The Function Of Plasma Membrane H~+-ATPase Gene SmPHA4 In Salvia Miltiorrhiza

H⁺-ATPase is a functional protein widely present in the plasma membrane（PM）and various intimal systems and plays a key role in cell metabolism.Cytoplasmic acidification of plant cells has been reported to be an important signal that is closely related to PM H⁺-ATPase.Our previous study found that salicylic acid（SA）induced cytosolic acidification and promoted the biosynthesis of phenolic acid compounds.However,the PM H⁺-ATPase-encoding genes have not yet been reported in Salvia miltiorrhiza,and their functions,especially those regarding the regulation of secondary metabolism,are not clear.Therefore,in this study,PM H⁺-ATPase-encoding genes were screened using the transcriptome and genome databases of S.miltiorrhiza.SmPHA4,a PM H⁺-ATPase-encoding gene,was successfully cloned.To explore its function,SmPHA4 was genetically transformed into Saccharomyces cereviscera,Arabidopsis thaliana,and S.miltiorrhiza hairy roots using genetic engineering techniques and was either subjected to overexpression or gene silencing.The main research contents and results are as follows.1.Based on the transcriptome and genome databases of S.Miltiorrhiza,nine PM H⁺-ATPase isoforms were identified and named SmPHA1?SmPHA9.Their coding sequences（CDS）were 1500–4000 bp long.The evolutionary tree analysis showed that the genetic relationship between PM H⁺-ATPase family members in S.miltiorrhiza was relatively dispersed.Sequence alignment analysis revealed that the middle functional region of the protein sequences of all the analyzed species were highly similar,indicating high conservation of the protein sequences among different species.Using the online transmembrane prediction analysis software,the PM H⁺-ATPase family in S.miltiorrhiza was found to be rich in transmembrane structures.Additionally,tissue expression analysis indicated that the PM H⁺-ATPase family exhibits tissue-discrepancy expression;the expression of the PM H⁺-ATPase family was the highest in roots,and successively decreased in the stem and mature and tender leaves of S.miltiorrhiza.Interestingly,the relative expression level of SmPHA4 was the highest in flowers,while that of SmPHA3/4/5/7 was more than 1000 times greater than the reference gene in roots.In addition,the expression of SmPHA3/6 was down-regulated whereas that of SmPHA4/5/7 was up-regulated following exogenous SA induction.Based on the high expression of SmPHA4 in roots and flowers of S.miltiorrhiza,SmPHA4 was used as the target gene,and its full-length CDS was obtained by using segmented amplification and seamless cloning.Furthermore,the subcellular localization analysis showed that the SmPHA4 protein was located in the cell membrane.2.The recombinant vector p YES2-SmPHA4 was constructed using the seamless cloning method and was successfully introduced into S.cerevisiae INVSC1.Its resilience was analyzed based on the response phenotype of recombinant S.cerevisiae to stress factors.In recombinant S.cerevisiae,SmPHA4 expression significantly reduced the salt resistance and antioxidant capacity;however,it significantly improved high（47°C）and low（-20°C）temperature tolerance.These results showed that SmPHA4 may be involved in osmotic regulation of cells.3.Transgenic Arabidopsis plants were obtained using the inflorescence impregnation method.Compared with the wild type,the expression of SmPHA4 not only reduced plant height and life cycle,but also decreased tolerance to drought.Transgenic S.miltiorrhiza hairy roots were obtained by Agrobacterium rhizogenes ATCC15834-mediated transformation.The findings showed that overexpression of SmPHA4 significantly reduced peroxidase（POD）activity but had no significant effect on superoxide dismutase（SOD）,polyphenol oxidase（PPO）,and Na⁺/K⁺-ATPase activities.In addition,POD,PPO,and Na⁺/K⁺-ATPase activities increased significantly,whereas SOD activity was downregulated upon the inhibition of SmPHA4 expression.These results indicated that SmPHA4 is involved in the regulation of plant adaptation to environmental stress.4.The hairy roots of SmPHA4-OE lines were light yellow whereas those of SmPHA4_1468-1768-RNAi lines were red,and their culture medium and resulting solution after extraction with 70%methanol also showed the corresponding colors.Overexpression of SmPHA4 not only increased the biomass of hairy roots but also increased their water content.The biomass of hairy roots decreased,but the p H of the hairy root culture medium significantly increased upon the inhibition of SmPHA4 expression.These results indicated that SmPHA4 is involved in regulating the growth of hairy roots of S.miltiorrhiza.5.The content of secondary metabolites in transgenic hairy roots was analyzed using high-performance liquid chromatography（HPLC）.The results showed that the accumulation of salvianolic acid B and rosmarinic acid was significantly increased,whereas that of tanshinones was significantly decreased in SmPHA4-OE lines.The biosynthesis of rosmarinic acid and salvianolic acid B was inhibited,but the tanshinone content was significantly increased upon the inhibition of SmPHA4 expression.These results indicate that SmPHA4 regulates the biosynthesis of secondary metabolites in S.miltiorrhiza.6.The transcription of SmMYC2a and SmMYC2b was upregulated in SmPHA4-OE lines and downregulated in SmPHA4_1468-1768-RNAi lines.The promotional effect of SmPHA4 on the biosynthesis of rosmarinic acid and salvianolic acid B may be closely related to the upregulated expression of SmMYC2a and SmMYC2b.Similarly,SmPHA4 might negatively regulate tanshinone biosynthesis in hairy roots of SmPHA4_1468-1768-RNAi lines by upregulating the expression of related synthase genes in the tanshinone metabolic pathway,such as DXS2,AACT1,KSL1,and SmCYP76AH1.According to the ions dynamic transport determined by non-invasive micro-test technology（NMT）,it was found that overexpression of SmPHA4 enhanced H⁺influx and reduced Ca²⁺influx,while downregulation upon the inhibition of SmPHA4 expression promoted H⁺efflux and enhanced Ca²⁺influx.Therefore,the regulation of SmPHA4 on the biosynthesis of secondary metabolites of Salvia miltiorrhiza could be involved in cytoplasmic acidification and Ca²⁺signal transduction by regulating the direction and strength of H⁺and Ca²⁺flux.This study systematically analyzed the function of SmPHA4 and proved that SmPHA4is involved in the regulation of adaptation to adversity,plant root growth,and secondary metabolite biosynthesis.Moreover,it preliminarily clarified the regulation mechanism of secondary metabolite biosynthesis by SmPHA4 in S.miltiorrhiza.The results of this study are crucial for elucidating the functions of PM H⁺-ATPase and provide the theoretical basis for the molecular mechanism underlying the regulation of secondary metabolism in S.miltiorrhiza.In summary,this study highlighted PM H⁺-ATPase functions and provided new insights into candidate genes that may be involved in modulating the biosynthesis of secondary metabolites in S.miltiorrhiza.