Bradford reagent

Protein was extracted from both the treated and control B. napus seeds via trichloroacetic acid (TCA)/acetone precipitation as described by Damerval et al. (1986) (link) and Gan et al. (2010) (link) with minor modifications. Briefly, the seeds were ground in a precooled mortar in the presence of liquid nitrogen. The powders were precipitated with 10 volumes of precooled acetone (containing 10% TCA and 0.07% dithiothreitol (DTT)), and the mixtures were incubated at –20°C for 1 h. After centrifuging at 20 000 ×g for 30 min and removing the supernatant, the pellet of each sample was rinsed and incubated in ice-cold acetone (containing 0.07% DTT) for 1 h at –20°C. This step was repeated three times. Finally, each pellet was air-dried and resuspended in 500 μl of lysis buffer (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 1 M phenylmethanesulfonyl fluoride (PMSF), 50 mM DTT, 0.5% Triton X-100, and 0.5% ampholine) and then vortexed for 1 h at room temperature. After centrifugation at 20 000 ×g for 30 min, each supernatant was collected in a fresh tube, and the protein concentrations were quantified using Bradford reagent (Aidlab, China) according to the manufacturer’s instructions. The protein samples were stored at –80°C.

Glyoxalase I activity in total protein extracts from yeast was measured as described previously (Singla-Pareek et al., 2003 (link); Mustafiz et al., 2010 (link)). Each of the measurements was performed using three replicates. The enzymatic production of S-lactoylglutathione (extinction coefficient 3.37 mM^–1 cm^–1) was measured at 240 nm at 15 s intervals for 2 min. Specific activity of GLYI is expressed as μmol S-lactoylglutathione formed/hr/mg protein (Junaid et al., 2004 (link)). The yeast protein was extracted using Y-PER Yeast Protein Extraction Reagent (Thermo Scientific, USA). Bradford reagent (Aidlab, China) was used to determine the protein concentrations. ImageJ software was used to determine the relative expression levels of BnGLYI-2 and BnGLYI-3.
Superoxide dismutase (SOD) activity was assessed by monitoring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) according to the method of Beauchamp and Fridovich (1971) (link). One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the rate of NBT reduction at 560 nm (Beauchamp and Fridovich, 1971 (link)). SOD activity is expressed as units per milligram of protein.