**2.3.3 Other stress-response programs**

The accumulation of excitation energy produced when the rate of absorption of photons exceeds the rate of utilization of excitation energy in photosynthetic electron transport leads to an accumulation of reduced electron acceptors that eventually produce excited states of chlorophyll (i.e. triplet state). This process is presumed to predominantly occur in the PSII reaction center where quenching by carotenoids is less effective. Triplet state chlorophyll readily reacts with oxygen to give rise to singlet oxygen, a highly destructive excited oxygen species causing photo-oxidations (Triantaphylidés & Havaux, 2009) (Figure 1). Superoxide, H2O2 (produced via reduction or dismutation of superoxide) and hydroxyl radicals, all of which are more reactive than ground state triplet O2, can also be produced by numerous

Friend or Foe? Exploring the Factors that Determine the Difference

distinct pathways (Laloi et al., 2006; op den Camp et al., 2003).

Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 165

Identifying ROS specific signaling pathways leading to changes in nuclear gene expression is hampered by the fact that several chemically distinct ROS are generated simultaneously during stress within the plastid compartment. This problem was partially solved by the generation of the *flu* mutant of Arabidopsis that generates singlet oxygen in plastids in a controlled and non-invasive manner (Meskauskiene et al., 2001; op den Camp et al., 2003). Thus, this mutant accumulates excess protochlorophyllide in the dark that, upon illumination, acts as a photosensitizer capable of generating singlet oxygen (Flors & Nonell, 2006; Hideg et al., 2006; op den Camp et al., 2003). Light induced generation of singlet oxygen has revealed a rapid change in nuclear gene expression that differs substantially from nuclear gene expression profiles activated by superoxide or H2O2, further supporting the proposal that superoxide/H2O2- and singlet oxygen-dependent signaling occur via

The high reactivity of singlet oxygen, together with its unlikely ability to leave the plastid compartment, suggested that its physiological impact depended on the generation of more stable second messengers within the plastid, which were assumed to activate a signaling cascade outside of the plastid compartment. Two components of singlet oxygen signaling, the EXECUTER 1 and 2 proteins localized in the chloroplast, were recently identified as additional signaling components of singlet oxygen (Wagner et al., 2004; Lee et al., 2007) (Figure 1). However, the mechanisms involved in singlet oxygen sensing and signal transduction to the nucleus remain to be characterized, although experimental evidence supporting a positive role for abscisic acid (ABA), ethylene-, salicylic acid (SA)- and JAdependent signaling pathways in the singlet oxygen- induced response was recently reported (Danon et al., 2005; Ochsenbein et al., 2006). In addition, approximately 50 genes encoding putative transcription factors have been identified to be rapidly and strongly induced within 30 min of the release of singlet oxygen. These include ethylene responsive factors, WRKY transcription factors, zinc-finger proteins and several DNA-binding proteins. Many genes involved in putative signal-transduction pathways and calcium regulation, such as protein kinases, calcium and calmodulin-binding proteins, were also identified

(Danon et al., 2005; Laloi et al., 2006; Lee et al., 2007; op den Camp et al., 2003).

the state of chloroplasts to the nucleus.

As mentioned above, H2O2 activates a response program different from singlet oxygen which is also more stable. Its experimentally proven capacity to diffuse across the chloroplast envelope is believed to be a pivotal step in a model that involves an H2O2 dependent activation of a mitogen-activated protein kinase (MAPK) cascade in the cytosol that subsequently affects gene expression in the nucleus (Kovtun et al., 2000; Vranova et al., 2002; Apel & Hirt, 2004; Mittler et al., 2004) (Figure 1). However, the polar nature of H2O2, which would be expected to limit its capacity to diffuse across hydrophobic membranes unaided, has been a strong argument used to question this model. Alternatively, it has been proposed that H2O2 transport is mediated by aquaporin channels (Bienert et al., 2007; Dynowski et al., 2008). This proposal, is supported by the hypersensitivity to H2O2 observed in yeast cells expressing Arabidopsis aquaporins in the plasma membrane, but has yet to be demonstrated in plants. Another nebulous aspect of the model is the mechanism by which H2O2, produced in multiple cell sites and in response to various different stresses and stimuli, acquires the specificity needed to act as a reliable signal conveying information on

Further experimental support for the presence of independent redox signaling pathways acting via differentiated signaling cascades came from the characterization of the so called

pathways in photosynthetic cells. It is generally accepted that PSI is the major site of superoxide generation in the photosynthetic electron transport (PET) chain (Asada, 2006) (Figure 1). In addition to production linked to PET and respiratory electron transport (RET) chains, the photorespiration pathway is a major producer of H2O2 (Peterhansel et al., 2010). Photorespiration is due to the oxygenase activity of ribulose-1, 5-bisphosphate carboxylaseoxygenase (Rubisco), which produces 2-phosphoglycolate. This small molecule is metabolized through a sequence of reactions that includes H2O2 production by glycolate oxidase. Thus, the implementation of protective/metabolizing systems to prevent the deleterious effects of ROS accumulation in plants, are essential to maintain the process of photosynthesis in the oxygen-rich atmosphere of this planet.

The perturbation of the equilibrium between ROS production and scavenging that is frequently produced in plants under stress, can result in a transient increase in ROS levels that is closely associated with the emergence of various disorders such as cell death, disease, and aging (Neill et al., 2002; Overmyer et al., 2003). ROS exert this effect either by reacting with, and irreversibly damaging, a large variety of bio-molecules or by altering the expression of genes that affect signal transduction pathways in a highly selective, specific, and sometimes antagonistic, manner (Apel & Hirt, 2004; Danon et al., 2005; Gadjev et al., 2006; Laloi et al., 2007; Lee et al., 2007). Strong evidence suggesting that H2O2 either directly or indirectly antagonizes singlet-oxygen-mediated signaling was obtained recently using a ingenious experimental approach in which Arabidopsis *flu* mutant plants, which generate singlet oxygen in plastids during a dark-to-light transition (see below), were found to produce a more intense stress responses when the H2O2 concentration was reduced noninvasively by the over-expression of a thylakoid ascorbate peroxidase (Murgia et al., 2004; Laloi et al., 2007). In addition, low molecular weight antioxidants (e.g., ascorbate, glutathione) serve not only to limit the lifetime of the ROS signals but also participate in an extensive range of other redox signaling and regulatory functions (Foyer & Noctor, 2009).

Considering the above, ROS are considered to be primary diffusible and reactive mediators of signaling linked to electron transport status. For instance, singlet oxygen was considered for many years as a highly toxic molecule with very limited diffusion. However, the utilization of specific probes capable of detecting singlet oxygen in the aqueous phase of isolated thylakoid suspensions and the cytoplasm of high light stressed cells of *C. reinhardtii*, strongly suggested that singlet oxygen can diffuse significant distances from its site of production to activate specific gene expression, such as the nuclear-encoded glutathione peroxidase homolog GPXH (Fisher et al., 2007). However, the physiological relevance of these findings remains questionable considering that the fraction of mobile singlet oxygen was probably small, was detectable only at very high light intensities and has been observed only in this species. H2O2 is also recognized as an important signaling molecule. Its role as a signal conveyor was reinforced by data, generated using spin trapping electron paramagnetic resonance spectroscopy and H2O2-sensitive fluorescence dyes, that showed that up to 5% of the total H2O2 produced inside the chloroplasts was able to diffuse out of the chloroplasts, and in the process evade the effective antioxidant systems located inside this organelle. Moreover, H2O2 diffusion was shown to increase concomitantly with light intensity and time of illumination (Mubarakshina et al., 2010). Additional observations have suggested that glutathione, whose synthesis is affected by changes in photosynthesis, may also act as a plastid signal that controls expression of stress defense genes in the nucleus (Wachter et al., 2005; Mullineaux & Rausch, 2005; Rausch et al., 2007).

pathways in photosynthetic cells. It is generally accepted that PSI is the major site of superoxide generation in the photosynthetic electron transport (PET) chain (Asada, 2006) (Figure 1). In addition to production linked to PET and respiratory electron transport (RET) chains, the photorespiration pathway is a major producer of H2O2 (Peterhansel et al., 2010). Photorespiration is due to the oxygenase activity of ribulose-1, 5-bisphosphate carboxylaseoxygenase (Rubisco), which produces 2-phosphoglycolate. This small molecule is metabolized through a sequence of reactions that includes H2O2 production by glycolate oxidase. Thus, the implementation of protective/metabolizing systems to prevent the deleterious effects of ROS accumulation in plants, are essential to maintain the process of

The perturbation of the equilibrium between ROS production and scavenging that is frequently produced in plants under stress, can result in a transient increase in ROS levels that is closely associated with the emergence of various disorders such as cell death, disease, and aging (Neill et al., 2002; Overmyer et al., 2003). ROS exert this effect either by reacting with, and irreversibly damaging, a large variety of bio-molecules or by altering the expression of genes that affect signal transduction pathways in a highly selective, specific, and sometimes antagonistic, manner (Apel & Hirt, 2004; Danon et al., 2005; Gadjev et al., 2006; Laloi et al., 2007; Lee et al., 2007). Strong evidence suggesting that H2O2 either directly or indirectly antagonizes singlet-oxygen-mediated signaling was obtained recently using a ingenious experimental approach in which Arabidopsis *flu* mutant plants, which generate singlet oxygen in plastids during a dark-to-light transition (see below), were found to produce a more intense stress responses when the H2O2 concentration was reduced noninvasively by the over-expression of a thylakoid ascorbate peroxidase (Murgia et al., 2004; Laloi et al., 2007). In addition, low molecular weight antioxidants (e.g., ascorbate, glutathione) serve not only to limit the lifetime of the ROS signals but also participate in an extensive range of other redox signaling and regulatory functions (Foyer & Noctor, 2009). Considering the above, ROS are considered to be primary diffusible and reactive mediators of signaling linked to electron transport status. For instance, singlet oxygen was considered for many years as a highly toxic molecule with very limited diffusion. However, the utilization of specific probes capable of detecting singlet oxygen in the aqueous phase of isolated thylakoid suspensions and the cytoplasm of high light stressed cells of *C. reinhardtii*, strongly suggested that singlet oxygen can diffuse significant distances from its site of production to activate specific gene expression, such as the nuclear-encoded glutathione peroxidase homolog GPXH (Fisher et al., 2007). However, the physiological relevance of these findings remains questionable considering that the fraction of mobile singlet oxygen was probably small, was detectable only at very high light intensities and has been observed only in this species. H2O2 is also recognized as an important signaling molecule. Its role as a signal conveyor was reinforced by data, generated using spin trapping electron paramagnetic resonance spectroscopy and H2O2-sensitive fluorescence dyes, that showed that up to 5% of the total H2O2 produced inside the chloroplasts was able to diffuse out of the chloroplasts, and in the process evade the effective antioxidant systems located inside this organelle. Moreover, H2O2 diffusion was shown to increase concomitantly with light intensity and time of illumination (Mubarakshina et al., 2010). Additional observations have suggested that glutathione, whose synthesis is affected by changes in photosynthesis, may also act as a plastid signal that controls expression of stress defense genes in the nucleus

photosynthesis in the oxygen-rich atmosphere of this planet.

(Wachter et al., 2005; Mullineaux & Rausch, 2005; Rausch et al., 2007).

Identifying ROS specific signaling pathways leading to changes in nuclear gene expression is hampered by the fact that several chemically distinct ROS are generated simultaneously during stress within the plastid compartment. This problem was partially solved by the generation of the *flu* mutant of Arabidopsis that generates singlet oxygen in plastids in a controlled and non-invasive manner (Meskauskiene et al., 2001; op den Camp et al., 2003). Thus, this mutant accumulates excess protochlorophyllide in the dark that, upon illumination, acts as a photosensitizer capable of generating singlet oxygen (Flors & Nonell, 2006; Hideg et al., 2006; op den Camp et al., 2003). Light induced generation of singlet oxygen has revealed a rapid change in nuclear gene expression that differs substantially from nuclear gene expression profiles activated by superoxide or H2O2, further supporting the proposal that superoxide/H2O2- and singlet oxygen-dependent signaling occur via distinct pathways (Laloi et al., 2006; op den Camp et al., 2003).

The high reactivity of singlet oxygen, together with its unlikely ability to leave the plastid compartment, suggested that its physiological impact depended on the generation of more stable second messengers within the plastid, which were assumed to activate a signaling cascade outside of the plastid compartment. Two components of singlet oxygen signaling, the EXECUTER 1 and 2 proteins localized in the chloroplast, were recently identified as additional signaling components of singlet oxygen (Wagner et al., 2004; Lee et al., 2007) (Figure 1). However, the mechanisms involved in singlet oxygen sensing and signal transduction to the nucleus remain to be characterized, although experimental evidence supporting a positive role for abscisic acid (ABA), ethylene-, salicylic acid (SA)- and JAdependent signaling pathways in the singlet oxygen- induced response was recently reported (Danon et al., 2005; Ochsenbein et al., 2006). In addition, approximately 50 genes encoding putative transcription factors have been identified to be rapidly and strongly induced within 30 min of the release of singlet oxygen. These include ethylene responsive factors, WRKY transcription factors, zinc-finger proteins and several DNA-binding proteins. Many genes involved in putative signal-transduction pathways and calcium regulation, such as protein kinases, calcium and calmodulin-binding proteins, were also identified (Danon et al., 2005; Laloi et al., 2006; Lee et al., 2007; op den Camp et al., 2003).

As mentioned above, H2O2 activates a response program different from singlet oxygen which is also more stable. Its experimentally proven capacity to diffuse across the chloroplast envelope is believed to be a pivotal step in a model that involves an H2O2 dependent activation of a mitogen-activated protein kinase (MAPK) cascade in the cytosol that subsequently affects gene expression in the nucleus (Kovtun et al., 2000; Vranova et al., 2002; Apel & Hirt, 2004; Mittler et al., 2004) (Figure 1). However, the polar nature of H2O2, which would be expected to limit its capacity to diffuse across hydrophobic membranes unaided, has been a strong argument used to question this model. Alternatively, it has been proposed that H2O2 transport is mediated by aquaporin channels (Bienert et al., 2007; Dynowski et al., 2008). This proposal, is supported by the hypersensitivity to H2O2 observed in yeast cells expressing Arabidopsis aquaporins in the plasma membrane, but has yet to be demonstrated in plants. Another nebulous aspect of the model is the mechanism by which H2O2, produced in multiple cell sites and in response to various different stresses and stimuli, acquires the specificity needed to act as a reliable signal conveying information on the state of chloroplasts to the nucleus.

Further experimental support for the presence of independent redox signaling pathways acting via differentiated signaling cascades came from the characterization of the so called

Friend or Foe? Exploring the Factors that Determine the Difference

proposed role played by metabolite exchange in redox signaling .

ABA resulting from a depressed carotenoid biosynthesis (Kleine et al., 2009).

Fig. 1. Proposed plastid signal transduction pathways (redox, *metabolic* and ABAdependent) involved in the regulation of acclimation responses to stress, including state transitions, long-term responses (LTR) and/or the activation/ repression of specific target genes in the chloroplast and nucleus. Redox signals are generated within the electron transport chain (purple) or by generation of reactive oxygen species (ROS) (black). The plastoquinone pool (PQ/PQH2, in purple letters) is the origin for at least two redox

Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 167

cytosol, but that could also export both ATP and NADPH from chloroplasts into the cytosol (Figure 1). Interestingly, the altered nuclear gene expression detected in the *tpt* (Biehl et al., 2005) and *cue1* mutants, the latter affected in the phosphoenolpyruvate/phosphate translocator (PPT) in the inner chloroplast envelope (Streatfield et al., 1999) supports the

Finally, the role of ABA as a signal relying information on the chloroplast status to the nucleus can be explained by evoking the multiple effects that photosynthetic activity rates have on the biosynthetic ABA pathway that is partially localized in the chloroplast (Baier & Dietz, 2005). For instance, oxidative stress conditions leading to increased ABA through inductive effects on the synthesis of its xanthophyll precursor in the chloroplast, could provide a link between the redox state and ROS levels in the plastid and gene expression in the nucleus. In this respect, it has been speculated that the repressed photosynthetic gene expression produced by norflurazon treatment might be associated with reduced levels of

*redox-imbalanced* (*rimb*) mutants, which were detected using an Arabidopsis reporter gene line expressing luciferase under control of the redox-sensitive 2-cysteine peroxiredoxin A (2CPA) promoter (Heiber et al., 2007). Valuable information shedding light on the nature of redox signaling should be expected when the identity of the RIMB genes and their biochemical function is determined.
