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Characterization and Subcellular Compartmentation of Recombinant 4-Hydroxyphenylpyruvate Dioxygenase from Arabidopsis in Transgenic Tobacco1

Isabelle Garcia, Matthew Rodgers, Régis Pepin, Tzung-Fu Hsieh, and Michel Matringe*

Unité Mixte Centre National de la Recherche Scientifique/Rhône-Poulenc (Unité, Mixte de Recherche 41) (I.G., R.P., M.M.); Département des Biotechnologies, Rhône-Poulenc Agrochimie, 14-20 rue Pierre Baizet, 69263 Lyon cedex 09, France (M.R.); and Department of Biology, Texas A&M University, College Station, Texas 77843 (T.-F.H.)

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.

1   This study was conducted under the BIO Avenir program financed by Rhône-Poulenc with a contribution from the Ministère de la Recherche et de l'Enseignement Supérieur.
*   Corresponding author; e-mail michel.matringe{at}ladargoire.rhone-poulenc.com; fax 33-4-72-85-22-97.

Plant Physiol. (1999) 119: 1507-1516
Copyright Clearance Center:   0032-0889/99/119//10
© 1999 American Society of Plant Physiologists




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