Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis

Abstract

Tocopherols are essential components of the human diet and are synthesized exclusively by photosynthetic organisms. These lipophilic antioxidants consist of a chromanol ring and a 15-carbon tail derived from homogentisate (HGA) and phytyl diphosphate, respectively. Condensation of HGA and phytyl diphosphate, the committed step in tocopherol biosynthesis, is catalyzed by HGA phytyltransferase (HPT). To investigate whether HPT activity is limiting for tocopherol synthesis in plants, the gene encoding Arabidopsis HPT, HPT1, was constitutively overexpressed in Arabidopsis. In leaves, HPT1 overexpression resulted in a 10-fold increase in HPT specific activity and a 4.4-fold increase in total tocopherol content relative to wild type. In seeds, HPT1 overexpression resulted in a 4-fold increase in HPT specific activity and a total seed tocopherol content that was 40% higher than wild type, primarily because of an increase in gamma-tocopherol content. This enlarged pool of gamma-tocopherol was almost entirely converted to alpha-tocopherol by crossing HPT1 overexpressing plants with lines constitutively overexpressing gamma-tocopherol methyltransferase. Seed of the resulting double overexpressing lines had a 12-fold increase in vitamin E activity relative to wild type. These results indicate that HPT activity is limiting in various Arabidopsis tissues and that total tocopherol levels and vitamin E activity can be elevated in leaves and seeds by combined overexpression of the HPT1 and gamma-tocopherol methyltransferase genes.

Figure 1

The tocopherol biosynthetic pathway in plants. Dashed arrows represent multiple steps. Enzymes are indicated by circled numbers: 1, HGA phytyltransferase (HPT); 2, p-hydroxyphenyl pyruvate dioxygenase (HPPD); 3, HGA dioxygenase; 4, geranylgeranyl diphosphate reductase (GGDR); 5, geranylgeranyl diphosphate synthase (GGPS); 6, 1-deoxy-d-xylulose-5-phosphate synthase (DXPS); 7, 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase (MPBQ); 8, tocopherol cyclase (TC); and 9, γ-tocopherol methyltransferase (γ-TMT).

Figure 2

Tocopherol composition and levels in leaves of 4-week-old wild-type and homozygous 35S::HPT1 Arabidopsis plants. Thirty milligrams of leaf tissue was extracted, and individual tocopherols were separated and quantified by reverse-phase HPLC. Each line is represented as an average ± sd of tocopherol levels in three plants analyzed in duplicate. Total tocopherol levels in picomoles per milligram of leaf are indicated above the error bar of each line. Total tocopherol levels of 35S::HPT1 lines were significantly higher than wild-type levels (P < 0.01). β- and δ-tocopherols were not detected.

Figure 3

Tocopherol composition and levels in seed of wild-type and homozygous 35S::HPT1 Arabidopsis plants. Seed was extracted and analyzed for tocopherols by normal phase HPLC. Tocopherol levels are expressed as an average ± sd of three analyses per each line in a representative experiment. Total tocopherol levels in picomoles per milligram of seed are indicated above the error bar of each line. Arabidopsis seeds accumulate predominantly γ-tocopherol, which increased up to 40% in 35S::HPT1 compared with wild type. The levels of α-tocopherol did not change significantly, whereas δ-tocopherol increased up to 2-fold in some transgenic lines. β-Tocopherol was not detected. With the exception of 35S::HPT1-16, statistical significance for total tocopherol levels of all transgenic lines relative to wild type was P < 0.001.

Figure 4

Tocopherol composition and levels in leaves of 5-week-old wild-type, 35S::γ-TMT, 35S::HPT1, and double 35S::γ-TMT/ 35S::HPT1 overexpressers. Leaf tissue (approximately 70 mg) was extracted, and individual tocopherols were separated and quantified by normal phase HPLC. Each line is represented as an average ± sd from three plants. Total tocopherol levels of 35S::HPT1 and 35S::γ-TMT/35S::HPT1 plants were significantly higher than wild-type levels (P < 0.005). The excess γ-tocopherol in double transgenics was methylated to α-tocopherol.

Figure 5

Tocopherol composition and levels in seed of wild-type, 35S::γ-TMT, 35S::HPT1, and 35S::γ-TMT/35S::HPT1 overexpressers. A, α-, β-, γ-, and δ- tocopherols; B, β- and δ-tocopherols. Tocopherol analysis of mature dry seed was performed as described in “Materials and Methods.” Each line is presented as an average of three independent measurements of three replicates. Total tocopherol levels of 35S::HPT1 and 35S::γ-TMT/35S::HPT1 lines were significantly higher than wild type (P < 0.05). The majority of γ- and δ-tocopherols was converted to α- and β-tocopherols in 35S::γ-TMT/35S::HPT1 overexpressers.