Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco

作     者：Garcia, I Rodgers, M Pepin, R Hsieh, TF Matringe, M 

作者机构：Rhone Poulenc CNRS UMR 41 F-69263 Lyon 09 France Rhone Poulenc Agrochim Dept Biotechnol F-69263 Lyon 09 France Texas A&M Univ Dept Biol College Stn TX 77843 USA 

出 版 物：《PLANT PHYSIOLOGY》 (植物生理学)

年 卷 期：1999年第119卷第4期

页      面：1507-1516页

核心收录：

学科分类：0710[理学-生物学] 071001[理学-植物学] 07[理学] 

主　　题：Recombinant Proteins Arabidopsis Enzymatic activity immunodetection COMPARTMENT 4-hydroxyphenylpyruvate 4-Hydroxyphenylpyruvate Dioxygenase Biochemical characteristics leaf extracts Coding Sequence Tobacco Products Dioxygen Dioxygenases Subcellular 

摘      要：4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We. performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.