Cucumber Root Plasma Membrane Characterization

Cucumber seeds (Cucumis sativus L. var. Krak), germinated in darkness at 25 °C for 48 h, were transferred to a nutrient medium containing 10 μM Cd or Cu for 6 d. After 3 d, some of the plants exposure to heavy metals were transferred to control conditions (nutrient solution without 10 μM Cd or Cu) for another 3 d (3/3 plants). The nutrient solution (pH 5,5) contained: 1.7 mM KNO₃, 1.7 mM Ca(NO₃)₂, 0.33 mM KH₂PO₄, 0.33 mM MgSO₄, and the microelements 75 μM ferric citrate, 10 μM MnSO₄, 5 μM H₃BO₄, 1 μM CuSO₄, 0.01 μM ZnSO₄, and 0.05 μM Na₂MoO₄. The plants were grown hydroponically with a 16 h photoperiod (180 μmol m⁻² s⁻¹) at 25 °C during the day and 22 °C at night. The relative humidity in the light and dark was 70%.
PM vesicles were isolated from cucumber root microsomes by phase partitioning according to the procedure of Larsson (1985) , as modified by Kłobus (1995) . An 8 g phase system containing 6.2% (w/w) Dextran T500, 6.2% (w/w) polyethylene glycol 3350, 330 mM sorbitol, 5 mM KCl, and 5 mM Bis-Tris propane (BTP)/MES (pH 7.5) was used. The PMs obtained by this procedure were composed mainly of right-side-out vesicles and were used to determine the hydrolytic ATPase activity. Some of the vesicles were turned to the inside-out-oriented form by the method of Johansson et al. (1995) (link) and used for measurements of ATP-dependent H⁺ transport in the PM.
The hydrolytic activity of the vanadate-sensitive ATPase (PM H⁺-ATPase) was determined according to the procedure of Gallagher and Leonard (1982) (link), as modified by Sze (1985) . The reaction mixture contained 50 μg of protein (PM), 33 mM TRIS-MES (pH 7.5), 3 mM ATP, 2.5 mM MgSO₄, 50 mM KCl, 1 mM NaN₃, 0.1 mM Na₂MoO₄, and 50 mM NaNO₃, with or without 200 μM Na₃VO₄ and 0.02% Triton X-100. PM H⁺-ATPase activity was expressed as the difference between the activity measured in the absence and presence of Na₃VO_4. The amount of P_i released during the reaction was determined according to the method of Ames (1966) with 0.2% (w/v) SDS included to prevent precipitation (Dulley, 1975 (link)).
H⁺ transport activity was measured spectrophotometrically as the change in acridine orange absorbance at 495 nm (A₄₉₅) according to the method of Kłobus and Buczek (1995) . The assay medium contained PM vesicles (about 50 μg of protein), 25 mM BTP-MES (pH 7.5), 330 mM sorbitol, 50 mM KCl, 0.1% BSA, 10 μM acridine orange, and 0.05% Brij 58. Proton transport was initiated by the addition of 3 mM Mg-ATP. For every combination, passive proton movement through the membrane was determined without ATP in the reaction medium.
To evaluate expression of the genes encoding the PM H⁺-ATPase, CsHA2 (GenBank accession no. EU735752), CsHA3 (EF375892), CsHA4 (HO054960), CsHA8 (HO054964), CsHA9 (HO054965), and CsHA10 (HO054966), real-time PCR was performed using the LightCycler^® 2.0 system from Roche Diagnostics. For the normalization of expression of each CsHA gene, a gene encoding TIP41-like protein (GW881871) was used as the internal standard. Total RNA was isolated from 50 mg of frozen root tissue using Tri Reagent (Sigma) according to the manufacturer’s instructions. Total RNA yield was determined using a NanoDrop Spectrophotometer ND-1000 (Thermo Scientific) and the A_260/280 ratio showed the expected values between 1.9 and 2.0. To avoid any DNA contamination, the RNA samples were treated with RNase-free DNase I (Fermentas) and then reverse transcribed into first-strand cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instructions. The cDNA was then used as the template for PCR amplification with a RealTime 2× PCRMaster Mix SYBR^® (A&A Biotechnology) kit. The gene-specific primers used for PCR were carefully designed using the LightCycler Probe Design program. Expression of CsHAs was analysed with the following primer pairs: 5′-ACCCGAGTCGACAAACATCT-3′ (forward) and 5′-CTTGGCACAGCAAAGTGAAA-3′ (reverse) for CsHA2; 5′-AAGTTTCTGGGGTTCATGTGGAAT-3′ (forward) and 5′-GTAACAGGAAGTGACTCTCCAGTC-3′ (reverse) for CsHA3; 5′-CTACAGCTTGGTAACATACATTC-3′ (forward) and 5′-GTTGTAGTCCATGTAATGTCCTC-3′ (reverse) for CsHA4; 5′-CTCATGCGCAAAGAACATTAC-3′ (forward) and 5′-CTGAATTGTGTCAATGTCAAGTC-3′ (reverse) for CsHA8; 5′-AAACCAGAAGTGCTGGAG-3′ (forward) and 5′-CTCAGCACCCTCACTAGTAA-3′ (reverse) for CsHA9; 5′-GACATAATCAAGTTTGCAATCAGATA-3′ (forward) and 5′-TTCTGTATAAGTTGTGCGGT-3′ (reverse) for CsHA10; and 5′-CAACAGGTGATATTGGATTATGATTATAC-3′ (forward) and 5′-GCCAGCTCATCCTCATATAAG-3′ (reverse) for TIP41-like protein. The following amplifications conditions were applied: 30 s at 95 °C; 45 cycles of 10 s at 95 °C, 10 s at 58 °C, and 12 s at 72°C, and a final melting for 15 s at 65°C.
For Western blot analysis, 10 μg of PM protein was incubated in SDS buffer containing 2% (w/v) SDS, 80 mM dithiothreitol, 40% (w/v) glycerol, 5 mM PMSF, 10 mM Tris/HCl (pH 6.8), 1 mM EDTA, and 0.05% (w/v) bromophenol blue for 30 min at room temperature and separated by 7.5% SDS-PAGE (Laemmli, 1970 (link)). After 1 h of electrophoresis at 25 mA, the proteins were electrotransferred (60 V, 200 mA) for 1.5 h to nitrocellulose using an SV10-EB10 blotting apparatus (Sigma-Aldrich). The transfer buffer contained 25 mM Tris/HCl (pH 8.3), 150 mM glycine, and 10% (v/v) methanol.
To identify the PM H⁺-ATPase, the blots were incubated overnight (8 °C) with monoclonal antibody against PM H⁺-ATPase (46E5B11D, kindly provided by W. Michalke, Universität Freiburg, Germany). The antiserum was diluted 2000-fold. After repeated washing, the nitrocellulose membrane was incubated at room temperature for 1 h with 1:4000-diluted secondary antibody (anti-mouse, conjugated to horseradish peroxidase (HRP); Sigma-Aldrich) and visualized by staining with 3,3′-diaminobenzidine (DAB).
Phosphorylation of the PM H⁺-ATPase was detected with a rabbit polyclonal anti-phosphothreonine antibody (Abcam) used at a concentration of 2 μg/ml after overnight incubation (8 °C). The membranes were rinsed and incubated for 1 h at room temperature with 10 000-fold secondary antibody conjugated to HRP (polyclonal goat anti-rabbit IgG; Abcam). The results were visualized by staining with DAB.
Detection of 14-3-3 protein was performed with a rabbit polyclonal anti-14-3-3 antibody (Abcam). The antiserum was diluted 1000-fold. After overnight incubation (8 °C), the membranes were rinsed and incubated for 1 h at room temperature with 10 000-fold secondary antibody conjugated to HRP (polyclonal goat anti-rabbit IgG; Abcam). The results were visualized by staining with DAB.
Detection of HSPs was performed with rabbit polyclonal anti-HSP17.6, anti-HSP17.7, anti-HSP70, and anti-HSP101 (Agrisera). The antisera were diluted 500-, 1000-, 6000-, and 500-fold, respectively. After overnight incubation (8 °C), the membranes were rinsed and incubated for 1 h at room temperature with 6000-fold secondary antibody conjugated to HRP (polyclonal goat anti-rabbit IgG; Abcam). The results were visualized by staining with DAB.
The activity of PM oxidoreductase was assayed according to the method of Kłobus (1995) . Reduction of ferricyanide by NADH in PM vesicles was measured spectrophotometrically as the change in A₄₂₀. The assay medium contained PM vesicles (about 50 μg of protein), 25 mM BTP-Mes (pH 7.5), 250 mM sorbitol, 50 mM KCl, 3.75 mM MgSO₄, 0.5 mM NADH, 0.02% Triton X-100, and 0.5 mM K₃Fe(CN)_6.To measure H₂O₂ levels, 1 g of cucumber root was ground with a mortar and pestle in liquid nitrogen. Next, 3 ml of 50 mM Mops (pH 7.2) was added. Samples were centrifuged at 10 000 g for 10 min. The supernatant was used for measurement of H₂O₂. The reaction mixture contained 50 mM Mops, 0.2 μg/l of pyranine, 30 U/ml of peroxidase (VI-A; Sigma), and supernatant. The H₂O₂ level was determined fluorometrically (excitation at 405 nm and emission at 510 nm) using a TD-20/20 Fluorometer (Turner Designs).
Catalase (EC 1.11.1.6) activity was determined as described by Aebi (1984) (link). The decomposition of H₂O₂ was followed by measuring the decrease in A₂₄₀ for 150 s and was calculated per 60 s. The reaction mixture consisted of 50 mM phosphate buffer (pH 7.0), plant extract, and 10 mM H₂O₂. One unit of catalase is defined as the amount of enzyme that breaks down 1 μmol of H₂O₂/min.
Ascorbate peroxidase (APX) activity was determined in a mixture containing 100 mM potassium phosphate (pH 7.0), 0.5 mM ascorbate, 0.2 mM H₂O₂, and enzyme extract (Chen and Asada, 1989 ). Oxidation of ascorbate was followed by measuring the decrease in A₂₉₀. The conversion was assumed as the molar absorption coefficient value of 2.8 mM⁻¹ cm⁻¹.
Protein was measured according to the method of Bradford (1976) (link) in the presence of 0.02% Triton X-100 with BSA as the standard.
For each of at least three independent protein and RNA extractions, measurements of enzyme activity and gene expression were obtained in triplicate and the means ±SD of these values are presented in the figures. The quantitative PCR data were analysed by the ΔΔC_T method using LightCycler Software 4.1 (Roche).