Redox- and Reactive Oxygen Species-Dependent Signaling into and out of the Photosynthesizing Chloroplast^,

The photosynthesizing chloroplast functions as a conditional source of redox and ROS information which tunes processes inside the chloroplast and hence impacts on signaling events in the cytosol and nucleus.

Abstract

Photosynthesis is a high-rate redox metabolic process that is subjected to rapid changes in input parameters, particularly light. Rapid transients of photon capture, electron fluxes, and redox potentials during photosynthesis cause reactive oxygen species (ROS) to be released, including singlet oxygen, superoxide anion radicals, and hydrogen peroxide. Thus, the photosynthesizing chloroplast functions as a conditional source of important redox and ROS information, which is exploited to tune processes both inside the chloroplast and, following retrograde release or processing, in the cytosol and nucleus. Analyses of mutants and comparative transcriptome profiling have led to the identification of these processes and associated players and have allowed the specificity and generality of response patterns to be defined. The release of ROS and oxidation products, envelope permeabilization (for larger molecules), and metabolic interference with mitochondria and peroxisomes produce an intricate ROS and redox signature, which controls acclimation processes. This photosynthesis-related ROS and redox information feeds into various pathways (e.g. the mitogen-activated protein kinase and OXI1 signaling pathways) and controls processes such as gene expression and translation.

GENERATION OF REACTIVE OXYGEN SPECIES IN THE CHLOROPLAST

Photosynthetic electron transport requires the absorption of light by chlorophylls. Excitation of pigments and electron transfer reactions in an oxygen-rich environment lead to the production of reactive oxygen species (ROS) such as singlet oxygen (¹O₂), superoxide anion radicals (O₂·⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (Fig. 1). ¹O₂ is produced by the reaction of excited chlorophyll in its triplet state (³Chl) with molecular oxygen in its ground state, which is the triplet state ³O₂. In the reaction center of PSII, ³Chl is generated by charge recombination of the primary radical pair (P680⁺ pheophytin⁻), with pheophytin acting as the primary electron acceptor. When light absorption exceeds the capacity of photosynthetic electron transport, the quinone acceptors of PSII (primary electron-accepting plastoquinone of PSII [Q_A], secondary electron-accepting plastoquinone of PSII, and the plastoquinone pool) are reduced and the probability of ¹O₂ generation increases. The midpoint potential of Q_A determines whether charge recombination proceeds via the dangerous route of repopulation of the primary radical pair (P680⁺ pheophytin⁻), generating (with a certain probability) ³P680 and ¹O₂, or via the safe pathway of charge recombination directly into the ground state of P680 (Rutherford and Krieger-Liszkay, 2001). The yield of ¹O₂ formation is reduced when Q_A adopts the high-potential form by shifting the midpoint redox potential of Q_A to a more positive value. PSII centers with high-potential Q_A have been observed under various physiological conditions in vivo: (1) in green algae prior to photoactivation through the light-dependent assembly of the manganese cluster (Johnson et al., 1995) and (2) in leaves of higher plants exposed to high light (Krieger-Liszkay et al., 2008). Light-harvesting complexes contain many more chlorophylls than the PSII reaction center. However, they are less susceptible to damage by ¹O₂ than the reaction center, because the antennas are protected by nearby carotenoids, including xanthophylls, which efficiently quench ³Chl. In the light, O₂·⁻ is generated mainly at the acceptor side of PSI via the Mehler reaction (Mehler, 1951). O₂·⁻ can damage the [4Fe-4S] cluster of PSI (PSI photoinhibition). It has been suggested that the phylloquinone acceptor A₁ is a major contributor to O₂·⁻ generation inside the thylakoid membrane in vivo (Kozuleva et al., 2014; Takagi et al., 2016). Takagi et al. (2016) demonstrated that the ROS production site rather than the quantity of ROS is crucial for PSI photoinhibition. Reduced ferredoxin does not play a role in O₂·⁻ generation at PSI, since the reaction of reduced ferredoxin with oxygen is rather slow (Hosein and Palmer, 1983).

Figure 1.

Sources of ROS in the photosynthesizing chloroplast. The interplay between photosynthetic redox metabolism (top) and ROS generation and associated signaling (bottom) is shown. Safe redox metabolism allows the overreduction of photosynthetic electron transport (PET) to be avoided by balancing metabolism and efficient redox regulation. Reducing power is exported via the malate valve, and export of dihydroxyacetone phosphate (DHAP) occurs via the triosephosphate phosphate translocator (TPT). Increasing production of ROS also feeds into redox regulation, enables redox and ROS signaling, and ultimately causes oxidative damage. FTR, Ferredoxin-dependent thioredoxin reductase; PQ, plastoquinone; PQH₂, plastohydroquinol.

In algae and cyanobacteria, oxygen reduction represents a significant electron transport pathway, while in higher plants, its importance as an alternative electron sink is thought to be low (Shirao et al., 2013). Thus, gymnosperms have a higher capacity for performing the Mehler reaction (approximately 10% of the maximum electron flow) than angiosperms, particularly before the redox-regulated enzymes of the Calvin cycle become active (Shirao et al., 2013). O₂·⁻ also can be produced in vitro at the level of the cytochrome b₆f complex (Baniulis et al., 2013) and at the acceptor side of PSII (Pospíšil, 2012). However, the capacities of these oxygen reduction pathways seem to play minor roles in the intact electron transport chain in thylakoids.

Furthermore, plastid terminal oxidase (PTOX) participates in controlling O₂·⁻ levels. PTOX catalyzes the oxidation of plastoquinol and transfers the electrons to oxygen to form water. However, depending on the plastoquinol concentration, PTOX can generate O₂·⁻ in a side reaction (Yu et al., 2014).

Photorespiration is a major source of photosynthesis-associated ROS. During the recycling of by-products of the oxygenase activity of Rubisco, H₂O₂ is produced in peroxisomes. H₂O₂ also is generated inside the chloroplast by the dismutation of O₂·⁻, either spontaneously or via superoxide dismutase (SOD) catalysis. Furthermore, H₂O₂ can be formed by the reduction of O₂·⁻ by plastoquinol (Mubarakshina and Ivanov, 2010). The reactivity of H₂O₂ is mostly restricted to thiolates (Mock and Dietz, 2016). In the presence of transition metals such as Fe²⁺ or Cu⁺ (either in their free form or coordinated as metal centers to proteins), H₂O₂ is converted to the highly reactive hydroxyl radical. In the intact chloroplast, several enzymes detoxify ROS. O₂·⁻ is dismutated to H₂O₂ by copper/zinc-SOD or iron-SOD. H₂O₂ is mainly detoxified by ascorbate peroxidase (APX) and chloroplast-located peroxiredoxins (PRXs; Dietz, 2016). While APX activity requires ascorbate, PRX activity usually depends on rereduction by glutathione/glutaredoxin or thioredoxin (TRX). The high chloroplastic ascorbate (20–300 mm) and glutathione (0.5–3.5 mm) concentrations enable efficient H₂O₂ detoxification. Glutathione is a substrate for thiol peroxidases and glutathione S-transferases, which detoxify reactive lipid species formed by ¹O₂. ¹O₂ can be scavenged by tocopherol, plastoquinone, carotenoids, and ascorbate (Miret and Munné-Bosch, 2015). Apart from ascorbate, these scavengers are compartmentalized and protect the photosystems in the light. However, they have limited capacity and may become exhausted under stress due to their limited regeneration capacity.

EFFECTS OF REDOX AND ROS ON CHLOROPLAST PROCESSES

The complexity of the chloroplast redox regulatory network appears to exceed that of other cell compartments. Three main experimental approaches have been used to identify and characterize redox-regulated processes and individual players in the chloroplast: (1) determining the redox sensitivity of processes in thylakoid preparations and isolated chloroplasts; (2) analyzing isolated redox-sensitive proteins; and (3) performing redox proteomics using a wide range of methods, such as in gel, chromatographic, in silico, and in vivo applications (Akter et al., 2015; Mock and Dietz, 2016). The Calvin cycle and adjacent pathways (such as starch synthesis) are established examples of redox-regulated chloroplast processes. Recently, evidence for redox regulation has emerged for many other processes, including PET. Cyclic electron flow (CEF) via ferredoxin-dependent quinone reductase is sensitive to antimycin A and is stimulated by thiol-reducing conditions, with a redox midpoint potential of −306 mV (Strand et al., 2016). In a converse manner, the NADPH dehydrogenase-dependent CEF pathway is activated by oxidizing conditions in vivo after H₂O₂ infiltration or in mutants with enhanced production of chloroplast H₂O₂ (Strand et al., 2015). In contrast to these observations, TRX m4 appears to inhibit CEF, based on the analysis of mutants affected in TRX m4 (Courteille et al., 2013). These findings indicate that alternative CEF pathways are inversely controlled by redox and ROS. In addition, the Mehler reaction appears to be subjected to redox regulation. O₂·⁻ production in isolated thylakoids depends on the photoperiod under which the plants are grown (Michelet et al., 2013). This difference is abolished upon the addition of NADPH (Michelet et al., 2013) and in mutants affected in NADPH-dependent TRX reductase C (Lepistö et al., 2013).

The partitioning between linear electron flow, CEF, and the Mehler reaction, which appears to be redox controlled, tightly links the light reactions with the CO₂-fixing pathway. When CO₂ fixation is restricted by the low availability of electron acceptors or the inadequate activity of redox-regulated enzymes (e.g. after the dark/light transition), TRXs assume the reduced state and linear and cyclic electron flow is shut down. O₂·⁻/H₂O₂ is then generated by the Mehler reaction, and the H₂O₂ is decomposed by APX, PRX, and GPX. However, above a certain threshold of accumulation, H₂O₂ may oxidize the thiols, rendering enzymes of the Calvin cycle inactive and simultaneously activating the NDH-dependent CEF (Strand et al., 2015).

Redox and ROS not only affect the function of PET but also affect the assembly and repair of PSII (Nath et al., 2013) and chloroplast development. We will not discuss this topic in detail, but it should be mentioned that defects in the chloroplast antioxidant defense system often conditionally affect early leaf and chloroplast development, while later stages of development only respond to more severe stresses (Baier and Dietz, 1999; Kangasjärvi et al., 2008; Awad et al., 2015).

TRANSMISSION OF REDOX AND ROS SIGNALS TO THE CYTOSOL

Over the past several years, our conceptual understanding of the organellar ROS and redox regulatory network has advanced. On the one hand, this topic concerns the interplay between ROS-generating systems and chloroplast antioxidant defense. On the other hand, the transcriptomic changes that occur in response to specific redox- and ROS-induced disturbances of chloroplast metabolism have been defined. For example, op den Camp et al. (2003) compared O₂·⁻/H₂O₂- and ¹O₂-dependent transcript regulation, and Fey et al. (2005) analyzed plastoquinone redox state-dependent signaling. Recent meta-analyses have provided additional information about regulatory patterns specific to various cellular ROS signatures (Rosenwasser et al., 2013) and signaling pathways (Gläßer et al., 2014). A comparison of the transcriptomic responses to diverse retrograde signaling pathways has led to the identification of a core module of target genes. The data indicate that ROS and redox signaling participate in early retrograde signaling (Gläßer et al., 2014). Conditional H₂O₂ generation in chloroplasts or peroxisomes elicits distinct changes in gene expression (Sewelam et al., 2014). Chloroplast H₂O₂ causes the up-regulation of transcripts involved in early signaling (including transcription factors), secondary signaling, mitochondrial retrograde signaling, and the biosynthesis of defense compounds in a time-dependent manner (Sewelam et al., 2014).

Both ¹O₂ and H₂O₂, which are produced inside the chloroplast, have been shown to act as specific signals that trigger responses outside of the organelle. The transmission of redox signals from the chloroplasts to the cytoplasm or other cellular compartments involves the transport of ROS, metabolites, and oxidation products and possibly yet unknown transduction pathways (Dietz, 2015). Detailed analyses have revealed that the transcriptional response of Arabidopsis (Arabidopsis thaliana) to increased ¹O₂ formation is distinct from the response to other ROS such as O₂·⁻ and H₂O₂ (op den Camp et al., 2003). ¹O₂ signal transduction forms an integral part of a complex signaling network that is modified by other signaling routes involving hormones such as jasmonic acid. Due to its high reactivity, ¹O₂ should be detected using specific sensors in the thylakoids or via ¹O₂ reaction products in the stroma. Such sensors could activate a signal transduction pathway directed to the nucleus. Putative components of the ¹O₂ signal transduction pathway include the proteins EX1 and EX2 in Arabidopsis (Kim et al., 2012) and the chloroplast-located protein PSBP2 in Chlamydomonas reinhardtii (Brzezowski et al., 2012). The exact functions of these proteins are unknown, but they play a crucial role in the ¹O₂-mediated changes in gene expression and the stimulation of phytohormone biosynthesis in the flu mutant, as shown by the loss of ¹O₂ response in the ex1 single or ex1/ex2 double mutant. Furthermore, oxidation products of carotenoids (such as β-cyclocitral) and lipids (such as the oxylipins 13-hydroxy octadecadienoic acid and/or 13-keto-octadecatrienoic acid) have been shown to act as signaling compounds (López et al., 2011; Ramel et al., 2013).

High levels of ¹O₂ generated in PSII not only react with proteins, carotenoids, or lipids inside the chloroplast but also may act directly on cytosolic compounds. ¹O₂ is detectable in the cytosol in C. reinhardtii (Fischer et al., 2007). Some of the generated ¹O₂ may diffuse out of the chloroplast, or ¹O₂ may be generated from lipid peroxides at the outer envelope membrane of the chloroplast in a secondary reaction.

H₂O₂ is much less reactive than ¹O₂, and considerable amounts are released from the chloroplast. Aquaporin-like proteins may allow for H₂O₂ efflux from the chloroplast, since H₂O₂ release is sensitive to inhibitors of aquaporins. Aquaporins in the plasma membrane and tonoplast facilitate the transmembrane diffusion of H₂O₂, but the presence of aquaporins in the chloroplast envelope remains to be demonstrated (for review, see Bienert and Chaumont, 2014).

The malate shuttle enables efficient equilibration of the redox state between different compartments such as the chloroplast and cytosol (Fig. 1). The key enzyme, NADP-malate dehydrogenase (NADP-MDH), is activated by TRX m if NADPH accumulates, and increasing electron pressure pushes the TRX system into a more reduced state (Hebbelmann et al., 2012). A transient increase in H₂O₂ levels upon light stress depends on the reversible thiol-dependent inhibition of catalase activity (Michelet et al., 2013). Mutants affected in NADP-MDH lack the transient down-regulation of catalase activity and the concomitant transient increase in H₂O₂ levels (Heyno et al., 2014). Catalase in higher plants is localized to the peroxisome. Thus, the data imply that the redox state of the chloroplast is transmitted to peroxisomes via the malate valve. Information about the redox and ROS states of the chloroplast, particularly under stress, also is transmitted to the cytosol via metabolically coupled compounds such as cyclocitral (Ramel et al., 2012), 3′-phosphoadenosine 5′-phosphate (PAP; Estavillo et al., 2011), and methylerythritol cyclodiphosphate (MEcPP; Walley et al., 2015). PAP participates in the expressional control of a subset of genes in the high-light acclimation response. The precise role of ROS and redox in regulating the PAP and MEcPP pathways remains to be established.

EXTRAPLASTIDIC SIGNAL PROCESSING

Chloroplast energization and ROS metabolism are intimately linked to the cytosolic mitogen-activated protein kinase (MAPK) pathway (Fig. 2). The MAPK pathway coordinates defense reactions and stress acclimation responses in eukaryotes (Meng and Zhang, 2013; Moustafa et al., 2014). MAPK signaling is under the control of redox and ROS stimuli (Apel and Hirt, 2004) and, in turn, regulates redox and ROS homeostasis (Pitzschke et al., 2006). Thus, MEKK1, a MAPKKK, has been implicated in adjusting cell redox homeostasis and hormone signaling (Nakagami et al., 2006). Artificial activation of NtMEK2 in transgenic tobacco (Nicotiana tabacum) initiates a cell death program similar to the hypersensitive response (Liu et al., 2007). Strong NtMEK2 expression induced by activating a construct harboring this gene fused to a steroid-inducible promoter inhibited photosynthesis within 2 h, and subsequently, the chloroplasts started to accumulate O₂·⁻ in a light-dependent manner. This, in turn, damaged the chloroplasts, depolarized the plasma membrane, and led to the loss of cell constituents. Enhanced electrolyte leakage began 6 h after DEX treatment, ultimately leading to cell death (Liu et al., 2007).

Figure 2.

Connection between chloroplast redox/ROS generation and cytosolic signaling pathways. The focus is given to the MAPK and OXI1 pathways. The high-energy metabolite DHAP and H₂O₂ are exported and feed into the MAPK pathway. ¹O₂ activates the OXI1-dependent pathway. Both processes induce changes in gene expression for acclimation, defense, and eventual cell death. The MAPK pathway also feeds back into the chloroplast and affects PET. See text for details.

Another example of MAPK signaling in the context of the chloroplast was established in light intensity shift experiments. After transfer of Arabidopsis plants to high light, MPK6 phosphorylation occurred quite rapidly (i.e. within 20 s). Phosphorylation corresponds to signaling activation (Vogel et al., 2014). In this study, rapid MAPK phosphorylation depended on the presence of the TPT and, thus, on metabolite export. The Calvin cycle intermediate dihydroxyacetone phosphate rapidly accumulates in leaf chloroplasts when the rate of CO₂ fixation increases (e.g. in response to light; Dietz and Heber, 1984). The TPT catalyzes DHAP export to the cytosol and allows phosphorylation energy and reducing power to be transmitted to the cytosol (Fig. 1). The TPT-mediated export of metabolites provides retrograde information to the cytosol and nucleus and stimulates, for instance, the expression of an AP2/ERF transcription factor network (Vogel et al., 2014).

¹O₂ and O₂·⁻/H₂O₂ generated in the chloroplast trigger signaling pathways that ultimately induce changes in gene expression (see above). The Ser/Thr protein kinase OXI1 functions as an important signaling element in both respiratory burst-released H₂O₂- and chloroplast-emitted ¹O₂-dependent signaling (Rentel et al., 2004; Shumbe et al., 2016). The H₂O₂ signaling pathway feeds into the MAPK module, which controls gene expression for defense and hypersensitive responses (Fig. 2). In a converse manner, the ¹O₂-triggered activation of OXI1 affects the jasmonate pathway, thereby affecting the ¹O₂-linked cell death pathway (Shumbe et al., 2016). The OXI1 signaling pathway functions independently of the ¹O₂/EX-dependent pathway of cell death observed in flu mutants (op den Camp et al., 2003). In the cytosol, other sensor proteins, such as the small zinc finger protein MBS (Shao et al., 2013), also are required for the induction of responsive genes.

In addition to ROS, oxidation products, and metabolites, specific regulatory proteins are released from the chloroplast. General or specific permeabilization of the envelope for the release of regulatory proteins from the chloroplast, which depends on, for instance, the ROS and redox state, might be a potent mechanism involved in the conditional release of WHIRLY1 from the chloroplast to the cytosol and the subsequent regulation of target genes such as PR1 and PR2 (Isemer et al., 2012; Foyer et al., 2014). Another example is the envelope-tethered PHD-type transcription factor with transmembrane domain (PTM), which is released from the plastid surface upon treatment of leaves with the plastid translation inhibitor lincomycin, the carotenoid biosynthesis inhibitor norflurazon, or high light and controls the expression of ABI4 by binding to its promoter (Sun et al., 2011). The release of PTM from the envelope might involve ROS signals. An intriguing pathway for signal transmission comes from an experiment involving transplastomic tobacco and lettuce (Lactuca sativa) expressing GFP fused to the 16-amino acid protein transduction domain of PDX-1 in the plastid genome (Kwon et al., 2013). Upon treatment with the pathogen Erwinia carotovora or the superoxide-inducing agent methyl viologen, the chloroplast-localized GFP was released to the cytosol. The addition of the superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulfonate suppressed the liberation of GFP from the plastid (Kwon et al., 2013). Chloroplast ROS also trigger other processes apart from gene expression. Protein synthesis is thought to be another major target of retrograde signaling in the chloroplast (Dietz, 2015). Changes in de novo protein synthesis poorly reflect changes in transcript levels during light-shift experiments (Oelze et al., 2014). The regulation of translation in the context of retrograde signaling awaits scrutiny, but both the MAPK pathway (Bollig et al., 2003) and redox/ROS (Moore et al., 2016) likely affect the activity of components and, therefore, the different steps of initiation, elongation, and termination. Several regulatory factors have been identified that act downstream of chloroplast-released ROS, as summarized recently (Chan et al., 2016). ROS appear to trigger the interaction of the regulatory protein RADICAL-INDUCED CELL DEATH1 with the transcription factor RAP2.4a, which in turn up-regulates the expression of chloroplast antioxidant genes such as 2-CYSTEINE PEROXIREDOXIN (Hiltscher et al., 2014). This pathway may establish a feedback loop for the control of chloroplast ROS level. Topoisomerase VI constitutes another example of an identified cytosolic component that mediates chloroplast ¹O₂ signaling by binding to the promoter of the triple-A ATPase gene, thereby stimulating its expression (Simková et al., 2012). Simultaneously, topoisomerase VI suppresses the expression of H₂O₂-stimulated genes. These examples provide early insights into the complex sensory and regulatory system controlled by chloroplast redox and ROS cues.

REVERSE COUPLING OF REDOX SIGNALING TO THE CHLOROPLAST

In photosynthetic tissues, chloroplasts are closely associated with other cellular organelles such as mitochondria, peroxisomes, the endoplasmic reticulum (ER), and the nucleus to fulfill various metabolic processes, including photorespiration and the biosynthesis of secondary metabolites and lipids. Among these organelles, one of the best-known relationships occurs during photorespiration, a metabolic process shared between the chloroplast, peroxisome, and mitochondria. Oikawa et al. (2015) demonstrated that the interaction between chloroplasts and peroxisomes is light dependent. Upon exposure to light, peroxisomes change from a spherical to an elliptical shape to increase the surface area that interacts with the chloroplast. Moreover, in PET-deficient genotypes, peroxisomes tend to be spherical rather than elliptical. These findings indicate a role for redox control in the dynamic morphological changes that occur in peroxisomes during their interaction with chloroplasts, which are required for efficient photorespiration.

The activities of chloroplasts, peroxisomes, and mitochondria are coordinated via retrograde and anterograde signaling and by metabolite exchange. A topic attracting increasing attention is the reverse coupling of redox and ROS signals from other organelles to the chloroplast. Meticulous control over the redox state of the NAD(P)H system is needed for efficient photosynthesis. This level of control is realized by balancing photosynthesis, mitochondrial respiration, and photorespiration. Excess reducing power produced in chloroplasts is exported to the cytosol to regenerate an electron acceptor for PET. Because NAD(P)H cannot readily pass through the envelope, plants use mechanisms such as malate/OAA and TPT shuttles to transfer reducing power through their membranes (Noguchi and Yoshida, 2008). Both phosphorylating and nonphosphorylating mitochondrial electron transport (MET) dissipate excess reductants. In particular, external NADPH dehydrogenases, alternative oxidase (AOX), and uncoupling protein in the mitochondria are vital for efficient photosynthesis (Dutilleul et al., 2003; Yoshida et al., 2006; Sweetlove et al., 2006; Bailleul et al., 2015). Nonphosphorylating pathways are especially essential under conditions that favor high turnover of Gly decarboxylation during photorespiration, since this process produces large quantities of NADH that must be reoxidized to NAD⁺. This regeneration is achieved partly by the conversion of OAA to malate by mitochondrial MDH (Tomaz et al., 2010) and partly by oxidation through MET. Overall, increased photorespiration creates a high electron flux through the MET, eventually building up a strong proton motive force across the inner mitochondrial membrane (Sweetlove et al., 2006) and increasing mitochondrial ROS production. In turn, the high proton motive force inhibits MET flow, and unless the proton gradient is dissipated or its buildup is prevented, NADH cycling is decelerated. Insufficient availability of NAD⁺ for photorespiratory Gly decarboxylation retards photorespiration and decreases carbon assimilation due to reduced recycling of 2-phosphoglycolate to 3-phosphoglycerate, which limits the availability of ribulose-1,5-bisphosphate for the Calvin cycle (Raghavendra and Padmasree, 2003). Thus, reverse redox coupling of the mitochondria to chloroplasts and the reduction state of MET ultimately exert control over the rate of photosynthesis by controlling the availability of NAD(P)⁺ and ROS release. Genetic disturbance of mitochondrial metabolism causes an overenergization of chloroplasts. In a mutant screening for plants unable to up-regulate the expression of AOX needed for safe energy dissipation, cyclin-dependent kinase E1 (CDKE1) was identified as a regulator of AOX (Blanco et al., 2014). Interestingly, CDKE1 also regulates the expression of light-harvesting proteins and might help coordinate the retrograde control of chloroplasts and mitochondria.

In addition to shuttling redox equivalents, ROS produced in these organelles play a role in direct signaling, producing organelle-specific signatures, such as transcriptomic changes (Gadjev et al., 2006; Rosenwasser et al., 2013). ROS usually spread over short distances in the cell, depending on their half-life, concentration, and the strength of cytoplasmic antioxidant systems. Thus, cum grano salis, one may consider ROS as rather unreliable second messengers among organelles over greater distances, although the situation is different if the organelles are in close physical contact (e.g. among chloroplasts, mitochondria, and peroxisomes). In the latter case, cytoplasmic antioxidant systems are bypassed and a direct route for ROS transfer (i.e. for H₂O₂) is formed.

Stromule formation between chloroplasts is another mechanism for establishing interorganellar communication through the use of physical contacts. These thin, long, stroma-filled tubules (0.4–0.8 µm in diameter and up to 65 µm long) originate from plastids and have been observed in all plants species investigated to date (Gray et al., 2001). Their exact function remains elusive (Natesan et al., 2005). Recently, Brunkard et al. (2015) demonstrated that in chloroplasts and leucoplasts, stromules form more frequently in the light than in darkness. Moreover, inhibitors of PET rapidly increase the frequency of stromule formation in chloroplasts, suggesting that redox plays a role in controlling stromule formation. Interestingly, stromule formation was shown to depend on NADPH-dependent TRX reductase C, and silencing its expression doubled the rate of stromule formation. Isolated chloroplasts were able to form stromules, suggesting that stromules intrinsically emerge independent of cytosolic factors (Brunkard et al., 2015), but their emergence might be supported by interaction with microtubules (Kwok and Hanson, 2003). In addition to light and direct changes in redox state, which mimic abiotic stress conditions, stromule formation responds to temperature (Holzinger et al., 2007) and abscisic acid (Gray et al., 2012). These observations implicate the involvement of ROS and redox state in stromule formation.

To date, chloroplasts have been found to associate with the plasma membrane, ER, mitochondria, nucleus, and other plastids via stromules (Kwok and Hanson, 2003; Hanson and Sattarzadeh, 2013). Chloroplast stromules also might function in innate immunity. During this response, numerous stromules surround the nuclei, and the intensity of their connection correlates with the accumulation of chloroplast-derived defense protein and (interestingly) H₂O₂ in the nucleus (Caplan et al., 2015). The association of the ER with the chloroplast is an example of the physical interaction of the chloroplast with another organelle. Using a transorganellar assay, Mehrshahi et al. (2013) provided evidence for a chloroplast-ER membrane hemifusion model. In this assay, tocopherol cyclase was targeted to the ER of the vte1 mutant, which lacks chloroplast tocopherol cyclase and hence tocopherols. The experiment revealed that ER-localized tocopherol cyclase complements the vte1 mutation, providing tocopherol levels similar to that of the wild type, which confirms that metabolites move from the chloroplast to the ER via membrane connections. The model suggests the formation of a hemifused bilayer composed of the inner leaflets of the ER and plastid envelope, which would allow nonpolar metabolites to diffuse through the membrane from one compartment to the other (Mehrshahi et al., 2013). Interestingly, retraction and directional changes in stromule branches occur in tandem with the movement of neighboring ER tubules (Schattat et al., 2011). This observation provides additional evidence for the establishment of interacting surfaces, which serve as conduits for the bidirectional exchange of ions, lipids, and metabolites, including ROS and redox-active compounds, between the two organelles. Unlike chloroplasts, the ER encounters a more oxidizing milieu due to its role in disulfide bond formation and oxidative protein folding. Using redox-sensitive GFP, Birk et al. (2013) measured a chloroplast redox potential below −300, while the potential of the ER was 100 mV higher than this. In the ER, disulfide bonds are formed by a TRX-like protein, protein disulfide isomerase (PDI). Reduced PDI must be oxidized by ER OXIDOREDUCTASE1, a flavoenzyme tightly associated with the lumenal face of the ER membrane (Dixon et al., 2003). Electrons from thiol oxidation are transferred to oxygen to form H₂O₂. Accumulation of unfolded proteins (which can be induced by the ER stress agent tunicamycin) causes excessive production of H₂O₂ in the ER. This, in turn, can trigger ROS signaling via NOX in the plasma membrane and can alter the glutathione redox state (Ozgur et al., 2014). Thus, the hemifused membrane model and the coordinated movement of stromules and ER tubules may enable the ER to send out redox signals to chloroplasts via H₂O₂, especially under high temperatures, where protein folding is significantly affected and the unfolded protein response is induced (Liu and Howell, 2010). Interestingly, the unfolded protein response in the ER was linked recently to retrograde signaling from the chloroplast. MEcPP is a signaling metabolite released from perturbed chloroplasts. Elevated MEcPP levels up-regulate the expression of genes involved in the unfolded protein response, such as the transcription factor gene bZIP60, possibly sensitizing the cell for episodes of increased stress (Walley et al., 2015).

SUMMARY AND OUTLOOK

Retrograde signaling from the photosynthesizing chloroplast to the cytosol, mitochondrion, and nucleus is now understood to involve a network of diverse signals categorized as redox/ROS, metabolites, hormones, and signaling pathways, which feed into diverse regulatory circuits and processes (Dietz, 2015; Chan et al., 2016; see “Outstanding Questions”). This review describes ROS signaling as a central component of the retrograde signaling network. ROS signaling is tied to metabolite signaling. Figure 3 summarizes three scenarios in which different redox/ROS signal intensities are encountered: upon changing environmental conditions for photosynthesis (Fig. 3A), during moderate abiotic and combinatorial stress (Fig. 3B), and during severe stress leading to cell death (Fig. 3C). Small increases in ROS release drain electrons from the thiol-redox regulatory network and allow for metabolic adjustments. Elevated levels of ¹O₂ react with lipids and carotenoids in the chloroplast. H₂O₂ is released from the plastid. The transcription factor PTM is freed from the envelope. The conditional ROS pattern triggers changes in gene expression and protein synthesis to optimize the defense response. Extreme stress, such as severe drought or pathogen infection, enhances lipid peroxidation, causing membrane permeabilization and eventual cell death. Each case involves redox- and ROS-dependent signaling into and out of the photosynthesizing chloroplast.

Figure 3.

Scenarios of chloroplast redox/ROS signaling of increasing strength. The scheme illustrates three scenarios of redox/ROS disturbances. A, Under conditions of environmentally induced, moderate metabolic changes, the redox milieu is altered but ROS are still kept in check. Retrograde signaling relies on redox and other mechanisms. B, Under moderate stress, which may include combinatorial stresses, ROS-dependent metabolites such as cyclocitral, the regulatory proteins EXECUTER1 (EX1) and EX2, and ROS themselves are released or activate retrograde signaling. The membrane-anchored transcription factor PTM is shed from the outer envelope. Gene expression and protein translation are altered to enable defense and acclimation. C, Extreme stress alters PET and metabolism, leading to the accumulation of high levels of ROS. β-Cyclocitral participates in extreme light stress signaling, which often is accompanied by the loss of integrity. The envelope is permeabilized and molecules are released to the cytosol. These changes may activate the cell death program.

Advances

• Redox state and ROS generation reflect the state of photosynthesis. Therefore, redox and ROS information from the photosynthesizing chloroplasts is transmitted to the cytosol and controls rapid adjustments to transcription and translation.

• A major target is the MAPK pathway, which triggers or modulates cytosolic and nuclear activities.

• The buildup of interfaces between organelles (e.g. by changing the structures of peroxisomes and stromules) is a conditionally dynamic process that likely facilitates the exchange of redox metabolites and ROS.

• The structure of the redox-dependent regulatory network of the cellular compartments depends on ROS as final electron acceptors (oxidants). For this reason, ROS-generating systems are an essential component of the plant cell.

Outstanding Questions

• There is a need to understand the mechanisms by which the different ROS signals are conditionally transmitted from the plastid to the cytosol and how they feed into the regulatory network of the cell.

• The use of compartment-specific cell-imaging probes will unveil whether ROS are delivered directly from the plastids to the neighboring compartments in vivo.

• The role and specificity of the MAPK pathway in modulating ROS- and redox-dependent retrograde signaling need to be explored in detail.

• The long-term goal is to achieve an integrated view of the interaction between the different retrograde pathways. It is proposed that ROS signals function as amplifiers in determining the ultimate response strength.

Glossary

ROS

reactive oxygen species

¹O₂

singlet oxygen

O₂·⁻

superoxide anion radical

H₂O₂

hydrogen peroxide

³Chl

chlorophyll in its triplet state

Q_A

primary electron-accepting plastoquinone of PSII

PTOX

plastid terminal oxidase

SOD

superoxide dismutase

APX

ascorbate peroxidase

PRX

peroxiredoxin

PET

photosynthetic electron transport

CEF

cyclic electron flow

TRX

thioredoxin

PAP

3′-phosphoadenosine 5′-phosphate

MEcPP

methylerythritol cyclodiphosphate

TPT

triosephosphate phosphate translocator

DHAP

dihydroxyacetone phosphate

PTM

PHD-type transcription factor with transmembrane domain

ER

endoplasmic reticulum

MET

mitochondrial electron transport

AOX

alternative oxidase