Preliminary Analysis On The Mechanism Of Na~+/K~+-ATPase Regulating Salt Tolerance Of Pyropia Haitanensis

Soil salinization has become an important factor hindering agricultural development,and the main harm of high salt stress to crops is the ion toxicity caused by Na⁺.Therefore,the study of Na⁺exclusion system is crucial for understanding the salt tolerance mechanism of plants.Intertidal macroalgae have stronger salinity tolerance than terrestrial plants because they grow in environments with dramatic salinity changes.The study found that in addition to Na⁺/H⁺antiporter SOS1 in plants,intertidal macroalgae,Porphyra/Pyropia,also contains a Na⁺transporter Na⁺/K⁺-ATPase that homologous to animals,which may be one of the key reasons for the stronger salt tolerance of intertidal macroalgae such as Porphyra/Pyropia.However,the research on Na⁺/K⁺-ATPase of macroalgae is still very limited.It has been verified that the Na⁺/K⁺-ATPase of Porphyra yezoensis can improve the salt tolerance of rice,but the molecular mechanism of its response to salt stress is still unclear.Therefore,in this study,the genes encoding the Na⁺/K⁺-ATPase of P.haitanensis were screened by genome,and the salt-tolerant function of the Na⁺/K⁺-ATPase of P.haitanensis was analyzed by bioinformatics,non-invasive micro-test technology,yeast two-hybrid and heterologous expression.The results will help to enrich the molecular mechanism of intertidal macroalgae in response to salt stress and provide new ideas for the breeding of salt-tolerant crops.The main results are as follows:1.The ion fluxes and contents of P.haitanensis under 100‰hypersaline stress were measured by non-invasive micro-test technology（NMT）and induced coupled plasma-optical emission spectrometer（ICP-OES）,respectively,to analyze the role of Na⁺/K⁺-ATPase in maintaining K⁺/Na⁺homeostasis in response to salt stress.The results showed that hypersaline stress led to a significant efflux of Na⁺,indicating that P.haitanensis was actively resistant to salt stress.After the addition of Na⁺/K⁺-ATPase inhibitor,a large amount of Na⁺influxed and K⁺was significantly effluxed,resulting in a significant decrease in K⁺/Na⁺ratio.In addition,after adding inhibitor,the cells shrank seriously,the maximum photochemical quantum yield（Fv/Fm）decreased significantly,the contents of superoxideb（O₂^-）and malondialdehyde（MDA）increased significantly,and the degree of oxidative damage increased.This revealed the important role of Na⁺/K⁺-ATPase in the resistance of P.haitanensis to hypersaline stress,and enriched the diversity of K⁺/Na⁺homeostasis in P.haitanensis under hypersaline stress.2.The full-length sequence of the gene encoding the Na⁺/K⁺-ATPase of P.haitanensis was cloned by PCR amplification,and named PhNKA2.The PhNKA2 gene was 3534 bp in length and encoded 1177 amino acids.It contains four conserved domains:the N-terminal of cation transport ATPase,E1-E2 ATPase,hydrolase and C-terminal of cation transport ATPase,which are typical Na⁺/K⁺-ATPase conserved domains.In addition,the protein contains 69 phosphorylation sites.The hydrolase domain and phosphorylation sites give it ATP binding and hydrolysis functions.E1-E2ATPase is responsible for protein conformational transformation during ions transport.Subcellular localization predicted that PhNKA2 was localized to the plasma membrane and had 9transmembrane proteins.Phylogenetic tree analysis showed that PhNKA2 had high homology with red algae Porphyra umbilicus,Gracilariopsis chorda,Chondrus crispus,Porphyridium purpureum,animal Bufonidae,Drosophila melanogaster and Rattus norvegicus.PhNKA2 was clustered into a branch with Porphyra yezoensis,indicating that PhNKA2 was more conserved in Porphyra/Pyropia.The expression pattern of PhNKA2 under 100‰hypersaline stress was analyzed by real-time quantitative PCR.The results showed that PhNKA2 was significantly induced by hypersaline stress.In addition,the enzyme activity of Na⁺/K⁺-ATPase was also significantly enhanced under hypersaline stress,which indicated that PhNKA2 was involved in the process of response to hypersaline stress from the perspective of gene and enzyme activity.3.Five proteins that interact with PhNKA2 were screened by yeast two-hybrid technology.They are deubiquitinating enzyme（Usp5）,methionine sulfoxide reductase B2（MSRB2）,glycine decarboxylase T protein（GDCST）,dihydropteroate synthase（Dhps）,actin（AC）.The results of q RT-PCR showed that they were all involved in the response of P.haitanensis to hypersaline stress.Among them,Usp5 may reverse the ubiquitination of PhNKA2 through the ubiquitin-proteasome system,so that PhNKA2 is not degraded by the proteasome,thereby functioning;MSRB2 may reduce oxidative damage by reducing methionine sulfoxide（Met SO）to maintain the stability of PhNKA2;GDCST may provide sufficient energy for the driving function of PhNKA2 by enhancing photorespiration and carbon metabolism;PhNKA2 can activate Dhps to maintain the osmotic balance under hypersaline stress by regulating the balance of lysine metabolism.AC provides energy for the conformational variant of PhNKA2 to exert the function of removing Na⁺and preserving K⁺.4.PhNKA2 was transferred into C.reinhardtii to further verify the salt-tolerant function of Na⁺/K⁺-ATPase in P.haitanensis.The results showed that the transcription level of PhNKA2 in C.reinhardtii was significantly up-regulated and maintained at a high level when treated with 225m M Na Cl hypersaline stress for 15 min.The gene expression level reached the peak at 120 min,which was about 5.5 times that of the control group and the the biomass of transgenic C.reinhardtii under hypersaline stress was always higher than that of wild-type C.reinhardtii.In addition,the Na⁺efflux capacity and K⁺influx capacity of transgenic C.reinhardtii were significantly stronger than those of wild-type C.reinhardtii,indicating that PhNKA2 improved the high salt tolerance of C.reinhardtii,which further verified the ability of PhNKA2 to regulate Na⁺efflux and K⁺retention under hypersaline stress.In summary,in this study,the gene PhNKA2 encoding Na⁺/K⁺-ATPase in P.haitanensis was cloned for the first time.It can quickly respond to hypersaline stress and maintain a high K⁺/Na⁺ratio by removing Na⁺and preserving K⁺.In addition,PhNKA2 and its interacting proteins regulate the response of P.haitanensis to hypersaline stress through photorespiration,amino acid metabolism and energy conversion.The maintenance of K⁺/Na⁺homeostasis regulated by PhNKA2 may be one of the important reasons why P.haitanensis has stronger salt tolerance than terrestrial plants and freshwater algae.