Life and death under salt stress: same players, different timing?

Journal of Experimental Botany, Jul 2014

Salinity does not only stress plants but also challenges human life and the economy by posing severe constraints upon agriculture. To understand salt adaptation strategies of plants, it is central to extend agricultural production to salt-affected soils. Despite high impact and intensive research, it has been difficult to dissect the plant responses to salt stress and to define the decisive key factors for the outcome of salinity signalling. To connect the rapidly accumulating data from different systems, treatments, and organization levels (whole-plant, cellular, and molecular), and to identify the appropriate correlations among them, a clear conceptual framework is required. Similar to other stress responses, the molecular nature of the signals evoked after the onset of salt stress seems to be general, as with that observed in response to many other stimuli, and should not be considered to confer specificity per se. The focus of the current review is therefore on the temporal patterns of signals conveyed by molecules such as Ca2+, H+, reactive oxygen species, abscisic acid, and jasmonate. We propose that the outcome of the salinity response (adaptation versus cell death) depends on the timing with which these signals appear and disappear. In this context, the often-neglected non-selective cation channels are relevant. We also propose that constraining a given signal is as important as its induction, as it is the temporal competence of signalling (signal on demand) that confers specificity.

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Life and death under salt stress: same players, different timing?

Journal of Experimental Botany Life and death under salt stress: same players, different timing? Ahmed Ismail 2 Shin Takeda 1 Peter Nick 0 0 Molecular Cell Biology, Botanical Institute, Karlsruhe Institute of Technology (KIT) , Germany 1 Bioscience and Biotechnology Center, Nagoya University, Chikusa , Nagoya 464-8601 , Japan 2 Department of Horticulture, Faculty of Agriculture, Damanhour University , Damanhour , Egypt Salinity does not only stress plants but also challenges human life and the economy by posing severe constraints upon agriculture. To understand salt adaptation strategies of plants, it is central to extend agricultural production to salt-affected soils. Despite high impact and intensive research, it has been difficult to dissect the plant responses to salt stress and to define the decisive key factors for the outcome of salinity signalling. To connect the rapidly accumulating data from different systems, treatments, and organization levels (whole-plant, cellular, and molecular), and to identify the appropriate correlations among them, a clear conceptual framework is required. Similar to other stress responses, the molecular nature of the signals evoked after the onset of salt stress seems to be general, as with that observed in response to many other stimuli, and should not be considered to confer specificity per se. The focus of the current review is therefore on the temporal patterns of signals conveyed by molecules such as Ca2+, H+, reactive oxygen species, abscisic acid, and jasmonate. We propose that the outcome of the salinity response (adaptation versus cell death) depends on the timing with which these signals appear and disappear. In this context, the oftenneglected non-selective cation channels are relevant. We also propose that constraining a given signal is as important as its induction, as it is the temporal competence of signalling (signal on demand) that confers specificity. ABA; adaptaion; calcium; cell death; cross-talk; jasmonate; salinity; signal on demand; proton influx; ROS - r eviw stress-dependent losses of crop yield are estimated to range between 65 and 87% (Buchanan et  al., 2002) . Hunger and malnutrition still represent the primary health risk, exceeding the impact of AIDS, malaria, and tuberculosis, and leaving 1 billion people in the world without enough food to be healthy (FAO, 2011) . This underlines the importance of plant stress for society. Salinity, in particular, has been a threat to agriculture in some areas in the world for more than 3000 years (Flowers, 2006) ; it affects more than 80 million ha of arable land worldwide (reviewed by Munns and Tester, 2008) , with estimated annual global costs equivalent to US$11 000 million in 2011 (FAO, 2011) . High salinity is commonly caused by high concentrations of sodium (Na+) and chloride (Cl–) ions in the soluble fraction of the soil resulting in both hyperionic and hyperosmotic conditions, which in turn impair the ability of plants to take up water and micronutrients. This not only leads to increased concentration of ions to levels that are toxic to plants but also causes degraded soil structure. Plants have acquired different adaptive mechanisms to control the negative impacts of salinity and are classified into two groups with respect to their adaptability to salinity: halophytes are efficient in adaptation and therefore are able to inhabit saline environments, whereas glycophytes cannot cope with saline soils and therefore are excluded from saline habitats (reviewed by Flowers et al., 1977; Hasegawa et al., 2000) . In this review, we present and discuss recent advances in our understanding of the mechanisms by which plants respond and adapt to salt stress. In particular, we focus on the temporal pattern of signals that are crucial for adaptation and the role of these temporal patterns for the orchestration of cross-talks between signalling pathways. We propose that the correct timing of these cross-talks decides which salinity-triggered signalling will culminate in successful adaptation. Salinity can trigger two qualitatively different modes of cellular response Salinity can challenge plants to a degree that may even lead to cell death. Salt stress causes membrane disorganization, metabolic toxicity, formation of reactive oxygen species (ROS), inhibition of photosynthesis, and reduced nutrient acquisition (reviewed by Hasegawa et al., 2000; Tuteja, 2007) . When stress intensity reaches non-permissive levels, these processes can culminate in cell death. Growth responses to salinity are comprised of two phases (Munns, 1993) . Rapid and often transient changes in growth occur within minutes and are attributed to the osmotic effects of salt ions in the rhizosphere. Hormonal signals originating from the roots are assumed to regulate the growth reduction during this phase. The second phase of growth reduction is the result of a salt-specific effect and needs some time (days, weeks, or months) to develop. This second phase is not a mere consequence of water stress alone (Munns, 2002) . Cellular damage in this second phase is due to salt accumulation in transpiring leaves, leading to levels that exceed the ability of the cells to sequester salts into the vacuole (Munns, 2002; reviewed by Läuchli and Grattan, 2007; Munns, 1993, 2005) . It should be kept in mind that the resulting cell death may be deleterious for the individual cell but adaptive for the plant as a whole, as plants can dump ions to toxic levels in their older leaves and then remove the salt by simple abscission (Munns and Tester, 2008) . Although the ionic effects of salinity stress occur later than the osmotic effects, influx of Na+ has been observed from very early time points after the onset of salt stress. For maize—an extreme glycophyte—Na+ ions accumulate in chloroplasts within 4 h, even preceding any changes of water potential in the challenged leaves (De Costa et al., 2007; Zörb et al., 2009) . As we will discuss in this review, this early influx of Na+ might act as a signal to trigger salinity adaptation and thus would not be a mere manifestation of cellular toxicity leading to cell death. Na+ might play a dual role: as an early signal triggering ‘salinity signalling’ (which can result in successful adaptation), and as a late noxious factor that, upon accumulation, will lead to cell death. It is thus possible to order the complex salinitytriggered events in terms of a two-mode model for the cellular response. As we will elaborate in the following section, the relationship between (adaptive) salt signalling and (destructive) salt accumulation depends on the timing of the events triggered by salinity stress. Salinity signals: same inputs, different outputs Conceptual framework: temporal signatures define response quality Plants respond to salinity challenges at the level of both cells and the whole organism. It is important to identify factors responsible for the adaptability to stress and to understand the underlying mechanisms connecting these factors to cellular signalling pathways in order to improve plant growth and productivity under stressful conditions. However, to dissect the biological function of the individual stress signals is difficult; the events involved in stress adaptation overlap, at least partially, with those accompanying stress-dependent cellular damage. In addition, in the case of salinity stress, plants experience two stress qualities at a similar time: osmotic and ionic stresses. What determines the fate of a cell under salinity stress? How can virtually the same signalling molecules cause adaptation in one plant but trigger cell death in another? Below, we propose and elaborate a model where not the molecular nature of signals but also their temporal signatures define the cellular response to salinity. The level of explanation will be explicitly cellular; for the sake of scientific reduction we do not consider the systemic level, although it is evident that the cellular events described below are integrated into interactions of different tissues and organs (readers are referred to the comprehensive review by Munns and Tester, 2008) . The central idea of this model is that the two response modes (adaptation versus cell death) depend on the relative timing of two signal chains: one triggered by calcium and the other triggered by oxidative burst in the apoplast (Fig.  1). A delay in generation and dissipation of a salinity-triggered calcium-dependent signal relative to a signal conveyed by ROS will lead in the unconstrained activation of jasmonate (JA) signalling culminating in cell death. In contrast, the same molecular signal carrier (calcium) can, if properly timed, initiate adaptive processes such as sequestration and extrusion of sodium, and induce efficient constraint of JA signalling through the activation of abscisic acid (ABA) signalling. In the following sections, we will consider, step by step, the details of the individual signalling events (Fig.  1). For each step, we will try to define: (i) by which events the signal is generated (referred to as the ‘on’ state), (ii) by which events the signal is dissipated (referred to as the ‘off ’ state), (iii) what is the appropriate target of the signal, and (iv) what is the inappropriate target of this signal in case of a delayed time signature. Non-selective cation channels (NSCCs): the earliest players but often overlooked Ions are selectively conducted through channels in the plasma-membrane channels depending on electrochemical potential. In plants, these ion fluxes are tightly controlled by the gating of these channels (reviewed by Yeo, 1998) . However, in saline conditions, a rapid influx of Na+ from the soluble phase of the soil into the cortical cytoplasm of plant roots occurs through NSCCs, and, later, through the highaffinity K+ transporter (HKT1) (Essah et al., 2003; reviewed by Tester and Davenport, 2003) . NSCCs have been classified according to their voltage dependence or to their responsiveness to certain ligands and physical stimuli, and their physiological roles under salinity have been described previously (reviewed by Demidchik and Maathuis, 2007; Kronzucker and Britto, 2011) . Hyperpolarization-activated (HA)-NSCCs, which are activated later and weakly selective for monovalent cations (Davenport and Tester 2000; reviewed by Demidchik et al., 2002) , might be the predominant type of channel in plasma membranes of sensitive species. In contrast, more efficient NSCCs [depolarization-activated (DA)-NSCCs and voltage-insensitive (VI)-NSCCs] might take the lead in tolerant species. This idea was tested for cells of grapevine, where otherwise very similar pairs of species can be compared, such as the salt-tolerant Vitis rupestris and the salt-sensitive Vitis riparia (Ismail et  al., 2012) .Vitis rupestris inhabits rocky, sunny slopes, and therefore has evolved a considerable osmotic tolerance. In contrast, V.  riparia occurs in alluvial woods and performs poorly under osmotic stress. In the drought-sensitive V.  riparia, sodium influx was observed to be slow, consistent with the hypothesis that HA-NSCCs might be the predominant type of channel (Ismail et al., 2014) . In contrast, the vigorous and rapid influx observed in V. rupestris indicates that the more efficient DA-NSCCs and/or VI-NSCCs are the major types of channel. Interestingly, the role of NSCCs and their kinetic activities not only determine the pattern of Na+ influx but also modulate the cytoplasmic signatures of two crucial signalling elements, Ca2+ and H+. In the sensitive V. riparia, gradual and low kinetics of Na+ influx were observed, while in the tolerant V.  rupestris, NSCCs catalysed a fast and strong Na+ influx reaching its maximal amplitude after only 2 min (the first time point measured). This initial and rapid Na+ uptake could serve as an calcium-dependent signalling from processes leading to ion sequestration/ extrusion towards processes constraining the ABA status, such that the parallel activation of the JA status will be released from ABA-dependent control. (This figure is available in colour at JXB online.) efficient signal to activate adaptation to the osmotic part of salinity (Fig. 2A, ① ; reviewed by Munns and Tester 2008) . Furthermore, the elevated intra- and extracellular Na+ are partially able to inhibit the K+ outward rectifiers and thereby prevent the loss of cellular K+, maintaining cellular K+/Na+ homeostasis (Shabala et al., 2006) . Thus, in the tolerant line, rapid influx of Na+ by the more effective NSCCs has advantageous effects, at least during the first phase of the salt stress response. The slower influx in sensitive cells will be less efficient in adjusting the water potential such that cells will lose water (Fig.  3, ①). These differences in sodium content are very short lived and therefore their connection with differential responses at the later stages must be conveyed by signals of adifferent molecular nature. Cells cannot tolerate the harmful effects of Na+ accumula- sodium entry is later and does not contribute to protective tion in the cytoplasm due to impaired enzyme activities and signalling. Conversely, in the Arabidopsis mutants hkt1-1 and cytotoxicity (ion-specific effects). To circumvent such toxicity, hkt1-2, Na+ entry was suppressed to some extent, but these cells exclude Na+ from the cytosol and/or sequester sodium mutants were found to be endowed with enhanced salinity inside the vacuole. To block additional influx of sodium, tolerance (Rus et al., 2001; reviewed by Schroeder et al., 2013) . VI-NSCCs are rapidly deactivated by cAMP or cGMP pro- To arrest further influx of sodium is not sufficient, howposed to be generated by turgor-sensitive membrane-located ever; ions have to be removed from the cytoplasm. The roles cyclases (Fig. 2B, ③), which results in improved plant salinity of the salt overly sensitive (SOS) pathway in expelling Na+ tolerance (Maathuis and Sanders, 2001). A  second route of out of the cell or caging it inside the vacuole via an SOS/NHX entry is the high-affinity K+ transporter HKT1. However, this interaction (Fig. 2B, ③) seems to be the central mechanism for Na+ exclusion (reviewed by Ji et al., 2013) , although the role of the SOS pathway for plant salt tolerance has been discussed controversially, as reviewed by Kronzucker and Britto (2011). It has been questioned whether extrusion of sodium through the plasma membrane by the SOS1 exporter represents an effective strategy, as Na+ ions will re-enter the cell though the NSCCs (if these were not deactivated by cAMP/cGMP) and HKT1 channels, respectively, and the accumulation of sodium in the apoplast would generate a more negative water potential such that the cell would lose additional water (reviewed by Shavrukov, 2013) . A  further caveat was found when the role of SOS1 in stress tolerance was examined at a whole-plant level: In Arabidopsis thaliana, SOS1 is expressed preferentially in the rhizodermis near to the root tip, i.e. in cells that are still weakly vacuolated, and in xylem parenchyma cells, from which Na+ is uploaded to xylem vessels for long-distance transport from the root to the shoot (Shi et  al., 2002; reviewed by Munns and Tester, 2008) . Mutants impaired in the expression of sos1 in both Arabidopsis (Shi et al., 2002) and tomato (Olías et al., 2009) accumulate more sodium in the shoots under salinity stress. Therefore, Na+ exclusion by SOS1 seems to be important as a mechanism by which cells get rid of excess Na+. However, if not accompanied by other adaptive responses, mere enhancement of SOS1 activity (e.g. by overexpression) may not be sufficient to improve salt tolerance (Oh et al., 2010; reviewed by Kronzucker and Britto, 2011;,Ji et  al., 2013) . As will be given in detail in the section on ‘Ca2+ ions’, SOS1 is activated via a calcium-dependent pathway. Simultaneously, the very long cytoplasmic tail of this transporter has been proposed to sense Na+ directly (reviewed by Zhu, 2002) . However, the SOS system integrates a second strategy for sodium dissipation: sequestration into the vacuole. As will be outlined in more detail in the following section on calcium, SOS2 and -3 will activate the NHX1 transporter that pumping sodium from the cytoplasm into the vacuole (Fig. 2B, ③). Compared with sodium extrusion, this strategy is efficient in removing noxious sodium from the cytoplasm but at the same time lowers the water potential of the entire protoplast, such that additional water loss to the environment is prevented, and not only the ionic but also the osmotic component of salinity stress is encountered. It should be mentioned that NHX1 is not the only transporter able to sequester sodium into the vacuole. The tonoplast harbours slow-activating and fast-activating channels that can facilitate sodium uptake to the vacuole and improve salinity tolerance in leaves of quinoa (Bonales-Alatorre et al., 2013) . In the case of a non-tolerant cell, the SOS system is activated in only a sluggish manner (the reason is linked to the reduced activity of NSCCs, as will be pointed out in the subsequent section) such that sodium will remain in the cytoplasm and accumulate there (Fig. 3B) to toxic levels. To summarize the main points on sodium as a salinity signal are: 1. The ‘on’ state is generated by the rapid influx of sodium (along with calcium ions and protons) through DA-NSCCs and/or VI-NSCCs (Fig.  2A, ①). This rapid sodium/ calcium peak represents the first signal that allows the discrimination of salinity stress from mere osmotic stress (e.g. as a consequence of drought), i.e. here is the point where the ionic component in salinity signalling bifurcates from osmotic signalling. In parallel, the osmotic challenge will result in activation of mechanosensitive calcium channels yielding additional influx of calcium ions and protons (Fig. 2A, ②). 2. The rapid elevation in the cytoplasmic concentration of sodium/calcium activates a signalling (which is explained in detail in the subsequent sections) that will initiate a rapid dissipation of the sodium signal (Fig.  2B, ③): to achieve the ‘off ’ state, on the one hand, additional influx of sodium ions is prevented by deactivation of the NCCS through cAMP/cGMP-dependent signals (Fig. 2B, ③), as well as deactivation of the slower HKAT channel. On the other hand, sodium ions are removed from the cytoplasm either by extrusion (SOS1), or by sequestration into the vacuole (SOS2/3 and NHX1). 3. The appropriate target for the sodium signal is actually the concomitant influx of calcium (Fig.  2A, ②), which will carry on signalling even after the sodium signal has been dissipated by the mechanisms given in (2). 4. In the case of a delayed sodium/calcium influx through the HA-NCCS (Fig. 3A, ①), activation of the SOS system as well as the block of sustained sodium influx will not be efficient. This not only results in accumulation of cytoplasmic sodium to toxic levels (Fig. 3B), but will also lead to sustained accumulation of calcium that, as pointed out in the subsequent section, will go astray and channel towards overactivation of the JA pathway. Ca2+ ions: promiscuous but choosy Calcium ions (Ca2+) are considered the most prominent ubiquitous second messenger in cells ranging from bacteria and plants up to specialized neurons (reviewed by Clapham, 1995) . The normal cytoplasmic Ca2+ (Ca2+cyt) level is ~100– 200 nM, while in membrane-enclosed organelles it is ~1–2 mM (reviewed by White, 2000) . Ca2+cyt signals are shaped by influx or efflux of ions from the extracellular space (cell wall or apoplast in plants) through a couple of different channels in the plasma membrane, some of which seem to be mechanosensitive, whereas others are voltage gated and might be identical to the NCCS (for a recent review, see Swarbreck et  al., 2013) . Different channels are localized at the surface of intracellular compartments (such as vacuoles, chloroplasts, or mitochondria). Slow vacuolar channels, such as TPC1, are targets of different signalling molecules including Ca2+, calmodulin (CaM), and nucleotides, and play a crucial role in raising cytosolic Ca2+ under a wide range of environmental and developmental cues (Pottosin et  al., 2009; reviewed by Hedrich and Martena, 2011; Peiter, 2011) . The spatial pattern of Ca2+ signals (e.g. cytosol, nucleus, organelles, or other specific regions of the cell), the temporal propagation of Ca2+ levels, the amplitude of the signal, and the frequency of Ca2+ oscillations are all informative aspects of Ca2+ signals, which are perceived by adaptor proteins or Ca2+-modulated proteins that regulate downstream signalling events (reviewed by Bouché et al., 2005; Kudla et al., 2010 ). Interestingly, Ca2+ signals participate in virtually all developmental, hormonal, and stress cues (reviewed by Reddy et al., 2011) . The apparent ambiguity of this signal is even amplified by the fact that nitric oxide (NO), a small, uncharged, short-lived, water- and lipid-soluble, highly diffusible, ubiquitous, volatile, highly reactive free radical, can act as a Ca2+-mobilizing messenger (reviewed by Neill et al., 2003; Besson-Bard et al., 2008; Siddiqui et al., 2011) . Under salinity, the earliest cellular response seems to be a rapid increase in free cytosolic Ca2+ within 1–5 s via influx through either NSCCs or a mechanosensitive calcium channel in the plasma membrane, which can be amplified through release from internal stores, especially the vacuole (Knight et al., 1997; Donaldson et al., 2004) . NaCl-induced cytosolic Ca2+, in turn, activates the plasma-membrane ATPases mediated by Ca2+/CaM-dependent protein kinases, restoring membrane voltage after Na+-induced depolarization, maintaining membrane integrity and ionic homeostasis, promoting H+ influx, and inhibiting both K+ and H+ efflux (Klobus and Janicka-Russak, 2004; Shabala et  al., 2006; reviewed by Wolf et  al., 2012) . Moreover, cytosolic Ca2+ activates salt overly sensitive 3 (SOS3), a member of the calcineurin B-like (CBL) family known as CBL4, to interact with SOS2 (a CBL-interacting protein kinase, CIPK24). The SOS3/SOS2 complex, in turn, activates SOS1 (a plasma-membrane Na+/H+ antiporter) through its phosphorylation (Fig. 2B, ③). The activated SOS1 extrudes Na+ from the cell, thus reducing its harmful effects on cellular metabolism (reviewed by Zhu, 2002; Harper et  al., 2004; Munns and Tester, 2008) . SOS1 directly signals to a putative K+ transporter by-passing SOS2 and SOS3 and therefore was proposed to be necessary for safeguarding the K+ permeability of the plasma membrane during salinity stress (Qi and Spalding, 2004; Shabala et  al., 2005) . In addition, the Na+/H+ exchanger (NHX) class of transporters (Fig.  2B, ③)—the plant homologue of the yeast Na+/H+ exchanger (Apse et  al., 1999; Gaxiola et  al., 1999) that catalyses the electroneutral exchange of Na+ or K+ with H+, maintaining intracellular pH and Na+ and K+ homeostasis in all eukaryotes (reviewed by Martinoia et  al., 2012) —is interconnected to Ca2+ signals via SOS/NHX interaction (Qiu et al., 2004). To date, six members of the NHX gene family have been identified in A. thaliana and classified according to their intracellular localization into vacuolar (NHX1–4) and endosomal (NHX5 and -6) compartments (Bassil et al., 2011) . The generation of salt-resistant tomato by overexpression of NHX1 was considered one of the milestones of green genetic engineering (Zhang and Blumwald, 2001) . Despite a long history of biotechnological application, the actual reason for salinity tolerance conferred by NHX1 is still under debate. Overexpression of Arabidopsis NHX1 or tomato NHX2 in tomato did not yield a consistent elevation of vacuolar Na+ (Rodriguez-Rosales et al., 2008; Leidi et al., 2010) and the protective effect was attributed to improved potassium partitioning to the vacuole. On the other hand, the Arabidopsis double mutant nhx1 nhx2 showed a similar salinity sensitivity to the wild type but a reduced vacuolar pool of K+ at simultaneously elevated sequestration of Na+. Additionally to steering the SOS pathway, calcium can activate gene expression. For instance, the CaM-binding transcription activators CAMTA1–4, and CAMTA6 are all salt induced (Yang and Poovaiah, 2002) . In addition, a specific CaM isoform in soybean (GmCaM4) interacts directly with a MYB2 transcription factor, enhancing the transcription rate of MYB2-dependent genes, such as P5CS1, conferring salt adaptation to Arabidopsis overexpressing GmCaM4 (Yoo et al., 2005). However, calcium ions can also act directly without the need for a protein adaptor: the Arabidopsis salt stress-responsive gene 1 (AtNIG1), a basic helix–loop–helix (bHLH)-type transcription factor, is the first known Ca2+binding transcription factor involved in the plant response to salt stress (Kim and Kim, 2006) . Interestingly, Ca2+ can also stimulate the NADPH oxidase (Fig. 2B, ⑥), a primary source of stress-related oxidative burst in plants, and thus generates a further important stress signal (Dubiella et al., 2013) . Similar to sodium, the protective function of calcium signalling can turn deleterious when cytosolic calcium levels remain high over a longer period. Under these circumstances, Ca2+ can activate degradative processes or cell death by precipitating phosphate (depleting, among others, ATP as ‘cellular currency’), cause aggregation of proteins and nucleic acids, and impair the integrity of lipid membranes (reviewed by Clapham, 1995; Case et al., 2007) . For example, ROS-activated sustained Ca2+ influx (feedback stimulated by threshold levels of H2O2) was followed by programmed cell death in soybean (Levine et al., 1996). Therefore, plants have adopted different strategies to restore Ca2+cyt levels after the completion of Ca2+ signalling, and the balance between reactions that cause elevated Ca2+cyt (‘on’ reaction) and reactions through which the Ca2+ signal is damped by buffering, pumping, and exchanging machineries (‘off ’ reactions) determines the intracellular Ca2+ levels at any time point (reviewed by Berridge et al., 2003; Bouché et al., 2005; Clapham, 2007; Bose et  al., 2011) . The central plant vacuoles (equivalent to lysosomes of animal cells with regard to their degradation and autophagy functions) represent the major Ca2+ store in a mature plant cell. High-capacity vacuolar Ca2+ exchangers (CAXs) play crucial roles in ion homeostasis and signal transduction (Hirschi, 2001) . In the resting state, these CAX pumps are complemented by auto inhibited Ca2+-ATPase pumps of the PIIB-type. However, upon Ca2+cyt elevation, Ca2+/CaM will bind to the N-terminal autoinhibitory domain, releasing them from autoinhibition and restoring Ca2+cyt by a feedback regulation (Fig.  2B, ④) that is an energy-consumptive process (reviewed by Pittman, 2011) . In A.  thaliana, there are six members of CAX (CAX1–6) that seem to function specifically with regard to different cues. For instance, the cax1 mutant displayed enhanced freezing tolerance, while cax3 resulted in higher salinity sensitivity (Catalá et al., 2003; Zhao et al., 2008; reviewed by Bose et al., 2011) . Interestingly, CAX1 is also interconnected to the SOS pathway and activated via SOS2, restoring Na+/Ca2+ homeostasis, whereas elevated expression of deregulated CAX1 caused salinity sensitivity (Cheng et al., 2004) . Failure to dissipate a cytosolic calcium signal in a timely fashion will strongly interfere with the signalling status of important stress hormones such as JA and ABA (Fig.  3C). Details on the function of these hormones are given in the sections below—however, here their regulation by calcium is considered. Calcium released from the vacuole through the TPC1 channel can activate JA synthesis (Fig.  3B, ③; Bonaventure et al., 2007) , whereas CBL proteins impede both the synthesis and the signal transduction of ABA (Fig.  3C, ④; Pandey et al., 2004). To summarize the main points on calcium as a salinity signal are: 1. The ‘on’ state is generated by the rapid influx of calcium through different channels, probably including the NCCS carrying sodium ions (Fig. 2A, ①), and a mechanosensitive calcium channel triggered by membrane load caused by osmotic water loss (Fig. 2A, ②). 2. The ‘off ’ state is restored by rapid dissipation of calcium by either binding to SOS3 (Fig.  2B, ③) or by sequestration by the CAX transporters that are controlled through SOS2, which means that calcium activates its own removal from the cytoplasm (Fig. 2B, ④). 3. The appropriate target for the calcium signal is on the one hand the SOS system driving the elimination of sodium ions from the cytoplasm (Fig. 2B, ③), and the CAX transporters (Fig.  2B, ④) that will contribute to the shut-off of the calcium signal. Calcium-triggered activation of the NADPH oxidase will relay the signal to the next player, apoplastic ROS (Fig. 2B, ⑥). 4. In case of a delayed sodium/calcium influx (Fig. 3A, ①), activation of the SOS system as well as the block of sustained sodium influx will not be efficient. As a result, the sequestration of calcium into the vacuole will be slowed down, and the calcium signal is conveyed to other calciumadaptor proteins, such as CBL9 (Fig.  3B, ④). This will impede the ABA ‘status’ as a dynamic product of synthesis and signalling (Fig.  3C, ④).This situation might even become accentuated by sustained calcium released from the vacuole through the slowly activated TPC1 channels (Fig. 3B, ③). Proton influx: a signal enhancer? Protons (H+) play crucial roles for cell signalling either directly or in cross-talk with phytohormones or Ca2+ (Gao et al., 2004a) . In addition, protons directly regulate enzymatic conformations and thus metabolic activities (Roberts et  al., 1980) . However, intracellular pH can also act as a second messenger for several signalling pathways. For instance, a cytoplasmic alkalinization is able to convey methyl-JA (MeJA) and ABA signalling during stomatal closure of A.  thaliana (Suhita et al., 2004) , and is also involved in plant responses to salinity and drought stresses, indole-3-acetic acid, and gravity (Gao et al., 2004a; Fasano et al., 2001; reviewed by Kurkdjian and Guern, 1989) . Proton influx can occur concomitantly with calcium, and the resulting apoplastic alkalinization has been used extensively as a robust reporter for the rapid activation of calcium influx channels by elicitors (Felix et al., 1993, 1999) or abiotic stresses including salinity stress (Ismail et al., 2012, 2014; Geilfuß and Mühling, 2013 ). With respect to the downstream signals, it should be noted that pH controls the ratio between the active and the inactive enantiomer of the bioactive JA conjugate JA-Ile (Fonseca et al., 2009) . A comparison of two Vitis cell lines differing in salt tolerance (see ‘Conceptual framework’ section) showed that efficient adaptation in V. rupestris correlated with a more rapid and more persistent apoplastic alkalinization compared with the salt-susceptible V. riparia (Ismail et al., 2014) . Apoplastic alkalinization, in turn, might promote adaptive events such as activation of wall-consolidating enzymes such as pectin methyl esterase, or, on the other hand, inhibition of expansins that render the wall softer (reviewed by Wolf et al., 2012) . It should also be considered that depletion of protons in the apoplast will release anionic binding sites to complex sodium ions. In addition, the elevated steady-state level of apoplastic superoxide as a further relevant signal (see following section) will be enhanced if the level of protons is low. Furthermore, stress-induced pH changes in the xylem sap might act as a root signal through ABA anions that redistribute and accumulate due to the low membrane-permeability of the charged anion, promoting stomatal closure (Taiz and Zeiger, 2010). The impact of proton activity on the enzymes or other proteins in the cytoplasm is very critical, where Ca2+-induced H+ influx might feedback on Ca2+ signalling by affecting Ca2+ affinity for CaM (reviewed by Busa and Nuccitelli, 1984) . Moreover, the activities of the important transporters NHX1, CAX1, and CAX2 are inhibited by cytosolic protons (Pittman et  al., 2005; reviewed by Padan et  al., 2001) . Thus, the influx of protons would promote the temporary accumulation of the concomitant calcium and sodium signals. However, this fluctuation of cytoplasmic pH will remain transient, because protons are rapidly extruded by powerful proton ATPases at the plasma membrane and especially the V-ATPase at the tonoplast (Fig. 2B, ⑤), and at the same time are complexed by the high buffering capacity of the cytosol (reviewed by Kurkdjian and Guern, 1989) . In summary, although proton influx does not act as an independent signal, it can act as an enhancer of early sodium and calcium signals: 1. The ‘on’ state is generated by influx together with calcium through the NSCC (Fig. 2A, ①) and the mechanosensitive calcium channels (Fig. 2A, ②). 2. The ‘off ’ state is restored by rapid buffering of protons in the cytosol and active extrusion through the proton ATPases at the plasma membrane, and, most importantly, by vacuolar sequestering by the V-ATPase (Fig. 2B, ⑤). 3. The appropriate target is the inhibition of NHX1 and CAX activities acting as an amplifier of the initial sodium and calcium signal (Fig. 2B, ③, ④). At the same time, the depletion of protons from the apoplast will increase the lifetime of ROS (Fig. 2B, ⑥), and improve the matrix buffering for sodium ions. 4. In the case of a delayed sodium/calcium influx (Fig.  3A, ①), apoplastic protons will be available for quenching ROS, thus dampening a further important signal (Fig. 3B, ⑤). In the cytosol, the initial sodium/calcium signal would not be enhanced, and later acidification might even interfere negatively with hormonal signalling. ROS: bifunctional in the response to salinity stress ROS are continuously produced in plant compartments such as mitochondria, chloroplasts, and peroxisomes as unavoidable by-products of aerobic metabolism such as photosynthesis, photorespiration, and respiration (reviewed by Abogadallah, 2010; Apel and Hirt, 2004) . As aerobic metabolism is based on electron flow across membranes, even mild damage of mitochondrial or plastidic membranes will result in uncontrolled intracellular oxidative burst (Fig.  3C, ⑦).The term ROS comprises both free radical (O2•–, superoxide radicals; OH•, hydroxyl radical; HO2•, perhydroxy radical; and RO•, alkoxy radical), and non-radical (molecular) forms (H2O2, hydrogen peroxide; and 1O2, singlet oxygen) (reviewed by Gill and Tuteja, 2010). The different ROS vary not only in their chemical nature but also in their toxicity. The superoxide O2•– is considered the earliest ROS, while OH• is among the most highly reactive ROS known. The accumulation of ROS causes oxidative damage to DNA, proteins, carbohydrates, and lipids. However, they also could function as signalling molecules regulating responses of development and various aspects of stress. Therefore, they must be closely regulated by orchestrated mechanisms (reviewed by Miller et al., 2010) . For different stimuli, the elevated levels of ROS are sensed at the plasma membrane, for instance by two-component signalling systems (membrane-localized histidine kinases) that, in turn, activate the mitogen-activated protein kinase (MAPK) signalling cascades. Under salinity challenge, different MAPK elements are activated such as MAPK4, MAPK6, and MAPKK1 (reviewed by Taj et  al., 2010) . Arabidopsis overexpressors for AtMAPKK2 exhibited constitutive MAPK4 and MAPK6 activity, constitutively unregulated expression of stress-induced marker genes, and increased freezing and salt tolerance. Transcriptomic analysis of this mutant showed altered expression of 152 genes involved in transcriptional regulation [such as STZ (slt tolerance zinc finger protein), WRKY and MYB], signal transduction (such as a MAPKK5related protein and a putative calmodulin), cellular defence (such as lipoxygenase and the ACC synthase AtACS-6), and stress metabolism (including a flavonol synthase and P5CS, a gene encoding a key enzyme of proline biosynthesis) (Teige et  al., 2004) . Although MAPK4 regulates the cross-talk between SA and JA, supporting the JA/ethylene signalling pathway (Brodersen et al., 2006) , growth assays and northern blot analysis of transcripts did not detect differences between the mpk4 Arabidopsis mutant compared with the wild type under salinity, cold, or heat shock, although differences were noted for pathogen challenge (Petersen et  al., 2000) , indicating that, in the context of abiotic stress, the alternative MKK2/MAPK6 cascade is relevant. However, MAPK signalling can also act as an antagonist for abiotic stress signalling—for instance, AtMAPK1 negatively regulates a putative Na+/H+ antiporter, leading to salinity sensitivity (reviewed by Chinnusamy et  al., 2006) . In addition to the MAPK pathways, ROS can modulate gene expression by modifying transcription factors (reviewed by Apel and Hirt, 2004) . A third mechanism is the reversible oxidation of critical thiols in key signalling enzymes (reviewed by Forman and Torres, 2002) . However, ROS production needs to be tightly controlled to act as a signal, otherwise an excessive oxidative burst would result in cell death. In fact, ROS are a hallmark of plant-specific forms of programmed cell death, so called necroptosis (reviewed by Coll et al., 2011) . Interestingly, animals and plants share common apoptosis signal transduction pathways triggered by oxidative stress, where H2O2-induced lipoxygenase activities that are able to introduce molecular oxygen into the fatty acid moieties of phospholipids lead to increasing mitochondrial membrane lipid peroxidation and, subsequently, cytochrome c release (reviewed by Maccarrone et al., 2001). Singlet oxygen, on the other hand, is used as a substrate of lipoxygenases triggering a metabolic pathway that will generate a further important stress signal, JA (Fig. 3C, ⑥; Farmer and Mueller, 2013) . Also, ABA synthesis is activated by ROS (Xiong and Zhu, 2003) . Salinity- or drought-stressed plants close their tomata, which in turn limits water loss (favourable effect) and the influx of CO2 (unfavourable effect) (Hsu and Kao, 2003) . Consequently, carbon reduction and photosynthetic NADPH consumption by the Calvin cycle decrease, resulting in electron leakage from photosystem I to O2 as an alternative electron acceptor, initiating the Mehler reaction (reviewed by Türkan and Demiral, 2009) . The resultant O2•– is considered the earliest ROS that consequently gives rise to other ROS, including the most noxious OH•. Additionally, the peroxisomal glycolate oxidase during photorespiration, plasma-membrane located NADPH oxidases, amine oxidases, and cell-wall-bound peroxidases are important sources of ROS that are active to a certain extent even under normal conditions but are activated in response to stress (reviewed by Mittler, 2002). Plants must strictly maintain ROS homeostasis to mitigate the toxicity of ROS. Therefore, plants have employed different scavenging machineries that tightly control ROS levels, both enzymatic and non-enzymatic. Plant enzymatic antioxidant mechanisms include superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), guaicol peroxidase (GOPX), and glutathione S-transferase (GST). The metalloenzyme SOD is the most effective intracellular enzymatic antioxidant and acts by dismutating superoxide to H2O2, which in turn can be detoxified by APX, GPX, and CAT. The non-enzymatic antioxidants comprise ascorbic acid (ASH), glutathione (GSH), phenolic compounds, alkaloids, non-proteinogenic amino acids, and α-tocopherols (reviewed by Apel and Hirt, 2004; Gill and Tuteja, 2010) . The quelling of ROS accumulation can also be achieved by other signals, such as NO. NO has the ability to neutralize Fenton-type oxidative damage by scavenging superoxide, therefore preventing the formation of oxidants (such as O2•–, H2O2, and alkyl peroxides), which makes it easier to recover a redox homeostasis (Lamattina et  al., 2003) . For example, pre-treatment with 1 mM sodium nitroprusside, a NO donor, results in enhancement of the antioxidant defence and methylglyoxal detoxification systems in salt-stressed wheat seedlings (Hasanuzzaman et al., 2011) . In addition, NO is considered a redox regulator of the NPR1/TGA1 system, a key redoxcontrolled regulators in plant systemic acquired resistance in plants (Lindermayr et al., 2010) . As an additional regulator, hydrogen sulfide (H₂S) has emerged as a signalling molecule in plants that increases GSH levels, alters enzyme activities, and interacts with NO and ROS metabolism (reviewed by Paul and Snyder, 2012; Lisjak et  al., 2013) . As NO is acting as a secondary messenger of ABA signalling (reviewed by Hancock et  al., 2011) , this molecule provides cross-talk between oxidative and phytohormonal signalling. This crosstalk is even bilayered, because also ROS deriving from the activity of the NADPH oxidase in the plasma membrane are essential for ABA induced signalling (Kwak et al., 2003). As with most stress signals, ROS are ambiguous, switching between activation of adaptive events and causing oxidative damage. Again, it depends on timing and regulation as to whether they act as a ‘signal on demand’ or go wild as cellular terminators: 1. The ‘on’ state is generated by metabolic disbalance of oxidative processes such as respiration or photosynthesis, but in the context of signalling, calcium-triggered activation of the membrane-bound NADPH oxidase seems to be central(Fig.  2, ⑥). The elevated steady-state level of the resulting apoplastic ROS is increased due to the apoplastic depletion of protons, such that these ROS can enter the cytoplasm, probably through aquaporins. 2. The ‘off ’ state is on the one hand provided by enzymatic and non-enzymatic antioxidants (Fig. 2C, ⑥), and on the other by dissipation into stress signalling (e.g. MAPK cascades) and activation of phytohormone synthesis. 3. The appropriate targets are signal cascades such as the MAPK pathway leading to the activation of adaptive genes but also the activation of JA and ABA synthesis (Fig. 2C, ⑦). 4. In the case where ROS accumulate at later stages (Fig. 3B, ⑤), or are formed as a consequence of mitochondrial damage (Fig. 3C, ⑦) this will result not only in a pertinent hyperactivation of JA synthesis due to excessive lipid peroxidation but also in autocatalytic oxidative burst (Fig. 3C, ⑥), as the membrane damage will impair the functionality of electron transport in both mitochondria and plastids. ABA: commitment for adaptive responses? The phytohormone ABA, synthesized via the terpenoid pathway, regulates numerous plant biological processes ranging from development, including the inhibition of growth/germination and bud dormancy, to adaptive stress responses, such as drought, salt, ozone, and pathogen infection, and therefore is seen as a stress-related hormone (reviewed by Xiong and Zhu, 2003) . The perception and signalling pathways of ABA have been studied extensively in A.  thaliana and other species using biochemical and molecular genetic approaches (Ishibashi et al., 2012; reviewed by Cutler et al., 2010; Raghavendra et  al., 2010) . In 2009, the long search for the ABA receptor succeeded with the identification of pyrabactin resistance 1 (PYR1), a member of the PYR/ PYR1-like (PYL)/regulatory component of the ABA receptor (RCAR) group of proteins. This novel ABA-binding protein was demonstrated as a soluble ABA receptor by two independent research groups, considered as a breakthrough for the understanding of ABA signalling (Ma et  al., 2009; Park et  al., 2009) . These receptors, now termed PYR/PYL/ RCAR, represent a family of soluble proteins of about 150– 200 aa that share a conserved START domain. The ABAfree ‘open-lid’ conformation of PYR1 is converted to a more compact and symmetric closed-lid dimer upon binding to ABA (Nishimura et  al., 2009, 2010) . Plants constrain ABA signalling through clade A protein phosphatases 2C (PP2C) [mainly ABI1, ABI2, and HOMOLOGY TO ABI (HAB1 and HAB2), which negatively regulate (dephosphorylate) downstream kinases. However, in response to environmental or developmental signals, ABA is synthesized and bound to PYR1, and this receptor, in turn, binds to PP2Cs inducing a conformational change resulting in its inhibition, and thus terminating the inhibition of the downstream ABAactivated kinases (OST1/SnRK2.6/SRK2E, SnRK2.2, and SnRK2.3). The released SnRK2s are able to phosphorylate downstream factors, such as the majority of osmotic stressresponsive genes harbouring ABA-responsive promoter elements/complexes (ABREs) and bZIP transcription factors (such as ABI5), ion channels (SLAC1, KAT1), and the NADPH oxidase AtrbohF (reviewed by Hubbard et al., 2010; Umezawa et al., 2010; Joshi-Saha et al., 2011) . ABA activates genes that encode enzymes for the biosynthesis of compatible osmolytes (as shown for water-stress-induced betaine in pear leaves; Gao et  al., 2004b) , proline (Strizhov et  al., 1997) , and cellular chaperones (dehydrins and LEA-like proteins) that protect proteins and membranes under stress (Liu et  al., 2013; reviewed by Hasegawa et  al., 2000, Shinozaki and Yamaguchi-Shinozaki, 2007) . In addition, ABA causes induction of Ca2+cyt via ROS or IP3 recruitment (Murata et al., 2001; Taiz and Zeiger, 2010) . Furthermore, ABA and JA play pivotal roles in controlling stomatal closure, which is considered a fast response in stressed plants, although plants cannot keep stomata closed over a long period as they need to fix CO2 for survive. Interestingly, ABA and JA transduction pathways leading to stomatal closure share overlapping signalling elements. Several ABA mutants with NCED (the key regulatory gene in ABA biosynthesis) overexpression showed better drought adaptation, while ABA-deficient aba mutants of Arabidopsis perform poorly under drought or salt stress or even die (reviewed by Zhu 2002; Bartels and Sunkar, 2005) . Direct comparison of two genetically similar grapevine cell lines differing in their osmotic sensitivity under salt stress revealed that salt susceptibility was accompanied by a delayed accumulation of ABA (Ismail et al., 2014). By keeping in mind that some osmotic stress-responsive genes are ABA independent and are activated via JA signalling including MYC and MYB elements (Ishitani et al., 1997; reviewed by Bartels and Sunkar, 2005) , although both rd22BP1/ AtMYC2 and AtMYB2 proteins were firstly identified as ABA-inducible transcriptional activators under drought (Abe et  al., 2003) , ABA was concluded not to be the only adaptive signal, as will be discussed in the next section. On the other hand, the ABA transient increase under demanding conditions points to the importance not only of the activation of signalling and biosynthesis but also the suppression strategy. Indeed, ABA enhancement in an NCED mutant resulted in accumulation of its catabolite, phaseic acid, via (+)-ABA 8′-hydroxylase activation that catalyses the first step in the oxidative degradation of ABA, in addition to other catabolic pathways (Qin and Zeevaart, 2002; reviewed by Cutler and Krochko, 1999) . Oxidative degradation might be complemented by other ABA inactivation strategies, such as ABA conjugation. As the phosphatase activity of ABI1 and ABI2 increases in response to ABA, dephosphorylation of ABA signalling elements will constitute a negative-feedback loop (Merlot et al., 2001) . A further (negative) feedback loop is provided by ABA-dependent inhibition of ABI5 degradation and simultaneous ABI-dependent promotion of the RING E3 ABI3-INTERACTING PROTEIN 2 (AIP2) that in turn suppresses ABI3, which interacts with ABI5, enhancing its activity (Vierstra, 2009) . Both synthesis (Xiong and Zhu, 2003) and signalling (Kwak et  al., 2003) of the ABA pathway are promoted by ROS, whereas calcium, through calcineurin B, constrains both synthesis and signalling of ABA (Pandey et al., 2004) . ABA seems to be the first step that, by its molecular nature, is committed to adaptation, as both calcium and ROS modulate the ABA status (defined as dynamic equilibrium between ABA content and signalling activity, Fig.  2C, ⑦), and this point seems to be important for the decision between adaptation and cell death: 1. The ‘on’ state is activated through both synthesis and activation of signalling by ROS (Fig. 2C, ⑦). 2. The ‘off ’ state is achieved by the ABA signalling pathway itself due to the induction of negative regulators (ABI proteins) by ABA (Fig. 2C, ⑧). 3. The appropriate targets are adaptive genes harbouring ABA-inducible adaptive genes that encode osmoprotectans (such as the LEA proteins) but also signalling components that adjust a sustainable ABA status as a balance between constraint (through the ABIs) or promotion (through the NADPH oxidase RboH generating ROS) (Fig. 2C, ⑥, ⑧). 4. In the case of delayed calcium signatures, calcium will, through CBL proteins, impair the ROS-dependent activation of the ABA pathway (Fig. 3C, ④). Due to this delay, the concurrent JA pathway will become dominant, culminating in cell death. JAs: a dangerous switch Jasmonic acid and related compounds, collectively named jasmonates (JAs), are ubiquitously occurring lipid-derived compounds, and function as a master switch in plant responses to several abiotic and biotic stresses such as wounding (mechanical stress), drought and salt stress, ozone and pathogen infection, and insect attack (reviewed by Wasternack, 2007; Wasternack and Hause, 2013) . Similar to ABA, the synthesis of JA is triggered by ROS, as the first committed step of synthesis, the peroxidation of linoleic acid by lipoxygenases, requires singlet oxygen (Farmer and Mueller, 2013) . In contrast to the ABA pathway, which is negatively regulated by CBL proteins, there is evidence that the JA pathway is stimulated (reviewed by Hu et al., 2009) . In addition to their role as a general stress signal, JAs regulate many aspects of plant development and growth such as seed germination, fruit ripening, production of viable pollen, root growth, tendril coiling, photomorphogenesis, leaf abscission, and senescence (Creelman and Mullet, 1995, 1997a, b; Conconi et al., 1996; Rao et al., 2000; Riemann et al., 2003, 2013; Haga and Iino, 2004; Ma et al., 2006; Robson et al., 2010; reviewed by Wang et al., 2011) . Among JA conjugates and derivatives, (+)-7-isojasmonoyl-l -isoleucine (JA-Ile) formed by the enzyme JAR1 (Jasmonate-Resistant 1) was found to be an endogenous bioactive form of JA (Fonseca et  al., 2009) . Under non-stress conditions, JA-Ile is maintained at low levels, and this allows a multimeric protein complex to inactivate JA signalling in plant cells. This machinery is composed of JAZ repressor proteins that bind and repress the transcriptional activator MYC2, via recruiting the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a transcriptional repressor called Novel Interactor of JAZ/ TIFY (NINJA) (Chini et  al., 2007; Thines et  al., 2007 ; Pauwels et al., 2010; reviewed by Kazan and Manners 2008 , 2012, 2013). In response to developmental or environmental cues (including salinity), the levels of JA-Ile are elevated. JA-Ile binds to COI1 and promotes the interaction of JAZ proteins with COI1, leading to SCFCOI1-mediated ubiquitination of the JAZ factors, followed by their degradation via the 26S proteasome. This results in the release of MYCtype transcription factors from repression by the JAZ factors and thereby will induce transcription of early JA-responsive genes including the JAZ genes themselves. In Arabidopsis, derepression of AtMYC2 is induced under dehydration and saline conditions. In addition, Arabidopsis plants in which AtMYC2 is overexpressed exhibited less electrolyte leakage under osmotic stress (Abe et  al., 2003) . When treated with MeJA, protective proteins against oxidative stress (which is a true companion of many abiotic stresses including drought or salt) accumulate in the wild type but at reduced levels in a myc2 null mutant. These protective proteins include the HSP20-like chaperone protein, the fibrillin precursor protein, a luminal binding protein (BiP2), and GST (Guo et al., 2012) . JA-dependent activation of OsbHLH148 upregulates rice OsDREB1A, a functional orthologue of Arabidopsis DREB1A (Fig. 2C, ⑨), which plays critical roles in improving drought, salinity, and freezing tolerance but in an ABAindependent manner (Dubouzet et al., 2003; Seo et al., 2011) . These results, at first sight, suggest a role for JA signalling in conferring tolerance to drought and salinity, or oxidative stress. However, a closer look reveals that JA signalling is tightly controlled, as the transcription of JAZ genes is induced by JA. The newly synthesized JAZ proteins interact with and restore the repression of MYC2, which in turn deactivates the JA signal transduction pathway (Chini et al., 2007; Thines et al., 2007 ; reviewed by Wager and Browse, 2012). This negativefeedback loop and the resulting transient action of JA indicate that unfavourable effects of overshooting JA signalling have to be strictly avoided (Fig.  2C, ⑨). Indeed, MeJA was found to induce cell death in Arabidopsis protoplasts (Zhang and Xing, 2008) and Vitis cell suspensions (Repka et al., 2004) in a concentration- and time-dependent manner (Zhang and Xing 2008) . The MeJA-induced cell death correlates with ROS production, alterations of mitochondrial dynamics, and photosynthetic collapse (Fig. 3C, ⑥). The above-mentioned direct comparison of the two salt-stressed grapevine cell lines showed that the salt-sensitive V.  riparia, where the accumulation of ABA was delayed, accumulated tenfold higher levels of the bioactive JA-Ile, whereas in the more salt-tolerant V. rupestris, ABA accumulated earlier and strongly suppressed formation of the JA-Ile signal (Ismail et al., 2014) . This more rapid and more massive induction of JA-Ile was accompanied by a pronounced oxidative burst in V. riparia accompanied by synthesis of high amounts of δ-viniferin, a metabolic indicator for ensuing programmed cell death (Chang and Nick, 2012) . The importance of JA tuning is corroborated by analysis of the rice mutant rice salt sensitive 3 (rss3), where root growth is more severely inhibited under salinity compared with the wild type (Toda et  al., 2013) . This growth phenotype is accompanied by elevated expression of JA-dependent genes. RSS3 binds to JAZ and non-MYC-type bHLH transcription factors, and has been proposed to repress an exaggerated JA response in the root tip (Toda et  al., 2013) . Collectively, these data suggest that fine-tuning JA signalling is important for the growth and viability of plants under salinity stress. The existence of a multimeric transcriptional co-repression complex machinery to inactivate JA signalling (Chini et  al., 2007; Thines et al., 2007 ; Pauwels et al., 2010), in addition to JA-dependent repression of MYC2 via the MEK2/MAPK6 pathway (Petersen et al., 2000) , is evidence that suppression of hazardous side effects of JA signalling is crucial for survival. It should be kept in mind that ABA and JA signalling are antagonistic on several levels—partially by mutual competition for shared signalling factors such as MYC2 (Anderson et al., 2004, Fig. 2C, ⑨) . The ambiguity of the ROS signal seems to be perpetuated at the level of JA—early activation of this pathway seems to be beneficial for salt adaptation, but sustained JA signalling is clearly deleterious and culminates in cell death: 1. The ‘on’ state is triggered through oxidative cleavage of membrane lipids and is therefore stimulated by ROS (Fig. 2C, ⑦). 2. The ‘off ’ state is achieved by tight negative feedback of the JA signal pathway itself with JAZ/TIFY proteins as central players (Fig. 2C, ⑨). 3. Under adaptive conditions, the JA pathway will initially be under tight constraint, probably triggered by a short transient peak of ROS; some JA will be formed, but signalling will be rapidly shut off, such that concurrent activation of ABA signalling is ahead (Fig. 2A, ⑨). 4. In the case of delayed calcium signatures, impaired ABA signalling (Fig.  3C, ④) in combination with sodiumdependent mitochondrial membrane damage (Fig. 3C, ⑦) will lead to excessive accumulation of JA accompanied by accelerated oxidative burst, membrane damage, and eventually cell death (Fig. 3C, ⑥). The ubiquitin–26S proteasome system (UPS): executors of cross-talk? In eukaryotes, such as plants, the UPS constitutes a tightly regulated and highly specific machinery that is devoted to specific proteolysis (reviewed by Sullivan et al., 2003) . Plants utilize the UPS to modulate almost all aspects of their biology including growth, development, and stress responses (Santner and Estelle, 2010) . The crucial roles of UPS are reflected in the number of genes encoding UPS components. A  genomic analysis of A.  thaliana showed that over 1400 genes (or >5% of the proteome) code for UPS components (Smalle and Vierstra, 2004) . Interestingly, several enzymes in the UPS are hormonal receptors. Moreover, the UPS controls the levels of essential downstream signalling proteins in hormonal signal transduction (Santner et  al., 2009) . Thus, the plant UPS not only removes abnormal proteins that arise due to biosynthetic errors or normal proteins with the wrong configuration (reviewed by Vierstra, 2009), but, in addition to this canonical function, controls signal specificity by removal of specific repressors. Furthermore, the UPS has been reported to be critically involved in plant programmed cell death, as the disruption of proteasome function by gene silencing of the proteasome subunits activates programmed cell death in plant cells (Kim et al., 2003) . As discussed above, salt-stressed plants increase the levels of different hormones such as JA activating specific branches of the UPS. The elevated levels of JA-Ile promote the proteolytic degradation of JAZ proteins via UPS releasing JA transcription factors (reviewed by Wager and Browse, 2012; Fig. 2C, ⑨) . Conversely, in ABA signalling, the synthesis of AIP2 is increased, which in turn suppresses the ABA transcriptional regulator ABI3. However, ABA blocks degradation of ABI5 (Fig.  2C, ⑧), a central regulator of ABA signalling during post-germinative growth (Vierstra, 2009) . It is possible to modulate stress tolerance through the UPS. In fact, the functional ubiquitin-specific protease UBP16 was found to increase salt tolerance by stabilization of SERINE HYDROXYMETHYLTRANSFERASE1 (SHM1), which can reduce oxidative burst and therefore repress cell death and at the same time positively regulates plasma-membrane Na+/H+ antiporter activity (Zhou et  al., 2012) . Arabidopsis thaliana ABA insensitive RING protein 3 (AtAIRP3/LOG2) is a positive regulator of the ABA-mediated drought and salinity adaptation by targeting RD21 (Responsive to Dehydration 21), which might accelerate cell death progression during senescence and stress conditions (Kim and Kim, 2013) . On the other hand, two A. thaliana C3HC4 RING domain-containing proteins, named DREB2A-INTERACTING PROTEIN1 (DRIP1) and DRIP2, function as E3 ubiquitin ligases, and negatively Life and death under salt stress: same players, different timing? | 2975 regulate drought-responsive gene expression by mediating DREB2A ubiquitination (Qin et al., 2008) . The convergence of different hormonal pathways on shared elements of the UPS provides a molecular framework that allows the integration of different signals, for instance the relative status of JA versus ABA signalling (Fig. 2C, ⑧,⑨). In other words, it might be this machinery that is dedicated to specific destruction, where plant cells decode the ‘meaning’ of different concurrent signal pathways. Concluding remarks To endow crops with enhanced stress tolerance, it is important to understand the underlying mechanisms. The advances of the last decade have revealed numerous molecular details of salinity-triggered responses and mechanisms of adaptation. In addition, complex and obviously precisely tuned cross-talk between different pathways seems to be relevant. How is the specificity of this cross-talk achieved? Both stress-tolerant and -sensitive plants utilize the same signalling molecules. However, it is important to conceptually discriminate signals from the molecules that convey these signals. The central message transported in this review is that the timing of stress signals is decisive. Stress signals are activated transiently and they are subsequently turned off. Whether a plant cell will adapt to salt stress or whether it will yield to cell death appears to be dependent on the correct timing of these transient signalling events. Tolerant plants are tolerant because they can orchestrate cross-talks between different signalling pathways (signal on demand). This is likely to be achieved by controlling the temporal signature and amplitude of the signalling. When this temporal control turns loose, such that a signalling event persists longer or initiates later, this will lead to inappropriate cross-talk with downstream events of other pathways. These downstream events would otherwise not be competent for the respective signal, simply because they proceed at a time point that is later. We have elaborated this heterochronous shift of signalling for the interaction between calcium and JA versus ABA signalling. A delay in the activation and, consequently, also in the deactivation, of the calcium signature will channel ROS-triggered signalling towards the JA pathway, which, in consequence, will run out of control, culminating in cell death. The same molecule (calcium), occurring at the right time, will be recruited for the activation of sodium extrusion and sequestering, such that ROS-triggered signalling will be channelled to (protective) ABA signalling constraining the JA pathway and thus leading to efficient adaptation. Thus, a deeper understanding of the temporal patterns in signalling will help us to dissect adaptive from damagerelated events. But this conclusion also calls for a specific experimental approach to stress physiology: to identify temporal signatures in stress adaptation, we need approaches that are both comparative and integrative—comparative in the sense that systems that are biologically very similar but differ in the outcome of their stress response are investigated side by side under the same conditions, and integrative in the sense that the different stages of the stress response are studied in the same system in parallel with respect to their time course. Knowledge of the molecular players of stress adaptation is a necessary prerequisite but is t sufficient. We need to consider and investigate molecular activities rather than mere molecules. Acknowledgements AI was supported by the German Egyptian Research Long term Scholarship ʿGERLSʾ program, which is jointly funded by the Ministry of Higher Education and Scientific Research ‘MHESR’, Egypt and the Deutscher Akademischer Austauschdienst ‘DAAD’, Bonn, Germany. Abe H , Urao T , Ito T , Seki M , Shinozaki K , Yamaguchi-Shinozaki K. 2003 . Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling . Plant Cell 15 , 63 - 78 . Abogadallah GM . 2010 . Antioxidative defense under salt stress . Plant Signaling & Behavior 5 , 369 - 374 . Anderson JP , Badruzsaufari E , Schenk PM , Manners JM , Desmond OJ , Ehlert C , Maclean DJ , Ebert PR , Kazan K. 2004 . 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Ahmed Ismail, Shin Takeda, Peter Nick. Life and death under salt stress: same players, different timing?, Journal of Experimental Botany, 2014, 2963-2979, DOI: 10.1093/jxb/eru159