RRC ID 72016
著者 Krajewski MP, Kanawati B, Fekete A, Kowalski N, Schmitt-Kopplin P, Grill E.
タイトル Analysis of Arabidopsis glutathione-transferases in yeast.
ジャーナル Phytochemistry
Abstract The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows screening for GST substrates and identifying substrate preferences within the plant GST family. To this end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The resulting yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members demonstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently, the established yeast system was explored for its potential to screen the Arabidopsis GST family for conjugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased levels of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi, tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast extracts allowed the simultaneous detection and semiquantification of anilazine conjugates as well as catabolites.
巻・号 91
ページ 198-207
公開日 2013-7-1
DOI 10.1016/j.phytochem.2012.04.016
PII S0031-9422(12)00195-1
PMID 22633844
MeSH Arabidopsis / enzymology* Dinitrobenzenes / chemistry Dinitrobenzenes / metabolism Glutathione Transferase / analysis* Glutathione Transferase / deficiency Glutathione Transferase / metabolism Molecular Structure Saccharomyces cerevisiae / genetics* Saccharomyces cerevisiae / metabolism Triazines / chemistry Triazines / metabolism
IF 3.044
リソース情報
シロイヌナズナ / 植物培養細胞・遺伝子 pda00113 pda13232 pda02701 pda09379 pda08837 pda06713 pda07676 pda10641 pda00597 pda04959 pda00221 pdz10553 pda10643 pda12172 pda02708 pda02125 pda09025 pda07148 pda00742 pda00319 pda03430