Photobleaching of Chlorophyll in Light-Harvesting Complex II Increases in Lipid Environment

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.

The rise and fall of the photoinhibition-related energy dissipation qI

“…The fact that the formation of the quenching site within the core of PSII precedes D1 cleavage leaves several options regarding the quenching mechanism, including non-photochemical energy dissipation and charge separation-based quenching. It was recently shown that protein oxidation events take place early upon photoinhibition (Kale et al, 2017) and that they can induce quenching in pigment-protein complexes (Lingvay et al, 2020); furthermore, it was observed that oxygen influences the F M level during photoinhibition (Gong and Ohad, 1991). We thus investigated whether oxygen is necessary for (1) the quenching itself, or (2) for the formation of the q I site.…”

“…Formation of 1 O 2 is well established upon photoinhibition and ROS have been shown to damage the protein scaffold (Kale et al, 2017). Pigment oxidation also results in a shortening of chlorophyll excited state lifetime (Lingvay et al, 2020). Finally, singlet oxygen concentration increases linearly with light intensity, as does the rate of q I formation (Fig.…”

Measuring ROS and redox markers in plant cells

“…RSC Chemical Biology in reconstituted membrane of pigment of light harvesting complex II from pea (Pisum sativum). 75 Spin traps Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid) and 4-POBN (a-(4-pyridyl-1-oxide)-N-tert-butylnitrone, Fig. 3B) have been used for O 2 À and OH detection respectively, in microsomal and thylakoid membranes, roots, and plant cell suspension culture.…”

“…DMPO has been successfully used to detect 1 O 2 production from extracted thylakoids 73 of spinach and derivatives of DMPO have been successfully implemented in plant tissues: DEPMPO, a phosphorylated analogue, forms a stable abduct with ˙OH in the apoplastic fluid of Zea mays roots, 74 while TEMPD (2,2,6,6-tetramethyl-4-piperidone monohydrate) forms an adduct with 1 O 2 to form paramagnetic 4-oxo-TEMPO and has been used to follow light induced 1 O 2 production in reconstituted membrane of pigment of light harvesting complex II from pea ( Pisum sativum ). 75 …”

Singlet oxygen damages the function of Photosystem II in isolated thylakoids and in the green alga Chlorella sorokiniana

“…The most lightsensitive site in the photosynthetic apparatus is the PSII complex in which electron transport activity is impaired and the D1 (and D2) reaction centre protein is degraded, see (Aro et al 1993). In addition to PSII, PSI (Sonoike and Terashima 1994;Sonoike et al 1995;Suorsa et al 2012;Tiwari et al 2016;Lima-Melo et al 2019), as well as the Chl containing light harvesting antenna structures can also be damaged by light (Zolla and Rinalducci 2002;Rinalducci et al 2004Rinalducci et al , 2008Lingvay et al 2020). The detrimental effects of light linearly depend on light intensity (Tyystjarvi and Aro 1996) and can occur at all light intensities (Keren et al 1997;Kou et al 2012), therefore plants have evolved a protective repair mechanism, by which light induced loss of photosynthetic activity can be restored.…”