УДК 581.1
© 2010 г. Donghui Liu*,***, Lida Zhang*, Chengxiang Li*, Ke Yang*,
Yueyue Wang*, Xiaofen Sun**, Kexuan Tang*,**
* Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, Shanghai, P. R. China
** State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Fudan University, Shanghai, P. R. China
*** Department of Biochemistry and Molecular Biology, Shenyang Medical College, Shenyang , Liaoning, P. R. China
Received December 9, 2008
Effects of mechanical wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in Artemisia annua L. leaves were investigated. HPLC-ELSD analysis indicated that there was a remarkable enhancement of the artemisinin content in 2 h after wounding treatment and the content reached the maximum value at 4 h (nearly 50% higher than that in the control plants). The expression profile analysis showed that many important genes (HMGR, ADS, CPR, and CYP71AV1) involved in the artemisinin biosynthetic pathway were induced in a short time after wounding treatment. This study indicates that the artemisinin biosynthesis is affected by mechanical wounding. The possible mechanism of the control of gene expression during wounding is discussed.
Abbreviations: HPLC-ELSD - high performance liquid chromatography-evaporative light scattering detection; MeJa - methyl jasmonate; MS - Murashige and Skoog nutrient medium.
Corresponding author: Kexuan Tang. Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, Shanghai 200240, P. R. China. Fax: 86-21-3420-5828; e-mails: kxtang1@yahoo.com; kxtang1@163.com

Key words: Artemisia annua - mechanical wounding - gene expression profile - artemisinin
Artemisinin also known as qinghaosu, an endoperoxide sesquiterpene lactone isolated from the aerial parts of Artemisia annua L. plants, is an effective antimalarial agent, particularly effective against multidrug-resistant strains of the malarial parasite Plasmodium [1, 2]. The pathway of artemisinin biosynthesis is complicated, and the endogenous production of artemisinin is very low (0.01-0.8%) [3]; therefore, a better understanding of the biochemical mechanism leading to the synthesis of artemisinin and its regulation by both exogenous and endogenous factors is essential for facilitating an increased yield. There are some reports on the accumulation of artemisinin in A. annua under the stress conditions, such as temperature [4], light [5], and water stress [6]. However, little information is available about the effects of wounding on the genes expression involving in the artemisinin biosynthesis and artemisinin production in A. annua. In the present study, we used the middle leaves to test the effects of wounding stress on the gene expression profile involved in the artemisinin biosynthesis pathway in A. annua and on the change in the artemisinin content measured by using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD).


Plant material, growth condition, and wounding treatment. Seeds of Artemisia annua L. collected from Sichuan Province, China, were surface-sterilized with 20% (v/v) sodium hypochlorite for 20 min, sown onto germination MS basal medium supplemented with sucrose (30 g/l) and phytagel (Sigma, United States) (2.6 g/l) in a growth chamber with a photoperiod of 16 h and light intensity of 80 000 lx (metal halide source) at 26ºC, and grown for 4 weeks. When about 5 cm in height, the seedlings were transplanted into plastic pots containing peat-moss and perlite at the ratio of 1 : 1. To adjust to the new surrounding, the plants were watered by normal nutrient solution (33 mg/l NH4NO3, 38 mg/l KNO3, 8.8 mg/l CaCl2 ( 2H2O, 7.4 mg/l MgSO4 ( 7H2O, 3.4 mg/l KH2PO4) every 5 days. When about 40 cm in height (before the flowering), 20 plants were selected and divided randomly into two groups with ten plants in each group. Plants of one group were subjected to wounding stress by damaging the leaves and stems with surgical blade and pliers, and then two samples of the middle leaves were collected at different time intervals (0, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h, 48 h, and 72 h). Another group of plants was used as control plants, which were harvested at the same time. One sample was frozen in the liquid nitrogen immediately after collecting and stored at -70ºC for RNA extraction. The other sample was dried at 50ºC and used for measuring artemisinin contents with HPLC. Experiments were repeated three times with three recordings each.
Sample preparation and quantification of artemisinin using HPLC-ELSD. The middle leaves of the treated A. annua plants were collected and dried at 50ºC, and then dried leaves were grounded into powder. 0.1 g of powder was weighed and extracted with ethanol (1 ml) in the ultrasonic processor (twice, 15 min each), then centrifuged for 10 min at 5000 rpm to remove the suspended particles. The final supernatant was filtered with a 0.25 (m filter. After preparation, the samples were analyzed by the Waters Alliance 2695 HPLC system coupled with a Waters 2420 ELSD detector. The HPLC condition was Waters C18 column using water : methanol (40 : 60, v/v) mixture as a mobile phase at a flow rate of 1 ml/min. ELSD condition was optimized at nebulizer-gas pressure of 50 psi and drift tube temperature of 45ºC, and the gain was set at 7. The artemisinin purchased from "Sigma" was used as the standard control in the measurement. For each sample, the injection volume was 20 (l, and the results were analyzed with the Empower data system. The measurement was repeated three times.
RNA extraction and semi-quantitative RT-PCR analysis. Total RNA was isolated from different wound-treated and control leaves by using Total RNA Isolation Kit ("Watson Biotechnologies", China). DNA contamination was removed using DNase I ("Takara", China) following the protocol provided by the manufacturers. The first strand cDNA was synthesized from 5 (g of DNase-treated RNA using oligo(dT)18 primer ("Promega", United States) and Reverse Transcriptase XL (AMV) ("Takara") following the manufacturer's protocol, and finally we obtained a 20 (l of cDNA solution.
The expression of several key genes involved in the artemisinin biosynthetic pathway were studied, including genes for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, NCBI accession no. AF142473), farnesyl diphosphate synthase (FPS, NCBI accession no. U36376), amorpha-4,11-diene synthase (ADS, NCBI accession no. DQ448294), amorpha-4,11-diene C-12 oxidase (CYP71AV1, NCBI accession no. DQ268763), and its redox partner cytochrome P450 reductase (CPR, NCBI accession no. EF104642). In addition, the ubiquitin-conjugating gene (UBC) was used as an internal reference. For each of the genes under study, a primer pair was designed to obtain a PCR amplification product between 100 and 500 bp. The sequences of the primers used for PCR amplifications of HMGR, FPS, ADS, CPR, CYP71AV1, and UBC were HMGR-F: 5'-TTGTGTGCGAGGCAGTAAT-3' and HMGR-R: 5'-CCTGACCAGTGGCTATAAAGA-3'; FPS-F: 5'-TCATTGTCTATTCACCGCCG-3' and FPS-R: 5'-CACCGCTTGGACTGCTTTGCT-3'; ADS-F: 5'-AATGGGCAAATGAGGGACAC-3' and ADS-R: 5'-TTTCAAGGCTCGATGAACTATG-3'; CPR-F: 5'-AGCCTCTTTGCCACCTCCT-3' and CPR-R: 5'-GAACAGACTCCCTTGTGAACG-3'; CYP71AV1-F: 5'-CACCCTCCACTACCCTTG-3' and CYP71AV1-R: 5'-GACACATCCTTCTCCCAGC-3'; UBC-F: 5'-CACACTTGAGGTTGAGTCCAG-3' and UBC-R: 5'-CATAACATTTGCGGCAGATAG-3'. The PCR mixture (50 (l) contained 2(l of the RT reaction mixture described above, 1 (l of each dNTP (10 mM), 1.75 nM MgCl2, 2 (l of each primer (10 (M), and 1U of Taq polymerase ("Sangon", Shanghai, China). Semi-quantitative RT-PCR was performed under the following condition: denaturing at 94ºC for 5 min, followed by 27-30 cycles of amplification (94ºC for 15 s, 54ºC for 15 s, and 72ºC for 30 s). To ensure that the PCR was linear, the optimal amount of cDNA and the number of cycles were determined for each primer pair. PCR products were analyzed on 1% agarose gels containing ethidium bromide and photographed under UV light. 3 (l of PCR products were used as the samples for semi-quantitative RT-PCR analysis. The PCR products were visualized on an UVP transilluminator with the image analysis being assisted with Labworks ("Media Cybernetics", United Kingdom). To obtain a legible visual impression, the pictures were submitted to multi-expose or expose delay.


The Content of Artemisinin in A. annua after Wounding Treatment

The artemisinin content in A. annua leaves after wounding treatment was determined by HPLC-ELSD. The results indicated that artemisinin content increased during the first 4 h as compared with the control (fig. 1). It should pay attention to the phenomenon that the artemisinin content increased rapidly after 30 min of wounding treatment and then there was a little decrease at the time of 1 h. The artemisinin content decreased gradually after 4 h and approached nearly to the normal level at 72 h. The highest content of artemisinin was by about 50% higher than that in the control plants. The reason for this fact is probably that there are some artemisinin molecules stored in A. annua plants, which could be used for the rapid response to the environmental stress. After releasing the stored artemisinin, the A. annua plants begin to biosynthesize new artemisinin molecules immediately in 2 to 4 h and used them to adapt the wounding stress. The artemisinin content in the control samples was unchanged (fig. 1).

The Influence of Wounding Treatment on the Gene Expression Profiles

From the results above, it can be found that there are some time points during the wounding treatment, at which the artemisinin content changes greatly, such as at 0.5 h, 1 h and 4 h. Therefore, we analyzed the expression changes of some key genes involved in the artemisinin biosynthesis in A. annua leaves after wounding treatment. The expression of HMGR, ADS, CPR, and CYP71AV1 genes was significantly enhanced (fig. 2), and the gene expression reached the highest levels at 2, 8, 4, and 4 h after the treatment, respectively. However, the expression of FPS was not changed obviously during the treatment. The results demonstrated apparently that the expression of HMGR, ADS, CPR, and CYP71AV1 were affected strongly by wounding treatment, especially at the time soon after treatment, and FPS gene expression was not clearly responding to mechanic wounding (fig. 2).
HMGR catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction, which is the rate-limiting step in the synthesis of cholesterol and other isoprenoids [7]. Chappell et al. [8] reported that elicitor-inducible HMGR activity was required for sesquiterpene accumulation in tobacco cell suspension cultures. ADS, which catalyzes the cyclization of the ubiquitous precursor farnesyl diphosphate (FDP) to the sesquiterpene skeleton, was postulated to be the key enzyme in the biosynthesis of artemisinin [9]. CPR acts as cytochrome P450 reductase and is responsible for the biosynthesis of secondary metabolites in plants. For example, CPR is essential for the activity of G10H (a key enzyme for the biosynthesis of terpenoid indole alkaloids) and other cytochrome P450 monooxygenases in Catharanthus roseus [10]. The CPR promoter fragment extends from -1510 to -8 bp, and the region from -632 to -366 bp contains the main transcription-enhancing cis-regulatory sequences [11]. It had been approved that the CPR gene is regulated by the ORCA3 factor, a member of the AP2/ERF domain transcription factor family in C. roseus, and the expression of ORCA3 gene is rapidly induced by yeast extract (YE) and methyl jasmonate (MeJA) [12]. MeJA is reported to be involved in the wounding signal transduction in plants [13]. On the other hand, FPS and CYP71AV1 were demonstrated to play important roles in the biosynthesis of artemisinin [14, 15]. However, our results showed that the FPS gene expression was not affected by wounding, and this fact suggests that there are probably other factors controlling the differential expression of FPS and CYP71AV1 in A. annua plants. Therefore, based on the fact that some genes (HMGR, ADS, CPR, and CYP71AV1) were activated during the wounding treatment in A. annua and the analysis above, it could be deduced that there are probably transcription factors, which coordinate the wound signal transduction with the gene expression involved in the artemisinin biosynthesis in A. annua. The further study should pay attention to the characterization and functional analysis of transcription factors involved in the artemisinin biosynthesis in A. annua.

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Fig. 1. Contents of artemisinin in A. annua after wounding treatment.
Data represent the means ± SE of 10 randomly selected individual plants from each treatment.
1 - treatment; 2 - control.

Fig. 2. Expression profiles and digit quantities of some genes involved in the artemisinin biosynthesis in A. annua leaves after wounding treatment.
The gene expression was analyzed by using semi-quantitative RT-PCR method with 3 (l of the PCR products. HMGR, FPS, CPR, CYP71AV1, and ADS are genes involved in artemisinin biosynthetic pathway, and the expression of these genes was quantified and then digital displayed in (a), (b), (c), (d), and (e), respectively. UBC was used as the internal reference gene. Data represent the mean values ± standard error (SE) of three replicates.