.
4× stock solution of NIB was prepared without spermine, spermidine and DTT, and stored at 4°C. 1× NIB was prepared from the 4× stock and supplemented immediately before use with spermine, spermidine and DTT from the stocks of 100 mM spermine, 100 mM spermidine and 1 M DTT in deionized H2O.
1× NIB was used for nuclei isolation from tobacco and potato leaves, whereas for apple leaf tissue, 1× NIB was further supplemented with 1% PVP (MW 40,000) and 0.1% protease inhibitor cocktail.
10% (v/v) Triton X-100 in deionized H 2 O
60% (v/v) Percoll solution in 1×NIB
2.5 M sucrose
Nuclei storage buffer: 20% glycerol, 20 mM HEPES-KOH (pH 7.2), 5 mM MgCl2, 1 mM DTT. Store at −20°C.
1× phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4.
Electrophoresis buffer: 8.75 M urea, 2.5 M thiourea, 5% (w/v) CHAPS.
Tobacco plants (Nicotiana tabacum L. cultivar Samsun nn) were grown from seeds. Potato plants (Solanum tuberosum L. breeding line v2-108 ) were propagated in tissue culture, plantlets transferred to soil in pots and multiplied by rooting stem cuttings. Plants were grown under controlled conditions in a growth chamber at 23°C with photoperiod of 16 h (110 μE m-2 s-1) under illumination of fluorescent lamps (tubes of 58 W/830 and 36 W/77 in turns) and relative humidity of 40%. Plants were watered twice a week and fertilized weekly with 1% N:P:K = 16:9:22 fertilizer. The fresh leaves of tobacco (BBCH growth stage code: 1004 ; leaf length of 8–11 cm) and potato (BBCH growth stage code: 19 ; leaflet lengths of 2–5 cm) plants were sampled in the morning (2–4 hours to the light period) for isolation of nuclei. The leaves of young apple (Malus domestica Borkh. cultivar Orlovim) (BBCH growth stage code: 19 ; leaf length of 5–7 cm) were collected in June (before noon) from the young shoots of field grown trees in the genetic resources collection of the Institute of Horticulture, Lithuania and stored at −70°C after quick freezing in liquid nitrogen.
Isolation of nuclei
NOTE: All solutions, glassware, tubes, and equipment should be precooled to 0-4°C and kept on ice all the time. Homogenizer heads and centrifuge rotors should be cooled down to the same temperature.
Homogenization of leaf tissue
Solanaceae Remove the midvein from leaves and chop 3–5 g of them into small pieces with a blade. Treat with ice-cold diethyl ether (2–3 ml/g) for 3–5 min in a glass container. Rinse ether-drained leaves with ice-cold NIB (3–5 ml/g fresh weight) and discard rinse. Grind in 5–10 volumes of ice-cold NIB in 50 ml Falcon tube with homogenizer set on its lowest speed (8000 rpm) 3–5 times, each 5 s, until the tissue is completely disrupted.
NOTE: According to our observations, nuclei of potato are more fragile than those of tobacco and should be handled with special care.
Grind frozen apple leaves (2 g) in liquid nitrogen into powder with mortar and pestle and resuspend in ten volumes of NIB + 1% PVP (approximately 20 ml).
NOTE: diethyl ether treatment was not used for homogenization of apple leaves.
Solanaceae Slowly decant homogenates through two layers of pre-wetted cheesecloth and then through one layer of pre-wetted Miracloth.
Slowly decant homogenates through two layers of pre-wetted cheesecloth. Remove the debris from cheesecloth, resuspend in another 20 ml of NIB + 1% PVP and pass through the same cheesecloth. Filter through one layer of pre-wetted Miracloth.
NOTE: It is important to use as small a pad of cheesecloth and Miracloth as possible to reduce the loss of sample.
Lysis of contaminating organelles
Add 10% Triton X-100 dropwise to the solution until a final concentration of 0.5% (tobacco and potato) or 1% (apple) is reached. Gently agitate the solution for 20 min at +4°C.
Centrifuge the homogenate at 1000 × g (tobacco and potato) or 1800 × g (apple) for 10 min. Slowly resuspend the pellet with Pasteur pipette in 10 ml (tobacco and potato) or 5 ml (apple) of NIB.
Assembly of density gradient
Solanaceae Place 5 ml of 2.5 M sucrose into the chilled 50 ml Falcon tube. Carefully overlay with Pasteur pipette 5 ml of 60% Percoll solution. Be very careful not to mix the sucrose and Percoll layers.
Carefully with Pasteur pipette overlay 3 ml of 60% Percoll solution on 3 ml of 2.5 M sucrose in a chilled 15 ml Falcon tube.
Isolation of nuclei using Percoll/sucrose density gradient centrifugation
Carefully load the crude preparation of nuclei on the top of the density gradient by drawing the extract into a Pasteur pipette and slowly releasing the solution onto the side of the tube above the 60% Percoll layer (Figure 1B).
Subject the gradient to centrifugation in a swinging bucket rotor at 1000 × g (tobacco and potato) or 1200 × g (apple) for 30 min at 4°C. Use a slow break speed.
Remove the liquid above the gradient. Collect the 60% Percoll layer that contains most of the nuclei carefully with a Pasteur pipette (Figure 1B). Be careful not to disturb the dark green band located at the interface between Percoll and sucrose layers, which contains most of the contaminating debris and organelles.
Solanaceae Dilute the Percoll suspension containing tobacco nuclei slowly with 5 volumes of NIB and 0.5% Triton X-100 using a Pasteur pipette and incubate for 10 min under gentle shaking. Dilute the Percoll suspension containing potato nuclei with 5 volumes of NIB. Centrifuge at 1000 × g for 10 min.
Dilute the Percoll suspension with 3–5 volumes of NIB and centrifuge at 1800 × g for 10 min.
Purification of nuclei on 35% Percoll cushion
Resuspend the pellet of nuclei in 5 ml of NIB and overlay the solution on 5 ml (tobacco and potato) or 3 ml (apple) of 35% Percoll solution. Centrifuge at 1000 × g (tobacco and potato) or 1200 × g (apple) for 10 min.
Wash the nuclei by resuspending the pellet in 5 ml of NIB and centrifugate as previously (washing step). Proceed to nuclear protein isolation or alternatively, resuspend the nuclei in 500 μl of nuclei storage buffer, freeze in liquid N2 and store at −70°C until use.
Protein extraction and quantification
Total proteins are extracted using TRizol reagent according to the manufacturer’s instructions (Invitrogen). Protein concentration can be determined using Bradford reagent.
Western blot analysis
Proteins solubilized in electrophoresis buffer are analyzed on a 12.5% SDS polyacrylamide gel by electrophoresis and electroblotted onto a Hybond-P polyvinylidene fluoride membrane. To test the purity of nuclear proteins, membranes can be probed with rabbit anti-histone (H3, 1:15.000 dilution), anti-lumenal-binding protein 2 (BiP2, 1:2.000 dilution), and anti-plastocyanin (PC, 1:6.000 dilution) polyclonal antibodies. Signals are detected by incubation with donkey anti-rabbit IgG Horseradish Peroxidase –linked whole antibody (1:20.000 dilution) using the enhanced chemiluminescence (ECL) system and SuperSignal West Pico Chemiluminescent Substrate.
DAPI staining and microscoping
Nuclei were stained with DAPI (1 μg/ml in 1× PBS, 10 μl of nuclei in storage solution was mixed in 10 μl of DAPI solution) and analysed using a Leitz Laborlux S microscope with an epifluorescence extension and DAPI filter.
The nuclear proteome has recently gained importance in basic and applied research, because identification of novel nuclear proteins helps to understand protein functions and plant responses to different environmental cues. Comparative analysis of nuclear proteomes of different plant species may also provide evolutionary insights. The protocol described here allows enrichment of the nuclear fraction and reduction of contaminating non-nuclear proteins. Collection of nuclei from a 60% Percoll layer of the density gradient following low-speed centrifugation enhances the procedure significantly and allows completion of the whole process in 2 hours. Western blot analysis of organelle-specific marker proteins and microscopic observations indicated that the fractions of nuclear proteins were highly enriched and contained no or barely detectable amounts of proteins from chloroplasts and the endoplasmic reticulum. It is suggested that the procedure described in this study may be widely applicable for isolation of nuclei from leaves of species in the families of Solanaceae and Rosaceae.
Financial support from the European Social Fund under the Global Grant measure to DB (grant VP1-3.1-ŠMM-07-K-01-041), The Center of International Mobility (CIMO), Finland, to MLR for a scholarship of SS, and from the Academy of Finland to JPTV (grant 1253126) is gratefully acknowledged.
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