EPR detection of reactive oxygen in the photosynthetic apparatus of higher plants under light stress
╔va Hideg and Imre Vass

Institute of Plant Biology, Biological Research Center, Szeged, Hungary



INTRODUCTION

Plants performing oxygenic photosynthesis have developed a balanced system of enzymatic and non-enzymatic defence against reactive oxygen species (ROS), e.g. singlet oxygen, hydrogen peroxide or various oxygen free radicals. This way, although molecular oxygen and its highly reactive forms are continuously produced in illuminated chloroplasts, whose thylakoid membranes containing both highly unsaturated fatty acids – which can participate in free radical cascades –and excessive concentration of chlorophyll – a potential photosensitising dye –, the antioxidant system is usually sufficient to prevent damage under normal metabolic conditions. However, this balance is frequently disturbed in plants subject to unfavourable environmental conditions and/or pollutants. Activated oxygen has been implicated in the damage of plants upon numerous types of natural and artificial stress conditions (Asada et al 1994, Demmig-Adams and Adams 1994, Foyer et al 1994, Krause 1994, Hideg 1997).

Surprisingly, light — the obligatory driving force of higher plant photosynthesis — is among these stress factors. Under conditions when photochemically active radiation (PAR) is in excess, either due to unusually high intensity irradiation or as a consequence of lowered photon utilising capacity, reactive molecules capable of initiating membrane, protein and pigment damage are photoproduced. The complex set of these reactions is known as photoinhibition (PI, Powles 1984). If stress conditions are not severe, plants may prevent the above situation by facilitating energy utilisation, e.g. down regulating the energy input or by dissipating the excess energy (Allen 1992, Demmig-Adams and Adams 1992). Surplus PAR, however, may exhaust the adaptation systems (reviewed by Demmig-Adams 1992, Foyer and Harbison 1994, Krause 1994) and result in the overexcitation of photosynthesis. PI results in a net, in vivo decrease of photosynthetic activity. It is generally accepted, that the primary target of damage is photosystem (PS) II.

PS II is a pigment protein complex with a reaction centre consisting of a heterodimer of two membrane spanning proteins, D1 and D2. These either bind or contain the redox cofactors involved in the electron transport (Namba and Satoh 1987). The bound components are: the primary electron donor chlorophyll dimer (P680), the primary electron acceptor pheophytin (Pheo), the subsequent quinone electron acceptors QA and QB. Electrons from the catalytic cleavage of water by a manganese containing cluster bound to the lumenal side of D1/D2 are transferred to P680 via TyrZ, a redox active residue on D1. From P680, electrons are delivered to a mobile pool of plastoquinone molecules by subsequent redox reactions via Pheo, QA and QB.

Oxygen evolving thylakoid membrane, PS II and other sub-thylakoid membrane preparations provide good models for studying light stress. These in vitro studies have revealed the occurrence of two major routes, the so called acceptor side induced and donor side induced photoinhibition (API and DPI, respectively). Both API and DPI result in the impairment of PS II electron transport followed by the selective degradation of the D1 reaction centre protein (Mattoo et al 1984) and, to a lesser extent, of the D2 protein (Schuster et al 1988). Prolonged PI results in more general membrane damage, characterised by the appearance of lipid peroxidation products (Hundal 1992, Hideg et al 1994a) (Scheme 2). The two forms of PI are distinguished on the basis of differences in the primary site of electron transport malfunctioning, fragmentation pattern of the subsequent D1 protein degradation, as well as in the light intensity and oxygen requirement of the two process (for review see Aro et al 1993 and references therein). A third, alternative pathway of PI has been suggested to operate under low light intensities. ROS are also likely involved in this process, but their predicted amount is below the dection level of methods available at present (Keren et al 1995, 1997).
Both models of PI assume the formation of active oxygen (Aro et al 1993, Telfer and Barber 1994 and references therein). In API, which is caused by excess PAR in the presence of oxygen, singlet oxygen production has been predicted as a result of increased reaction centre chlorophyll triplet formation, which is a consequence of the non-physiological over-reduction of the first quinone electron acceptor in photosystem II (Vass and Styring 1992, Vass et al 1992, Aro et al 1993). DPI occurs when electron flow from water to P680 is insufficient. There is a consensus that the damage is triggered by the strong oxidants (P680+, TyrZ+) created by primary charge separation and whose lifetime is prolonged as a result of inoperative water splitting (Thompson and Brudwig, 1988, Telfer and Barber 1989). In such case, both electron transport and protein damage proceed in the absence of oxygen even upon illumination with relatively lower intensities of PAR (Jegersch÷ld and Styring, 1991).
Similarly to PI by excess PAR, UV-B (280-320 nm) irradiation causes a multitude of physiological and biochemical changes in plants, although these two types of light stress are different at several points. Increased doses of UV-B radiation reaching the Earth's surface as a consequence of stratospheric ozone depletion have increased interest in this form of light stress in the past decade. It is well established that UV-B results in the rapid inactivation of photosynthetic electron transport, altered pigment composition, destruction of the membrane structure and it may cause the dimerisation of thymine bases and lesions in DNA (for reviews see Tevini and Teramura 1989, Vass 1997). The increased synthesis of flavinoids (Bornman 1989) – effective quenchers of singlet oxygen, hydroxyl, superoxide and peroxy radicals – as well as the increased expression of genes for flavinoid biosynthesis (Strid 1993) imply the involvement of ROS in the process. In the thylakoid membrane, the primary target of UV-B is PS II: damage by UV-B involves functional impairment of PS II electron transport (Kulandaivelu and Noorudeen 1983, Renger et al 1989, Hideg et al 1993) and degradation of PS II reaction centre proteins, primarily D1 (Renger et al 1989, Greenberg et al 1989, Friso et al 1994a) and D2 (Friso et al 1994b).

Our in vitro studies have confirmed production of ROS in the above light stress conditions. The aim of the present work is to review these studies and to show possible outlooks on in vivo applications. Figures 4, 5 and 6 contain unpublished data, which will be published in the printed, journal version of the First Internet Conference on Photochemistry and Photobiology.