Signaling by Singlet Oxygen and Hydrogen Peroxide in Eukaryotic Cells and Plants
In plants cells, ^gO2 is known to function predominantly as a plastid ROS signal which activates nuclear gene expression (Li et al., 2012). Because of its high reactivity, :AgO2 has a very short lifetime in cells (about 200 ns, see Gorman and Rodgers, 1992), other sources report times down to 70 ns (Prasad et al., 2015). For a report on markedly longer lifetime in cells, see (Skovsen et al., 2005). The short lifetime leads to a rather limited diffusion radius. However it seems as if the diffusion radius is about 10 nm, especially as longer lifetime is reported in Lipid membranes and therefore singlet oxygen is able to cross cell membranes (Schmitt et al., 2014a). However, for long range signaling, the involvement of additional components is necessary as the diffusion radius of :AgO2 is surely limiting. An important pathway is signal transfer from the site of formation within the chloroplast through the cytosol to the nucleus, which is termed chloroplast-to-nucleus signaling (Kreslavski et al., 2012b, Schmitt et al., 2014a).
Generally like in cyanobacteria, so also in plants, two types of fundamentally different responses to :AgO2 stress are known: i) development of increased tolerance and ii) induction of programmed cell death.
A :AgO2 signaling pathway in C.reinhardtii was shown to give rise to gene expression that leads to increased tolerance to ROS (acclimation). This phenomenon comprises enhanced expression of genes for ROS protection and detoxification, e.g. of a glutathione peroxidase-homologous gene (gpxh/gpsx), and also the expression of a Д-class glutathione-S- transferase gene (gsts) greatly increases (Fischer et al., 2012). The effect on components participating in signaling was analyzed in a ^gO2-resis- tant mutant (SOR 1). The results obtained revealed the involvement of reactive electrophilic species that are formed by ^gO2-induced lipid peroxidation (Fischer et al., 2012). It was found that the SOR1 gene encodes a leucine zipper transcription factor, which controls the expression of numerous genes of stress response and detoxification. It was inferred from these results that reactive electrophilic species play a key signaling role in acclimation of C.reinhardtii cells to :AgO2 stress (Kreslavski et al., 2012b).
In many cases, 1AgO2 signaling induces programmed cell death, in particular under biotic stress. Much information on the genetic control of this phenomenon has been gathered from investigations on the A. thali- ana mutant flul which is defective in the feedback control of the Chl biosynthesis pathway. This mutant, which accumulates the photosensitizer protochlorophyllide in the dark, generates 1AgO2 within the first minute of illumination after a dark-to-light shift (op den Camp et al., 2003). The 1AgO2 formation taking place in the vicinity of the thylakoid membrane (Przybyla et al., 2008) can be manipulated by altering the degree of light exposure and the preceding dark period. In contrast to wild-type plants, the 1AgO2 production in flul is not associated with excess excitation of PSII (Mullineaux and Baker, 2010). The studies on the flul mutant revealed that :AgO2 can trigger the activation of programmed cell death and that two chloroplast-located proteins, EXECUTER1 and 2 (EXE1 and EXE2), control this process (Przybyla et al., 2008; Wagner et al., 2004; Lee et al., 2007) (see Figure 71). EXE1 and EXE2 act as suppressors (Wagner et al., 2004; Lee et al., 2007), but their mode of function in signaling of the 1AgO2-induced programmed cell death is not yet resolved.
As a consequence of the special mode of 1AgO2 formation in the flul mutant, a cell death in their leaves can be induced either due to direct oxidative destruction (necrosis) under a large excess of ROS or at a slower rate of 1AgO2 formation via signaling the activation of a programmed cell death pathway.
On the basis of data obtained on C.reinhardtii cells, 1AgO2 was inferred to be able to leave chloroplasts directly into the cytosol and even to reach the nucleus, thereby inducing the expression of nuclear gene gpxh, which encodes glutathione peroxidase (Fischer et al., 2007). Since the fraction of “mobile” 1AgO2 is extremely small, a direct effect of 1AgO2 was manifested only under high light and so far observed only in cells of this microalga (Fischer et al., 2007). It appears much more likely that oxidation products of special molecules are formed, which act as second signal messengers and are transferred via the cytosol and to the nucleus. This idea is confirmed by experimental data obtained on both, cells of the unicellular green alga C.reinhardtii and multicellular leaves of the higher plant A.thaliana. In the latter, plant 6-cyclocitral was shown to be formed by the oxidation of 6-carotene under ROS stress and identified as a stress signal that acts as a second messenger in 1AgO2 signaling (Ramel et al., 2012). Likewise, oxidation of polyunsaturated fatty acids due to interac?tion with :AgO2 in the lipid fraction of thylakoid membranes leads to formation of reactive electrophilic species, which are able to exit into the cytosol (Galvez-Valdivieso and Mullineaux, 2010). Via autocatalytic cascades, lipoperoxide radicals can result in generation of :AgO2 in the cytosol (Flors et al., 2006) and trigger the EXE1/EXE2-mediated pathway of programmed cell death (Wagner et al., 2004).
The enzymatic peroxidation of lipids is catalyzed by lipoxygenases. These enzymes play an essential role in response to pathogen infection and wounding (Feussner and Wasternack, 2002; Overmyer and Brosche, 2003; Hoeberichts and Woltering, 2003). Specific lipoxygenase pathways lead to formation of lipoxide species which are likely to be different when induced by chemically different ROS like :AgO2 versus O 2/H2O2. Studies on the flul mutant of A. thaliana revealed that 70 genes are up-regulated by :AgO2 but not by O 2 /H2O2, the latter being formed at PS I via the methylviologen mediation reaction (op den Camp et al., 2003).
The signaling pathway(s) of :AgO2 leading to cell apoptosis tightly in- teract(s) with other signaling pathways involving hormones and other ROS. The 1AgO2 species activates the signaling pathways controlled by salicylic and jasmonic acid resulting in changes of the expression of numerous genes which are related to anti-stress defense systems. An example of regulatory interaction is the decrease in cell injury and death induced by :AgO2 due to its conversion into H2O2 (Laloi et al., 2007).
This effect is possible because the cell is able to scavenge 1AgO2 via the increase of the amount of lipid-soluble antioxidants and also the acceleration of reduction of photodamaged D1 protein in the PS II reaction center. This pathway counteracts cell apoptosis along the EXE1 and EXE2 pathways (Mullineaux and Baker 2010).
H2O2 stress in plants induces the expression of many chloroplast and nuclear genes (Figure 68) (Foyer and Noctor, 2009; Bechtold et al., 2008). It was found that several genes are down-regulated, while others are up- regulated (Vandenabeele et al., 2004). In particular, H2O2 was shown to activate several genes encoding antioxidant and signaling proteins: ascorbate peroxidase (APX), glutathione reductase, catalase, mitogen- activated protein kinase (MAPK), and phosphatases (see Figure 54, Figure 68, Figure 71) (Mullineaux et al., 2000; Vranova et al., 2002). H2O2 of chloroplast origin can serve as a redox signal which triggers the expression of the gene encoding cytoplasmic APX2 (Davletova et al., 2005). Likewise H2O2 of extracellular/plasmamembrane origin has been shown to be important for APX2 expression (Bechtold et al., 2008; Galvez- Valdivieso et al., 2009). Furthermore, H2O2 is also involved in inducing the expression of some light-responsive genes.
Figure 68. Cellular and physiological processes regulated by H2O2.
Plant treatment with H2O2 stimulates the expression of the gene APX2 and of genes ZAT10 and ZAT12, which encode transcription factors (Davletova et al., 2005; Rossel et al., 2007). Both factors ZAT10 and ZAT12 mediate different subsets of the high light-inducible or - repressible gene set, including genes coding for APX2 and APX1, respectively.
It has been suggested that H2O2 molecules produced in the chloro- plasts can exit the organelles by diffusion, likely via water channels (aquaporins), and induce signaling processes in the cytoplasm (Mubarakshina et al., 2010), i.e. triggering the MAPK cascade (Pfannschmidt et al., 2009), by which nuclear genes are activated in the cell, in particular the gene encoding cytoplasmic APX (Apel and Hirt, 2004; Yabuta et al., 2004; Vranova et al., 2002). H2O2, produced on the cytoplasmic membrane or in the apoplast, can also play a signaling role, possibly by functioning together with abscisic acid (Bechtold et al., 2008; Yabuta et al., 2004).
A new genetic approach for analyzing the signaling effect of H2O2 in plants has been reported (Maruta et al., 2012). This method is based on chemically inducible RNAi. It has been shown that silencing the expression of ascorbate peroxidase bound to the thylakoid membrane (t-APX) in A. thaliana leaves leads to both, an increase of the fraction of oxidized proteins in chloroplasts and to effects on the expression of a large set of genes. Among these, the transcription levels of a central regulator of cold acclimation are suppressed and the levels of salicylic acid (SA) increase together with the response to SA. The results reveal synergistic and antagonistic effects of H2O2, when chloroplasts are exposed to high light.
Another striking feature is the finding that growth of A.thaliana plants under short-day illumination gives rise to a diminished expression of several genes which are involved in sensing and hormone synthesis (Thimm et al., 2004). It was found that the level of ROS production is higher by a factor of about two in leaves from short-day (8 h light) tobacco than in leaves from long-day (16 h light) plants. Based on these results, an unknown regulatory protein was proposed to exist which changes the relative extent of cyclic and pseudo-cyclic photosynthetic electron transport, thereby affecting the ROS content in chloroplasts (Michelet and Krieger-Liszkay, 2012). These findings suggest that light sensor(s) participate in this phenomenon.
The expression of ROS sensitive genes was shown to depend on diurnal and circadian conditions, thus illustrating a role of the biological clock in transcriptional regulation of these genes. Likewise H2O2, generation and scavenging exhibit a diurnal rhythm. These findings indicate that an important functional relation exists between ROS signaling and circadian output, which provides a mechanistic link for plant response to oxidative stress (Lai et al., 2012). The components involved and the underlying mechanism(s) of these mutual interactions of signal networks are not yet resolved and represent challenging topics for future research.