For protein extraction 4 g of frozen hairy roots were grounded in a mortar with a pestle, previously pre-cooled with liquid nitrogen.
   The resultant powder was homogenized with 6 mL of extraction buffer 7 M urea (U0631, Sigma), 2 M thiourea (T8656, Sigma), 4% NP-40 (N0896, Sigma), 1% ditiotreitol (DDT; D9163, Sigma), 1% ampholytes (pH 3-10; 163-1152, Bio-Rad), 1 mM PMSF (P7626, Sigma), 10 mM EDTA, 40 mM Tris-HCl (T6066, Sigma) and 0.05% PVPP (P6755, Sigma). The homogenates were centrifuged at 15,000 x g for 15 min.
   The proteins from the supernatant were precipitated with 25 mL of cold (-20ºC) 10% trichloroacetic acid (TCA; dissolved in acetone; T9159, Sigma) containing 0.07% ß-mercaptoethanol (M7154, Sigma). The sample was kept at -20ºC for 2 h to allow for complete precipitation of the proteins.
   After centrifugation (15 min, 3,000 x g), the samples were washed thrice with10 mL acetone (-20ºC; A1820, Fisher Scientific) containing 0.07% ß-mercaptoethanol to remove the TCA. The precipitate was lyophilized for 1 h and then solubilized in 2 mL of lysis buffer: 7 M urea, 2 M thiourea, 4% NP-40, 1% DTT and 2% carrier ampholytes (pH 10-3 and pH 5-7).
   This mixture was repeatedly shaken, and after 1 h it was centrifuged (15 min, 16,000 x g). The supernatant was recovered and stored in aliquots at -80ºC.

   The isoelectrofocus (IEF) was carried out in glass capillary tube of 130 mm length and 1.5 mm diameter.
   Briefly, the gel solution consisted of 10% NP-40, 30% (w/v) acrylamide (A3553, Sigma), 9.5 M urea, 10% ammonium persulfate (A3678, Sigma), 2% Temed (T9281, Sigma) and 2% ampholytes in relation 1:3 (pH 3-10 and pH 5-7) was disposed into the glass capillary tubes. After one hour the mixture was completed polymerized.
   The pre-running step was carried out at 200 V for 10 min, 300 V for 15 min and 400 V for 15 min in a Bio-Rad electrophoretic unit (Model 175 tube cell unit) in the presence of the running buffer containing phosphoric acid (0260-02, JT Baker) and sodium hydroxide (3722-01, JT Baker).
   After the pre-running step, 250 µg of protein sample was loaded on the gel. The electrophoresis was carried out at 600 V for 15 h, followed by 750 V for 3 h. After the running, each focused gel was equilibrated into 10 mL of equilibration buffer which contains 6 M urea, 30% (v/v) glycerol (G5516, Sigma), 2.5% (w/v) SDS (L4390, Sigma), 125 mM Tris-HCl (pH 6.8; T-6066, Sigma), 1% (w/v) DTT (D9163, Sigma) and 0.01% bromophenol blue (B0126, Sigma). Each gel was then agitated gently at room temperature for 15 min.
   The capillary gel, from the first dimension, was horizontally collocated on the top of a SDS-PAGE for the second dimension. This was carried out as described Laemmli {Laemmli, 1970 8350 /id /d} on a SE 600 series electrophoresis unit (Hoefer). In summary, the second dimension was performed using polyacrylamide at 15% (w/v), running at 150 V for 6 h. The 2D-PAGE gels were silver stained by method of Blum et al. {Blum, 1987 3771 /id}. The gels were incubate per 2 h with the fix solution which composition was of 60% methanol (67561, JT Baker), 39% acetic acid (64197, Fermont) and 1% formaldehyde (F1269, Sigma). The gel was then washed three times with 30% ethanol and sensitize with 0.02% (w/v) sodium thiosulfate (3946-05, JT Baker). The gels were stained with 0.2% silver nitrate (S5187, Sigma) and 0.075% formaldehyde (F1269, Sigma) per 20 min and revealed with a solution containing 6% (w/v) sodium carbonate (3604-05, JT Baker), 0.0004% sodium thiosulfate (3946-05, JT Baker), and 1% (w/v) formaldehyde (F1269, Sigma). After 5 min the spots were visualized and the reaction was stop with 2.5% EDTA (E9884, Sigma). The 2D-PAGE gels experiments were repeated at least three times to confirm reproducibility, an important factor in this study