Complementation of the Arabidopsispds1Mutation with the Gene Encodingp-Hydroxyphenylpyruvate Dioxygenase

Abstract

Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid, indicating that it encodes 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 complements this mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene.

The Arabidopsis Spontaneous Cell Death1 gene, encoding a ζ-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development, photoprotection and retrograde signalling

“…Phytoene undergoes four desaturation reactions to form the pink pigment lycopene, catalyzed by two enzymes, phytoene desaturase (PDS) and z-carotene desaturase (ZDS) [15]. In addition to PDS and ZDS, other components, such as Phytoene Desaturation 1 (PDS1, a p-hydroxyphenylpyruvate dioxygenase) [17] and PDS2, are also involved in the desaturation reactions [18]. Lycopene is a branch point for two separate pathways.…”

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis

“…Phytoene desaturase (PDS3) plays an important role in carotenoid biosynthesis. Two Arabidopsis PDS-related mutants, pds1 and pds2, with albino phenotypes, were found to accumulate high levels of phytoene, the substrate of PDS3, in their leaves [14,15]. However, mutations in PDS1 and PDS2 genes only affected the availability of the electron carrier plastoquinone required for the phytoene desaturase function, but not the PDS3 enzyme itself.…”

“…First-strand cDNA synthesis was performed in a 20-µL-reaction volume consisting of 3 µg of total RNA, oligodT [12][13][14][15][16][17][18] and SuperScript TM reverse transcriptase (GIBCO BRL). The reaction was allowed to proceed at 42 ºC for 50 min before being terminated by treatment at 70 ºC for 15 min.…”

“…Genetic screen for phytoene desaturation defect resulted in the isolation of two related mutants, pds1 and pds2, both defective in phytoene desaturation and exhibiting the albino phenotype [14,15]. The fact that the PDS1 and PDS2 genes encode 4-hydroxyphenylpyrurate dioxygenase and prenylphytyltransferase respectively [14,15], suggests that plastoquinone is a critical intermediate electron carrier for electron transport in the phytoene desaturation reaction. Another Arabidopsis mutant, immutans, accumulates phytoene in variegated white tissues.…”

Alterations in Tocopherol Cyclase Activity in Transgenic and Mutant Plants of Arabidopsis Affect Tocopherol Content, Tocopherol Composition, and Oxidative Stress

“…Homogentisate, the precursor for the synthesis of tocopherol, tocotrienol, and plastoquinone, is synthesized by p-hydroxyphenylpyruvate dioxygenase (HPPD; Fig. 1; Norris et al, 1998) (Collakova et al, 2003a). Final methylation by g-tocopherol methyltransferase (g-TMT, VTE4) results in the production of a-tocopherol.…”