SE140:/S1/M1/D1

Sample Set Information

ID	TSE1241
Title	Metabolomic screening applied to rice FOX Arabidopsis lines leads to the identification of a gene-changing nitrogen metabolism.
Description	Plant metabolomics developed as a powerful tool to examine gene functions and to gain deeper insight into the physiology of the plant cell. In this study, we screened Arabidopsis lines overexpressing rice full-length (FL) cDNAs (rice FOX Arabidopsis lines) using a gas chromatography–time-of-flight mass spectrometry (GC–TOF/MS)-based technique to identify rice genes that caused metabolic changes. This screening system allows fast and reliable identification of candidate lines showing altered metabolite profiles. We performed metabolomic and transcriptomic analysis of a rice FOX Arabidopsis line that harbored the FL cDNA of the rice ortholog of the Lateral Organ Boundaries (LOB) Domain (LBD)/Asymmetric Leaves2-like (ASL) gene of Arabidopsis, At-LBD37/ASL39. The investigated rice FOX Arabidopsis line showed prominent changes in the levels of metabolites related to nitrogen metabolism. The transcriptomic data as well as the results from the metabolite analysis of the Arabidopsis At-LBD37/ASL39-overexpressor plants were consistent with these findings. Furthermore, the metabolomic and transcriptomic analysis of the Os-LBD37/ASL39-overexpressing rice plants indicated that Os-LBD37/ASL39 is associated with processes related to nitrogen metabolism in rice. Thus, the combination of a metabolomics-based screening method and a gain-of-function approach is useful for rapid characterization of novel genes in both Arabidopsis and rice.
Authors	Albinsky D, Kusano M, Higuchi M, Hayashi N, Kobayashi M, Fukushima A, Mori M, Ichikawa T, Matsui K, Kuroda H, Horii Y, Tsumoto Y, Sakakibara H, Hirochika H, Matsui M, Saito K.
Reference	Mol Plant. 2010 Jan;3(1):125-42. doi: 10.1093/mp/ssp069. Epub 2009 Aug 26.
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Sample Information

ID	S1
Title	Arabidopsis thaliana (ecotype Col-0)
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy 3702
Compound - ID
Compound - Source
Preparation	For the metabolite measurements, qRT–PCR, and microarray analysis, the plant material was sterilized, grown, and harvested according to the following procedure. Surface-sterilized T2 seeds of the rice FOX Arabidopsis lines and the Arabidopsis At-LBD37/ASL39-overexpressor lines were inoculated on solid Murashige and Skoog (MS) medium containing 1% sucrose and 20 mg l−1 hygromycin. Seeds of the plants of the activation-tagged lines of the AS2 and ASL1 genes (Chalfun-Junior et al., 2005; Iwakawa et al., 2002; Nakazawa et al., 2003), and the LBD38/ASL40 gene originating from the same mutant collection, as well as seeds of the LBD39/ASL41-overexpressing Arabidopsis FOX line F15720, were inoculated under the same growth conditions using the above-mentioned procedure. SALK lines (Alonso et al., 2003) were obtained from the Arabidopsis Biological Resource Center (ABRC). The homozygous SALK lines 057939 and 058867, which harbor a T-DNA insertion site in the ORF and the 5′ UTR of LBD37/ASL39, respectively, were grown on MS medium supplemented with 50 mg l−1 kanamycin. The plants were grown under long-day (16 h light) and low-light conditions (50 μmol s−1) at 22°C for 18 d in growth chambers (SANYO; MLR-350h/MLR-350 HT) until sampling. The plates were prepared in duplicate using 60 seeds of six independent lines; each plate contained five seeds of a particular line and five seeds of the empty-vector control line ‘BIG’. The plates were numbered, placed in a growth chamber in a defined position, and rotated twice a week to ensure equal light reception. At 18 DAS, the seedlings were harvested without roots, weighed, immediately deep-frozen in liquid nitrogen, and stored at –80°C until further analysis. For assays using homozygous T3 lines, the seeds were treated using the above-mentioned procedure and inoculated on solid hygromycin-free MS medium. Plant growth and harvesting were performed using the above-mentioned protocol. The soil-grown plants in the greenhouse were grown under the same conditions as those used for the plants grown in growth chambers.
Sample Preparation Details ID
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Analytical Method Information

ID	M1
Title	GC-TOF MS
Method Details ID	MS1
Sample Amount	55.6 μg
Comment

Analytical Method Details Information

ID	MS1
Title	GC-TOF MS
Instrument	GC:Agilent 6890N MS:LECO Pegasus 4D MS system
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	The samples were extracted, derivatized, and subsequently analyzed by using GC–TOF/MS, as described previously (Kusano et al., 2007). Briefly, we used extracts of 5-mg fresh weight (FW) plant samples for the GC–TOF/MS analysis. The extracts were derivatized, and 55.6 μg of the derivatized samples were injected into the column by using a CTC CombiPAL autosampler (CTC analytics, Zwingen, Switzerland).
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Data Analysis Information

ID	D1
Title	Data Processing
Data Analysis Details ID	DS1
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS1
Title	Data processing
Description	The obtained non-processed data were imported to the MATLAB software (version 7.0.4; Mathworks, USA). All data-alignment procedures and statistical analyses were performed by using previously described protocols (Kusano et al., 2007).
Comment_of_details

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