Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis.
Tocopherols, synthesized by photosynthetic organisms, are micronutrients with antioxidant properties that play important roles in animal and human nutrition. Because of these health benefits, there is considerable interest in identifying the genes involved in tocopherol biosynthesis to allow transgenic alteration of both tocopherol levels and composition in agricultural crops. Tocopherols are generated from the condensation of phytyldiphosphate and homogentisic acid (HGA), followed by cyclization and methylation reactions. Homogentisate phytyltransferase (HPT) performs the first committed step in this pathway, the phytylation of HGA. In this study, bioinformatics techniques were used to identify candidate genes, slr1736 and HPT1, that encode HPT from Synechocystis sp. PCC 6803 and Arabidopsis, respectively. These two genes encode putative membrane-bound proteins, and contain amino acid residues highly conserved with other prenyltransferases of the aromatic type. A Synechocystis sp. PCC 6803 slr1736 null mutant obtained by insertional inactivation did not accumulate tocopherols, and was rescued by the Arabidopsis HPT1 ortholog. The membrane fraction of wild-type Synechocystis sp. PCC 6803 was capable of catalyzing the phytylation of HGA, whereas the membrane fraction from the slr1736 null mutant was not. The microsomal membrane fraction of baculovirus-infected insect cells expressing the Synechocystis sp. PCC 6803 slr1736 were also able to perform the phytylation reaction, verifying HPT activity of the protein encoded by this gene. In addition, evidence that antisense expression of HPT1 in Arabidopsis resulted in reduced seed tocopherol levels, whereas seed-specific sense expression resulted in increased seed tocopherol levels, is presented.
Savidge
Beth
B
Monsanto Company, Calgene Campus, 1920 Fifth Street, Davis, California 95616, USA.
Weiss
James D
JD
Wong
Yun-Hua H
YH
Lassner
Michael W
MW
Mitsky
Timothy A
TA
Shewmaker
Christine K
CK
Post-Beittenmiller
Dusty
D
Valentin
Henry E
HE
D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D016375:Antisense Elements (Genetics); D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins; D001426:Bacterial Proteins / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002734:Chlorophyll / Q000378:metabolism; D019295:Computational Biology; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D009154:Mutation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism; D012639:Seeds / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D024505:Tocopherols / Q000737:chemistry / Q000378:metabolism*; D024502:alpha-Tocopherol / Q000737:chemistry / Q000378:metabolism; D024503:beta-Tocopherol / Q000737:chemistry / Q000378:metabolism; D024504:gamma-Tocopherol / Q000737:chemistry / Q000378:metabolism
D016375:Antisense Elements (Genetics); D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D002734:Chlorophyll; D024504:gamma-Tocopherol; D024503:beta-Tocopherol; D019883:Alkyl and Aryl Transferases; C446531:HPT1 protein, Arabidopsis; D024502:alpha-Tocopherol; D024505:Tocopherols