Signaling Network In The Perception Of Salt Stress And Ionic Homeostasis Regulation In Populus Euphratica

Soil salinity causes increasingly agricultural and environmental problems on a worldwide scale, especially in arid areas. Understanding physiological and molecular mechanisms of salt tolerance is important for the improvement of plant productivity under salinity conditions. Populus euphratica Oliv. (P. euphratica) is a valuable tree species used for afforestation on saline and alkaline desert sits, and plays very important roles in stabilizing sand dunes, and in agriculture shelter belt construction in north-west China. P. euphratica has a higher capacity to tolerate salinity, and in recent years it has been widely considered as a model woody plant to address tree-specific questions—especially physiological, biochemical and molecular mechanisms in salt tolerance.Maintenance of intracellular K+/Na+ homeostasis is crucial for plants to adapt to saline environments. The regulation of K+/Na+ homeostasis is complicated by a complex signaling network. For instance, numerous signaling molecules, such as Ca2+, hydrogen peroxide (H2O2) and nitric oxide (NO), play a regulating role in K+/Na+ homeostasis in model plants. Recently, a novel signaling molecule, extracellular ATP (eATP), has been reported to be involved in the responses of plant cells to biotic stress. However, the roles of eATP in mediating plant salt tolerance and K+/Na+ homeostasis are largely unknown. At present, many investigations related to steady ion relations always focus on salt accumulation and cellular ion compartmentation, however, the signaling network in the mediation of K+/Na+homeostasis in P. euphratica is still unclear.In this study, we attempt to elucidate the signaling network in the perception of salt stress and K+/Na+ homeostasis regulation in P. euphratica. Using hydroponic seedlings of salt-resistant P. euphratica and salt-sensitive P. popularis 35-44 (P. popularis), we investigated the NaCl-induced alterations of cellular and tissue ion fluxes in roots (Na+, H+ and K+) by means of scanning ion-selective micro-electrode technique (SIET). We explored the contributions of plasma membrane (PM) H+-coupled transporters and channels to the K+/Na+ homeostasis mediation in P. euphratica. Callus cells were initiated from P. euphratica and P. popularis shoots and used to address the perception of poplar to salt stress. The contributions of H2O2 and NO to K+/Na+ homeostasis and antioxidant defense were elucidated in the two contrasting poplars by means of EDAX (energy dispersive X-ray analysis) and Confocal laser scanning microscopy. Using EDAX, confocal and SIET, we designed a variety of pharmacological experiments to clarify the differential response of P. euphratica to osmotic and ion-specific effects of NaCl. We confirmed the involvement of PM H+-coupled transporters, H2O2 and Ca2+ in the mediation of K+/Na+ homeostasis in P. euphratica cells, and a cellular signaling model upon ion specific effect was proposed. The role of salt-induced eATP (extracellular ATP) signaling in K+/Na+ homeostasis control and antioxidant defence were also explored in NaCl-stressed P. euphratica cells. Finally, we found that an excess eATP induced PCD (programmed cell death) in P. euphratica cells and a signaling pathway of eATP-PCD was proposed. Taken together, we proposed a signaling network to elucidate the perception and defense when P. euphratica cells were subjected to NaCl salinity.The main experimental results and conclusions are as follows:1. Compared to P. popularis, P. euphratica roots exhibited a greater capacity to retain K+ and to restrict Na+ accumulation after exposure to a long-term (LT) salinity (50 mM NaCl,3 weeks) by means of EDAX and ion-flux measurements. Our SIET data show that P. euphratica roots retained a lesser K+ efflux under both a short-and long-term of salt stress, as compared to P. popularis. Salt shock (SS)-induced K+ efflux in the two species was markedly restricted by K+ channels blocker, TEA (tetraethylammonium chloride), but enhanced by sodium orthovanadate, the inhibitor of plasma membrane (PM) H+-ATPase, suggesting that the K+ efflux is mediated by depolarization-activated channels, e.g. KORCs (outwarding rectifying K+ channels), and NSCCs (non-selective cation channels). P. euphratica roots were more effectively to exclude Na+ than P. popularis in a LT experiment, resulting from the Na+/H+ antiport across the PM. Moreover, pharmacological evidence implies that the greater ability to control K+/Na+ homeostasis in salinised P. euphratica roots is associated with the higher H+ pumping activity, which provides an electrochemical H+ gradient for Na+/H+ exchange, and simultaneously decreases the NaCl-induced depolarization of PM, thus reducing Na+ influx via NSCCs and K+ efflux through DA-KORCs and DA-NSCCs. Exogenously applied Ca2+ was favorable for poplar roots to maintain K+/Na+ homeostasis and the effect was more pronounced in the salt-sensitive species. Ca2+ application markedly limited salt-induced K+ efflux but enhanced the apparent Na+ efflux, thus enables the two species, especially the salt-sensitive poplar, to retain K+/Na+ homeostasis in roots exposed to prolonged NaCl treatment.2. Compared to P. popularis, P. euphratica roots (0-3000μm from the apex) exhibited a higher capacity to extrude Na+ after a short-term exposure to 50mM NaCl (24h) and a long term in a saline environment of 100mM NaCl(15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na+ efflux and a correspondingly increased H+ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na+/H+ exchange in root tissues and cells was inhibited by amiloride (a Na+/H+ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H+ -ATPase inhibitor). These results indicate that the Na+ extrusion in stressed P. euphratica roots is the result of an active Na+/H+ antiport across the plasma membrane. In comparison, the Na+/H+ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na+ at both the tissue and cellular levels. The pattern of NaCl-induced fluxes of H+ and Na+ differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.3. We found the species difference in the cellular response to NaCl treatment. Using callus cells of a salt-tolerant P. euphratica and a salt-sensitive P. popularis, the effects of NaCl stress on hydrogen peroxide (H2O2) and nitric oxide (NO) production and the relevance to ionic homeostasis and antioxidant defense were investigated. Results show that P. euphratica exhibited a greater capacity to tolerate NaCl stress in terms of cell viability, membrane permeability and K+/Na+ relations. NaCl salinity (150mM) caused a rapid increase of H2O2 and NO in P. euphratica cells. Moreover, salinised P. euphratica cells retained a high and stable level of H2O2 and NO during the period of 24-h salt stress. However, there were no evident increase of H2O2 and NO in P. popularis after the onset of salinity and an increase of H2O2 was only seen after a prolonged period of salt treatment. Noteworthy, P. eupratica cells increased activities of superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase under salinity stress, but these antioxidant enzymes were significantly inhibited by the salt treatment in P. popularis cells. Pharmacological experiments proved that the NaCl-induced H2O2 and NO was interdependent and contributed to the mediation of K+/Na+ homeostasis and antioxidant defense in P. euphratica cells. Given these results, we conclude that the increased H2O2 and NO enable P. euphratica cells to regulate ionic and ROS (reactive oxygen species) homeostasis under salinity stress in the longer term.4. With regard to the cellular response to ion-specific effects, we investigated the signalling of H2O2, cytosolic Ca2+([Ca2+]cyt) and the PM H+-coupled transport system in K+/Na+ homeostasis control in NaCl-stressed calluses of P. euphratica. SIET data showed an obvious Na+/H+ antiport in salinized cells; Meanwhile, NaCl stress caused a net K+ efflux, because of the salt-induced membrane depolarization. H2O2 levels, upwards regulated by salinity, contributed to ionic homeostasis, because H2O2 restrictions by DPI or DMTU caused an enhanced K+ efflux and decreased Na+/H+ antiport activity. NaCl induced a net Ca2+ influx and a subsequent rise of free Ca2+ in the cytosol ([Ca2+]cyt), which is involved in H2O2-mediated K+/Na+ homeostasis in salinized P. euphratica cells. NaCl, Cl- (choline Cl) and Na+(Na2SO4) caused a net H+ influx, which was presumably able to trigger the production of stress signals. When callus cells were pretreated with inhibitors of the Na+/H+ antiport system, the NaCl-induced elevation of H2O2 and [Ca+]cyt was correspondingly restricted, leading to a greater K+ efflux and a more pronounced reduction in Na+/H+ antiport activity. Results suggest that the PM H+-coupled transport system mediated H+ translocation upon salt treatment and brought about an alternation of pH. The pH variation triggers the stress signalling of H2O2 and Ca2+, which results in a K+/Na+ homeostasis via mediations of K+ channels and the Na+/H+ antiport system in the PM of NaCl-stressed cells. Accordingly, a signalling pathway in the response of P. euphratica cells to ion-specific effects is proposed.5. We investigated the cellular response to osmotic effects and an extracellular ATP (eATP) signaling pathway was established in P. euphratica. It is well known that eATP plays a versatile signaling role in animals, and now emerging evidence shows that it regulates higher plant growth, development and biotic responses. Whether eATP is involved in plant salinity sensing and adaptation is still unknown. Thus, using callus cells of P. euphratica, we attempt to clarify this issue in the present study. NaCl (200 mM) and iso-osmotic mannitol induced a rapid increase of ATP level in extracellular medium within 20 minutes. Hexokinase and glucose system (H-G system) could hydrolysis ATP rapidly and thus blocked the salt-and mannitol-induced elevation of eATP. Pharmacological studies show that eATP plays a regulating role the salt resistance of P. euphratica. Application of antagonists of animal PM P2 receptors and H-G system significantly decreased the cell viability in stressed P. euphratica cells, but enhanced H2O2 accumulation after exposure of 24 h. Under salt stress conditions, the treatment of suramin (P2 receptor antagonist) and H-G system increased Na+ accumulation in the cytoplasma but decreased Na+ compartmentation in the vacuole. Meanwhile, the inhibitors of eATP (suramin, H-G system) enhanced K+ efflux and PM depolarization in salt-stressed P. euphratica cells. After the application of antagonist of animal PM P2 receptors and H-G system, the early responses of H2O2 and Ca2+ induced by NaCl and mannitol were impaired, whereas there were no corresponding changes in H+ fluxes. Therefore, our results suggests that NaCl (osmotic effect) induced a release of endogenous ATP, which is perceive by purinoceptors in the PM, leading to the induction of downstream signals, e.g. H2O2 and cytosolic Ca2+, that are required for the regulation of K+ and Na+ transporters and antioxidant defence. Consequently, K+/Na+ and ROS homeostasis of P. euphratica were maintained during a prolonged period of salt stress.6. The physiological mechanism of PCD that induced by eATP was explored.. It has shown that eATP plays a crucial role in mediating the salinity tolerance of P. euphratica cells. However, the NaCl-induced eATP was a transient response and eATP returned to pretreatment levels after 20 min of salt and mannitol treatment. We suppose that eATP may exert adverse effects on the woody species since an excessive eATP induces PCD in animal cells. In our study, exogenously applied ATP (high dose,0.5 to 2 mM) resulted in a dose- and time-dependent reduction of viability and the agonist-treated cells displayed hallmark features indicative of PCD, such as cytoplasmic shrinkage, chromatin condensation and DNA fragmentation. A sequence of events accounting for ATP-induced PCD is proposed as evidenced using a variety of pharmacological agents. Extracellular ATP (eATP) caused an elevation of Ca2+ in the cytosol ([Ca2+]cyt), resulting from a transient influx of Ca2+ across the plasma membrane (PM) and a subsequent release of Ca2+ from the vacuole. The long-term sustained [Ca2+]cyt resulted in an evident Ca2+ uptake in the mitochondria, leading to a H2O2 accumulation therein. Noteworthy is that P. euphratica exhibited an increased mitochondrial transmembrane potential (△Ψm) and the release of cytochrome c took place without the opening of permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, which is essential for the activation of caspase-like proteases and the subsequent PCD execution, was found to be correlated with the increased△Ψm. NO is implicated as a downstream component of [Ca2+]cyt but plays a negligible role in eATP-stimulated cell death. We speculate that ATP is assumed to bind P2-like receptors in the PM, leading to the induction of downstream intermediate signals because the proposed sequence of events in PCD signaling chain were terminated by an animal P2 receptor antagonist suramin.In conclusion, at tissue and cellular levels, our data confirmed that the salt tolerance of P. euphratica is partly due to its strong ability on K+/Na+ homeostasis control. The Na+ extrusion is mainly ascribed to the strong activity of PM Na+/H+ antiport system (PM H+-ATPase and Na+/H+ antiporter) and thus, P. euphratica could restrict the radial transport of Na+ and decrease the accumulation of Na+ in the shoots and leaves. The maintenance of K+ homeostasis is mainly due to the higher activity of PM H+-ATPase, which decreased the magnitude of PM depolarization and consequently reduced the K+ loss through depolarization-activated K+ channels. At cellular level, the putative PM ATP receptors and H+-coupled ion transporters could sense osmotic and ionic effects of NaCl, respectively, and then independently activates H2O2 and Ca2+ signaling pathways. H2O2 and Ca2+ contribute to the up-regulation of K+- and Na+-related antiport system and ion channels in the PM and tonoplast, leading to a K+/Na+ homeostasis in salinised cells. In P. euphratica cells, the NaCl-induced release of ATP was hydrolysed by ecto-apyrase and the occurrence of eATP-induced PCD was avoided during a prolonged salt treatment. Finally, P. euphratica cells could survive the saline conditions under a long-term of salt stress.