Revised scheme for the mechanism of photoinhibition and its application to enhance the abiotic stress tolerance of the photosynthetic machinery.

Appl Microbiol Biotechnol DOI 10.1007/s00253-014-6020-0

MINI-REVIEW

Revised scheme for the mechanism of photoinhibition and its application to enhance the abiotic stress tolerance of the photosynthetic machinery Yoshitaka Nishiyama & Norio Murata

Received: 25 June 2014 / Revised: 5 August 2014 / Accepted: 6 August 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract When photosynthetic organisms are exposed to abiotic stress, their photosynthetic activity is significantly depressed. In particular, photosystem II (PSII) in the photosynthetic machinery is readily inactivated under strong light and this phenomenon is referred to as photoinhibition of PSII. Other types of abiotic stress act synergistically with light stress to accelerate photoinhibition. Recent studies of photoinhibition have revealed that light stress damages PSII directly, whereas other abiotic stresses act exclusively to inhibit the repair of PSII after light-induced damage (photodamage). Such inhibition of repair is associated with suppression, by reactive oxygen species (ROS), of the synthesis of proteins de novo and, in particular, of the D1 protein, and also with the reduced efficiency of repair under stress conditions. Gene-technological improvements in the tolerance of photosynthetic organisms to various abiotic stresses have been achieved via protection of the repair system from ROS and, also, by enhancing the efficiency of repair via facilitation of the turnover of the D1 protein in PSII. In this review, we summarize the current status of research on photoinhibition as it relates to the effects of abiotic stress and we discuss successful strategies that enhance the activity of the repair machinery. In addition, we propose several potential methods for activating the repair system by gene-technological methods. Keywords Abiotic stress . Photoinhibition . Photosystem II (PSII) . Repair . Stress tolerance . Gene technology Y. Nishiyama (*) Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering and Institute for Environmental Science and Technology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan e-mail: [email protected] N. Murata National Institute for Basic Biology, Okazaki 444-8585, Japan

Introduction Photosynthetic organisms, including cyanobacteria, microalgae, and plants encounter various types of abiotic stress in their habitats, for example, strong light, high concentrations of salt, drought (water deficiency), and low and high temperatures. These abiotic stresses have negative effects on photosynthetic activity, which result in reductions in both growth and productivity. Within the photosynthetic machinery, photosystem II (PSII) is particularly sensitive to such abiotic stresses (Nishiyama et al. 2006; Takahashi and Murata 2008). PSII is located in the thylakoid membranes of photosynthetic organisms and is a pigment-protein complex in which light energy is converted into chemical energy. However, PSII is very sensitive to light stress and is rapidly inactivated under strong light, a phenomenon that is referred to as photoinhibition of PSII (Powles 1984; Aro et al. 1993). The extent of photoinhibition of PSII is strongly and synergistically influenced by other types of abiotic stress. For example, the combination of strong light and low temperature has a much greater negative effect on PSII than strong light alone (Allakhverdiev and Murata 2004). Therefore, in order to improve the tolerance of PSII to abiotic stress, it is necessary to understand the effect of strong light alone on PSII, as well as the synergistic effects on PSII of strong light and other types of abiotic stress. After PSII has been inactivated by light, its activity is efficiently restored by a repair system that involves the degradation and removal of the D1 protein in the photodamaged PSII; the synthesis de novo of proteins and, in particular, of the precursor to the D1 protein (pre-D1); and the processing of pre-D1 (Aro et al. 1993, 2005). The light-induced inactivation of (or photodamage to) PSII and the repair of photodamaged PSII occur simultaneously (Fig. 1a). Thus, photoinhibition of PSII becomes apparent when the rate of photodamage to PSII exceeds the rate of its repair.

Appl Microbiol Biotechnol Fig. 1 Schematic representation of the dynamics of photoinhibition of photosystem II (PSII). The extent of photoinhibition is a result of the balance between the rates of photoinactivation of PSII (photodamage) and its repair (a). To elucidate the mechanism of photoinhibition, it is essential to monitor photodamage and repair separately. Photodamage can be monitored in the presence of an inhibitor of protein synthesis, such as lincomycin or chloramphenicol (b). Repair can be monitored after the activity of PSII has fallen to a low level, such as 10 % of the original activity (c)

(a) Normal conditions

Photoinactivation (photodamage)

Active PSII

Inactive PSII Repair

(b) In the presence of an inhibitor of protein synthesis


Active PSII

X

Inactive PSII

Repair

(c) Under weak light


Active PSII

Inactive PSII Repair

To understand the mechanism of photoinhibition and the effects of other abiotic stresses on photoinhibition, it is necessary to monitor the processes of photodamage and repair separately, and methods for the separate monitoring of photodamage and repair have been established in cyanobacteria (Samuelsson et al. 1985; Gombos et al. 1994; Wada et al. 1994) and in a green alga (Lidholm et al. 1987). Photodamage can be monitored in the presence of an appropriate inhibitor of prokaryotic protein synthesis, for example lincomycin or chloramphenicol, which blocks repair (Fig. 1b), while repair can be monitored in terms of the recovery of PSII activity after transfer of the photosynthetic organism from strong to weak light (Fig. 1c). Similar methods have been successfully applied to the study of photoinhibition in plants (Greer et al. 1986; Moon et al. 1995). Exploitation of this method has revealed several new aspects of the mechanism of photoinhibition, as well as the effects of abiotic stress on photoinhibition. For example, the rate of photodamage to PSII is proportional to the intensity of incident light (Tyystjärvi and Aro 1996; Anderson and Chow 2002; Tyystjärvi 2008), whereas the rate of repair of PSII reaches a plateau value at relatively low intensities of light (Allakhverdiev and Murata 2004). These findings have enabled us to interpret photoinhibition as follows: Photoinhibition is not apparent under moderate-intensity light.

However, under strong light, when the rate of photodamage exceeds the rate of repair, photoinhibition becomes apparent (Nishiyama et al. 2006; Takahashi and Murata 2008). By contrast, other types of abiotic stress, such as low temperature and high concentrations of salt, inhibit the repair of photodamaged PSII rather than accelerating photodamage to PSII (Nishiyama et al. 2006; Takahashi and Murata 2008). Thus, an increasing body of evidence has revealed that the repair of PSII is the critical event that determines the tolerance of PSII to abiotic stress. Therefore, efforts to improve the tolerance of the photosynthetic machinery to abiotic stress have focused on enhancing the repair of PSII by genetechnological methods. Photoinhibition of PSII has been studied extensively since the first report of this phenomenon by Kok (1956), and there have been many reports of gene-technological improvements in the tolerance, in terms of PSII activity, of photosynthetic organisms to photoinhibition and other forms of abiotic stress. Such gene-technological methods, described in detail below, can be categorized into two types: one involves reductions in levels of reactive oxygen species (ROS), leading to activation of the translational machinery; and the other involves acceleration of the turnover of the D1 protein. Results of many previous studies were interpreted within t h e f r a m e w o r k o f a h y p o t h e t i c a l m e c h a n i s m of

Appl Microbiol Biotechnol

photoinhibition, in which abiotic stresses, including oxidative stress, accelerate photodamage to the reaction center of PSII. However, in a “new” scheme that more accurately explains the mechanism of photoinhibition, as well as the effects of abiotic stresses on it, abiotic stresses act by inhibiting the repair of PSII rather than accelerating photodamage itself. Thus, with the abandonment of the earlier hypothesis, all the results relating to gene-technological improvements in tolerance have to be re-evaluated. In this review, we summarize recent progress in studies of the effects of abiotic stress on photoinhibition and several successful strategies for improving the stress tolerance of PSII by gene-technological methods. We also reevaluate earlier successful strategies in terms of our improved understanding of photoinhibition and we propose new approaches that might enhance the tolerance of photosynthesis to abiotic stress.

not to acceleration of the photodamage to PSII (Nishiyama et al. 2001; Allakhverdiev and Murata 2004). Intracellular production of 1O2 in the presence of a photosensitizer, such as rose bengal or ethyl eosin, results in deceleration of the repair of PSII without any change in the rate of photodamage to PSII (Nishiyama et al. 2004). Moreover, elimination of molecular oxygen, a precursor to 1O2, from cyanobacterial cells has no effect on the rate of photodamage (Nishiyama et al. 2004). These observations indicate that ROS, including 1 O2, act primarily by interfering with the repair of PSII. Figure 2 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 light by chlorophylls, generates ROS, such as H2O2 and 1O2. The difference between the two schemes becomes evident at the next step, namely, the action of ROS. In the earlier scheme, ROS inactivate the PSII reaction center directly (Keren et al. 1997; Hideg et al. 2007; Krieger-Liszkay et al. 2008). In the currently accepted

The mechanism of photoinhibition of PSII The mechanism of photoinhibition of PSII has been the focus of intensive study for more than three decades. In particular, the role of ROS in photoinhibition has received considerable attention. ROS are produced as by-products of photosynthetic reactions. Hydrogen peroxide (H2O2) and singlet oxygen (1O2) are produced as a result of the photosynthetic transport of electrons and excitation energy (Asada 1999). The production of ROS increases dramatically under strong light, which generates an excess of electrons that cannot be fully consumed by the Calvin cycle (Takahashi and Murata 2008). In earlier studies, ROS and, in particular, 1O2 were considered to be the mediators of photodamage to PSII. For example, according to the “acceptor-side” and “charge-recombination” hypotheses, the D1 protein, a protein in the reaction center of PSII, might be damaged directly by 1O2 that is produced via energy transfer from the triplet chlorophyll to molecular oxygen during charge recombination at the reaction center (Vass et al. 1992; Hideg et al. 1994; Keren et al. 1997). However, most of the earlier studies that supported these hypotheses were performed with isolated thylakoid membranes or PSII complexes, which lack the repair system, or with leaves in which photodamage was not examined separately from repair. The separate monitoring of photodamage and repair in cyanobacterial cells, by contrast, revealed that ROS act primarily by inhibiting the repair of PSII and not by damaging PSII (Nishiyama et al. 2001, 2004). Addition of H2O2 or methyl viologen, which accelerates the production of H2O2, to cultures of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter, Synechocystis) results in an increase in intracellular concentrations of ROS and in enhanced photoinhibition (Nishiyama et al. 2001). The enhanced photoinhibition is due to depression of the repair of photodamaged PSII and

Earlier scheme

Current scheme

Strong light absorbed by Chl

Excess light energy

Generation of ROS, H2O2, 1O2, etc.

Inhibition of protein synthesis Damage to PSII Inhibition of PSII repair

Decrease in PSII activity (stimulation of photoinhibition) Fig. 2 Earlier and current schemes for the mechanism of photoinhibition by strong light. In both schemes, excess light energy due to strong light generates ROS, such as H2O2 and 1O2. The difference between the two schemes is evident in the action of ROS. Whereas the earlier scheme assumes that ROS, generated by excess light energy, damages PSII directly, the current scheme suggests that ROS inhibit the repair of PSII, with resultant stimulation of the photoinhibition of PSII. In the current scheme, PSII is inactivated directly by light (not shown in this scheme, see text)


scheme, ROS act primarily by inhibiting protein synthesis, which is essential for the repair of PSII. The end result is, however, the same in both schemes, namely, the photoinhibition of PSII (for a review, see Murata et al. 2012). The above-mentioned change in perspective led to a reconsideration of the mechanism of photodamage to PSII which, according to the earlier scheme, was assumed to be caused by the direct action of ROS. The most explicit results that led to clarification of the mechanism originated from action spectra of photodamage. In early studies with thylakoid membranes from spinach (Jones and Kok 1966) and more recent studies with thylakoid membranes from a thermophilic cyanobacterium (Ohnishi et al. 2005) and from pumpkin (Hakala et al. 2005), as well as with intact leaves of Arabidopsis (Takahashi et al. 2010), the action spectrum of photodamage to PSII consistently revealed that UV and blue light are much more effective than red light and that UV light is the strongest effector of damage to PSII. The action spectrum of photodamage resembles the absorption spectrum of manganese compounds rather than that of chlorophyll (Hakala et al. 2005). By contrast, the action spectrum of photodamage to the reaction center, from which the oxygen-evolving complex has been eliminated, is very similar to the absorption spectra of chlorophylls (Ohnishi et al. 2005). The two types of action spectrum led to the proposal of a new model for photodamage to PSII, namely, the two-step model. In this model, the oxygen-evolving complex, most probably the manganese cluster, is damaged first by absorption of light, in particular, light in the UV and blue regions. The damage to the oxygenevolving complex triggers rapid secondary damage to the reaction center upon absorption of visible light by chlorophylls. Although details of each step remain to be clarified, the two-step model suggests that ROS, including 1O2, are not the primary cause of photodamage to PSII. Thus, photodamage to PSII depends exclusively on light and is unavoidable when PSII is exposed to light (Nishiyama et al. 2006; Murata et al. 2007). To minimize photodamage, PSII needs to be protected from exposure to strong light, in particular, light in the UV and blue regions. Plants have developed a variety of strategies for avoiding or reducing exposure to light, such as the movement of leaves and chloroplasts and the shielding of PSII with UV-absorbing compounds (Takahashi and Badger 2011). However, it should be noted here that on the definitive mechanism of photodamage, either via the two-step model or via the earlier one-step models, such as the acceptor-side model (Vass et al. 1992), remains a matter of dispute (Vass 2012; Tyystjärvi 2013). For example, the earlier models were supported by studies with Synechocystis in which photodamage was affected by mutations in the D1 protein to change the redox potential of pheophytin (Rehman et al. 2013); by overexpression of flavodiiron proteins Flv4 and Flv2 (Bersanini et al. 2014); and by knockout of the genes for sigma factors (Hakkila et al. 2014).

Various abiotic stresses inhibit the repair of PSII Monitoring photodamage and the repair of PSII separately revealed that repair is inhibited by factors that also affect photoinhibition (Nishiyama et al. 2006; Takahashi and Murata 2008; Tyystjärvi 2008). For example, oxidative stress (H2O2), salt stress, and cold stress each decrease the maximum rate of repair (Allakhverdiev and Murata 2004). Three main steps are conspicuously sensitive to abiotic stress, namely, degradation of the D1 protein in photodamaged PSII, synthesis of pre-D1 and processing of pre-D1 to the mature D1 protein, as described below. Salt stress inhibits protein synthesis and the degradation of D1 protein In natural environments, salt stress often occurs in combination with light stress, and the results of several previous studies of the effects of salt stress on photoinhibition of PSII indicated that salt stress enhances the extent of photoinhibition in Chlamydomonas reinhardtii (Neale and Melis 1989) and in the cyanobacterium Spirulina platensis (Lu and Zhang 1999). However, these studies failed to clarify whether salt stress accelerated photodamage to PSII or inhibited the repair of PSII. Our results in Synechocystis demonstrated that salt stress, due to NaCl, inhibited the repair of photodamaged PSII but did not directly accelerate photodamage to PSII (Allakhverdiev et al. 2002; Allakhverdiev and Murata 2004). Furthermore, salt stress inhibited the synthesis of pre-D1 protein in Synechocystis cells (Allakhverdiev et al. 2002; Allakhverdiev and Murata 2004). In addition, salt stress suppressed not only the synthesis of the D1 protein de novo but also the synthesis of almost all other proteins. An investigation of the reactions involved in the synthesis de novo of the D1 protein revealed that salt stress suppressed the synthesis of D1 at the translational level (Allakhverdiev et al. 2002). Another study in Synechococcus elongatus PCC 7942 (hereafter, Synechococcus 7942) demonstrated that salt stress inhibited not only protein synthesis but also the degradation of the D1 protein in photodamaged PSII (Ohnishi and Murata 2006). However, to our knowledge, there have been no studies of the effects of salt stress on the processing of pre-D1. Low-temperature stress inhibits protein synthesis, degradation of D1 protein, and processing of pre-D1 Low-temperature (cold) stress was reported to enhance the extent of photoinhibition of PSII under strong light both in cyanobacteria (Samuelsson et al. 1987) and in plants (Greer et al. 1986; Öquist and Huner 1991; Öquist et al. 1993). We examined the effects of low-temperature stress on photodamage and repair in cyanobacteria by monitoring the two processes separately (Gombos et al. 1994; Wada et al.


1994; Allakhverdiev and Murata 2004) and in plants (Moon et al. 1995). We found that low-temperature stress inhibited the repair of PSII but did not affect photodamage to PSII. Labeling of proteins in Synechocystis cells demonstrated that the synthesis de novo of the D1 protein is suppressed at low temperatures (Allakhverdiev and Murata 2004). The extent of suppression depends on temperature and it seems likely that low-temperature stress suppresses the synthesis de novo of a variety of proteins (Allakhverdiev and Murata 2004). Low-temperature stress also inhibits the processing of preD1 that generates the mature D1 protein, which is necessary for the assembly of the active PSII complex (Kanervo et al. 1997). When the repair in Synechocystis was examined at 10 °C in light at 70 μmol photons m−2 s−1, both processing of pre-D1 and degradation of D1 in photodamaged PSII ceased completely, whereas a low rate of synthesis of the pre-D1 protein was detected (Allakhverdiev et al. 2003; Mohanty et al. 2007). These results suggested that processing of pre-D1 protein might be the step that is most sensitive to low temperature among the many steps involved in the repair of PSII. Moderate heat inhibits the repair of PSII Application of heat stress directly inactivates the oxygenevolving complex of PSII in thylakoid membranes from cyanobacteria (Mamedov et al. 1993) and from leaves (Nash et al. 1985; Enami et al. 1994), as well as in intact cyanobacterial cells (Fork et al. 1987; Nishiyama et al. 1993) and intact leaves (Berry and Björkman 1980). However, moderate heat stress, which does not directly inactivate the oxygen-evolving complex, stimulates photoinhibition in cyanobacteria (Allakhverdiev et al. 2007) and in plant leaves (Berry and Björkman 1980; Havaux 1992; Yang et al. 2007). Quantitation of the extent of photodamage and repair separately demonstrated that moderate heat stress decelerated the repair of photodamaged PSII but did not affect the photodamage to PSII in symbiotic algae in reef-building corals (Takahashi et al. 2004, 2009b) and in tobacco leaves (Yang et al. 2007). To our knowledge, the effects of moderate heat stress on individual steps in PSII repair, namely, degradation of the D1 protein, synthesis of pre-D1 and processing of pre-D1, have not been analyzed. CO2-limitation stress inhibits the repair of PSII Early observations suggested that depletion of CO2 enhanced the extent of photoinhibition of PSII in cyanobacteria (Kaplan 1981; Miller and Canvin 1989). It was assumed for many years that suppression of the fixation of CO2 accelerates photodamage to PSII via the excessive reduction of QA (Melis 1999). However, a more recent study demonstrated that, in C. reinhardtii, suppression of CO2 fixation by

application of exogenous glycolaldehyde, an inhibitor of phosphoribulokinase, or by a missense mutation in the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), did not accelerate the photodamage to PSII but did inhibit the repair of PSII (Takahashi and Murata 2005, 2006). These results suggested that CO2 limitation might inhibit the repair of PSII rather than accelerating photodamage to PSII. A putative mechanism for the inhibition of repair by CO2 depletion or by suppression of CO2 fixation is discussed below. In the context of the effect of CO2 depletion, it is noteworthy that salt and drought stress close stomata and suppress the fixation of CO2 (Barrs 1971; Wingler et al. 2000). Thus, these stresses might be expected to accelerate the production of ROS. Indeed, a recent study revealed that overexpression of plasma membrane H+-ATPase, which regulates the opening of stomata, promoted the uptake of CO 2 and mitigated photoinhibition of PSII in Arabidopsis (Wang et al. 2014).

Abiotic stress accelerates the production of ROS, inactivates EF-G, and inhibits the repair of PSII During photoinhibition, strong light drives the photosynthetic transport of electrons, and when the quantity of electrons generated exceeds the capacity of the Calvin cycle to absorb them, the excess electrons reduce oxygen to produce the superoxide anion radical (·O2−), which is converted to H2O2 by the action of superoxide dismutase (SOD). Strong light also generates excess energy in PSII, producing 1O2. Both the superoxide radical and singlet oxygen, indicated in red in Fig. 3, are strong oxidants and both oxidize elongation factor G (EF-G), which is essential for translation, with resultant inactivation of translation. Abiotic stress depresses the fixation of CO2, leading to generation of H2O2 How might abiotic stress increase the production of ROS, leading to the oxidation of EF-G? Rubisco catalyzes the production of glycerate-3-phosphate (3-PGA) from ribulose1,5-bisphosphate and CO2. 3-PGA is then converted to triosephosphates, such as glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, at the expense of NADPH and ATP, which are produced via the photosynthetic transport of electrons. When CO2 fixation is depressed or blocked, the production of 3-PGA decreases, with resultant decreased utilization of NADPH, and the level of NADP+ falls. Since NADP+ is a major acceptor of electrons in photosystem I (PSI), depletion of NADP+ accelerates the reduction of O2 to generate ROS (Asada 1999). There are several reports showing that interruption of the fixation of CO2 enhances the

Appl Microbiol Biotechnol CO2 limitation

NaCl

Cold

Moderate heat

Inactivation of Rubisco

Suppression of CO2 fixation

Strong light

EF–G (red.) Active

Excess electrons

(· O2– )

Excess energy

1O 2

H2O2

Catalase α-Tocopherol

NPQ

Zeaxanthin

Ascorbate peroxidase

EF–G (ox.) Inactive

3O 2

Heat

Violaxanthin de-epoxidase

H2O & O2

Fig. 3 A hypothetical scheme for the effects of abiotic stress on stimulation of the strong light-induced oxidation and inactivation of elongation factor G (EF-G) and the counteractive effects of overexpression of specific enzymes. Red boxes and arrows indicate the pathway for the light-induced oxidation and inactivation of EF-G, which is active in its reduced form and inactive in its oxidized form in the translational

machinery. Blue boxes and arrows indicate the ways in which abiotic stresses increase the accumulation of excess electrons, which leads to the production of excess ROS. Green boxes and arrows indicate the actions of specific enzymes that counteract the stress-induced stimulation of the production of H2O2 and 1O2. The arrows all indicate positive effects (see text for details). NPQ non-photochemical quenching

production of H2O2 (Radmer and Kok 1976; Radmer and Ollinger 1980; Asada and Badger 1984; Allakhverdiev et al. 2005), which, in turn, inhibits the synthesis of proteins and, in particular, the synthesis of the D1 protein (for details, see Takahashi and Murata 2008). Salinity (salt stress) inhibits CO2 fixation in plants (Plaut et al. 1991; Mota-Cadenas et al. 2010) and a high concentration of NaCl inactivates Rubisco in vitro (Solomon et al. 1994). Therefore, it seems reasonable to postulate that the primary target of salt stress is Rubisco and that inhibition of the fixation of CO2 by salt stress induces the generation of ROS (Fig. 3, in blue). The fixation of CO2 is also sensitive to moderate heat stress (Weis 1981, 1982; Feller et al. 1998; Law and Crafts-Brandner 1999). It has been reported that moderate heat stress accelerates the production of H2O2 (Tchernov et al. 2004; Yang et al. 2007) and it has also been suggested that various kinds of stress might suppress the fixation of CO2 and, thereby, stimulate the generation of ROS (Fig. 3; see also Murata et al. 2007; Takahashi and Murata 2008).

of the D1 protein is markedly suppressed by elevated levels of H2O2 and 1O2 (Nishiyama et al. 2001, 2004; Allakhverdiev and Murata 2004). Moreover, the synthesis not only of the D1 protein but also of almost all other proteins is suppressed at elevated levels of ROS (Nishiyama et al. 2001, 2004; Allakhverdiev and Murata 2004). Such global suppression of protein synthesis suggests that the protein-synthetic machinery might be a specific target of inactivation by ROS during photoinhibition. Exploration of the specific details of the inactivation by ROS of the synthesis of the D1 protein revealed that translation of the transcript of the psbA gene, which encodes pre-D1, is specifically inactivated by H2O2 and 1 O2 (Nishiyama et al. 2001, 2004). Moreover, analysis of polysomes suggested that the elongation step in the translation of psbA mRNA might be the primary target of ROS (Nishiyama et al. 2001, 2004). The effects on translation of the oxidation of EF-G, an essential participant in the elongation of nascent polypeptides, were examined in vitro in a translation system that had been prepared from Synechocystis cells (Kojima et al. 2007). Addition of the reduced form of EF-G to the H2O2-inactivated translation system restored the ability of the system to translate psbA2 mRNA, whereas the oxidized form of EF-G did not have a similar effect (Kojima et al. 2007). The critical influence of the redox state of EF-G on translation suggests that EF-G might be a primary target of inactivation by ROS within

ROS oxidize EF-G and inhibit the synthesis of proteins and the repair of PSII The ROS-induced inhibition of protein synthesis and of the repair of PSII has been investigated at the molecular level in cyanobacteria. In cyanobacterial cells, the synthesis de novo


the translational machinery. The inactivation of EF-G by ROS has been attributed to the oxidation of two specific cysteine residues in EF-G, namely, Cys105 and Cys242, with subsequent formation of an intramolecular disulfide bond (Kojima et al. 2009). Thus, it is likely that oxidation of EF-G is responsible for the ROS-induced inactivation of protein synthesis in cyanobacteria (Nishiyama et al. 2011). The particular sensitivity of EF-G to ROS was also evident in an analysis of the translational machinery from Escherichia coli: EF-G of E. coli was inactivated by H2O2 via oxidation of specific two cysteine residues and the subsequent formation of an intramolecular disulfide bond (Nagano et al. 2012). The sensitivity of protein synthesis to oxidation of EF-G specifically might be a phenomenon that is conserved in a wide variety of organisms.

Gene-technological improvements in the protection of EF-G against ROS-induced oxidation As noted above, ROS are produced by the photosynthetic machinery as inevitable by-products of photosynthesis and the main action of ROS is the inhibition of protein synthesis. The red boxes and arrows in Fig. 3 show pathways for the production and actions of ROS. ROS such as ·O2− and H2O2 are produced as a result of an excess of electrons from the photosynthetic transport of electrons, while 1O2 is produced as a result of an excess of excitation energy (Asada 1999). ROS oxidize EF-G and inactivate translation and the synthesis of proteins. In Fig. 3, the blue boxes and arrows show the effects of abiotic stress in the inactivation of Rubisco and suppression of the utilization of electrons, with the consequent stimulation of the production of H2O2 and eventual oxidation and inactivation of EF-G. The green boxes and arrows in Fig. 3 show the protective system for scavenging ROS and limiting the accumulation of ROS, which allows maintenance of EF-G in its reduced and active form. In this section, we summarize strategies that have been successfully exploited to protect EFG from oxidation (see also Table 1). Overexpression of catalase and ascorbate peroxidase for improved scavenging of H2O2 Intracellular levels of ROS depend on a balance between the rate of production and the rate of scavenging of ROS. Therefore, it is reasonable to postulate that intracellular levels of ROS should be decreased upon overexpression of genes for scavengers of ROS and that a resultant decrease in levels of ROS should result in the protection of the repair of PSII under strong light and other kinds of stress. Enzymes, such as catalase and ascorbate peroxidase, that convert H2O2 to H2O and O2 have been found in microorganisms and plants (Fig. 3).

In the cyanobacterium Synechococcus 7942, we demonstrated recently that overexpression of VktA, a highly active catalase derived from Vibrio rumoiensis, protected PSII against photoinhibition (Jimbo et al. 2013). Examination of photodamage and repair separately revealed that overexpression of VktA accelerated the repair of PSII and the synthesis of proteins, such as the D1 protein, under strong light. Overexpression of the highly active catalase appeared to protect the protein-synthetic machinery and, in particular, EF-G from oxidation by H2O2, thereby enhancing the repair of PSII. Similar effects of the overexpression of catalase were also observed in higher plants. Overexpression of the bacterial catalase KatE, derived from E. coli, in the chloroplasts of tobacco plants rendered the transgenic plants more resistant to photooxidative stress than the parental strain (Shikanai et al. 1998). In particular, the transgenic plants grew better than wild-type plants under a combination of strong light and abiotic stress, with photoinhibition of PSII being mitigated by the expression of KatE in the transgenic plants (Shikanai et al. 1998; Miyagawa et al. 2000; Foyer and Shigeoka 2011). Al-Taweel and colleagues demonstrated that decreases in intracellular levels of ROS, achieved by the introduction of KatE, protected the repair of PSII, without any effects on photodamage to PSII, under strong light in the presence of high concentrations of NaCl (Al-Taweel et al. 2007). The overexpression of KatE counteracted the inhibitory action of salt stress on the synthesis of the D1 protein de novo (AlTaweel et al. 2007). In the chloroplasts of higher plants, ascorbate peroxidase (APX) is the predominant scavenger of H2O2 (Asada 1999; Shigeoka et al. 2002). There are two types of APX in the chloroplasts of both tobacco and Arabidopsis, namely, tAPX and sAPX. The former is associated with thylakoid membranes and the latter is present in the stroma (Maruta et al. 2010). Overexpression of tAPX in the chloroplasts of tobacco plants (Yabuta et al. 2002) and of Arabidopsis plants (Murgia et al. 2004) mitigated the photoinhibition of PSII under oxidative stress due to methyl viologen, and overexpression of APX of pea in the chloroplasts of cotton plants mitigated the photoinhibition of PSII under chilling conditions (Kornyeyev et al. 2001, 2003). These studies confirmed that the respective ROS-scavenging enzymes protected PSII from photoinhibition and suggested that overexpression of ascorbate peroxidase might decrease levels of H2O2. It is reasonable to assume that a depressed level of H2O2 might tend to maintain EF-G in its reduced and active form, with consequent support of protein synthesis (Fig. 3 and Table 1) Accumulation of α-tocopherol decreases levels of 1O2 Vitamin E (α-tocopherol) is a particularly efficient scavenger of intracellular 1O2, converting it to the much less active 3O2 (Fig. 3; Neely et al. 1988; Di Mascio et al. 1990), and Havaux

LeVDE fusA

Violaxanthin de-epoxidase

Elongation factor G (EF-G)

Arabidopsis

Synechocystis 6803

codA

FNR1

Tobacco

Tobacco

Spinach Pea

Tobacco

Arthrobacter globiformis Escherichia coli

Chlamydomonas reinhardtii Synechococcus 7942

Tobacco

Synechococcus 7942

Synechococcus 7942

Oxidative stress

Heat stress

NaCl and low temperature

NaCl stress

Light stress

Chilling

Heat stress Oxidative stress

Low temperature NaCl stress

Strong light

Oxidative stress

Light stress

Reference

Photoinhibition

Photoinhibition

Photoinhibition

Photoinhibition Photoinhibition Protein synthesis

Han et al. 2010 Ejima et al. 2012

Schroda et al. 1999

Guo et al. 2007

Nakamoto et al. 2000; Sakthivel et al. 2009

Photoinhibition

Repair of PSII

Repair of PSII

Rodriguez et al. 2007

Yang et al. 2007

Holmström et al. 2000

D1 protein synthesis Ohnishi and Murata 2006

Repair of PSII

Photoinhibition

Photoinhibition Photoinhibition

D1 protein synthesis Gombos et al. 1997; Repair of PSII Allakhverdiev et al. 2001

Repair of PSII

Protein/D1 synthesis Kojima et al. 2007

Repair of PSII

Li et al. 2012b

Murgia et al. 2004

Yabuta et al. 2002

Kornyeyev et al. 2003

Shikanai et al. 1998; Miyagawa et al. 2000; Al-Taweel et al. 2007

Protein/D1 synthesis Jimbo et al. 2013

Level of analysis

Strong light and oxidative stress Photoinhibition

Oxidative stress

Oxidative and chilling stress

Chilling

Drought Oxidative stress NaCl stress

Strong light

Tobacco, Nicotiana tabacum; Pea, Pisum sativum L.; Spinach, Spinacia oleracea; Tomato, Solanum lycopersicum; Sweet pepper, Capsicum grossum

Ferredoxin-NADP reductase

+

Choline dehydrogenase and betA and betB betaine aldehyde dehydrogenase Betaine aldehyde dehydrogenase BADH

Choline oxidase

Sweet pepper Chlamydomonas reinhardtii

CaHSP26 HSP70B

Heat shock protein 70B

Synechococcus vulcanus

hspA

Small heat shock protein

Synechocystis 6803

desA

Δ12 Fatty acid desaturase

Synechocystis 6803

Synechococcus 7942

Synechocystis 6803 and Chlamydomonas Chlamydomonas reinhardtii reinhardtii Tomato Tomato

Tobacco Arabidopsis

Spinach

fusA (Cys-replaced) Synechocystis 6803

VTE2

Homogentisate phytyltransferase

Gossypium hirsutum

Tobacco

Synechococcus 7942

Transformed organism Abiotic stress tested

Pea

Escherichia coli

katE

APX

Vibrio rumoiensis

Origin

vktA

Name

Gene

Ascorbate peroxidase

Catalase

Enzyme

Table 1 Summary of successful gene-technological efforts to improve the tolerance of PSII to abiotic stress under light-stress conditions



and colleagues showed that α-tocopherol protects PSII against photoinhibition in Arabidopsis (Havaux et al. 2005). It was proposed that α-tocopherol might mitigate photodamage (Shikanai et al. 1998; Niyogi et al. 2005; Krieger-Liszkay et al. 2008) but recent studies have demonstrated that it acts by protecting the repair of PSII rather than by mitigating photodamage to PSII. In an α-tocopherol-deficient mutant of Synechocystis, the repair of PSII and the synthesis de novo of proteins, such as the D1 protein, were suppressed under strong light, but photodamage to PSII was unaffected (Inoue et al. 2011). Li and colleagues expressed the VTE2 gene for homogentisate phytyltransferase from Synechocystis in C. reinhardtii (Li et al. 2012b). The gene product plays an important role in the synthesis of tocopherols (Table 1). In the transformed Chlamydomonas cells, levels of tocopherols were more than tenfold higher than wild-type levels. Although the transformation of carotenoid-deficient mutant Chlamydomonas cells with the VTE2 gene enhanced tolerance to light stress, the transformation of wild-type cells did not increase their tolerance to light stress to any significant extent. Gene-technological increases in levels of α-tocopherol might be useful under certain specific conditions but not in general. In this context, it is noteworthy that replacement of αtocopherol by β-tocopherol in a xanthophyll-deficient mutant of Chlamydomonas significantly enhanced the tolerance of PSII in intact cells to photooxidative stress (Sirikhachornkit et al. 2009). Increases in levels of zeaxanthin enhance non-photochemical quenching of excess energy Thermal dissipation of excitation energy is a protective mechanism whereby excess light energy, absorbed by PSII, is eliminated as heat. It is the main component of nonphotochemical quenching (NPQ). Thermal dissipation in plants requires zeaxanthin, a carotenoid that is involved in the xanthophyll cycle, and the action of PsbS, a chlorophyllbinding protein within PSII (Bugos and Yamamoto 1996; Li et al. 2000; Niyogi et al. 2005). Li and colleagues proposed that the thermal dissipation of excitation energy might protect PSII from photoinhibition by decreasing the rate of photodamage to PSII (Li et al. 2002). However, more recent studies, in which photodamage and repair were monitored separately in NPQ-defective mutants of Arabidopsis, revealed that defects in NPQ decelerated the repair of PSII, with much smaller effects on photodamage to PSII than had been anticipated (Sarvikas et al. 2006, 2010; Takahashi et al. 2009a). Deceleration of the repair of PSII in the NPQ-deficient mutants was attributed to suppression of the synthesis de novo of proteins, such as the D1 protein (Takahashi et al. 2009a). Thus, thermal dissipation appears to play a role in preventing the generation of ROS by reducing

the transport of electrons rather than by protecting PSII from photodamage. The apparent protection of PSII from photoinhibition by thermal dissipation of excess energy might actually correspond to interference with the ROS-induced suppression of protein synthesis and, thus, to the resultant protection of the repair of PSII (Fig. 3). Han and colleagues overexpressed the LeVDE gene for violaxanthin de-epoxidase in tomato (Han et al. 2010). The transgenic plants contained zeaxanthin at levels higher than those in wild-type plants and levels of NPQ were also higher than those in wild-type plants. Photoinhibition was weaker in the transgenic plants than in parental plants. Although photodamage to and repair of PSII were not monitored separately by Han and colleagues, it is probable that elevated levels of zeaxanthin decreased the amount of effective light energy, with a resultant decrease in the production of ROS and the consequent mitigation of the oxidation and inactivation of EF-G (Fig. 3 and Table 1). Overexpression of glutathione reductase might lower levels of ROS Foyer and colleagues demonstrated that overexpression of a bacterial glutathione reductase in chloroplasts of poplar increased levels of the reduced form of glutathione and enhanced resistance to photoinhibition (Foyer et al. 1995). Logan and colleagues summarized the effects of overexpression of glutathione reductase on photoinhibition of PSII (Logan et al. 2006). However, while overexpression of glutathione reductase in several plant species protected PSII against photoinhibition under chilling conditions (Payton et al. 2001; Kornyeyev et al. 2001, 2003; Le Martret et al. 2011) and under oxidative conditions due to the presence of methyl viologen (Aono et al. 1995), a distinct effect on protection against photoinhibition was not always evident under certain conditions (Tyystjärvi et al. 1999; Logan et al. 2003). Although photodamage to and repair of PSII were not monitored separately in most of the above-mentioned studies, it is possible that the reduced form of glutathione, generated by glutathione reductase, might scavenge ROS and protect EF-G from oxidation and inactivation, thereby mitigating the photoinhibition of PSII. Overexpression and genetic engineering of EF-G to maintain its translational activity As noted above, ROS suppress protein synthesis, inhibiting the repair of PSII, and the sensitivity of protein synthesis to ROS is attributable to the specific oxidation of EF-G and the resultant formation of an intramolecular disulfide bond (Kojima et al. 2009). Thus, it is likely that overexpression or mutation of EF-G might protect protein synthesis and the repair of PSII against photooxidative damage.


We demonstrated that overexpression of the EF-G of Synechocystis in Synechococcus 7942 cells enhanced the synthesis de novo of all proteins, including the D1 protein, under oxidative conditions (Table 1; Kojima et al. 2007). More recently, we showed that expression in Synechocystis of mutated EF-G, in which Cys105, one of the targets of ROS, was replaced by serine, resulted in the protection of PSII from photoinhibition (Table 1; Ejima et al. 2012). This protection was attributable to the enhanced repair of PSII via acceleration of the synthesis of the D1 protein, which might have been due to a reduction in the sensitivity of protein synthesis to oxidative stress.

Gene-technological enhancement of the protection of the repair machinery against abiotic stress The repair of PSII involves the proteolytic degradation and removal of the D1 protein from photodamaged PSII, synthesis de novo of pre-D1, the assembly of PSII and the processing of pre-D1 to yield the D1 protein (Aro et al. 1993, 2005; Mulo et al. 2008). The mechanism of inhibition of the repair of PSII under most or, perhaps, all forms of abiotic stress involves the ROS-induced oxidation of EF-G and the inhibition of protein synthesis and, in particular, the synthesis of pre-D1. Therefore, genetechnological efforts to decrease levels of ROS have proved most effective in improving the tolerance of PSII to abiotic stress (see Fig. 3 and Table 1). Nonetheless, steps in the repair of PSII that are not directly related to the redox regulation of EF-G might be rate-limiting, and gene-technological modification of these steps might also improve the capacity for the repair of PSII, with resultant enhanced tolerance to abiotic stress. Figure 4 shows some examples of such inhibitory and protective mechanisms that might be involved in the regulation of PSII activity during photoinhibition (see also Table 1). Engineered increases in the unsaturation of fatty acids in membrane lipids counteract low temperature-induced inhibition of the processing of pre-D1 Thylakoid membranes of cyanobacterial cells and of chloroplasts contain four major glycerolipids: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG) (Nishida and Murata 1996). The molecular motion of these glycerolipids is determined, for the most part, by the extent of unsaturation of the fatty acids that have been esterified to the glycerol backbones of the glycerolipids (Szalontai et al. 2000). The extent of

unsaturation is, in turn, determined by the activity of fatty acid desaturases, which introduce double (unsaturated) bonds at specific positions in fatty-acyl chains that have been esterified to glycerolipids (Los and Murata 1998). Changes in the unsaturation of fatty acids might be expected to affect various functions of membrane-bound proteins, such as the photochemical and electron transport reactions that occur in thylakoid membranes. The unsaturation of fatty acids varies among cyanobacterial strains. For example, the fatty acids in membrane lipids of Synechocystis contain four double bonds, at the Δ6, Δ9, Δ12, and Δ15 positions, whereas those of Synechococcus 7942 contain only one double bond, at the Δ9 position (Murata and Wada 1995). In 1993, Öquist and colleagues reported that lowtemperature stress enhanced photoinhibition of PSII under strong light (Öquist et al. 1993). We examined the effects of low-temperature stress on photodamage and repair separately in cyanobacteria (Gombos et al. 1994; Wada et al. 1994) and in plants (Moon et al. 1995). We found that low-temperature stress inhibits the repair of PSII but does not affect photodamage to PSII. A further study demonstrated that low-temperature stress inhibits the processing of pre-D1 to generate the mature D1 protein (Fig. 4; Kanervo et al. 1997). We postulated that a decrease in membrane fluidity at low temperature might be involved in this process. The unsaturation of fatty acids in membrane lipids, which controls the fluidity of the membrane, might compensate for the low temperature-induced inhibition of the processing of pre-D1. The pre-D1 protein is synthesized on and inserted into the membrane simultaneously (Zhang and Aro 2002), and processing of pre-D1 also occurs on the thylakoid membrane. Therefore, it is likely that the processing of pre-D1 is strongly affected by the fluidity of membranes, which decreases as the temperature falls and increases upon the desaturation of fatty acids. The first successful attempt at genetically engineering an increase in the unsaturation of fatty acids was made using Synechococcus 7942, which was transformed with the desA gene for the Δ12 desaturase of Synechocystis (Wada et al. 1990). Wild-type Synechococcus 7942 cells contain monounsaturated and saturated fatty acids. By contrast, the desA+transformed Synechococcus cells also contained diunsaturated fatty acids (for details, see Murata and Wada 1995). The desA+-transformed Synechococcus cells were more resistant to photoinhibition at low temperatures than were wild-type cells (Wada et al. 1990, 1994; Gombos et al. 1997). Separate measurements of photodamage to and repair of PSII revealed that, at low temperature, repair was markedly accelerated by the introduction of the second double bond. The synthesis of the D1 protein at low temperature occurred much more rapidly in the desA+-transformed cells than in the wild-type cells. These findings demonstrated that an increase in the


Strong light Active

Inactive D2

D2 D1 Photodamage

Degradation of D1

LPA19 Processing

FtsH

Cold Insertion Membrane fluidity

D2

D2 pD1

Cold

Fatty acid desaturase

Light & abiotic stress

NaCl

pD1

ROS

Translation Glycine betaine

HSPs

CodA BADH

ATP

Abiotic stress

mRNA

Electron transport

Transcription FNR

psbA gene Fig. 4 A hypothetical representation of the effects of abiotic stresses, such as low temperature (cold) and salt stress (NaCl), and counteractive mechanisms in the photodamage and repair cycle of PSII during photoinhibition. In this scheme, translation is inhibited by ROS, which are generated by strong light under abiotic stress, such as high salt, cold, moderate heat, and CO2 limitation (see Fig. 3). Both translation and transcription are supported by the presence of ATP at high levels, which are achieved by the photosynthetic transport of electrons, which can be, in turn, inhibited by various abiotic stresses and stimulated by FNR (see text for details). Heat shock proteins (HSPs) might be involved in the

stimulation of translation. Salt and cold stress inhibit the proteolytic degradation of the D1 protein by FtsH. In addition, cold stress inhibits the processing of pre-D1. Overexpression of fatty acid desaturases increases levels of unsaturation of fatty acids in membrane lipids, fluidizes the membrane, and counteracts the effects of low-temperature stress. LPA19 is essential for the processing of pre-D1 and might compensate for the inhibitory effects of cold stress on the processing of pre-D1. Glycine betaine has pleiotropic effects: it stimulates translation and transcription and also counteracts the negative effects of salt and cold stress on the D1-degrading action of FtsH

unsaturation of fatty acids enhanced the ability of Synechococcus 7942 to tolerate strong light by accelerating the synthesis and processing of pre-D1 (Gombos et al. 1997). Under salt stress, photoinhibition of PSII was less prominent and the repair of PSII was faster in desA+-transformed cells than in wild-type cells (Allakhverdiev et al. 2001).

“heat shock proteins (HSPs).” Synechocystis contains a number of genes for HSPs, namely, Hsp18 (small heat shock protein, or sHSP), Hsp40 (DnaJ), Hsp70 (DnaK), Hsp90 (HtpG), and Hsp100 (ClpB) and, in addition, it also contains genes for Hsp60 (GroEL) and Hsp10 (GroES), which chaperone the folding and maintenance of the highly ordered structures of proteins (http://genome.kazusa.or.jp/cyanobase). It is assumed that the HSPs enhance the tolerance of cyanobacteria to heat stress. Transcriptomic analysis with microarrays has revealed that the expression of heat shock genes is induced not only by heat stress but also by many other kinds of abiotic stress, such as salt stress, low-temperature

Expression of heat shock proteins increases the resistance of PSII to photoinhibition When microorganisms are exposed to heat stress (heat shock), they synthesize several unique proteins, known collectively as


stress, osmotic stress, and oxidative stress (Los et al. 2007; Kanesaki et al. 2010). Proteomic analysis has also revealed that various abiotic stresses induce the expression of HSPs (Fulda et al. 2006; Slabas et al. 2006). These findings have suggested that HSPs play an important role in protection not only against heat stress but also against other forms of abiotic stress. Schuster and colleagues were the first to suggest that a small HSP (HSP22) in C. reinhardtii might protect PSII against heat stress under illumination (Schuster et al. 1988). Eriksson and Clark demonstrated that targeted mutagenesis of the clpB gene for Hps100 in Synechococcus 7942 decreased the ability of cells to acclimate to high temperature, as monitored in terms of photosynthetic activity (Eriksson and Clarke 1996). HtpG (Hsp90) also contributes to the protection of photosynthesis against cold stress and light stress (Hossain and Nakamoto 2002, 2003). Gene-technological improvements in tolerance of PSII to light stress were achieved by overexpression of HSPs in photosynthetic organisms. Nakamoto and colleagues demonstrated that expression in Synechococcus 7942 of the hspA gene for a small HSP from Synechococcus vulcanus moderated the photoinhibition of PSII during heat stress (Nakamoto et al. 2000). Sakthivel and colleagues observed that constitutive expression of the hspA gene in cyanobacteria conferred tolerance to oxidative stress due to exogenously applied H2O2 (Sakthivel et al. 2009). Guo and colleagues demonstrated that overexpression of a small HSP from chloroplasts of sweet pepper in tobacco plants alleviated photoinhibition under lowtemperature conditions (Guo et al. 2007; Li et al. 2012a). In the various studies mentioned above, photodamage and repair were not examined separately. Only a single study by Schroda and colleagues demonstrated that overexpression of a chloroplast-localized HSP70B in C. reinhardtii enhanced the protection of PSII against light stress by accelerating the repair of PSII (Schroda et al. 1999). It remains to be determined whether HSPs have a direct effect on translation and/or an indirect effect on translation via changes in the redox state of EF-G. Katano and colleagues generated a strain of Synechococcus 7942 with a missense mutation in the peptide-binding domain of membrane-bound DnaK3 (Katano et al. 2006). The mutant grew poorly at elevated temperatures and it accumulated an intermediate of translational form of pre-D1 on membranebound polysomes. When Katano and colleagues isolated a mutant strain in which the original mutation was suppressed, they found the suppressor mutation in the rpl24 gene that encodes the 50S ribosomal protein L24. Their findings indicate that DnaK3 is important for the structural integrity of ribosomes at high temperature and is involved in the synthesis of pre-D1. HSPs might also contribute to the stabilization of FtsH (Silva et al. 2003; Nixon et al. 2005) and accelerate the degradation of the D1 protein in photodamaged PSII under

salt stress and other kinds of abiotic stress. FtsH is a protease with a highly ordered structure and might dissociate and, thus, might become inactive under such stress. It is also possible that the overexpression of HSPs in the above-mentioned transgenic plants might stabilize the translational machinery and reduce its susceptibility to salt-induced dissociation. Engineered synthesis of glycine betaine protects PSII against abiotic stress-induced photoinhibition: pleiotropic effects Glycinebetaine (hereafter, betaine) is a fully N-methylsubstituted derivative of glycine, which is found in a large variety of halotolerant microorganisms and higher plants, as well as in animals (Rhodes and Hanson 1993). Betaine belongs to a group of compounds that are known collectively as “compatible solutes.” They are small organic metabolites that are very soluble in water and non-toxic at high concentrations. Betaine accumulates in certain halotolerant bacteria, cyanobacteria, microorganisms, and plants in response to various kinds of abiotic stress (Chen and Murata 2002, 2008, 2011; Takabe et al. 2006). Levels of betaine are generally correlated with the extent of stress tolerance (Rhodes and Hanson 1993). Betaine effectively stabilizes the quaternary structures of enzymes and complex proteins and maintains the highly ordered state of membranes at non-physiological temperatures and salt concentrations (Papageorgiou and Murata 1995). We transformed Synechococcus 7942 with the codA gene for choline oxidase from the soil bacterium Arthrobacter globiformis (Deshnium et al. 1995). This enzyme catalyzes the synthesis of betaine from choline. The codA-transformed Synechococcus 7942 cells synthesized betaine in vivo in the presence of exogenously supplied choline and accumulated betaine at levels of 60–80 mM. The codA transformation also enhanced the tolerance of the cells to low temperatures (Deshnium et al. 1997), salt stress (Ohnishi and Murata 2006), and moderate heat-stress conditions (Allakhverdiev et al. 2007). We found that salt stress due to NaCl stimulated the photoinhibition of PSII, while betaine in codA-transformed cells protected PSII against photoinhibition (Ohnishi and Murata 2006). Further studies demonstrated that the salt stress inhibited the repair of PSII and betaine reversed the inhibitory effect of salt stress and, also, that salt stress inhibited the synthesis of pre-D1 and the degradation of D1 in photodamaged PSII. By contrast, betaine protected PSII against the salt stress-induced inhibition of the degradation of the D1 protein and the synthesis of pre-D1. Our observations suggested that betaine might counteract the inhibitory effects of salt stress on both translation and the degradation of the D1 protein. We also transformed plants, namely, Arabidopsis, rice, and tomato, with the codA gene. The resultant transgenic plants


synthesized and accumulated betaine from endogenous choline and exhibited enhanced tolerance to various abiotic stresses, such as high salt, high and low temperatures, and freezing (Sakamoto and Murata 2000, 2001, 2002; Chen and Murata 2002, 2008, 2011). Holmström and colleagues examined the effects of betaine in transgenic tobacco plants on photoinhibition under high-salt and low-temperature conditions and inferred, from their results, that the recovery of PSII was enhanced by the presence of betaine (Holmström et al. 2000). Yang and colleagues examined the effects of the synthesis of betaine on photoinhibition under moderate heat stress in tobacco plants that had been transformed to synthesize betaine in vivo (Yang et al. 2005, 2007). They found that moderate heat stress inhibited Rubisco activity and, as a result, limited the fixation of CO2. These conditions are known to accelerate the generation of ROS, which, in turn, inhibit the repair of PSII. It is likely that betaine protects Rubisco against moderate heat and decreases the production of ROS, thereby limiting inhibition of the repair of PSII under moderate heat conditions. It seems likely that, in the transgenic cyanobacteria and plants discussed above, betaine had pleiotropic effects on the protection of PSII against photoinhibition under abiotic stress conditions. A common aspect of the protective effect of betaine in such transgenic organisms seems to be the stabilization of the quaternary structure of complex proteins. For example, since Rubisco is a highly ordered complex protein and is, thus, susceptible to protein-destabilizing conditions, such as high salt and heat, betaine might stabilize the structure and activity of Rubisco, thereby suppressing the production of ROS and counteracting the inhibitory effects of salt and moderate heat on protein synthesis (Fig. 3). Moreover, since the ribosome is a highly ordered complex of proteins and rRNA and since RNA polymerase is also a highly ordered five-subunit complex, the translational and transcriptional machineries might be complexes that can be dissociated and inactivated by abiotic stress. Betaine might protect and accelerate both transcription and translation (Fig. 4). Betaine also might accelerate the degradation of D1 under high-salt and low-temperature conditions. FtsH, the enzyme that degrades the D1 protein in photodamaged PSII, is a highly ordered enzyme, which is likely to be sensitive to high-salt conditions. Betaine might also stabilize the structure and activity of FtsH and, thus, maintain the process required for the repair of PSII (Fig. 4).

Potential strategies for gene-technological improvements in the tolerance of PSII to abiotic stress The repair of PSII is a complex multi-step process and, in addition to the strategies mentioned above, there are many

other potential methods for protecting it from abiotic stress under photoinhibitory conditions, as described below. Gene-technological stimulation of the synthesis of ATP enhances the protection of protein synthesis Various attempts have been made to characterize the roles of electron transport and the synthesis of ATP in the synthesis of the D1 protein. Mattoo and colleagues proposed that both electron transport and the synthesis of ATP are important for the synthesis of D1 in Spirodela oligorhiza (Mattoo et al. 1984). Studies in intact chloroplasts from spinach showed that the level of stromal ATP was correlated with the rate of lightdependent synthesis of the D1 protein (Kuroda et al. 1992). Kuroda and colleagues also demonstrated that electron transport via PSI was essential for the light-dependent translational elongation of the D1 protein (Kuroda et al. 1996). We postulated that stimulation of the synthesis of ATP might be a potential method for improvement of the tolerance of PSII to abiotic stresses (Fig. 4). We examined the effects of electron transport and the synthesis of ATP on the synthesis of proteins and, in particular, the pre-D1 protein in Synechocystis cells (Allakhverdiev et al. 2005). Our results demonstrated: (1) that inhibition of the non-cyclic transport of electrons by 3-(3′,4′-dichlorophenyl)1,1-dimethylurea (DCMU) decreased the intracellular level of ATP, levels of psbA transcripts, and the synthesis of pre-D1; (2) that inhibition of ATP synthesis by either N,Ndicyclohexylcarbodiimide (DCCD) or nigericin plus valinomycin decreased the level of ATP, suppressed the transcription and translation of psbA genes, and completely inhibited the synthesis of D1 and other proteins; and (3) that stimulation of the cyclic transport of electrons by addition of exogenous phenazine methosulfate (PMS) to DCMU-treated Synechocystis cells fully restored the capacity for transcription and translation of psbA genes and synthesis of the D1 protein. These observations suggested that the transcription of psbA genes and translation of their transcripts might be the primary target of the inhibition that results from a reduction in the intracellular level of ATP (Fig. 4). It seems likely that the requirement for ATP reflects the requirement for the energy that is essential for transcription and translation. The addition of each amino acid to a polypeptide chain during translation requires at least one molecule of ATP for aminoacylation of the cognate tRNA and two molecules of GTP for the binding of that aminoacyl-tRNA to the ribosome and the subsequent translocation of the peptidyl tRNA (Gold 1988). In addition, transcription also requires large amounts of ATP and related high-energy compounds: the addition of a nucleotide during the polymerization of RNA consumes one molecule of ATP or some other nucleotide triphosphate, whose synthesis requires ATP. Therefore, the synthesis of one molecule of pre-D1, with 360 amino acid


residues, consumes approximately 2,000 molecules of ATP. The strong correlation between the synthesis of ATP and the transcription and translation of psbA genes suggests that the supply of ATP might be an important and rate-limiting factor in the repair of PSII. As mentioned above, abiotic stresses inhibit the fixation of CO2, with the consequent depression of both electron transport and of the synthesis of ATP. Zheng and colleagues showed that salt stress inhibited the synthesis of ATP in wheat chloroplasts (Zheng et al. 2009). Tezara and colleagues demonstrated that water-deficit stress lowered the level of ATP in sunflower leaves (Tezara et al. 1999). Thus, stimulation of ATP synthesis by gene-technological methods might enhance the ability of photosynthetic organisms to tolerate abiotic stresses. Success has been reported in the overexpression of ferredoxin-NADP+ oxidoreductase (FNR), which transfers electrons from ferredoxin to NADP+ and regulates both the cyclic and the linear transport of electrons. Defects in FNR in Arabidopsis resulted in the increased sensitivity of PSII to photoinhibition (Lintala et al. 2012). Rodriguez and colleagues observed that overexpression of pea FNR in tobacco plants rendered the transgenic plants more resistant to photoinhibition of PSII (Rodriguez et al. 2007). Overexpression of PGR5 (PROTON GRADIENT REGULATION 5), which stimulates the cyclic transport of electrons (Shikanai 2007), might also be promising as an approach to the improvement of stress tolerance by increasing levels of ATP (Fig. 4). Stabilization of Rubisco might improve the protection of PSII against moderate heat and salt stress The activity of Rubisco is susceptible to abiotic stresses, such as high salt and moderate heat (Yang et al. 2007; Delfine et al. 1998). When the fixation of CO2 is limited by inactivation of Rubisco under stress conditions, the production of ROS is stimulated and levels of ROS increase, resulting in the inhibition of protein synthesis and the repair of PSII and, thus, in an increase in the extent of photoinhibition (Fig. 2). If the stability of Rubisco under stress conditions could be enhanced, the extent of photoinhibition of PSII might be reduced. Rubisco activase is associated with Rubisco and regulates the fixation of CO2 by Rubisco. Rubisco activase has been found in plants, algae, and cyanobacteria and it is essential for the activity of Rubisco. However, it dissociates from Rubisco under moderate heat stress (Feller et al. 1998; Eckardt and Portis 1997; Crafts-Brandner and Salvucci 2000). Under such environmental stress, inhibition of the repair of PSII upon interruption of the fixation of CO2 might be expected to accelerate photoinhibition. Consistent with this hypothesis, moderate heat stress (Takahashi et al. 2004; Yang et al. 2007), low-temperature stress (Allakhverdiev and Murata 2004; Grennan and Ort 2007) and salt stress (Allakhverdiev

et al. 2002; Al-Taweel et al. 2007) stimulate photoinhibition via inactivation of the fixation of CO2, production of ROS, and inhibition of the repair of PSII. Expression of the rca gene for Rubisco activase, to increase the level of Rubisco activase, should enhance CO2-fixation activity and thereby reduce the production of ROS. Expression of Rubisco and Rubisco activase from heattolerant plants, which should be stable under moderate heat stress, in mesophilic plants might improve the tolerance of the photosynthetic machinery of the mesophilic plants exposed to a combination of light plus moderate heat, low-temperature, or salt stress. Gene-technological activation of the CO2-concentrating system might protect PSII against photoinhibition under abiotic stress Activation of the CO2-concentrating system (CCM) in cyanobacteria and microalgae might reverse the effects of CO2 deficiency, which can induce the production of ROS, the oxidation of EF-G, and the inhibition of PSII repair (Fig. 3). Cyanobacteria, such as Synechocystis and Synechococcus 7942, have two CO 2 -uptake systems, NdfD3F3 and NdhD4F4, and three HCO3− transporters, BCT1, SbtA, and BicA (Kaplan et al. 2008; Price 2011; Fukuzawa et al. 2012). NdfD3F3 is a high-affinity CO2-uptake system and SbtA is a high-affinity HCO3− transporter. If such a system were overexpressed in cyanobacterial and microalgal cells, intracellular levels of inorganic carbon should rise. The resulting acceleration of CO2 fixation might then suppress the production of ROS and the inhibition of the repair of PSII. Gene-technological activation of photorespiration to protect PSII against a combination of light and abiotic stress When the supply of CO2 is limited, Rubisco oxygenates ribulose-1,5-bisphosphate (RuBP) and produces glycerate-3-phosphate and glycolate-2-phosphate (Ogren 1984). The Calvin cycle uses glycerate-3-phosphate directly but photorespiration converts glycolate-2phosphate into glycerate-3-phosphate and CO2 with the consumption of two electron equivalents (Ogren 1984). In the absence of a functional photorespiratory pathway, accumulation of the intermediates in the pathway (e.g., glyoxylate), which inhibit Rubisco, and depletion of intermediates in the Calvin cycle block the Calvin cycle under CO2-limiting conditions (Takahashi et al. 2007). Therefore, the photorespiratory pathway might provide a route towards decreasing the production of ROS and protecting PSII from photoinhibition (Osmond 1981, 1997; Osmond and Grace 1995; Wingler et al. 2000). Gene-technological evidence for this possibility was


obtained by Takahashi and colleagues, who demonstrated that mutants of Arabidopsis thaliana with impairment of enzymes involved in the photorespiratory pathway exhibited increased photoinhibition of PSII that resulted from suppression of the repair of photodamaged PSII and not from acceleration of the photodamage to PSII (Takahashi et al. 2007). Suppression of the repair process was attributable to an increase in the production of ROS and inhibition of the synthesis of the D1 protein at the translational level. The rate-limiting step in the photorespiratory pathway under abiotic stress has not yet been identified. When the ratelimiting step has been identified, the gene for the relevant enzyme will be a good target for gene-technological improvements in the stress tolerance of PSII.

CtpA, a processing enzyme, and LPA19 are potential targets for improvements in the stress tolerance of PSII The carboxy-terminal protease CtpA is an enzyme that processes pre-D1 to yield mature D1 protein (Yamamoto et al. 2001). As noted above, the processing of pre-D1 is particularly sensitive to low temperature (Kanervo et al. 1997). CtpA from cold-tolerant organisms is likely to be active at low temperatures. Therefore, expression of the ctpA gene from cold-tolerant organisms in mesophilic organisms, such as Synechocystis, might enhance the recovery of PSII from photoinhibition at low temperatures. Wei and colleagues demonstrated that LPA19, a homolog of Psb27, contributes to the processing of pre-D1 in Arabidopsis (Wei et al. 2010). It seems plausible that overexpression of LPA19 in chloroplasts might render transgenic plants tolerant to low-temperature stress in the light.

Conclusion The tolerance of the photosynthetic machinery to abiotic stress can be improved by genetic manipulation of the repair of PSII. Results obtained in efforts to achieve this goal can be interpreted in terms of the recently proposed mechanism of photoinhibition of PSII. Gene-technological methods that lead to enhanced repair of PSII should allow the development of strains of important microalgae, agricultural crops, and sources of biofuel with improved productivity in current harsh environments and in those that emerge as climate change accelerates. Acknowledgments This work was supported, in part, by JSPS KAKENHI Grant Numbers 24570039 and 25119704 (to Y.N.).

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