The Role Of Extracellular ATP-Mediated Salt Signaling In The Regulation Of K~+/Na~+ Homeostasis In Two Poplars With Contrasting Salt Tolerance

Soil salinity together with secondary salinization are one of the most serious problems for agro forestry and ecological environment.It is of particular interest for exploring salt-tolerance mechanism and finding reliable biotechnologies for enhancing plant salt adaptation.Maintenance of intracellular K+/Na+ homeostasis is crucial for plants to adapt to saline environments.Increasing the Na+ efflux and reducing the K+loss is the main strategy for mediating K+/Na+ homeostasis under salt stress.The response to salt stress is regulated by a series of complex signaling networks,such as Ca2+,H2O2,NO,ethylene,and H2S.Studies have shown that extracellular ATP(eATP)can be involved in mediating the ion homeostasis of salt-tolerant Populus euphratica cells under salt stress.However,whether eATP mediates K+/Na+ homeostasis in salt-sensitive poplar and plant tissue under salt stress is still unknown.Moreover,the interaction between eATP and signal molecules on the effect of K+/Na+ homeostasis is still unclear under the salt stress.In this study,at tissue and cellular levels,we attempted to clarify whether eATP mediates K+/Na+homeostasis in two contrasting poplar species(a salt-resistant Populus euphratica and a salt-sensitive Populus popularis)differing in salt tolerance.Roots of the two species were used as a model system for investigating the interaction between eATP and signal molecules on the K+/Na+ homeostasis of poplar under salt stress.In this study,we used the noninvasive micro-test technology(NMT)to record the effect of eATP on NaCl-altered Na+,H+,and K+ fluxes in callus cells and roots of salt-resistant and salt-sensitive poplar species.Similarly,through the dynamic changes of Na+and K+fluxes,the interaction between eATP and signal molecules on regulation of ion homeostasis was still be found in salt-stressed poplars.The main experimental results and conclusions are as follows:1.eATP mediates cells and roots K+/Na+ homeostasis in two contrasting poplar species under NaCl stress.(1)NaCl(100 mM NaCl)resulted in a Na+ efflux and a correspondingly increased H+ influx in P.euphratica cells and roots,but the effect was not seen in P.popularis.ATP(50 μM)enhanced exchange of Na+ with H+ in salt-stressed cells and roots of two species,especially in P.popularis.Pharmacological experiments showed that the ATP-stimulated Na+ efflux and H+ influx were significantly inhibited by amiloride(a Na+/H+ antiporter inhibitor)or sodium orthovanadate(a plasma membrane H+-ATPase inhibitor),indicating that the ATP induction of Na+ extrusion resulted from an active Na+/H+ antiport across the plasma membrane(PM).Non-hydrolysing analogues of ATP,ATPyS(50 μM),produced an effect similar to that of the hydrolysable form.However,ADP-and AMP-stimulated cells and roots exhibited behaviors different from those invoked by ATP and ATPyS treatments.eATP signaling in Na+homeostasis was blocked by the antagonists of animal P2 receptors,PPADS and suramin.Moreover,ATP-stimulated Na+ extrusion in NaCl stressed cells and roots were inhibited by LaC13(an inhibitor of Ca2+-permeable channels)and DPI(an inhibitor of PM NADPH oxidase).Of note,exogenous H2O2 and Ca2+application markedly increased the Na+efflux induced by eATP in NaCl treated P.euphratica and P.popularis roots,indicating that ATP signalling was positively mediated via second messengers,H2O2 and Ca2+,in the two poplars differing in salt tolerance.(2)NaCl accelerated K+efflux in the two species.For cells,a more pronounced effect was found in the salt-sensitive poplar.The salt induced K+ efflux was markedly restricted by the K+ channel blocker TEA(tetraethylammonium chloride)and was enhanced by plasma membrane H+-ATPase inhibitor,sodium orthovanadate,indicating that the K+efflux is mediated by depolarization-activated outward rectifying K+ channels and non-selective cation channels.Exogenously applied ATP markedly reduced the salt-induced K+efflux in the two poplars.ATP benifited poplar cells and roots,especially the saltsensitive P.popularis,in maintaining K homeostasis under external salinity.This was likely the result of activated H+pump in the PM,which restricted the K+efflux through the inhibition of depolarization-activated K+ channels in both species.Similarly,PPADS and Suramin inhibited the regulation of K+homeostasis by eATP.Moreover,ATP-stimulated reduction of K loss in NaCl stressed cells and roots were inhibited by LaC13 and DPI.Moreover,exogenous H2O2 and Ca2+ application markedly decreased the K+ efflux induced by eATP in NaCl treated P.euphratica and P.popularis roots,indicating that eATP signalling was positively mediated via H2O2 and Ca2+,in the two poplars differing in salt tolerance2.eATP interacts with salt signals to regulate K+/Na+ homeostasis(1)Clear interactions occur between eATP and stress signals to accelerate Na+ homeostasis in P.euphratica and P.Popularis roots under salt stress.In the NaCl-treated roots,the ATP-increased Na+extrusion was enhanced by Ca2+and H2O2.eATP in return promoted the effect of Ca2+ and H2O2 in Na+efflux although a less pronounced effect was observed in Ca2+-treated P.euphratica roots.However,the ATP enhancement was diminished by LaCl3 or DPI.Collectively,the results indicated that eATP interacted with H2O2 and Ca2+signaling to maintain Na+ homeostasis in salt stressed poplar roots.eATP was assumed to bind P2-like receptors in the PM,leading to an increase in H2O2 and a transient elevation in[Ca2+]cyt.[Ca2+]cyt enhanced Na+extrusion through the Ca2+-SOS3-SOS2-SOS1 signaling pathway ATP-elicited Na+efflux in salt-treated roots of P.euphratica and P.popularis was enhanced by the external application of ACC(the ethylene precursor)but markedly inhibited by AgNO3(ethylene inhibitor),indicating that ethylene contributed to the ATP induction of Na+ efflux in salinized poplars Moreover,the Na+ extrusion elicited by ACC was enhanced by ATP.Under NaCl stress,the H2S donor,NaHS,accelerated Na+ efflux treated with ATP in the two species,and vice versa.The Na+ efflux induced by ATP was obviously inhibited by HT(a scavenger of H2S),in both species.This indicated that the gas signal molecule,H2S,interacts with ATP to accelerate Na+ exclusion in salt-stressed P.euphratica and P.popularis roots.We speculated that H2S interacted with ATP to enhance Na+ efflux through the activation of SOS pathway in two contrasting poplar species under salt stress.However,the ATP-elicited Na+ efflux in salt-treated poplar roots was decreased after the application of SNP,and vice versa,indicating that NO is redundant in the presence of eATP,which already exert a more pronounced effect on Na+/H+ antiport.(2)In the two poplar species,the reduction of K+ efflux elicited by ATP were enhanced by supplement of exogenous Ca2+,H2O2,ACC,or NaHS in P.euphratica and P.popularis roots,and vice versa.However,the agonist-mediated responses were blocked with pharmacological agents,LaC13,DPI,HT,or AgNO3.The effect of eATP on K+homeostasis was mediated by cytosolic Ca2+and H2O2.Ca2+enhanced the activation of PM H+-ATPase and decreased K+ loss through K+ permeable channels.In addition,H2O2 is also thought to up-regulate activity of PM H+-ATPase under NaCl stress.Therefore,eATP interacts with H2O2 and Ca2+and the signaling cascades leads to up-regulation of PM H+-ATPase under salt stress,then decreased K+loss through depolarization-activated K+channels.The ethylene-and H2S-mediated responses were strengthened by ATP and vice versa.This indicates that ATP interacts with ethylene and H2S to activate H+ pumping activity,thus relieve the NaCl-induced PM depolarization and limit the efflux of K+.In this study,an additive effect was not observed in the SNP-and eATP-treated roots.The K-efflux in SNP-treated was lowered by the addition of ATP,but SNP addition increased K+efflux in ATP-treated P.popularis.Thus it can be inferred that NO was redundant in the presence of eATP.as the predominant signal eATP had a more effective effect to restrict the salt-elicited K+ efflux.In conclusion,our data confirmed that eATP mediated K+/Na+ homeostasis at cellular and organ levels under salt stress.The eATP effect was more pronounced in salt-sensitive P.popularis than in salt-resistant P.euphratica.The beneficial effects of eATP were presumably resulted from the enhanced plasma membrane Na+/H+ antiport driven by the H+-ATPase.Noteworthy,eATP interacts with a variety of salt signals to regulate root K+/Na+ homeostasis under salt stress.eATP enhanced the K+/Na+homeostasis mediated by Ca2+,H2O2,ethylene,and H2S under a short-term salinity,and vice versa.