Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination

Abstract

Tocopherols (vitamin E) are lipophilic antioxidants synthesized by all plants and are particularly abundant in seeds. Despite cloning of the complete suite of tocopherol biosynthetic enzymes and successful engineering of the tocopherol content and composition of Arabidopsis thaliana leaves and seeds, the functions of tocopherols in plants have remained elusive. To address this issue, we have isolated and characterized two VITAMIN E loci (VTE1 and VTE2) in Arabidopsis that when mutated result in tocopherol deficiency in all tissues. vte1 disrupts tocopherol cyclase activity and accumulates a redox-active biosynthetic intermediate, whereas vte2 disrupts homogentisate phytyl transferase activity and does not accumulate pathway intermediates. Mutations at either locus cause significantly reduced seed longevity compared with the wild type, indicating a critical role for tocopherols in maintaining viability during quiescence. However, only vte2 mutants exhibited severe seedling growth defects during germination and contained levels of lipid hydroperoxides and hydroxy fatty acids elevated up to 4- and 100-fold, respectively, relative to the wild type. These data demonstrate that a primary function of tocopherols in plants is to limit nonenzymatic lipid oxidation during seed storage, germination, and early seedling development. The vte mutant phenotypes also explain the strong selection for retention of tocopherol biosynthesis during the evolution of seed-bearing plants.

Figure 1.

The Tocopherol Pathway in Arabidopsis. The enzymes are numbered as follows: 1, hydroxyphenylpyruvate dioxygenase; 2, homogentisate phytyl transferase; 3, 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase; 4, tocopherol cyclase; 5, γ-tocopherol methyltransferase. The gray lines indicate the locations in the pathway blocked by vte1 and vte2. Bold arrows represent the steps leading to γ-tocopherol, the most abundant tocopherol produced in wild-type Arabidopsis seed. HPP, hydroxyphenylpyruvate; HGA, homogentisic acid; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinol; SAM, S-adenosyl l-Met.

Figure 2.

Germination of Wild-Type and Tocopherol-Deficient Arabidopsis Seeds. (A) Germination of unaged 2-month-old seeds. (B) Germination of 2-month-old seeds subjected to accelerated aging treatment (40°C for 72 h at 100% RH). Germination was defined as the emergence of the root radical and was scored daily. The zero day time point is seed that have been imbibed for 5 d at 4°C. Closed circle, Col (wild type); open circle, vte2-2 (Col); open triangle, vte1-1 (Col); closed square, Ws (wild type); open square, vte2-2 (Ws). The data in (A) and (B) are means, and error bars are sd (n = 4).

Figure 3.

Tocopherol-Deficient Mutants in Arabidopsis. Large panels show representative 6-d-old seedlings grown on media without a carbon source; bars = 5 mm. Top insets are 14-d-old soil-grown plants; bars = 5 mm. Bottom insets are representative cotyledons from 6-d-old soil-grown seedlings; bars = 2 mm. The arrowheads indicate defective cotyledons in the vte2 mutants. (A) Col (wild type). (B) Ws (wild type). (C) vte2-1 (Col). (D) vte2-2 (Ws). (E) vte1-1 (Col).

Figure 4.

Root Growth of Wild-Type and Tocopherol-Deficient Arabidopsis Seedlings. (A) Root growth without an exogenous carbon source. (B) Root growth in the presence of 2% sucrose. Root growth was measured each day in seedlings grown vertically on Petri plates containing 0.5× MS media with or without sucrose. Closed circle, Col (wild type); open circle, vte2-1 (Col); open triangle, vte1-1 (Col); closed square, Ws (wild type); open square vte2-2 (Ws). The data in (A) and (B) are means, and error bars are sd (n = 15).

Figure 5.

Fatty Acid Composition in Wild-Type and Tocopherol-Deficient Arabidopsis Seed and Seedlings. (A) mol % of 20:1 (eicosenoic acid). (B) mol % of 18:2 (linoleic acid). (C) mol % of 18:3 (linolenic acid). The zero day time point is seed that have been imbibed for 5 d at 4°C. Col (black bars); vte2-1 (right hatched bars); vte1-1 (white bars); Ws (gray bars); vte2-2 (left hatched bars). The differences between vte2 alleles and their respective wild-type backgrounds were statistically significant (Student's t test, ^* P < 0.05 and ^** P < 0.01). The data in (A) to (C) are means, and error bars are sd (n = 4).

Figure 6.

Lipid Peroxidation in Wild-Type and Tocopherol-Deficient Arabidopsis Seedlings. Lipid peroxidation in germinating seedlings as measured by the FOX assay (Griffiths et al., 2000). Hydrogen peroxide was used to construct a standard curve, and LOOH levels were expressed as picomole LOOHs per seedling (A) or picomole LOOHs per milligram of fresh weight (B). Closed circle, Col (wild type); open circle, vte2-1 (Col); open triangle, vte1-1 (Col); closed squre, Ws (wild type); open square, vte2-2 (Ws). The zero day time point is seed that have been imbibed for 5 d at 4°C. The differences between vte2 alleles and their respective wild-type backgrounds were statistically significant (Student's t test, P < 0.01) for all time points except zero days. Data are means, and error bars are sd (n = 4).

Figure 7.

LOH Levels in Wild-Type and Tocopherol-Deficient Arabidopsis Seedlings. Normal phase HPLC analysis (Feussner et al., 1995) of LOHs and LOOHs from saponified lipids of 6-d-old seedlings. The identity of peaks I, III, IV, and V from vte2-1 were confirmed by GC-MS. I, 13-HODE cis/trans; II, 13-HOTE; III, 13-HODE trans/trans; IV, 9-HODE cis/trans; V, 9-HODE trans/trans. mAU, milliabsorbance units. (A) Columbia, wild type (black line), vte1-1 (dashed line), and vte2-1 (gray line). Inset, chiral phase HPLC analysis (Feussner et al., 1995) to determine the enantiomeric composition of peak IV, 9-HODE cis/trans. The ratio of R and S enantiomers is indicated. (B) Ws, wild type (black line) and vte2-2 (gray line). Inset, chiral phase HPLC analysis (Feussner et al., 1995) to determine the enantiomeric composition of peak IV, 9-HODE cis/trans. The ratio of R and S enantiomers is indicated.