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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

Title Rice (Oryza sativa ‘Nipponbare’)
Organism - Scientific Name Oryza sativa ‘Nipponbare’
Organism - ID NCBI taxonomy 4530
Compound - ID
Compound - Source
Preparation We used wt plants (Oryza sativa L. ssp. japonica ‘Nipponbare’), the control line FOX3, and transgenic lines transformed using the high-speed Agrobacterium-mediated transformation system (Toki et al., 2006).

qRT–PCR analysis of the T0 generation of 19 independent transformant lines revealed high LBD37/ASL39 expression in the RK16331–4 and RK16331–13 transformant lines; therefore, these lines were selected for further analysis.

The samples for the metabolite- and transcript-profiling experiments and the qRT–PCR experiments were prepared using the following procedure. The husks were separated from the seeds, and the brown rice seeds were sterilized by treating them with 70% ethanol for approximately 20 s. Subsequently, the seeds were stirred in 10 ml of sterilizing solution for 20 min by using an agitator (Rotator RT-50; Taitech Co., Ltd, Japan). After sterilization, the seeds were washed four times in 10 ml of distilled water by using the agitator. The sterilized seeds were inoculated on a solid half-strength MS medium containing 3% sucrose and 50 mg l−1 hygromycin (pH 5.8). After a 4-d incubation under dark conditions at room temperature, the plates were transferred to the growth chamber (MLR-350h/MLR-350 H; Sanyo, Japan). The following conditions were used for growth: 12 h of light at 30°C and 12 h of darkness at 25°C; relative humidity, 40%; light strength, 200–250 μmol s−1. Rice seedlings with a shoot length of 6–8 cm were hydroponically grown in half-strength hydroponic-culture solution (Makino et al., 1983) in a greenhouse (12 h of light at 30°C and 12 h of darkness at 25°C) for a week. Then, the seedlings were grown in a normal hydroponic culture solution in the greenhouse until the generation of the eighth leaf blade, after which the fifth and sixth leaves were harvested, bulked, and used for metabolite profiling, qRT–PCR, and transcriptomic analysis. The ages (DAS) of the RK16331–4 and RK16331–13 LBD37/ASL39-overexpressor plants and the controls differed at the time of sampling: the plants of lines RK16331–4 were sampled between 48 and 55 DAS; plants of lines RK16331–13, between 41 and 55 DAS; and the control plants, between 31 and 51 DAS.

Sample Preparation Details ID

Analytical Method Information

Method Details ID MS1
Sample Amount 55.6 μg

Analytical Method Details Information

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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