SE144:/S1/M1

Sample Set Information

ID	SE144
Title	Metabolomics data reveal a crucial role of cytosolic glutamine synthetase 1;1 in coordinating metabolic balance in rice.
Description	Rice plants grown in paddy fields preferentially use ammonium as a source of inorganic nitrogen. Glutamine synthetase (GS) catalyses the conversion of ammonium to glutamine. Of the three genes encoding cytosolic GS in rice, OsGS1;1 is critical for normal growth and grain filling. However, the basis of its physiological function that may alter the rate of nitrogen assimilation and carbon metabolism within the context of metabolic networks remains unclear. To address this issue, we carried out quantitative comparative analyses between the metabolite profiles of a rice mutant lacking OsGS1;1 and its background wild type (WT). The mutant plants exhibited severe retardation of shoot growth in the presence of ammonium compared with the WT. Overaccumulation of free ammonium in the leaf sheath and roots of the mutant indicated the importance of OsGS1;1 for ammonium assimilation in both organs. The metabolite profiles of the mutant line revealed: (i) an imbalance in levels of sugars, amino acids and metabolites in the tricarboxylic acid cycle, and (ii) overaccumulation of secondary metabolites, particularly in the roots under a continuous supply of ammonium. Metabolite-to-metabolite correlation analysis revealed the presence of mutant-specific networks between tryptamine and other primary metabolites in the roots. These results demonstrated a crucial function of OsGS1;1 in coordinating the global metabolic network in rice plants grown using ammonium as the nitrogen source.
Authors	Kusano M, Tabuchi M, Fukushima A, Funayama K, Diaz C, Kobayashi M, Hayashi N, Tsuchiya YN, Takahashi H, Kamata A, Yamaya T, Saito K.
Reference	Plant J. 2011 May;66(3):456-66.
Comment

PubMed

Sample Information

ID	S1
Title	Oryza sativa L. ‘Nipponbare’
Organism - Scientific Name	Oryza sativa
Organism - ID	NCBI taxonomy:4530
Compound - ID
Compound - Source
Preparation	We used the rice variety Oryza sativa L. ‘Nipponbare’. Homozygous lines of OsGS1;1 knockout mutants were generated by inserting the endogenous retrotransposon Tos17 (Hirochika, 2001) into the OsGS1;1 gene. We selected the NC2373 and ND8037 lines, which carried a Tos17 insertion only in the OsGS1;1 open reading frame (Tabuchi et al., 2005). The knockout lines exhibited very low fertility and inhibited grain filling; therefore, heterozygous seeds were used to obtain homozygous plants. Wild type and two independent heterozygous lines were grown as described previously (Yamaya et al.,1995). Briefly, in May 2010, July 2008 and September 2005 and 2008,WT and heterozygous seeds were soaked in water (Dataset S1) at 30°C for 36–48 h under dark conditions and the germinated seeds were transferred to a nylon net that floated on culture medium in a plastic container. When the endosperm had been fully utilised,seedlings were transferred to different nutrition conditions to measure different N availabilities. The WT and heterozygous plants were cultivated in a greenhouse with the following conditions for metabotyping in June (14 h of light at 26°C and 10 h of darkness at 23°C; without controlling humidity; light strength, 810 μmol m-2 sec-1), while we used another greenhouse for other experiments (12 h of light at 30°C and 12 h of darkness at 25°C; without control humidity). To identify homozygous mutants, the second LB of each plant was harvested for PCR analysis.
Sample Preparation Details ID
Comment

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	Metabotyping and metabolite profiling analysis
Instrument	GC:Agilent 6890N MS:LECO Pegasus III
Instrument Type
Ionization	EI
Ion Mode	positive
Description	We used the three different tissues (LB, LS and roots) to perform metabolite phenotyping and profiling using GC-TOF-MS (Kusano et al., 2007a). Metabotyping was conducted using six independent seedlings for WT, two independent knockout lines (NC2373 and ND8037) and the corresponding null lines, respectively. Extracts from 5 mg (fresh weight, FW) samples were used for metabotyping. Extracts of 55.6 μg FW samples was injected into the GC-TOF-MS.We performed data pre-treatment and normalisation as described by Kusano et al. (2007a) and Redestig et al. (2009). For metabolite profiling, a total of 16–20 biological replicates were collected,extracted, derivatised and analysed. Extracts from 5 mg FW samples were used. Subsequently, extracts of 83.3 μg FW samples for the ammonium experiment and extracts of 55.6 μg FW samples for the water experiment were injected .
Comment_of_details

PubMed