Photoinhibition is light-induced inactivation of PSII. Hypotheses about the photoreceptor(s) of photoinhibition include the Chl antenna of PSII, manganese of the oxygen-evolving complex (OEC), uncoupled Chl and iron-sulfur centres. We measured the action spectrum of photoinhibition in vivo from wild-type Arabidopsis thaliana L. and from the npq1-2 and npq4-1 mutants defective in non-photochemical quenching (NPQ) of excitations of the PSII antenna. The in vivo action spectrum was found to resemble closely the in vitro action spectra published for photoinhibition. We compared the action spectrum with absorbance spectra of model compounds of the OEC complex and other potential photoreceptors of photoinhibition. The comparison suggests that both manganese and Chl function as photoreceptors in photoinhibition. In accordance with the function of two types of photoreceptors in photoinhibition, NPQ was found to offer only partial protection against photoinhibition at visible wavelengths. The low protective efficiency of NPQ supports the conclusion that the Chl antenna of PSII is not the only photoreceptor of photoinhibition. Comparison of the action spectrum of photoinhibition with the emission spectrum of sunlight shows that the UV part of sunlight is responsible for the major part of photoinhibition under natural conditions.

Recombination of the primary radical pair of photosystem II (PSII) of photosynthesis may produce the triplet state of the primary donor of PSII. Triplet formation is potentially harmful because chlorophyll triplets can react with molecular oxygen to produce the reactive singlet oxygen ( 1 O 2 ). The yield of 1 O 2 is expected to be directly proportional to the triplet yield and the triplet yield of charge recombination can be lowered with a magnetic field of 100-300 mT. In this study, we illuminated intact pumpkin leaves with strong light in the presence and absence of a magnetic field and found that the magnetic field protects against photoinhibition of PSII. The result suggests that radical pair recombination is responsible for significant part of 1 O 2 production in the chloroplast. The magnetic field effect vanished if leaves were illuminated in the presence of lincomycin, an inhibitor of chloroplast protein synthesis, or if isolated thylakoid membranes were exposed to light. These data, in turn, indicate that 1 O 2 produced by the recombination of the primary charge pair is not directly involved in photoinactivation of PSII but instead damages PSII by inhibiting the repair of photoinhibited PSII. We also found that an Arabidopsis thaliana mutant lacking α-tocopherol, a scavenger of 1 O 2 , is more sensitive to photoinhibition than the wild-type in the absence but not in the presence of lincomycin, confirming that the target of 1 O 2 is the repair mechanism.

Photoinhibition of Photosystem II (PSII) in lincomycin-treated leaves begins as a first-order reaction, but fluorescence measurements have suggested that after prolonged illumination, the number of active PSII centres stabilizes to 15-20% of control. The stabilization has been interpreted to indicate that photoinhibited PSII centres protect the remaining active centres against photoinhibition (Lee, Hong and Chow, Planta 212:332-342, 2001). In an attempt to study the mechanism of this protection, we measured the reaction kinetics of photoinhibition in lincomycin-treated pumpkin (Cucurbita pepo L.) and pepper (Capsicum annuum L.) leaves in vivo. The light-saturated rate of PSII oxygen evolution, assayed from thylakoids and isolated from the treated leaves, was used as a direct measure of the number of remaining active PSII centres, and the fluorescence parameters F (V)/F (M) and (F (V)/F (M))/F (0) (=1/F (0) - 1/F (M)) were measured for comparison. To our surprise, no stabilization of PSII activity was observed and photoinhibition followed first-order kinetics until PSII activity had virtually declined to zero. A series of in vitro experiments was carried out to see whether stabilization of PSII activity occurs if a particular combination of light intensity and wavelength range is applied, or if a specific PSII preparation is used as experimental material. The results of the in vitro experiments confirmed the in vivo result about persistent first-order kinetics. We conclude that photoinhibited PSII centres offer no measurable protection against photoinhibition.

The light response curves of the acceptor and donor side mechanisms of photoinhibition of Photosystem II were calculated, using Arabidopsis as a model organism. Acceptor-side photoinhibition was modelled as double reduction of QA, noting that non-photochemical quenching has the same effect on the quantum yield of QA double reduction in closed PSII centres as it has on the quantum yield of electron transport in open centres. The light response curve of acceptor-side photoinhibition in Arabidopsis shows very low efficiency under low intensity light and a relatively constant quantum yield above light saturation of photosynthesis. To calculate the light response curve of donor-side photoinhibition, we built a model describing the concentration of the oxidized primary donor P680 + during steady-state photosynthesis. The model is based on literature values of rate constants of electron transfer reactions of PSII, and it was fitted with fluorescence parameters measured in the steady state. The modelling analysis showed that the quantum yield of donor-side photoinhibition peaks under moderate light. The deviation of the acceptor and donor side mechanisms from the direct proportionality between photoinhibition and photon flux density suggests that these mechanisms cannot solely account for photoinhibition in vivo, but contribution of a reaction whose quantum yield is independent of light intensity is needed. Furthermore, a simple kinetic calculation suggests that the acceptor-side mechanism may not explain singlet oxygen production by photoinhibited leaves. The theoretical framework described here can be used to estimate the yields of different photoinhibition mechanisms under different wavelengths or light intensities.

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S(0)-S(4)), and dark incubation leads to 25% S(0) and 75% S(1). This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor Q(B)(-). We measured flash-induced oxygen evolution to understand how S(3) and S(2) are converted to lower S states when the electron required to reduce the manganese cluster does not come from Q(B)(-). Thylakoid samples preconditioned to make the concentration of the S(1) state 100% and to oxidize tyrosine Y(D) were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S(2) and S(3) are converted to lower S states via recombination from S(2)Q(B)(-) and S(3)Q(B)(-) and by a slow change of the state of oxygen-evolving complex from S(3) and S(2) to S(1) and S(0) in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S(2)Q(B) -->S(0)Q(B), and S(3)Q(B) -->S(1)Q(B). The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S(3)Q(B) -->S(2)Q(B) -->S(1)Q(B) in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H(2)O(2) acts as an electron donor for the reaction S(2)Q(B) -->S(0)Q(B) but added H(2)O(2) did not enhance this reaction.

Lincomycin-treated pumpkin leaves were illuminated with either continuous light or saturating single-turnover xenon flashes to study the dependence of photoinactivation of photosystem II (PSII) on the mode of delivery of light. The flash energy and the time interval between the flashes were varied between the experiments, and photoinactivation was measured with oxygen evolution and the ratio of variable to maximum fluorescence (Fv/Fm). The photoinhibitory efficiency of saturating xenon flashes was found to be directly proportional to flash energy and independent of the time interval between the flashes. These findings indicate that a low-light-specific mechanism, based on charge recombination between PSII electron acceptors and the oxygen-evolving complex, is not the main cause of photoinactivation caused by short flashes in vivo. Furthermore, the relationship between the rate constant of photoinactivation and photon flux density was similar for flashes and continuous light when Fv/Fm was used to quantify photoinactivation, suggesting that continuous-light photoinactivation has a mechanism in which the quantum yield does not depend on the mode of delivery of light. A similar quantum yield of photoinhibition for flashes and continuous light is compatible with the manganese-based photoinhibition mechanism and with mechanisms in which singlet oxygen, produced via a direct photosensitization reaction, is the agent of damage. However, the classical acceptor-side and donor-side mechanisms do not predict a similar quantum yield for flashes and continuous light.