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Sample Set Information

ID TSE1326
Title Omics-based approaches to methionine side chain elongation in Arabidopsis: characterization of the genes encoding methylthioalkylmalate isomerase and methylthioalkylmalate dehydrogenase.
Description Glucosinolates (GSLs) are secondary metabolites in Brassicaceae plants synthesized from amino acids. Methionine-derived GSLs (Met-GSLs) with diverse side chains of various lengths are the major GSLs in Arabidopsis. Methionine chain elongation enzymes are responsible for variations in chain length in Met-GSL biosynthesis. The genes encoding methionine chain elongation enzymes are considered to have been recruited from the leucine biosynthetic pathway in the course of evolution. Among them, the genes encoding methylthioalkylmalate synthases and aminotransferases have been identified; however, the remaining genes that encode methylthioalkylmalate isomerase (MAM-I) and methylthioalkylmalate dehydro-genase (MAM-D) remain to be identified. In a previous study based on transcriptome co-expression analysis, we identified candidate genes for the large subunit of MAM-I and MAM-D. In this study, we confirmed their predicted functions by targeted GSL analysis of the knockout mutants, and named the respective genes MAM-IL1/AtleuC1 and MAM-D1/AtIMD1. Metabolic profiling of the knockout mutants of methionine chain elongation enzymes, conducted by means of widely targeted metabolomics, implied that these enzymes have roles in controlling metabolism from methionine to primary and methionine-related secondary metabolites. As shown here, an omics-based approach is an efficient strategy for the functional elucidation of genes involved in metabolism.
Authors Sawada Y, Kuwahara A, Nagano M, Narisawa T, Sakata A, Saito K, Hirai MY.
Reference Plant Cell Physiol. 2009 Jul;50(7):1181-90. doi: 10.1093/pcp/pcp079. Epub 2009 Jun 3.

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Analytical Method Details Information

Title UPLC-ZQ-MS (GSL analysis)
Instrument UPLC-ZQ-MS (Waters)
Instrument Type
Ionization ESI
Ion Mode negative
Description 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.

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.


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