The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism

Abstract

When light energy absorbed by plants becomes excessive relative to the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly deepoxidized to zeaxanthin (violaxanthin cycle). The protective function of this phenomenon was investigated in a mutant of Arabidopsis thaliana, npq1, that has no functional violaxanthin deepoxidase. Two major consequences of the npq1 mutation are the absence of zeaxanthin formation in strong light and the partial inhibition of the quenching of singlet excited chlorophylls in the photosystem II light-harvesting complexes. Prolonged exposure of whole plants to bright light resulted in a limited photoinhibition of photosystem II in both npq1 and wild-type leaves, although CO(2) fixation and the linear electron transport in npq1 plants were reduced substantially. Lipid peroxidation was more pronounced in npq1 compared with the wild type, as measured by chlorophyll thermoluminescence, ethane production, and the total hydroperoxy fatty acids content. Lipid peroxidation was amplified markedly under chilling stress, and photooxidative damage ultimately resulted in leaf bleaching and tissue necrosis in npq1. The npq4 mutant, which possesses a normal violaxanthin cycle but has a limited capacity of quenching singlet excited chlorophylls, was rather tolerant to lipid peroxidation. The double mutant, npq4 npq1, which differs from npq4 only by the absence of the violaxanthin cycle, exhibited an increased susceptibility to photooxidative damage, similar to that of npq1. Our results demonstrate that the violaxanthin cycle specifically protects thylakoid membrane lipids against photooxidation. Part of this protection involves a mechanism other than quenching of singlet excited chlorophylls.

Figure 1

V cycle and NPQ in the npq1 mutant of A. thaliana. (A) Conversion of V to A and Z, as monitored by the (A + Z)/(A + Z + V) ratio, in leaves of the wild type and npq1 mutant suddenly exposed to a strong, white light of PFD of 1,500 μmol of photons·m⁻²·s⁻¹. (B) Quenching of F_m induced by the strong, white light and subsequent relaxation of the fluorescence quenching in the dark. The F_o and F_m fluorescence levels at time 0 were identical in the wild type and the mutant.

Figure 2

Inhibition of PSII photochemistry and of photosynthetic CO₂ fixation during strong light stress. (A) F_v/F_m during a strong light stress (1,500 μmol of photons·m⁻²·s⁻¹) of wild-type and npq1 Arabidopsis leaves. The hatched areas indicate the night periods. Each experimental point is the mean value of 4–15 measurements. Chl fluorescence was measured only on green leaves or the green parts of partially bleached npq1 leaves (see Fig. 3). (A + Z)/(V + A + Z) values: wild type, 0.07 and 0.53 before and after light stress (60 hr); npq1, 0 and 0.025 before and after light stress (60 hr). (B) Net photosynthetic CO₂ fixation by one Arabidopsis plant (▵, wild type; ▴, npq1 mutant) exposed to strong, white light. At time 0, plants were shifted from 300 μmol of photons·m⁻²·s⁻¹ to 1,500 μmol of photons·m⁻²·s⁻¹, and the rate of CO₂ fixation was measured every day during 1 hr from 11:30 a.m. to 12:30 p.m. Transpiration of npq1 (●) and wild-type (○) plants was comparable, indicating that stomatal closure was not responsible for the lower CO₂ fixation activity of npq1.

Figure 3

Wild-type and npq1 mutant plants after 4 days at 1,600 μmol of photons·m⁻²·s⁻¹.

Figure 4

Lipid peroxidation in wild-type and npq1 leaves during strong light stress. (A) Typical TL curve of an Arabidopsis leaf (wild type), showing the lipid-peroxidation-related TL band at ca. 135°C. (B) Amplitude of the 135°C TL band during a strong light treatment (1,500 μmol of photons·m⁻²·s⁻¹) of wild-type and npq1 Arabidopsis leaves. The gray areas indicate the night periods. Each experimental point corresponds to one TL measurement performed on six discs taken from different leaves. (C) Comparison of the effects of a strong light treatment (3 days at 1,500 μmol of photons·m⁻²·s⁻¹) on the amplitude of the 135°C TL band, the production of ethane, and the accumulation of hydroperoxy fatty acids (HFA) in npq1 and wild-type Arabidopsis leaves. Open bars, before light stress; solid bars, after light stress. Data are mean values of three to four separate experiments. See legend of Fig. 2 for (A + Z)/(V + A + Z) data.