I. Garcia et al., Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco, PLANT PHYSL, 119(4), 1999, pp. 1507-1516
4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homo
gentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and mol
ecular oxygen. In plants this enzyme activity is involved in two distinct m
etabolic processes, the biosynthesis of prenylquinones and the catabolism o
f tyrosine. We report here the molecular and biochemical characterization o
f an Arabidopsis 4HPPD and the compartmentation of the recombinant protein
in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open r
eading frame of 1338 bp. Southern analysis strongly suggested that this Ara
bidopsis 4HPPD is encoded by a single-copy gene. We investigated the bioche
mical characteristics of this 4HPPD by overproducing the recombinant protei
n in Escherichia coli JM105. The subcellular localization of the recombinan
t 4HPPD in chlorophyllous tissues was examined by overexpressing its comple
te coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobac
terium tumefaciens transformation. We. performed western analyses for the i
mmunodetection of protein extracts from purified chloroplasts and total lea
f extracts and for the immunocytochemistry on tissue sections. These analys
es 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.