ROS, Calcium, and Electric Signals: Key Mediators of Rapid Systemic Signaling in Plants
The systemic response of plants to pathogen infection (systemic acquired resistance [SAR]), or wounding has been extensively studied with a network of numerous compounds and signals implicated (for review, see Dempsey and Klessig, 2012; Shah and Zeier, 2013). In recent years a new type of systemic response, termed systemic acquired acclimation (SAA), has emerged as an important acclimation response of plants to abiotic stresses (e.g. Karpiński et al., 1999; Szechyńska-Hebda et al., 2010; Suzuki et al., 2013). This response is characterized by a rapid spread of the systemic signal(s) that can reach the systemic tissue within minutes from the application of abiotic stress to a local tissue. A number of different signaling mechanisms were implicated in this response, including the reactive oxygen species (ROS) wave (Miller et al., 2009), the calcium (Ca2+) wave (Choi et al., 2014), and electric signals (Szechyńska-Hebda et al., 2010). In this review we will focus on recent findings regarding each of these signals, as well as their integration, and attempt to propose a model for the propagation of rapid systemic signals during SAA and SAR. Due to space limitations, we will not address many other important aspects of ROS signaling that have been covered by a number of recent excellent reviews (for review, see Foyer and Noctor, 2013; Vaahtera et al., 2014; Considine et al., 2015; Dietz, 2015; Mignolet-Spruyt et al., 2016). The ROS wave is an autopropagating wave of ROS production mediated via RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) activation in each cell along its systemic path (Fig. 1). It was initially discovered by Miller et al. (2009), and was extensively reviewed by Mittler et al. (2011), Gilroy et al. (2014), and Mittler and Blumwald (2015). It can reach rates of up to 8.4 cm/min and is directly linked to the calcium wave (for review, see Gilroy et al., 2014) and possibly to electric signals (Suzuki et al., 2013; Fig. 2). It is required for SAA, but by itself it likely does not convey specificity to the systemic response of plants to different abiotic stresses (Suzuki et al., 2013; for review, see Mittler et al., 2011). The ROS wave is currently thought to be integrated with additional metabolic/signaling pathways and to enable rapid SAA responses and acclimation of plants, improving their overall fitness (Suzuki et al., 2013; for review, see Mittler et al., 2011; Mittler and Blumwald, 2015). Activation of the ROS wave by a local heat stress was shown, for example, to enhance the acclimation of systemic tissues to heat stress, and to be regulated by temporal and special interactions with ABA signaling (Suzuki et al., 2013; for review, see Mittler and Blumwald, 2015). In addition, local application of high light resulted in the activation of a ROS wave that enabled systemic tissues to withstand light stress, and was accompanied by the accumulation of photorespiratory amino acids, including Gly and Ser, in nonstressed systemic tissues (Suzuki et al., 2013). Integration of the ROS, Ca2+, and electric waves in and between cells via the function of RBOH proteins and superoxide dismutases (SODs; yellow), Ca2+-dependent protein kinases (CPK/CBL-CIPKs; green), calcium channels such as TPC1 and H2O2-activated plasma membrane calcium channels (red), and GLRs and/or plasma membrane H2O2 channels (blue). Activation of RBOHD is shown to be mediated by CPK/CBL-CIPKs, Cys-rich receptor kinases (CRKs), or directly by Ca2+. Activation of Ca2+ channels is shown to be mediated by H2O2 or by calcium via calcium-induced calcium release. Activation of GLRs is proposed to be mediated by H2O2 and/or calcium levels via calcium-induced calcium release. The level of ROS in cells is proposed to be regulated by NO-ROS and NO-RBOH interactions, retrograde signaling, and ROS removal/production in the chloroplast (Cp), mitochondria (Mt), and peroxisomes (Px). The regulation of gene expression in the nuclei is shown to be mediated via redox/ROS changes, LESION SIMULATING DISEASE1 (LSD), ENHANCED DISEASE SUSCEPTIBILITY1 (EDS), MITOGEN-ACTIVATED PROTEIN KINASEs (MPKs), WRKY, and RRTF1. The electric signal is depicted as a sinus-like wave that travels along the plasma membrane and through the plasmodesmata (PD). Copropagation of the ROS, calcium, hydraulic, and electric waves during rapid systemic signaling. The ROS wave is shown as a series of red arrows, the calcium wave is shown as a dashed green arrow, the hydraulic wave is shown as a dotted blue arrow, and the electric wave is shown as a dotted black arrow. Different sections along the path of the signal (yellow arrows) are also shown to have alternating levels of NPQ and ROS/APX1 levels, and JA is shown to accumulate in cells along the systemic path. The local tissue is shown to have alterations in ROS, calcium, and membrane depolarization potential, and the systemic tissue is shown to have accumulation of ROS and abscisic acid (ABA). Black arrows indicate accumulation or suppression in the level of a particular chemical or transcript/protein, and dashed, dotted, and wide red arrows indicate direction of the signal. The integration of different signaling pathways with ROS-dependent systemic responses has been extensively studied during pathogen-induced SAR, and research focusing on phloem-mobile SAR signals have identified several biologically active molecules, including methyl salicylate, a glycerol-3-P (G3P) derivative, a lipid-transfer protein (DIR1), azelaic acid (AzA), dehydroabietinal, jasmonic acid (JA), and pipecolic acid (Návarová et al., 2012; for review, see Dempsey and Klessig, 2012; Shah and Zeier, 2013). Among these, methyl salicylate, AzA, dehydroabietinal, and G3P were shown to induce SAR when applied to local tissues (Kachroo and Robin, 2013), and JA was shown to rapidly accumulate throughout the path of the systemic signal (Glauser et al., 2009). Recent studies demonstrated that the synthesis of these molecules could be regulated by ROS. Therefore, Arabidopsis (Arabidopsis thaliana) plants deficient in RBOHD or RBOHF showed lower accumulation of AzA and G3P, and exogenous application of G3P was able to rescue SAR in these plants (for review, see Wang et al., 2014). These findings could suggest that the ROS wave is also activated during SAR. Some of the most recent findings in SAR that may shed more light on how the ROS wave is mediated during SAA come from recent studies that focused on the interdependence of ROS signaling on nitric oxide (NO). The noa1/nia1 double mutant, deficient in NO accumulation, showed, for example, a fully compromised SAR accompanied by lower ROS accumulation in systemic tissues, that could be rescued by exogenous application of H2O2 (for review, see Wang et al., 2014). In addition, RBOHD was shown to be up-regulated through an NO-dependent process elicited by oligogalacturonides in response to pathogen attack (Rasul et al., 2012). Although these findings indicated that ROS could act downstream to NO in the SAR pathway, Arabidopsis deficient in RBOHD or RBOHF were unable to accumulate NO in response to pathogen attack (for review, see Wang et al., 2014; Wendehenne et al., 2014), suggesting that NO and ROS could operate in a feedback loop (Fig. 1). NO can react with reduced glutathione, by an S-nitrosylation reaction, to form S-nitrosoglutathione (GSNO; for review, see Considine et al., 2015; Del Río, 2015), and GSNO could function as a pool of NO ready to be used in ROS-NO interactions during SAR/SAA systemic signaling (Fig. 1). ROS, NO, and GSNO function as master-switches regulating various processes, including the SAR pathway and stomatal responses (for review, see Wang et al., 2014; Mittler and Blumwald, 2015; Considine et al., 2015). NO-dependent redox-based posttranslational modifications, such as addition of a glutathione to a protein Cys thiol in a glutathionylation reaction or a NO moiety to form a S-nitrosothiol, were shown to act as regulators of different processes (for review, see Zaffagnini et al., 2012; Considine et al., 2015), and these could also function during SAR or SAA to amplify or dampen the signal. In contrast to the up-regulation of RBOHD through an NO-dependent process enhanced by oligogalacturonides (Rasul et al., 2012), S-nitrosylation of RBOHD at Cys-890 was found to inhibit its enzymatic activity by impeding FAD binding (Yun et al., 2011), indicating a possible dual role for NO in the regulation of RBOHD. NO-mediated S-nitrosylation was also shown to inhibit ROS scavenging enzymes such as catalase and ascorbate peroxidase (Ortega-Galisteo et al., 2012; de Pinto et al., 2013), suggesting that NO could also promote an increase in H2O2 by reducing its decay (Suzuki et al., 2013). Moreover, glutaredoxin S12 in the chloroplast and Gly decarboxylase in the mitochondria were shown to be targets of glutathionylation, and these posttranslational modifications might also contribute to the regulation of ROS levels in cells (Palmieri et al., 2010; for review, see Zaffagnini et al., 2012). Although the involvement of NO in SAA has not been thoroughly studied, the regulation of RBOHD via NO signaling could implicate NO as a key player in modulating the ROS wave (Fig. 1). Furthermore, peroxisomes, mitochondria, and chloroplasts, known as sources of ROS, were also shown to generate NO that might regulate signal transduction involved in various biological processes (for review, see Del Río, 2015). Recent studies uncovered a possible integration between a hub of regulatory genes involved in programmed cell death, retrograde signaling, and hormone regulation, and ROS-dependent systemic responses to high light stress. Thus, genes such as LESION SIMULATING DISEASE1, ENHANCED DISEASE SUSCEPTIBILITY1, PHYTOALEXIN DEFICIENT4, ETHYLENE INSENSITIVE2, and MITOGEN-ACTIVATED PROTEIN KINASE 4 were shown to play important roles in the simultaneous regulation of SAA via ROS- and salicylic acid-dependent pathways (Mühlenbock et al., 2008; Szechyńska-Hebda et al., 2010; Wituszyńska et al., 2013; Gawroński et al., 2014; for review, see Karpiński et al., 2013; Mignolet-Spruyt et al., 2016). In addition, PHOTOSYSTEM II SUBUNIT S-dependent local and systemic wave-like regulation of nonphotochemical quenching (NPQ) and chlorophyll fluorescence decay time, important dissipation and quenching mechanisms of light absorbed in excess, were also proposed to be dependent on ROS and salicylic acid signaling during SAA (Szechyńska-Hebda et al., 2010; Gawroński et al., 2013, 2014; Ciszak et al., 2015; Fig. 2). Although a possible cross talk between ROS and retrograde signaling involving some of the genes indicated above has been proposed, and could explain how the systemic signal is integrated with redox and photosynthesis/respiration control, further studies are required to uncover how these pathways and the RBOHD-dependent ROS wave are coordinated during SAA and possibly SAR (for review, see Mignolet-Spruyt et al., 2016). For example, it is not clear if retrograde signaling functions downstream to rapid systemic signaling, or whether it is directly involved in attenuating or amplifying the signal. A recent study demonstrated that REDOX RESPONSIVE TRANSCRIPTION FACTOR1 (RRTF1) might function as a key regulator of systemic ROS-dependent responses during high light stress (Matsuo et al., 2015). RRTF1-dependent systemic signaling was shown to be regulated by WRKY transcription factors (Matsuo et al., 2015), whose expression is enhanced during RBOHD-dependent SAA (Miller et al., 2009), but its rate of systemic response is slower than that of the ROS wave. RRTF1 could therefore play a role in the regulation of gene expression downstream to the ROS wave during SAA (Fig. 1). Taken together, SAA might be regulated by a complex network linking NO, retrograde signaling, and RBOH-dependent ROS signaling that could function to amplify or attenuate the systemic signal (Fig. 1). Further studies are of course required to determine the mode of integration of NO and ROS signals in SAA that could be a key aspect of systemic signaling during abiotic stress. Along with ROS, changes in cytosolic Ca2+ have also been linked to rapid, systemic signaling activity throughout the plant. Calcium is a ubiquitous regulator involved in a wide of processes and responses to and abiotic stresses (for review, see et al., 2010; and 2014). Ca2+ through has been proposed as of this Ca2+-dependent response network et al., in addition to this for a of Ca2+ signals, is also for the systemic spread of a cytosolic Ca2+ This Ca2+ to local of such as wounding or stress to responses and 2). Thus, local stress has been shown to a cytosolic Ca2+ increase that from of local in and in Arabidopsis (Choi et al., 2014; et al., 2014). This Ca2+ through the and cell in the (Choi et al., 2014), but to spread more the et al., 2014). directly with cell can mechanisms to in the and cell wounding and signals et al., 2013; et al., 2014) that are likely to a Ca2+ wave et al., that from to with at of the tissues used for of the signal et al., 2014). signaling in the has been linked to responses (for review, see et al., 2016). of Ca2+ channels at the and to cells has also been proposed as a key player in systemic propagation of Ca2+ linked to these responses et al., also see in et al., 2014). Although the channels involved in the processes above at the level a recent the role of the and channels have to shed some light on mechanisms (Fig. 1). Thus, propagation of and Ca2+ waves are dependent on the by TPC1 (Choi et al., 2014; et al., 2015). in the of this et al., et al., 2011; et al., also to up-regulation of a hormone linked to systemic responses (e.g. et al., the that the regulation of this might be linked to these of systemic response The of a TPC1 Ca2+ response to a of and abiotic stresses when at the level et al., is with a role for this as a systemic of Ca2+ Thus, the spread of Ca2+ increase is in the in response to local and Ca2+ are et al., 2014) or et al., 2015). TPC1 also as an of the complex of interactions we can to contribute to and/or these systemic propagation Thus, in addition to its Ca2+ TPC1 on its cytosolic may be in Ca2+ et al., 2011; et al., and and and cytosolic Ca2+ levels are thought to activity through on the et al., for review, see and 2011). In addition, roles for a of other regulatory likely on the from (e.g. and see also in and and ROS et al., 2009), to et al., 2012), proteins et al., and an of interactions that (for review, see and 2011). The of these regulatory mechanisms may in activity a cytosolic signal increase it to a to rapidly amplify the signal. whether of these other regulators an important role in modulating the Ca2+ wave has to be In addition, TPC1 is to a of including Ca2+ (e.g. and for review, see and 2011; 2011), whether it directly Ca2+ or by modulating other to be In addition to the GLRs have also emerged as key in systemic signaling The GLRs are a of channels et al., 2013; for review, see et al., that linked to rapid signaling. Thus, in and attenuate systemic signaling et al., 2013; et al., 2014; 2016). has been to play a role in the spread of the signal the that the of channels to signal to some and to to the systemic signal can of the and Ca2+ signals does with between of et al., 2015). of likely between but it is of if for example, in response to of a indicating a more in its the spread of the Ca2+ increase in the of the by local of with in the of propagation et al., 2014). such in the pathways of within the or response between different an important to be more fully the double signaling from to it does not in the et al., the of the likely of the systemic signaling The GLRs have binding and are by a of amino (e.g. et al., 2008; et al., 2012). whether such amino acid an important role in their in propagation of systemic signals to be It is also important to that the role for these channels in processes such as regulation et al., and hormone signaling et al., may be on their in systemic signal and functions in signaling will of The of of Ca2+ and other systemic signals has also propagation a hydraulic signal through the could the signal (e.g. et al., 2014). In this a wave of that in act to an Ca2+ to cells or (Fig. This the cell for further and Although Ca2+ are linked to signaling in plants (for review, see and 2013), and an of likely such as et al., 2014), kinases et al., 2014), and the and channels (for review, see et al., have been the possible roles of such and channels in rapid systemic signaling have to be fully Integration of the different waves that rapid systemic signaling during are shown to the as well as a hydraulic wave that in the calcium wave via The calcium and ROS waves are shown to be linked via RBOH and the ROS and electric waves are shown to be linked through RBOHD and and the calcium and electric waves are shown to be linked via channels such as GLRs or The different waves are shown to the propagation of each other and to a response in the systemic tissue. further of proposed of the Ca2+ wave propagation from the of a role for ROS and the in signal These suggest a for propagation of a Ca2+ increase ROS by activation could channels Ca2+ at the plasma Ca2+ channels and in the plasma membrane have been identified at the (e.g. et al., et al., et al., and levels (e.g. the or et al., 2010; et al., 2014; Fig. to the channels to systemic signaling have not been such an Ca2+ has the cytosolic Ca2+ increase could be through a Ca2+ involving and a loop of RBOH activation further of Ca2+ and propagation to cells (Fig. 1). are known to be by Ca2+-dependent signaling directly through their et al., 2008; et al., and through posttranslational such as Ca2+-dependent protein activity (e.g. et al., 2012; et al., 2013; et al., 2013; et al., 2014; et al., 2014). In such a model ROS and Ca2+ to a whether Ca2+ or ROS of the other of a as the response of ROS production that are linked to ROS-dependent Ca2+ (Fig. 1). to ROS-dependent could a for of the to the wave-like of the Ca2+ changes from cell to For example, the ROS production has elicited rapid to the and the of responses in a slower of ROS to the cell (e.g. through et al., could to of important as et al., and of the signal in that The propagation to cells to a wave-like of the Ca2+ Further regulatory that could contribute to this increase and in Ca2+ are also at in the For example, have been to attenuate Ca2+ in the in et al., 2014). the known between Ca2+, stress response, and NO (for review, see et al., and the above of between ROS, NO, and signaling, of the interactions between these may be a to the systemic signal propagation and signals in plants are et al., by (e.g. and spread rapidly in various plants from a to several by (e.g. through the and regulated by hydraulic (for review, see and and that on the pool and et al., 2009). cell to signals can along the cell membrane likely via plasmodesmata (Fig. 1). between cells via plasmodesmata has been shown in species and Arabidopsis is known the of plasmodesmata that regulate signals is that the of a local on cell membrane could to depolarization of the cell membrane of an cell et al., In resistance be high for signals to than For signal throughout the the are more a pathway to their and plasma membrane In addition, channels that are in the plasma membrane of the are with propagation of by and abiotic stresses (for review, see et al., 2014). may also be involved in systemic signal for example, in wounding the in changes, to changes in the cells that membrane changes via et al., for review, see et al., 2014; et al., 2014). of the signal between and is thought to be mediated by cells (Szechyńska-Hebda et al., 2010; et al., 2013). signal propagation along the tissue in the of was shown by et al., 2014). the of the channels various of signals is currently it was that genes channels could signals et al., 2013; Fig. 1). numerous studies have shown a systemic of signals on various processes in The most are in and These on of et al., 2010). the elicited by are and it was discovered that are required for to be that is able to et al., 2016). additional of in is via regulation of the reaction and et al., 2011). of a was shown to that a of of II and et al., 2014). of on were found in other species such as and et al., 2013). In addition, studies of or pathogen attack indicated that signaling an important role in the of systemic such as the activation of various genes (for review, see and and including JA et al., 2013), and et al., 2010). it was demonstrated that chloroplast ROS, and NPQ play an important role in signaling in plants and Szechyńska-Hebda et al., 2010). signaling was also shown to generate a wave-like systemic in ROS, and and systemic changes in ROS and gene expression were found to be in a contrast to the waves of NPQ changes (Fig. Szechyńska-Hebda et al., 2010). Although changes in light or from light to was found to induce systemic signals that were by cytosolic Ca2+ and were light (Szechyńska-Hebda et al., 2010; for review, see et al., the involved in and regulating these waves during SAA at (Fig. 1). A number of different waves are proposed to be involved in rapid systemic signaling during SAA and SAR (Fig. and are some of the mechanisms these waves could be and regulate each other (Fig. The integration of the Ca2+ wave with the ROS wave could be mediated via the of such as kinases and RBOHD (Fig. 1). The recent of GLRs as of systemic electric signals et al., 2013; et al., 2014; and the findings that in the of RBOHD electric signaling is (Suzuki et al., could to a between ROS signaling and electric signaling mediated via GLRs and Fig. 1). GLRs could therefore be regulated by interactions and regulate Ca2+ or electric In addition, hydraulic waves that could at the local tissue to wounding (for review, see et al., 2014), rapid changes in stomatal and/or local stress (for review, see Mittler and Blumwald, 2015), could also be or integrated Ca2+ and ROS waves by that Ca2+ and/or ROS production (e.g. et al., 2009). A model could therefore the different waves (Fig. shown in local could directly Ca2+ or a hydraulic wave that be a Ca2+ signal via The Ca2+ wave in be integrated with the ROS wave via the activation of RBOH proteins by Ca2+ binding or RBOH that enhanced ROS production in will further or inhibit Ca2+ channels such as TPC1 and/or plasma membrane The ROS wave could be integrated with the electric wave via activation of GLRs or other channels that will membrane and regulate electric signals could of course further via Ca2+ The Ca2+ wave could be integrated with the electric wave via GLRs and TPC1 in a calcium-induced calcium The waves could therefore amplify and regulate each other and the systemic signal the to the systemic tissue (Fig. It be that the different waves the of an activated between and not the of a particular systemic from cell to the The ROS wave is mediated by the of an RBOH activation between the Ca2+ wave by the of a Ca2+ activation as through regulation of GLRs or and the electric signal is mediated by the of an activation or each of these processes likely a complex of regulators that each the activated cell along the path of the signal on therefore a particular of in response to a signal that is to it by the cells in the production of a signal that is to the cells it in the pathway (Fig. 1). The signal not be the and of the wave could feedback or the course a new activated is within the different cells along the pathway, each of these cells could be a particular or hormone (e.g. JA or et al., Suzuki et al., 2013), or a particular or that more to abiotic stress (e.g. the accumulation of Gly and Suzuki et al., 2013). This autopropagating wave signal therefore be more a of each other the the systemic tissue to a particular that is from cell to cell it the systemic It is possible that the integration of the different signals with to their and specificity to the or that an or signal is for a response in the systemic tissue (Suzuki et al., 2013; for review, see Mittler et al., 2011). Further studies are of course to this important systemic acquired resistance systemic acquired acclimation nitric oxide glycerol-3-P azelaic acid S-nitrosoglutathione nonphotochemical quenching