Signaling by Superoxide and Hydrogen Peroxide in Cyanobacteria
Various mechanisms are involved in the signal function of ROS. At first, ROS-induced modifications of proteins can lead to changes of either structure or activity or both, in particular via oxidation of thiol groups.
Illustrative examples are the suppression of CO2 fixation and the blockage of the elongation factor EF-G in cyanobacteria and iron- containing clusters in enzymes (Spadaro et al., 2010). The oxidation of EF-G represents a rather general signaling scheme. Such a reaction chain as represented by the EF-G oxidation can be understood as a chemical inactivation process that is switched on and off by the oxidative potential. High oxidative potential in cyanobacteria leads to the oxidation of the two residues 105Cys and 242Cys in EF-G, and subsequent formation of a disulfide bridge between the two cysteine residues blocks the elongation of translation (Kojima et al., 2007). Replacement of these conserved cysteine residues by serine makes EF-G insensitive to ROS (Kojima et al., 2009). The mechanism of translation blockage under the influence of oxidative stress via post-translational redox regulation of the elongation factor state is a universal way of cell protection against ROS. Thus, EF-G is a primary target for ROS action and a key regulator of the translation efficiency (Nishiyama et al., 2011; Murata et al., 2012). This H2O2-induced blockage of the translation machinery interrupts the repair of photodamaged PS II, thus eventually leading to the disappearance of PS II and, consequently, the interruption of the linear electron transport chain. Studies on the effect of other stress factors (heat, drought, salinity) on photoinhibition have shown that the suppression of PS II repair determines the PS II sensitivity of cyanobacteria to environmental conditions (Allakhverdiev and Murata, 2004; Murata et al., 2007, 2012; Nishiyama et al., 2011).
The two Cys residues oxidatively linked to an S-S bridge by H2O2 are highly conserved in EF-G of cyanobacteria and of chloroplasts in algae and higher plants. Therefore, it seems very likely that ROS induces similar effects in the chloroplasts of plant cells. The translation of the D1 protein in chloroplasts is also regulated by redox components at both initiation and elongation steps (Zhang et al., 2000). A marked difference to cyanobacterial D1 is the possibility to phosphorylate D1 in plants. This property permits a regulation process of the circadian rhythm of degradation and metabolism. In this context, it is important to know the extent of how ROS affect the phosphorylation pattern of D1 in plants. This question remains to be answered in future studies.
Depending on the lifetime, different types of ROS molecules can either directly act as signal molecules or generate signal chains by formation of oxidation products.
It must be emphasized that fundamental differences exist between prokaryotic cyanobacteria and eukaryotic plants. In cyanobacteria, the photosynthetic and respiratory electron transport reactions take place in the intracytoplasmic (thylakoids) and cytoplasmic membrane, respectively, and close interactions exist between both photosynthesis and respiration (Peschek, 2008). On the other hand, eukaryotic plant cells contain semi-autonomous organelles (chloroplasts, mitochondria, peroxisomes, nucleus) with specific functional activities. This differentiation requires a more complex signaling system for “cross-talk” between these organelles. As a consequence, the mechanisms of “handling” stress-induced ROS and the modes of protection are markedly different between cyanobacteria and plants, and even within the plant kingdom. Therefore, a generalized and unified scheme cannot be presented at the moment and only selected characteristic examples of signaling are presented.
In cyanobacteria and plants, the O 2 radical is assumed to be predominantly produced at the acceptor side of PS I (Asada, 1999). The lifetime of O 2 is mainly determined by the presence of SOD and does not exceed a few microseconds in cells (Gechev et al., 2006). The signaling function of O 2 has been investigated by analyses of gene expression using DNA microarrays (Scarpeci et al., 2008) and studies on O 2 accumulation in plants deficient in Cu/Zn-SOD (Rizhsky et al., 2003). The results are in favor of a signaling role of this radical but details of the pathway(s) are not well known today.
O 2 can react with NO under formation of peroxinitrite. This species is likely to be synthesized in chloroplasts, where it can fulfill signaling functions (Foyer and Shigeoka, 2011). Under normal pH conditions, O 2 is deprotonated in animal cells (pH = 7.4 in blood cells) due to its pKa value of 4.8. However, at sufficiently low pH values (e.g. sometimes existing in the thylakoid lumen, see Joliot and Joliot, 2005), O2 anion radicals become protonated and the neutral hydroperoxyl radical (HO2*) can cross membranes (Sagi and Fluhr, 2006) (see chapter 3.1). The formation of H2O2 occurs mainly via the formation of O 2 followed by SOD-catalyzed dismutation (Asada, 1999, 2006) and in the process of photorespiration (Foyer and Noctor, 2009). H2O2 is markedly less reactive than :AgO2 (Halliwell and Gutteridge, 1985) (see chap. 3.1) and characterized by a much longer lifetime in the order of 1 ms (Henzler and Steudle, 2000; Gechev and Hille, 2005). Therefore, H2O2 is a most promising candidate to function as an intra- and intercellular messenger (Vranova et al., 2002; Hung et al., 2005; Bienert et al., 2007; Foyer and Shigeoka, 2011; Mittler et al., 2011). Numerous results on H2O2
signaling were reported for both, prokaryotic cyanobacteria and eukaryotic plants.
Eubacteria, including cyanobacteria, actively use characteristic two- component systems of signal perception and transduction (Kreslavski et al., 2013a).
Such two-component regulatory systems are typically composed of a sensory histidine kinase (Hik) and a response regulator form the central core of the phosphate signaling system in cyanobacteria (Los et al., 2010; Kreslavski et al., 2013a). The sensory histidine kinase perceives changes in the environment with its sensory domain. A subsequent change of its conformation often leads to autophosphorylation of the conservative histidine residue in a Hik from a donor ATP molecule from which a phosphate group is then transferred to the conserved aspartate in a receiver domain of the response regulator protein (RRP). After phosphorylation, the RRP also changes its conformation and gains (positive regulation) or loses (negative regulation) the ability to bind to DNA. The RRP usually binds the promoter region(s) of genes for proteins that are involved in the stress signal network or are linked to stress protection pathways (Kreslavski et al., 2013 a).
Hik33 of Synechocystis is the multisensory protein, which perceives cold, salt, and oxidative stresses. The mechanisms by which Hik33 recognizes the stresses are still not fully clear. It is assumed that changes in the physical mobility of membrane lipids and changes in the surface charge on the membrane, associated with changing mobility, are activators for Hik33. Activation may be also caused by depolarization of the cytoplasmic membrane upon cold stress or due to changes in charge density of the membrane surface under stress (Nazarenko et al., 2003; Kreslavski et al., 2013a).
Sensory histidine kinases are also important for the functioning of genes involved in photosynthesis and/or regulated by high light intensity. Experiments with the Synechocystis mutant deficient in Hik33 (this mutant is also named DspA, see Hsiao et al., 2004) revealed that low or moderate light intensity causes retardation in growth and decrease in photosynthetic oxygen evolution in mutant cells, compared to wild-type cells, under photoautotrophic conditions. The addition of glucose neutralized these differences. However, mutant cells were more sensitive to light intensity and quickly died under strong light (Hsiao et al., 2004; Kreslavski et al., 2013a).
The defense of bacteria against oxidative stress and adaptive regulation mechanisms have been thoroughly analyzed in Escherichia (E.) coli and Bacillus (B.) subtilis. In eubacteria (heterotrophic, autotrophic, and chemotrophic), two global regulators, OxyR and PerR, are involved in the control of gene transcription induced by H2O2 addition (Zheng et al., 1998) (see Figure 69). Both these regulators have active thiol groups and can directly recognize changes in the redox state of the cytoplasm. The ferric uptake repressor (Fur) type protein PerR was found to be the central regulator of inducible stress response (Herbig and Helmann, 2001; Mongkolsuk and Helmann, 2002). In the cyanobacterium Synechocystis sp. PCC 6803, a gene (srl1738) encoding a protein similar to PerR was identified as being induced by H2O2 (Li et al., 2004) in methylviologen treated cells upon illumination (Kobayashi et al., 2004). It was concluded that the Fur-type protein Slr 1738 functions as a regulator in inducing the potent antioxidant gene sZZ1621, which encodes for a peroxiredoxin.
The regulator OxyR is absent in Synechocystis sp. PCC 6803. Studies on Synechocystis sp. PCC 6803 incubated for 20 min with 0.25 mM H2O2 proved how several histidine kinases can serve as H2O2 sensors (Kreslavski et al., 2013a). Mutations of genes Hik34, Hik16, Hik41, and Hik33 encoding histidine kinases led to blockage of the H2O2-induced gene expression (Zheng et al., 1998; Kanesaki 2007). Peroxidases were found to control 26 of 77 genes induced by H2O2. The histidine kinase Hik34 was shown to regulate the expression of the gene htpG under oxidative stress. This kinase was characterized as regulator of gene expression due to heat (Suzuki et al., 2005), salt, and hyperosmotic stress (Shoumskaya et al., 2005). In addition, Hik34 is subjected to autoregulation in the presence of H2O2. The pair of histidine kinases Hik16-Hik41 regulates the genes sll0967 and sll0939 with unknown functions not only in response to H2O2 but also under salinity and hyperosmotic stress. Hik33 controls 22 genes; among them are ndhD2 encoding NADH dehydrogenase, three hli (high-light-inducible) genes, pgr5 encoding ferredoxin-plastoquinone reductase, the genes nblA1 and nblA2 involved in phycobilisome degradation, and others. It should be noted that ROS induce also the expression of the genes hspA, dnaJ, dnaK2, clpB1, ctpA and sigB. These genes were activated by mechanisms without the involvement of histidine kinases, although Hik34 is acting as repressor of genes encoding heat shock proteins (Zheng et al., 1998; Kanesaki et al., 2002; Suzuki et al., 2005; Los et al., 2010).
In addition to histidine kinases, the transcription factor PerR participates in the response of Synechocystis sp. PCC 6803 to ROS. PerR is involved in the regulation of only six genes, of which four encode proteins with unknown function. Based on evidence for both PerR and Hik33 being components in the control of the induction of nbl gene expression due to oxidative stress, it seems possible that PerR interacts with a two-component regulatory system (Zheng et al., 1998; Kanesaki et al., 2002; Suzuki et al., 2005; Los et al., 2010).