Integrated metabolomic and transcriptomic analyses of high-tryptophan rice expressing a mutant anthranilate synthase alpha subunit.
Transgenic rice plants overexpressing a mutant rice gene for anthranilate synthase alpha subunit (OASA1D) accumulate large amounts of free tryptophan (Trp) with few adverse effects on the phenotype, except for poor germination and weak seedling growth. Metabolic profiling of 8-d-old seedlings of Nipponbare and two high-Trp lines, HW1 and HW5, by high performance liquid chromatography-photo diode array (HPLC-PDA) confirmed that, relative to Nipponbare, only the peak attributed to Trp was significantly changed in the profiles of the OASA1D lines. More detailed and targeted analysis using HPLC coupled with tandem mass spectrometry revealed that the OASA1D lines had higher levels of anthranilate, tryptamine, and serotonin than Nipponbare, but these metabolites were at much lower levels than free Trp. The levels of phenylalanine (Phe) and tyrosine (Tyr) were not affected by the overproduction of Trp. Transcriptomic analysis by microarray validated by quantitative Real-Time PCR (qRT-PCR) revealed that at least 12 out of 21 500 genes showed significant differential expression among genotypes. Except for the OASA1D transgene and a putative IAA beta-glucosyltransferase, these were not related to Trp metabolism. Most importantly, the overexpression of the OASA1D and the consequent accumulation of Trp in these lines had little effect on the overall transcriptome, consistent with the minimal effects on growth and the metabolome. Integrated analysis of the metabolome and transcriptome of these OASA1D transgenic lines indicates that the over-accumulation of free Trp may be partly due to the low activity of Trp decarboxylase or other metabolic genes that directly utilize Trp as a substrate.
Dubouzet
Joseph G
JG
CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan.
Ishihara
Atsushi
A
Matsuda
Fumio
F
Miyagawa
Hisashi
H
Iwata
Hiroyoshi
H
Wakasa
Kyo
K
D000878:Anthranilate Synthase / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D018506:Gene Expression Regulation, Plant; D012275:Oryza / Q000201:enzymology / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry