Alterations in tocopherol cyclase activity in transgenic and mutant plants of Arabidopsis affect tocopherol content, tocopherol composition, and oxidative stress

Abstract

Tocopherol belongs to the Vitamin E class of lipid soluble antioxidants that are essential for human nutrition. In plants, tocopherol is synthesized in plastids where it protects membranes from oxidative degradation by reactive oxygen species. Tocopherol cyclase (VTE1) catalyzes the penultimate step of tocopherol synthesis, and an Arabidopsis (Arabidopsis thaliana) mutant deficient in VTE1 (vte1) is totally devoid of tocopherol. Overexpression of VTE1 resulted in an increase in total tocopherol of at least 7-fold in leaves, and a dramatic shift from alpha-tocopherol to gamma-tocopherol. Expression studies demonstrated that indeed VTE1 is a major limiting factor of tocopherol synthesis in leaves. Tocopherol deficiency in vte1 resulted in the increase in ascorbate and glutathione, whereas accumulation of tocopherol in VTE1 overexpressing plants led to a decrease in ascorbate and glutathione. Deficiency in one antioxidant in vte1, vtc1 (ascorbate deficient), or cad2 (glutathione deficient) led to increased oxidative stress and to the concomitant increase in alternative antioxidants. Double mutants of vte1 were generated with vtc1 and cad2. Whereas growth, chlorophyll content, and photosynthetic quantum yield were very similar to wild type in vte1, vtc1, cad2, or vte1vtc1, they were reduced in vte1cad2, indicating that the simultaneous loss of tocopherol and glutathione results in moderate oxidative stress that affects the stability and the efficiency of the photosynthetic apparatus.

Figure 1.

Tocopherol synthesis in Arabidopsis. p-Hydroxyphenylpyruvate derived from the shikimate pathway is converted to homogentisate by action of HPPD. Attachment of a phytyl group by HPT1 (VTE2) results in the synthesis of MPQ. After methylation (VTE3), the second ring is formed by cyclization of DMPQ via the VTE1 reaction. The product of this reaction, γ-tocopherol, is converted to α-tocopherol by γ-TMT (VTE4). Side activities of VTE1 and γ-TMT in leaves resulting in the production of δ-tocopherol and β-tocopherol are indicated by dashed arrows.

Figure 2.

Overexpression of VTE1 results in accumulation of γ-tocopherol in leaves. The cDNA (under control of the cauliflower mosaic virus 35S promoter, 35VTE1) or genomic DNA of VTE1 (gVTE1) was introduced into Col-2 wild-type or vte1 mutant plants as indicated. A, The amounts of different forms of tocopherol (α, β, γ, δ) were determined by fluorescence HPLC. The numbers above the bars indicate total amount of tocopherol (nmol g⁻¹ fresh weight). Data show mean ± se of three experiments. B, HPLC chromatogram of tocopherols extracted from wild-type leaves. The numbers indicate tocopherol composition in percent. C, HPLC chromatogram showing tocopherol composition of line WT-35VTE1 number 40 with overexpression of the VTE1 cDNA under control of the 35S promoter. D, HPLC chromatogram of tocopherol and tocol standards.

Figure 3.

Expression of HPT1 and VTE1 in VTE1 overexpression lines. A, Expression of VTE1 was measured by western analysis with anti-VTE1 antiserum. B, Accumulation of mRNA in transgenic lines was measured by northern hybridization using VTE1 cDNA as a probe. C, HPT1 mRNA was detected after hybridization with a HPT1 probe amplified from genomic DNA by PCR. D, 25S rRNA bands of the northern gel after ethidium bromide staining.

Figure 4.

Accumulation of tocopherol and induction of VTE1 under high light stress and in oxidative stress mutants. A, Total tocopherol was determined by fluorescence HPLC. The black and the gray parts of the stacked bars indicate γ-tocopherol and α-tocopherol, respectively. The amounts of β-tocopherol and δ-tocopherol were below detection limit. Data represent the means and se of at least five measurements each. Asterisk, Total amount of tocopherol is significantly different to wild type after t test analysis (P < 0.05). B, Induction of VTE1 expression in Arabidopsis plants under high light conditions and in the antioxidant mutants vtc1 and cad2 as determined by northern analysis. C, 25S rRNA (photo of northern gel) as loading control.

Figure 5.

The growth phenotype of antioxidant mutants. A, Growth curves were recorded by measuring fresh weight of the aerial part of the plants wild type (WT), vte1, vtc1, and vte1vtc1 (mean ± se of five experiments). B, Growth curves for WT, vte1, cad2, and vte1cad2.

Figure 6.

Glutathione and ascorbate content in antioxidant mutants. A, Dehydroascorbate and ascorbate and (C) total glutathione were measured in leaves of 5-week-old single and double mutant plants in a VTE1 overexpression line (WT 35VTE1) and in vte1 carrying a genomic DNA construct (vte1 gVTE1). B, The redox status of ascorbate is presented as the ratio of ascorbate to the sum of ascorbate and dehydroascorbate. D, The redox status of glutathione is indicated by the ratio of (total glutathione − 2 × GSSG) to total glutathione. Data represent the means and se of at least five measurements each. Asterisk or white circle indicates significantly different to wild type or vte1, respectively, after t test analysis (P < 0.05).

Figure 7.

Light response curves of fluorescent ΦPSII and NPQ ΦPSII (A and B) and NPQ (C, D, and E) were determined by chlorophyll fluorescence analysis. Data represent mean ± se derived from fluorescence measurements of at least five leaves each. The following lines were analyzed: Wild-type Col-2, vte1 single mutant (Col-2 background), vtc1 single mutant (Col-0), cad2 single mutant (in Col-0), vte1vtc1 double mutant, vte1cad2 double mutant, and vte1 transformed with a genomic fragment containing the entire VTE1 gene.

Figure 8.

Photosynthetic pigment content in antioxidant mutants. A, Total chlorophyll in leaves was measured spectrophotometrically. The numbers indicate the chlorophyll a to chlorophyll b ratio. B, The content of neoxanthin, lutein, β-carotene, and xanthophyll cycle components (violaxanthin, antheraxanthin, zeaxanthin; V+A+Z) in leaves was determined by HPLC analysis. Data represent the mean and se of at least five experiments. Asterisk, white circle, or black circle indicates significantly different as compared to wild type, vte1 or cad2, respectively, after t test analysis (P < 0.05).