Regulation Of Artemisinin Biosynthesis By AabHLH113 Integration Of JA And ABA Signaling

Artemisinin,a sesquiterpene lactone compound extracted from Artemisia annua L.,is the first-line antimalaria drug.Currently,artemisinin still relies on extraction from A.annua,but the content of artemisinin in A.annua is low.Therefore,it is of great value to study the regulatory mechanism of artemisinin biosynthesis,so as to increase the content of artemisinin.Studies have shown that the plant hormones jasmonic acid(JA)and abscisic acid(ABA)promote artemisinin biosynthesis,and these two hormones have a certain synergistic effect in promoting artemisinin biosynthesis.However,less has been reported about how JA and ABA signaling synergistically regulate artemisinin biosynthesis.Therefore,mining and identifying the transcription factors(TFs)induced by JA and ABA in A.annua will further fill the theoretical gap of JA and ABA co-regulation of artemisinin biosynthesis,and provide new candidate genes for the breeding A.annua germplasm with high artemisinin content.In this study,a bHLH-type TF AabHLH113 induced by JA and ABA was identified from A.annua,which plays an important role in integrating JA and ABA signals to regulate artemisinin biosynthesis.In this study,the molecular mechanism of AabHLH113 regulating artemisinin biosynthesis was revealed by molecular biology,biochemistry,and transgenic technology.The main results are:1.AabHLH113 was induced by JA and ABA and highly expressed in artemisinin biosynthesis tissuesFirstly,we analyzed the co-expression of the bHLH genes family with the key enzyme genes of artemisinin biosynthesis in A.annua.We found that six bHLH TF genes showed similar expression patterns to ADS,CYP71AV1 and ALDH1.All of them were highly expressed in buds,flowers,and young leaves where are the main tissues artemisinin produced.Further studies showed that both JA and ABA could significantly promote the expression of AabHLH113.JA and ABA play important regulatory roles in artemisinin biosynthesis.Therefore,it is suggested that AabHLH113 plays an important role in synergistic regulation of artemisinin biosynthesis by integrating JA and ABA signaling.Studies showed that artemisinin is synthesized and accumulated mainly in the glandular hairs of the flowers and leaves of A.annua.In this study,the qRT-PCR analysis showed that AabHLH113 was expressed at a high level in the flowers and leaves of A.annua,which also confirmed the transcriptome analysis results of AabHLH113.In order to further study the tissue localization of AabHLH113,the promoter sequence of AabHLH113 was cloned and p AabHLH113-GUS transgenic A.annua was created.GUS staining indicated that AabHLH113 could be expressed in glandular hairs of A.annua leaves.Subcellular localization experiments showed that AabHLH113 was a nuclear localization gene,which is consistent with the common localization results of TFs.In conclusion,JA and ABA induced AabHLH113 may be involved in regulating artemisinin biosynthesis.2.Overexpression/inhibition of AabHLH113 can promote/inhibit artemisinin biosynthesisIn order to study the biological function of AabHLH113 in artemisinin biosynthesis,transgenic A.annua strains with overexpression(OE-AabHLH113)and inhibition expression(RNAi-AabHLH113)were created.qRT-PCR results showed that the expression levels of AabHLH113 than the control group significantly were increased in OE-AabHLH113 strains,by about 7.25 to 25.71 times.In addition,the expression levels of artemisinin biosynthesis key enzyme genes(ADS,CYP71AV1,DBR2 and ALDH1)were increased by 3.00～3.64,1.85～2.90,1.84～2.86 and 2.46～4.07 times in OE-AabHLH113 strains,respectively.The results of metabolite detection showed that the content of artemisinin and dihydroartemisinic acid were increased by 1.71～2.06 and 1.47～2.23 times in OE-AabHLH113 strains,respectively.On the contrary,the expression levels of AabHLH113 in RNAi-AabHLH113 strains were decreased by about 53～81%compared with the control group.At the same time,the expression of four key enzyme genes also decreased by about 8～44%,12～34%,12～74% and 15～60%,respectively.Analysis of metabolites of RNAi-AabHLH113 A.annua showed that the content of artemisinin and dihydroartemisinic acid were decreased significantly,about 14～36% and 26～53% compared with the wild type,respectively.These results suggest that AabHLH113 plays a positive regulatory role in artemisinin biosynthesis.3.AabHLH113 regulates artemisinin biosynthesis by directly regulating the expression of DBR2 and ALDH1The catalytic reaction mediated by ADS,CYP71AV1,DBR2 and ALDH1 are rate-limiting steps of artemisinin biosynthesis,and the functional genes corresponding to these four enzymes are also important targets for regulation studies of artemisinin biosynthesis.In order to further study the molecular mechanism of AabHLH113 regulating artemisinin biosynthesis,the Dual-LUC(dual-luciferase),Y1H(yeast-one hybrid)and EMSA(Electrophoretic mobility shift assays)assays were executed.The results of Dual-LUC assays showed that AabHLH113 could significantly activate the transcription activities of ADS,DBR2 and ALDH1 promoters in tobacco leaves,and the activities were increased by about2.8,3.8 and 2.0 times compared with the control,respectively.However,AabHLH113 did not activate CYP71AV1 promoter transcription activity.In order to further investigate the mode of action of AabHLH113 in activating the transcription of ADS,DBR2 and ALDH1 promoters,Y1 H and EMSA assays were conducted in this research.The results of Y1 H assays showed that AabHLH113 bind E-box cis-elements on the promoters of DBR2 and ALDH1.However,AabHLH113 could not be combined with ADS promoter any E-box cis-elements.In order to further confirm the binding effect of AabHLH113 on the promoters of DBR2 and ALDH1,EMSA assays were performed.The results showed that AabHLH113 bind to E-box cis-elements of DBR2 and ALDH1 promoters in vitro.In addition,the results of Y1 H and EMSA assays showed that the binding effect of AabHLH113 to these fragments disappeared after the core sequence of E-box cis-elements on the promoters of DBR2 and ALDH1 were mutated,which further verified the specificity of the binding.These results suggest that AabHLH113 directly bind and activate DBR2 and ALDH1 promoters,thereby promoting artemisinin biosynthesis.4.AabHLH113 integrates JA and ABA signaling to regulate artemisinin biosynthesisIn order to investigate how JA and ABA signaling induce the expression of AabHLH113,we evaluated the effects of JA or ABA induced TFs on the transcription activities of AabHLH113 promoter by Dual-LUC assays to screen out potential TFs that regulate the expression of AabHLH113.Dual-LUC assays results showed that JA-induced AabHLH112 and ABA-induced Aab ZIP1 significantly enhanced the transcription activities of AabHLH113 promoter by 6.4 and 6.7 times,respectively.In order to investigate whether Aab ZIP1 and AabHLH112 can directly regulate the expression of AabHLH113.In this research,we analyzed the promoter sequence of AabHLH113 and found that there was an ABRE type G-box(CACGTG)cis-element on AabHLH113 promoter.G-box is a cis-element that can be specifically recognized by b ZIP family and bHLH family TFs.The Y1 H assays results showed that both Aab ZIP1 and AabHLH112 directly bind the G-box cis-element on the AabHLH113 promoter.The EMSA assays also confirmed that Aab ZIP1 directly bind to the G-box cis-element on the AabHLH113 promoter.The assays results after mutation also further verified the specificity of Aab ZIP1 and AabHLH112 binding to the AabHLH113 promoter.The qRT-PCR results showed that the expression levels of AabHLH113 were significantly increased in transgenic A.annua plants overexpressing Aab ZIP1(OE-Aab ZIP1)and AabHLH112(OE-AabHLH112).These results suggest that both AabHLH112 and Aab ZIP1 can directly positively regulate AabHLH113 expressing.In conclusion,AabHLH113 plays a key role by integrating JA and ABA signaling to regulating artemisinin biosynthesis.