Effect And Mechanism Of Hormones On Growth And Rosmarinic Acid Synthesis Of Origanum Vulgare Suspension Cells

Origanum vulgare（O.vulgare）is an herb of the genus Origanum in Lamiaceae,with antibacterial,antioxidant,anti mutation and other biological activities.This plant has various active components,such as aromatic oil,polyphenols,and flavones.Rosmarinic acid（RosA）is a natural phenolic acid compound and a symbolic secondary metabolite in O.vulgare.Plant cell culture is expected to be used in RosA production because of its many advantages,such as short growth cycle,high stability,easy extraction and purification of target substances and so on.In our laboratory,a fine O.vulgare calli line was screened and cultivated,and a stable cell suspension culture was established,from which six polyphenols such as rosmarinic acid-3-O-glucoside,RosA,and methyl rosmarinate were identified.In order to further improve the accumulation of RosA in the suspension cells,the effects of hormones on cell growth and RosA synthesis were studied in the present paper.And the hormones were screened and optimized.Moreover,integrated detection and analysis of transcriptome and metabolome were used to analyze the mechanism of ABA on RosA synthesis in the O.vulgare cells.In addition,the significantly differentially expressed WRKY70 and WRKY75 genes were bioinformatic analyzed.Overexpression vectors were constructed by homologous recombination technique and sequenced,then the function of WRKY70 gene was verified by transient expression.The main results and conclusions were as follows:（1）Effects,screening and optimization of hormones on the growth of O.vulgare suspension cells.The effects and screening tests of seven hormones,including kinetin（KT）,6-Benzylaminopurine（6-BA）,zeatin（ZT）,thidiazuron（TDZ）,2,4-dichlorophenoxyacetic acid（2,4-D）,1-naphthylacetic acid（NAA）,and 3-indolybutyric acid（IBA）,on the growth of cells were carried out by 1+1 model（cytokinin+auxin）.Results indicated that 0.5～2.0mg/L 6-BA and 4.0 mg/L NAA showed the best promotion among cytokinins and auxins respectively.A comprehensive experiment was used to optimize the concentration of 6-BA and NAA in the combined effect.The optimized results showed that the optimized hormone ratio was 0.5 mg/L 6-BA and 4.0 mg/L NAA,and the maximum biomass was 20.25 g/L.（2）Effects,screening and optimization of hormones on the RosA synthesis in the O.vulgare suspension cells.Based on the growth hormone screening and optimization combination test of O.vulgare cells,the effects of ten hormones,including KT,6-BA,ZT,TDZ,2,4-D,NAA,IBA,gibberellin（GA₃）,abscisic acid（ABA）,ethephon（ETH）,methyl jasmonate（Me JA）and spermine（Spm）,on the RosA systhesis in the cultured O.vulgare cells were studied by 2+1 model（“2”is 0.5 mg/L 6-BA+4.0 mg/L NAA;“1”is another hormone added on the basis of growth hormones）.Results indicated ZT,2,4-D,and GA₃inhibited the cell growth and RosA synthesis;KT,TDZ,IBA,ETH and Me JA had no effect on cell growth,but inhibited RosA synthesis;ABA and Spm promoted cell growth and RosA accumulation.Under the action of 0.3 mg/L ABA,the RosA content and RosA yield were12.14 mg/g and 285.98 mg/L,respectively,3.06 times and 3.10 times that of the control group.The optimal ABA adding time was determined on day 0,the RosA content and yield in cells were 12.99 mg/g and 283.54 mg/L,which were 3.18 times and 3.47 times than that of the control.（3）Transcriptome detection and analysis of the ABA treated and untreated cells.Through differential expression analysis,it was found that there were 785 DEGs,including 430 up-regulated genes and 355 down-regulated genes;Through KEGG pathway analysis,it was found that 12 genes were related to ABA synthesis and signal transduction,among which ZEP1,ABA2,PYL4,PP2C,and other genes up-expressed;A total of 11 genes related to RosA synthesis pathway were screened,among which HPPR（TRINITY＿DN36914＿c0＿g1）changed the most significantly,with the expression up-regulated by 4.18 times;In addition,a total of 9 transcription factors are related to ABA signal transduction and phenylpropane synthesis,among which the expression of 7 transcription factors such as WRKY70,MYB61and ERF110 are up-regulated.At the same time,the results of RT-q PCR showed that the expression of 9 genes related to RosA synthesis and regulation,such as 4CL,HPPR,and CYP was basically consistent with the data obtained by transcriptome sequencing.（4）Metabolome detection and analysis of O.vulgare suspension cells trested by ABA.733 and 524 substances were obtained in positive and negative ion mode from the O.vulgare cells,respectively;The metabolite differences between the two groups were studied by PCA and OPLS-DA analysis,which showed that the regulatory effect of ABA was obvious.Through KEGG enrichment analysis,a total of 233 differential metabolites were enriched into 86 metabolic pathways.The pathways related to RosA metabolism including phenylpropanoid biosynthesis pathway,phenylalanine metabolism pathway,tyrosine metabolism pathway and plant hormone signal transduction pathway.In addition,the contents of L-phenylalanine,L-tyrosine,cinnamic acid,p-coumaric acid,4-hydroxyphenylpyruvate,and RosA were increased in varying degrees.（5）Integrated detection and analysis of transcriptome and metabolome were conducted to interpret the regulation mechanism of ABA on the RosA biosynthesis in the O.vulgare suspension cells.Through the analysis of KEGG database,a total of 16 differential metabolites and 37 related transcripts were obtained.Further significant difference analysis showed that 6 metabolites and 11 related transcripts related to RosA synthesis pathway changed significantly,among which,the expression of p-coumaric acid,Cinnamic acid,4-hydroxyphenylpyruvate and RosA and related transcripts showed the same increasing trend.It could be inferred that the RosA synthesis in ABA-treated O.vulgare cells mainly activated the metabolic pathways of L-phenylalanine and L-tyrosine.In the two branch pathways,the up regulation of the expression of 4CL,TAT,HPPR,RAS and CYP led to the increase of the content of downstream metabolites,resulting in the increase of RosA accumulation in O.vulgare suspension cells.（6）Cloning and functional verification analysis of DEDs in O.vulgare:WRKY70 and WRKY75 genes were cloned from O.vulgare by RT-PCR and homologous cloning technique.The overexpression vector of WRKY70 and WRKY75 gene was constructed by homologous recombination technique and sequenced.WRKY70 and WRKY75 proteins are unstable hydrophilic proteins without signal peptides,shear sites,and transmembrane structure.They were mainly composed of random coil,and playing the biological functions in the nucleus.In addition,the function of WRKY70 gene was verified by transient expression technique.The HPLC results showed that the RosA content of the WRKY70 overexpressed leaves was2.76 times than that of the control.The above results showed that the up-regulation of WRKY70 gene could significantly promote the RosA synthesis.In conclusion,ABA could significantly increase the RosA yield of the O.vulgare suspension cells,which was 3.47 times than that of control.Moreover,integrated detection and analysis of transcriptome and metabolome showed that ABA promoted the expression of regulatory genes（such as WRKY70,MYB61,ERF110）,and then increased the expression of structural genes（such as 4CL,HPPR,CYP）,eventually led to the RosA synthesis.WRKY70 and WRKY75 were two significantly up-regulated genes related to RosA biosynthesis.The transient expression experiment indicated that the up-regulation of WRKY70 gene was verified by to deeply understand the expression of WRKY70 gene could significantly promote the RosA synthesis.This study provided a theoretical basis for the regulation of RosA production in O.vulgare cell suspension culture.