Mechanistic Study On Salicylic Acid-and Methyl Jasmonate-induced Expression Of Artemisinin Biosynthetic Genes

Malaria is the most serious public health problem of insect-borne diseases in the world, which is widely prevalent in some tropical and subtropical developing countries. While conventional antimalarials such as chloroquine are gradually phased out due to resistance to the parasite, the World Health Organization (WHO) has recommended artemisinin and derivatives as the most effective antimalarials. However, artemisinin content in the natural medicinal plant Artemisia annua is very low, only about 0.4% to 1.0%, and cannot meet the urgent need for antimalarial drugs in global endemic regions. To explore the molecular mechanism behind enhanced artemisinin production, the present study has devoted to elucidate the induced gene expression mode of the promoter of a key enzyme gene for artemisinin biosynthesis, pADS, from a viewpoint of the environmental stress-induced gene expression and by using a rapid phenotyping platform of pADS-GUS in transgenic tobacco. We found that GUS activity was significantly increased after treatment by salicylic acid (SA)/methyl jasmonate (MJ), indicating that SA/MJ may induce the up-regulation of ADS gene. Furthermore, we investigated the effect of SA/MJ on the transcriptional levels of three key artemisinin biosynthetic genes by semi-quantitative RT-PCR. Consequently, it was clear that SA/MJ induced ADS gene, but not induced CYP71AV1 and CPR genes. In order to reveal whether SA/MJ signaling is relevant to Ca2+, we assessed the alteration of transcriptional levels of artemisinin biosynthetic genes in cultured Artemisia annua plants upon treatment by SA/MJ with a blocker of Ca2+channel, LaCL3, by semi-quantitative RT-PCR. It was aware that only ADS mRNA declined, but CYP71AV1 and CPR mRNAs remained stable post treatments, suggesting SA/MJ-induced ADS gene expression being dependent on Ca2+. Further studies indicated that 1O2 was rapidly released from leaves of A. annua following treatment by SA/MJ, whereas 1O2 was declined from leaves of A. annua as contacting with the 1O2 donor, Rose Bengal (RB). The measurement of artemisinin content showed that treatment by SA/MJ significantly boosted artemisinin content in cultured A. annua plants. In contrast, RB led to decrease of artemisinin content. It can be concluded that only endogenous rather than exogenous 1O2 promote artemisinin biosynthesis. Simultaneously, it was also found that glutathione (GSH) content, H2O2 level and catalase (CAT) activity were elevated accordingly, which subsequently induced the expression of artemisinin biosynthetic genes and ultimately enhanced artemisinin production. The innovative outcomes of the present study are represented by that discovery of SA/MJ accelerating artemisinin biosynthesis by simulating environmental stresses; validation of SA/MJ inducing artemisinin biosynthetic gene expression; clarification of SA/MJ affecting the cellular redox state; and distinction of endogenous from exogenous 1O2 impacting artemisinin yield. This study has depicted the mechanistic episode underlying SA/MJ rendering artemisinin overproduction on the molecular level, which should be beneficial to provide the scientific guide to the cultivation, field management, harvest and processing of A. annua and to pave a new avenue towards solving the problem of artemisinin shortage.