Plant material, growth conditions and treatments
Alfalfa (Medicago sativa cv. Aragon) seedlings were surface sterilized for 5 min in 5% (v/v) commercial bleach. After rinsing several times with sterile water, seeds were soaked overnight at 4ºC and germinated on 1.5% (w/v) agar in square Petri dishes (10 x 10 mm), in complete darkness for 48 h at 28ºC. Homogeneous selected seedlings were transferred to a semi-hydroponic system using a perlite inert substrate in plastic trays submerged in two modified Hoagland nutrient solutions (Ortega-Villasante et al. 2005): One of them with low NO3− concentration (LN; 2 mM) (macronutrients [mM]: 1.0 KH2PO4, 0.5 MgSO4, 0.1 NaCl, 0.9 Ca(NO3)2, 0.63 KNO3, 0.47 Mg(NO3)2) and the other one with high NO3− concentration (HN; 12 mM) (macronutrients [mM]: 1.0 KH2PO4, 0.5 MgSO4, 0.1 NaCl, 5.4 Ca(NO3)2, 3.8 KNO3, 2.8 Mg(NO3)2); both supplemented with micronutrients [µM]: 45.0 Fe (EDDHA), 18.0 MnSO4, 3.0 ZnSO4, 6.0 CuSO4, 23.5 H3BO3, 2.0 Mo7O24(NH4)6). The plants grew for 12 days in a controlled environment chamber (16 h light (lamps of 120 Wm− 2)/8 h darkness) at 25/18ºC respectively and relative humidity, 75%. 0, 6 and 30 µM Hg was then supplied as HgCl2, and plants were collected after 7 days. Samples were thoroughly washed in deionized H2O, fresh weight measured and stored at -80ºC until analysis.
Mercury analysis
Solid samples of roots and shoots were air dried and ground with mortar and pestle. Dried plant material (100 mg) was acid digested in 2 mL of the digestion mixture (HNO3:H2O2,:H2O, 0.6:0.4:1 v:v) in an autoclave (Presoclave-75 Selecta, Barcelona, Spain) at 120ºC and 1.5 atm for 30 min (Ortega-Villasante et al. 2007). Mercury concentration was measured by atomic absorption spectrophotometry using the Advanced Mercury Analyser 254 Leco (St. Joseph, Michigan, MI, USA) with a detection limit of 0.5 µg kg− 1. Certified Reference Materials (CRM) were used to determine the accuracy of the measurements and validation.
Nitrogen in plants
The nitrogen (N) in plant tissue was determined by Kjeldahl digestion, performed in a wet digestor system B-440 (Buchi, Switzerland). Dry plant material was ground to powder using a mortar and pestle, and 0.05 g was transferred to the digestion tubes together with 10 ml of 98% H2SO4 (v/v) and 10 g of K2SO4. The solution was heated at 410 ºC for 1.5 h, and cooled for 30 min. NH4+ was distilled after the addition of 25 ml of 32% NaOH (v/w) in a K-355 distillation unit (Buchi, Switzerland). The concentration of NH3 released in the resulting alkaline mixture was calculated by back titration of 2% H3BO3 (v/v) buffer adjusted to pH 4.65 with 0.02 M HCl, following the specifications of a KF Titrino Plus 870 equipment (Metrohm, Switzerland).
Lipid peroxidation and chlorophyll analysis
Lipid peroxidation was estimated by measuring the concentration of the by-product malondialdehyde, which reacts with thiobarbituric acid. The resulting chromophore absorbs at 535 nm, and the concentration was calculated directly from the extinction coefficient of 1.56 x 105 M− 1 cm− 1 (Rellán-Álvarez et al. 2006). Ground frozen tissue (0.1 g) was transferred to a screw-capped 1.5 ml Eppendorf tube, and homogenized in 1 ml of TCA–TBA–HCl reagent (15% (w/v) trichloroacetic acid (TCA), 0.37% (w/v) 2-thiobarbituric acid (TBA), 0.25 M HCl, and 0.01% buthylated hydroxytoluene). Samples were incubated at 90 ºC for 30 min, chilled in ice, and centrifuged at 12000 g for 10 min. Absorbance was measured in a UV-2401 PC spectrophotometer (Shimadzu, Tokyo, Japan). For chlorophyll (Chl) determination, 0.05 g of frozen leaves were homogenized with 10 ml 80% (v/v) acetone using a mortar and pestle. Homogenates were filtered through a paper filter and absorbance was measured in a UV-2401 PC spectrophotometer (Shimadzu) at 645 and 663 nm. Total chlorophyll concentration was calculated according to the procedure of Porra (2002).
Analysis of biothiols
0.1 g of frozen tissue was ground with 300 µl of 0.25 N HCl, the homogenate centrifuged for 15 min at 12000 g and 4ºC, and the clear supernatant was transferred to a boron-silica glass injection vial. 100 µl were injected in a Mediterranea Sea18 column (5 µm, 250 x 4.6 mm; Teknokroma, Spain), and biothiols were eluted using an Agilent 1200 HPLC system (Santa Clara, CA, USA) with the elution program and detection procedure with Ellman´s reagent (5,5-dithio-bis (2 nitrobenzoic acid) at 412 nm absorbance as described by Ortega-Villasante et al. (2005). Biothiol concentration was calculated relative to the acetyl cysteine (N-AcCys) internal standard (25 nmol). Several biothiol standards were prepared in analytical-grade type I water (Milli-Q Synthesis, Millipore), diluted to 0.1 mM and injected alone or as internal standards for retention time peak identification, of the following compounds: GSH, homoglutathione (hGSH), cysteine (Cys), homophytochelatin-2 (γ-(Glu-Cys)2-Ala; hPC2), and hompphytochelatin-3 (γ-(Glu-Cys)3-Ala; hPC3).
Determination of nitrate reductase activity in vitro
The in vitro nitrate reductase (NR) activity was analysed following the procedure described by Ramón et al. (1989). Intact frozen tissue (0.5 g) was homogenised in 1 ml or 0.5 ml enzymatic Extraction Solution respectively for shoot and root, freshly prepared by mixing 10 ml extraction buffer (30 mM 3-(N-morpholino) propanesulfonic acid (MOPS) at pH 7.5, 5 mM Na2-EDTA, 10 mM DTT, 10 mM ascorbic acid, 0.6% PVP, 10 µl 100 mM PMSF) and 1 ml protease inhibitors cocktail Sigma-Aldrich (P2714; St. Louis, MO, USA). After centrifugation (14000 x g) for 15 min at 4ºC, the supernatant was kept in ice until NR activity assay, measured by adding 0.1 ml of enzymatic extract, 0.1 ml 100 mM KNO3, 0.5 ml reaction buffer (100 mM KH2PO4/K2HPO4, 1 mM EDTA, pH 7.5) and 0.1 ml of 1 mg ml− 1 NADH. Formed NO2− was analysed after 15 min incubation at 28ºC, and the reaction was stopped by the addition of 2 ml of freshly prepared colorimetric reagent (1% (v/v) sulphanilamide in 3 M HCl, and 0.02% (w/v) N-(1-naphthyl) ethylenediamide dihydrochloride mixed in a 1:1 ratio). Samples were centrifuged at 10000 g for 15 min, and absorbance read at 540 nm using a UV-2401 PC spectrophotometer (Shimadzu).
Glutathione reductase and ascorbate peroxidase
Glutathione reductase (GR) and ascorbate peroxidise (APX) activities were determined in gel after separation of protein extracts by non-denaturing electrophoresis in 10% polyacrylamide gels. Extracts were prepared from 0.5 g of intact frozen samples in 1 ml enzymatic Extraction Solution (see above). After centrifugation (14000 g) for 15 min at 4ºC, the supernatant was stored as single use 100–200 µl aliquots at 80ºC. Protein concentration in the extracts was preliminarily determined with the BioRad Protein Assay reagent (BioRad, Hercules, CA, USA), and the final loading for activity staining was adjusted after denaturing gel electrophoresis and Coomassie-blue staining (Laemmli 1970). Protein loading for GR and APX analysis was 15 µg and 5 µg of shoot and root extracts, respectively. Gel slabs were incubated in GR staining solution (250 mM Tris-HCl buffer at pH 7.5, supplemented with 0.2 mg ml− 1 thizolyl blue tetrazolium bromide, 0.2 mg ml− 1 2,6-dichlorophenol indophenol, 0.5 mM NADPH and 3.5 mM oxidised glutathione (Sobrino-Plata et al. 2009). APX was detected as described by Jiménez et al. (1998). Gel slabs were incubated for 20 min with 2 mM ascorbate and 2 mM H2O2 in 50 mM Na-phosphate buffer at pH 7.0, and APX were revealed after incubation with 0.5 mM nitroblue tetrazolium (NBT) and 10 mM TEMED in 50 mM phosphate buffered at pH 7.8.
Protein immunodetection
Proteins of interest were immunodetected after denaturing gel electrophoresis and Western-blotting (Laemmli 1970), using α-cytosolic APX (AS06180), α-GR (AS06181), α-NR (AS08310), α-NTR1.1 (AS122611), and α-NTR2.1 (AS122612) (Agrisera, Vännäs, Sweden). After electrophoresis (20 µg total protein), proteins were blotted onto a nitrocellulose membrane (BioTraceNT Pall Corporation, East Hills, NY, USA) using a semi-dry procedure (Trans Blot SD Semi-Dry Electrophoretic Transfer Cell, BioRad), after incubation in transfer buffer (48 mM Tris-HCl, 39 mM glycine, 1.3 mM SDS, and 20% methanol at pH 8.3). Membranes were blocked with 1% bovine serum albumin in Tris-buffer-saline (TBS), and incubated overnight at 4ºC with the primary antibodies diluted 1/1000. Then, membranes were incubated with the secondary antibody (α-rabbit IgG goat:horseradish peroxidase, Ref. A00160, GenScript, Piscataway, NJ, USA) diluted 1/10000 for 2 h. Proteins were detected by incubating with LumiSensor™ Chemiluminescent HRP Substrate Kit (Ref. L00221V300, GenScript).
Statistical analysis
Results were mean of at least three independent replicates ± standard deviation, with significant differences between treatments at p < 0.05. ANOVA statistical analysis was performed with post-hoc Duncan test using SPSS 17.0 (SPSS Inc., Chicago, USA). For image analysis of in gel enzymatic activity and Western-blot immunodetection, images were obtained with a ChemiDoc™ XRS + System (BioRad). Band intensity were measured and relative fold-change to control samples was calculated. Only relevant differences of gels representative of three independent assays are shown.