SE45:/S01/M01

Sample Set Information

ID	SE45
Title	Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis
Description	Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally, and improving gene annotation remains a major challenge in plant science. As metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation in the plant metabolism, it provides clues to the function(s) of genes of interest. We chose 50 Arabidopsis mutants, including a set of characterized and uncharacterized mutants, that resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry. To make the data set available as an efficient public functional genomics tool for hypothesis generation, we developed the Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO). It allows the evaluation of whether a mutation affects metabolism during normal plant growth and contains images of mutants, data on differences in metabolite accumulation, and interactive analysis tools. Nonprocessed data, including chromatograms, mass spectra, and experimental metadata, follow the guidelines set by the Metabolomics Standards Initiative and are freely downloadable. Proof-of-concept analysis suggests that MeKO is highly useful for the generation of hypotheses for genes of interest and for improving gene annotation. MeKO is publicly available at http://prime.psc.riken.jp/meko/.
Authors	Atsushi Fukushima, Miyako Kusano, Ramon Francisco Mejia, Mami Iwasa, Makoto Kobayashi, Naomi Hayashi, Akiko Watanabe-Takahashi, Tomoko Narisawa, Takayuki Tohge, Manhoi Hur, Eve Syrkin Wurtele, Basil J. Nikolau, Kazuki Saito, RIKEN CSRS
Reference	Fukushima et al. (2014) Plant Physiology 165:948-961 (PMID: 24828308)
Comment	The raw data files are available at DropMet web site in PRIMe database, MetaboLights (Salek et al., 2013) (accession no. MTBLS47), and The MetabolomeExpress (Carroll et al., 2010).

PubMed

MeKO

The raw data files are available at DROP Met web site in PRIMe database of RIKEN.

Related data are deposited in MetaboLights.

Related data are deposited in MetabolomeExpress.

Sample Information

ID	S01
Title	Arabidopsis (Aerial parts,ecotype: Col-0 or Col)
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	All mutants were purchased from the Arabidopsis Biological Resource Center (ABRC, https://abrc.osu.edu/).
Sample Preparation Details ID	SS01
Comment

The mutants used in this study

Line	AGI or ID	Background	Germplasm / StockID
aat2-2	AT5G19550	Col	CS3971
aba1-5	AT5G67030	Col	CS155
aba1-6	AT5G67030	Col-0	CS3772
aba2-1	AT1G52340	Col	CS156
aba2-3	AT1G52340	Col	CS156
aba3-1	AT1G16540	Col	CS157
amt1-1	AT4G13510	Col	CS6168
cim10	CIM10	Col	CS6574
cim11	CIM11	Col	CS6575
cim13	CIM13	Col	CS6576
cim14	CIM14	Col	CS6577
cim6	CIM6	Col	CS6571
cim7	CIM7	Col	CS6572
cim9	CIM9	Col	CS6573
cla-1S	AT4G15560	Col	CS16003
cob-2	AT5G60920	Col	CS8542
eto1-1	AT3G51770	Col	CS3072
eto3	AT3G49700	Col	CS8060
fad5-1	AT3G15850	Col	CS206
fad6-1	AT4G30950	Col	CS207
fah1-2	AT4G36220	Col	CS6172
fur1-1	AT4G05120	Col	CS3729
fus6-1S	AT3G61140	Col	CS16057
gsr1-1	GSR1	Col	CS6391
ixr1-1	AT5G05170	Col	CS6201
ixr1-2	AT5G05170	Col	CS6202
mur1-1	AT3G51160	Col	CS6243
mur1-2	AT3G51160	Col	CS6244
mur2-1	AT2G03220	Col	CS8565
mur3-2	AT2G20370	Col	CS8567
mur4-2	AT1G30620	Col	CS8569
mur5-1	MUR5	Col	CS8572
mur6-1	MUR6	Col	CS8573
mur7-1	MUR7	Col	CS8574
mur8-1	MUR8	Col	CS8575
mur9-1	MUR9	Col	CS8576
mur11-1	MUR11	Col	CS8579
pac-1S	AT2G48120	Col	CS16058
pad2-1	AT4G23100	Col	CS3804
pad3-1	AT3G26830	Col	CS3805
pad4-1	AT3G52430	Col	CS3806
pap1-D	AT1G56650	Col	CS3884
phyB-9	AT2G18790	Col	CS6217
prc1-1	AT5G64740	Col	CS297
rsw1-1	AT4G32410	Col	CS6554
rsw2-1	AT5G49720	Col	CS6555
rsw3-1	AT5G63840	Col	CS6556
sng1-1	AT2G22990	Col	CS3737
tbr-1	AT5G06700	Col	CS3741
vtc1-1	AT2G39770	Col-0	CS8326

Sample Preparation Details Information

ID	SS01
Title	Sample Preparation
Description	<Growth condition> Sterilized seeds were stratified at 5°C for 2 days and then sown on Murashige and Skoog medium containing 1% sucrose. Seedlings of Arabidopsis Col-0 and the mutants were cultivated in controlled growth chambers at 22°C under 16-h light and 8-h dark conditions for 18 days (light strength approximately 80 photosynthetic photon flux). <Sampling and sampling date> The leaves were harvested September- through December 2008 <Metabolism quenching method> The aerial part of each sample was harvested. All plant materials were frozen immediately in liquid nitrogen to quench enzymatic activity. The all raw data are also available in MetaboLights (Salek et al., 2013) (accession no. MTBLS47), and The MetabolomeExpress (Carroll et al., 2010).
Comment_of_details

Related data are deposited in MetaboLights.

Related data are deposited in MetabolomeExpress.

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 IV TOF mass spectrometer
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	<Sample processing and extraction> Each sample was extracted at a concentration of 5 mg flesh weight (FW) of tissues per ml extraction medium (methanol / chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds: • [2H4]-succinic acid • [13C5,15N]-glutamic acid • [2H7]-cholesterol • [13C3]-myristic acid • [13C5]-proline • [13C12]-sucrose • [13C4]-hexadecanoic acid • [2H4]-1,4-butanediamine • [2H6]-2-hydoxybenzoic acid • [13C6]-glucose using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4°C. Each isotope compound was adjusted to a final concentration of 15 ng µl-1 for each 1-µl injection. After 5-min centrifugation at 15,100 × g, a 100 µl aliquot of the supernatant was drawn and transferred into a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo Electron Corporation, Waltham, MA, USA). For methoximation, 30 µl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA with 1% TMCS at 37°C with shaking. For methoximation, 30 µl of methoxyamine hydrochloride (20 mg ml-1 in pyridine) were added to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA at 37°C with shaking. After silylation 30 µl of n-heptane were added. All derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen. <GC-TOF/MS conditions> Using the splitless mode by an CTC CombiPAL auto-sampler (CTC analytics, Zwin-gen, Switzerland), 1 µl of each sample was injected into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) featuring a 30 m x 0.25 mm inner diameter fused-silica capillary column with a chemically bound 0.25-µl film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA) for metabolome analysis. Helium was delivered as the carrier gas at a constant flow rate of 1 ml min-1. The temperature program for metabolome analysis started with a 2-min isothermal step at 80°C followed by temperature ramping of 30°C to a final temperature of 320°C, which was maintained for 3.5 min. The transfer line and the ion source temperatures were 250 and 200°C, respectively. Ions were generated with a 70-eV electron beam at an ionization current of 2.0 mA. Data acquisition was on a Pegasus IV TOF MS instrument (LECO, St. Joseph, MI, USA) at an acquisition rate of 30 spectra s-1 in the mass range of a mass-to-charge ratio of m/z = 60-800. The solvent delay was 237 sec. Alkane standard mixtures (C8-C20 and C21-C40) were purchased from Sigma-Aldrich (Tokyo, Japan) and used for calculating the retention index (RI). The normalized response for calculating the signal intensity of each metabolite from the mass-detector response was obtained with each selected ion current unique in each metabolite MS spectrum to normalize the peak response. For quality control, we injected methylstearate in every 6 samples. Quality control (QC) samples were prepared by mixing 100 μl of extracts of each wild-type sample. We run six QC samples per batch (30 samples in total).
Comment_of_details