Enzyme Activity Assays

The extraction and activity of the EPSPS enzyme was carried out following the methodology described by Dayan et al. (2015). Five grams of foliar tissue from the different Lolium spp. populations were ground with liquid N₂ to a fine powder. Samples were transferred to 50 mL Falcon tubes containing 25 mL of extraction buffer (100 mM MOPS, 5 mM EDTA, 10% glycerol, 50 mM KCl, and 0.5 mM benzamidine) with 70 μL of ß-mercaptoethanol (10 mM) and 1% in polyvinylpolypyrrolidone (PVPP) to extract the EPSPS. Samples were vortexed for 5 min, avoiding foaming, and then centrifuged (18,000 g, 30 min, 4°C). Supernatant was filtered using a cheesecloth in a cold beaker and then slowly added (NH₄)₂SO₄, was then slowly added while supernatant was under continuous stirring until a 45% solution of (NH₄)₂SO₄ (w/v) was obtained. After addition of (NH₄)₂SO₄ the sample was stirred for 30 min and then centrifuged (15,000 g, 30 min, 4°C). This step was repeated once more to obtain a 70% (NH₄)₂SO₄ (w/v) solution to precipitate the fraction that contained the EPSPS activity. Supernatants were discarded, and pellets were resuspended in 1–2 mL assay buffer (100 mM MOPS, 1 mM MgCl₂, 10% glycerol (v/v), 2 mM sodium molybdate, 200 mM NaF). Samples were dialysed using Slide-A-Lyzer dialysis cassettes (1000-MWC, Thermo Scientific, Meridian, IL, United States) overnight in 2 L of dialysis buffer (100 mM MOPS and 5 mM EDTA) at 4°C on a stir plate. The final pH of the buffers was adjusted to 7.0 with HCl or NaOH. The concentration of total protein soluble (TPS) was determined by Bradford assay (Bradford, 1976). Basal and enzyme EPSPS activities were determined in a continuous assay quantifying the inorganic phosphate (Pi) released from shikimate-3-phosphate with the EnzCheck phosphate assay kit (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. The glyphosate concentrations used were: 0, 0.1, 1, 10, 100 and 1,000 μM. The amount Pi released was measured for 10 min at 360 nm in a spectrophotometer (DU-640, Beckman Coulter Inc. Fullerton, CA, United States). The EPSPS activity was calculated by determining the amount of Pi (μmol) released in μg of TSP^–1 min^–1. EPSPS enzyme activity was expressed as percentage of enzyme activity in presence of glyphosate with respect to the control (basal activity without glyphosate). The experiment was repeated for all populations, and each glyphosate concentration had three technical replicates.

The in vitro ACCase enzyme activity was performed following the protocol described by De Prado et al. (2005). Six grams of fresh weight were taken from new leaves of the different Lolium spp. populations for the extraction step. The samples were ground using liquid N₂ and then added to 24 mL extraction buffer (0.1 M Hepes-KOH at pH 7.5, 0.5 M glycerol, 2 mM EDTA and 0.32 mM PMSF). After mixing for 3 min with a magnetic stirrer and then filtered using a cheesecloth, the samples were centrifuged (24,000 g, 30 min, 4°C). the supernatant was fractioned with (NH₄)₂SO₄ and centrifuged (12,000 g, 10 min at 4°C). The pellets were resuspended in 1 mL S400 buffer (0.1 M Tricine-KOH at pH 8.3, 0.5 M glycerol, 0.05 M KCl, 2 mM EDTA and 0.5 mM DTT). The homogenate was applied to a desalting column (PD-10 columns, Sephadex G-25 M, Amersham Biosciences AB, SE-751 84, Uppsala, Sweden) and eluted in 2 mL of S400 buffer. The specific protein concentrations were determined by Bradford assay (Bradford, 1976). The enzyme activity was measured through the ATP-dependent incorporation of NaH[¹⁴C]O₃ into [¹⁴C]malonyl-CoA. The reaction was conducted at 34°C in 7 mL scintillation vials with 0.1 M Tricine-KOH at pH 8.3, 0.5 M glycerol, 0.05 M KCl, 2 mM EDTA, 0.5 mM DTT, 1.5 mM MgCl₂, 15 mM NaH[¹⁴C]O₃ (1.22 MBq μmol^–1), 50 μL of enzyme extract, 5 mM acetyl-CoA in a final volume of 0.2 mL. The reaction was stopped after 5 min by adding 30 μL of HCl 4N. After the drying step, 0.5 mL of ethanol-water solution (1:1, v/v) was added to the vial, followed by 5 mL of scintillation cocktail (Ultima Gold, Perkin-Elmer, BV BioScience Packard). Radioactivity was determined by liquid scintillation spectrometry. One unit of ACCase activity was defined as 1 μmol malonyl CoA formed min^–1. The diclofop-acid concentrations were: 0, 0.1, 1, 10, 100, 1,000 and 10,000 μM. The experiment was repeated with three replicates for each herbicide concentration.

ALS activity in presence of different iodosulfuron concentrations was determined in vitro following the protocol described by Rojano-Delgado et al. (2015). Three grams of young leaf tissue were powdered using liquid N₂. Polyvinylpyrrolidone (0.5 g) was added to the fine powder as well as extraction buffer [1M K-phosphate buffer solution (pH 7.5), 10 mM sodium pyruvate, 5 mM MgCl₂, 50 mM thiamine pyrophosphate, 100 μM flavin adenine dinucleotide, 12 mM dithiothreitol and glycerol–water (1:9, v/v)] in a proportion of 1:2 tissue–buffer. Samples were agitated for 10 min at 4°C, filtered through a cheesecloth, and centrifuged (20,000 g for 20 min). The supernatant was immediately used for the enzyme assays. For the ALS enzyme activity, 90 μL of enzyme extract was used with 110 μL of freshly prepared assay buffer [0.08 M K-phosphate buffer solution (pH 7.5), 0.5 M sodium pyruvate, 0.1 M MgCl₂, 0.5 mM thiamine pyrophosphate and 1,000 μM flavin adenine dinucleotide]. The iodosulfuron concentrations assayed were: 0, 0.1, 1, 10, 50, 100, 500, 1,000 and 5,000 μM. Aliquots of 250 μL of a solution 0.04 M K₂HPO₄ at pH 7.0 were added. This mixture was incubated for 1 h at 37°C. Afterward, the reaction was stopped by adding 50 μL of H₂SO₄/water 1:10 (v/v). To decarboxylate acetolactate to acetoin, the tubes with the mixture were heated for 15 min at 60°C. A colored complex (λ 520 nm) formed after the addition of 250 μL of creatine (5 g L^–1 freshly prepared in water) and 250 μL of 1-naphthol (50 g L^–1 freshly prepared in 5 N NaOH) prior to incubation at 60°C for 15 min. Total protein content was determined by the Bradford method (Bradford, 1976), based on measurement of the absorbance at 595 nm of an acidic solution of Coomassie Brilliant Blue G-250 after binding to proteins. The experiment was repeated and with three replicates for each concentration of the herbicide.