SE46:/S01/M02

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	M02
Title	LC-q-TOF-MS
Method Details ID	MS02
Sample Amount
Comment

Analytical Method Details Information

ID	MS02
Title	LC-q-TOF-MS
Instrument	LC, Waters Acquity UPLC system; MS, Q-ToF Premier mass spectrometer
Instrument Type	UPLC-QTOF-MS
Ionization	ESI
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. LC-Q-TOF/MS analysis Frozen leaves were homogenized in 5-μl extraction solvent (methanol/H2O [4:1 v/v]) per mg fresh weight of tissues by using a mixer mill at a frequency of 20 Hz for 3 min at 4°C. After centrifugation at 12,000 × g, the cell debris was discarded, and the extracts were centrifuged again. These supernatants were immediately used for flavonoid analysis. For flavonoid profiling, Waters Acquity UPLC™ system (Waters Co., Massachusetts, USA) fitted with a Q-ToF Premier mass spectrometer (Micromass MS Technologies, Manchester, UK) was used. Ultra-performance liquid chromatography (UPLC) was carried out on a UPLC™ BEH C18 column (100-mm length × 2.1-mm inner diameter, 1.7-μm particles, Waters Co.) at a flow rate of 0.5 ml/min at 35°C. The elution gradient comprised solvent A (0.1% trifluoroacetic acid in H2O) and solvent B (0.1% trifluoroacetic acid in acetonitrile) and the elution profile – 0 min, 100% A; 5 min, 11% B; 20 min, 13% B; 24 min, 100% B – using linear gradients in between the time points. A photodiode array (PDA) detector was used for the detection of UV-visible absorption in the range of 210–500 nm. The TOF mass analyzer was used for the detection of flavonoid glycosides [M+H]+ and fragment ions peak in a positive ion mode scan. The desolvation temperature was 450°C with a nitrogen gas flow rate of 600 l/h, capillary spray at 3.2 kV, source temperature at 150°C, and cone voltage at 35 V. The peaks in the plant extracts were identified based on retention times, UV visible absorption spectra, and mass fragmentation by tandem MS analysis as reported (Yonekura-Sakakibara et al. 2007). The amounts of each kaempferol glycoside and sinapoyl derivative were calculated using kaempferol (at 340 nm) or sinapic acid (at 340 nm) as a standard, respectively.
Comment_of_details

Yonekura-Sakakibara et al. 2007 (PubMed)