SE164:/S1/M1

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.
Comment

PubMed

Sample Information

ID	S1
Title	Arabidopsis thaliana
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	As knockout mutants of AtLeuC1 and AtIMD1, two allelic lines of the respective genes [SALK_029510 (atleuc1-1) and SALK_065789 (atleuc1-2) for AtLeuC1, and SALK_063423 (atimd1-1) and SALK_ 069991 (atimd1-2) for AtIMD1] were used. In atleuc1-1, atimd1-1 and atimd1-2, expression of the corresponding gene was almost completely repressed. In atleuc1-2, transcript levels for AtLeuC1 were reduced but not completely repressed (Supplementary Fig. S1B). Knockout lines of AtBCAT4 (SALK_013627, bcat4-1) have already been reported (Schuster et al. 2006). As MAM1, MAM3 and AtLeuD1 knockout lines, SALK_012677 (mam1), SALK_004536 (mam3) and SALK_048320 (atleud1-1) were used. Homozygous lines of the T-DNA insertion mutants were selected by genomic PCR according to the T-DNA Express: Arabidopsis iSect Tool manual (http://signal.salk.edu/). Wild-type Arabidopsis thaliana (accession Colombia) and mutants (T-DNA insertion lines) were grown in a pre-fabricated room-type chamber at 22°C and a 16 h photoperiod in soil (PRO-MIX BX, Premier Horticulture, Rivière-du-Loup, QC, Canada) for seed collection, or on agar-solidified 1/2 Murashige–Skoog medium containing 1% sucrose for GSL analysis and widely targeted metabolomics.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M1
Title	UPLC-ZQ-MS
Method Details ID	MS1
Sample Amount
Comment

Analytical Method Details Information

ID	MS1
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.
Comment_of_details