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Cent. Eur. J. Biol. • 8(12) • 2013 • 1153-1163 DOI: 10.2478/s11535-013-0243-4 Central European Journal of Biology Redox signals as a language of interorganellar communication in plant cells Review Article Tomasz Kopczewski, Elżbieta Kuźniak* Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection, University of Łódź, 90-237 Łódź, Poland Received 29 April 2013; Accepted 18 July 2013 Abstract: Plants are redox systems and redox-active compounds control and regulate all aspects of their life. Recent studies have shown that changes in reactive oxygen species (ROS) concentration mediated by enzymatic and non-enzymatic antioxidants are transferred into redox signals used by plants to activate various physiological responses. An overview of the main antioxidants and redox signaling in plant cells is presented. In this review, the biological effects of ROS and related redox signals are discussed in the context of acclimation to changing environmental conditions. Special attention is paid to the role of thiol/disulfide exchange via thioredoxins (Trxs), glutaredoxins (Grxs) and peroxiredoxins (Prxs) in the redox regulatory network. In plants, chloroplasts and mitochondria occupying a chloroplasts and mitochondria play key roles in cellular metabolism as well as in redox regulation and signaling. The integrated redox functions of these organelles are discussed with emphasis on the importance of the chloroplast and mitochondrion to the nucleus retrograde signaling in acclimatory and stress response. Keywords: Antioxidants • Chloroplasts • Glutaredoxins • Peroxiredoxins • Plastoquinone • Reactive oxygen species • Redox homeostasis • Thioredoxins © Versita Sp. z o.o. 1. Introduction Plants have evolved different mechanisms which are responsible for acclimation to the constantly fluctuating environmental conditions. Attacks of pathogens and herbivores, water deficit and flooding, low and high temperature – these and many other stress factors induce defensive responses. However, to respond adequately to the changing environment, a plant must sense and process external stimuli and integrate signaling pathways into a comprehensive signaling network. Reactive oxygen species (ROS) and modifications of redox metabolites are integral to stress and stress defense mechanisms [1]. This review focuses on ROS and redox signaling in plant cells and on the importance of the intercompartmental redox communication in the acclimatory response to environmental stresses. 2. Reactive oxygen species and redox homeostasis Molecular oxygen occurs in nature in two main allotropic varieties: as dioxygen (O2) and trioxygen (ozone, O3). Oxygen in the ground state, which has two unpaired electrons in two orbitals π*, is called triplet oxygen (3O2). This form of oxygen is relatively inactive. ROS are formed by several different mechanisms (Figure 1) [2]. ROS are synthesized in different parts of a plant cell: in chloroplasts, mitochondria, peroxisomes, apoplast and plasma membrane. Generation of ROS during photosynthesis, cellular respiration and photorespiration is normally maintained at a safe level. However, under stress, intensification of ROS production occurs, leading to potentially harmful effects like lipid peroxidation, carbonylation of proteins, formation of disulfide and dityrosine bridges, modification of amino acids in polypeptide chains, DNA damage as well as modifications of the structure of pigments [3]. On the other hand, ROS can also act as signaling molecules involved in many physiological processes. ROS and related redox signals make an important contribution to the cell cycle, cell and organ expansion, cell wall remodeling, photosynthesis, stomatal opening, senescence, programmed cell death (PCD) as well as plant responses to abiotic and biotic stresses [4,5]. Photosynthetic organisms have evolved enzymatic and non-enzymatic antioxidants and redox buffering * E-mail: [email protected] 1153 Unauthenticated Download Date | 6/17/17 2:32 PM Redox signals in plant cells Figure 1. ROS formation and the antioxidant system in a plant cell. The activity of PS I and PS II in chloroplasts, ETC functioning as well as GO (in peroxisomes), Ac-CoA O (in glyoxysomes) and NADPH oxidase (in membranes) activities are associated with ROS formation in a plant cell. Enzymatic (CAT, APX, GPX, SOD) and non-enzymatic (e.g. AA, car, a-toc) antioxidants prevent excessive accumulation of ROS. AA – ascorbate, Ac-CoA O – acyl-coenzyme A oxidase, APX – ascorbate peroxidase, a-toc – a-tocopherol, car – carotenoids, CAT – catalase, DHA – dehydroascorbate, ETC – mitochondrial electron transport chain, GO – glycolate oxidase, GPX – glutathione peroxidase, GSH – glutathione, GSSG – glutathione disulfide, PS I – photosystem I, PS II – photosystem II, ROS – reactive oxygen species, SOD – superoxide dismutase. According to [1,7-9], modified. systems to prevent ROS-mediated cellular oxidation and to maintain cellular redox balance [6-9] (Figure 1). This review focuses selectively on low molecular weight antioxidants and redox enzymes that are important for antioxidant defense and regulate redox signaling. The total pool of redox-active compounds which are found in a cell in reduced and oxidized forms creates the cellular redox buffer and NADPH/NADP+, ascorbate/ dehydroascorbate (AA/DHA), glutathione/glutathione disulfide (GSH/GSSG) and thioredoxin reduced/thioredoxin oxidized (Trxred/Trxox) pairs are considered the most important ones [10]. AA and GSH are major components of the soluble redox buffering system and they contribute significantly to the redox environment of a cell. AA is the most abundant antioxidant present in plant cells at millimolar concentration (10-300 mmol L-1), thus it is the major contributor to the cellular redox state and redox signaling [11]. Besides its antioxidant function, AA regulates cell growth and differentiation and also takes part in cell wall expansion and cell division [12]. The shift of AA/DHA equilibrium towards the oxidized state inhibits cell cycle progression [13]. Moreover, the guard cell AA redox state controls stomatal movement through H2O2 signaling [14]. AA is also the substrate or cofactor for enzymes, e.g. prolyl hydroxylase catalyzing the synthesis of hydroxyproline and violaxanthin deepoxidase in the xanthophyll cycle [15]. The vitamin c-1 (vtc1) mutant of Arabidopsis thaliana with a reduced level of AA is characterized by increased sensitivity to abiotic stresses and enhanced basal resistance to biotrophic pathogens. It has been demonstrated that in the vtc mutants an abundance of AA modified plant response to stresses via redox mechanisms [16]. AA is also involved in the xanthophyll cycle that protects chloroplasts from photooxidatve damage and contributes to excess excitation energy dissipation in the antennae complexes of photosystem II (PS II) [17]. However, it has been shown that in the non-photochemical quenching 1 (npq1) mutant of A. thaliana with inactive violaxanthin deepoxidase and in the wild form of this plant, the levels of photoinhibition associated with high-light stress were similar. Moreover, fluctuating light conditions in npq1 mutants give more negative effects than high light. These facts show that the role of the xanthophyll cycle in protection of photosystems is not fully elucidated [18]. AA cooperates tightly with GSH (γ-Glu-Cys-Gly) in the Halliwell–Asada cycle, comprised of three interdependent redox couples: AA/DHA, GSH/GSSG and NADPH/NADP+. They undergo subsequent reduction/oxidation reactions catalysed by ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) that are responsible for H2O2 scavenging and keeping AA and GSH in the reduced state at the expense of NADPH. The cycle is located in all cellular compartments in which ROS detoxification is required. It serves as the main antioxidant pathway in 1154 Unauthenticated Download Date | 6/17/17 2:32 PM T. Kopczewski, E. Kuźniak plant cells and is also involved in redox-regulated plant defense against environmental stresses [11]. GSH, the main thiol buffer in plant cells, acts as an antioxidant by scavenging ROS but it also plays a pivotal role in cellular redox regulation. Similarly to AA, changes in the GSH pool and its redox status could act as redox signals integrated into the network that regulates gene expression, including antioxidant defense genes [11,19]. Protein S-glutathionylation, a process of mixed disulfide formation between a free thiol of a protein and GSH, has been suggested to be a mechanism for transducing GSH-related redox signals under the conditions of oxidative stress. This process plays an important regulatory role in controlling metabolism, gene transcription and protein turnover [20]. One of the best characterized mechanisms of redox signaling in photosynthetic organisms is that mediated by thiol/disulfide regulation which is realized by redox proteins, namely thioredoxins (Trxs), glutaredoxins (Grxs) and peroxiredoxins (Prxs). The thiol/disulfide turnover participates in turning on/off the function of proteins and in modifying the activity of transcription factors and enzymes [21]. As the Trxs system is linked to photosynthetic light reactions, its role will be addressed in the next section. Grxs are small proteins belonging to the superfamily of Trxs proteins which utilize the reducing power of GSH to reduce disulfide bonds of target proteins. They can reduce disulfide bonds by a monothiol or dithiol mechanism. Restoration of the reduced form of Grxs is provided by GSH in the presence of NADPH and GR or thioredoxin reductase [22,23]. It has been shown that three classes of Grxs (monothiol, dithiol and CC-type) occur in plants. Monothiol Grxs contain one cysteine (Cys) residue occurring most frequently in the CGFS motif. These Grxs were primarily identified in yeasts but they also occur in plants. Monothiol Grxs are involved in the biogenesis of mitochondrial iron-sulfur clusters, protection of proteins against oxidation damage, Ca2+ signaling, regulation of iron homeostasis and in protein kinase C-mediated stress responses [24]. Grxs recognize glutathionylated substrates, including proteins and catalyze their reduction, called deglutathionylation (removal of GSH from glutathionylated proteins). The reversible protein S-glutathionylation has now emerged as a redox post-translational mechanism protecting protein thiols against irreversible oxidation, controlling protein function and regulating the signaling and metabolic pathways [20]. Besides regulating protein structure and function, the glutathionylation-deglutathionylation cycle is implicated in transducing redox signals [20]. It has been found that plant cytosolic triose phosphate isomerase and some Trxs were regulated by glutathionylation. A similar regulatory mechanism controls the activity of L-galactono-1,4-lactone dehydrogenase catalyzing the final step of AA biosynthesis in the mitochondria [25]. These enzymes are potential targets of Grxs for deglutathionylation. In chloroplasts, where ROS production is intense, glutathionylation is a very important mechanism of redox regulation [26]. Grxs, like Trxs, may operate as disulfide reductases. However, they differ with respect to specificity versus target proteins and mode of catalysis. It has been shown that Grxs reduce glutathionylated proteins more efficiently than Trxs whereas the reduction of intramolecular disulfides is more characteristic of Trxs than of Grxs [26]. Prxs are thiol-dependent non-heme peroxidases decomposing peroxides which cooperate with Trxs and Grxs in the antioxidant system and the thiol-disulfide regulatory network. They are classified as broad spectrum peroxidases able to detoxify H2O2, alkyl hydroperoxides and peroxinitrite. Based on the number of catalytic Cys residues, their position in amino acid sequence and the mechanism of catalyzed reaction, four classes of Prxs have been distinguished in plants, namely 1-Cys Prx, 2-Cys Prx, Prx Q and Prx II [27]. They are distributed in many isoforms in several subcellular compartments. Prxs contain one or two Cys residues at the active site and function as dimers (2-Cys Prxs) or monomers (1-Cys Prxs, Prxs Q and Prxs II). In the common catalytic cycle, represented by 2-Cys Prxs and Prxs Q, the catalytic Cys thiol is oxidized by peroxides to sulfenic acid and then reduced by a second Cys thiol forming an intra- or intermolecular disulfide bond. 1-Cys Prxs have a single catalytic Cys residue and do not create disulfide bridges (Figure 2A). Prxs II have an ability to form heterodimers with Grxs (Figure 2B) [27]. To complete the catalytic cycle Prxs interact with Trxs, Grxs, GSH, AA and cyclophilins as well as NADPH thioredoxin reductase C (NTRC) as electron donors [27]. The biological function of Prxs in plants goes far beyond the peroxide detoxification system. Depending on the Prxs type they sense the redox state, transmit redox information to binding partners and function as chaperones. In plants, they are involved in balancing hydroperoxide production during photosynthesis and respiration as well as in the redox regulation of cell division and elongation, germination, organ development and stress adaptation [28]. Interactions of redox-active compounds and the capacity of redox buffers determine the cellular redox state. Under optimal conditions the inherent redox balance in individual organelles is maintained at different levels compatible with their specific metabolic 1155 Unauthenticated Download Date | 6/17/17 2:32 PM Redox signals in plant cells Figure 2. Reaction cycle of peroxiredoxins. Prxs catalyze the reduction of hydroperoxides. (A) Classical 2-Cys Prx is a homodimer with two reduced Cys residues per subunit. During the catalytic cycle the peroxide substrate is reduced to the corresponding alcohol and the sulfenic acid derivative of the catalytic Cys of one subunit interacts with the resolving Cys of the other subunit forming an intermolecular disulfide bridge. The oxidized 2-Cys Prx is regenerated by electron donors such as Grxs. (B) 1-Cys Prxs possess one catalytic Cys on both subunits. The oxidized 1-Cys Prxs are preferentially regenerated by Trxs. Grx – dithiol glutaredoxin, GSH – glutathione, GSSG – glutathione disulfide, Prx(s) – peroxiredoxin(s), ROOH – hydrogen peroxide (R=H) or lipid peroxide (R = hydrocarbon group), ROH – water (R=H) or alcohol/phenol (R=hydrocarbon group), Trx(s) – thioredoxin(s). According to [27], modified. requirements. The redox buffering capacity of compartments such as apoplast, endoplasmic reticulum and vacuole are considered much weaker than that of chloroplasts and mitochondria [29]. Redox signals are key regulators of plant metabolism, morphology, development and defense responses. As each cellular compartment has different antioxidants and redox buffering capacity, at each location the redox state and redox-based signaling can be controlled independently [30]. Specific compartment-based redox signaling, including that involved in chloroplast-to-nucleus and mitochondria-to-nucleus retrograde communication, and regulation of gene expression can be achieved by changes in the redox status of a given compartment via modifying ROS production and/or antioxidant defenses [10,30]. The cellular balance between redox buffers and ROS called redox homeostasis is strongly affected by environmental cues [31]. Increased ROS production and changes in redox potential caused by abiotic and biotic stressors are important factors in cell redox signaling influencing the specificity of a response. Amplitude, duration and localization of particular redox signals specifically indicate what the nature of stress is and what defense response has to be activated [32]. 3. Redox signaling related to photosynthesis and mitochondrial respiration ROS are produced in many parts of a plant cell, however their synthesis is mainly associated with activities of electron transport chains in chloroplasts and mitochondria [33]. Under stressful conditions an imbalance between light energy absorbed through PS II and the ultimate consumption of the photosynthetic electrons through metabolic pathways leads to increased formation of ROS and to photooxidative stress. Recently it has been shown, however, that redox signals generated in the photosynthetic electron transport chain have a strong impact on the cellular signaling network. Chloroplasts act as sensors for changes in the environment which are communicated via redox signaling. A variation in the redox status of the photosynthetic apparatus generates redox signals involved in mediating stress-induced metabolic changes [19]. It has been proposed that 1156 Unauthenticated Download Date | 6/17/17 2:32 PM T. Kopczewski, E. Kuźniak perturbation of the photosynthetic electron transfer produces three types of redox signals [34]. Class 1 signaling originates from specific redox pairs in the photosynthetic electron transport chain, e.g. the reduced and oxidized plastoquinone (PQ), class 2 signaling depends on the redox state of stromal Trxs, NAD(P)H, GSH and AA whereas class 3 signaling is mediated by ROS [35,36]. Here we focus on the redox signals from the PQ and Trxs pools. PQ, 5,6-dimethyl-para-quinone with a hydrophobic chain attached at position 2 and variable number of isoprenoid units, carries the electrons from PS II to the cytochrome b6f (cyt b6f) complex and serves as an indicator of photosynthetic electron transport [37]. The redox state of PQ, depending directly on the photosynthetic electron flux, controls the size of photosynthetic antennae and regulates chloroplast protein expression. The PQ redox state regulates the activity of thylakoid protein kinase STATE TRANSITION7 (STN7) involved in the reversible phosphorylation of light harvesting complex II (LHCII) antenna proteins. STN7 is required for state transition that optimizes electron transfer under fluctuating light conditions [38]. It has been shown that the reduction of PQ into plastoquinol (PQH2) induces expression of psaA and psaB genes encoding proteins of photosystem I (PS I) while increased concentration of PQ activates the transcription of PS II genes (psbA, psbD). Moreover, changes in the PQ pool affect the level of nuclear and chloroplast mRNA during acclimatory response to environmental conditions [39]. Trxs are low molecular redox proteins with enzymatic activity, discovered both in non-photosynthetic and photosynthetic organisms. Three types of Trxs (h, f, m) have been identified in plants. In plant cells, Trxs were recognized as redox state creators and proteins affecting the activity of numerous target molecules via thiol/disulfide exchanges. Thioredoxin h (Trx h) is present in the cytosol. Thioredoxins f and m are located in chloroplasts and they are reduced in the light via ferredoxin and ferredoxin thioredoxin reductase (FTR) [40]. They were primarily considered as regulators of several Calvin cycle enzymes, e.g. fructose-1,6-bisphosphatase, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase and ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) activase. All these enzymes are activated in the light by Trxs and remain inactive in the dark. However, current reports show that Trxs also regulate the activity of enzymes associated with other metabolic pathways, e.g. some electron transport chain components and enzymes of the sulfur assimilation pathway. Moreover, biosynthesis of amino acids, lipids and tetrapyrolles is controlled by Trxs. Trxs also influence the activity of some enzymes involved in translation and DNA metabolism. They are also versatile redox regulators interacting with other redox transmitters, such as GSH and Grxs [40,41]. Trxs may also be part of enzyme complexes. The Arabidopsis thaliana Tetratricopeptide Domaincontaining thioredoxin (AtTDX) is a Trx-like protein consisting of a Trx motif and three tetratricopeptide (TPR) repeat domains. The Trx motif is a disulfide reductase responsible for redox properties of AtTDX while TPR exhibits chaperone activity [42]. Moreover, a new Trx-like protein (TrxZ) that is a subunit of plastid RNA polymerase complex has been identified recently. It is recognized as redox active protein linking redox regulations and plastid transcription [43]. Recent studies highlighted the importance of a co-operation between PQ and Trxs redox signaling in tailoring the photosynthetic apparatus to the illumination conditions. The light-induced changes in the PQ and Trxs pools affect gene expression and metabolic enzyme activities indispensable to adapt plant productivity to the light environment [44]. Although in the photosynthesizing cells mitochondria are not regarded as the main source of ROS, perturbations of the mitochondrial redox state have important implications for cellular redox homeostasis. Firstly, electron flow through the mitochondrial electron transport chain may control AA biosynthesis as L-galactono-1,4lactone dehydrogenase is associated with NADH dehydrogenase complex I (complex I) and is required for its assembly and this enzyme uses oxidized cytochrome c as the only electron acceptor [45]. Secondly, in Nicotiana sylvestris the cytoplasmic malesterile mutant (CMSII) lacking functional complex I re-adjustment of mitochondrial, chloroplastic and peroxisomal antioxidant defense as well as enhanced alternative oxidase-mediated, cyanide insensitive respiration were observed [46]. Thus, loss of complex I function in mitochondria changed the whole cell redox homeostasis. Moreover, the malate valve also serves as a redox-based mechanism linking mitochondria and chloroplasts. The chloroplastic NADP-malate dehydrogenase, which is Trx-controlled, uses excess NADPH to convert oxaloacetate to malate in order to regenerate the electron acceptor NADP and to prevent photoinhibition. However, the effectiveness of this mechanism depends on the malate gradient between chloroplasts and cytosol, and the increase in the cytosolic malate pool is coupled with a higher mitochondrial malate level [47]. 1157 Unauthenticated Download Date | 6/17/17 2:32 PM Redox signals in plant cells 4. Chloroplasts – mitochondria communication and retrograde signaling In plant cells, the main cellular compartments, e.g. chloroplasts, mitochondria, peroxisomes and cytosol, exist in a subtle metabolic equilibrium and disturbances in any of these locations generate perturbations in the whole cellular metabolism. Special attention has been paid to the interaction between mitochondrial metabolism and photosynthetic carbon assimilation. In the photosynthesizing cells, chloroplasts and mitochondria, both involved in the production of ATP, are also connected by metabolite exchange and the photorespiration pathway (Figure 3). The exchange of metabolites between chloroplasts and mitochondria requires the presence of translocators in their inner envelope membrane and is connected with the formation of inter-organelle communication channels [48,49]. Numerous studies showed that the transfer of photosynthetically active cells from darkness to light is associated with an increase in photosynthetic activity Figure 3. in comparison with respiration which emphasizes the interaction between chloroplasts and mitochondria [50]. Moreover, the balance between triose-phosphate and 3-phosphoglycerate in chloroplasts as well as between malate and oxaloacetate in mitochondria reflects the redox status of these organelles and may modulate photosynthesis and respiration [49]. However, the chloroplasts-mitochondria interaction is still not known well and the current knowledge refers mainly to communication based on carbon signals [51]. Other signals, e.g. AA, nitric oxide (NO) and the cytosolic pH have also been proposed to be involved in the biochemical cross-talk between chloroplasts and mitochondria. It has been suggested that AA, an important antioxidant synthesized in mitochondria, influences the redox conditions and ROS scavenging in chloroplasts [12,52]. Moreover, alkalinization of cytosol in mesophyll cells resulting from the activity of illuminated chloroplasts may modulate the cytosolic, chloroplast and mitochondrial metabolism [49]. In the context of photorespiration, mitochondria help to dissipate excess redox equivalents exported from chloroplasts in the Interactions between chloroplasts, mitochondria and the nucleus. Chloroplasts and mitochondria act as sensors of environmental changes. Extensive metabolic communication is established between chloroplasts and mitochondria to maintain energy flow through these organelles and to optimize different cell functions. Moreover, redox signals generated in chloroplasts and mitochondria within the electron transport chains or by generation of ROS and ROS-scavenging systems initiate signaling pathways that activate nuclear gene expression. This retrograde signaling coordinates the nuclear gene expression with the functional state of chloroplasts and mitochondria but also with the acclimatory response that helps the plant to respond optimally to environmental stress. Cyt b6f – cytochrome b6f, EX1 – EXECUTER 1, EX2 – EXECUTER 2, Fd – ferredoxin, Gc – glycolate, Mg-protoIX – Mg-protoporphyrin IX, mitETC – mitochondrial electron transport chain, PC – plastocyanin, PGc – 2-phosphoglycolate, PQ – plastoquinone, PQH2 – plastoquinol, PS I – photosystem I, PS II – photosytem II, RuBP – ribulose-1,5-bisphosphate, STN7 – STATE TRANSITION7 protein kinase, Trxox – thioredoxin oxidized, Trxred – thioredoxin reduced. According to [69], modified. 1158 Unauthenticated Download Date | 6/17/17 2:32 PM T. Kopczewski, E. Kuźniak light via the malate valve, as described earlier [47]. The photorespiratory pathway is also an internal source of CO2 which can help to sustain carbon assimilation under limiting CO2 conditions [49]. However, further experimental evidence is necessary to establish the roles of these signals in inter-organelle communication. Chloroplasts and mitochondria also cooperate in retrograde signaling, which serves to communicate the developmental and functional state of chloroplasts and mitochondria to the nucleus to regulate nuclear gene expression [13,53-55]. While the expression of nuclear-encoded proteins and their import to organelles is called anterograde signaling, the information transfer from organelles to a nucleus that affects nuclear gene expression is defined as retrograde signaling. Chloroplasts and mitochondria produce retrograde signals to alter nuclear gene expression in order to coordinate their biogenesis and function under stressful conditions [56]. There are four retrograde signaling pathways: tetrapyrrole biosynthesis, organellar protein synthesis, redox state and ROS [51]. A broad range of ROS/redox signals generated in chloroplasts and mitochondria is targeted to the nucleus (retrograde signaling) to induce gene expression. Singlet oxygen (1O2) produced in chloroplasts in light via chlorophylls that act as photosensitizers is a signaling molecule in plants [51]. The primary effect of 1O2 generation is not the control of chloroplast biogenesis as only about 15% of the 1O2-responsive genes encode plastid proteins. It was discovered that 1O2 is responsible for the activation of PCD genes [57]. 1O2 has a relatively short half-life (t = 200 ns) and its scavenging or transformation often outruns its physiological actions [58]. Therefore, it has been suggested that other molecules must be involved in the transduction of 1 O2-dependent signals. Two proteins associated with the thylakoid membranes, namely EXECUTER1 (EX1) and EXECUTER2 (EX2) are mediators of 1O2 signals [17]. EX1 is responsible for the perception of 1O2 produced in chloroplasts and EX2 controls the activity of EX1. Evidence for the concerted action of EX1 and EX2 comes from EXECUTER1 EXECUTER2 fluorescent (ex1 ex2 flu) mutant of A. thaliana. Inactivation of genes encoding EX1 and EX2 suppressed the 1O2-induced genes completely [17]. H2O2, which is the product of further reduction of O2•-, also plays an important role in retrograde signaling. This molecule is implicated in modulation of nuclear gene expression via mitogenactivated protein kinase (MAPK) signaling cascades [59]. In Arabidopsis, transcriptome analysis has revealed that 1-2% of the transcriptome and one third of the transcription factor mRNA are altered after H2O2 treatment [60,61]. Moreover, analysis of the flu mutant of A. thaliana showed that H2O2 activates a different set of genes in comparison with 1O2 and it antagonizes the 1 O2-mediated stress responses [57]. Mg-protoporphyrin IX (Mg-protoIX), an intermediate in chlorophyll biosynthesis, is also responsible for retrograde signaling [62,63]. This compound is identified as a negative regulator of photosynthetic gene expression in the nucleus and chloroplasts. Mg-protoIX is accumulated under stress and after norflurazon (inhibitor of the last stage of chlorophyll biosynthesis) treatment and therefore signals from Mg-protoIX are considered to be associated with inhibition of photosynthesis and generation of responses against stress [51,58,64,65]. Studies with genome uncoupled (gun) mutants failing to repress the expression of photosynthesis-associated nuclear genes when treated with norflurazon indicated that the accumulation of Mg-protoIX triggers plastid-to-nucleus signaling mediated by ABA INSENSITIVE4 (ABI4) transcription factor. GUN1 protein integrates signals related to Mg-protoIX, photosynthetic electron flow and oxidative stress which are generated in chloroplasts and is implicated in their transmission to the nucleus. In response to GUN1-derived signals, ABI4 transcription factor represses photosynthetic nuclear genes by preventing DNA binding of factors needed for their expression [51,66,67]. However, the role of Mg-protoIX in retrograde signaling needs further elucidation as tetrapyrrole profiling in Arabidopsis seedlings showed no correlation between Mg-protoIX accumulation and the expression of nuclear genes [68]. Photosynthetic electron transport chain components have also been postulated to be redox-dependent retrograde signaling molecules that communicate the chloroplast status to the nucleus. As discussed above, redox signals generated in chloroplasts are transmitted by PQH2 and by the elements on the reducing side of PS I, particularly by ferredoxin (Fd) (Figure 3). The thylakoid protein kinase STN7, playing a role in the reversible phosphorylation of LHCII and its temporary migration to PS I as well as in long-term adaptation of plants to changes in light quality or intensity [39,70], has been proposed to transfer the changes in chloroplast redox status to the nucleus [58,71]. Moreover, electrons from Fd may be transported to Trxs and GSSG. Together with other compounds such as Grxs and Prxs, Trxs can participate in the modification of functions of target proteins. Especially chloroplast and nuclear transcription factors may be activated or deactivated with the involvement of Trxs, Grxs and Prxs [63]. The latest data showed the role of 3’-phosphoadenosine-5’-phosphate (PAP) in the regulation of nuclear-gene expression associated with 1159 Unauthenticated Download Date | 6/17/17 2:32 PM Redox signals in plant cells stress response in plants. The level of chloroplastic PAP is controlled by SAL1 – a phosphatase that catalyzes the conversion of PAP into AMP [72]. In high light- or drought-stressed Arabidopsis plants, PAP accumulates in chloroplasts and it can migrate into the nucleus where it inhibits some 5’ to 3’ exoribonucleases (XRNs). This leads to the activation of transcription factors (ZAT12, DREB2A and others) and to the regulation of stress-responsive genes (e.g. APX2, ELIP2) [56]. Interestingly, SAL1 is dual targeted to chloroplasts and mitochondria. This suggests that the PAP-mediated chloroplast retrograde signaling can be linked to the mitochondrial pathway [73]. Another recent study has suggested that methylerythritol cyclodiphosphate (MEcPP), a precursor of isoprenoids synthesized in the chloroplastic methylerythritol phosphate (MEP) pathway, also has a function in plastid retrograde signaling during stress [74]. In chloroplasts, MEcPP is normally converted into hydroxy-2-methyl-2-(E)-butenyl4-diphosphate (HMBPP) and hydroxymethylbutenyl diphosphate synthase (HDS) catalyzes this process [75]. It has been shown that constitutively expressed hydroperoxide lyase (ceh1) mutant of A. thaliana that has a mutation in the HDS gene responsible for the conversion of MEcPP into HMBPP in the MEP pathway accumulates MEcPP. Ceh1 displays changes in stress-related gene expression associated with salicylic acid-mediated defense leading to increased resistance to Pseudomonas syringae. These data clearly indicate that accumulation of MEcPP in chloroplasts is associated with regulation of stress-responsive nuclear gene expression and MEcPP can be considered as an important compound involved in retrograde signaling. Chloroplastic retrograde regulations are, to date, the best studied retrograde signaling pathways in plants. The mitochondrial retrograde regulations are well known in fungi and animals whereas in plants they remain elusive. However, it has been evidenced that changes in mitochondrial functions, especially the dysfunction of mitochondrial electron transport chain, trigger altered nuclear gene expression. These genes encode proteins, e.g. alternative oxidase, alternative NAD(P)H dehydrogenase, indispensible for the recovery of mitochondrial function, and enzymes involved in maintaining the prooxidant/antioxidant homeostasis, namely catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST) [55]. Recently, three functional targets of mitochondrial retrograde signaling, i.e. protein synthesis, photosynthesis light reactions and plant-pathogen interactions, have been identified based on a comprehensive transcriptional analysis [76]. Although the identity of signals in the mitochondrial retrograde signaling pathway remains largely unknown, these results further support the concept of the role of mitochondria in retrograde signaling in plants and chloroplast-mitochondria cooperation in this pathway. In addition, it has been shown that ABI4 transcription factor is a target of retrograde signaling from mitochondria and chloroplasts [77]. 5. Conclusions Plants can sense and respond to changes in the environment. To acclimate to the current environmental conditions they activate a diverse set of responses including changes in gene expression and regulations at the metabolic and physiological levels involving redox signaling. ROS and ROS-antioxidant interactions are sources of different types of redox signals which regulate acclimation processes. In plants, chloroplasts and mitochondria are the main intracellular ROS producers and generators of redox signals. Recent studies have revealed the central role of chloroplasts as a source and target of redox regulation. A lot of chloroplastic metabolites, both antioxidants and components of the photosynthetic electron transport chain, were defined as important sensors creating the cellular redox status. NADPH, AA, GSH, PQ and Fd form a specific redox system that, together with antioxidant enzymes and thiol/disulfide exchange systems, e.g. Trxs, Grxs and Prxs, regulates gene expression and allows a plant to respond adequately to fluctuating environmental conditions. Signals from both mitochondria and chloroplasts cooperate in the regulation of nuclear gene expression. 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