Preliminary Study On The Mechanism Of H₂O₂ And Ca²⁺ Regulating K⁺/Na⁺balance In Pyropia Haitanensis In Response To Salt Stress

Pyropia haitanensis,a kind of warm temperate macroalgae unique to China,has important economic,ecological and research value.Due to the complex and changeable environment in the intertidal zone,P.haitanensis often suffers from salt stress between low tide water loss and high tide water rehydration.Previous studies have shown that K+/Na+ balance plays an important role in salt tolerance of salt-tolerant plants,seaweed and microalgae.It has been found in studies on the mechanism of the phyllodesome in response to salt stress that the P.haitanensis can withstand both 110 and 5 salinity stress by maintaining a K+/Na+homeostasis,but how the P.haitanensis of the P.haitanensis can perceive salt stress and conduct signal transduction remains unclear.Studies have confirmed that Ca2+and H2O2,the second messengers,regulate the response mechanism of plants to salt stress.Therefore,in this study,we took the Z-61 strain of the P.haitanensis as the research object.Firstly,we determined whether the algal body could produce calcium signal and synthesize H2O2 under salt stress by using the non-damage micromeasurement technology.Combined with the pharmacological method of exogenous addition of Ca2+ and H2O2 and scavenger of messenger related channel protein inhibitor and the determination of enzyme activity,the way of P.haitanensis producing calcium signal and H2O2 signal under salt stress was determined.Then,through the detection of dynamic ion transport process and pharmacological experiments,the regulation of stress messenger to maintain K+/Na+balance and the interaction between Ca2+and H2O2 in P.haitanensis was explored.Finally,the similarities and differences of ion transport and signal regulation in response to high salt stress and hyposaline salt stress were analyzed,which provided a new theoretical basis for elucidate the signal transduction mechanism in response to salt stress.The main results are as follows:(1)Under high salt stress,H2O2 regulates the K+/Na+balance of P.haitanensis thalli.The specific results are as follows:P.haitanensis rapidly produces H2O2 through NADPH oxidase and superoxide dismutase,causing Na+efflux.After the removal of H2O2,the efflux of Na+ was significantly weakened,which initially indicated that H2O2 may alleviate Na+toxicity caused by salt stress by enhancing the Na+/H+ reverse transport system.At the same time,H2O2 can also cause significant efflux of K+.After removing H2O2,the efflux of K+is significantly inhibited.This may be due to the depolarization of the plasma membrane caused by H2O2,which leads to the opening of the K+outward channel DA-KORCS,which in turn causes the loss of K+.The results of this study indicate that H2O2 regulates the transportation of Na+ and K+ in P.haitanensis under high salt stress.(2)Under hypersaline salt stress,Ca2+is also involved in the homeostasis regulation of P.haitanensis thalli.Hypersaline salt stress causes a large influx of Ca2+in P.haitanensis.Ca2+can increase the salt tolerance of the thalli by enhancing the antioxidant system and easing the decrease in the maximum photochemical quantum yield Fv/Fm.However,after inhibiting the influx of Ca2+,the efflux of thalli Na+ was significantly inhibited under hypersaline salt stress,indicating that Ca2+played an important role in the efflux of Na+.Combined with the previous research results,it was found that Ca2+ can reverse transport by regulating Na+/H+ The system promotes Na+efflux.At the same time,the calcium signal produced by the thalli under high salt stress can induce NADPH oxidase to produce reactive oxygen species,and the production of H2O2 can cause a significant influx of Ca2+,confirming the related regulatory relationship between the two stress messengers.But what is interesting is that,unlike the effect of H2O2,exogenous Ca2+significantly alleviates the K+loss induced by salt stress,which indicates that under hypersaline salt stress,the effects of H2O2 and Ca2+ on K+transport of P.haitanensis thalli may be independent.Therefore,the interaction between the two and the mechanism of regulating the ion balance of P.haitanensis is worthy of further study.(3)Under hyposaline salt stress,the thalli of P.haitanensis can quickly discharge Na+ions to cope with the external hypotonic environment,but can reduce the efflux of K+ to maintain normal physiological metabolism,which is consistent with the previous research results.However,the process of P.haitanensis leaflet excretion of Na+ and K+ under hyposaline salt stress is different from that under high salt,and is not the result of the combined action of plasma membrane Na+/H+ antiporter and plasma membrane H+-ATPase.In addition,under hyposaline salt stress,H2O2 actively regulates the osmotic balance between the inside and outside of the cell by increasing the efflux of Na+,while Ca2+can reduce the loss of K+,thereby maintaining the steady state of high K+/Na+.Therefore,under high salt stress and hyposaline salt stress conditions,P.haitanensis thalli can adopt different ways to maintain ion balance.In summary,the P.haitanensis thalli can quickly activate the stress messenger H2O2 and Ca2+under high salt stress to regulate their homeostasis;there is a clear difference between the ways in which P.haitanensis thalli maintain ion balance under hypersaline salt stress and hyposaline salt stress.This study provides new insights for further clarifying the salt tolerance mechanism of thalli.