EPR detection of reactive oxygen in the photosynthetic apparatus of
higher plants under light stress
Éva 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 (Jegerschöld 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.
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