Analysis of OJIP transients during photoinactivation of photosystem II indicates the presence of multiple photosensitizers in vivo and in vitro

Generally, excessive excitation absorbed by the pigments is considered the cause of PSII photodamage. Previous studies of action spectra of PSII photodamage concluded that shorter wavelengths induce more damage, supporting the hypothesis of the existence of more than one photosensitizer. However, the relative influence of different photosensitizers is still inconclusive. In this work, we have revisited this question by inducing PSII photodamage in vivo and in vitro at two different wavelengths (460 and 660 nm) where the net absorption cross section was the same using equal irradiance. To correlate PSII photodamage with each wavelength band, we followed its time course using the OJIP transient of the chlorophyll fluorescence to determine the possible contributions of photoinhibition by different photosensitizers. We found evidence that at least two sites of photoinactivation of PSII exist. Additional key words: absorptance; chlorophyll fluorescence; manganese cluster; photodamage; spinach; two-photon excitation microscopy. Received 28 August 2019, accepted 6 December 2019. +Corresponding author; e-mail: alonso.zavaleta@anu.edu. au Abbreviations: Chl – chlorophyll; HWHM – half width half maximum; LED – light-emitting diodes; NPQ – nonphotochemical quenching; ROS – reactive oxygen species; UV-Vis – ultraviolet and visible. Acknowledgements: This research was financed in part by the BioSolar Cells open innovation consortium, supported by the Dutch Ministry of Economic Affairs, Agriculture and Innovation (I. Iermak). A. Zavafer acknowledges Australian Research Council (Discovery grant DP120100872). A. Zavafer is grateful to his PhD sponsors National Council of Science and Technology of Mexico (CONACYT) and the Mexican Secretariat of Public Education (SEP). A.Zavafer and I. Iermak are grateful to Mr. John Philippi for all the technical help during the development of this project and to Prof. Reto J. Strasser for providing an M-PEA fluorometer. A. Zavafer thanks Prof. Shunichi Takahashi for supplying the LED boxes. M. Szabó acknowledges funding from the Hungarian Academy of Sciences (PREMIUM-2017-38) and The National Research, Development and Innovation Office (NKFI FK 128977). The authors would like to thank Prof. Eldon Ball for help in improving this work. and Murata 2008, Oguchi et al. 2011b, Vass 2011, 2012; Schreiber and Klughammer 2013, Tyystjärvi 2013) that: (1) the most affected component of photosynthesis is PSII (Ohad et al. 1990, Aro et al. 1993); (2) the photodamage rate (usually expressed as the rate coefficient kPI) is directly proportional to irradiance (Tyystjärvi and Aro 1996); (3) UV wavelengths induce preferential damage to the Mn4O5Ca cluster of PSII (Hakala et al. 2005); (4) the quantum efficiency of photodamage is higher at shorter wavelengths (Zavafer et al. 2015a); and (5) after the initial event of photodamage (inactivation of the PSII), the subunit D1 has to be degraded and a new D1 synthesized in order to recover photosynthetic activity (Ohad et al. 1990, Aro et al. 1993). Available hypotheses can be classified based on photoIntroduction Plants depend on light to survive but light absorption has the side effect of photodamage to the photosynthetic machinery (van Gorkom and Schelvis 1993). Photodamage correlates directly with the light irradiance, which means that even at low irradiances there is still some degree of photodamage (Tyystjärvi and Aro 1996, Oguchi et al. 2011a,b; 2013). For example, Tyystjärvi and Aro (1996) demonstrated that the rate of photoinhibition at 100 μmol(photon) m–2 s–1 is 10% of the rate of photoinhibition at 1,000 μmol(photon) m–2 s–1. While the exact mechanism of PSII photodamage is far from established, there is a consensus (Melis 1999, Nishiyama et al. 2006, Murata et al. 2007, Takahashi