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Artemisinin is an effective antimalarial drug isolated from the medicinal plant Artemisia annua L. Due to its increasing market demand and the low yield in A. annua, there is a great interest in increasing its production. In this paper, in an attempt to increase artemisinin content of A. ANNUA by suppressing the expression of β-caryophyllene synthase, a sesquiterpene synthase competing as a precursor of artemisinin, the antisense fragment (750 bp) of β-caryophyllene synthase cDNA was inserted into the plant expression vector pBI121 and introduced into A. annua by Agrobacterium-mediated transformation. PCR and Southern hybridization confirmed the stable integration of multiple copies of the transgene in 5 different transgenic lines of A. annua. Reverse transcription PCR showed that the expression of endogenous CPS in the transgenic lines was significantly lower than that in the wild-type control A. annua plants, and β-caryophyllene content decreased sharply in the transgenic lines in comparison to the control. The artemisinin content of one of the transgenic lines showed an increase of 54.9 % compared with the wild-type control. The present study demonstrated that the inhibition pathway in the precursor competition for artemisinin biosynthesis by anti-sense technology is an effective means of increasing the artemisinin content of A. annua plants., (© Georg Thieme Verlag KG Stuttgart · New York.)

Considerable difference in artemisinin and its direct precursors, artemisinic acid and dihydroartemisinic acid, was detected between two chemotypes within the species Artemisia annua (A. annua). These two chemotypes showed differential metabolic response to methyl jasmonate (MeJA) elicitation. Exogenous application of MeJA resulted in an accumulation of dihydroartemisinic acid and artemisinin in Type I plants. In Type II plants, however, artemisinic acid and artemisinin level decreased dramatically under MeJA elicitation. Squalene and other sesquiterpenes, (e.g., caryophyllene, germacrene D), were stimulated by MeJA in both chemotypes. The effect of MeJA elicitation was also studied at the transcription level. Real time RT-PCR analysis showed a coordinated activation of most artemisinin pathway genes by MeJA in Type I plants. The lack of change in cytochrome P450 reductase (CPR) transcript in Type I plants indicates that the rate-limiting enzymes in artemisinin biosynthesis have yet to be identified. Other chemotype-specific electron donor proteins likely exist in A. annua to meet the demand for P450-mediated reactions in MeJA-mediated cellular processes. In Type II plants, mRNA expression patterns of most pathway genes were consistent with the reduced artemisinin level. Intriguingly, the mRNA transcript of aldehyde dehydrogenase1 (ADHL1), an enzyme which catalyzes the oxidation of artemisinic and dihydroartemisinic aldehydes, was upregulated by MeJA. The differential metabolic response to MeJA suggests a chemotype-dependent metabolic flux control towards artemisinin and sterol production in the species A. annua., (© Georg Thieme Verlag KG Stuttgart · New York.)

A key point in the biosynthesis of the antimalarial drug artemisinin is the formation of dihydroartemisinic aldehyde which represents the key difference between chemotype specific pathways. This key intermediate is the substrate for several competing enzymes, some of which increase the metabolic flux towards artemisinin, and some of which--as we show in the present study--may have a negative impact on artemisinin production. In an effort to understand the biosynthetic network of artemisinin biosynthesis, extracts of A. annua flowers were investigated and found to contain an enzyme activity competing in a negative sense with artemisinin biosynthesis. The enzyme Red1 is a broad substrate oxidoreductase belonging to the short chain dehydrogenase/reductase family with high affinity for dihydroartemisinic aldehyde and valuable monoterpenoids. Spatial and temporal analysis of cDNA revealed Red1 to be trichome specific. The relevance of Red1 to artemisinin biosynthesis is discussed., (© Georg Thieme Verlag KG Stuttgart · New York.)

Due to the high demand and low yield of the anti-malarial drug artemisinin in natural populations of Artemisia annua (Quinghao), an attempt has been made to enhance the artemisinin content through 4 cycles of recurrent selection (C(0)-C(3)) using selected genotypic and phenotypic traits. Based on their phenotypic and genotypic characteristics, the top 5% plants of each cycle were selected, and their seedlings were planted in poly-cross block to produce seeds for the subsequent generation. A significant increase in the artemisinin content (0.15% in C (0) to 1.16% in C (3), i.e., about 40% genetic gain over the generation) was observed. This enhancement was directly correlated with the plant height and branching intensity in all four cycles. Similarly, the PCV (phenotypic coefficient of variation) and GCV (genotypic coefficient of variation) have been observed to have a higher value for artemisinin content. The DNA marker (MAP 12) with relation to artemisinin was also identified for high yielding genotypes in all four cycles of selection. Over the four cycles of recurrent selection, the plant developed an oval appearance (Variety: CIM-Arogya) and a high artemisinin content (1.16%)., (© Georg Thieme Verlag KG Stuttgart-New York.)

To dissect and penetrate complexicity regarding the tissue-specific and environment-induced expression modes of cytosolic and plastidial terpene biosynthetic genes in A. annua, corresponding mRNAs relevant to terpene biosynthesis were quantitatively compared among distinctive organs and during different growth stages. Although all examined mRNAs gradually elevate from June to August in tested organs, a putative artemisinin biosynthesis responsible DBR2 mRNA represents the most abundant transcript anyplace and anytime. Apart from others, senescent leaves endow global activation of artemisinin biosynthetic genes and ultimately lead to enhanced artemisinin production. Direct measurement of (1)O (2) burst from senescent leaves strongly supports an involvement of (1)O (2) in conversion from precursor(s) to artemisinin. In the context of environmental stresses, physical and chemical stress signals that include those invoking (1)O (2) burst were evaluated as if inducing artemisinin biosynthetic genes. The quantitative data have reiterated a common pattern of modulating artemisinin production in A. annua by triggering (1)O (2) burst during senescence and under chilling acclimatization. In conclusion, a missing link concatenating senescence-coupled (1)O (2) generation to (1)O (2)-induced upregulation of artemisinin biosynthetic genes has been re-established, which would provide a fertile base for future endeavors pursuing further enhancements of artemisinin production., (Georg Thieme Verlag KG Stuttgart. New York.)

Artemisinin has been proven to be an effective antimalarial compound, especially for chloroquine-resistant and cerebral malaria. However, its biosynthesis pathway is still not completely clear. In order to get new clues about artemisinin biosynthesis, metabolic profiling by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) was applied to compare the secondary metabolites of two Artemisia annua L., genotype SP18 and 001, for some phenotypic and agricultural trait differences, including artemisinin content, existed between the two genotypes. Samples at 7 time points of three growth stages were studied. The data of profiles were subjected to multivariate analysis with partial least squares discriminant analysis (PLS-DA). The results indicated that there were clear differences in terpenoids and artemisinin metabolism between different growth stages and genotypes. Twenty-one compounds, including artemisinin and its related precursors, were selected as the marker compounds of the PLS-DA between the two genotypes. Among them, artemisinic acid, arteannuin B, borneol, beta-farnesene and an unidentified sesquiterpenoid (peak 48) were abundant in 001, while camphor, methyl artemisinic acid and lanceol accumulated mainly in SP18. The relationship between these differences and artemisinin biosynthesis in the two genotypes of A. annua were discussed., (Georg Thieme Verlag KG Stuttgart. New York.)

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Artemisinin is highly effective against multidrug-resistant Plasmodium falciparum and it has been widely used as part of the artemisinin-based combination therapies against malaria. To elucidate the biosynthetic pathway of artemisinin and to clone related genes in Artemisia annua, differentially expressed genes between blooming flowers and flower buds were isolated and characterized by a combined approach of suppression subtractive hybridization (SSH) and metabolite analysis. A total of 350 cDNA clones from a subtractive cDNA library were randomly picked, sequenced and analyzed and 253 high-quality sequences were obtained. BLASTX comparisons indicated that about 9.9 % of the clones encoded enzymes involved in isoprenoid (including artemisinin) biosynthesis. The expression of 4 gene transcripts involved in artemisinin biosynthesis was examined by RT-PCR and the results confirmed the higher expression of these transcripts in blooming flowers than in flower buds. In addition, 2 putative transcript factors transparenta testa glabra 1 (TTG1) and ENHANCER OF GLABRA3 (GL3), which promote trichome initiation, were presented in the library. Finally, this study demonstrated that the increase of expression level of the putative TTG1 gene correlated with the improvement of glandular trichome density and artemisinin production in A. annua leaves. The subtractive cDNA library described in the present study provides important candidate genes for future research in order to increase the artemisinin content in A. annua., (Georg Thieme Verlag KG Stuttgart, New York.)

An effective and affordable treatment against malaria is still a challenge for medicine. Most contemporary drugs either are too expensive to produce or are not effective against resistant strains of the malaria parasite Plasmodium falciparum. The plant Artemisia annua L. is the source of artemisinin, an effective drug against malaria for which no resistant strains of the bacterium have been reported. However, the artemisinin content of A. annua is very low, which makes its production expensive. Here we report the use of transgenic technology to increase the artemisinin content of A. annua. We report the production of transgenic plants of A. annua into which we transferred 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) gene from Catharanthus roseus (L.) G. Don using Agrobacterium-mediated gene transfer technology. Transgene integration and copy number were assessed by PCR and Southern hybridization, which confirmed the stable integration of multiple copies of the transgene in 7 different transgenic lines of A. annua. The leaf tissue of three of the A. annua transgenic lines possessed significantly higher HMGR activity compared with wild-type controls, and this activity was associated exclusively with microsomal membranes. The artemisinin content of the shoots of one of the transgenic lines depicted an increase of 22.5 % artemisinin content compared with wild-type control A. annua plants., (Georg Thieme Verlag KG Stuttgart-New York.)

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.

The flowering promoting factor1 ( fpf1) from Arabidopsis thaliana was transferred into Artemisia annua L. via Agrobacterium tumefaciens. The fpf1 gene was firstly inserted in the binary vector pBI121 under the control of CaMV 35S promoter to construct the plant expression vector pBIfpf1, then leaf explants of A. annua were infected with A. tumefaciens LBA4404 containing pBIfpf1, and induced shoots. Transgenic plants were obtained through the selection with kanamycin. PCR, PCR-Southern and Southern blot analyses confirmed that the foreign fpf1 gene had been integrated into the A. annua genome. RT-PCR and RT-PCR-Southern analyses suggested that the foreign fpf1 gene had expressed at the transcriptional level. Under short-day conditions, the flowering time of fpf1 transgenic plants was about 20 days earlier than the non-transformed plants; however, no significant differences were detected in artemisinin content between the flowering transgenic plants and the non-flowering non-transgenic plants. These results showed that flowering is not a necessary factor for increasing the artemisinin content, furthermore, there may be no direct linkage between flowering and artemisinin biosynthesis.