Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis

Abstract

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.

Figure 1

Tocol and tocopherol structure.

Figure 2

Tocopherol biosynthetic pathway.

Figure 3

Protein alignment of HPT1 and Slr1736 and conserved putative catalytic domain. A, Alignment of HPT1 and Slr1736 using the ClustalW algorithm. B, Alignment of putative catalytic domain of Slr1736 and Arabidopsis putative prenyltransferases identified using UbiA as a query in a PSI-BLAST search. Gray shading indicates identity. Underlined sequence corresponds to the putative catalytic domain. Asterisks indicate amino acids required for catalytic activity in heme-O-synthase, which are conserved in HPT1. The putative chloroplast target peptide processing site is indicated by an arrowhead.

Figure 4

HPLC chromatographic analysis of ethanol extracts of wild-type Synechocystis sp. PCC 6803, slr1736 null mutant, and slr1736 null mutant complemented with HPT1. A, Wild-type Synechocystis sp. PCC 6803 transformed with control vector. Peak 4 corresponds to α-tocopherol at 4.6 min. B, slr1736 null mutant lacking tocopherol peak at 4.6 min. C, slr1736 null mutant complemented with Arabidopsis HPT1 with tocopherol signal apparent at 4.6 min. A compound eluting at 8.5 min represents the tocol internal standard (peak 5). Peak 1 corresponds to the solvent front, and peaks 2 and 3 are two unknown compounds.

Figure 5

HPT activity of Synechocystis sp. PCC 6803 wild-type and slr1736 null mutant membrane fractions. A, Formation of [³H]2-methyl-6-phytylplastiquinone in membrane fractions of Synechocystis sp. PCC 6803 wild-type cells from [³H]HGA and phytyl-DP. B, Lack of accumulation of [³H]2-methyl-6-phytylplastiquinone formation by membrane fractions from Synechocystis sp. PCC 6803 slr1736 null mutants.

Figure 6

Tocopherol content of T₂ seed from pNapin::HPT1 Arabidopsis plants. A, Total tocopherol levels (ng mg⁻¹ seed) in individual pools of segregating T₂ seed derived from 36 independent transgenic events containing the pCGN10822 construct (pNapin::HPT1) compared with vector (VC) and wild-type (WT) control populations. B, Total tocopherol levels (ng mg⁻¹ seed) in individual pools of T₂ seed derived from 86 independent transgenic events harboring the pCGN10803 construct (e35S::HPT1_antisense) are compared with control populations. Error bars on control samples represent the 95% confidence interval with the sample size indicated as n. The gray bar in the background includes the 95% confidence interval of both controls. Seed tocopherol levels of wild-type (░⃞), vector control (□), and T₂ transgenic lines (▪).