Reagents
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)
Liquid nitrogen
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)
Equipment
Blades
Cheesecloth
Falcon tubes (15 ml, 50 ml)
Funnels
Glass container
Homogenizer (UltraTurrax T25, IKA)
Hybond-P polyvinylidene fluoride membrane (GE Healthcare)
Leitz Laborlux S microscope with an epifluorescence extension (Leitz Ploemopak) and DAPI filter
Miracloth
Mortar and pestle
Orbital shaker incubator
Pasteur pipettes
Swinging rotor centrifuge (e.g., Eppendorf 5804R)
Solutions
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.
Plant material
Tobacco plants (Nicotiana tabacum L. cultivar Samsun nn) were grown from seeds. Potato plants (Solanum tuberosum L. breeding line v2-108 [38]) 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 [39]; leaf length of 8–11 cm) and potato (BBCH growth stage code: 19 [39]; 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 [39]; 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.
Protocol
An overview of the main steps of the protocol is presented in Figure 1.
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.
Rosaceae
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.
Filtration
Solanaceae Slowly decant homogenates through two layers of pre-wetted cheesecloth and then through one layer of pre-wetted Miracloth.
Rosaceae
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.
Centrifugation
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.
Rosaceae
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.
Washing
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.
Rosaceae
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.