ROS and redox signalling in the response of plants to abiotic stress

Authors


G. Miller. e-mail: gad.miller@biu.ac.il

ABSTRACT

The redox state of the chloroplast and mitochondria, the two main powerhouses of photosynthesizing eukaryotes, is maintained by a delicate balance between energy production and consumption, and affected by the need to avoid increased production of reactive oxygen species (ROS). These demands are especially critical during exposure to extreme environmental conditions, such as high light (HL) intensity, heat, drought or a combination of different environmental stresses. Under these conditions, ROS and redox cues, generated in the chloroplast and mitochondria, are essential for maintaining normal energy and metabolic fluxes, optimizing different cell functions, activating acclimation responses through retrograde signalling, and controlling whole-plant systemic signalling pathways. Regulation of the multiple redox and ROS signals in plants requires a high degree of coordination and balance between signalling and metabolic pathways in different cellular compartments. In this review, we provide an update on ROS and redox signalling in the context of abiotic stress responses, while addressing their role in retrograde regulation, systemic acquired acclimation and cellular coordination in plants.

INTRODUCTION

Plants are constantly subjected to changes in their environment, causing them to alter their metabolism in order to maintain a steady-state balance between energy generation and consumption. This balance largely depends on a delicate signalling network that coordinates three of the most critical processes in plant life: photosynthesis, dark respiration and photorespiration, activities linked in terms of electron transfer, substrate, reductants and energy (Noctor, De Paepe & Foyer 2007; Foyer & Noctor 2009; Foyer et al. 2009; Pfannschmidt et al. 2009). Metabolic pathways in plant organelles are sensitive to changes in environmental conditions, and metabolic imbalances can induce an oxidative stress in cells by promoting the generation and accumulation of reactive oxygen species (ROS), causing oxidation of cellular components, hindering metabolic activities and affecting organelle integrity. Oxidized metabolites and carbonylated proteins have traditionally been regarded as markers for oxidative stress; however, these compounds are now suggested to function in signalling under oxidative conditions (Buchanan & Balmer 2005; Møller, Jensen & Hansson 2007; Mueller & Berger 2009; Møller & Sweetlove 2010). The electron transport chains in chloroplasts and mitochondria are associated with carbon metabolism through reducing power and energy such as NAD(P)H and ATP (Couée et al. 2006; Reumann & Weber 2006; Rhoads & Subbaiah 2007; Foyer & Noctor 2009; Häusler et al. 2009). Therefore, a close relationship between the different organelles linking their redox state can affect carbon metabolism and energy balance in cells.

Alterations in carbon metabolism and energy balance during stress have been reported in both chloroplasts and mitochondria (Baxter et al. 2007; Takahashi & Murata 2008), and a high level of metabolic coordination is required to maintain energy flow through these organelles under all growth conditions to avoid excessive generation of ROS and oxidative damage.

A tight control is needed to balance these activities and maintain coordination, including reversible redox regulation of proteins by dithiol-disulfide exchange, regulation of phosphoproteins, activation of signalling pathways by ROS-responsive regulatory genes and buffering of ROS by ROS-scavenging enzymes and antioxidant molecules (Mittler et al. 2004; Noctor et al. 2007; Foyer & Noctor 2009; Foyer et al. 2009; Martíet al. 2009; Pfannschmidt et al. 2009; Chibani et al. 2010; Miller et al. 2010; Dietz 2011). In addition, coordination of chloroplasts and mitochondrial activities partially requires close physical interaction between organelles to allow efficient exchange of metabolites and inter-organellar signals (Baker et al. 2006; Noguchi & Yoshida 2008; Nunes-Nesi et al. 2008). Furthermore, under stress conditions, the balance of activity can affect exchange of signals between chloroplasts or mitochondria with the nucleus, namely retrograde or anterograde signalling (Woodson & Chory 2008), or induce activation of intercellular signals such as systemic signals (Karpinski et al. 1999).

In this review, we provide an update on recent findings related to redox and ROS signalling in chloroplasts and mitochondria with respect to abiotic stress and acclimation. We address subjects such as cellular coordination, and intra- and intercellular signalling. This review does not attempt to cover all aspects of redox regulation and ROS signalling pathways related to photosynthesis and respiration. For more details on organellar redox regulation and signalling, we would like to refer our readers to more extensive reviews (see Mittler et al. 2004; Pogson et al. 2008; Woodson & Chory 2008; Foyer & Noctor 2009; Mullineaux 2009; Galvez-Valdivieso & Mullineaux 2010).

REDOX SIGNALLING IN CHLOROPLASTS AND MITOCHONDRIA DURING ABIOTIC STRESS

The chloroplast redox state, manifested through the plastquinone (PQ) pool, is involved in the regulation of several important biological processes including nuclear and plastid gene expression, hormone signalling and stress responses. Expression of nuclear genes encoding plastid proteins has been shown to correlate with redox shifts in the PQ, glutathione (GSH) and ascorbate pools in coordination with chloroplast development and light signals (Foyer & Noctor 2009). Under high light (HL) or fluctuating light conditions, changes in the redox state of the PQ pool are known to be correlated with expression of antioxidant and defense genes, including pathogen defense genes, and phosphorylation of thylakoid proteins (Karpinski et al. 1999; Zer & Ohad 2003; Mühlenbock et al. 2008; Li et al. 2009). Chloroplast kinases and phosphatases have emerged as essential instruments in maintaining optimized photosynthetic activity and redox state during environmental changes (Dietzel & Pfannschmidt 2008; Schliebner et al. 2008; Pesaresi et al. 2009, 2010). Acclimation to changes in light quality and light intensity requires balancing energy distribution within the photosynthetic machinery to efficiently drive photosynthesis and to protect the thylakoid from damage. In the short term, activation of state transition that balances and maintains the photosynthetic energy distribution between photosystem II (PSII) and photosystem I (PSI) is mediated by the redox state of the PQ pool (Zer & Ohad 2003; Dietzel & Pfannschmidt 2008). In the long term, imbalances in energy distribution between the two photosystems are counteracted by adjusting photosystem stoichiometry, changing the abundance of reaction centre and light harvesting proteins within hours or days [long-term response (LTR)] (Zer & Ohad 2003; Dietzel & Pfannschmidt 2008; Pesaresi et al. 2009, 2010). In Arabidopsis, the protein kinase STN7 is required for the PQ redox-state dependent regulation of both state transition and LTR activities (Pesaresi et al. 2009). STN7 regulates short-term responses via phosphorylation of a thylakoid-bound phosphoprotein TSP9 (Fristedt et al. 2009; Pesaresi et al. 2009). LTR is essential for acclimation to low light stress condition, but is not required for acclimation to HL and is not involved in photo-inhibition (Reinhold et al. 2008; Wagner et al. 2008). STN8 is another STN7-like protein kinase that is required for the quantitative phosphorylation of PSII, which includes the phosphorylation of D1 and D2 proteins. STN8 is not essential for the protection of PSII under HL intensities (Bonardi et al. 2005; Vainonen, Hansson & Vener 2005); however, it is required for the specific HL-induced phosphorylation of a 40 kDa protein in the thylakoid membrane, suggesting a role in HL response (Vainonen et al. 2008).

Mitochondrial respiration is also important for neutralizing excess of photosynthetic reducing power, preventing oxidative damage of thylakoid membranes and other cellular components (Møller 2001; Raghavendra & Padmasree 2003; Dinakar et al. 2010). Mitochondrial function during increased respiratory activity and photorespiratory metabolism is sensitive to oxidative damage, similar to the damage induced by the oxidizing herbicide paraquat, chilling or drought. Oxidized lipids such as polyunsaturated fatty acids generated under these conditions were shown to inhibit tricarboxylic acid (TCA) cycle activity causing perturbation in carbon and nitrogen metabolisms (Taylor, Day & Millar 2002, 2004; Mueller 2004; Møller et al. 2007; Ito et al. 2009). The activities of alternative oxidase (AOX), type II NAD(P)H dehydrogenase and uncoupling proteins in the inner mitochondrial membrane are essential for optimizing the flow of electrons through the electron transport chain, preventing the over-reduction of the mtETC and the generation of excess ROS. These are therefore regarded as regulators of mitochondrial redox state and ROS generation (Padmasree & Raghavendra 1999; Noctor et al. 2007; Rasmusson & Wallström 2010). Recently, the significance of mitochondrial function to the regulation of photosynthesis has been addressed. Under HL conditions, excess reductants exported from chloroplasts are dissipated in the mitochondria through the function of type II NAD(P)H dehydrogenase and AOX (Noguchi & Yoshida 2008; Nunes-Nesi et al. 2008). Reductants are transferred from the chloroplast to the cytosol, and then to the mitochondria through the malate-oxaloacetate shuttle that is required for the import of respiratory substrates into mitochondria (Scheibe et al. 2005; Noguchi & Yoshida 2008).

Like chloroplasts, phosphorylation of mitochondrial proteins is thought to play a major role in mitochondrial redox regulation as indicated by recent proteomics studies that identified conserved- and plant specific-phosphoproteins, as well as phosphorylated proteins associated with mtETC complexes, ATP synthesis and TCA cycle activity (Bykova, Egsgaard & Møller 2003; Ito et al. 2009). However, further identification and characterization of mitochondrial protein kinase and phosphatase activities, as well as their target proteins, is needed for understanding the involvement of reversible phosphorylation in redox regulation and signalling in mitochondria.

Redox regulation and ROS metabolism are interlinked and involved in optimizing the function of mitochondria, chloroplast and other organelles (Dinakar et al. 2010). Gradients in redox state of NAD(P)H, photorespiration metabolism, shared pathways involving carbon and nitrogen metabolism, and bypass shunts such as the γ-aminobutyrate shunt, can influence the energy flow and redox fluctuations between chloroplasts, mitochondria and cytosol (Fait et al. 2007; Noctor et al. 2007; Foyer & Noctor 2009), and interconnect these organelles to a wider cellular redox-network. This network requires tight regulation and high level of coordination especially under abiotic stress conditions that increase ROS metabolism and cause fluctuations in the redox state of both mitochondria and chloroplasts.

Elucidating redox regulation and the relative contribution of each organelle to the network would require the monitoring of redox states in chloroplast and mitochondria simultaneously. ROS network genes and redox regulatory enzymes such as the ascorbate-glutathione cycle enzymes monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and glutathione peroxidase (GPX) or the thiol peroxidase type II peroxiredoxin (PrxR)E are co-expressed both in the chloroplast and mitochondria (Mittler et al. 2004 and current TAIR annotation; Rouhier & Jacquot 2005). These dual-targeted genes could be considered as functional redox state reporters that would indicate the oxidative division of labour or load between these two powerhouses under different physiological conditions. In future studies, using these dual-targeted proteins fused to fluorescent proteins (FPs) in combination with organelle-localized redox-sensitive FPs and microscopic imaging could be useful for monitoring and elucidating the redox network in plant cells.

ROS AND REDOX RETROGRADE SIGNALLING IN ABIOTIC STRESS RESPONSES

Retrograde (organelle to nucleus) mechanisms have evolved to coordinate and communicate gene expression, metabolism and development between the organelles and the nucleus, which can subsequently modulate anterograde (nucleus to organelle) control (Woodson & Chory 2008). Retrograde signalling can be largely divided into two categories: developmental control of organelle biogenesis, and operational control to adjust and acclimate to fluctuating environmental conditions (Pogson et al. 2008). Organellar redox state and ROS metabolism in chloroplast and mitochondria are sources for retrograde signals, which during stress conditions play an important role in the acclimation of plants (Rhoads & Subbaiah 2007; Pogson et al. 2008; Woodson & Chory 2008). Deviation from regular redox homeostasis can be sensed in the chloroplast or mitochondria and transmitted to the nucleus by retrograde signalling cascades. Alternatively, redox imbalances in these organelles could be transmitted into the cytoplasm by metabolic coupling (Baier & Dietz 2005; Mullineaux 2009; Galvez-Valdivieso & Mullineaux 2010).

‘Classic’ chloroplast retrograde signalling

The chloroplast is the organelle most prone to oxidative damage because of its bivalent oxygen chemistry, the demand for reductive power and excess excitation energy at PSII (Baier & Dietz 2005). Retrograde signalling in plants has been studied in seedlings bleached by norflurazon, an inhibitor of carotenoid biosynthesis that causes photooxidative damage in the light or by inhibition of plastid protein synthesis with lincomycin or chloramphenicol, eventually leading to cell death. In addition, mutants with segments of undeveloped chloroplasts have also been used (Nott et al. 2006; Koussevitzky et al. 2007). Three different processes in young seedlings of Arabidopsis were shown to induce chloroplast-derived signals to the nucleus altering the expression of nuclear genes, depending on the presence of GUN1 in the chloroplast and ABI4 in the nucleus; (1) accumulation of the chlorophyll biosynthesis intermediate Mg-Protoporphyrin IX and its methylester (Mg-Proto IX and Mg-Proto IX-ME) alters gene expression in both Arabidopsis and Chlamydomonas; (2) inhibition of plastid gene expression (PGE), probably at the protein translation stage; and (3) changes in the redox state of the photosynthetic electron transfer (PET) chain (Koussevitzky et al. 2007; Woodson & Chory 2008). GUN1 (genome uncoupled 1) encodes a chloroplast-localized pentatricopeptide-repeat (PPR) containing protein that plays a yet unknown role in retrograde signalling (Koussevitzky et al. 2007).

The role of Mg-ProtoIX and Mg-ProtoIX-ME in retrograde signalling has been recently challenged, and it was suggested that rather than the accumulation of Mg-ProtoIX per se, the signal might originate from rapid changes in the flux through the tetrapyrrole pathway, the activity of the Mg-chelatase or accumulation of Mg-ProtoIX in specific sites (Mochizuki et al. 2008; Moulin et al. 2008; Galvez-Valdivieso & Mullineaux 2010). In a recent proteomic analysis of Mg-ProtoIX-binding proteins, approximately 35% of the identified proteins were related to various stress responses, including three glutathione S-transferases (AtGST10, AtGSTT1 and AtGSTF3) and four peroxidases (ATP15, APX1, PER22 and ATP3), and it was speculated that these peroxidases might have a role in Mg-ProtoIX degradation (Kindgren et al. 2010). This lends further support to the role of Mg-ProtoIX in both retrograde and ROS signalling.

Unlike PGE-dependent signals, the ROS-related retrograde signalling pathways are postulated to be primarily used for stress signalling rather than genome coordination (Woodson & Chory 2008), and occur as part of the plant's normal response to environmental cues (Mullineaux 2009). Nevertheless, the GUN1-ABI4 retrograde signalling pathway could exert influence on abiotic stress response and acclimation as it was previously shown that gun1 and abi4 seedlings displayed altered expression patterns of the stress-induced zinc finger proteins Zat10 and Zat12 under HL, and increased sensitivity to heat stress (Koussevitzky et al. 2007; Miller et al. 2007). These results suggest a role for GUN1-ABI4 pathway in coordination of abiotic stress responses in Arabidopsis seedlings; however, the physiological relevance of this pathway to the normal development, stress response and adaptation in mature plants is still unknown.

Chloroplast ROS retrograde signalling (singlet oxygen signalling)

The most studied ROS-dependent retrograde signalling pathway in higher plants is the singlet oxygen pathway, which is independent of Mg-ProtoIX and GUN1-mediated signalling. Singlet oxygen (1O2) has long been considered as a toxic ROS molecule causing considerable damage to chloroplasts by promoting oxidation of lipids containing polyunsaturated fatty acids (Foyer & Noctor 2009). Generation of 1O2 occurs at PSII by excited triplet-state chlorophyll at the P680 reaction centre and in the light-harvesting complex when the electron transport chain is over-reduced (Asada 2006). In addition, many chlorophyll precursors (porphyrin- and haem-containing compound) are photoactive and generate 1O2 in the presence of light (Duke et al. 1991; Jimenez-Banzo et al. 2008; Mullineaux 2009).

The singlet oxygen signalling pathway has been extensively studied in Arabidopsis using the conditional flu mutants that accumulate protochlorophyllide, a potent photosensitizer, during dark adaptation and upon re-exposure to light produces 1O2 (op den Camp et al. 2003; Apel & Hirt 2004; Wagner et al. 2004; Laloi et al. 2007; Lee et al. 2007). Using this mutant, the biological activity of 1O2 as a signal molecule, and not just a toxin, was revealed in microarray experiments that identified distinct sets of genes specifically activated by 1O2 (op den Camp et al. 2003; Gadjev et al. 2006). 1O2 activates a genetic programme leading to growth inhibition and lethality through a distinct pathway that was identified using a second site mutation in the EXECUTER1 (exe1/flu double mutant) gene that was fully suppressed by a third mutation in EXECUTER 2 (Wagner et al. 2004; Lee et al. 2007).

Singlet oxygen signalling communicates with other ROS signals such as hydrogen peroxide. H2O2 has a positive role in reducing the likelihood of 1O2 formation, and treatment with exogenous H2O2 was shown to promote the oxidation of quinone A (QA), the primary plastoquinone (PQ) electron acceptor, which increases the photosynthetic electron transport flow and decreases the generation of 1O2 during stress; thus, the water–water cycle functions also as a relaxation system to suppress the photoproduction of 1O2 (Karpinska, Wingsle & Karpinski 2000; Asada 2006; Møller et al. 2007). Accordingly, a flu mutant overexpressing thylakoid-ascorbate peroxidase (tAPX) strongly reduced the activation of nuclear gene expression through the EXECUTER singlet oxygen pathway (Laloi et al. 2007).

New Arabidopsis flu-like mutants exhibiting flu-independent 1O2-dependent cell death pathway (Samol et al. 2011), as well as a new group of flu-suppressors mutants identified in genetic screens, point to the complexity of 1O2-activated cell death (Coll et al. 2009; Meskauskiene et al. 2009). However, it is important to note that in spite of the progress that has been achieved in the study of 1O2 signalling, its physiologic context and significance is still not clear.

Mitochondrial retrograde signalling

Mitochondrial retrograde regulation (MRR) signalling is poorly understood compared with the chloroplast to nucleus retrograde signalling. MRR in plants has been studied primarily in instances of mitochondrial dysfunction that often result in male sterility or embryo lethality (for further reading, see Rhoads & Subbaiah 2007; Woodson & Chory 2008). ROS generation and accumulation seem to be intimately involved in MRR (Rhoads et al. 2006; Rhoads & Subbaiah 2007), although redox alternations of mitochondrial electron transport activity revealed a distinct pattern in Arabidopsis gene expression compared with other oxidative stress treatments (Rhoads & Subbaiah 2007).

The study of mitochondrial redox and ROS signalling involves increased expression of nuclear-encoded alternative oxidase 1 (AOX1) as the marker of retrograde responses in plants treated with mtETC inhibitors such as rotenone (complex I – NADPH dehydrogenase), antimycin A (AA; Complex II) and monofluoroacetate (MFA; TCA cycle) that increase AOX expression (Maxwell, Wang & McIntosh 1999; Vanlerberghe, Robson & Yip 2002; Rhoads & Subbaiah 2007), or antioxidants that prevent AOX induction (Maxwell et al. 1999; Vanlerberghe et al. 2002).

Although mitochondrial retrograde signalling has been implicated in ROS signalling, O2 sensing, heat shock, pathogen sensing and programmed cell death (PCD) (Rhoads & Subbaiah 2007; Woodson & Chory 2008), no distinct protein component of any plant mitochondrial retrograde signalling pathway has been identified (Woodson & Chory 2008). MRR-deficient Arabidopsis (mrrd) mutants were identified by their inability to induce luciferase activity driven by the AOX1a promoter in response to AA treatment; however, some of these mutants can still respond to MFA (Zarkovic, Anderson & Rhoads 2005; Rhoads & Subbaiah 2007). Six redox imbalance (rimb) mutants were isolated by screening an Arabidopsis reporter line expressing luciferase under the control of a 2-cystein peroxiredoxin A (2CPA) promoter showing reduced levels of several chloroplast antioxidant enzymes despite the higher oxidation status of the PQ, ascorbate and 2CPA pools under photooxidative conditions (Heiber et al. 2007). Recently, functional analysis of the Arabidopsis AOX1a promoter sequence identified a repressor B cis-acting element that is a target for ABI4, and AOX1a promoter activity was fully de-repressed in abi4 (Giraud et al. 2009). The abi4 mutants are insensitive to transcriptional derepression of AOX1a by the mitochondrial complex I inhibitor rotenone, indicating a role for ABI4 in redox regulation and mitochondria to nucleus retrograde signalling (Giraud et al. 2009). AOX1a redox-transcriptional repression was shown to be mediated by the ABI4 transcription factor (Giraud et al. 2009). This work placed ABI4 at the crossroads between mitochondrial and chloroplast retrograde signalling pathways and perhaps as a convergence point for mitochondria-plastid-nucleus coordination.

Role of the cytosol in the chloroplast and mitochondrial signalling pathways

Diffusion, leakage or active transport of ROS from the chloroplast or mitochondria under conditions such as HL intensities or increased temperatures can cause impairment of normal metabolism and even cell death. Interestingly, although 1O2 has a very short half-life of 200 ns, it was suggested that it might diffuse further from the site of its production (Fischer et al. 2007). ROS leakage as a form of signalling has also been regarded as retrograde signalling (Mullineaux & Karpinski 2002; Baier & Dietz 2005; Nott et al. 2006; Mullineaux 2009; Pfannschmidt et al. 2009). In this respect, the cytosolic ROS scavenging system could act as a buffer or a modifier of these signals between plastids or mitochondria and the nucleus.

Mutants deficient in cytosolic ascorbate peroxidase (APX) activities in Arabidopsis (Davletova et al. 2005; Rossel et al. 2006), or in cytosolic glutathione metabolism (Ball et al. 2004), highlight the need for cytosolic removal of ROS generated during photosynthesis under photo-oxidative stress conditions. Although knock-out APX1 plants exhibit increased sensitivity to photo-oxidative stress (Davletova et al. 2005; Miller et al. 2007), they acquired increased acclimation under hyper osmotic conditions (Miller et al. 2007), which could be exerted by a cytosolic (plastids or mitochondria) H2O2 signal transmitted to the nucleus.

Heat stress-induced apoptotic-like (AL) PCD in a green Arabidopsis cell line was amplified under conditions suppressing chloroplast development (i.e. dark, norflurazon or lincomycin), as well as addition of antioxidants (Doyle, Diamond & McCabe 2010). In a follow up to this work, Doyle & McCabe (2010) recently reported that application of catalase suppressed this type of AL-PCD, suggesting a complex mode of regulation that may involve cross-talk between plastid-derived ROS signals, and apoplastic and even cytosolic H2O2 signals in the regulation of AL-PCD.

Photorespiratory generation of H2O2 in peroxisomes can induce oxidative stress that would activate PCD under long day and HL stress if not controlled by catalase activity (Chaouch et al. 2010; Mhamdi et al. 2010). Introduction of a second mutation to catalase-deficient Arabidopsis plants can modify PCD, causing H2O2 photorespiratory signal and enhanced resistance of the double mutants to pathogens under long daylight (Chaouch et al. 2010; Mhamdi et al. 2010). Most recently, Vanderauwera et al. (2011) reported that the introduction of a mutated cytosolic APX1 into the catalase2 mutant background reversed the HL- and thermo-sensitivity of the catalase mutant and enhanced the oxidative stress tolerance of the double mutant. These results showed that acclimation to conditions of HL was dependent on an interaction between a photorespiration-derived ROS signal and a cytosolic ROS signal.

Because ROS are proposed to function in signalling between the different organelles and the nucleus, the cytosol should be considered as a hub where cross-talk between divergent ROS signals occurs. Therefore, the role of the cytosolic ROS metabolic systems in modifying or fine-tuning and further relaying these signals should be better defined.

CONSIDERING A ROLE FOR ORGANELLAR MOVEMENT IN ROS SIGNALLING AND STRESS ACCLIMATION

When we consider intracellular signalling or metabolic networks, we often imagine the signal, or metabolite, as being delivered from one location to another along a chain or a cascade until it reaches its target destination. We usually do not take into account the fact that organelle movement and positioning relative to the cell interior is also affected by environmental cues and that this movement could be an important factor in cell signalling, influencing the specificity of the response.

The ability of organelles to move inside the cell is an important feature of the normal function of cells and their homeostasis that is also affected by the energetic balance within the cell and its physiological state. Movement of organelles is essential for their ability to interact among their own kind as well with other types of cellular compartments and has been reported to be affected by environmental stimuli such as drought, salinity, light, nutrient deficiency, temperature and mechanical stress (Britz 1979; Nagai 1993; Wada, Kagawa & Sato 2003; Islam, Niwa & Takagi 2009). However, there is little information about the effects of environmental stresses on regulation of organelle movement. More so, the reports drawing a relationship among ROS generation, or the redox state of the major producers of cellular ROS, chloroplast, mitochondria and peroxisome, and their movement and positioning are scarce and mostly related to cell death (Zhang & Xing 2008; Bi et al. 2009; Rodríguez-Serrano et al. 2009). The importance of organelle movement in maintaining normal ROS homeostasis is illustrated in rice seedlings deficient in the blue light receptor PHOT1a that regulates movement of chloroplasts, which led to increased H2O2 accumulation in leaves, reduced activity of ROS scavengers in the chloroplast, decrease in photosynthesis and reduced growth under control light conditions (Goh et al. 2009).

The chloroplast, mitochondria, peroxisomes and cytosol are closely linked through shared metabolic pathways including amino acid and sugar metabolism, photorespiration, the malate-oxaloacetate shuttle, fatty acid β-oxidation and more (Baker et al. 2006; Noguchi & Yoshida 2008; Nunes-Nesi et al. 2008; Dinakar et al. 2010), which require a high degree of cooperation and coordination in order to maintain normal cellular homeostasis, energy balance and redox state. For these types of metabolic connections to be efficiently executed, physical association between these organelles and their movement might be critical. For example, mitochondrial imaging frequently shows close proximity between mitochondria and chloroplasts. This was assumed to facilitate exchange of metabolites and respiratory gases (Gardeström 1996).

Choloroplast movement

The most extensively analysed type of organelle movement in a variety of plants, algae, ferns and mosses is the chloroplast in response to light stimuli (Islam et al. 2009). Chloroplasts can gather together at an illuminated area of the cell to maximize light absorption and photosynthesis (known as accumulation response), or on the other hand, can escape from HL intensity to avoid photo-damage (known as the avoidance response; Wada et al. 2003). Chloroplast light avoidance response or accumulation response depends on light availability and quality, and denotes chloroplast movement away from light towards the plasma membrane or towards light, respectively. These two opposite responses are dependent on two photoreceptors that also function in the light-induced stomata opening, phototropin1 (PHOT1) and phototropin2 (PHOT2) (Kinoshita et al. 2001; Wada et al. 2003). In Arabidopsis, under low fluence rate, both PHOT1 and PHOT2 control the accumulation response, whereas at high fluence rate, PHOT2 switches its function and mediates the avoidance response (Wada et al. 2003). In contrast, in several cryptogam species, both blue and red light mediate chloroplast movement (Wada et al. 2003). Chloroplast relocation is necessary for plants growing under fluctuating light conditions and is an efficient way to protect the photosystem (PS) and avoid photo damages (Wada et al. 2003).

CO2 diffusion through leaf tissue may be one of the most important physiological factors influencing chloroplast movement and positioning (Walczak & Gabrys 1980). Chloroplasts tend to physically interact with the plasma membrane to maximize the diffusion of CO2 from the intercellular airspace to the stroma, the site of fixation (Evans & von Caemmerer 1996; Terashima et al. 2006).

In addition to phototropins, zeaxanthin might also constitute part of the light perception systems. In response to strong blue light, chloroplast avoidance was accompanied by accumulation and cis-trans isomerization of zeaxanthin (Tlałka, Runquist & Fricker 1999). Interestingly, ascorbic acid enhanced chloroplast movement as well as zeaxanthin production under dark conditions. Ascorbic acid is essential to reduce the epoxy group of violaxanthin through the function of violaxanthin de-epoxidase (VDE) in zeaxanthin synthesis, and both ascorbic acid and VDE were shown to be required for non-photochemical quenching under HL (Müller, Li & Niyogi 2001; Müller-Moulé, Conklin & Niyogi 2002). Taken together, these results suggest a possible link between chloroplast movement, antioxidants and ROS metabolism via the zeaxanthin synthesis pathway.

Mitochondrial movement

There is an unequal distribution of the mitochondrial genome, whereby only a small percentage of the mitochondria of diploid cells contain the full mitochondrial genome (Logan 2010). This was suggested as one mean by which plant mtDNA could be protected from oxidative damage (Logan 2006), especially given that mtDNA has been demonstrated to be more susceptible to ROS damage than nuclear DNA (Mandavilli, Santos & Van Houten 2002). It was therefore hypothesized that a division of labour among mitochondria is needed to balance between two opposing demands; ATP synthesis on the one hand and minimizing ROS damage on the other hand. With different population serving each role, mitochondria with lower DNA content could be more bioenergetically active without direct risk of ROS-induced mtDNA damage, whereas those with higher mtDNA contents act as genetic vaults (Logan 2010).

Paraquat and H2O2 induce a change in mitochondrial morphology in Arabidopsis leaf epidermal cells and mesophyll protoplasts. The effect of the metabolic status of the mitochondrion on mitochondrial morphology and motility has been suggested to help ensure that the mitochondria are located where they are needed (Logan 2006). Furthermore, in animal cells, it was recently hypothesized that pathways regulating mitochondrial movement are mediated by microtubles and can move mitochondria to regions better suited for their physiological state; for example, by moving them from a region exposed to oxidative damage to a more protected location (Cox & Spradling 2009).

Interactions between organelles

Peroxules are thin peroxisomal extensions of a transient nature that can extend over the surface of the chloroplast and curve around it in a dynamically very rapid (seconds) manner connecting with other peroxisomes (Sinclair et al. 2009). Peroxisome morphology and motility can change under conditions that promote production of hydroxyl radicals, which induce a rapid switch between globular motile organelles with extended tubular-beaded shape and immotile peroxisomes with extended peroxules (Sinclair et al. 2009).

In chloroplasts, stromules are stroma-filled tubules comprising thin extensions of the stroma surrounded by the double envelope membrane (Hanson & Sattarzadeh 2008). Stromules can often connect together different chloroplast bodies and other cellular structures, and were observed to penetrate into grooves of the nucleus (Kwok & Hanson 2004).

Chloroplasts, mitochondria and peroxisomes have high rates of ROS metabolism that fluctuate in response to developmental and environmental cues. It is therefore reasonable to assume that their intracellular distribution and their relative position with respect to each other, as well as other cellular structures at a given time, could influence ROS signalling and its signatures. Close association between chloroplast, mitochondria and peroxisomes could increase cellular metabolic coordination under stress conditions and contribute to plant stress acclimation, as was reported by Rivero, Shulaev & Blumwald (2009) for transgenic tobacco plants experiencing water deficit. In this work, transgenic tobacco plants with induced accumulation of cytokinins displayed improved water efficiency and drought tolerance that was associated with a tighter association between mitochondria, chloroplasts and peroxisomes, as well as increased catalase abundance (Rivero et al. 2009). In another study, accumulation of peroxisomes and mitochondria at the penetration site of a fungus has been observed, presumably to scavenge ROS production at the infected site of the fungus Erysiphe cichoracearum (Koh et al. 2005).

Changes in the spatial arrangement of chloroplast, mitochondria and peroxisome in the plant cell at a given time are subjected to patterns dictated by environmental constraints, and could hypothetically influence the perception of ROS and redox signals generated by these organelles. It is conceivable to postulate that this kind of added level of complexity in the ROS signalling network would also contribute to specificity of the signal's nature. It would be challenging to address this issue in the future by combining dynamic real-time imaging, with molecular biology and plant stress biology.

SYSTEMIC SIGNALLING IN RESPONSE TO HL

The involvement of chloroplast redox regulation or cellular ROS signalling in the activation of systemic signals that respond to abiotic stress cues, primarily HL (or excessive excitation energy), has gained increasing attention over the last few years (Rossel et al. 2007; Miller et al. 2009; Szechyńska-Hebda et al. 2010).

More than a decade ago, Karpinski et al. (1999) described the induction of acclimation mechanisms to HL intensity, termed systemic acquired acclimation (SAA), which depends on the PQ pool redox state and was mediated by the generation of H2O2. There is a great deal of overlap between pathogenesis-related defence and response to intense light conditions (Mullineaux et al. 2000; Mullineaux & Baker 2010; Straus et al. 2010), although SAA is independent of salisylic acid (SA) and jasmonic acid accumulation and perception (Rossel et al. 2007). Accordingly, HL-acclimated systemic leaves showed higher tolerance to pathogen infection, indicating a cross-talk between light acclimation and pathogen responses (Karpinski et al. 2003; Griebel & Zeier 2008; Mühlenbock et al. 2008).

Induction of cell death by reduced PQ pools is associated with production of ROS and ethylene, as well as increased expression of ascorbate peroxidase 2 (APX2) and pathogenesis-related 1 (PR1) in systemic tissues (Mullineaux & Baker 2010; Straus et al. 2010). Enhanced disease susceptibility 1 (EDS1) that is required for systemic resistance against virulent pathogens and SA production controls cell death propagation in response to chloroplastic O2 signal (Mateo et al. 2004; Straus et al. 2010). Szechyńska-Hebda et al. (2010) demonstrated that systemic signalling in response to HL is regulated by plasma membrane electric signals, called photo-electrophysiological signaling (PEPS). Expression of APX1 and APX2 in systemic leaves was enhanced by PEPS that was activated in a light wavelength-specific manner. PEPS was shown to be regulated by changes in the redox state of PSII, non-photochemical quenching and GSH metabolism under HL condition. PEPS propagation speed and localization are dependent on the function of APX2. Interestingly, intact vascular tissues are required for PEPS-dependent systemic induction of APX1 and APX2. Previously, APX2 was shown to be exclusively expressed in the bundle sheath cell layer (Fryer et al. 2003) where H2O2 accumulation was specifically observed in response to HL (Mullineaux, Karpinski & Baker 2006). These results suggest that PEPS that is propagated through bundle sheath cells may change the cellular redox status and ROS signalling in systemic leaves, and regulate light acclimation system.

The potential involvement of electric signals was also suggested in the RbohD-dependent rapid systemic signalling pathway activated by wounding, heat stress, salinity, cold or HL (Miller et al. 2009). This type of systemic signalling required accumulation of H2O2 in the extracellular spaces of cells in an auto-propagating manner, suggesting the existence of a ROS wave that propagates through the entire plant.

Systemic activation of cold stress responses was recently demonstrated, and included activation of cold responsive genes such as CBF1, CBF2 and RCI2A. Although the involvement of ROS or redox regulation was not reported (Gorsuch et al. 2010), the transcriptional activation of Zat10 and Zat12, which are highly sensitive to oxidative conditions in addition to the recently reported cold-induced systemic induction of Zat12 expression (Rossel et al. 2007; Miller et al. 2009), hints to the involvement of ROS signalling in systemic acclimation to cold.

Recently, we have provided evidence that the NADPH-oxidase homolog RbohD is required for initiation and propagation of a rapid systemic signal(s) that travels at the rate of approximately 8 cm per minute (Miller et al. 2009).

An intimate relationship exists between redox signals, ROS production and activation of systemic signals associated with acclimation responses to abiotic stresses (Karpinski et al. 1999; Mullineaux et al. 2006; Mühlenbock et al. 2008; Szechyńska-Hebda et al. 2010). As it unfolds from the pioneering work of Karpinski et al. (1999), and following studies, the chloroplast plays a central role in redox/ROS-mediated systemic signalling and acclimation responses.

It is tempting to speculate that upon exposure of plants to HL, close interactions of the chloroplast with the plasma membrane would mediate direct cross-talk between the oxidative burst and photosynthesis-derived ROS signals that in turn could facilitate or at least influence the systemic signals triggered under these conditions.

CONCLUDING REMARKS

Networks of ROS/redox signalling in the chloroplast and mitochondria play essential roles in the acclimation of plants to abiotic stresses. These signals contribute to a delicate balance of homeostasis within each organelle, as well as to cross-talk between different cellular components by regulating important biological pathways such as gene expression, energy metabolism and protein phosphorylation under stress conditions.

Signals from chloroplast and mitochondria are involved in signal transduction pathways in the cell in response to different stimuli (Fig. 1a). Redox/ROS signalling from chloroplasts might also be one of the key regulators for signal transductions in the cell because it regulates both retrograde signalling and systemic signalling under stress conditions. On the other hand, the function of mitochondrial signalling is less understood compared with that of chloroplasts despite its contributions to the regulation of retrograde signalling. To elucidate the role of mitochondria in these signal transduction pathways, key regulators need to be identified in future studies. Changes in redox state in the chloroplast control systemic signalling via activation of electric signals on plasma membrane under HL conditions, and activation of these signals, are accompanied by ROS generation (Szechyńska-Hebda et al. 2010). RbohD was shown to be required for ROS-dependent signal propagation in response to abiotic stresses, such as HL, heat and wounding (Fig. 1a,b; Miller et al. 2009). There is a great overlap in the regulatory mechanisms between systemic responses dependent on RbohD and chloroplast redox signalling, suggesting a cross-talk between these mechanisms (Fig. 1). However, previous studies did not provide direct evidences of interaction between these responses. Integration of RbohD-dependent systemic signalling with electric signals on the plasma membrane, redox signals from the chloroplast, as well as signal specificity should be addressed in future studies.

Figure 1.

Local and systemic signal transduction pathways in plant cells during abiotic stress. (a) A hypothetical model for local and systemic abiotic stress-response signal transduction in plants. RbohD is required for rapid signal propagation from cell to cell in response to different environmental stimuli, such as HL, heat or wounding, and H2O2 generated by dismutation of superoxide is required for this signal transduction (Miller et al. 2010). Under abiotic stress conditions, changes in the redox state of PSII and the PQ pools in the chloroplast activate electric signalling at the plasma membrane involved in systemic responses (Szechyńska-Hebda et al. 2010). Calcium signalling is required for both mechanisms, and the electric signal that is dependent upon the chloroplast redox state is also accompanied by ROS generation, implicating a cross-talk between the RbohD-dependent signal, electric signals on the plasma membrane and ROS\redox signalling from chloroplast. Mitochondrial retrograde signalling (Rhoads & Subbaiah 2007; Pogson et al. 2008; Woodson & Chory 2008) could also play a key role in these responses as part of a cross-talk network that senses different metabolic/environmental states and activates rapid systemic signalling. (b) Imaging of rapid systemic signal transduction in Arabidopsis using a luciferase reporter gene expressed under the control of the Zat12 promoter in response to abiotic stresses (Miller et al. 2009). In response to exposure of local tissues to HL, Zat12 : Luc was activated in tissues that were not directly subjected to stress stimuli (systemic tissues). HL, high light; PQ, plastquinone; PSII, photosystem II; ROS, reactive oxygen species.

ACKNOWLEDGMENTS

Supported by funding from The National Science Foundation (IBN-0420033, NSF-0431327, IOS-0639964 and IOS-0743954), The Nevada Agricultural Experimental Station, NIH Grant Number P20 RR-016464 from the INBRE Program of the National Center for Research Resources, and EU grant FP7 – MARRIE CURIE 447.

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