Genetic dissection of phytoene desaturation in Arabidopsis thaliana

Carotenoids are C₄₀ tetraterpenoids synthesized by nuclear-encoded, multi-enzyme complexes located in the plastids of higher plants. In order to further understand the components and mechanisms involved in carotenoid biosynthesis, our laboratory has identified Arabidopsis thaliana mutants that disrupt this pathway. Here, I report the identification and characterization of three non-allelic albino mutations, pds1, pds2, and pds3 (pds = b phytoene desaturation), that are disrupted in phytoene desaturation, an early step in carotenoid biosynthesis. pds1 and pds2 have been more thoroughly characterized than pds3. Surprisingly, neither pds1 nor pds2 maps to the locus encoding the phytoene desaturase enzyme, indicating the products of at least three loci are required for phytoene desaturation. Electron transport chain components are hypothesized to be involved in phytoene desaturation and the analysis of pds1 and pds2 provide the first genetic evidence that plastoquinone is an essential component in carotenoid biosynthesis. Both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene, affecting distinct steps of the common plastoquinone/tocopherol biosynthetic pathway. The pds1 mutation affects the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) as it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and p-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Additionally, I report the isolation of an Arabidopsis HPPD cDNA, the first from a higher plant, which encodes a 50 kD polypeptide with between 29 and 40% identity to non-plant HPPDs. Alignment of the Arabidopsis HPPD with non-plant HPPDs identifies 38 identical amino acid residues, including six tyrosine and histidine residues thought to form the ferric iron center of the enzyme. When expressed in E. coli, the Arabidopsis HPPD catalyzes the accumulation of two compounds, homogentisic acid and ochronotic pigment, a polymerized oxidation product of homogentisic acid. Additionally, the Arabidopsis HPPD locus and the pds1 locus co-segregate. Finally, and most-significantly, the constitutive expression of the Arabidopsis HPPD cDNA in the pds1 mutant background complements the pds1 mutant. Taken together, these data suggest that pds1 is a mutation in the HPPD gene