Metabolic flux analysis in plants using dynamic labeling technique: application to tryptophan biosynthesis in cultured rice cells.
The concept and methodology of using dynamic labeling for the MFA of plant metabolic pathways are described, based on a case study to develop a method for the MFA of the tryptophan biosynthetic pathway in cultured rice cells. Dynamic labeling traces the change in the labeling level of a metabolite in a metabolic pathway after the application of a stable isotope-labeled compound. In this study, [1-(13)C] l-serine was fed as a labeling precursor and the labeling level of Trp was determined by using the LC-MS/MS. The value of metabolic flux is determined by fitting a model describing the labeling dynamics of the pathway to the observed labeling data. The biosynthetic flux of Trp in rice suspension cultured cell was determined to be 6.0+/-1.1 nmol (gFWh)(-1). It is also demonstrated that an approximately sixfold increase in the biosynthetic flux of Trp in transgenic rice cells expressing the feedback-insensitive version of anthranilate synthase alpha-subunit gene (OASA1D) resulted in a 45-fold increase in the level of Trp. In this article, the basic workflow for the experiment is introduced and the details of the actual experimental procedures are explained. Future perspectives are also discussed by referring recent advances in the dynamic labeling approach.
Matsuda
Fumio
F
Plant Functions and Their Control, CREST, Japan Science and Technology Agency, 3-4-5 Nihonbashi, Chuo, Tokyo 103-0027, Japan.
Wakasa
Kyo
K
Miyagawa
Hisashi
H
D000878:Anthranilate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D007553:Isotope Labeling; D008954:Models, Biological; D012275:Oryza / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000096:biosynthesis* / Q000737:chemistry
D010940:Plant Proteins; D021122:Protein Subunits; D014364:Tryptophan; D000878:Anthranilate Synthase