Transcriptional Regulation Mechanism For Artemisinin Biosynthesis In Circumstance Of Natural Modulation And Artificial Simulation

As important raw meterials in medicine,food,chemical industry and agriculture,plant secondary metabolites possess high commercial values.Terpenoids,such as anti-malarial agent artemisinin and anti-tumor drug taxol,constitute the most major components of plant secondary metabolites.It was widely accepted that the formation of terpenoids with a common structural five-carbon unit occur via two distinctive pathways in all plants:the mevalonate(MVA) pathway in the cytosol and the 1-deoxy-D-xylulose-5-phosphate(DXP) pathway in plastids.Until now,however,we have only known little about the molecular mechanism underlying the regulated gene expression during terpenoid biosynthesis, although most of intermediates on the terpenoid biosynthesistic pathways and their conversion processes have been typically elucidated.In order to establish a platform for investigating the expression patterns of terpenoid biosynthetic genes,Artemisia annua L,a Compositae Artemesia plant,was used as a model to systematically and comprehensively study the transcriptional regulation on the biosynthesis of artemisinin and other relative terpenoids in the natural and artificial environments by RTFQ-PCR and phenotyping the GUS reporter.Meanwhile,based on our previous work,a hypothesis proposing that singlet oxygen(¹O₂) maybe an inductor for artemisinin biosynthesis was confirmed experimentally.This paper is aimed to set up a model to disclose the common anabolism pathway of terpenoids and construct a platform to reveal the expression mode of artemisinin biosynthesis genes,to elucidate the mechanism of conditioned stimulants,temporal and spatial modulators for artemisinin production,and finally to find out feasible approaches to enhanced artemisinin production.The main topics and results of this paper are as follows:Eight MVA and DXP pathway genes(HMGR,FPS,DBR2,DXS,DXR,FS,CS and EPS) were cloned from A.annua and sequenced,among which the sequence ofDBR2 gene was summitted to GenBank for the first time(accession No.EU848577).With inclusion of ADS,CYP71AV1,CPR and SQS genes previously cloned,there were 12 genes that encode the terpenoids biosynthetic key enzymes in A.annua available for further researches of functional genomics and metabolic engineering aming at high-yield artemisinin.To unravel the diverse expression patterns of terpenoid biosynthetic genes during the developmental stage in A.annua,the transcriptional profiles of nine MVA and DXP pathway genes(HMGR,FPS,ADS,CYP71AV1,CPR,DBR2,DXS,DXR and SQS) were quantitatively assayed by RTFQ-PCR.As results,the expression levels of all tested genes were extremely low in June,and raised dramatically in July,and reached their peak values before flowering(in August),but dropped gradually after blooming(in September). Especially,the great elevation of ADS and CYP71AV1 mRNA levels were detected in July, which were 40 and 18 times higher than those in June.The developmental expression pattems of other genes on the DXP pathway were consistent with those of artemisinin biosynthetic genes on the MVA pathway.Therefore,these results suggested that artemisinin was probably biosynthesized via both pathways.In the aspect of tissue-specific gene expression patterns in A.annua,artemisinin biosynthetic genes,comprising ASD,CYP71AV1,CPR and DBR2,illustrated no difference in roots,stems,leaves and flowers during flowering.Such result revealed that artemisinin biosynthetic genes in those tissues showed no obvious tissue specificity.However, artemisinin biosynthetic mRNA levels significantly elevated in senescent leaves that had tured brown.The increments of CYP71AV1,ADS,DBR2 mRNA levels were 51.2,25.5 and 14.8 times higher than those in green leaves.Accordingly,artemisinin content in senescent leaves was also promoted to 1.5 times higher than that in green leaves.In conclusion,the fashions regarding expression of artemisinin biosynthetic genes and accumulation of artemisinin might be directly regulated by the growth and developmental procedures of plants themselves,especially by the senescent process.Because senescent leaves emitted more amounts of ¹O₂ than green leaves,it is speculated that ²O₂ may be directly involved in the regulation of artemisinin biosynthetic genes and non-enzymatic conversion from artemisinin precursors to artemisinin,thereby playing a major role in facilitating artemisinin biosynthesis and accumulation.To further compile evidence on the modulatory mechanism of artemisinin biosynthesis, transgenic tobacco(Nicotiana tabacum) plants that introduced an ADSP-GUS fusion gene were cultivated in the present study and showed a typical GUS phanotype in whole plants, indicating that a basal level of ADSP-driven GUS expression didn't exhibit tissue specificity.The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature(4℃) and ultraviolet irradiation were 2.2 and 1.6 times higher than that in the control.It is suggested that the ADS gene is induced by adverse environmental stresses at least including cold and irradiation,and may be more sensitive to ¹O₂ generated by a variety of stress conditions.The successful establishment of the stress-inducible GUS reporter phenotyping system provided convenience for future high-throughput screening of inducers that stimulate over-expression of artemisinin biosynthetic genes.The transcriptional patterns of artemisinin biosynthetic genes under the circumstances of 12 artificial simulations that enhance generation of ¹O₂ were monitored by using RTFQ-PCR technology.The artificial simulative elements included low-temperature(4℃), high-temperature(50℃),ultraviolet irradiation,hypoxia(flooding and rehydration), drought(dehydration),high salt(NaCl),fungal elicitor(yeast extract) and plant signal molecules(SA,MJ,GA₃ and ABA).The results showed that the expression levels of artemisinin biosynthetic genes(including those on the common and specific biosynthetic pathways) generally up-regulated,in which ADS and DBR2 genes were more sensitive than others to those stimuli.The ADS transcript level in A.annua plantlets treated with cold, ultraviolet,dehydration and yeast extract were 35.6,14.2,15.5 and 5.5 times higher than that in the control,and the DBR2 mRNA level upon the treatment by flooding,rehydration and yeast extract were 13.8,38.2 and 20.7 times higher than that in the control.It is revealed for the first time that artemisinin biosynthetic genes are highly induced by oxidative stresses,especially by ¹O₂,based on the simulation outcomes under the oxidative stress conditions,including the natural senescence process,which enables the induction of sensitive genes by ¹O₂ release.Low temperature-induced expression of previously identified expressed sequence tags (ESTs) by our group were quantitatively analyzed,which included seven sequences,i.e., peroxidase 1 gene(POD1),chitinase gene(CH1),calmodulin gene(CaM), ubiquitin-conjugating enzyme gene(UCE),drought/low temperature and salt responsive protein gene(D/LTSRP),RNA-binding glycine rich protein gene(RGP),and auxin-repressed/dormancy-associated protein gene(AR/DAP).The quantitative results showed that D/LTSRP,UCE,CaM,AR/DAP and POD1 mRNA levels after 4℃treatment for 48h were 7.5,5.0,2.6,2.3 and 1.5 times higher than that of the control,suggesting that these genes may be involve in the regulation throughout the metabolic networks that relevant to initiation and transduction of the low-temperature signal in A.annua.This possibility provided a clue to reveal the mechanism underlying the signal transduction pathway and cascade regulation network for artemisinin biosynthesis.A specific MVA pathway inhibitor,lovastatin and a specific DXP pathway inhibitor, fosmidomycin,were used to perturb the biosynthetic flux in A.annua seedlings.The quantitative analysis at the transcriptional level showed that a mutual compensation mechanism was present in both pathways.Treatment of seedlings with fosmidomycin resulted in a transient decline of mRNA levels on the DXP pathway.After the initial drop, the mRNA levels of DXP pathway genes recovered to those corresponding to control levels, whereas mRNA levels of MVA pathway genes first increased but then decreased.As a response to lovastatin exposure,the expression activities of both pathway genes were inhibited,further suggesting that crosstalk between cytosolic and plastidial pathways for terpenoid biosynthesis might persist,and that both MVA and DXP pathways were likely involved in artemisinin production.After supplement with miconazole,an inhibitor of sterols biosynthesis,the levels of ADS and DBR2 mRNAs increased two and five times compared to the control,suggesting that carbon source was partially shifted from sterols to sesquiterpenes.The expression patterns of squalene synthase gene(SQS) and other sesquiterpene synthase genes(ADS,CS,FS and EPS) in transgenic A.annua plants that were integrated with one or more copies of anti-sense squalene synthase genes(asSQS) were surveyed.The results showed that the SQS gene in transgenic plantlets was significantly inhibited by the generation of asSQS mRNA,and decreased about two to six times compared to the wild-type control,whereas ADS mRNA in two of transgenic plantlets increased 3.5 and 3 times.After intermittent cold-treatment,SQS and FS mRNAs exhibited no changes in transgenic and wild-type plants,whereas ADS and EPS mRNA increased 1.5 to 5 times,and CS mRNA dramatically decreased about 50-100 folds.The main innovations of this paper were summarized as follows:it is the first time to survey the expression profile of metabolic regulation on terpenoid biosynthetic genes including artemisinin biosynthetic genes at the transcription level.It provided a model for the follow-up studies on the functional genomics and metabolic engineering of other terpenoids like taxol and vinblastine.Secondly,through monitoring the expression kinetics of these genes in different subcellular spaces and during different development stages, present study has preliminarily revealed a unique mode of developmental and tissuesspecific regulation on artemisinin biosynthesis,and first discovered artemisinin biosynthesis being modulated by the senescence process.On such basis,using various environmental stress conditions that simulate the senescence process,this research has elucidated that artemisinin biosynthetic genes were inducible by various environmental stresses.It was confirmed that excessive release of ¹O₂ during the senescence process is the main cause to increase the expression of artemisinin biosynthetic genes and enhance the production of artemisinin.It was also proved the hypothesis that ¹O₂ is mostly the inducer for artemisinin biosynthesis,and further clarified that artemisinin may be a by-product generated from its precursor dihydroartemisinic acid in the process of ¹O₂ scavenging.