Abstract
Excess light causes damage to the photosynthetic apparatus of plants and algae primarily via reactive oxygen species. Singlet oxygen can be formed by interaction of chlorophyll (Chl) triplet states, especially in the Photosystem II reaction center, with oxygen. Whether Chls in the light-harvesting antenna complexes play direct role in oxidative photodamage is less clear. In this work, light-induced photobleaching of Chls in the major trimeric light-harvesting complex II (LHCII) is investigated in different molecular environments – protein aggregates, embedded in detergent micelles or in reconstituted membranes (proteoliposomes). The effects of intense light treatment were analyzed by absorption and circular dichroism spectroscopy, steady-state and time-resolved fluorescence and EPR spectroscopy. The rate and quantum yield of photobleaching was estimated from the light-induced Chl absorption changes. Photobleaching occurred mainly in Chl a and was accompanied by strong fluorescence quenching of the remaining unbleached Chls. The rate of photobleaching increased by 140% when LHCII was embedded in lipid membranes, compared to detergent-solubilized LHCII. Removing oxygen from the medium or adding antioxidants largely suppressed the bleaching, confirming its oxidative mechanism. Singlet oxygen formation was monitored by EPR spectroscopy using spin traps and spin labels to detect singlet oxygen directly and indirectly, respectively. The quantum yield of Chl a photobleaching in membranes and detergent was found to be 3.4 × 10–5 and 1.4 × 10–5, respectively. These values compare well with the yields of ROS production estimated from spin-trap EPR spectroscopy (around 4 × 10–5 and 2 × 10–5). A kinetic model is proposed, quantifying the generation of Chl and carotenoid triplet states and singlet oxygen. The high quantum yield of photobleaching, especially in the lipid membrane, suggest that direct photodamage of the antenna occurs with rates relevant to photoinhibition in vivo. The results represent further evidence that the molecular environment of LHCII has profound impact on its functional characteristics, including, among others, the susceptibility to photodamage.
Highlights
Plants have to cope with variable light conditions – maintaining efficient light harvesting while avoiding photodamage (Li et al, 2009)
The formation of singlet oxygen (1O2) during light exposure of chloroplast thylakoid membranes has been directly followed by spin-trapping electron paramagnetic resonance (EPR) spectroscopy and associated with the acceptor-side inhibition of photosystem II (PSII) and the D1 protein degradation (Hideg et al, 1994a,b)
Photobleaching of Chls in light-harvesting complex II (LHCII) in different molecular environments was observed by monitoring the changes in absorption in the course of irradiation with intense white light
Summary
Plants have to cope with variable light conditions – maintaining efficient light harvesting while avoiding photodamage (Li et al, 2009). Prolonged exposure to excess light causes photoinhibition, that is decrease in photosynthetic activity, followed by chlorosis – bleaching of chlorophylls (Chl) – and death. Most of the Chls are located in the light-harvesting antenna, including the core antenna, CP43 and CP47, and LHCII monomers and trimers (van Amerongen and Croce, 2013). It is believed that the antenna has negligible role in the production of ROS because the 3Chl states are effectively quenched by carotenoids (Cars) bound to the complexes (Breton et al, 1979; Sonneveld et al, 1979; Frank and Cogdell, 1996). The formation of 1O2 during light exposure of chloroplast thylakoid membranes has been directly followed by spin-trapping EPR spectroscopy and associated with the acceptor-side inhibition of PSII and the D1 protein degradation (Hideg et al, 1994a,b)
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