COMPLEMENTATION OF THE ARABIDOPSIS PDS1 MUTATION WITH THE GENE ENCODING P-HYDROXYPHENYLPYRUVATE DIOXYGENASE

Plastoquinone and tocopherols are the two major quinone compounds in h igher plant chloroplasts and are synthesized by a common pathway. In p revious studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. Mutation of the PDS1 locus disrupt s the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the f irst committed step in the synthesis of both plastoquinone and tocophe rols in plants. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the produc t of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. This paper reports the isolatio n of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional ch aracterization by expression in both plants and Escherichia coli. pHPP D encodes a 50-kD polypeptide with homology to previously identified H PPDases, including 37 highly conserved amino acid residues clustered i n the carboxyl region of the protein. Expression of pHPPD in E. coli c atalyzes the accumulation of homogentisic acid, indicating that it enc odes a functional HPPDase enzyme. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage . Constitutive expression of pHPPD in a pds1 mutant background complem ents this mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pdsl allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Together, these data conclusively demonstrate that p dsl is a mutation in the HPPDase structural gene.