Brassica juncea cv. Arrid was obtained from Derek Potts of Viterra, Saskatoon, SK. B. juncea cv. Vulcan and Sinapis alba seed were obtained from Kevin Falk, Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK. Seed was produced on plots near Saskatoon in 2006.
Modifying the AITC ground seed assay
The method to extract AITC from ground seed and determine its concentration is essentially that of Raquet [17]. Glucosinolates in ground seed are converted to isothiocyanates by continuously stirring 5 g of seed in 100 mL of water at 37°C for 2 h. AITC in ground seed is then recovered by adding 20 mL of 95% ethanol and a few boiling chips. Sixty millilitres of the distillate was collected in a flask containing 10 mL of 33.5% ammonium hydroxide solution and 20 mL of 0.1 N silver nitrate was added. The final volume was adjusted to 100 mL with distilled water and incubated overnight in the dark at room temperature. The resulting black precipitate was removed by filtration with Whatman grade No. 4 filter paper (GE Health Care, Piscataway, NJ) and two titrations were performed, each using 50 mL of this filtrate. The filtrate (50 mL) was acidified with 5 mL of concentrated nitric acid (analytical grade, Sigma-Aldrich, Oakville, ON, Canada) and was titrated with 0.1N ammonium thiocyanate (analytical grade, Sigma-Aldrich) after adding 5 mL of 8% FeNH4(SO4)2.12H2O indicator (Sigma-Aldrich). Percent volatile oil was calculated by:
We examined the effects of varying the incubation time, solvent and temperature on AITC release by ground B. juncea seed. The effects of various durations (0-5 min at 30 s intervals and 5, 15, 30, 60 and 120 min), temperatures (7 to 97°C) and solvents (water, 0.02 N sodium hydroxide, 0.02 N hydrochloric acid, 0.02 N acetic acid) on percent volatile oil released were examined. Four replicates of each treatment were performed.
C. elegans culture and AITC treatment
C. elegans N2 strain was grown on 10% bacteriological agar (Sigma-Aldrich) layered with 1 mL of autoclaved 1% (w/v) Baker's yeast in sterile 10-mm diameter plates. Cultures were incubated in the dark at room temperature and sub-cultured to fresh plates every 15 days.
Two-week-old cultures of C. elegans were treated with 0-10 μM of commercially prepared AITC (Sigma-Aldrich) and/or 0.0-144.5 μg/mL B. juncea cv. Arrid ground seed and incubated in the dark at room temperature for 2 h. After treatment and incubation, cultures were centrifuged at 400 × g for 10 min at 4°C and the pellet was stored at -80°C pending further analysis.
RNA isolation and quantitative RT-PCR analysis
Total RNA was extracted from C. elegans lysates stored at -80°C using an RNeasy Mini kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacturer's instructions. The integrity of RNA was confirmed by agarose gel electrophoresis and RNA was quantified using a Nano drop spectrophotometer (Thermo, Fisher Scientific, Ottawa, ON, Canada). Following DNase treatment, the mRNA was reverse transcribed at 42°C for 30 min using the QuantiTect Reverse Transcription kit (Qiagen Inc.) as per the manufacturer's instructions. This cDNA was used for quantitative real-time RT-PCR (qRT-PCR) analysis for the expression of HSP70A (GenBank Accession No. M18540) using the QuantiFast SYBR Green kit (Qiagen Inc.) as per the manufacturer's instructions. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH; GenBank Accession X04818) was used as the reference housekeeping gene. The reactions were performed using the following primer pairs: 5'-ATGAGTAAGCATAACGCTGTT-3' and 5'-ACAGTGTTATGTGGGTTCATG-3' for a 200 bp HSP70A fragment and 5'-AACCATGAGAAGTACGAC-3' and 5'-CTGTCTTCTGGGTTGCGG-3' for a 212 bp GAPDH fragment. A negative control reaction consisted of all the components of the reaction mixture except cDNA. Real-time PCR analysis was performed using a MX3005P LightCycler (Stratagene, La Jolla, CA, USA) and the following program: initial denaturation at 94°C for 5 min; 45 cycles of denaturation at 94°C for 15 s; annealing at 57°C for 30 s; and elongation at 68°C for 60 sec. Relative expression levels were calculated after correction for expression of GAPDH using MxPro software.
Quantification of HSP70 by an enzyme-linked immunosorbant assay (ELISA)
As HSP70A is a cytoplasmic protein known to be expressed in response to stress or toxicity, studies on the effects of AITC on C. elegans focused on expression of this protein. A 52 amino acid residue fragment at the C-terminal end of HSP70A (GenBank accession AAA28078) showed > 98% identity to the human HSP70 (GenBank accession NP_005337). A goat antibody raised against the human peptide of this peptide fragment was purchased from AbCam (Cambridge, MA, USA).
C. elegans samples stored at -80°C were ground to a fine powder under liquid nitrogen using a sterile mortar and pestle. Ground tissues were incubated in 200 μL of freshly prepared lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40; 1 mM phenylmethylsulfonyl fluoride; 5 μg/mL antipain; 5 μg/mL aprotinin; 5 μg/mL leupeptin; 7.5% polyvinylpolypyrrolidone) on ice for 30 min. The lysate was stored at -80°C until further use. Total protein was quantified using the Bradford dye-binding assay (Bio-Rad Laboratories, Hercules, CA, USA) as per the manufacturer's instructions.
Levels of induced HSP70 were measured in protein extracts from AITC-treated worms using a sandwich ELISA kit (Stressgen Biotechnologies, Ann Arbor, MI, USA) as per the manufacturer's instructions. Absorbance at 450 nm was measured using a NOVOstar microplate reader (BMG Labtech, Durham, NC, USA). The HSP70 concentrations of the samples were quantified by interpolating absorbance readings from the standard curve.