SE142:/S1/M2

Sample Set Information

ID	TSE1243
Title	Physiological roles of the beta-substituted alanine synthase gene family in Arabidopsis.
Description	The beta-substituted alanine (Ala) synthase (Bsas) family in the large superfamily of pyridoxal 5'-phosphate-dependent enzymes comprises cysteine (Cys) synthase (CSase) [O-acetyl-serine (thiol) lyase] and beta-cyano-Ala synthase (CASase) in plants. Nine genomic sequences encode putative Bsas proteins in Arabidopsis thaliana. The physiological roles of these Bsas isoforms in vivo were investigated by the characterization of T-DNA insertion mutants. Analyses of gene expression, activities of CSase and CASase, and levels of Cys and glutathione in the bsas mutants indicated that cytosolic Bsas1;1, plastidic Bsas2;1, and mitochondrial Bsas2;2 play major roles in Cys biosynthesis. Cytosolic Bsas1;1 has the most dominant contribution both in leaf and root, and mitochondrial Bsas2;2 plays a significant role in root. Mitochondrial Bsas3;1 is a genuine CASase. Nontargeted metabolome analyses of knockout mutants were carried out by a combination of gas chromatography time-of-flight mass spectrometry and capillary electrophoresis time-of-flight mass spectrometry. The level of gamma-glutamyl-beta-cyano-Ala decreased in the mutant bsas3;1, indicating the crucial role of Bsas3;1 in beta-cyano-Ala metabolism in vivo.
Authors	Watanabe M, Kusano M, Oikawa A, Fukushima A, Noji M, Saito K.
Reference	Plant Physiol. 2008 Jan;146(1):310-20. Epub 2007 Nov 16.
Comment

PubMed

Sample Information

ID	S1
Title	Plant Materials and Growth Conditions
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy 3702
Compound - ID
Compound - Source
Preparation	Arabidopsis (Arabidopsis thaliana ecotype Col-0) plants were used as the wild type in this study.
Sample Preparation Details ID	SS1
Comment

Sample Preparation Details Information

ID	SS1
Title	Sample Preparation
Description	Plants were cultured on germination medium (GM)-agar medium containing 1% Suc (Valvekens et al., 1988) in a growth chamber at 22°C under 16-h-light (approximately 2,500 lux)/8-h-dark cycles for 2 weeks. The leaves and roots of the plants were harvested, immediately frozen with liquid nitrogen, and stored at −80°C until use. Identical plant materials were analyzed for their gene expression, enzyme activities, and metabolite profiles.
Comment_of_details

Analytical Method Information

ID	M2
Title	CE-TOF/MS
Method Details ID	MS2
Sample Amount
Comment

Analytical Method Details Information

ID	MS2
Title	CE-TOF/MS
Instrument	CE:Agilent CE capillary electrophoresis system (Agilent Technologies) TOF-MS:Agilent G3250AA LC/MSD TOF system (Agilent Technologies) CE-MS:Agilent G1603A
Instrument Type
Ionization	ESI
Ion Mode	Positive
Description	Sixteen plants were planted on a single plate separated into fourths to minimize the differences in growth conditions. Four wild-type plants were planted on one-fourth, and four bsas mutant plants were planted on each of the remaining fourths. Five plates were replicated for each bsas mutant. Each sample was extracted with a concentration of 25 mg fresh weight of tissues per microliter of the extraction medium (methanol:chloroform:water [3:1:1; v/v/v]) by using a Retsh mixer mill MM 310 at a frequency of 30 Hz−1 for 3 min at 4°C. After centrifugation for 5 min at 15,100g, 400 μL of the supernatant of each plate were put together in accordance with each section of fourths. Four hundred microliters of the 2-mL supernatant were used for GC-TOF/MS analysis, and another 400 μL were used for CE-TOF/MS analysis. Analysis of metabolites by CE-TOF/MS was performed using an Agilent CE capillary electrophoresis system (Agilent Technologies), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies), an Agilent 1100 series binary HPLC pump, and the Agilent G1603A CE-MS adapter and Agilent G1607A CE-ESI-MS sprayer kit. Agilent G2201AA ChemStation software for CE and Analyst QS software for TOF/MS were used. For cationic compounds, separations were carried out using a fused silica capillary (50 μm i.d. × 100 cm total length) filled with 1 m formic acid as the electrolyte. The sample solutions were injected at 50 mbar for 15 s (15 nL). Prior to each run, the capillary was flushed with electrolyte for 5 min. The applied voltage was set at 30 kV. The capillary temperature was maintained at 20°C, and the sample tray was cooled below 4°C. Fifty percent (v/v) methanol-water containing 0.5 μm reserpine was delivered as the sheath liquid at 10 μL min−1. ESI-TOF/MS was conducted in the positive ion mode and the capillary voltage was set at 4 kV. A flow rate of heated dry nitrogen gas (heater temperature 300°C) was maintained at 10 psig. In TOF/MS, the fragmentor, skimmer, and Oct RFV voltage were set at 110, 50, and 160 V, respectively. In acquiring a fragment ion mass spectrum, the fragmentor voltage was increased to 210 V. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. The methanol dimer ion ([2M + H]+; m/z 65.0597) and reserpine ([M + H]+; m/z 609.2806) provided the lock mass for exact mass measurements. Exact mass data were acquired at a rate of 1.5 cycles s−1 over a 50 to 1,000 m/z range. Analysis of anionic compounds and nucleotides was carried out as described previously (Soga et al., 2002a, 2002b).
Comment_of_details