Chilling Stress-induced Overexpression Of Artemisinin Biosynthetic Genes And Mechanism Of Ca²⁺-dependent Signal Transduction

Artemisinin is an antimalaria chemical monomer that was isolated from the herbaceousplant Artemisia annua and structurally identified by Chinese scientists,and artemisininderivatives and compound drugs have been recommended by the World HealthOrganization (WHO)as the first-line therapeutical agent for malaria.To explore theroute toward artemisinin overproduction and validate the hypothesis of environmentalstress-mediated artemisinin biosynthesis,we have transcently treated the in vitrocultural A.annua plantlets by low temperature and tested their transcripts ofamorphadiene synthase gene (ADS),cytochrome P450 monooxygenase gene(CYP71AV1)and cytochrome P450 reductase gene (CPR)to investigate the chillingstress-induced expression pattern of artemisinin biosynthetic genes and elucidate themechanism of signal transduction during artemisinin biosynthesis.For this purpose,total RNAs were extracted from the pre-chilled A.annua plantlets,ADS,CYP71AV1 and CPR mRNAs were amplified using RT-PCR.Upon cDNAcloning and sequencing,the sequence data derived from these three genes wereaccessed in GenBank.Determination of ADS,CYP71AV1 and CPR mRNA levels bythe semi-quantitative reverse transcription polymerase chain reaction (SQRT-PCR)hasshown that ADS and CYP71AV1 gene were strongly induced by chilling stress,whileCPR gene was seemingly not significantly affected.Moreover,the chilling-induced expression of ADS and CYP71AV1 genes wassuppressed by Ca²⁺ channel inhibitor and Ca²⁺ chelate,implying that the Ca²⁺-calmodulin (CaM)-mediated signal transduction pathway might be involved in the lowtemperature-induced expression of ADS and CYP71AV1 genes. To further understand the impact of low temperature on the accumulation of artemisinin biosynthetic enzymes,immunoquantification of ADS,CYP71AV1 andCPR by ELISA has indicated that ADS and CYP71AV1 are slightly higher in chillingexposed plantlets than in untreated plantlets and exhibit significant difference (P<0.05),and are much higher in field-grown flowering plants than in untreated plantlets anddemonstrate very significant difference (P<0.05),suggesting that biosynthesis of ADS and CYP71AV1 was not only affected by the environmental conditions,but alsomodulated by the developmental rhythm.In contrast,CPR remains unfluctuationamong the untreated plantlets,chilling-exposed plantlets and field-grown floweringplants,which is accordance with variation of the CPR mRNA level.Furthermore,a transient pre-chilling treatment can also lead to an elevated artemisinincontent up to the range of 66.7%-95.6%.The present work has investigated the mannerof low temperature-induced expression and regulation of tested genes in A.annuaplantlets and disclosed the gateway of artemisinin biosynthetic gene expression drivenby the low temperature-initiated signal transduction,which should be beneficial to ourfurther understanding of the mechanism of artemisinin biosynthesis and annotation onthe rule of artemisinin accumulation.Once the innate regulatory and homeostaticcontrol outcomes of artemisinin biosynthetic gene expression are elucidated,we wouldeventually find out a highly desirable way toward the tremendous enhancement forartemisinin production by the metabolic engineering.