Quantitative Transcript Profiling Reveals Down-Regulation of A Sterol Pathway Relevant Gene and Overexpression of Artemisinin Biogenetic Genes in TransgenicArtemisia annuaPlants

Abstract

To investigate the dynamic fluctuation of terpenoid relevant transcriptomics in transgenic ARTEMISIA ANNUA plants that express the genomic integrated antisense squalene synthase gene ( ASSS), we have quantified the transcript levels of the sterol anabolic SS gene as well as artemisinin biogenetic amorphadiene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 reductase (CPR) genes by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). The SS mRNA level in transgenic plants sharply droped to 7.4 % - 55.3 % (i. e., 44.7 - 92.6 % reduction as the wild-type control), strongly implying that the expression of endogenous SS gene is significantly suppressed by the exogenous ASSS gene. In a synchronous fashion, ADS, CYP71AV1 and CPR mRNA levels elevated with the decline of SS mRNA level in transgenic plants, and the maximal ADS, CYP71AV1 and CPR mRNA levels in transgenic plants were 3.0-, 4.4- and 2.5-fold, respectively, higher than those in the control. Without a lethal effect but with a distinguishable impact on the organogenesis and morphology of transgenic plants, the down-regulation of SS gene has also led to the coordinated overexpression of ADS, CYP71AV1 and CPR genes together with the overproduction of artemisinin although no fully perfect correlation among the available experimental data has been shown.

An ecological implication of glandular trichome-sequestered artemisinin: as a sink of biotic/abiotic stress-triggered singlet oxygen

“…Direct measurement of 1 O2 burst from chilling and senescent leaves has provided strong support to an involvement of 1 O2 in the non-enzymatic conversion to artemisinin, but whether artemisinin biosynthesis genes are also inducible under the stress circumstance that triggers 1 O2 emission remains unclear. In the context of senescence and chilling, it was observed that 1 O2 burst from chilled and senescent leaves can really induces artemisinin biosynthesis genes, reiterating that 1 O2 is a common signal that modulate artemisinin accumulation either in senescent A. annua leaves or after A. annua leaves acclimatized to chilling (Feng et al 2008;Zeng et al 2008b).…”

“…Although artemisinin production was reported to be influenced by natural stress (Chen and Zhang 1987;Martinez and Staba 1988;Ferreira et al 1995), no direct evidence indicating the involvement of 1 O2 in artemisinin biosynthesis has been provided for many years. Feng et al (2008) The sustained release of 1 O2 is recognized from a gradual decline of absorbance at 440 nm (A440) due to the bleaching of the selective 1 O2 acceptor N, N-dimethyl-p-nitrosoaniline.…”

“…(a) A time-course measurement of A440 that correlates reversely with the amount of 1 O2 emitted by the chilling-treated transgenic plant sample, T47, and untreated T47. (▲) is the blank sample; (♦) is the untreated T47 sample; and (□) is the chilled T47 sample (Feng et al 2008). (b) The time course of 1 O2 emission from green, yellow and brown leaves of A. annua plants ).…”

“…During last two decades, a lot of initiatives to engineer artemisinin biosynthesis genes towards enhanced artemisinin production has been lunched (Farhi et al 2013). The transgenic endeavors in A. annua include 'pulling carbon flux' to artemisinin via the introduction of a single or multiple artemisinin biosynthesis genes Aquil et al 2009;Banyai et al 2010;Elfahmi and Chahyadi 2014;Nafis et al 2011;Shen et al 2012;Yuan et al 2014) or 'shutting carbon flux' from steroids or other isoprenoids to artemisinin through anti-sense/RNA interference (Chen et al 2011;Feng et al 2009;Yang et al 2008;Zhang et al 2009). In the former situation, artemisinin biosynthesis is enhanced due to the increase of the copy numbers of artemisinin biosynthesis genes; in the latter one, other isoprenoid biogenesis is suppressed due to the knockdown of one or more genes (Lange and Ahkami 2013).…”

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua

“…7). Researchers have reported that a co-suppression phenomenon can often occur in overexpressing transgenic plants (Yang et al, 2008;Banyai et al, 2010).…”