Study On The Regulatory Mechanism Of Transcription Factors Involved In The Patchoulol Biosynthesis Pathway

Pogostemon cablin(Blanco)Benth.(Patchouli)is one of the most famous genuine medicinal material in Lingnan area.It is one of the ten most widely used Guangdong herbs and the common aromatic for resolving dampness in the traditional Chinese medicine(TCM).Additionally,there is a vastly demand of patchouli oil which extracted from P.cablin as it is an important raw material for pharmaceutical and chemical industries.Patchouli plants are the only natural source of patchouli oil and its main component patchouli alcohol(patchoulol).Patchoulol is a typical sesquiterpenoid secondary metabolite in patchouli with various biological activities,such as neuroprotection,anti influenza virus,anti-various fungi,anti-tumor,anti-inflammatory,etc.Therefore,patchoulol content is assigned as the index for qualifying patchouli medicine and patchouli oil in Chinese Pharmacopoeia.At present,the study on the biosynthesis and dynamic accumulation of patchouli alcohol has attracted much attention.With the application of various detection and analysis technologies of functional genomics,plant metabolism and multi-omics,the secondary metabolism mode of terpenoids and the biosynthesis pathway of patchoulol in P.cablin have been gradually characterized.It is reported that the Patchoulol synthase(PatPTS)is the key enzyme of the biosynthesis and accumulation of patchouli oil,as it can directly catalyze the formation of patchoulol and at least 13 additional sesquiterpene products from farnesylpyrophosphate(FPP).Although the genes involved in patchoulol biosynthesis were identified,attempts in the transcriptional regulation involved in patchoulol biosynthesis pathway have not been performed,especially about the key enzyme PatPTS and the related transcriptional regulation mechanism and interactions have not been revealed.Herein,we established the first full-length patchouli transeriptome and yeast hybrid full-length cDNA library of patchouli for subsequent study on identifying transcription factors(TFs)that regulate the biosynthesis of patchoulol.A series of TFs were identified as PatPTS promoter-binding proteins by yeast one-hybrid(Y1H)screening using the promoter of PatPTS as the bait.Additionally,we also combined plant genetic engineering and metabolism analysis to further study the gene functions of these identified TFs.The methods and conclusions are summarized below.1 Construction and analysis of full-length transcriptome and yeast hybrid library of P.cablin1.1 Establishment and analysis of full-length transcriptome of P cablin via PacBio Iso-SeqThe clean data which obtained for PacBio sequencing were finally assembled into 82,335 full length UniTransModels,of which,65,826(79.95%)UniTransModels were successfully matched to known proteins in at least one out of the seven data bases,including Nr,SwissProt,KE GG,KOG,GO,Nt,and Pfam.Among them,103 transcripts were annotated as 16 encoding enzymes involved in patchouli alcohol biosynthesis,and 3,176 TFs belonging to 90 different families were also annotated.1.2 Construction and characterization of yeast hybrid cDNA library of P.cablinA high quality yeast hybrid cDNA library of patchouli was constructed using CloneMiner II cDNA Library Construction Kit(Invitrogen).Based on the plating assay,the average titer of the library was 4.3×106 CFU/mL,and the total number of colony-forming units(CFU)was 8.6×106.The recombination rate of the cDNA library was calculated to be>95%,indicating that the yeast hybrid cDNA library could be used for further research.2 Screening and analysis of TFs that binding to PatPTS promoter2.1 Cloning and analysis of PatPTS and its promoterBased on the full-length transcript of PatPTS from the transcriptome,we have successfully obtained 1659 bp ORF,3392 bp gDNA and 854 bp promoter sequences of PatPTS.It is indicated that that promoter contained many cis-elements related to TFs binding,tissue-specific,stress,hormone,and light response genes by searching the PlantCARE and New PLACE databases.2.2 Screening and identification of TFs that bind to PatPTS promoterIn order to isolate genes encoding proteins that bind to the PatPTS promoter,the P.cablin cDNA library was screened by using yeast one-hybrid system.After sequences annotation and Y1H repeated verification,6 transcription factors including PatDREB,PatERF061,PatSWC4,PatASIL2,PatGATA17 and PatGIS2 were identified which bind to the PatPTS promoter.3 Expression and function analysis of TFs that regulate patchoulol biosynthesis3.1 Cloning and transcriptional regulation analysis of MYC2 TFs in P.cablin(1)Previous studies also indicated that MYC2 TFs play essential roles in plant terpenoid biosynthesis.Based on the full-length sequences of MYC from the P.cablin transcriptome,we successfully clone 8 MYC genes and bioinformatic analysis showed that the proteins of PatMYC2a,PatMYC2b1 and PatMYC2b2 contain highly conserved domains of MYC transcription factors,including bHLH-MYC-N,bHLH-Zip,and ACT-like.Subcellular localization and yeast-hybrid assays showed that these three PatMYC2s are nuclear proteins and had transcriptional activation activities.Furthermore,bHLH-MYC-N was found to be the transcriptional activation functional domain of PatMYC2a and PatMYC2b2.RT-qPCR revealed that PatMYC2a,PatMYC2bl and PatMYC2b2 were expression in these tissues and the expression pattern of these three PatMYC2s were similar after temperature and light treatment(2)Dual-luciferase(dual-LUC)assays in Nicotiana benthamiana leaf cells indicated that PatMYC2a and PatMYC2b2 could activate the activity of PatPTS promoter.Transient overexpression of PatMYC2a or PatMYC2b2 could increase the patchoulol production in P.cablin leaves by up-regulating the expression level of PatPTS and most genes in MVA pathway,including PatIPPI,PatMVD,PatPMK,PatHMGS and PatAACT.3.2 Regulatory mechanism study of two DREB transcription factors,PatDREB and PatERF061,governing patchoulol biosynthesis(1)Two members of AP2/ERF family,PatDREB and PatERF061,which identified through Y1H screening,both were contained the AP2 conserved domain that comprised of about 60 amino acids,and belonged to DREB subfamily.PatDREB belongs to DREB-A4 group,while PatERF061 belongs to DREB-A6 group.PatDREB was localized to the nucleus,while PatERF061 was mainly expressed in the nucleus and cytoplasm.RT-qPCR revealed that PatDREB and PatERF061 were expressed in all tissues of patchouli and the expression level were induced by MeJA,but their expression patterns were different.(2)Yeast two hybrid(Y2H)and Bimolecular Fluorescent Complimentary(BiFC)assays revealed that both PatDREB and PatERF061 interact with PatJAZ4 and PatERF061 was validated that could coexpressed in nucleus with PatJAZ.Dual-LUC assays showed that both PatDREB and PatERF061 could significantly activate the promoter of PatPTS.To test whether PatJAZ4 affects the transcriptional activation activity of PatDREB and PatERF061,we performed dual-LUC assays in the present or absent of MeJA in N.benthamiana leaf cells.The results indicated that the transcriptional function of PatERF061 was negatively regulated by PatJAZ4,and also demonstrate that MeJA can reverse the PatJAZ4-mediated interference with the transcriptional function of PatERF061.Exogenous MeJA could repress the transcriptional activity of PatDREB,while PatJAZ4 had no obvious effect on that.(3)Transient overexpression in the patchouli leaf showed that PatDREB and PatERF061 could increase the patchoulol production by up-regulating the expression of genes in the biosynthesis pathway.In accordance with the dual-luc assays' results,transient co-overexpression of PatERF061 and PatJAZ4 in P.cablin leaves reduced the content of patchoulol compared with PatERF061 overexpressed plants.3.3 PatSWC4,a MYB-related transcription factor,positively regulates patchoulol biosynthesis in P.cablin(1)PatSWC4,which identified by Y1H screening,belongs to the MYB_related TF family and contains two specific domains including the SANT/Myb-like domain of DAMP 1 and DNA methyltransferase 1-associated protein(DMAP1).PatSWC4 is a nucleus localized protein,it expressed in all organs tested and its expression level could induce by MeJA.Y1H and dual-LUC assays revealed that PatSWC4 could bind to the promoter of PatPTS gene and increase its transcriptional activity,and MeJA could significantly enhance this activation.Y2H assay showed that PatSWC4 could interact with PatJAZ4 and PatJAZ11,respectively.Dual-LUC assay further indicated that the transcriptional function of PatSWC4 was negatively regulated by PatJAZ4 and MeJA could reverse PatJAZ4-mediated interference with the transcriptional function of PatSWC4.Transient overexpression of PatSWC4 gene in P.cablin leaves markedly increased the production of patchoulol,and RT-qPCR analysis further revealed that genes in the patchoulol biosynthesis pathway were significantly up-regulated at the transcriptional level.(2)Y2H and dual-LUC assays indicated that PatSWC4 could interact with PatDREB and PatERF061 respectively to form a complex.The complex could activate the promoter activity of PatPTS,while was repressed by MeJA.PatJAZ4 could respectively interact with PatSWC4,PatDREB,and PatERF061.Their interactions repressed the activation activity of PatPTS promoter,while could be eliminated or reversed by MeJA.Transient co-overexpression of PatSWC4-PatDREB and PatSWC4-PatERF061 respectively could significantly promote the production of patchoulol.3.4 PatASIL2,a trihelix transcription factor,regulates patchoulol biosynthesis in P.cahlin(1)PatASIL2 was a member of the SIP1 clade of trihelix family that contains one trihelix DNA binding domain in the N-terminal region and one a-helical domain in the C-terminal.Bioinformatics analysis revealed a Myb/SANT-like DNA-binding domain(pfam13837)and two putative nuclear localization signals(NLSs)in PatASIL2.PatASIL2 is a nuclear-localized protein and expressed in all organs tested.The expression level of PatASIL2 was induced by MeJA.Y1H and dual-LUC assays indicated that PatASIL2 could bind to the promoter of PatPTS and repress its transcriptional activity,and this repression could be eliminated by MeJA.Transient overexpression of PatASIL2 could significantly promote the production of patchoulol,and qRT-PCR analysis further revealed that most genes in the patchoulol biosynthesis pathway were significantly up-regulated at the transcriptional level,while the expression of PatPTS was down-regulated.(2)Y2H and BiFC assays indicated that PatASIL2 could interact with PatDREB and PatERF061,respectively.Transient co-overexpression of PatASIL2-PatDREB and PatASIL2-PatERF061 respectively could increase patchoulol production in P.cablin.3.5 Two zinc finger proteins may regulate patchoulol biosynthesisTwo zinc finger proteins,PatGATA17 and PatGIS2,were identified by Y1H screening.PatGATA17 belongs to Zf-GATA TF family,which could bind to the promoter of PatPTS and activate its transcriptional activity,thereby promoted patchoulol production.In addition,PatGATA17 is a nuclear-localized protein with transcriptional activation activities,whose expression level is higher in the root and mature leaves and could be induced by MeJA and low tempreture.PatGIS2 is a member of Zf-CCHC family,which also could target the PatPTS promoter and repress its transcriptional activity,thereby decreased patchoulol production.In conclusion,this study constructed the first full-length transcriptome and yeast hybrid library of P.cablin.Six TFs were identified as PatPTS regulators by Y1H screening using PatPTS promoter as the bait and their regulation functions in patchoulol biosynthesis were further revealed.PatDREB,PatERF061,PatSWC4,and PatASIL2 were found to be MeJA-responsive factors,and might act as the downstream transcription factors in the JA signal pathway.Protein interaction indicated that the two DREBs could form transcriptional activator complexes with MYB-like TFs(PatSWC4 and PatASIL2),respectively,that modulate patchoulol biosynthesis.At the same time,PatGATA17 and PatGIS2 were identified as Zinc-finger proteins which participate in patchoulol biosynthesis.Our findings not only first identify a series of TFs that participate in the JA-induced biosynthesis of patchoulol but also establish a foundation for future studies on the regulatory network governing patchoulol biosynthesis and improving the patchoulol production through genetic and metabolic engineering.