Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana

Abstract

Vitamin E is considered a major antioxidant in biomembranes, but little evidence exists for this function in plants under photooxidative stress. Leaf discs of two vitamin E mutants, a tocopherol cyclase mutant (vte1) and a homogentisate phytyl transferase mutant (vte2), were exposed to high light stress at low temperature, which resulted in bleaching and lipid photodestruction. However, this was not observed in whole plants exposed to long-term high light stress, unless the stress conditions were extreme (very low temperature and very high light), suggesting compensatory mechanisms for vitamin E deficiency under physiological conditions. We identified two such mechanisms: nonphotochemical energy dissipation (NPQ) in photosystem II (PSII) and synthesis of zeaxanthin. Inhibition of NPQ in the double mutant vte1 npq4 led to a marked photoinhibition of PSII, suggesting protection of PSII by tocopherols. vte1 plants accumulated more zeaxanthin in high light than the wild type, and inhibiting zeaxanthin synthesis in the vte1 npq1 double mutant resulted in PSII photoinhibition accompanied by extensive oxidation of lipids and pigments. The single mutants npq1, npq4, vte2, and vte1 showed little sensitivity to the stress treatments. We conclude that, in cooperation with the xanthophyll cycle, vitamin E fulfills at least two different functions in chloroplasts at the two major sites of singlet oxygen production: preserving PSII from photoinactivation and protecting membrane lipids from photooxidation.

Figure 1.

Photoinhibition and Photooxidation of Arabidopsis Leaf Discs Exposed to High Light Stress. Leaf discs (the wild type and vte1 mutant) were exposed to high light (1000 μmol photons m⁻² s⁻¹) at 10°C. Open circles, the wild type; closed circles, vte1. Data are mean values of three to five separate experiments ± sd, except when there is no error bar (two replicates). a.u., arbitrary units. (A) PSII photoinhibition was measured by the decrease in the Fv/Fm chlorophyll fluorescence ratio. Fv/Fm was ∼0 for times >15 h. (B) and (C) Photooxidative stress was estimated from the extent of lipid peroxidation measured by TL. (B) presents TL traces from wild-type and vte1 leaf discs exposed for 24 h to the high light stress (1000 μmol photons m⁻² s⁻¹/10°C), showing the lipid peroxidation–related band peaking at ∼130°C. The sharp peak at ∼65°C, which tends to disappear with oxidative stress, is specific to unstressed cruciferous plants. The origin of this band is unknown (Havaux, 2003).

Figure 2.

Photodestruction of Fatty Acids and Chlorophylls in Arabidopsis Leaf Discs Exposed to High Light Stress. Total fatty acid ([A]; in micrograms per leaf disk of 1.2 cm in diameter) and total chlorophyll ([B]; in μg cm⁻²) in leaf discs (the wild type and vte1 mutant) exposed to high light stress (1000 μmol photons m⁻² s⁻¹) at 10°C. Open circles, wild type; closed circles, vte1. Data are mean values of three separate experiments ± sd, except when there is no error bar (two replicates).

Figure 3.

Singlet Oxygen Toxicity in Arabidopsis Leaf Discs. Leaf discs (the wild type and vte1 mutant) floating on eosin were illuminated with white light of PFD 200 μmol m⁻² s⁻¹. Oxidative stress (lipid peroxidation) was monitored by TL. Open circles, wild type; closed circles, vte1. Each experimental point corresponds to a different sample. These data are representative of two independent experiments.

Figure 4.

Photoinhibition and Photooxidation of Arabidopsis Plants Exposed to High Light Stress at Low Temperature. PSII photoinhibition was measured by the decrease in the chlorophyll fluorescence ratio Fv/Fm, and photooxidation (lipid peroxidation) was measured by TL in wild-type and vte1 mutant plants (open and closed circles, respectively) exposed to high light stress at low temperature (8°C/1000 μmol photons m⁻² s⁻¹; photoperiod, 8 h). Data are mean values of a minimum of 10 measurements (Fv/Fm) or three measurements (TL). This experiment was done three times with qualitatively similar results. a, significantly different with P < 0.05 (t test).

Figure 5.

Photoinhibition and Photooxidation of Arabidopsis Plants Exposed to High Light Stress at High Temperature. PSII photoinhibition was measured by the decrease in the chlorophyll fluorescence ratio Fv/Fm, and photooxidation (lipid peroxidation) was measured by TL in wild-type and vte1 mutant plants (open and closed circles, respectively) exposed to high light stress at high temperature (30°C/1300 μmol photons m⁻² s⁻¹; photoperiod, 8 h). Data are mean values of a minimum of 10 measurements (Fv/Fm) or three measurements (TL) ± sd, except when there is no error bar (two replicates). This experiment was done three times with qualitatively similar results. a, significantly different values with P < 0.001 (t test).

Figure 6.

Accumulation of Zeaxanthin and Tocopherols in Wild-Type and vte1 Leaves Exposed to High Light Stress at Low or High Temperature. (A) Zeanthin in wild-type and vte1 leaves exposed to high light stress at low or high temperature (1000 μmol photons m⁻² s⁻¹ at 8°C [circles] or 1300 μmol photons m⁻² s⁻¹ at 30°C [triangles], respectively). (B) Tocopherols in wild-type and vte1 leaves exposed to high light stress at low or high temperature (1000 μmol photons m⁻² s⁻¹ at 8°C [circles] or 1300 μmol photons m⁻² s⁻¹ at 30°C [triangles], respectively). Samples were taken after 2-h illumination, and the measured concentrations of zeaxanthin corresponded to the steady state level of this xanthophyll. Closed symbols, vte1; open symbols, wild type. Tocopherol was not detected in vte1 under any conditions. Data are mean values of three to five separate experiments ± sd. a, significantly different from wild-type values under similar conditions using t test with P < 0.01. b, different from wild-type value under similar conditions in a t test with P < 0.12. HL, high light.

Figure 7.

NPQ in Wild-Type and vte1 Arabidopsis Leaves after High Light Stress at Low Temperature or after High Light at High Temperature. (A) NPQ in wild-type and vte1 Arabidopsis leaves after high light stress at low temperature (6°C and 1100 μmol m⁻² s⁻¹ for 6 d). (B) NPQ in wild-type and vte1 Arabidopsis leaves after high light at high temperature (30°C and 1500 μmol m⁻² s⁻¹). Data are mean values of three separate experiments ± sd. NPQ in unstressed wild-type leaves is also shown in (A). NPQ in unstressed vte1 leaves (data not shown) was very similar to NPQ in wild-type leaves.

Figure 8.

Protein Gel Blots of a Series of Enzymes Involved in the Protection of Thylakoid Membrane Lipids or Proteins against Oxidation. The following proteins were analyzed: PRXs PRXQ, BAS1, and PRXE II, the thioredoxin CDSP32, and the Msrs MsrA, MsrB1, and MsrB2. Control leaves (WTc and vte1c) are compared with leaves stressed for 7 d in high light at low temperature (WTs and vte1s). Amounts of soluble protein loaded were as follows: PRXQ, 25 μg; BAS1, 4 μg; PRXEII-E, 4 μg; CDSP32, 25 μg; MsrBs and MsrA, 25 μg.

Figure 9.

Wild-Type, vte1, npq1, and vte1 npq1 Plants Grown for 7 d in High Light at Low Temperature.

Figure 10.

Photoinhibition and Photooxidation of Wild-Type, vte1, npq1, or npq1 vte1 Leaves during Growth of Whole Plants in High Light at Low Temperature. PSII photoinhibition as measured by the Fv/Fm (A) and photooxidative stress measured by thermoluminescence (B). Open circles, wild type; closed circles, vte1 npq1; triangles, vte1; squares, npq1. Number of replicates was 10 for (A) and 3 for (B). a and b, significantly different with P < 0.001 and P < 0.003, respectively (t test).

Figure 11.

Total Fatty Acid, MDA, and Total Chlorophyll in Leaves of Wild-Type, vte1, npq1, and vte1 npq1 Plants Grown for 7 d in High Light at Low Temperature. (A) Total fatty acid. (B) MDA. (C) Total chlorophyll. Open bars, before stress; closed bars, after stress. The fatty acid content was not measured in npq1 leaves. Data are mean values of three separate experiments ± sd. a, significantly different from all the other mean values with P < 0.03 (t test)

Figure 12.

Protein Gel Blots of the PSII Reaction Center Protein D1 in Wild-Type, vte1, vte1 npq1, and npq1 Leaves Grown for 0 or 7 d in High Light at Low Temperature. (A) A Coomassie blue–stained gel showing equal protein loading. (B) Protein gel blot of the PSII reaction center protein D1. (C) Relative intensity of the D1 bands measured in four different protein gel blots. a, significantly different from control value, t test with P < 0.01. Amount of thylakoid membrane proteins loaded for the D1 blot was 4.5 μg. Data are mean values of four separate experiments ± sd. DM, vte1 npq1; c, control; s, high light.

Figure 13.

Photoinhibition and Photooxidation of Wild-Type, vte1 npq1, and vte1 npq4 Leaves during Growth of Whole Plants in High Light at Low Temperature. (A) PSII photoinhibition as measured by the Fv/Fm chlorophyll fluorescence parameter. Data are mean values of 8 to 10 measurements ± sd. Open circles, wild type; closed circles, vte1 npq1; closed triangles, vte1 npq4. a, significantly different with P < 0.001 (t test). (B) Lipid peroxidation, as measured by the high-temperature TL band, in vte1 npq1 and vte1 npq4 leaves after 7 d in high light at low temperature. (C) vte1 npq4 plants after 7 d in high light at low temperature.