SE46:/S01/M01/D01

Sample Set Information

ID	SE46
Title	Unbiased characterization of genotype-dependent metabolic regulations by metabolomic approach in Arabidopsis thaliana
Description	Metabolites are not only the catalytic products of enzymatic reactions but also the active regulators or the ultimate phenotype of metabolic homeostasis in highly complex cellular processes. The modes of regulation at the metabolome level can be revealed by metabolic networks. We investigated the metabolic network between wild-type and 2 mutant (methionine-over accumulation 1 [mto1] and transparent testa4 [tt4]) plants regarding the alteration of metabolite accumulation in Arabidopsis thaliana. In the GC-TOF/MS analysis, we acquired quantitative information regarding over 170 metabolites, which has been analyzed by a novel score (ZMC, z-score of metabolite correlation) describing a characteristic metabolite in terms of correlation. Although the 2 mutants revealed no apparent morphological abnormalities, the overall correlation values in mto1 were much lower than those of the wild-type and tt4 plants, indicating the loss of overall network stability due to the uncontrolled accumulation of methionine. In the tt4 mutant, a new correlation between malate and sinapate was observed although the levels of malate, sinapate, and sinapoylmalate remain unchanged, suggesting an adaptive reconfiguration of the network. Gene-expression correlations presumably responsible for these metabolic networks were determined using the metabolite correlations as clues. Two Arabidopsis mutants, mto1 and tt4, exhibited the following changes in entire metabolome networks: the overall loss of metabolic stability (mto1) or the generation of a metabolic network of a backup pathway for the lost physiological functions (tt4). The expansion of metabolite correlation to gene-expression correlation provides detailed insights into the systemic understanding of the plant cellular process regarding metabolome and transcriptome.
Authors	Miyako Kusano, Atsushi Fukushima, Masanori Arita, Par Jonsson, Thomas Moritz, Makoto Kobayashi, Naomi Hayashi, Takayuki Tohge, Kazuki Saito, RIKEN PSC
Reference	Kusano, Fukushima et al. (2007) BMC Syst Biol 1:53 (PMID: 18028551)
Comment

PubMed

Related data are deposited in MetaboLights.

Related data are deposited in MetabolomeExpress.

Sample Information

ID	S01
Title	Arabidopsis thaliana
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	Wild-type A.thaliana plants accession Columbia (Col-0) and the mutants mto1 (Inaba et al. 1994) and tt4 (Shikazono et al. 2003) of Col-0 background were obtained from Dr. Naito, Hokkaido University, and Dr. Kitamura, Japan Atomic Energy Research Institute, respectively. The sterilized seeds were stratified at 5°C for 2 d, and were successively sown on Murashige and Skoog (MS) medium containing 1% sucrose. Plants were cultivated in controlled growth chambers at 22°C in 16-h light and 8-h dark conditions for 18 d. The aerial regions were harvested with 20 different biological replicates, 6 h after the onset of the bright phase.
Sample Preparation Details ID
Comment

Inaba et al. 1994 (PubMed)

Shikazono et al. 2003 (PubMed)

Analytical Method Information

ID	M01
Title	GC-TOF/MS
Method Details ID	MS01
Sample Amount	1 μL
Comment

Analytical Method Details Information

ID	MS01
Title	GC-TOF/MS
Instrument	GC Agilent 6890N gas chromatograph / MS Pegasus III TOF mass spectrometer
Instrument Type
Ionization	EI
Ion Mode	positive
Description	Among these harvested plants, the plants that had fresh weight over 13 mg were used for GC-TOF/MS analysis (WT, n = 17; mto1, n = 13; and tt4, n = 20). For liquid chromatography-quadrupole-time-of-flight/mass spectrometry (LC-Q-TOF/MS) analysis, 3 biological replicates were prepared. All the plant materials were frozen immediately in liquid nitrogen to quench the enzymatic activity. Extraction and sample preparation for GC-TOF/MS analysis Each sample was extracted with a concentration of 25 mg fresh weight of tissues per μl extraction medium (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4°C. Each isotope was adjusted to a final concentration of 22.5 ng per μl injection [49,50]. After centrifugation for 5 min at 15,100 × g, 400 μl of the supernatant was used for further analysis. The extracts were evaporated to dryness in a Savant SPD2010 SpeedVac Concentrator (Thermo Electron Corporation, Waltham, MA, USA). For methoximation, 20 μl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 30 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 20 μl of MSTFA with 1% TMCS at 37°C with shaking. Twenty μl of n-heptane was added following silylation. All the derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen. The analysis of metabolites by GC-TOF/MS was performed as described previously.
Comment_of_details

PubMed

Data Analysis Information

ID	D01
Title	Data processing (GC-MS)
Data Analysis Details ID	DS01
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS01
Title	Data processing (GC-MS)
Description	Processing of GC-TOF/MS data Nonprocessed MS data from GC-TOF/MS analysis were exported in NetCDF format to MATLAB 6.5 (Mathworks, Natick, MA, USA), where all data-pretreatment procedures, such as smoothing, alignment, time-window setting, and H-MCR, were carried out (Jonsson et al. 2006). The resolved MS spectra were matched against reference mass spectra using the NIST mass spectral search program for the NIST/EPA/NIH mass spectral library (version 2.0) and our custom software for peak annotation written in JAVA. Peaks were identified or annotated based on retention indices (RIs) and the reference mass spectra comparison to the Golm Metabolome Database (GMD) (Kopka et al. 2005) and our in-house spectral library. The metabolites were identified by comparison with RIs from the library databases (GMD and our own library) and with those of authentic standards, and the metabolites were defined as annotated metabolites on comparison with mass spectra and RIs from these two libraries. The amount of S-adenosyl-methionine was calculated by the sum of the mass numbers at m/z 188 and 236 using Leco ChromaTOF software version 2.32 (LECO, St. Joseph, MI, USA) since this compound was not adequately detected by H-MCR.
Comment_of_details

Jonsson et al. 2006 (PubMed)

Kopka et al. 2005 (PubMed)