Post-transcriptional Regulation On Artemisinin Biosynthesis And Carotenoid Biosynthetic Inhibitor-enhanced Artemisinin Production

Artemisinin is a kind of novel antimalarial agent that was first invented by the Chinese scientists and wellknown in the world.Currently,direct extraction of artemisinin from the medicinal herb Artemisia annua remains a principal means for commercialization of artemisinin.Therefore,how to enhance artemisinin production in planta by virtue of elucidating the regulatory mechinery of artemisinin biosynthesis has been eagerly and extensively explored.In this prelimilary research,an enzyme-linked immunosorbent assay (ELISA)-based immunoquantitative platform for quantifying the concentration of artemisinin biosynthesis relevant enzymes was established by employing Escherichia coli as a host for expression of the recombinant antigen genes and injecting mice by affinity-purified antigens for raising high titers(1:12800) of specific antisera.Upon application of home-made polyclonal antibodies,detailed investigations on development- and tissue-specific expression patterns of artemisinin biosynthetic genes were conducted at the post-transcriptional level(i.e.translationally) in the wild grown A.annua plants.Consequently, ADS and CYP71AV1 enzymes possess higher amounts in leaves and stems than in roots, demonstrating the tissue-specific regulation modes regarding those genes.In contrary,CPR enzyme keeps always homeostasis among different tissues or under normal and adverse conditions,implying a constitutive expression pattern of this gene.Furthermore,in the blooming A.annua plants,the lowest and highest concentrations of ADS and CYP71AV1 enzymes were detected in green leaves and dry leaves,roots and flowers,respectively, indicating a development-specific feature of ADS and CYP71AV1 genes.To understand if there present the interactions of matrix and information between differentiated subcellular compartments,an inhibitor of carotenoid biosynthesis,fluridon, was used to block the plastidial terpene biosynthetic pathway.The impact of fluridon on the cytosolic artemisinin biosynthesis was evaluated by the quantitative polymerase chain reaction.Upon briefly exposure to fluridon(within 6 h),the levels of ADS,CYP71AV1, DBR2 mRNAs transiently elevated,whereas the level of CPR mRNA was unaffected. When exposure to fluridon for 7d,however,ADS and CYP71AV1 mRNAs returned to the normal levels.Correspondingly,ADS and CYP71AV1 enzymes exhibited a dynamic fashion of "first elevation followed by decline" shortly after treated by fluridon,while both enzymes showed an elevated level in leaves that had been incubated with fluridon for 7d. Simultanuously,a combination of fluridon with chilling stress enabled the significant elevation of ADS and CYP71AV1 mRNA levels,while such treatment was unable to reverse decline of ADS and CYP71AV1 enzymes.As a conclusion,fluridon was capable of up-regulating the expression level of ADS and CYP71AV1 genes by suppressing the plastidial carbon flux,but such an enhancement was transiently affacted and automatically modulated,which meant that the elevated levels of mRNAs and enzymes in subjected cells would rapidly drop to a base-line level after integrating and adapting the variation of metabolic networks.To further reveal the bona fide mechanism underlying the up-regulation of ADS and CYP71AV1 genes by fluridon,the dynamic fluctuation of ¹O₂ release by leaves of sterol-suppressed transgenic plants upon exposure to fluridon was determined in a time-course way,from which a markable release of more ¹O₂ in fluridon-treated leaves than chilled samples and a similar extent of ¹O₂ release in combination of fluridon with chilling were observed.Furthermore,as co-treated by fluridon and chilling,transgenic plants gave rise to the highest artemisinin content(12.53 mg/g dry weight on average or 17.5 mg/g dry weight in maximum).In summary,fluridon and chilling stress might coordinately upregulate artemisinin biosynthetic genes and facilitated conversion to artemisinin from its precursor,and artificial modulation had diminished the limitation of tissue- and development-specific regulation on artemisinin biosynthetic genes under the natural circumstance.