The Studies On Molecular Mechanism Of TCP Transcription Factors Regulating Artemisinin Biosynthesis In Artemisia Annua L.

Sweet wormwood(Artemisia annua L.)is traditional Chinese medicine.Artemisinin is the most important medicinal ingredient in A.anuua.The artemisinin-based combination therapy(ACT)has been proposed by the World Health Organization(WHO)to treat malaria.The artemisinin content in the wild A.annua plant is very low.Therefore,to enhance the artemisinin content in A.annua is an international hotspot research.Although exogenous phythormones such as JA(Jasmonate)and ABA(Abscisic Acid)can enhance the artemisinin content,the molecular regulatory mechanism of artemisinin biosynthesis by these phytohormones is largely unknown.Although there are several transcription factors including AaORA(Octadecanoid-derivatie Responsive AP2-domain protein)that regulate artemisinin biosynthesis,but the specific molecular mechanism is still unknown.TCP(TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR)transcription factors are unique in plants,and they play important roles in plant secondary metabolites.TCP regulate flavonoid biosynthesis,chlorophyll and carotenoid content in Arabidopsis.However,whether TCP transcription factors can regulate artemisinin biosynthesis in A.annua needs to be elucidated.In this research,based on the transriptome data and AaORA(Octadecanoid-derivatie Responsive AP2-domain protein)Y2H protein screening,AaTCP14 was identified as a novel interactor of AaORA.Then,the hierarchical cluster analysis of different tissues was used to identify AaTCP15 as the homologous gene of AaTCP14.Various approaches including transgenic A.annua plants analysis,Dual-luciferase(Dual-LUC)assays,Co-immunoprecipitation(Co-IP)assays,Electrophoretic mobility shift assay(EMSA),High performance liquid chromatography(HPLC)analysis were adopted to study the molecular mechanism of hormone-regulated TCP transcription factors that are involved in artemisinin biosynthesis from protein interacting network and transcriptional regulatory cascade layers.The main results are as follows: 1.Jasmonate promotes artemisinin biosynthesis by activating the AaTCP14-AaORA complex in A.annuaIn this research,AaTCP14 and AaORA which is the key regulator in artemisinin biosynthesis could form AaTCP14-AaORA transcriptional complex,the JA-response AaTCP14-AaORA complex was found to positively regulate artemisinin biosynthesis through activating artemisinin biosynthetic genes DBR2(Double Bond Reductase 2)and ALDH1(Aldehyde Dehydrogenase 1).Furthermore,JA signaling repressor AaJAZ8 was identified to repress the AaTCP14-AaORA complex formation by interacting with both AaTCP14 and AaORA,and to decrease activation of the DBR2 promoter,thus impeding artemisinin biosynthesis.By contrast,in the presence of JA,AaJAZ8(Jasmonate Zim-Domain 8)was recognized by JA and degraded by the 26 S proteasome,releasing AaTCP14-AaORA complex which synergistically activated the DBR2 promoter to enhance artemisinin biosynthesis.This research combined positive regulators AaMYC2(Myelocytomatosis Protein 2)and AaGSW1(Glandular Trichome-Specific WRKY 1)and negative regulator AaJAZ8 in artemisinin biosynthesis to build different regulatory networks based on AaTCP14-AaORA complex.This reaearch also elucidated dynamic regulation mechanism of JA signaling in artemisinin biosynthesis,indicated relationships among multiple regulatory factors involved in JA-response to modulate artemisinin biosynthesis,and enriched the JA-regulated network of transcription factors that mediated artemisinin biosynthesis.2.The AaMYC2-AaGSW1-AaTCP15/AaORA transcriptional cascade positively regulates artemisinin biosynthesis in A.anuuaIn this research,it was found that AaTCP15 was a key point in JA and ABA signaling pathway to enhance artemisinin biosynthesis.AaTCP15 formed AaMYC2-AaGSW1-AaTCP15/AaORA transcriptional cascade to regulate artemisinin biosynthesis through JA signaling pathway.AaTCP15 enhances artemisinin biosynthesis through binding to and activating the DBR2 and ALDH1 promoters.JA responsive AaMYC2,AaGSW1 and AaERF1 transcription factors enhance artemisinin biosynthesis through directly activating the key enzyme genes in artemisinin biosynthesis or indirectly activating AaTCP15 activity.AaGSW1 binds to and activates the promoters of AaTCP15 and AaORA which facilitated the formation of AaTCP15-AaORA complex.AaORA enhances artemisinin biosynthesis in multiple layers.On the one hand,AaORA promotes the transcriptional activation activity of AaTCP15 by interaction at the protein level.On the other hand,AaORA activates the AaTCP15 promoter at the transcriptional level.The ABA responsive AabZIP1(Basic Leucine Zipper 1)transcription factor enhances artemisinin biosynthesis through directly activating the key enzyme genes in artemisinin biosynthesis or indirectly activating AaTCP15 activity.This study expands the molecular network of artemisinin biosynthesis through JA and ABA signaling pathway,and lays solid foundation to apply regulatory network to enhance artemisinin content and develop high artemisinin content cultivar.