Reactive Oxygen Species and Autophagy in Plants and Algae

Reactive oxygen species (ROS) and autophagy have been historically associated with cell death. However, more recent evidence indicates that both ROS and autophagy play important roles in signaling and cellular adaptation to stress. As a catabolic process, autophagy allows eukaryotic cells to recycle intracellular components including entire organelles during development or under stress conditions such as nutrient limitation. Degradation and recycling of macromolecules via autophagy provides a source of building blocks (amino acids, lipids, and sugars) that allow temporal adaptation of cells to adverse conditions. In addition to recycling, autophagy is required for the degradation of damaged or toxic material that can be generated as a result of ROS accumulation during oxidative stress. The mitochondrial electron-transport chain and the peroxisomes are primary sources of ROS production in most eukaryotes. The plant cell contains an additional organelle, the chloroplast, with an intense electron flow that leads to high rates of ROS production. Studies in plants and algae have demonstrated that autophagy is structurally and functionally conserved in photosynthetic organisms and plays an important role in the cellular response and adaptation to different stress conditions that involve the generation of ROS, such as oxidative and drought stresses, pathogen infection, or photooxidative damage. These findings suggested a strong link between autophagy and ROS in photosynthetic eukaryotes. Here, we review recent studies in plants and algae describing redox control of autophagy and discuss the conserved regulatory proteins that may transmit redox signals to the autophagic machinery. During autophagy (also known as macroautophagy), cytoplasmic components are nonselectively enclosed within a double-membrane vesicle known as the autophagosome and delivered to the vacuole/lysosome for degradation of toxic components and recycling of needed nutrients (He and Klionsky, 2009; Mizushima et al., 2011; Liu and Bassham, 2012). This degradative process is widely conserved through evolution, and accordingly, autophagy-related (ATG) genes have been found in all eukaryotes. Originally identified in the budding yeast Saccharomyces cerevisiae (Tsukada and Ohsumi, 1993), homologs to ATG genes have been described from lower eukaryotes like fungi and algae to plants and metazoans (Meijer et al., 2007; Díaz-Troya et al., 2008b). Some ATG proteins have a structural function in autophagy, while others are important for the regulation of the process. For instance, the ATG5-ATG12-ATG16 protein complex and the ATG8-phosphatidylethanolamine (PE) conjugate are essential for autophagosome formation and completion, while ATG8-PE also mediates fusion of the autophagosome to the vacuole membrane (Mizushima et al., 2011). The ATG4 Cys protease processes nascent ATG8 at the C terminus to facilitate its covalent binding to PE, but it is also able to cleave PE from ATG8 for its recycling, playing a crucial role in autophagy regulation (Kirisako et al., 2000; see below). Another important regulatory protein in the autophagic process is the ATG1 kinase (Fig. 1), which is required for the generation of preautophagosome structures and autophagy initiation (He and Klionsky, 2009; Mizushima, 2010). In yeast, mammals, and flies, ATG1 function is controlled by phosphorylation events mainly through the Target of Rapamycin (TOR) kinase (Kamada et al., 2000; Chang and Neufeld, 2009; Jung et al., 2009; Fig. 1), a central controller of cell growth in all eukaryotes (Wullschleger et al., 2006) functionally conserved in plants and algae (Menand et al., 2002; Crespo et al., 2005; see below). An important upstream positive regulator of autophagy in mammalian cells is the evolutionarily conserved AMPK (for AMP-activated protein kinase) protein, which stimulates autophagic processes by inhibiting mTORC1 signaling at the level of Tuberous Sclerosis Complex2 (TSC2; Inoki et al., 2003) in response to energy (ATP) level limitation (Alers et al., 2012). It has recently been shown that in addition to this indirect pathway, AMPK regulates autophagy by direct phosphorylation of Ulk1 (the mammalian ATG1 homolog) in coordination with mTORC1 (Kim et al., 2011). Proposed model of autophagy regulation by ROS in plants and algae. The intracellular level of ROS is modulated by different signals, including high-light stress, nutrient limitation, ER stress, or pathogen infection. ROS ultimately lead to ATG1 activation and hence autophagy induction either via TOR signaling or a TOR-independent mechanism, denoted here as X. This unknown pathway might control autophagy through ATG1 regulation, as reported in mammalian cells for AMPK (SnRK1 in plants), or by a different mechanism as described for the ATG4 protease. See text for details. DPI, Diphenylene iodonium; NF, norflurazon; rap, rapamycin; tun, tunicamycin. The evolutionary conservation of structural and regulatory ATG proteins in photosynthetic eukaryotes strongly suggests the presence of similar regulatory mechanisms of autophagy in these organisms. In agreement with this hypothesis, TOR has been shown to inhibit autophagy in plants and algae (Liu and Bassham, 2010; Pérez-Pérez et al., 2010), whereas ATG1 activity appears to participate in autophagy regulation in plants (Suttangkakul et al., 2011). Nevertheless, our current knowledge of the molecular mechanisms and signals by which TOR and ATG1 control autophagy in photosynthetic organisms is still very limited. The purpose of this review will be to highlight recent findings connecting redox signals to the control of autophagy in plants and algae and to discuss how regulatory components in the autophagy process integrate these stress signals based on our current knowledge from other systems. Excessive ROS may cause irreversible oxidative damage to proteins, lipids, and nucleic acids and activate signaling pathways ultimately leading to cell death (Apel and Hirt, 2004). However, at low levels, ROS participate in prosurvival mechanisms, acting as second messengers that transmit initial stress signals allowing cells to react and adapt to different environmental cues (Mittler et al., 2011). The chloroplast is known to be one of the main sources of ROS in plants and algae. Superoxide anions (O2 •−) are generated as by-products of photosynthetic electron transport and readily converted into hydrogen peroxide (H2O2) inside the chloroplast through chemical and enzymatic reactions. Singlet oxygen (1O2) and hydroxyl radicals are also produced during photosynthesis and can cause oxidative damage. In addition to the chloroplast, ROS are produced in plant cells in mitochondria, peroxisomes, and at the plasma membrane by NADPH oxidases (NOX; Fig. 2). Among different types of ROS generated in the chloroplast, H2O2 is the most stable molecule and diffuses across membranes, in striking contrast to O2 •− and hydroxyl radicals, which are not usually released from functional chloroplasts. The highly reactive 1O2 is generated predominantly at the reaction center of PSII, but despite its short half-time (about 200 ns; Krieger-Liszkay, 2005), it can play a role in signaling either by diffusion outside the thylakoid membrane (Fischer et al., 2007) or through second messengers, such as the carotenoid oxidation product β-cyclocitral (Ramel et al., 2012). Indeed, besides its toxic effects, 1O2 was shown to trigger, in plants and algae, different signaling cascades leading to programmed cell death (Wagner et al., 2004) or to acclimation (Ledford et al., 2007) through regulation of the expression of specific genes (op den Camp et al., 2003; Gadjev et al., 2006; Fischer et al., 2009). Among ROS, H2O2 best fulfills the requirements of being a second messenger, as its stability, its membrane permeability, its reactivity that provides specificity for the oxidation of thiols, and its enzymatic production and degradation that provide specificity for time and place are required for signaling (Forman et al., 2010). H2O2 is recognized as an important signaling molecule in a wide range of organisms, including plants. H2O2 regulates numerous processes such as cell division, differentiation, and growth as well as apoptosis and plays a major role in the control of plant development and adaptation to biotic and abiotic stresses (Foyer and Noctor, 2009). Recent studies in photosynthetic organisms described the activation of autophagy in response to several stimuli that increase ROS generation, regardless of the origin and location of ROS production in the cell (Xiong et al., 2007; Liu et al., 2009; Pérez-Pérez et al., 2010, 2012). Different ROS sources control autophagy in plants and algae. ROS can be generated by plasma membrane-localized NOX and different organelles, including chloroplast, mitochondria, peroxisome, and ER. Excess ROS then induce autophagy, which contributes to down-regulate ROS production and remove damaged cellular components. See text for details. Treatment of Arabidopsis (Arabidopsis thaliana) plants with H2O2 results in severe oxidative stress and leads to the induction of autophagy (Xiong et al., 2007). Under these conditions, different types of irreversible oxidation of proteins occur, including carbonylation, sulfinic/sulfonic acid formation, or Tyr nitration. The finding that mutant plants defective in autophagy are hypersensitive to H2O2 and accumulate carbonylated proteins demonstrated that this degradative process is required for the cellular adaptation to oxidative stress. In close agreement, it has been shown that atg2 and atg5 Arabidopsis mutants accumulate high levels of H2O2 (Yoshimoto et al., 2009). Similar to H2O2, treatment of plants with the ROS-producing agent methylviologen (MV) also triggers autophagy (Xiong et al., 2007), although the molecular mechanism by which MV generates ROS differs from H2O2. MV is able to intercept electrons from PSI in the chloroplast or from the respiratory electron transport chain in the plant mitochondria and then reacts with oxygen to produce O2 •−, which in turn is rapidly converted to H2O2. Therefore, MV generates ROS in the chloroplast and/or in the mitochondria in plants and may induce autophagy by oxidative damage caused in different organelles. Treatment of cells of the green alga Chlamydomonas (Chlamydomonas reinhardtii) with H2O2 or MV also results in autophagy activation (Pérez-Pérez et al., 2010, 2012). Chlamydomonas can grow in the dark using acetate as a carbon source. Therefore, contrary to plants, darkness not induce autophagy in this alga (Pérez-Pérez et al., 2010). The of Chlamydomonas to grow a to the origin of redox signals in response to oxidative stress. As a ROS, H2O2 autophagy under or dark conditions, whereas MV photosynthetic electron flow to produce ROS and to this catabolic process (Pérez-Pérez et al., that ROS generated in the chloroplast may to the activation of cytoplasmic autophagic machinery. Nevertheless, treatment of Chlamydomonas cells with MV in the dark also in autophagy activation to ROS production in the mitochondria (Pérez-Pérez et al., 2012). also known as respiratory homologs or are at the plasma membrane and the production of O2 •− et al., These play a central role in the redox in plants by ROS with other signaling pathways and processes such as the hypersensitive response by pathogen or signaling in response to biotic and abiotic stresses et al., NOX activity has been in autophagy regulation in plants (Liu et al., 2009). of NOX with the chemical that induction of autophagy by nutrient or limitation or stress NOX Therefore, ROS generated by NOX to be required for the activation of autophagy in response to nutrient and stress, a role of ROS in the control of autophagy in plants. stress, which also triggers autophagy in plants, not to be by NOX (Liu et al., 2009). This finding indicates that autophagy is under and stresses, and while autophagy in and plants is by a pathway ROS, induction of this process by stress is by a has been in several NOX mutants (Liu et al., but the presence of genes in the Arabidopsis et al., 2003) may the of the specific NOX for autophagy regulation in plants. is a of autophagy in plant cells (Liu et al., ROS might autophagy induction in response to this biotic stress. infection, ROS are generated at the plasma membrane by although other organelles such as mitochondria, and/or peroxisomes ROS production et al., recent in Arabidopsis that NOX may also play an important role in intracellular ROS generated by these organelles to in redox and cell death associated with acid signaling et al., 2012). the role of ROS in the control of autophagy, NOX might participate in the activation of this catabolic process during the initial of pathogen infection. This be with and including NOX or autophagy in plants. link between ROS generation in the chloroplast, photooxidative and autophagy activation has recently been shown in The of mutants that the of leads to levels of ROS in the chloroplast and a increase in autophagic activity (Pérez-Pérez et al., 2012). The Chlamydomonas mutant the one of the in the carotenoid pathway, and grow in the presence of to the of et al., This mutant high levels of autophagy in the which are cells are to (Pérez-Pérez et al., 2012). can also be through the chemical of with the and The finding that treatment of Chlamydomonas cells with autophagy in the but not in the dark despite carotenoid under both conditions suggested that autophagy is associated with photooxidative damage caused by ROS production. this a activation of autophagy has been in Chlamydomonas cells to high-light stress (Pérez-Pérez et al., 2012). from the that the Chlamydomonas which specific and is high et al., high levels of autophagy to this stress (Pérez-Pérez et al., 2012). As NOX participate in the induction of autophagy nutrient limitation and stress in plants. However, it is unknown NOX might play a similar role in the redox regulation of autophagy in algae. being to be in (Mittler et al., an of the Chlamydomonas has recently the of NOX homologs in this et al., 2011; Pérez-Pérez et al., although it to be these proteins have NOX The of on the activation of autophagy photooxidative damage has been in Chlamydomonas and autophagy was but not with this (Pérez-Pérez et al., 2012). This finding a functional link between ROS and autophagy induction in Chlamydomonas and strongly suggested that plasma membrane-localized NOX in autophagy activation either or by to the of cellular ROS levels under stress conditions. It is that NOX to redox signals generated in response to other stresses in similar to the of signals reported for plant et al., et al., has been as a degradative process by nutrient limitation for the recycling of intracellular material that might be as building blocks to the of It is well that nutrient triggers autophagy by signaling in yeast and metazoans below). However, studies in different that nutrient limitation also ROS which in turn may et demonstrated that yeast mutant cells in autophagy accumulate ROS to a cells during and that ROS are a major to the respiratory function that leads to cell death. The that autophagy is required during for the of mitochondria function that is important for cell As Liu et that of NOX activity in plants autophagosome formation in response to or strongly that ROS may function as signaling to induce autophagy during nutrient stress. In mammalian et demonstrated that H2O2 produced in mitochondria as a signaling molecule during the accumulation of ROS appears to be essential for autophagy induction in cells et al., 2007). these findings that the of nutrients is a primary leading to autophagy activation in but this stress is associated with the production and accumulation of The accumulation of proteins in the is a stress that the expression of and proteins required for the of cell This signaling process is as the protein response and the activation of for protein control in the ER that function to the accumulation of proteins and 2007). As of the cellular response to ER stress, proteins are into the with and then via the a process that is known as degradation et al., ER stress is also a strong of autophagy, and this is conserved from yeast to et al., The of ER such as an of an autophagic response similar to other In photosynthetic a strong induction of autophagy by ER stress has been shown in Chlamydomonas (Pérez-Pérez et al., 2010), and based on the high conservation of both autophagy and ER stress it is to that a similar link in plants. the essential role of formation in the and of and membrane proteins, protein in the ER is as a highly process. studies have an between ER stress and ROS generation and 2007; and 2007), and the molecular mechanisms by which ROS are produced during have been in a recent review et al., 2009). These pathways in the ER such as which can H2O2, although mitochondrial ROS and NOX have also been to ROS generation during ER stress. it that ER ROS or In response to ER stress, cells may an autophagic process to remove proteins and including ROS, or to ER caused by during the as in yeast et al., to cell studies will be required to ROS production is a main regulatory mechanism in ER organisms have different mechanisms ROS In plants and algae, enzymatic and mechanisms have been reported for ROS of which are to these organisms to the presence of as ROS (Foyer and Noctor, 2009). such as and as well as membrane-localized and which are to plants and algae. These are a major of H2O2 and 1O2 produced in the mechanisms such as for H2O2 and for O2 •− to O2 and H2O2, and such as and for including H2O2 (Foyer and Noctor, 2009). and algae can activate several for the of different ROS, but in conditions, ROS can still be damage in the In this more mechanisms be in the cell in to remove damaged components and ROS under As autophagy is a primary mechanism to and it may also function to remove organelles. In yeast, ROS production during or the under respiratory conditions may to the of mitochondria by autophagy, a process known as et al., 2009; et al., 2009). In agreement with this the ROS is able to activity in yeast et al., 2009). an important H2O2 is in peroxisomes, and the specific degradation of this by autophagy has been reported in several yeast species and mammals, in a process et al., In plants, peroxisomes may play an important role in redox signaling and in the control of redox a major source of H2O2 but can also produce O2 •− radicals or et al., 2002; et al., 2009; 2011). has not been demonstrated in the role of degradation and its in redox signaling The chloroplast is a primary source of ROS in plants and algae, and it may be for degradation in a process Degradation of chloroplast material through autophagic processes has been reported in plants under conditions caused by darkness et al., et al., 2009; et al., 2010). degradation via chloroplast that can be in the et al., In addition to degradation of has been described in plants et al., 2009). However, are degradation pathways for chloroplast including autophagy and but autophagy not to be the main pathway, degradation is not in plants et al., 2009). In close agreement, direct between autophagy activation and degradation has been found in Chlamydomonas photooxidative damage (Pérez-Pérez et al., 2012). autophagy may ROS production by the degradation of specific organelles, but this mechanism to be in plants. on evidence in the autophagy is modulated by redox signals (Fig. through regulatory mechanisms that might be conserved from yeast and algae to and plants. how are redox and to the autophagic a has been to integrate redox signals for autophagy regulation, the ATG4 protease. However, the of this catabolic process, other regulatory such as the ATG1 and TOR might also participate in redox The kinase ATG1 is an essential of autophagy in all and its activity is required for the regulation of the process at the initiation level et al., 2009; Mizushima, 2010). Originally identified in yeast (Tsukada and Ohsumi, 1993), ATG1 is widely conserved through evolution, and have been described in lower and eukaryotes (Meijer et al., 2007). its regulatory ATG1 with other ATG proteins, including which stimulates the kinase activity of ATG1 and is also evolutionarily conserved (Kamada et al., 2000; et al., 2009). In yeast, the activity of the complex is by phosphorylation events on both proteins that are by at different signaling (for protein and AMPK 2010; et al., 2012). These the between ATG1 and the kinase activity of phosphorylation of ATG1 and/or by and results in autophagy et al., 2009; Mizushima, 2010), AMPK signaling stimulates this degradative process (Alers et al., 2012). The kinase complex as well as and signaling pathways are conserved in the model plant Arabidopsis et al., 2012). of ATG1 and genes are in the Arabidopsis and to autophagy in plants (Suttangkakul et al., 2011). However, the signaling pathways function in plants The TOR and have a central role in nutrient and energy in photosynthetic organisms et al., and signaling has been shown to autophagy in plants and algae. in TOR function by in Arabidopsis or treatment in Chlamydomonas in a increase of autophagic activity in both similar to the one in cells (Liu and Bassham, 2010; Pérez-Pérez et al., 2010). These findings a role of TOR in the control of autophagy in photosynthetic organisms but not that other conserved pathways such as signaling may also participate in the regulation of this process, as described in other systems. The the TOR pathway might redox signals in photosynthetic organisms to autophagy (Fig. TOR and energy to cell growth (Wullschleger et al., and evidence indicates that TOR may also be to redox signaling in lower and eukaryotes. Indeed, TOR is a controller of mitochondrial function and as is to redox upstream of TOR such as AMPK or integrate redox signals via 2011). are studies that ROS can both and TOR function at for autophagy regulation, ROS mainly leads to TOR and hence autophagy activation et al., 2010). signaling is conserved in plants et al., 2007; et al., 2006; et al., and algae et al., 2005; Díaz-Troya et al., but the of upstream of such as the strongly suggests that this signaling pathway might be in these organisms. a like ATG1 or that be to the regulation of autophagy by this pathway in plants and algae, it might be to place ROS upstream of TOR for the control of However, based on the role of TOR in autophagy regulation in photosynthetic organisms, with the between redox signaling and TOR reported in other it is to at under stress conditions, ROS might down-regulate TOR which in turn result in ATG1 activation and autophagy induction in plants and algae (Fig. mammalian ATG4 is the ATG protein activity has been shown to be a for redox regulation et al., 2007). As ATG4 has an essential function in This Cys protease plays a role in autophagosome the one ATG4 processes the C terminus of ATG8 (Kirisako et al., a crucial for the binding of ATG8 to PE by the ATG4 also ATG8 to recycle it from the autophagosome membrane (Kirisako et al., The function of ATG4 has been the role it has been in yeast that the and nonselectively ATG8-PE and that ATG4 to recycle ATG8-PE generated on to a of ATG8 that is required for autophagosome formation et al., 2012). The molecular mechanism by which the and of ATG4 are is not well It has been that H2O2 can mammalian ATG4 by its regulatory Cys to the of whereas conditions result in ATG4 activation et al., 2007). This model still that to be to a the in regulation of ATG4 by redox For instance, it is unknown and of ATG4 are or in time and/or ATG8 place in protein and is required for autophagosome formation, whereas has to autophagy is not and ATG8 to be of ATG4 to the of the also might regulatory mechanism of ATG4 structural studies with the protease that binding of ATG8 to ATG4 triggers in a regulatory and the of the protein, allowing ATG8 to the et al., 2009). These studies have been with and it to be the redox regulation of this protein is conserved in other systems. homologs of the ATG4 have been identified in the Arabidopsis and (Yoshimoto et al., et al., and in Arabidopsis results in a of the autophagy process to the of ATG8 to be (Yoshimoto et al., that plant ATG4 is essential in ATG4 that in ATG8 is in Chlamydomonas et al., 2008b). this ATG8 is by an protease which the of in the control of this process (Pérez-Pérez et al., 2010). The high evolutionary conservation of autophagy, including both structural and regulatory suggests that ATG4 might also be a central of redox signals in photosynthetic organisms. In signaling protein play a central as the in the intracellular redox to In response to ROS, Cys of proteins a of et al., 2012). Indeed, Cys can different of oxidation such as and acid but also protein or or These redox are mainly under the control of types of and Therefore, the mechanisms the induction of autophagy by ROS involve one or several proteins that redox activity and in the induction of In mammals, this of redox appears to control the activity of but the Cys are not that this mechanism is not and that other to be These proteins may be known components of the autophagic like or unknown to be these autophagy and the molecular mechanism will a major in the for reactive oxygen species hydrogen peroxide oxygen methylviologen NADPH protein response