Production and Scavenging of Reactive Oxygen Species in Chloroplasts and Their Functions

Abstract

The reaction centers of PSI and PSII in chloroplast thylakoids are the major generation site of reactive oxygen species (ROS). Photoreduction of oxygen to hydrogen peroxide (H2O2) in PSI was discovered over 50 years ago by Mehler (1951). Subsequently, the primary reduced product was identified to be superoxide anion (O2 −), and its disproportionation produces H2O2 and O2 (Asada et al., 1974). On the other hand, in PSII, oxygen of the ground (triplet) state (3O2) is excited to singlet state (1O2) by the reaction center chlorophyll (Chl) of triplet excited state (3P680*; Telfer et al., 1994; Hideg et al., 1998). The photoproduction of ROS is largely affected by physiological and environmental factors; the rate is enhanced under the conditions where photon intensity (P) is in excess of that required for the CO2 assimilation (A). Under the conditions of photon excess (P > A), the relaxation systems suppress the photoproduction of ROS in chloroplasts, such as photorespiration, the cyclic electron flows through either PSI or PSII, and the down-regulation of PSII quantum yield as regulated by the xanthophyll cycle and the proton gradient across thylakoid membrane. Prompt scavenging of the ROS produced in thylakoids prior to its diffusion from the generation site is indispensable to protect the target molecules in thylakoid and stroma. Here, the production of reduced and excited species of ROS and their scavenging system in chloroplasts are overviewed. The photoproduction and subsequent scavenging of ROS not only protect chloroplasts from the direct effects of ROS, but also relax the photon (electron) excess stress, and these physiological functions of ROS production and scavenging are discussed. The primary product of oxygen reduction, O2 − (Eq. 2), is disproportionated to H2O2 and O2 catalyzed with superoxide dismutase (SOD; Eq. 3). The H2O2 generated by SOD is reduced to water by ascorbate (AsA) catalyzed with APX, and AsA is oxidized to monodehydroascorbate radical (MDA; Eq. 4). Subsequently, MDA is directly reduced to AsA by either reduced ferredoxin (redFd; Miyake and Asada, 1994; Eq. 5) or NAD(P)H catalyzed with chloroplastic MDA reductase (Sano et al., 2005; Eq. 6). If MDA fails to reduce directly to AsA, it is spontaneously disproportionated to dehydroascorbate (DHA) and AsA. DHA is then reduced to AsA by reduced glutathione (GSH) catalyzed with DHA reductase (Shimaoka et al., 2003; Eq. 7). Finally, Fd and NADP+ for the regeneration of AsA (Eqs. 5–7) are reduced in PSI (Eq. 8). Thus, in any pathways of the regeneration of AsA, the half electrons derived from water in PSII (Eq. 1) are used for the univalent reduction of oxygen (Eq. 2) and another half for the generation of reductants (Eq. 8) to reduce H2O2 (Eqs. 4–7), which has been referred to as the water-water (W-W) cycle. Several plants contain Fe-SOD in addition to CuZn-SOD, but no Mn-SOD, in chloroplasts. In anaerobic bacteria, the reduced form of iron-sulfur protein, such as rubredoxin and neelaredoxin, can reduce O2 − to H2O2 (Abreu et al., 2001), but in chloroplasts no such iron-sulfur proteins are found. APX is classified as class I peroxidase similar to Cyt c peroxidase (CcP) and is different from class III peroxidase such as horseradish (Armoracia lapathifolia) peroxidase (Raven, 2003). Even though CcP and APX are classified in the same family, compound I of CcP is stable, but that of APX is not so stable in the absence of AsA (Miyake and Asada, 1996). Compound I of chloroplastic APX is more labile as compared with that of cytosolic APX (Amako et al., 1994). In contrast to plant APX, compound I of APX from a red alga is stable (Sano et al., 2001). APX is inhibited by thiol-modifying reagents, and the Cys residue participates in the binding of AsA to the heme (Raven, 2003). Chloroplastic APX is classified into thylakoid-bound (tAPX) and stroma-localized forms. tAPX binds in the vicinity of PSI (Miyake et al., 1993), and its anchor is the C-terminal, hydrophobic 50 residues. Except for Arabidopsis (Arabidopsis thaliana), a single gene codes tAPX and stroma-localized APX, and both isoforms are generated by alternative splicing (Yoshimura et al., 2002). Prokaryotes lack AsA and cyanobacteria scavenge H2O2 with the thioredoxin-peroxiredoxin (Prx) system (Yamamoto et al., 1999). Thioredoxin and Prx are localized in plant chloroplasts, but it is not known under what conditions this system functions as an alternative W-W cycle in place of APX. Prx Q associates to PSII complexes (Lamkemeyer et al., 2006). In addition to MDA, MDA reductase is able to reduce phenoxyl radicals, such as quercetin radicals to their parent phenols (Sakihama et al., 2000). Thus, this enzyme would participate also in the regeneration of antioxidants. In Arabidopsis, MDA reductase (Obara et al., 2001), stromal APX, and glutathione reductase gene products (Chew et al., 2003) have dual targeting into chloroplasts and mitochondria. In chloroplast stroma, no catalase has been found.