Detecting Singlet Oxygen Production in Leaves Under Stress

Abstract

The primary target of photoinhibition (PI) by excess photosynthetically active radiation (PAR) is the reaction centre of the Photosystem (PS) II complex of the photosynthetic apparatus which is located in the thylakoid membrane of higher plant chloroplasts. PI has been extensively studied in vitro, in isolated membrane preparations and a definitive sequence of events was agreed on. In this model, excess PAR leads to impairment of PS II electron transport which is followed by selective degradation of the D1 PS II reaction centre protein and by more general membrane protein and lipid damage [1–3]. Direct observation of reactive oxygen species by spin trapping EPR spectroscopy demonstrated that photoinhibition by excess PAR is indeed an oxidative stress [4–6]. One among the particular pathways of damage is recognised as acceptorside-induced PI, which occurs when photosynthetically active, oxygen evolving preparations are illuminated with excess PAR in the presence of oxygen. In this process, double reduction of the first PS II quinone acceptor QA results in increased reaction centre chlorophyll triplet formation [7] and, consequently, in singlet oxygen production. There are strong indications that the above reactive oxygen species are involved in the specific cleavage of the D1 protein: singlet oxygen generating substances cause Dl protein fragmentation to specific fragments as does acceptor-side-induced PI by excess PAR [8, 9]. Confirming this model, singlet oxygen was detected in vitro, in photoinhibited thylakoid membranes by spin trapping EPR spectroscopy [4, 10]. Recently, we found evidence for singlet oxygen production in broad bean leaves that were photoinhibited in vivo [11]. In these leaves, singlet oxygen production was proportional to the number of functionally impaired PS II reaction centres, providing direct experimental evidence that PI in vivo is, at least partly, governed by the process characterised as acceptor-side-induced mechanism in vitro.