The Plant Journal (2015) 81, 759–766
doi: 10.1111/tpj.12768
High light-induced hydrogen peroxide production in Chlamydomonas reinhardtii is increased by high CO2 availability Thomas Roach1,*,a, Chae Sun Na2,a and Anja Krieger-Liszkay1 Commissariat a l’Energie Atomique (CEA) Saclay, iBiTec-S, CNRS UMR 8221, Service de Bioe nerge tique, Biologie Structurale et Me canisme, 91191 Gif-sur-Yvette Cedex, France, and 2 Seed Bank of Wild Resource Plants, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea
1
Received 29 July 2014; revised 20 December 2014; accepted 12 January 2015; published online 24 January 2015. *For correspondence (e-mail: [email protected]). a Present address: Institute of Botany, Leopold-Franzens-Universita€ t-Innsbruck, Sternwartestraße 15, 6020 Innsbruck, Austria.
SUMMARY The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO2 availability putatively includes enhanced ROS production in the so-called Mehler reaction. Such conditions are thought to encourage O2 to become an electron acceptor at photosystem I, producing the ROS superoxide anion radical (O52 ) and hydrogen peroxide (H2O2). In contrast, here it is shown in Chlamydomonas reinhardtii that CO2 depletion under high light levels lowered cellular H2O2 production, and that elevated CO2 levels increased H2O2 production. Using various photosynthetic and mitochondrial mutants of C. reinhardtii, the chloroplast was identified as the main source of elevated H2O2 production under high CO2 availability. High light levels under low CO2 availability induced photoprotective mechanisms called non-photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE-deficient mutant npq4 produced more H2O2 than wild-type cells under high light levels, although less so under high CO2 availability, whereas it demonstrated equal or greater enzymatic H2O2-degrading capacity. The qT-deficient mutant stt7-9 produced the same H2O2 as wild-type cells under high CO2 availability. Physiological levels of H2O2 were able to hinder qT and the induction of state 2, providing an explanation for why under high light levels and high CO2 availability wild-type cells behaved like stt7-9 cells stuck in state 1. Keywords: photosynthesis, Chlamydomonas reinhardtii, CO2, Mehler reaction, NPQ, state transition, hydrogen peroxide.
INTRODUCTION During photosynthesis the reducing power released from photosystem I (PSI) via ferredoxin enables the reduction of NADP+ to NADPH, which is essential in the Calvin–Benson cycle to make sugars. Alternatively, PSI can reduce other electron acceptors. For example, in the Mehler reaction O2 is reduced by PSI to the superoxide anion radical (O52 ; Mehler, 1951), which is part of the water–water cycle (Takahashi and Asada, 1988; Miyake, 2010). In a dinoflagellate and in two diatom species investigated O2 can support up to 50% of PSII electron flow under high light levels (Waring et al., 2010; Roberty et al., 2014). It is a generally accepted concept that the Mehler reaction occurs when low CO2 and NADP+ levels impede the Calvin–Benson cycle (Asada, © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd
2000; Miyake and Yokota, 2000). By maintaining electron flow the Mehler reaction could theoretically enable the formation of a proton gradient (ΔpH) to support ATP synthesis and induce defence mechanisms against high light levels, but at the expense of producing O2 (Asada, 2000; Ort and Baker, 2002). Although O2 and its dismutation product hydrogen peroxide (H2O2) can be removed safely by antioxidant enzymes in the water–water cycle, the generation of these reactive oxygen species (ROS) has to be kept under control. Such ROS can damage the photosynthetic apparatus, contribute to photoinhibition and inhibit the Calvin–Benson cycle enzymes (Kaiser, 1976; Keren and Krieger-Liszkay, 2011). Under high light levels H2O2 is also 759
760 Thomas Roach et al. part of the signalling cascades that upregulate the expression of antioxidant enzyme genes and increase the production of low molecular-weight antioxidants (Karpinski et al., 1999; Urzica et al., 2012; Chang et al., 2013). Like chloroplasts, mitochondria produce H2O2, but at a rate approximately 20 times lower in the light (Foyer and Noctor, 2003), indicating the potential importance of the Mehler reaction to the cellular level of H2O2. The production of ROS can be restricted in the first place by regulatory mechanisms referred to as non-photochemical quenching (NPQ; as reviewed by Roach and Krieger-Liszkay, 2014). The two most rapidly reversible components of NPQ are high energy state quenching and state transitions, which are abbreviated to qE and qT, respectively. The DpH promotes the formation of zeaxanthin (Demmig-Adams and Adams, 1996) and the protonation of a key light harvesting complex (LHC)-type protein (Li et al., 2004; Tokutsu and Minagawa, 2013), which contibute to the induction of qE and enable a dissipation of excess light energy to heat. In higher plants this LHC-type protein is PsbS and does not bind pigments, whereas in Chlamydomonas reinhardtii it is LHCSR3 and binds chlorophyll a/b and xanthophylls (Bonente et al., 2011). Mutants deficient in PsbS or LHCSR3 are both referred to as npq4, show compromised qE, and are rendered sensi€ lheim et al., 2002) or high tive to naturally fluctuating (Ku light levels (Peers et al., 2009; Roach and Krieger-Liszkay, 2012). LHCSR3 is in low abundance in typical growth conditions of C. reinhardtii (e.g. 50 lmol quanta m 2 sec 1), but can be synthesised when cells are placed under high light levels (Peers et al., 2009). The other rapidly reversible NPQ mechanism, qT, is the temporary association of LHCII with either PSII or PSI, thereby changing the antenna size of the photosystems. In C. reinhardtii qT is more active than it is in higher plants (Rochaix et al., 2012). In ‘state 1’ LHCII is associated with PSII, whereas in ‘state 2’ approximately 70% of LHCII can detach from PSII in C. reinhardtii, of which approximately 20% can attach to PSI (Nagy et al., 2014). The redox state of the plastoquinone (PQ) pool controls qT. When the PQ pool is over-reduced LHCII becomes phosphorylated by Stt7 kinase, triggering its release from PSII and movement to PSI, whereas an oxidised PQ pool initiates the dephosphorylation of LHCII, inducing its return to PSII (Rochaix et al., 2012). Not only does this provide self-regulation of the redox state of the PQ pool, the movement of LHCII away from PSII also relieves excitation pressure (Tikkanen et al., 2012). The availability of mutants deficient in qE (npp4), qT (stt79), or qE and qT (npq4stt7-9) makes C. reinhardtii an excellent model for studying how NPQ protects against lightinduced ROS production. A further advantage is that this alga uses glycolate dehydrogenase rather than an oxidase for recycling the oxygenase products of RUBISCO (Plancke et al., 2014). Therefore, under low CO2 levels photorespira-
tory H2O2 production does not occur in C. reinhardtii, as it does in higher plants, which would otherwise confound measurements of the Mehler reaction. Furthermore, H2O2 passes through the chloroplast and out of the cell, making it readily quantifiable in the algal media (Collen and Pedersen, 1996; Takeda et al., 1997; Michelet et al., 2013). Taking advantage of these attributes, it is shown here that although CO2 limitation of cells induced NPQ, it lowered H2O2 production, despite that these conditions are thought to increase the rate of the Mehler reaction. Changes in CO2 availability led to relatively small variations in the NADP+/ NADPH ratio, indicating that the redox state of the stroma was maintained during CO2 depletion. By using respiratory mutants the source of H2O2 under high CO2 availability was shown to come from the chloroplast and not from the mitochondria. It is also demonstrated that under CO2 limitation NPQ afforded by qE and qT accumulatively prevented H2O2 production, whereas only qE remained protective under high CO2 availability. The Mehler reaction had a direct influence on qT. Not only did exogenous H2O2 inhibit LHCII phosphorylation needed for qT, physiological levels of H2O2 were able to hinder the induction of state 2. This explains why under high light levels and high CO2 availability the wild-type cells behaved like stt7-9 cells stuck in state 1. RESULTS Treating photoautotrophic C. reinhardtii in the exponential growth phase with high light levels (250 lmol quanta m 2 sec 1) led to CO2 depletion, reducing photosynthetic rates to
doi: 10.1111/tpj.12768
High light-induced hydrogen peroxide production in Chlamydomonas reinhardtii is increased by high CO2 availability Thomas Roach1,*,a, Chae Sun Na2,a and Anja Krieger-Liszkay1 Commissariat a l’Energie Atomique (CEA) Saclay, iBiTec-S, CNRS UMR 8221, Service de Bioe nerge tique, Biologie Structurale et Me canisme, 91191 Gif-sur-Yvette Cedex, France, and 2 Seed Bank of Wild Resource Plants, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea
1
Received 29 July 2014; revised 20 December 2014; accepted 12 January 2015; published online 24 January 2015. *For correspondence (e-mail: [email protected]). a Present address: Institute of Botany, Leopold-Franzens-Universita€ t-Innsbruck, Sternwartestraße 15, 6020 Innsbruck, Austria.
SUMMARY The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO2 availability putatively includes enhanced ROS production in the so-called Mehler reaction. Such conditions are thought to encourage O2 to become an electron acceptor at photosystem I, producing the ROS superoxide anion radical (O52 ) and hydrogen peroxide (H2O2). In contrast, here it is shown in Chlamydomonas reinhardtii that CO2 depletion under high light levels lowered cellular H2O2 production, and that elevated CO2 levels increased H2O2 production. Using various photosynthetic and mitochondrial mutants of C. reinhardtii, the chloroplast was identified as the main source of elevated H2O2 production under high CO2 availability. High light levels under low CO2 availability induced photoprotective mechanisms called non-photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE-deficient mutant npq4 produced more H2O2 than wild-type cells under high light levels, although less so under high CO2 availability, whereas it demonstrated equal or greater enzymatic H2O2-degrading capacity. The qT-deficient mutant stt7-9 produced the same H2O2 as wild-type cells under high CO2 availability. Physiological levels of H2O2 were able to hinder qT and the induction of state 2, providing an explanation for why under high light levels and high CO2 availability wild-type cells behaved like stt7-9 cells stuck in state 1. Keywords: photosynthesis, Chlamydomonas reinhardtii, CO2, Mehler reaction, NPQ, state transition, hydrogen peroxide.
INTRODUCTION During photosynthesis the reducing power released from photosystem I (PSI) via ferredoxin enables the reduction of NADP+ to NADPH, which is essential in the Calvin–Benson cycle to make sugars. Alternatively, PSI can reduce other electron acceptors. For example, in the Mehler reaction O2 is reduced by PSI to the superoxide anion radical (O52 ; Mehler, 1951), which is part of the water–water cycle (Takahashi and Asada, 1988; Miyake, 2010). In a dinoflagellate and in two diatom species investigated O2 can support up to 50% of PSII electron flow under high light levels (Waring et al., 2010; Roberty et al., 2014). It is a generally accepted concept that the Mehler reaction occurs when low CO2 and NADP+ levels impede the Calvin–Benson cycle (Asada, © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd
2000; Miyake and Yokota, 2000). By maintaining electron flow the Mehler reaction could theoretically enable the formation of a proton gradient (ΔpH) to support ATP synthesis and induce defence mechanisms against high light levels, but at the expense of producing O2 (Asada, 2000; Ort and Baker, 2002). Although O2 and its dismutation product hydrogen peroxide (H2O2) can be removed safely by antioxidant enzymes in the water–water cycle, the generation of these reactive oxygen species (ROS) has to be kept under control. Such ROS can damage the photosynthetic apparatus, contribute to photoinhibition and inhibit the Calvin–Benson cycle enzymes (Kaiser, 1976; Keren and Krieger-Liszkay, 2011). Under high light levels H2O2 is also 759
760 Thomas Roach et al. part of the signalling cascades that upregulate the expression of antioxidant enzyme genes and increase the production of low molecular-weight antioxidants (Karpinski et al., 1999; Urzica et al., 2012; Chang et al., 2013). Like chloroplasts, mitochondria produce H2O2, but at a rate approximately 20 times lower in the light (Foyer and Noctor, 2003), indicating the potential importance of the Mehler reaction to the cellular level of H2O2. The production of ROS can be restricted in the first place by regulatory mechanisms referred to as non-photochemical quenching (NPQ; as reviewed by Roach and Krieger-Liszkay, 2014). The two most rapidly reversible components of NPQ are high energy state quenching and state transitions, which are abbreviated to qE and qT, respectively. The DpH promotes the formation of zeaxanthin (Demmig-Adams and Adams, 1996) and the protonation of a key light harvesting complex (LHC)-type protein (Li et al., 2004; Tokutsu and Minagawa, 2013), which contibute to the induction of qE and enable a dissipation of excess light energy to heat. In higher plants this LHC-type protein is PsbS and does not bind pigments, whereas in Chlamydomonas reinhardtii it is LHCSR3 and binds chlorophyll a/b and xanthophylls (Bonente et al., 2011). Mutants deficient in PsbS or LHCSR3 are both referred to as npq4, show compromised qE, and are rendered sensi€ lheim et al., 2002) or high tive to naturally fluctuating (Ku light levels (Peers et al., 2009; Roach and Krieger-Liszkay, 2012). LHCSR3 is in low abundance in typical growth conditions of C. reinhardtii (e.g. 50 lmol quanta m 2 sec 1), but can be synthesised when cells are placed under high light levels (Peers et al., 2009). The other rapidly reversible NPQ mechanism, qT, is the temporary association of LHCII with either PSII or PSI, thereby changing the antenna size of the photosystems. In C. reinhardtii qT is more active than it is in higher plants (Rochaix et al., 2012). In ‘state 1’ LHCII is associated with PSII, whereas in ‘state 2’ approximately 70% of LHCII can detach from PSII in C. reinhardtii, of which approximately 20% can attach to PSI (Nagy et al., 2014). The redox state of the plastoquinone (PQ) pool controls qT. When the PQ pool is over-reduced LHCII becomes phosphorylated by Stt7 kinase, triggering its release from PSII and movement to PSI, whereas an oxidised PQ pool initiates the dephosphorylation of LHCII, inducing its return to PSII (Rochaix et al., 2012). Not only does this provide self-regulation of the redox state of the PQ pool, the movement of LHCII away from PSII also relieves excitation pressure (Tikkanen et al., 2012). The availability of mutants deficient in qE (npp4), qT (stt79), or qE and qT (npq4stt7-9) makes C. reinhardtii an excellent model for studying how NPQ protects against lightinduced ROS production. A further advantage is that this alga uses glycolate dehydrogenase rather than an oxidase for recycling the oxygenase products of RUBISCO (Plancke et al., 2014). Therefore, under low CO2 levels photorespira-
tory H2O2 production does not occur in C. reinhardtii, as it does in higher plants, which would otherwise confound measurements of the Mehler reaction. Furthermore, H2O2 passes through the chloroplast and out of the cell, making it readily quantifiable in the algal media (Collen and Pedersen, 1996; Takeda et al., 1997; Michelet et al., 2013). Taking advantage of these attributes, it is shown here that although CO2 limitation of cells induced NPQ, it lowered H2O2 production, despite that these conditions are thought to increase the rate of the Mehler reaction. Changes in CO2 availability led to relatively small variations in the NADP+/ NADPH ratio, indicating that the redox state of the stroma was maintained during CO2 depletion. By using respiratory mutants the source of H2O2 under high CO2 availability was shown to come from the chloroplast and not from the mitochondria. It is also demonstrated that under CO2 limitation NPQ afforded by qE and qT accumulatively prevented H2O2 production, whereas only qE remained protective under high CO2 availability. The Mehler reaction had a direct influence on qT. Not only did exogenous H2O2 inhibit LHCII phosphorylation needed for qT, physiological levels of H2O2 were able to hinder the induction of state 2. This explains why under high light levels and high CO2 availability the wild-type cells behaved like stt7-9 cells stuck in state 1. RESULTS Treating photoautotrophic C. reinhardtii in the exponential growth phase with high light levels (250 lmol quanta m 2 sec 1) led to CO2 depletion, reducing photosynthetic rates to
