Abstract
To interfere with the endogenous gene encoding squalene synthase (SS) responsible for sterol biosynthesis, an antisense SS cDNA ( asSS) was introduced into the genome of A. annua. Following the decline of SS mRNA and synchronous reduction of total sterols, mRNA coding for amorpha-4,11-diene synthase (ADS) tremendously elevated, whereas mRNAs for sesquiterpene cyclases encompassing 8-epicedrol synthase (EPS), β-caryophyllene synthase (CS) and β-farnesene synthase (FS) declined or kept homeostasis, suggesting a bias in redirection of the partially blocked metabolic flow to amorpha-4,11-diene. By phenotyping the chimeric ADS promoter-fused β-glucuronidase gene ( pADS-GUS), overexpression of the pADS-GUS reporter was detected upon chilling exposure and ultraviolet (UV) irradiation. In transgenic plants that expose to chilling, ADS and EPS were dramatically up-regulated, but CS was down-regulated and FS and SS were unaffected, implying further chilling-induced diversion of carbon flux among sesquiterpenes. Accordingly, ADS and cytochrome P450 reductase (CPR) were also chilling-inducible, while cytochrome P450 monooxygenase (CYP71AV1) reflected a complicated chilling profile. A maximal artemisinin content was accounted for 16.6 mg/g or 3.7-fold increase in a chilled transgenic line. Fascinatingly, an extraordinarily high artemisinin content of 23.5 mg/g or 5.2-fold increase was measured after 15-month storage of the ground powder from the identical chilled transgenic tissue, demonstrating non-enzymatic conversion from a putative artemisinin precursor to artemisinin. Moreover, a relevance of chilling exposure with singlet oxygen ( 1O 2) emission and artemisinin overproduction was established. These results indicated that the mevalonate pathway has been first skewed to amorpha-4, 11-diene and shunted ultimately to artemisinin through a synergy of genetic modification with environmental superinduction.
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