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A 50–100 mg aliquot of the rosette leaves … A 50–100 mg aliquot of the rosette leaves (3 weeks after germination) and approximately 2 mg of the mature seeds [collected by using the seed spoon 200 (Bio Medical Science, Tokyo, Japan)] of wild-type and mutant lines were used for GSL analysis. The 2 ml sample tubes containing 5 mm zirconia beads were pre-frozen with liquid nitrogen. The leaves or seeds were collected in the tubes, immediately frozen with liquid nitrogen and stored at –80°C until use.<br />
The frozen samples were homogenized using a mixer mill MM 200 (Retsch, Haan, Germany) at 20 Hz for 5 min. After homogenization, 10 µl of extraction buffer (MeOH : H2O = 4 : 1, 0.1% formic acid, 20 µM sinigrin as an internal standard) per mg of tissue (fresh weight) was added for extraction of metabolites. After centrifugation (10,000×g at 4°C), 100 µl of the supernatant was transferred to a new tube and concentrated to dryness using a Speedvac (Thermo Fisher Scientific, Waltham, MA, USA) vacuum centrifuge. The residue was dissolved in 50 µl of H2O and filtered using Pall Nanosep Centrifuge Filters (0.45 µm, GHP) (Pall Life Sciences, Ann Arbor, MI, USA). The filtered solution was analyzed using a UPLC-ZQ-MS (Waters, Milford, MA, USA). The GSLs were separated and detected under the following conditions: ACQUITY UPLC HSS T3 column (2.1 mm × 50 mm, Waters) at 30°C, flow rate 0.38 ml min–1, solvent A (0.1% formic acid in H2O), solvent B (0.1% formic acid in MeCN), gradient pattern (0–0.1 min, 100% solvent A; 2 min, 8% solvent B; 3 min, 20% solvent B; 5.5 min, 100% solvent B; 6.5 min, 100% solvent B), electrospray ionization-MS negative ion mode, capillary voltage 3.0 kV, cone voltage 40 V, source temperature at 150°C, desolvation gas 600 l h–1 at 300°C. The GSL contents were calculated using sinigrin as a standard. e calculated using sinigrin as a standard.
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