The Mechanism of Photoinhibition in Vivo: Re-Evaluation of the Roles of Catalase, Α-Tocopherol, Non-Photochemical Quenching, and Electron Transport☆

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Biochimica et Biophysica Acta 1817 (2012) 1127–1133

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Review The mechanism of photoinhibition in vivo: Re-evaluation of the roles of catalase, α-tocopherol, non-photochemical quenching, and electron transport☆

Norio Murata a, Suleyman I. Allakhverdiev b,c, Yoshitaka Nishiyama d,e,⁎

a National Institute for Basic Biology, Okazaki 444–8585, Japan b Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, Moscow 127276, Russia c Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia d Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338–8570, Japan e Institute for Environmental Science and Technology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338–8570, Japan

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Article history: Photoinhibition of photosystem II (PSII) occurs when the rate of light-induced inactivation (photodamage) of Received 22 December 2011 PSII exceeds the rate of repair of the photodamaged PSII. For the quantitative analysis of the mechanism of Received in revised form 11 February 2012 photoinhibition of PSII, it is essential to monitor the rate of photodamage and the rate of repair separately Accepted 17 February 2012 and, also, to examine the respective effects of various perturbations on the two processes. This strategy has Available online 24 February 2012 allowed the re-evaluation of the results of previous studies of photoinhibition and has provided insight into the roles of factors and mechanisms that protect PSII from photoinhibition, such as catalases and perox- Keywords: fi α fi Photoinhibition idases, which are ef cient scavengers of H2O2; -tocopherol, which is an ef cient scavenger of singlet oxy- Photosystem II gen; non-photochemical quenching, which dissipates excess light energy that has been absorbed by PSII; Protein synthesis and the cyclic and non-cyclic transport of electrons. Early studies of photoinhibition suggested that all of Reactive oxygen species these factors and mechanisms protect PSII against photodamage. However, re-evaluation by the strategy Repair mentioned above has indicated that, rather than protecting PSII from photodamage, they stimulate protein synthesis, with resultant repair of PSII and mitigation of photoinhibition. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. © 2012 Elsevier B.V. All rights reserved.

1. Introduction If we are to understand the full details of photoinhibition, we need to monitor the processes of photodamage and repair separately so that we Light is essential for photosynthesis but excess light energy is del- can identify the effects of light on each process (Fig. 1). Methods for the eterious to the photosynthetic machinery. When photosynthetic or- separate monitoring of photodamage and repair have been established ganisms are exposed to strong light, the activity of photosystem II in cyanobacteria [9,10] and in plants [5,11,12]. Photodamage can be (PSII) declines rapidly and this phenomenon is referred to as the monitored in the presence of an appropriate inhibitor of protein synthe- photoinhibition of PSII [1–3]. When photosynthetic cells are exposed sis, such as lincomycin or chloramphenicol, which blocks repair, while to light at any intensity, two distinct phenomena occur, namely, the repair can be monitored in terms of the recovery of PSII activity after light-induced inactivation (photodamage or photoinactivation) of transfer of the photosynthetic organism from strong to weak light. PSII and the repair of photodamaged PSII (Fig. 1). The extent of photo- Exploitation of this strategy has revealed several new aspects of the inhibition of PSII depends on the balance between the rate of photo- mechanism of photoinhibition. damage and the rate of repair [4–8]. Fig. 2 shows how the rate of photodamage and the rate of repair of PSII are related to the intensity of incident light. The rate of photodamage is proportional to light intensity and this relationship holds even Abbreviations: APX, ascorbate peroxidase; Chl, chlorophyll; EF-G, elongation factor under strong light, such as light at 3500 μmol photons m−2 s−1.By ′ ′ G; DCCD, N,N-dicyclohexylcarbodiimide; DCMU, 3-(3 ,4 -dichlorophenyl)-1,1- contrast, the rate of repair reaches a maximum value at 300 μmol pho- dimethylurea; Nig, nigericin; NPQ, non-photochemical quenching; PMS, N-methyl −2 −1 phenazonium methosulphate; PSI, photosystem I; PSII, photosystem II; ROS, reactive tons m s . It is noted that the light-dependent curve of the rate of oxygen species; Val, valinomycin repair is similar to that of the rate of degradation of the D1 protein, a ☆ This article is part of a Special Issue entitled: Photosynthesis Research for Sustain- key protein in the reaction center of PSII [13]. Under light at intensities ability: from Natural to Artiﬁcial. from approximately 50 to 200 μmol photons m−2 s−1, the rate of re- ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, pair is much higher than the rate of photodamage. Thus, almost all Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338–8570, Japan. Tel.: +81 48 858 3402; fax: +81 48 858 3384. PSII is in an active form and the extent of photoinhibition, that is to E-mail address: [email protected] (Y. Nishiyama). say, the relative level of inactive PSII, appears low. At light intensities

1 precursor to O2, from cyanobacterial cells has no effect on the rate of photodamage [26]. All these observations indicate that ROS, including Photoinactivation (photodamage) 1 O2, act primarily by interfering with the repair of PSII. Inevitably, (Strong light) when faced with these observations, researchers have been forced to re-evaluate the validity of the previously proposed ROS-dependent mechanism of photoinhibition. It should be noted that excess produc- 1 Active PSII Inactive PSII tion of O2 in the presence of very high concentrations of photosensitizers might result in the acceleration of photodamage to PSII [27]. Under such hyper-oxidizing conditions, it is likely that the repair of Repair PSII has already been inhibited. (Weak light) Fig. 3 shows differences between the earlier and the currently accepted schemes that explain the mechanism of photoinhibition of PSII. In both schemes, excess light energy, due to absorption of strong 1 light by chlorophyll (Chl), generates ROS, such as H2O2 and O2. The Fig. 1. The extent of photoinhibition is a result of the balance between the photoinac- difference between the two schemes appears at the next step, tivation (photodamage) of PSII and its repair. To understand the mechanism of photo- namely, the action of ROS. In the earlier scheme, ROS were considered inhibition, it is essential to measure photodamage and repair separately. Photodamage – can be monitored in the presence of lincomycin or chloramphenicol. Repair can be to inactivate the PSII reaction center directly [23,27 31]. In the cur- monitored after the activity of PS II has fallen to 10% of the original activity. rent scheme, ROS inhibit protein synthesis, which is essential for the repair of PSII. The end result is, however, the same in both schemes, namely, a decrease in the activity of PSII that is known as above 1000 μmol photons m−2 s−1, the rate of photodamage is higher photoinhibition. than the rate of repair. Thus, most of PSII is in an inactive form and the The ROS-induced inhibition of protein synthesis and of the repair extent of photoinhibition is large. At 900 μmol photons m−2 s−1,the of PSII has been investigated at the molecular level in cyanobacteria. rates of photodamage and repair are balanced such that approximately In cyanobacterial cells, the synthesis de novo of the D1 protein is a half of PSII is inactive and the extent of photoinhibition is approxi- markedly suppressed by elevated intracellular levels of H2O2 and 1 mately 50%. O2 [16,25,26]. Moreover, the synthesis of not only the D1 protein The separate monitoring of photodamage and repair has revealed but also of almost all other proteins is suppressed at elevated levels that photodamage is not, or is only rarely, affected by a variety of en- of ROS [16,25,26]. Such global suppression of protein synthesis sug- vironmental factors, such as oxidative stress, salt stress and cold gests that the protein-synthetic machinery might be a speciﬁc target stress, whereas repair is inhibited by many factors that are known of inactivation by ROS during photoinhibition. Exploration of the spe- to affect photoinhibition [7,8,14,15]. For example, oxidative stress ciﬁc details of the inactivation by ROS of the synthesis of the D1 pro-

(H2O2), salt stress and cold stress each decrease the maximum rate tein revealed that the translation of the transcript of the psbA gene, 1 of repair but have no effects on photodamage, with a resultant de- which encodes D1, is specifically inactivated by H2O2 and O2 crease in the compensation point (at which 50% of PSII is inactive) [25,26]. Moreover, analysis of polysomes suggested that the elonga- from 900 μmol photons m− 2 s− 1 to a much lower intensity [16]. tion step in the translation of psbA mRNA might be the primary target Previous studies revealed several factors that participate in the of ROS [25,26]. protection of PSII against photoinhibition. They include catalases The effects on translation of the oxidation of elongation factor G and peroxidases, which are efficient scavengers of H2O2 [17]; α- (EF-G), a critical participant in the elongation of nascent polypep- 1 tocopherol, an efficient scavenger of singlet oxygen ( O2) [18,19]; tides, were examined in vitro in a translation system that had been and non-photochemical quenching, which dissipates excess light en- prepared from Synechocystis. Addition of the reduced form of EF-G ergy [20,21]. These factors were formerly considered to mitigate – ) photodamage [22 24]. However, recent studies have demonstrated -1 that these factors act via the acceleration of repair of PSII rather than via a reduction in the rate of photodamage to PSII. In this review, 0.01 we summarize the results of recent studies of the actions of these factors and discuss how these actions differ from those proposed in previous reports.

2. The rate of repair of PSII is regulated by reactive oxygen species

Reactive oxygen species (ROS) are produced by the photosynthetic 0.005 machinery as inevitable by-products of photosynthesis. The superoxide − anion radical (•O2 ), H2O2 and the hydroxyl radical (•OH) are produced 1 as a result of the photosynthetic transport of electrons, while O2 is produced as a result of the transfer of excitation energy [17]. The effects of ROS on photodamage to PSII and on repair have been investigated in

depth both in cyanobacterial cells and in intact leaves. Addition of Initial rate of photodamage or repair (min 0

H2O2 or methyl viologen to suspensions of the cyanobacterium 0 200 400 600 800 1000 Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) Light intensity (µmol photons m-2 s-1) results in increased intracellular concentrations of ROS and enhanced extent of photoinhibition. The enhanced photoinhibition is due to de- Fig. 2. Dependence on light intensity of rates of photodamage to and repair of PSII in celeration of the repair of photodamaged PSII and not to acceleration Synechocystis. The initial rate of photodamage to PSII was measured in the presence 1 of lincomycin (dashed line; ﬁlled circles). For quantitation of the rate of repair, cells of the photodamage to PSII [16,25]. Intracellular production of O2 in were incubated, in the absence of lincomycin, in light at 2000 μmol photons m− 2 s− 1 the presence of photosensitizers, such as rose bengal and ethyl eosin, re- to induce 90% inactivation of PSII. Then cells were incubated in light at various intensi- sults in deceleration of the repair of PSII without any change in the rate ties. The initial rates of repair were calculated from the time courses of recovery exper- of photodamage to PSII [26]. Elimination of molecular oxygen, a iments (solid line; open circles). Adapted from Allakhverdiev and Murata [16]. N. Murata et al. / Biochimica et Biophysica Acta 1817 (2012) 1127–1133 1129

Earlier scheme Current scheme Table 1 Major differences between the earlier and current schemes for the mechanism of photodamage to photosystem II during photoinhibition.

Earlier scheme Current scheme Strong light absorbed by Chl Process One-step process Two-step process Photodamage to Step 1: Photodamage to oxygen-evolving complex reaction center II (slow and rate-limiting step) Excess light energy Step 2: Photodamage to reaction center II (fast step) Effective Light absorbed by Step 1: Light absorbed by the Mn cluster light Chl Step 2: Light absorbed by Chl Generation of ROS, H O , 1O , etc. 2 2 2 Adapted, with modiﬁcations, from the report by Murata et al. [38].

Inhibition of protein photodamage to PSII, as determined in the presence of inhibitors of Damage to PSII – synthesis and repair protein synthesis [16,26,39] and by kinetic analysis [40 42], was ex- actly proportional to the intensity of incident light. This proportional- ity was unaffected by ROS [26]. Furthermore, the action spectrum of photodamage to PSII was completely different from the absorption Decrease in PSII activity (photoinhibition) spectra of Chl and carotenoids [35,36,43–46]. Rather, it resembled the absorption spectra of manganese compounds [35,36,46]. In fact, Fig. 3. Earlier and current schemes for the mechanism of photoinhibition by strong various manganese compounds are susceptible to damage by light light. In both schemes, excess light energy due to strong light generates ROS, such as 1 [47–49]. Such results can be interpreted in the context of the two- H2O2 and O2. The difference between the two schemes is evident in the action of ROS. Whereas the earlier scheme assumed that ROS, generated by excess light energy, step mechanism, as distinct from the one-step mechanism and re- would directly damage PSII, the current scheme suggests that ROS inhibit the repair of searchers who have proposed the earlier and current explanations PSII, with resultant stimulation of the photoinhibition of PSII. of photodamage have not yet reached full agreement [37,50–52]. This review is an attempt to provide clarity and resolution. to the H2O2-inactivated translation system restored the ability of the system to translate psbA2 mRNA, whereas the oxidized form of EF-G 4. The roles of catalase and peroxidase in protection of the did not have similar ability [32]. The critical influence of the redox repair of PSII state of EF-G on translation suggests that EF-G might be a primary target of inactivation by ROS within the translational machinery. The in- Intracellular ROS can be eliminated by antioxidative systems that activation of EF-G by ROS has been attributed to the oxidation of two include ROS-scavenging enzymes and antioxidants [17]. In the chlo- specific cysteine residues in EF-G, namely, Cys105 and Cys242, with roplasts of higher plants, ascorbate peroxidase (APX) is the predomi- subsequent formation of an intramolecular disulfide bond [33]. Ex- nant scavenger of H2O2 [17]. Since APX is a rather unstable enzyme, pression of mutated EF-G with Cys105 replaced by serine in Synecho- especially in the absence of ascorbate, an attempt was made to over- cystis resulted in the protection of PSII from photoinhibition [34]. This express a bacterial catalase, namely, KatE from Escherichia coli, in the protection was attributable to the enhanced repair of PSII via acceler- chloroplasts of tobacco plants. Successful overexpression of KatE ren- ation of the synthesis of the D1 protein, which might have been due dered the transgenic plants more resistant to photo-oxidative stress to reduced sensitivity of protein synthesis to oxidative stress [34]. than the parental strain. In particular, the transgenic plants grew Overexpression of the EF-G of Synechocystis in the cyanobacterium better than wild-type plants under a combination of strong light Synechococcus elongatus PCC 7942 enhanced the synthesis de novo and drought stress, with photoinhibition of PSII being mitigated in of all proteins, including the D1 protein, under oxidative conditions, the transgenic plants [22,53]. suggesting that the oxidation of EF-G by ROS might be responsible There are two types of APX in the chloroplasts of both tobacco and for the suppression of protein synthesis in vivo [32]. Arabidopsis, namely, tAPX and sAPX. The former is associated with thylakoid membranes and the latter is soluble in the stroma [54]. 3. The mechanism of photodamage to PSII: conflicting hypotheses Overexpression of tAPX in the chloroplasts of tobacco plants mitigated the photoinhibition of PSII [55], while defects in either tAPX or sAPX in In the previously accepted scheme for the molecular mechanism Arabidopsis enhanced the photoinhibition of PSII [54].Althoughthese of photoinhibition, photodamage was interpreted as follows: photo- observations demonstrated that these ROS-scavenging enzymes protect synthetically active light produces ROS either by excessive reduction PSII from photoinhibition, it remained to be determined whether they of QA, the primary acceptor of electrons in PSII [28], or by charge re- affect photodamage or repair. combination between the acceptor side and the donor side of PSII A recent study revealed that decreases in intracellular levels of [29]. The resultant ROS then attack the photochemical reaction center ROS, achieved by introduction into tobacco plants of a katE transgene of PSII directly. for the bacterial catalase mentioned above, protected the repair of In the current scheme, photodamage occurs via a two-step pro- PSII, without any effects on photodamage to PSII, under strong light cess: the first step is the light-dependent destruction of the manga- in the presence of high concentrations of NaCl [56]. The katE trans- nese cluster of the oxygen-evolving complex of PSII and the second gene enhanced the synthesis of the D1 protein de novo during expo- step is the inactivation of the photochemical reaction center of PSII sure of plants to such stress conditions [56].InSynechocystis, by light that has been absorbed by Chl [35,36]. For details of this elevated intracellular levels of ROS, due to inactivation of genes for mechanism of photoinhibition, the reader is referred to articles catalase and thioredoxin peroxidase, resulted in deceleration of the Nishiyama et al. [7,37] and Murata et al. [38]. Table 1 summarizes repair of PSII, with suppression of the synthesis of the D1 protein de the differences between the earlier and the current explanations of novo [25]. Thus, enhanced capacity for scavenging ROS results in en- photodamage to PSII. hanced protection of the repair of PSII, via prevention of the suppres- Recent studies in plants and Synechocystis yielded results that can- sion of protein synthesis by ROS. not be interpreted by reference to the earlier scheme for the molecu- Fig. 4 shows the differences between the earlier and the current lar mechanism of photoinhibition, as follows. The initial rate of explanations of the protective effects of catalase and peroxidase. In 1130 N. Murata et al. / Biochimica et Biophysica Acta 1817 (2012) 1127–1133 both schemes, catalase and peroxidase decrease the intracellular con- Earlier scheme Current scheme centration of H2O2. In the earlier scheme for the mechanism of photoinhibition, lower levels of H2O2, due to the activities of catalase and peroxidase, were assumed to decelerate photodamage. By contrast, α-Tocopherol in the current scheme, these enzymes mitigate the inhibition of protein synthesis by H2O2, thereby accelerating the repair of PSII. As noted above, intervention by catalase and peroxidase enhances the 1 repair of PSII, and these enzymes are not involved in photodamage Decrease in level of O2 to PSII, suggesting that the current scheme reflects the true role of these H2O2-scavenging enzymes in the protection of PSII. Alleviation of inhibition 5. The role of α-tocopherol in protection of PSII of protein synthesis against photoinhibition Deceleration of damage to PSII α-Tocopherol is a particularly efficient scavenger of intracellular 1 Acceleration of repair O2 [18,19]. This antioxidant is found both in the envelope and in the thylakoid membranes of chloroplasts [57]. Studies of photosynthetic organisms that lack α-tocopherol or that have only low levels of α-tocopherol indicated that α-tocopherol protects PSII from Protection of PSII against photoinhibition photoinhibition [23]. Treatment of Chlamydomonas cells with inhibi- α Fig. 5. Earlier and current schemes for the protective effect of α-tocopherol on photo- tors of the biosynthesis of -tocopherol enhanced the photoinhibi- 1 inhibition. In both schemes, α-tocopherol is assumed to decrease the level of O2. The tion of PSII [58–60]. The vte1 mutant of Arabidopsis, which is unable 1 difference between the two schemes is evident in the action of O2. Whereas the earlier α 1 to synthesize -tocopherol, is more sensitive to the photoinhibition scheme assumed that a decrease in the level of O2 would decelerate damage to PSII, of PSII than the wild-type parental line [61]. The aforementioned ob- the current scheme suggests that such a decrease accelerates protein synthesis and servations were interpreted, according to the earlier scheme, as evi- consequent repair of PSII, with resultant protection of PSII against photoinhibition. dence that the protective effects of α-tocopherol might occur at the level of photodamage [23]. PSII, as measured in the presence of chloramphenicol, which blocks In a mutant of Synechocystis that lacks an intact hpd gene for 4- the repair of PSII, did not differ between the two lines of cells. By hydroxyphenylpyruvate dioxygenase, a key enzyme in the biosynthe- contrast, the repair of PSII from photodamage was suppressed in the sis of α-tocopherol, no detectable α-tocopherol is produced [62]. mutant cells. Addition of α-tocopherol to cultures of mutant cells Although, in plants, this enzyme is also involved in the biosynthesis returned the extent of photoinhibition to that in wild-type cells, with- of plastoquinone, levels of plastoquinone and other photosynthetic out any effect on photodamage. The synthesis de novo of various pro- pigments are normal in the hpd mutant of Synechocystis [62]. teins, including the D1 protein, was suppressed in the mutant cells Inoue and colleagues investigated the role of α-tocopherol in the under strong light. These observations indicate that α-tocopherol protection of PSII from photoinhibition using the hpd mutant of promotes the repair of photodamaged PSII by protecting, from inhibi- 1 Synechocystis [63]. The activity of PSII in mutant cells was more sensi- tion by O2, the synthesis de novo of the proteins that are required for tive to inactivation by strong light than the activity in wild-type cells, the repair of PSII. indicating that abnormally low levels of α-tocopherol enhance the Recent analysis of the vte1 mutant of Arabidopsis, which is unable extent of photoinhibition. However, the rate of photodamage to to synthesize α-tocopherol, indicated that the absence of α- tocopherol did not affect photodamage to PSII but suppressed the repair of PSII [64], supporting the hypothesis that α-tocopherol also protects Earlier scheme Current scheme the repair of PSII from ROS-induced inhibition in higher plants. Fig. 5 shows the differences between the earlier and current schemes for the protective effect of α-tocopherol on photoinhibition. 1 Catalase and peroxidase In both schemes, α-tocopherol scavenges O2, which is generated as a result of the photosynthetic transfer of excitation energy [17], de- 1 creasing intracellular levels of O2. However, the sites of regulation Decrease in level of H O by α-tocopherol differ between the two schemes. In the earlier 2 2 1 scheme, decreases in intracellular levels of O2 were assumed to decelerate photodamage. By contrast, in the current scheme, such decreases mitigate the inhibition of protein synthesis by 1O and Alleviation of inhibition 2 accelerate the repair of PSII. Thus, α-tocopherol influences repair of protein synthesis Deceleration of and not photodamage. damage to PSII 6. The role of non-photochemical quenching in protection of PSII Acceleration of repair against photoinhibition

The thermal dissipation of excitation energy is a protective mech- Protection of PSII against photoinhibition anism whereby excess light energy, absorbed by PSII, is eliminated as heat. This phenomenon is also referred to as non-photochemical Fig. 4. Earlier and current schemes for the protective effects of catalase and peroxidase quenching (NPQ) since thermal dissipation can be monitored in on photoinhibition. In both schemes, these enzymes are assumed to decrease the level terms of Chl ﬂuorescence. Thermal dissipation requires PsbS, a of H2O2. The difference between the two schemes is evident in the proposed action of chlorophyll-binding protein within PSII, and zeaxanthin, a carotenoid H O . Whereas the earlier scheme assumed that a decrease in the level of H O would 2 2 2 2 that is involved in the xanthophyll cycle [20,21,24]. decelerate damage to PSII, the current scheme suggests that such a decrease accelerates protein synthesis and consequent repair of PSII, with resultant protection of PSII In 2002, Li and colleagues proposed that thermal dissipation of ex- against photoinhibition. citation energy might protect PSII from photoinhibition by decreasing N. Murata et al. / Biochimica et Biophysica Acta 1817 (2012) 1127–1133 1131

Earlier scheme Current scheme systematically in Synechocystis [71]. The rate of photodamage, which was proportional to light intensity, was unaffected by the inhibition of electron transport in PSII, by the acceleration of electron transport Non-photochemical quenching (NPQ) in PSI, and by the inhibition of ATP synthesis. By contrast, the rate of repair fell upon inhibition of the synthesis of ATP either via PSI or via 35 PSII. Labeling of proteins in vivo with [ S]methionine revealed that the Decrease in excess light energy synthesis of the D1 protein was enhanced by the synthesis of ATP. Fig. 7 shows the effects of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), N-methyl phenazonium methosulphate (PMS), and ionophores on the synthesis of proteins de novo during the exposure Decreased generation of ROS of Synechocystis cells to light. In this experiment, protein synthesis 35 was examined by monitoring the incorporation of [ S]methionine into the proteins of thylakoid membranes. The synthesis of proteins Alleviation of inhibition during exposure of cells to light was markedly suppressed by of protein synthesis DCMU. However, the further addition of PMS, which donates electrons Deceleration of to PSI, fully restored the synthesis of all proteins. Further addition of damage to PSII N,N-dicyclohexylcarbodiimide (DCCD) or of nigericin and valinomycin Acceleration of repair together (Nig/Val) completely abolished the synthesis of all proteins, including the D1 protein. These results suggest that exposure of cells to light in the presence of PMS induces the synthesis of proteins, even though electron transport in PSII is inhibited by DCMU and, moreover, Protection of PSII against photoinhibition that conditions that allow the synthesis of ATP are prerequisite for the synthesis of proteins, including the D1 protein. It should be noted that Fig. 6. Earlier and current schemes for the protective effect of non-photochemical protein synthesis was totally eliminated in the presence of DCCD or of quenching (NPQ) on photoinhibition. In both schemes, NPQ is assumed to decrease the light energy associated with chlorophyll, thereby, decreasing the production of Nig/Val, in contrast to the synthesis of psbA transcripts, which occurred ROS. The difference between the two schemes is apparent in the action of ROS. Whereas under the same respective conditions, but at a reduced rate. the earlier scheme assumed that a decrease in levels of ROS would decelerate damage to In 2004, Munekage and colleagues also demonstrated the crucial PSII, the current scheme suggests that this decrease accelerates protein synthesis and role of the cyclic transport of electrons via PSI in the protection of Ara- consequent repair of PSII, with resultant protection of PSII against photoinhibition. bidopsis from photoinhibition under strong light at low temperature [72]. This cyclic system consists of two electron-transport pathways, the rate of photodamage to PSII [65]. However, recent studies, in the NAD(P)H dehydrogenase-dependent and the ferredoxin- which photodamage and repair were monitored separately in mu- dependent pathways [73]. Defects in either pathway resulted in the tants of Arabidopsis that were defective in NPQ, revealed that defects increased sensitivity of PSII to photoinhibition, suggesting that the cy- in NPQ decelerated the repair of PSII, with much less effects on photo- clic transport of electrons protects PSII from photoinhibition [74–76]. damage to PSII than what had been expected [46,66,67]. Deceleration As noted above, strong light induces photoinhibition and de- of the repair of PSII in the NPQ mutants was attributed to suppression creases the efficiency of photosynthesis. However, light is also impor- of the synthesis de novo of proteins, such as the D1 protein [66]. Thus, tant in the protection of PSII against photoinhibition. The repair of thermal dissipation appears to play a role in preventing the genera- PSII, supported by protein synthesis, occurs when EF-G in the translation of ROS by reducing the PSII-mediated transport of electrons rath- tional machinery is active in its reduced form, which is maintained by er than in protecting PSII from photodamage. The apparent protection electrons from PSI via thioredoxin [33]. Thus, light is essential for the of PSII from photoinhibition by thermal dissipation might actually maintenance of an active repair system. Protein synthesis requires correspond to prevention of the ROS-induced suppression of protein synthesis and to the resultant protection of the repair of PSII. The MS mechanism of the thermal dissipation of excitation energy in cyanobacteria is very different from that in higher plants [23,68–70], and the role of the thermal dissipation of excitation energy in photoinhi- +DCCD +DCMU+PMS +Nig/Val bition in cyanobacteria remains to be clarified. Control +DCMU +DCMU+PMS +DCMU+P Fig. 6 shows the differences between the earlier and current schemes for the protective effect of NPQ on photoinhibition of PSII. In both schemes, NPQ dissipates excess light energy, with resultant decreases in the intracellular concentration of ROS. In the earlier ex- planation of photoinhibition, lowering of levels of ROS was assumed to decelerate photodamage, whereas, in the current scheme, lowering D1 of levels of ROS mitigates the inhibition of protein synthesis by ROS and accelerates the repair of PSII. As noted above, the main site of regulation by NPQ is the repair of PSII and not photodamage to PSII, suggesting that the current scheme reflects the true function of NPQ in the protection of PSII. Fig. 7. Effects of the inhibition of electron transport, acceleration of the cyclic electron transport, inhibition of phosphorylation, and uncoupling of membrane energization on 35 7. The roles of the cyclic and the non-cyclic transport of electrons the synthesis of proteins de novo in Synechocystis. Cells were incubated with 10 nM [ S] in protection of PSII against photoinhibition: the dual effects of methionine (>1000 Ci mmol− 1) for 30 min in light at 500 μmol photons m− 2 s− 1 in light the presence of 20 μM DCMU, 20 μM PMS, 10 μM DCCD, and 2 μM nigericin plus 2 μM valinomycin (Nig/Val) as indicated, or in their absence (Control). After incubation, a portion of each suspension of cells was withdrawn for preparation of thylakoid mem- In 2005, our group examined the roles of electron transport and branes, which were then subjected to polyacrylamide gel electrophoresis. Equal ATP synthesis in changes in the rates of photodamage and repair of amounts of protein from thylakoid membranes were applied to each lane. The arrow PSII by monitoring photodamage and repair separately and indicates the position of the 32-kDa D1 protein. Adapted from Allakhverdiev et al. [71]. 1132 N. Murata et al. / Biochimica et Biophysica Acta 1817 (2012) 1127–1133

Light [11] B.Y. Moon, S. Higashi, Z. Gombos, N. Murata, Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 6219–6223. Electron transport [12] J. He, W.S. Chow, The rate coefficient of repair of photosystem II after photoinactivation, Physiol. Plant. 118 (2003) 297–304. [13] M. Edelman, A.K. Mattoo, D1-protein dynamics in photosystem II: the lingering enigma, Photosynth. Res. 98 (2008) 609–620. Electrons Synthesis of [14] Y. Nishiyama, S.I. Allakhverdiev, N. Murata, Inhibition of the repair of photosys- from PSI ATP tem II by oxidative stress in cyanobacteria, Photosynth. Res. 84 (2005) 1–7. [15] E. Tyystjärvi, Photoinhibition of photosystem II and photodamage of the oxygen evolving manganese cluster, Coord. Chem. Rev. 252 (2008) 361–376. [16] S.I. Allakhverdiev, N. Murata, Environmental stress inhibits the synthesis de novo Reduction of EF-G and of proteins involved in the photodamage-repair cycle of photosystem II in – activation of translation Synechocystis sp. PCC 6803, Biochim. Biophys. Acta 1657 (2004) 23 32. [17] K. Asada, The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 601–639. Acceleration of [18] W.C. Neely, J.M. Martin, S.A. Barker, Products and relative reaction rates of the protein synthesis oxidation of tocopherols with singlet molecular oxygen, Photochem. Photobiol. 48 (1988) 423–428. [19] P. Di Mascio, T.P. Devasagayam, S. Kaiser, H. Sies, Carotenoids, tocopherols and thiols as biological singlet molecular oxygen quenchers, Biochem. Soc. Trans. 18 Acceleration of repair (1990) 1054–1056. [20] R.C. Bugos, H.Y. Yamamoto, Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli, Proc. Natl. Acad. Sci. U. S. A. 93 Fig. 8. The repair of PSII is supported by light at moderate intensities in two ways. The (1996) 6320–6325. light-dependent transport of electrons induces the synthesis of ATP and the activation [21] X.-P. Li, O. Björkman, C. Shih, A.R. Grossman, M. Rosenquist, S. Jansson, K.K. of EF-G, both of which are necessary for the synthesis of proteins. The same process Niyogi, A pigment-binding protein essential for regulation of photosynthetic occurs in strong light, which also induces photodamage. light harvesting, Nature 403 (2000) 391–395. [22] T. Shikanai, T. Takeda, H. Yamauchi, S. Sano, K.I. Tomizawa, A. Yokota, S. Shigeoka, Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacte- large amounts of ATP, whose synthesis is driven by the cyclic and rial catalase in chloroplasts, FEBS Lett. 428 (1998) 47–51. non-cyclic transport of electrons, and light is essential for both [23] A. Krieger-Liszkay, C. Fufezan, A. Trebst, Singlet oxygen production in photosys- – types of electron transport. The dual effects of light in the repair of tem II and related protection mechanism, Photosynth. Res. 98 (2008) 551 564. [24] K.K. Niyogi, X.P. Li, V. Rosenberg, H.S. Jung, Is PsbS the site of non-photochemical PSII are summarized in Fig. 8. quenching in photosynthesis? J. Exp. Bot. 56 (2005) 375–382. [25] Y. Nishiyama, H. Yamamoto, S.I. Allakhverdiev, M. Inaba, A. Yokota, N. Murata, Oxidative stress inhibits the repair of photodamage to the photosynthetic Acknowledgements machinery, EMBO J. 20 (2001) 5587–5594. [26] Y. Nishiyama, S.I. Allakhverdiev, H. Yamamoto, H. Hayashi, N. Murata, Singlet oxygen This work was supported, in part, by a Grant-in-Aid for Scientific inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803, Biochemistry 43 (2004) 11321–11330. Research from the Ministry of Education, Culture, Sports, Science and [27] E. Hideg, P.B. Kos, I. Vass, Photosystem II damage induced by chemically generat- Technology of Japan (no. 21570033 to Y.N.); by a grant from the Asahi ed singlet oxygen in tobacco leaves, Physiol. Plant. 131 (2007) 33–40. Glass Foundation (to Y.N.); by grants from the Russian Foundation for [28] I. Vass, S. Styring, T. Hundal, A. Koivuniemi, E. Aro, B. Andersson, Reversible and Basic Research (nos. 11-04-01389a, 11-04-92690a and 12-04-9210a irreversible intermediates during photoinhibition of photosystem II: stable reduced QA species promote chlorophyll triplet formation, Proc. Natl. Acad. Sci. to S.I.A.), by a grant from the Russian Ministry of Science and Education U. S. A. 89 (1992) 1408–1412. (no. 16.740.11.0176 to S.I.A.), by a grant from the Molecular and Cell [29] N. Keren, A. Berg, P.J. van Kan, H. Levanon, I. Ohad, Mechanism of photosystem II Biology Programs of the Russian Academy of Sciences (to S.I.A.); and photoinactivation and D1 protein degradation at low light: the role of back electron flow, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 1579–1584. by the Cooperative Research Program of the National Institute for [30] C. Fufezan, A.W. Rutherford, A. Krieger-Liszkay, Singlet oxygen production in Basic Biology on the Stress Tolerance of Plants (to N.M.). herbicide-treated photosystem II, FEBS Lett. 532 (2002) 407–410. [31] I. Vass, K. Cser, Janus-faced charge recombinations in photosystem II photoinhibition, Trends Plant Sci. 14 (2009) 200–205. References [32] K. Kojima, M. Oshita, Y. Nanjo, K. Kasai, Y. Tozawa, H. Hayashi, Y. Nishiyama, Oxidation of elongation factor G inhibits the synthesis of the D1 protein of photo- [1] S.B. Powles, Photoinhibition of photosynthesis induced by visible light, Annu. Rev. system II, Mol. Microbiol. 65 (2007) 936–947. Plant Physiol. 35 (1984) 15–44. [33] K. Kojima, K. Motohashi, T. Morota, M. Oshita, T. Hisabori, H. Hayashi, Y. Nishiyama, [2] E.M. Aro, I. Virgin, B. Andersson, Photoinhibition of photosystem II. Inactivation, Regulation of translation by the redox state of elongation factor G in the cyanobac- protein damage and turnover, Biochim. Biophys. Acta 1143 (1993) 113–134. terium Synechocystis sp. PCC 6803, J. Biol. Chem. 284 (2009) 18685–18691. [3] E.M. Aro, M. Suorsa, A. Rokka, Y. Allahverdiyeva, V. Paakkarinen, A. Saleem, N. [34] K. Ejima, T. Kawaharada, S. Inoue, K. Kojima, Y. Nishiyama, A change in the Battchikova, E. Rintamäki, Dynamics of photosystem II: a proteomic approach sensitivity of elongation factor G to oxidation protects photosystem II from to thylakoid protein complexes, J. Exp. Bot. 56 (2005) 347–356. photoinhibition in Synechocystis sp. PCC 6803, FEBS Lett. 586 (2012) [4] G. Samuelsson, A. Lonneborg, E. Rosenqvist, P. Gustafsson, G. Öquist, Photoinhibi- 778–783. tion and reactivation of photosynthesis in the cyanobacterium Anacystis nidulans, [35] N. Ohnishi, S.I. Allakhverdiev, S. Takahashi, S. Higashi, M. Watanabe, Y. Nishiyama, Plant Physiol. 79 (1985) 992–995. N. Murata, Two-step mechanism of photodamage to photosystem II: step 1 occurs [5] D.H. Greer, J.A. Berry, O. Björkman, Photoinhibition of photosynthesis in intact at the oxygen-evolving complex and step 2 occurs at the photochemical reaction bean leaves: role of light and temperature, and requirement for chloroplast- center, Biochemistry 44 (2005) 8494–8499. protein synthesis during recovery, Planta 168 (1986) 253–260. [36] M. Hakala, I. Tuominen, M. Keränen, T. Tyystjärvi, E. Tyystjärvi, Evidence for the [6] E. Tyystjärvi, K. Ali-Yrkkö, R. Kettunen, E.M. Aro, Slow degradation of the D1 pro- role of the oxygen-evolving manganese complex in photoinhibition of photosys- tein is related to the susceptibility of low-light-grown pumpkin plants to photo- tem II, Biochim. Biophys. Acta 1706 (2005) 68–80. inhibition, Plant Physiol. 100 (1992) 1310–1317. [37] Y. Nishiyama, S.I. Allakhverdiev, N. Murata, Protein synthesis is the primary target [7] Y. Nishiyama, S.I. Allakhverdiev, N. Murata, A new paradigm for the action of re- of reactive oxygen species in the photoinhibition of photosystem II, Physiol. Plant. active oxygen species in the photoinhibition of photosystem II, Biochim. Biophys. 142 (2011) 35–46. Acta 1757 (2006) 742–749. [38] N. Murata, S. Takahashi, Y. Nishiyama, S.I. Allakhverdiev, Photoinhibition of photo- [8] S. Takahashi, N. Murata, How do environmental stresses accelerate photoinhibi- system II under environmental stress, Biochim. Biophys. Acta 1767 (2007) 414–421. tion? Trends Plant Sci. 13 (2008) 178–182. [39] E. Tyystjarvi, E.M. Aro, The rate constant of photoinhibition, measured in [9] Z. Gombos, H. Wada, N. Murata, The recovery of photosynthesis from low- lincomycin-treated leaves, is directly proportional to light intensity, Proc. Natl. temperature photoinhibition is accelerated by the unsaturation of membrane Acad. Sci. U. S. A. 93 (1996) 2213–2218. lipids: a mechanism of chilling tolerance, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) [40] J.M. Anderson, W.S. Chow, Structural and functional dynamics of plant photosys- 8787–8791. tem II, Philos. Trans. R. Soc. Lond. B Biol. Sci. 357 (2002) 1421–1430. [10] H. Wada, Z. Gombos, N. Murata, Contribution of membrane lipids to the ability of [41] Y.-I. Park, W.S. Chow, J.M. Anderson, Light inactivation of functional photosystem the photosynthetic machinery to tolerate temperature stress, Proc. Natl. Acad. Sci. II in leaves of peas grown in moderate light depends on photon exposure, Planta U. S. A. 91 (1994) 4273–4277. 196 (1995) 401–411. N. Murata et al. / Biochimica et Biophysica Acta 1817 (2012) 1127–1133 1133

[42] H.Y. Lee, Y.N. Hong, W.S. Chow, Photoinactivation of photosystem II complex and plastoquinone and tocopheryl derivatives in Chlamydomonas reinhardtii,Pest photoprotection by non-functional neighbours in Capsicum annuum L. leaves, Manag. Sci. 60 (2004) 669–674. Planta 212 (2001) 332–342. [60] J. Kruk, H. Holländer-Czytko, W. Oettmeier, A. Trebst, Tocopherol as singlet [43] L.W. Jones, B. Kok, Photoinhibition of chloroplast reactions. I. Kinetics and action oxygen scavenger in photosystem II, J. Plant Physiol. 162 (2005) 749–757. spectra, Plant Physiol. 41 (1966) 1037–1043. [61] M. Havaux, F. Eymery, S. Porﬁrova, P. Rey, P. Dormann, Vitamin E protects against [44] J. Jung, H.S. Kim, The chromophores as endogenous sensitizers involved in the photoinhibition and photooxidative stress in Arabidopsis thaliana, Plant Cell 17 photogeneration of singlet oxygen in spinach thylakoids, Photochem. Photobiol. (2005) 3451–3469. 52 (1990) 1003–1009. [62] D. Dähnhardt, J. Falk, J. Appel, T.A. van der Kooij, R. Schulz-Friedrich, K. Krupinska, [45] T. Tyystjärvi, I. Tuominen, M. Herranen, E.M. Aro, E. Tyystjärvi, Action spectrum of The hydroxyphenylpyruvate dioxygenase from Synechocystis sp. PCC 6803 is not psbA gene transcription is similar to that of photoinhibition in Synechocystis sp. required for plastoquinone biosynthesis, FEBS Lett. 523 (2002) 177–181. PCC 6803, FEBS Lett. 516 (2002) 167–171. [63] S. Inoue, K. Ejima, E. Iwai, H. Hayashi, J. Appel, E. Tyystjärvi, N. Murata, Y. [46] P. Sarvikas, M. Hakala, E. Pätsikkä, T. Tyystjärvi, E. Tyystjärvi, Action spectrum of Nishiyama, Protection by α-tocopherol of the repair of photosystem II during photoinhibition in leaves of wild type and npq1-2 and npq4-1 mutants of photoinhibition in Synechocystis sp. PCC 6803, Biochim. Biophys. Acta 1807 Arabidopsis thaliana, Plant Cell Physiol. 47 (2006) 391–400. (2011) 236–241. [47] M. Hakala, S. Rantamäki, E.M. Puputti, T. Tyystjärvi, E. Tyystjärvi, Photoinhibition [64] M. Hakala-Yatkin, P. Sarvikas, P. Paturi, M. Mäntysaari, H. Mattila, T. Tyystjärvi, L. of manganese enzymes: insights into the mechanism of photosystem II photoin- Nedbal, E. Tyystjärvi, Magnetic ﬁeld protects plants against high light by slowing hibition, J. Exp. Bot. 57 (2006) 1809–1816. down production of singlet oxygen, Physiol. Plant. 142 (2011) 26–34. [48] T.K. Antal, W. Lo, W.H. Armstrong, E. Tyystjärvi, Illumination with ultraviolet or [65] X.P. Li, P. Müller-Moulé, A.M. Gilmore, K.K. Niyogi, PsbS-dependent enhancement visible light induces chemical changes in the water-soluble manganese complex, of feedback de-excitation protects photosystem II from photoinhibition, Proc.

[Mn4O6(bpea)4]Br4, Photochem. Photobiol. 85 (2009) 663–668. Natl. Acad. Sci. U. S. A. 99 (2002) 15222–15227. [49] Z. Wei, C.W. Cady, G.W. Brudvig, H.J. Hou, Photodamage of a Mn(III/IV)-oxo [66] S. Takahashi, S.E. Milward, D.Y. Fan, W.S. Chow, M.R. Badger, How does cyclic mixed-valence compound and photosystem II: evidence that a high-valent electron flow alleviate photoinhibition in Arabidopsis? Plant Physiol. 149 (2009) manganese species is responsible for UV-induced photodamage of the oxygen- 1560–1567. evolving complex in photosystem II, J. Photochem. Photobiol. B 104 (2011) [67] P. Sarvikas, T. Tyystjärvi, E. Tyystjärvi, Kinetics of prolonged photoinhibition 118–125. revisited: photoinhibited photosystem II centres do not protect the active ones [50] N. Keren, A. Krieger-Liszkay, Photoinhibition: molecular mechanisms and physio- against loss of oxygen evolution, Photosynth. Res. 103 (2010) 7–17. logical significance, Physiol. Plant. 142 (2011) 1–5. [68] K. Sonoike, Y. Hihara, M. Ikeuchi, Physiological significance of the regulation of [51] R. Oguchi, I. Terashima, J. Kou, W.S. Chow, Operation of dual mechanisms that photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC both lead to photoinactivation of photosystem II in leaves by visible light, Physiol. 6803, Plant Cell Physiol. 42 (2001) 379–384. Plant. 142 (2011) 47–55. [69] T. Fujimori, Y. Hihara, K. Sonoike, PsaK2 subunit in photosystem I is involved in [52] I. Vass, Molecular mechanisms of photodamage in the photosystem II complex, state transition under high light condition in the cyanobacterium Synechocystis Biochim. Biophys. Acta 1817 (2012) 209–217. sp. PCC 6803, J. Biol. Chem. 280 (2005) 22191–22197. [53] Y. Miyagawa, M. Tamoi, S. Shigeoka, Evaluation of the defense system in chloro- [70] A. Wilson, G. Ajlani, J.M. Verbavatz, I. Vass, C.A. Kerfeld, D. Kirilovsky, A soluble plasts to photooxidative stress caused by paraquat using transgenic tobacco carotenoid protein involved in phycobilisome-related energy dissipation in plants expressing catalase from Escherichia coli, Plant Cell Physiol. 41 (2000) cyanobacteria, Plant Cell 18 (2006) 992–1007. 311–320. [71] S.I. Allakhverdiev, Y. Nishiyama, S. Takahashi, S. Miyairi, I. Suzuki, N. Murata, [54] T. Maruta, A. Tanouchi, M. Tamoi, Y. Yabuta, K. Yoshimura, T. Ishikawa, S. Systematic analysis of the relation of electron transport and ATP synthesis to Shigeoka, Arabidopsis chloroplastic ascorbate peroxidase isoenzymes play a dual the photodamage and repair of photosystem II in Synechocystis, Plant Physiol. role in photoprotection and gene regulation under photooxidative stress, Plant 137 (2005) 263–273. Cell Physiol. 51 (2010) 190–200. [72] Y. Munekage, M. Hashimoto, C. Miyake, K. Tomizawa, T. Endo, M. Tasaka, T. [55] Y. Yabuta, T. Motoki, K. Yoshimura, T. Takeda, T. Ishikawa, S. Shigeoka, Thylakoid Shikanai, Cyclic electron flow around photosystem I is essential for photosynthesis, membrane-bound ascorbate peroxidase is a limiting factor of antioxidative Nature 429 (2004) 579–582. systems under photo-oxidative stress, Plant J. 32 (2002) 915–925. [73] T. Shikanai, Cyclic electron transport around photosystem I: genetic approaches, [56] K. Al-Taweel, T. Iwaki, Y. Yabuta, S. Shigeoka, N. Murata, A. Wadano, A bacterial Annu. Rev. Plant Biol. 58 (2007) 199–217. transgene for catalase protects translation of D1 protein during exposure of [74] T. Endo, T. Shikanai, A. Takabayashi, K. Asada, F. Sato, The role of chloroplastic salt-stressed tobacco leaves to strong light, Plant Physiol. 145 (2007) 258–265. NAD(P)H dehydrogenase in photoprotection, FEBS Lett. 457 (1999) 5–8. [57] H.K. Lichtenthaler, Plastoglobuli and the fine structure of plastids, Endeavor 27 [75] Y. Munekage, M. Hojo, J. Meurer, T. Endo, M. Tasaka, T. Shikanai, PGR5 is involved (1968) 144–149. in cyclic electron flow around photosystem I and is essential for photoprotection [58] A. Trebst, B. Depka, H. Holländer-Czytko, A specific role for tocopherol and of in Arabidopsis, Cell 110 (2002) 361–371. chemical singlet oxygen quenchers in the maintenance of photosystem II struc- [76] N. Yeremenko, R. Jeanjean, P. Prommeenate, V. Krasikov, P.J. Nixon, W.F. Vermaas, ture and function in Chlamydomonas reinhardtii, FEBS Lett. 516 (2002) 156–160. M. Havaux, H.C. Matthijs, Open reading frame ssr2016 is required for antimycin [59] A. Trebst, B. Depka, J. Jäger, W. Oettmeier, Reversal of the inhibition of photo- A-sensitive photosystem I-driven cyclic electron flow in the cyanobacterium synthesis by herbicides affecting hydroxyphenylpyruvate dioxygenase by Synechocystis sp. PCC 6803, Plant Cell Physiol. 46 (2005) 1433–1436.