Tetraploid Artemisia annua plants were successfully inducted by using colchicine, and their ploidy was confirmed by flow cytometry. Higher stomatal length but lower frequency in tetraploids were revealed and could be considered as indicators of polyploidy. The average level of artemisinin in tetraploids was increased from 39% to 56% than that of the diploids during vegetation period, as detected by high-performance liquid chromatography-evaporative light scattering detector. Gene expressions of 10 key enzymes related to artemisinin biosynthetic pathway in different ploidy level were analyzed by semiquantitative polymerase chain reaction and significant upregulation of FPS, HMGR, and artemisinin metabolite-specific Aldh1 genes were revealed in tetraploids. Slight increased expression of ADS was also detected. Our results suggest that higher artemisinin content in tetraploid A. annua may result from the upregulated expression of some key enzyme genes related to artemisinin biosynthetic pathway.

Artemisinin is currently the best therapeutic against both drug-resistant and cerebral-malaria-causing strains of Plasmodium falciparum. Allene oxide cyclase (AOC) is the key enzyme in the biosynthetic pathway of jasmonates. In this study, a full-length cDNA of AOC gene (named as AaAOC) was cloned from Artemisia annua L. AaAOC was 1,007 bp, containing an open reading frame (750 bp) encoding 249 amino acids. Comparative and bioinformatic analyses revealed that the deduced protein of AaAOC was highly homologous to AOC from other plant species. Phylogenetic analysis indicated that AaAOC was clustered in a closely related subgroup with AOC of Camptotheca acuminata. Southern blot analysis revealed that AaAOC was a multicopy gene. Reverse transcriptasepolymerase chain reaction (RT-PCR) and QPCR analysis showed that AaAOC mRNA accumulated most abundantly in alabastrums, in which the content of artemisinin was previously proven to be the highest. RT-PCR analysis revealed that MeJA, ABA, and ethylene treatments significantly enhanced AaAOC transcript expression. And the results of HPLC showed that the contents of artemisinin were greatly increased after the treatments of ABA and MeJA.

Allene oxide synthase (AOS) is the first committed step in the biosynthetic pathway of Jasmonate. In this study, a full-length cDNA of AOS gene (named as AaAOS) was cloned from Artemisia annua. The gene was 1891 bp in size containing an open reading frame (1581 bp) encoding 526 amino acids. Comparative and bioinformatic analysis revealed that the deduced protein of AaAOS was highly homologous to AOSs from other plant species. Phylogenetic analysis indicated that the protein of AaAOS belonged to the dicotyledonous group, which was consistent with the category of A. annua. Southern blot analysis revealed that it was a low-copy gene. Quantitative Real-time PCR (qRT-PCR) analysis showed that AaAOS mRNA accumulated most abundantly in leaves and flowers. The qRT-PCR analysis revealed that MeJA, ABA and ethylene treatments significantly enhanced AaAOS transcript expression.