68. 植物培養細胞における代謝物のin vivo ^<15>N-標識とメタボロミクスへの応用

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[Objectives] Accurate quantification of nitrogen containing metabolites in living organisms and their flux analyses are one of the most important tactics in the field of plant metabolic engineering. However, practical methodologies have not been developed. This paper demonstrates analyses of nitrogen-containing metabolites turnover by means of in vivo ^<15>N enrichment in the plant cell culture followed by ^<15>N/^<14>N ratio observation using LC/MS. [Methods and Results] Arabidopsis thaliana culture cell T-87 was used as a model plant cell. Intracellular free amino acids were evaluated as target nitrogen-containing metabolites. Arabidopsis culture cell was precultured in a modified LS medium containing ^<14>N nitrogen sources. The precultured cells transferred to the modified LS medium containing ^<15>N nitrogen sources such as K^<15>NO_3, ^<15>NH_4^<15>NO_3 and cultivated for 24 hours. Amino acids fractions were extracted from cultured cells and were dansylated for further analysis. The derivatized amino acid fractions were subjected to LC/MS analysis to calculate ^<15>N/^<14>N ratio. The estimated ^<15>N labeled-efficiencies of glutamate, of glycine, and of aliphatic amino acids were high. Meanwhile, ones of asparagine, aspartate, and aromatic amino acids were rather low. In dark condition, the ^<15>N incorporation efficiencies of most amino acids were reduced, but one of aspartate was increased. This method was proved to be useful for the facile comparison of nitrogen containing metabolites turnover rates. A combination of the present method and time course sampling would be applicable for a prediction of nitrogen metabolic fluxes.
植物化学調節学会の論文
2004-10-13

68. 植物培養細胞における代謝物のin vivo ^<15>N-標識とメタボロミクスへの応用

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