SE185:/S1/M2

Sample Set Information

ID	TSE1343
Title	Metabolic profiling analysis of genetically modified rice seedlings that overproduce tryptophan reveals the occurrence of its inter-tissue translocation
Description	A metabolic profiling approach using high performance liquid chromatography-mass spectrometry (LC-MS) was applied to seedlings of transgenic rice plants (Oryza sativa) that over-express the OASA1D gene encoding a feedbackinsensitive a-subunit of anthranilate synthase (AS, EC 4.1.3.27). Analysis revealed that the seedlings accumulated tryptophan (Trp) at a high concentration without marked effects on the amounts of other major metabolites. Some minor indole metabolites showed a certain degree of increase in the amounts in Trp-accumulating tissues, while no active catabolic conversion of Trp was indicated. Analysis also revealed that the distribution of Trp in the plant was uneven, with the highest level being observed in young developing tissues, despite tissue-independent expression of the OASA1D gene under the control of ubiquitin promoter. Differences in AS activity and anthranilate content among organs were small. A feeding experiment with radiolabeled Trp clearly demonstrated one-way Trp movement from old to young leaves; thus, the uneven distribution of Trp in OASA1D plants is the manifestation of Trp translocation. The negligible effects of Trp accumulation in other metabolic pathways, low metabolic activity of Trp, and efficient translocation of Trp in rice plants expressing OASA1D transgene are favorable characters from the aspect of metabolic engineering of Trp production.
Authors	Matsuda, F., Ishihara, A., Takanashi, K., Morino, K., Miyazawa, H., Wakasa, K., and Miyagawa, H.
Reference	Plant biotechnology 27(1), 17-27, 2010-03-25
Comment

http://ci.nii.ac.jp/naid/10026411273

Sample Information

ID	S1
Title	Oryza sativa L. cv. Nipponbare
Organism - Scientific Name	Oryza sativa
Organism - ID	NCBI taxonomy:4530
Compound - ID
Compound - Source
Preparation	HW1 and HW5 lines were established from the original Agrobacterium-mediated transformation of rice calli (Oryza sativa L. cv. Nipponbare) with OASA1D (Wakasa et al. 2006). Two lines, HW1 and HW5, were grown in a non-containment greenhouse (T5). Seeds of T5 were then grown in an isolated field (T6). Seeds of both HW1 T5 generation and HW5 T6 generation were used in this study. After soaking in 5% sodium hypochlorite solution in water for 30 min, the seeds of transformed and untransformed rice were incubated at 28°C with a 16-h photoperiod per day. Rice seedlings were then grown in a pot containing Bonsol soil® (Sumitomo Chemical, Osaka) in a growth chamber under the same conditions for four weeks.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M2
Title	HPLC
Method Details ID	MS2
Sample Amount	10 μL
Comment

Analytical Method Details Information

ID	MS2
Title	Quantification of Trp and anthranilate (HPLC)
Instrument	LC, Hitachi L7000 series ; UV, Hitachi L7400
Instrument Type
Ionization
Ion Mode
Description	Tissue samples were cut into small pieces and extracted with ten volumes (w/v) of 2% acetic acid in water at 100°C for 10 min. The extracts were centrifuged at 15,000 g for 10 min, and filtered through a Cosmonice Filter S (pore size 0.5 mm, filter diameter 13 mm, Nacalai Tesque, Kyoto). The resulting filtrates were subjected to analyses. For the quantification of Trp, the filtrate (10 ml) was analyzed with an HPLC system (Hitachi L7000 series) coupled with a UV detector (Hitachi L7400). Chromatography was performed with a COSMOSIL 5C18AR-II column (1504.6 mm; particle size, five mm, Nacalai Tesque) and a solvent mixture of methanol and 0.1% phosphoric acid in water at a flow rate of 0.8 ml min1 at 35°C. The ratio of methanol to 0.1% phosphate in water was programmed as: 10:90, v/v, for 5 min, and then from 10:90 to 70:30 over 30 min. The detection wavelength was set at UV280 nm. The levels of anthranilic acid were determined by a previously reported method (Dubouzet et al. 2007).
Comment_of_details