The Molecular Mechanism Of AabZIP1 In The Regulation Of Sesquiterpenes Biosynthesis And Drought Tolerance In Artemisia Annua

Artemisia annua is a medicinal herb that has a long history of application in Chinese traditional medicine,which is rich in terpenoids mainly composed of sesquiterpenes.Artemisinin,a sesquiterpene lactone widely used in malaria treatment,was discovered in A.annua plants.In addition,other sesquiterpenes in A.annua have important physiological and ecological functions in response to biotic or abiotic stress,attracting pollinators and biological defense,playing important roles in plant defense and are valuable in industries of fine chemicals and pharmaceuticals.Abscisic acid(ABA)is an important endogenous hormone for plants to cope with abiotic stresses such as drought.In addition,it can also induce the biosynthesis of a variety of plant secondary metabolites.Previous research has shown that Aab ZIP1 is an important regulator linking the ABA signaling pathway and artemisinin biosynthesis in A.annua.Aab ZIP1 can directly binding to the promoters of artemisinin biosynthesis genes ADS and CYP71AV1 and activating their transcription,thereby promoting artemisinin biosynthesis.But it is not known whether Aab ZIP1 can regulate the transcription of DBR2 and ALDH1,another two genes involved in artemisinin biosynthesis.In this study,it was found that the biosynthesis of artemisinin,?-caryophyllene,cedrol and Germacrene A and the tolerance of drought stress were significantly increased by Aab ZIP1 overexpression.To further improve the theoretical model of Aab ZIP1 regulating the biosynthesis of artemisinin;to reveal its molecular mechanism regulating the biosynthesis of non-artemisinin sesquiterpenes and enhancing the drought resistance of A.annua plnats,this study uses comprehensive methods to carry out a series of work concerning gene cloning,expression analysis,protein-DNA binding,secondary metabolism analysis and drought resistance identification of transgenic plants.The results are as follows:1.Aab ZIP1 positively regulates artemisinin biosynthesis through the Aab ZIP1-Aa MYC2-DBR2/ALDH1 transcriptional regulatory moduleIn this study,we established a multilayer regulatory network of the Aab ZIP1-Aa MYC2-DBR2/ALDH1 module to regulate artemisinin biosynthesis.Dual-LUC assays showed that Aab ZIP1 enhanced the transcriptional activity of DBR2 and ALDH1 promoters in tobacco leaves.However,Y1 H results indicated Aab ZIP1 did not bind to any of the ABRE cis-elements in DBR2 and ALDH1 promoters.Based on these results,we infer that Aab ZIP1 regulates the expression of DBR2 and ALDH1 may require other transcription factors.Aa MYC2 is a key JA responsive TF that has been shown to regulate artemisinin biosynthesis.In addition,the expression of Aa MYC2 can also be induced by ABA treatment.It has been reported that Aa MYC2 upregulates the expression of CYP71AV1 and DBR2 by binding to their promoters,thus promoting artemisinin biosynthesis.In this study,four ABRE cis-elements in Aa MYC2 promoter were identified through promoter prediction software.Y1 H and EMSA results showed that Aab ZIP1 could directly bind to the ABRE motif in the Aa MYC2 promoter.Dual-LUC assays showed that the transcriptional activity of Aa MYC2 promoter was also activated by Aab ZIP1 in tobacco leaves.In addition,Aab ZIP1-overexpression(OE-Aab ZIP1)transgenic lines exhibited 3.1-5.1 folds' higher expression level of Aa MYC2 compared to the wild-type.By contrast,the expression levels of Aa MYC2 were decreased by 50.9%-85.2% in RNAi-Aab ZIP1 lines.Taken together,these results indicate that Aab ZIP1 directly activates the transcription of Aa MYC2 by binding to its promoter,thus promoting the expression of DBR2 and ALDH1.The q PCR results showed that the expressions levels of ADS,CYP71AV1,DBR2 and ALDH1 were significantly increased in the OE-Aab ZIP1 transgenic lines compared with the wild-type.By contrast,the expression levels of ADS,CYP71AV1,DBR2 and ALDH1 were significantly suppressed in the RNAi-Aab ZIP1 lines.The results showed that the average yield of artemisinin was 14.05 mg/g DW,and dihydroartemisinic acid was 1.66 mg/g DW in wild-type A.annua.In OE-Aab ZIP1 transgenic plants,the yield of artemisinin was about 19.62-23.76 mg/g DW,dihydroartemisinic acid was about4.08-4.67 mg/g DW.Compared with wild-type plants,the artemisinin yield was increased by 39.6%-69.1%,dihydroartemisinic acid yield was increased by 147.3%-183.0%.In RNAi-Aab ZIP1 transgenic plants,the yield of artemisinin was about 5.27-7.15 mg/g DW,dihydroartemisinic acid was about 0.63-0.81 mg/g DW.Compared with wild-type plants,the artemisinin yield was decreased by 49.1%-62.5%,dihydroartemisinic acid product was decreased by 50.9%-61.8%.Taken together,the above results indicate that Aab ZIP1 enhances the expression of artemisinin biosynthesis genes DBR2 and ALDH1 by directly activating the transcription of Aa MYC2,thereby promoting the biosynthesis of artemisinin.2.Aab ZIP1 regulates the biosynthesis of non-artemisinin sesquiterpenes in Artemisia annuaThe tissue expression patterns of terpene synthase(TPS)genes from A.annua were analyzed according to RNA-seq data.It was found that 29 TPS genes were expressed specifically or at a high level in the trichome.The phylogenetic tree analysis shows that there are 11 TPS genes closely related to the evolution of ADS involved in the artemisinin biosynthesis.Among these genes,Aa CPS,Aa ECS and Aa GAS have been reported to be involved in sesquiterpene biosynthesis,namely ?-caryophyllene synthase(CPS),epi-cedrol synthase(ECS)and germacrene A synthase(GAS).Therefore,Aa CPS,Aa ECS and Aa GAS were selected out as candidate genes to further study whether they are regulated by Aab ZIP1.Dual-LUC results showed that the transcriptional activity of Aa CPS,Aa ECS and Aa GAS promoters were all enhanced by Aab ZIP1,and their activities are increased by9.4,6.4 and 3.7-folds,respectively.Y1 H and EMSA results indicated that Aab ZIP1 can directly bind to Aa CPS and Aa GAS promoters,but not the Aa ECS promoter.In addition,the expression levels of Aa CPS,Aa ECS and Aa GAS were significantly increased in OE-Aab ZIP1 transgenic A.annua,compared to that in wild type plants.OE-Aab ZIP1 transgenic lines produced sesquiterpenes compounds at significantly higher levels than wild-type plants.Compared with wild-type plants,the ?-caryophyllene yield was increased by 57-69%,epi-cedrol yield was increased by 41-52%,germacrene A yield was increased by 37-50%.These results indicate that Aab ZIP1 promotes the biosynthesis of ?-caryophyllene and germacrene A by directly upregulating the transcription of Aa CPS and Aa GAS.In addition,it can also promote the accumulation of epi-cedrol by indirectly activating the transcription of Aa ECS.3.Aab ZIP1 regulates cuticular wax biosynthesis and drought resistance of Artemisia annua.To investigate whether Aab ZIP1 conferred drought resistance in A.annua plants,the OE-Aab ZIP1,RNAi-Aab ZIP1 and wild-type A.annua transgenic plants were subjected to drought tolerance test.After 2 days of drought stress,wild-type A.annua leaves exhibited slight wilting,while the leaves of the OE-Aab ZIP1 A.annua maintained flourishing.After 4 days of drought stress,wild-type leaves exhibited moderate wilting,while the leaves of OE-Aab ZIP1 plants remained green and flourishing.After 6 and 8 days,wild-type plants were severely withered,while OEAab ZIP1 plants showed only moderate wilting.Plants were re-watered at this point(day-8 of drought stress),and after 4 days of re-hydration,the OE-Aab ZIP1 plants recovered to normal condition,while the wild-type plants failed to recover and died from severe water deficiency.By contrast,the RNAi-Aab ZIP1 plants exhibited significantly lower drought tolerance than the wild-type plants.The morphology of cuticular waxes on the transgenic and wild-type A.annua leaves was examined by SEM.Wax crystal deposition was observed in OE-Aab ZIP1 leaves,compared with wild-type plants.By contrast,the RNAi-Aab ZIP1 plants exhibited much less wax deposition than the wild-type plants.Moreover,the contents of cuticular waxes in transgenic and wild-type plants were analyzed by GC-MS.OEAab ZIP1 transgenic lines produced cuticular wax compounds at significantly higher levels than wild-type plants.Compared with wild-type plants,the yield of hexacosanoic acid,heptacosane,hexacosanol and octacosanol were increased by 43%-119%,39%-88%,45%-56% and 43%-63% in three OE-Aab ZIP1 transgenic lines,respectively.By contrast,in the RNAi lines,the yield of hexacosanoic acid,heptacosane,hexacosanol,and octacosanol were decreased by 28%-47%,33%-71%,30%-84% and 65%-93%,respectively,compared with the wild type.The above results indicate that Aab ZIP1 promotes the accumulation of cuticular wax,which would contribute to enhanced drought tolerance in the OE-Aab ZIP1 transgenic A.annua plants.Dual-LUC assays showed that Aab ZIP1 significantly enhanced the transcriptional activity of the promoters of wax biosynthesis genes Aa CER1 and Aa CYP86A1 in tobacco leaves.Y1 H and EMSA results indicated that Aab ZIP1 can directly bind to Aa CER1 and Aa CYP86A1 promoters.q PCR analysis showed the expression levels of Aa CER1 and Aa CYP86A1 were also significantly increased by about 9.3-12.4 and 2.5-4.0 times,respectively,in OE-Aab ZIP1 lines when compared with wide-type plants.In the RNAi transgenic lines,the expression of Aa CER1 and Aa CYP86A1 were 51-83%and 34-65% lower respectively,compared with wide-type plants.Taken together,Aab ZIP1 promotes cuticular wax biosynthesis by directly activating the expression of Aa CER1 and Aa CYP86A1,thereby elevating the drought resistance of A.annua plants.In summary,this study revealed the molecular mechanism of Aab ZIP1 in the regulation of sesquiterpenes biosynthesis and tolerance to drought stress in A.annua,which is important for developing A.annua germplasm resources with high artemisinin content and strong tolerance to environmental stress.