The Solar Action Spectrum of Photosystem II Damage

The production of oxygen and the supply of energy for life on earth rely on the process of photosynthesis using sunlight. Paradoxically, sunlight damages the photosynthetic machinery, primarily photosystem II (PSII), leading to photoinhibition and loss of plant performance. However, there is uncertainty about which wavelengths are most damaging to PSII under sunlight. In this work we examined this in a simple experiment where Arabidopsis (Arabidopsis thaliana) leaves were exposed to different wavelengths of sunlight by dispersing the solar radiation across the surface of the leaf via a prism. To isolate only the process of photodamage, the repair of photodamaged PSII was inhibited by infiltration of chloramphenicol into the exposed leaves. The extent of photodamage was then measured as the decrease in the maximum quantum yield of PSII using an imaging pulse amplitude modulation fluorometer. Under the experimental light conditions, photodamage to PSII occurred most strongly in regions exposed to ultraviolet (UV) or yellow light. The extent of UV photodamage under incident sunlight would be greater than we observed when one corrects for the optical efficiency of our system. Our results suggest that photodamage to PSII under sunlight is primarily associated with UV rather than photosynthetically active light wavelengths.Plants absorb sunlight to power the productive photochemical reactions of photosynthesis. Absorption of sunlight may also lead to deleterious photochemistry that damages the photosynthetic machinery. The PSII protein complex is important in this regard as it seems to be most susceptible to photodamage that results in photoinhibition and ultimately suppresses photosynthetic CO₂ assimilation, growth, and productivity (Long et al., 1994; Takahashi and Murata, 2008). Although plants have photoprotection mechanisms (Niyogi, 1999) and can effectively repair photodamaged PSII through the PSII repair cycle (Aro et al., 1993), photoinhibition still occurs under stressful environmental conditions (Nishiyama et al., 2006; Murata et al., 2007; Takahashi and Murata, 2008).The onset of photoinhibition is strongly correlated with the absorption of excessive excitation energy for photosynthesis. Therefore, photodamage to PSII was most readily assumed to be attributed to the excess light absorbed by photosynthetic pigments (Melis, 1999). However, the extent of photodamage that is measured under conditions where the repair of photodamaged PSII is prevented by inhibiting chloroplast protein synthesis (i.e. lincomycin or chloramphenicol) is directly proportional to the intensity of light (Mattoo et al., 1984; Tyystjärvi and Aro, 1996; Nishiyama et al., 2001, 2004; Allakhverdiev and Murata, 2004; Chow et al., 2005). Furthermore, recent studies have demonstrated that interruption of the Calvin cycle (Hakala et al., 2005; Takahashi and Murata, 2005; Takahashi et al., 2007) and inhibition of electron transfer between Q_A and Q_B (Jegerschöld et al., 1990; Kirilovsky et al., 1994; Allakhverdiev et al., 2005) have no effect on the rate of photodamage to PSII, but in fact cause inhibition of the repair of photodamaged PSII due to suppression of the de novo synthesis of PSII proteins (Allakhverdiev et al., 2005; Takahashi and Murata, 2005, 2006). Thus, photodamage to PSII is paradoxically not associated with the excess light absorbed by photosynthetic pigments (Nishiyama et al., 2006; Murata et al., 2007; Takahashi and Murata, 2008).Studies of the effect of monochromatic light on the photodamage process have suggested that photodamage to PSII primarily occurs at the manganese cluster of the oxygen-evolving complex (OEC) through a direct photoexcitation of manganese (Hakala et al., 2005; Ohnishi et al., 2005). Release of manganese ions (Mn²⁺) from thylakoid membranes is accompanied by photodamage to PSII (Hakala et al., 2005; Zsiros et al., 2006), suggesting that disruption of the manganese cluster upon absorption of light might be a primary event in photodamage. It is likely that the reaction center of PSII is secondarily damaged by light absorbed by photosynthetic pigments after inactivation of the OEC (Hakala et al., 2005; Ohnishi et al., 2005), if an alternative electron transfer donor from lumenal ascorbate is not available (Mano et al., 2004; Tóth et al., 2009). These findings have lead to a recent photodamage model called the manganese (or two-step; Ohnishi et al., 2005) mechanism of photoinhibition (Tyystjärvi, 2008).Studies of the action spectrum of photodamage to PSII have shown that UV damages PSII more effectively than visible light (Jones and Kok, 1966; Jung and Kim, 1990; Hakala et al., 2005; Ohnishi et al., 2005). Thus, under identical light intensity, UV is the most damaging wavelength to PSII. However, inferring damage under natural sunlight is not straight forward as there is a need to account for the spectral distribution and intensity of sunlight. It is unclear which wavelengths of sunlight are most damaging to PSII and we cannot discount the premise that significant primary photodamage to PSII is caused by light absorbed by photosynthetic pigments (Vass and Cser, 2009). To identify which wavelengths of sunlight are most damaging to PSII, sunlight was spectrally dispersed via a prism onto an Arabidopsis (Arabidopsis thaliana) leaf infiltrated with chloramphenicol and decrease in the maximum quantum yield of PSII (F_v/F_m) was measured using an imaging pulse amplitude modulation (PAM) fluorometer. This simple but powerful approach revealed the in vivo spectral dependence of photodamage that had two peaks at UV and yellow wavelengths. Since the spectral efficiency of our optical system decreased below 400 nm, we calculated photodamage to PSII under incident sunlight. Our results show that photodamage to PSII was primarily associated with UV wavelengths and secondarily with yellow light wavelengths. This finding indicates that photodamage to PSII is less associated with light absorbed by photosynthetic pigments under sunlight and suggest that most of photodamage to PSII is potentially avoidable during photosynthesis.