SE142:/S1/M1/D1

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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

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

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

ID	MS1
Title	GC-TOF MS
Instrument	GC:Agilent 6890N MS:LECO Pegasus 4D MS system
Instrument Type
Ionization	EI
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. The analysis of metabolites by GC-TOF/MS, including the derivatization step and the processing of MS data, was performed as described elsewhere (Kusano et al., 2007a, 2007b).
Comment_of_details

ID	D1
Title	Statistical data analysis
Data Analysis Details ID	DS1
Recommended decimal places of m/z
Comment

ID	DS1
Title	Statistical Data Analysis
Description	The obtained data matrix (observations: samples, variables: 337 peaks, including peaks with mass spectral tags [137 annotated peaks from GC-TOF/MS plus 200 annotated or stably appearing peaks from CE-TOF/MS analysis; Supplemental Tables S4 and S5]) was used for statistical analysis. For statistical multivariate analysis, principal component analysis was performed with SIMCA-P 11.0 software, using log10-transformed and autoscaled data (Umetrics AB). Simple comparisons of means of obtained peak areas were performed by Welch's t test. A difference of P < 0.05 was considered to be significant. In terms of minimizing the effect of zero substitution, we scaled all normalized peak areas by 10,000 and then added 1 uniformly.
Comment_of_details