Journal Article

Isolation and Characterization of Homogentisate Phytyltransferase Genes from Synechocystis sp. PCC 6803 and Arabidopsis

Timothy A. Mitsky, Valentin, Henry E., Lassner, Michael W., Savidge, Beth, Yun-Hua H. Wong, Post-Beittenmiller, Dusty, Shewmaker, Christine K., James D. Weiss

2002

Overview

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.

• Publisher: American Society of Plant Biologists,American Society of Plant Physiologists,Oxford University Press
• Subject: Agronomy. Soil science and plant productions / algae / alkyl (aryl) transferases / Alkyl and Aryl Transferases / Alkyl and Aryl Transferases - genetics / Alkyl and Aryl Transferases - isolation & purification / alpha-Tocopherol / alpha-Tocopherol - chemistry / alpha-Tocopherol - metabolism / Amino Acid Sequence / amino acid sequences / Amino acids / Antisense Elements (Genetics) / Arabidopsis / Arabidopsis - enzymology / Arabidopsis - genetics / Arabidopsis Proteins / Arabidopsis thaliana / Bacterial Proteins / Bacterial Proteins - genetics / Baculoviridae / Baculoviridae - genetics / beta-Tocopherol / beta-Tocopherol - chemistry / beta-Tocopherol - metabolism / biochemical pathways / Biochemical Processes and Macromolecular Structures / Biological and medical sciences / Biosynthesis / Catalytic Domain / Catalytic Domain - genetics / chemical constituents of plants / chemistry / Chlorophyll / Chlorophyll - metabolism / Chlorophylls / Chloroplasts / Classical and quantitative genetics. Population genetics. Molecular genetics / Computational Biology / Cyanobacteria / Cyanobacteria - enzymology / Cyanobacteria - genetics / Economic plant physiology / enzyme activity / Enzymes / enzymology / Fundamental and applied biological sciences. Psychology / gamma-Tocopherol / gamma-Tocopherol - chemistry / gamma-Tocopherol - metabolism / gene expression / Gene Expression Regulation, Enzymologic / Generalities. Genetics. Plant material / genes / Genes. Genome / genetic complementation / Genetic Complementation Test / Genetic vectors / genetics / Genetics and breeding of economic plants / isolation & purification / Light-Harvesting Protein Complexes / Metabolism / Molecular and cellular biology / Molecular genetics / Molecular Sequence Data / mutants / Mutation / Nitrogen metabolism and other ones (excepting carbon metabolism) / nucleotide sequences / Nutrition. Photosynthesis. Respiration. Metabolism / Photosynthetic Reaction Center Complex Proteins / Photosynthetic Reaction Center Complex Proteins - metabolism / Plant physiology and development / plant proteins / Plants / Proteins / seeds / Seeds - enzymology / Seeds - genetics / Sequence Homology, Amino Acid / Terpenoids / Tocopherols / Tocopherols - chemistry / Tocopherols - metabolism / transgenic plants