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

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase ( AaICS1 ) gene from Artemisia annua 青蒿异分支酸合酶基因的克隆及功能分析 1 first-author 1 1 1 1 1 1 1 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhaojy@sjtu.edu.cn †E-mail:zhaojy@sjtu.edu.cn zhaojy@sjtu.edu.cn Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China Isochorismate synthase (ICS) is a crucial enzyme in the salicylic acid (SA) synthesis pathway. The full-length complementary DNA (cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1 , contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICS s from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis -acting regulatory elements, including TC-rich repeats, MYB binding site (MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A . annua using quantitative real-time polymerase chain reaction (qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis -acting elements in the promoter region of AaICS1 . Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography (HPLC) results from leaf tissue of transgenic A . annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway. 目的 2 研究青蒿异分支酸合酶的表达模式，评价其对青蒿素含量的影响。 创新点 2 该研究首次克隆了青蒿异分支酸合酶基因（ AaICS1 ），并发现 AaICS1 影响青蒿素的合成，为更有效地开发利用青蒿提供了新思路。 方法 2 根据青蒿转录组数据，利用聚合酶链式反应（PCR）克隆 AaICS1 基因和启动子，并进行多重序列分析和启动子作用元件预测。通过实时定量PCR（qRT-PCR）对 AaICS1 进行表达分析，用Southern杂交分析 AaICS1 的拷贝数。构建 AaICS1 过表达载体和干扰表达载体，转化青蒿获得转基因植株，用高效液相色谱法（HPLC）分析青蒿素含量。 结论 2 AaICS1 含一个总长为1710 bp的完整阅读框，编码570个氨基酸，与其它植物的ICS基因具有较高的相似性。Southern杂交结果表明 AaICS1 为单拷贝（图4），qRT-PCR结果显示该基因能够响应伤害、干旱、盐胁迫和水杨酸的处理，处理后基因表达量提高（图6），和启动子作用元件预测相符。qRT-PCR结果显示过表达转基因青蒿中 AaICS1 表达量提高，干扰转基因青蒿中该基因表达量降低（图7）。HPLC显示过表达 AaICS1 转基因植株中青蒿素含量提升，最高可达对照的1.9倍（图8）。 Salicylic acid Artemisia annua L. Quantitative real-time polymerase chain reaction (qRT-PCR) Isochorismate synthase 水杨酸 青蒿 实时定量聚合酶链式反应 异分支酸合酶 Introduction 1 The annual plant Artemisia annua L., belonging to the Asteraceae family, has attracted attention as the source of artemisinin, which is used as an alternative to quinoline drugs to treat malaria (Yin et al., 2012 2012 ). At present, A . annua is the sole commercial source of artemisinin, but the content of artemisinin in its flowers and leaves is just 0.05%–1.00% of the dry weight depending on the species (Duke et al., 1994 1994 ; Delabays et al., 2001 2001 ; Abdin et al., 2003 2003 ; Shen et al., 2012 2012 ; Patra and Srivastava, 2014 2014 ). Because of its low concentration, numerous efforts have been made to improve artemisinin production to facilitate the commercialization of A . annua . Salicylic acid (SA) is a type of phenolic phytohormone, which has been shown to be a significant player in plant growth and development. Compared with controls, 1.0 mmol/L SA could increase the content of artemisinic acid, artemisinin, and dihydroartemisinic acid (Pu et al., 2009 2009 ; Aftab et al., 2010 2010 ; Guo et al., 2010 2010 ). In addition, the artemisinin content of cell suspensions of A . annua treated with 20–50 mg/L (140–360 mmol/L) SA increased significantly, and the highest content was nearly 6-fold higher than that of controls (Baldi and Dixit, 2008 2008 ). Genes in the SA biosynthetic pathway are considered to be involved in SA production, plant development, and secondary metabolism (Loake and Grant, 2007 2007 ). In the biosynthesis of SA, isochorismate synthase (ICS; EC 5.4.4.2) catalyzes the conversion of isochorismate to yield SA (van Tegelen et al., 1999 1999 ). There are two very similar ICS genes in Arabidopsis thaliana , AtICS1 (AT1G74710) and AtICS2 (AT1G 18870) (Haas et al., 2004 2004 ). These genes are differently regulated, and only AtICS1 is induced by SA-mediated defense (Wildermuth et al., 2001 2001 ; Garcion et al., 2008 2008 ). From studies of the ics1 mutant, it is clear that the absence of ICS1 would lead to a low accumulation of SA (Wildermuth et al., 2001 2001 ). The effect of AaICS1 on the growth process is not fully understood. Therefore, it is important to determine how AaICS1 influences the growth of A . annua in terms of plant biochemistry and physiology. With the further aim to improve SA content and overcome the low yield of artemisinin, it is crucial to determine how AaICS1 affects the secondary metabolic products by way of prospective metabolic engineering in A . annua . In this study, we isolated the promoter and full-length complementary DNA (cDNA) of AaICS1 . The transcription profiles of AaICS1 under ambient stresses including wounding, drought, salinity, and SA treatments were analyzed. AaICS1 overexpression and RNA interference transgenic plants were also studied. Materials and methods 1 Plant materials and growth condition 2 Seeds of A . annua were provided by the School of Life Sciences, Southwest University, Chongqing, China. The parent sweet A . annua was used to provide cuttings for asexual reproduction. The branches were allowed to grow at (25±2) °C at 70%–80% relative humidity and with a 16-h photoperiod in plastic potted trays in the greenhouse. The cuttings were irrigated with distilled water. Total RNA was isolated from leaves, flowers, stems, and buds of A . annua using plant RNA isolation reagent (TaKaRa, Japan) in accordance with the manufacturer’s instructions. A NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA) was used to measure and examine the quality and concentration of RNA samples. Total RNA was used for reverse transcription to obtain cDNA. Generation of the full-length cDNA of AaICS1 2 Total RNA extracted from the leaves was reverse-transcribed to cDNA. The reverse transcription was performed in accordance with the following conditions: 20 min at 37 °C and 5 s at 85 °C. The cDNA was then stored as the polymerase chain reaction (PCR) template for the full length of AaICS1 amplification. Based on the data of transcriptome sequencing of A . annua in our lab and the nucleotide sequences of A . thaliana published by the National Center for Biotechnology Information (NCBI), two primers were designed ( AaICS1 -R and AaICS1 -F; Table 1 1 ). AaICS1 cDNA sequence was isolated under the following conditions: initial denaturation of template DNA for 5 min at 94 °C, followed by 35 cycles of amplification (94 °C for 30 s, 55 °C for 40 s, and 68 °C for 2 min) and 68 °C for 10 min. KOD-Plus-neo (TOYOBO, Japan) enzyme was used in the PCR. The final products were purified and ligated into pJET1.2 vectors (Thermo Scientific-Fermentas, USA) and sequenced. The primers involved in the study are listed in Table 1 1 . Sac I and Xho I sites are underlined Primer Purpose Primer sequence (5'→3')   AaICS1 -F Clone TCAAGCCTACTCACATGGGACAT   AaICS1 -R Clone CGTAAAATCTAAGGCTATCTACCATAAATC   AaICS1 -promoter-F Clone AATGATTTGGATGGAAAGGAGAC   AaICS1 -promoter-R Clone AATGGATTTTGGTTGTGGCTG   AaICS1 -Rt-F qRT-PCR AGAACTCCTGTTGGGATGACCGAAA   AaICS1 -Rt-R qRT-PCR TGTTGATTCGTGCTTGTCGTCTTCC   β-Actin -F qRT-PCR CCAGGCTGTTCAGTCTCTGTAT   β-Actin -R qRT-PCR CGCTCGGTAAGGATCTTCATCA   AaICS1 -southern-F Southern TCCAATAGAAGAGGAAGTAGAA   AaICS1 -southern-R Southern TTGATAGAACGCCAATCATGAAGT   AaICS1- pET-F Recombinant CGC GAGCTC ATGGCAGCCACAACCAAAATCCA   AaICS1 -pET-R Recombinant CCG CTCGAG CTTTTCGGACAACAGTGCCTTCTT Isolation of the promoter of AaICS1 2 Two primers ( AaICS1 -promoter-R and AaICS1 -promoter-F; Table 1 1 ) were designed according to the genome sequencing data of A. annua , to obtain the promoter sequences of AaICS1 . Genomic DNA was extracted from the leaves. PCR was performed for 5 min at 94 °C, followed by 35 cycles of amplification (94 °C for 30 s, 55 °C for 40 s, and 68 °C for 2 min 30 s) and 68 °C for 10 min. The PCR amplification was implemented using a KOD-Plus PCR kit (TOYOBO, Japan). Then the purified PCR products were ligated into a pJET1.2 vector and sequenced. Table 1 1 lists all the primers involved in this study. Bioinformatics and comparative genome analysis 2 Multiple sequence alignments were determined using the software Vector NTI Advance ® 11.5. Deduced amino acids and nucleotide sequences were predicted using the NCBI website. Phylogenetic analysis of AaICS1 and its homologues was carried out with MEGA 5.2 software. The cis -acting elements were forecasted after submitting AaICS1 5' sequence data to PLANTPAN, PLACE, and PlantCare databases. Analysis of AaICS1 expression by real-time PCR 2 Two-month-old A . annua cuttings were used for stress treatments. The leaves were punched using sterile blunt-nosed forceps to make wounds. Drought stress was induced by immersing the roots in 15% (0.15 g/ml) polyethylene glycol 6000 (PEG6000) during the experiment, with double distilled water added to the controls. For salt treatment, the cuttings were irrigated with 100, 200, or 300 mmol/L NaCl solutions while the control treatment was served with double distilled water. For SA treatment, the leaves of the cuttings were sprayed with either 0.1 or 1.0 mmol/L SA. The control treatment was served with double distilled water. Leaves were collected at different time during all stress treatments, then immediately frozen in liquid nitrogen and stored at −80 °C. Quantitative PCR was designed using a Lightcycler ® 96 PCR system (Roche, Germany) according to the manufacturer’s protocol (Zhang et al., 2013 2013 ).All samples were used to obtain the first-strand cDNA. Quantitative real-time PCR (qRT-PCR) was carried out with a Super Real PreMix Plus (SYBR Green) kit (Tiangen Biotech, Beijing, China). Every sample was denatured for 10 min at 95 °C, followed by 40 cycles of PCR amplification (15 s at 95 °C, 15 s at 58 °C, and 25 s at 72 °C) and a final extension for 10 min at 72 °C. Each sample was applied in triplicate. The reference gene was A . annua β-actin . All the primers involved in qRT-PCR are listed in Table 1 1 . Transformation vector construction 2 Transformation vectors were constructed using a Gateway ® cloning kit (Invitrogen, Waltham, MA, USA). The pHB vector was used for overexpression of the AaICS1 gene, and the pHELLSGATE 1.2 vector was used for the interference AaICS1 gene. Plant transformation 2 After immersing the A . annua seeds in 75% ethanol for 1 min and in 10% NaClO for 10 min, they were washed with sterile distilled water more than four times. These germ-free seeds were germinated on 4.4 g/L Phytagel-solidified MS 0 medium (Murashige and Skoog, 1962 1962 ) and cultured in a chamber under controlled conditions. Once the seedlings had grown to 4‒5 cm tall, the leaves were cut into 0.5 cm diameter discs that were co-cultivated for 3 d at 25 °C with Agrobacterium tumefaciens strain EHA105 (McCormick et al., 1986 1986 ). After two to three months of growth, the seedlings were transplanted into the greenhouse. Determination of artemisinin content by HPLC 2 Twenty-week-old greenhouse-grown plants were used to quantify artemisinin content using fresh weight pooled leaf samples (counted from the apical meristem) by HPLC. The treatment was carried out three times. Until used for artemisinin analysis, samples were stored at −80 °C. Crude artemisinin extract was isolated in accordance with the modified protocol from Graham et al. ( 2010 2010 ). Samples were put in 10-ml tubes, immersed in 3 ml of chloroform, and then shaken gently for 5 min. The solvent was evaporated at room temperature in a fume hood until the sample was absolutely dry. After adding 3 ml of methanol, the solution was mixed gently and treated with an ultrasonic processor. After filtering the solution through a 0.45-μm Sartorious ® membrane, 200 μl of sample was loaded into the HPLC. A Waters 2695 HPLC system coupled with a Waters 2420 evaporative light scattering detector (ELSD) was used to analyze the samples. The HPLC conditions were as follows: amethyst 5-μm C18-H 120A column, water/methanol (25:75, v/v) mobile phase, 1 ml/min flow rate. The ELSD was carried out under the optimized conditions of a drift-tube temperature of 40 °C and a nebulizer-gas pressure of 206.8 kPa (30 psi). The standard was authentic artemisinin (Sigma). We injected a volume of 20 μl of each sample and analyzed the results using Empower. Results 1 Cloning of the AaICS1 gene 2 The AaICS1 cDNA sequence was authenticated. Its opening reading-frame (ORF) was 1710 bp and encoded a 570-amino acid peptide (Fig. 1 1 ). The molecular weight was calculated as 63.81 kDa and the isoelectric point (pI) was predicted to be 6.88. Alignment revealed that AaICS1 had 57.7% homology with the A . thaliana ICS1 gene, suggesting that it could be the A . annua ICS1 gene. The AaICS1 sequence was deposited in the GenBank database under accession number KP100111. Motifs similar to the putative core promoter consensus sequences and the previously predicted cis -acting elements are boxed and their names given. Numbering starts from the potential transcription start sites. Stop and start codons are shown in grey Multialignment with other plant ICS polypeptides revealed that the deduced AaICS1 protein showed considerably high sequence homology (Fig. 2 2 ). The AaICS1 gene showed 63.7% sequence similarity to Populus tremuloides (ACX46384), 63.3% to Vitis vinifera (XP_002267681), 61.6% to Solanum lycopersicum (NP_001234794), 61.4% to Capsicum annuum (AAW66457), 61.1% to Glycine max (XP_003522193), and 61.0% to Fragaria vesca subsp. vesca (XP_004301164). Among AaICS1 polypeptide and ICS polypeptides from other species, there were different N-terminal domains among the first 30 amino acids, while the subsequent downstream residues in all ICS polypeptides were highly conserved. The ICS sequences are as follows: Populus tremuloides (ACX46384), Rubia cordifolia (ABK79678), Solanum lycopersicum (NP_001234794), Arabidopsis thaliana (AT1G74710), Vitis vinifera (XP_002267681), and Artemisia annua (KP100111). Matching amino acids are shown in capital letters against a light grey background. Less similar amino acids are shown in capital letters against a grey or dark grey background. Different amino acids are shown in black capital letters against a white background To investigate the evolutionary relationships of AaICS1 , several common species were chosen for species tree analysis. Based on the level of amino acids, an ICS phylogenic tree was constructed and calculated using MEGA 5.2 software. The AaICS1 gene was clustered with high probability and high bootstrap values with AtICS1 , AtICS2 , VvICS , SlICS , PtICS , and CrICS . The inferred phylogenic tree (Fig. 3 3 ) displayed conserved ICS orthologs with the relative genetic distance (0.05 amino acid substitution per site). The tree was constructed using AaICS1 and the following sequences: Arabidopsis thaliana (AT1G74710), Arabidopsis thaliana (AT1G18870), Brachypodium distachyon (XP_003578021), Glycine max (XP_003522193), Cicer arietinum (XP_004514127), Ricinus communis (XP_002511526), Artemisia annua (KP100111), Vitis vinifera (XP_002267681), Solanum lycopersicum (NP_001234794), Capsicum annuum (AAW66457), Fragaria vesca subsp. vesca (XP_004301164), Catharanthus roseus (Q9ZPC0), Populus tremuloides (ACX46384), and Rubia cordifolia (ABK79678) The secondary structure of the AaICS1 polypeptide was determined by SOPMA analysis. AaICS1 consists predominantly of random coils (40.07%), and α helices (39.54%) together with β turns (4.75%) and extended strands (15.64%). Prediction of AaICS1 promoter region 2 A 1470-bp DNA fragment upstream from the AaICS1 gene initiation codon (ATG) in A . annua was amplified from the genomic DNA. The sequence contained a predicted transcription start site (TSS, +1), which was found to be located 39 bp upstream of the start codon. At position −26, a TATA box was located with respect to TSS. Several cis -acting elements were found (Fig. 1 1 , Table 2 2 ). The AaICS1 promoter contained many conserved cis -acting fragments in the 5' flanking regions related to chorismic acid biosynthesis of angiosperms. cis -Element Position Signal sequence Putative function TATA-box 26 (+)  TATA Core promoter element around −30 of transcription start TGACG-motif 321 (+)  TGACG TGACG-motifs are involved in transcription of several genes by auxin and/or salicylic acid HES 598 (−)  AAAAAATTTC cis -Acting element involved in heat stress responsiveness TCA-element 507 (+)  TCAGAAGAGG 2 cis -Acting element involved in salicylic acid responsiveness 1098 (+)  CCATCTTTTT TC-rich repeats 888 (−)  GTTTTCTTAC cis -Acting element involved in defense and stress responsiveness MBS 1227 (+)  CAACTG 3 MYB binding site 1263 (+)  TAACTG 1395 (−)  CAACTG LTR 1343 (+)  CCGAAA cis -Acting element involved in low-temperature responsiveness A typical cis -acting element WRKY (TGAC) proven to be capable of activating the early genes in plant defence signaling (Eulgem et al., 1999 1999 ), was found at the position −322, −457 of the AaICS1 promoter. Analysis of the promoter boxes led to the finding of several cis -elements related to meristem-specific, stress, hormone, and numerous light response genes (Fig. 1 1 , Table 2 2 ). Bioinformatic analysis showed that several motifs were essential for some putative cis -acting motifs and tissue specific expression involved in MYB-like transcription factor binding sites. Expression analysis of AaICS1 in different tissues 2 It was considered important to determine the expression patterns of AaICS1 , since it is the first and rate-limiting enzyme in the isochorismate biosynthesis of SA. Southern blot analysis suggested that AaICS1 might be a single-copy gene (Fig. 4 4 ). RNAs extracted from leaves, stems, roots, flowers, and buds were used to examine expression levels of AaICS1 by quantitative PCR. The results showed that AaICS1 was most highly expressed in old leaves, followed by the buds and young leaves. The lowest expression was found in roots (Fig. 5 5 ). Molecular size markers are shown on the left The ratio of AaICS1 transcripts against β-actin was given as “1.0”. The y -axis indicates the relative expression levels of AaICS1 /β-actin in different tissues. Data are expressed as mean±standard deviation (SD) ( n =3) Expression analysis of AaICS1 under different treatments 2 Recent advances in studies of the signaling pathways of plants have indicated that the variation in plant metabolites induced by elicitors can be useful as a prediction tool for prospective pathway regulation and engineering. The expression of genes in the SA biosynthesis pathway can be induced by different environmental stresses, such as SA treatment (Hunter et al., 2013 2013 ; Sadeghi et al., 2013 2013 ). The predicted cis -acting elements in the AaICS1 5' flanking region suggested that AaICS1 might respond to SA, MeJA, heat, cold, wounding, drought, and other stresses. In this study, the expression patterns of AaICS1 under different treatments were obtained by quantitative PCR. To investigate the reliance of AaICS1 expression on SA, two different concentrations of SA (0.1 and 1.0 mmol/L) were applied to A . annua plantlets. AaICS1 transcription levels were monitored at different time points. AaICS1 was more effectively induced by 0.1 mmol/L SA. At 4 h, AaICS1 was noticeably induced and reached its highest level 8‒12 h after treatment, followed by a decline in expression. The higher concentration of SA had a more rapid effect on AaICS1 expression, peaking at 2 h then decreasing slightly (Fig. 6a 6a ). The results suggested a crucial role of SA in the defense response against abiotic stimuli and pathogens. The AaICS1 gene could be induced by exogenous SA, resulting in a greater supply of key precursors for defense compounds, like propanoids and flavonoids. (a) Salicylic acid (SA); (b) Salinity; (c) Wounding; (d) Drought. Data are expressed as mean±SD ( n =3 replicates) To investigate the salinity-dependence of AaICS1 , various salt concentrations (100, 200, and 300 mmol/L NaCl) were applied to A . annua plantlets. AaICS1 was markedly induced at 2 h with the 100 and 200 mmol/L salt treatments. The 200 mmol/L concentration led to a high expression of AaICS1 after 2 h, which then declined sharply. The 300 mmol/L treatment caused a low level of expression similar to that of the controls at all sampling times (Fig. 6b 6b ). To examine the activity of ICS in A . annua plants affected by wounding, we checked the expression patterns after leaf injury in a 48-h timeframe. AaICS1 was markedly induced at 2 h and then declined, but after 4 h expression was still significantly higher than that of the controls (Fig. 6c 6c ). The result indicated that AaICS1 , a key enzyme of the isochorismate pathway, had a great effect on the wounding response in A . annua . Quantitative PCR was used to analyze the expression level of AaICS1 under drought treatment. The transcript level of ICS was maximal after 24 h (Fig. 6d 6d ). The results indicated that all these stresses played a vital role in response to an internal or external stimulus. Expression of AaICS1 in transgenic plants 2 To determine whether overexpression of AaICS1 increases the artemisinin content, we analyzed the expression of AaICS1 in overexpressing and RNA interference transgenic plants of A . annua . The results showed that the expression of the AaICS1 gene in overexpressing transgenic plants was significantly higher than that of the controls (Fig. 7b 7b ). Expression levels in RNA interference transgenic plants were quite low compared with those of the controls (Fig. 7a 7a ). However, the expression of the AaICS1 gene in pHB- AaICS1 -6 and pHELLSGATE- AaICS1 -14 was not significantly different from that of the controls (Fig. 7 7 ). Researchers have reported that a co-suppression phenomenon can often occur in overexpressing transgenic plants (Yang et al., 2008 2008 ; Banyai et al., 2010 2010 ). (a) A . annua transformed with pHELLSGATE 1.2 AaICS1 ; (b) A. annua transformed with pHB- AaICS1 . CK represents non-transgenic plants (control). Data are expressed as mean±SD ( n =3). * P < 0.05, ** P < 0.01, vs. CK Estimation of artemisinin content by HPLC-ELSD 2 In the SA biosynthetic pathway of the transgenic A . annua , overexpression of the key enzyme genes could be an effective way to increase the artemisinin content (Han et al., 2006 2006 ; Aquil et al., 2009 2009 ; Banyai et al., 2010 2010 ; Nafis et al., 2011 2011 ). As SA treatment can improve the artemisinin content, the overexpression of a key gene in the SA biosynthesis pathway might increase endogenous SA, leading to improved artemisinin content (Fig. 8 8 ). (a) A. annua transformed with pHB- AaICS1 ; (b) A. annua transformed with pHELLSGATE 1.2 AaICS1 . CK represents the non-transgenic plants (control). DW represents dry weight. Data are expressed as mean±SD ( n =3). * P < 0.05, vs. CK The present experiments were conducted to observe a change of artemisinin content following the overexpression of AaICS1 in transgenic A . annua . The transgenic A . annua accumulated more artemisinin than the controls. In particular, the highest content of artemisinin was 1.9-fold greater than that of the controls. The AaICS1 RNA interference transgenic A . annua showed no significant difference in artemisinin content compared with the controls. Discussion 1 Artemisia annua L. is the sole commercial source of artemisinin. The current low yield of artemisinin means that market demand cannot be met, and it is necessary to improve the production of artemisinin. As SA can raise the artemisinin content in A . annua , identifying the genes involved in the biosynthesis pathway of SA could be beneficial. We isolated and characterized the AaICS1 gene and its promoter. Bioinformatic analysis showed that the AaICS1 gene belongs to ICS1 among plants (Fig. 2 2 ). The MYB binding site (MBS) and light responsive elements involved in fungal elicitor response were identified and located in the upstream putative cis -elements of AaICS1 (Fig. 1 1 ). Based on the present study, wounding, SA treatment, salt, and drought stress can induce the isochorismate pathway in A . annua . This implies that the AaICS1 gene might play a role in the regulation of isochorismate biosynthesis, and could increase the SA content, and thereby improve the artemisinin content. It has been recognized that accumulated secondary metabolites play crucial roles in stress-resistance of A . annua . ICS , as a vital gene in the isochorismate pathway, may stimulate the output of secondary metabolism products. Under different stress conditions, including salt (100, 200, and 300 mmol/L), SA (0.1 and 1.0 mmol/L), wounding, and drought, AaICS1 expression was induced in the leaves of A . annua (Fig. 6 6 ). We conclude that these elements had a great effect on stimulation in response to the internal or external environment. All these results imply that AaICS1 expression in A . annua is coordinated with environmental stresses. To conclude, we identified the promoter and the full-length cDNA of AaICS1 from A . annua . Bioinformatic analysis suggested that AaICS1 might have capabilities similar to those of other ICS polypeptides. Quantitative PCR analysis showed that AaICS1 mRNA accumulated mostly in buds and young leaves. Wounding, salt stress, drought, and SA greatly induced AaICS1 expression. AaICS1 might be a significant step and a potential target in the artemisinin pathway of A . annua . The cloning and identification of the AaICS1 gene will assist further transgenic studies aiming to promote SA and artemisinin content in A . annua . In summary, our study proved that overexpressing AaICS1 can improve artemisinin accumulation in A . annua plants. Not all transgenic plants had expression levels that were significantly different from those of the controls, and overexpressing a single gene might not be sufficient for raising the content of artemisinin. Similar results have been found in other studies of plant secondary metabolites (Zhang et al., 2004 2004 ). We conclude that genetic engineering may represent a useful approach for further plant metabolism studies. Contributors 2 Lu-yao WANG participated in the design of the study, performed the experiments, analyzed the data, and drafted the manuscript. Ying ZHANG contributed to the sample collection and helped finish figures. Xue-qing FU, Ting-ting ZHANG, and Jia-wei MA helped analyze the data and the results. Li-da ZHANG, Hong-mei QIAN, Ke-xuan TANG, and Shan LI participated in discussion of the results and helped revise the manuscript. Jing-ya ZHAO initiated the project, helped conceive the study, and revised the manuscript. References MZ Abdin , M Israr , RU Rehman , 等 . Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production . Planta Med , 2003 . 69 ( 4 ): 289 - 299 . 12709893 DOI: 10.1055/s-2003-38871 http://doi.org/10.1055/s-2003-38871 . Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production T Aftab , M Masroor , A Khan , 等 . Salicylic acid acts as potent enhancer of growth, photosynthesis and artemisinin production in Artemisia annua L . J Crop Sci Biotechnol , 2010 . 13 ( 3 ): 183 - 188 . DOI: 10.1007/s12892-010-0040-3 http://doi.org/10.1007/s12892-010-0040-3 . Salicylic acid acts as potent enhancer of growth, photosynthesis and artemisinin production in Artemisia annua L S Aquil , AM Husaini , MZ Abdin , 等 . Overexpression of the HMG-CoA reductase gene leads to enhanced artemisinin biosynthesis in transgenic Artemisia annua plants . Planta Med , 2009 . 75 ( 13 ): 1453 - 1458 . 19551613 DOI: 10.1055/s-0029-1185775 http://doi.org/10.1055/s-0029-1185775 . Overexpression of the HMG-CoA reductase gene leads to enhanced artemisinin biosynthesis in transgenic Artemisia annua plants A Baldi , VK Dixit . Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua . Bioresour Technol , 2008 . 99 ( 11 ): 4609 - 4614 . 17804216 DOI: 10.1016/j.biortech.2007.06.061 http://doi.org/10.1016/j.biortech.2007.06.061 . Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua W Banyai , C Kirdmanee , M Mii , 等 . Overexpression of farnesyl pyrophosphate synthase ( FPS ) gene affected artemisinin content and growth of Artemisia annua L . Plant Cell Tiss Organ Cult , 2010 . 103 ( 2 ): 255 - 265 . DOI: 10.1007/s11240-010-9775-8 http://doi.org/10.1007/s11240-010-9775-8 . Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L N Delabays , X Simonnet , M Gaudin . The genetics of artemisinin content in Artemisia annua L. and the breeding of high yielding cultivars . Curr Med Chem , 2001 . 8 ( 15 ): 1795 - 1801 . 11772351 DOI: 10.2174/0929867013371635 http://doi.org/10.2174/0929867013371635 . The genetics of artemisinin content in Artemisia annua L. and the breeding of high yielding cultivars MV Duke , RN Paul , HN Elsohly , 等 . Localization of artemisinin and artemisitene in foliar tissues of glanded and glandless biotypes of Artemisia annua L . Int J Plant Sci , 1994 . 155 ( 3 ): 365 - 372 . DOI: 10.1086/297173 http://doi.org/10.1086/297173 . Localization of artemisinin and artemisitene in foliar tissues of glanded and glandless biotypes of Artemisia annua L T Eulgem , PJ Rushton , E Schmelzer , 等 . Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors . EMBO J , 1999 . 18 ( 17 ): 4689 - 4699 . 10469648 DOI: 10.1093/emboj/18.17.4689 http://doi.org/10.1093/emboj/18.17.4689 . Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors C Garcion , A Lohmann , E Lamodière , 等 . Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis . Plant Physiol , 2008 . 147 ( 3 ): 1279 - 1287 . 18451262 DOI: 10.1104/pp.108.119420 http://doi.org/10.1104/pp.108.119420 . Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis IA Graham , K Besser , S Blumer , 等 . The genetic map of Artemisia annua L. identifies loci affecting yield of the antimalarial drug artemisinin . Science , 2010 . 327 ( 5963 ): 328 - 331 . 20075252 DOI: 10.1126/science.1182612 http://doi.org/10.1126/science.1182612 . The genetic map of Artemisia annua L. identifies loci affecting yield of the antimalarial drug artemisinin XX Guo , XQ Yang , RY Yang , 等 . Salicylic acid and methyl jasmonate but not Rose Bengal enhance artemisinin production through invoking burst of endogenous singlet oxygen . Plant Sci , 2010 . 178 ( 4 ): 390 - 397 . DOI: 10.1016/j.plantsci.2010.01.014 http://doi.org/10.1016/j.plantsci.2010.01.014 . Salicylic acid and methyl jasmonate but not Rose Bengal enhance artemisinin production through invoking burst of endogenous singlet oxygen BJ Haas , AL Delcher , JR Wortman , 等 . DAGchainer: a tool for mining segmental genome duplications and synteny . Bioinformatics , 2004 . 20 ( 18 ): 3643 - 3646 . 15247098 DOI: 10.1093/bioinformatics/bth397 http://doi.org/10.1093/bioinformatics/bth397 . DAGchainer: a tool for mining segmental genome duplications and synteny JL Han , BY Liu , HC Ye , 等 . Effects of overexpression of the endogenous farnesyl diphosphate synthase on the artemisinin content in Artemisia annua L . J Integr Plant Biol , 2006 . 48 ( 4 ): 482 - 487 . DOI: 10.1111/j.1744-7909.2006.00208.x http://doi.org/10.1111/j.1744-7909.2006.00208.x . Effects of overexpression of the endogenous farnesyl diphosphate synthase on the artemisinin content in Artemisia annua L LJR Hunter , JH Westwood , G Heath , 等 . Regulation of RNA-dependent RNA polymerase 1 and isochorismate synthase gene expression in Arabidopsis . PLoS ONE , 2013 . 8 ( 6 ): e66530 23799112 DOI: 10.1371/journal.pone.0066530 http://doi.org/10.1371/journal.pone.0066530 . Regulation of RNA-dependent RNA polymerase 1 and isochorismate synthase gene expression in Arabidopsis G Loake , M Grant . Salicylic acid in plant defence–the players and protagonists . Curr Opin Plant Biol , 2007 . 10 ( 5 ): 466 - 472 . 17904410 DOI: 10.1016/j.pbi.2007.08.008 http://doi.org/10.1016/j.pbi.2007.08.008 . Salicylic acid in plant defence–the players and protagonists S McCormick , J Niedermeyer , J Fry , 等 . Leaf disc transformation of cultivated tomato ( L . esculentum ) using Agrobacterium tumefaciens . Plant Cell Rep , 1986 . 5 ( 2 ): 81 - 84 . 24248039 DOI: 10.1007/BF00269239 http://doi.org/10.1007/BF00269239 . Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens T Murashige , F Skoog . A revised medium for rapid growth and bio assays with tobacco tissue cultures . Physiol Plant , 1962 . 15 ( 3 ): 473 - 497 . DOI: 10.1111/j.1399-3054.1962.tb08052.x http://doi.org/10.1111/j.1399-3054.1962.tb08052.x . A revised medium for rapid growth and bio assays with tobacco tissue cultures T Nafis , M Akmal , M Ram , 等 . Enhancement of artemisinin content by constitutive expression of the HMG-CoA reductase gene in high-yielding strain of Artemisia annua L . Plant Biotechnol Rep , 2011 . 5 ( 1 ): 53 - 60 . DOI: 10.1007/s11816-010-0156-x http://doi.org/10.1007/s11816-010-0156-x . Enhancement of artemisinin content by constitutive expression of the HMG-CoA reductase gene in high-yielding strain of Artemisia annua L N Patra , AK Srivastava . Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor . Appl Biochem Biotech , 2014 . 174 ( 6 ): 2209 - 2222 . DOI: 10.1007/s12010-014-1176-8 http://doi.org/10.1007/s12010-014-1176-8 . Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor GB Pu , DM Ma , JL Chen , 等 . Salicylic acid activates artemisinin biosynthesis in Artemisia annua L . Plant Cell Rep , 2009 . 28 ( 7 ): 1127 - 1135 . 19521701 DOI: 10.1007/s00299-009-0713-3 http://doi.org/10.1007/s00299-009-0713-3 . Salicylic acid activates artemisinin biosynthesis in Artemisia annua L M Sadeghi , S Dehghan , R Fischer , 等 . Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius . Plant Signal Behav , 2013 . 8 ( 11 ): e27335 24309561 DOI: 10.4161/psb.27335 http://doi.org/10.4161/psb.27335 . Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius Q Shen , YF Chen , T Wang , 等 . Overexpression of the cytochrome P450 monooxygenase ( cyp71av1 ) and cytochrome P450 reductase ( cpr ) genes increased artemisinin content in Artemisia annua (Asteraceae) . Genet Mol Res , 2012 . 11 ( 3 ): 3298 - 3309 . 23079824 DOI: 10.4238/2012.September.12.13 http://doi.org/10.4238/2012.September.12.13 . Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae) LJ van Tegelen , PR Moreno , AF Croes , 等 . Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus . Plant Physiol , 1999 . 119 ( 2 ): 705 - 712 . 9952467 DOI: 10.1104/pp.119.2.705 http://doi.org/10.1104/pp.119.2.705 . Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus MC Wildermuth , J Dewdney , G Wu , 等 . Isochorismate synthase is required to synthesize salicylic acid for plant defence . Nature , 2001 . 414 ( 6863 ): 562 - 565 . 11734859 DOI: 10.1038/35107108 http://doi.org/10.1038/35107108 . Isochorismate synthase is required to synthesize salicylic acid for plant defence RY Yang , LL Feng , XQ Yang , 等 . Quantitative transcript profiling reveals down-regulation of a sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants . Planta Med , 2008 . 74 ( 12 ): 1510 - 1516 . 18816428 DOI: 10.1055/s-2008-1081333 http://doi.org/10.1055/s-2008-1081333 . Quantitative transcript profiling reveals down-regulation of a sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants H Yin , A Kjaer , XC Fretté , 等 . Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L . Process Biochem , 2012 . 47 ( 11 ): 1559 - 1562 . DOI: 10.1016/j.procbio.2011.12.020 http://doi.org/10.1016/j.procbio.2011.12.020 . Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L F Zhang , X Lu , Z Lv , 等 . Overexpression of the Artemisia orthologue of ABA receptor, AaPYL9, enhances ABA sensitivity and improves artemisinin content in Artemisia annua L . PLoS ONE , 2013 . 8 ( 2 ): e56697 23437216 DOI: 10.1371/journal.pone.0056697 http://doi.org/10.1371/journal.pone.0056697 . Overexpression of the Artemisia orthologue of ABA receptor, AaPYL9, enhances ABA sensitivity and improves artemisinin content in Artemisia annua L L Zhang , R Ding , Y Chai , 等 . Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures . Proc Natl Acad Sci USA , 2004 . 101 ( 17 ): 6786 - 6791 . DOI: 10.1073/pnas.0401391101 http://doi.org/10.1073/pnas.0401391101 . Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures Compliance with ethics guidelines Lu-yao WANG, Ying ZHANG, Xue-qing FU, Ting-ting ZHANG, Jia-wei MA, Li-da ZHANG, Hong-mei QIAN, Ke-xuan TANG, Shan LI, and Jing-ya ZHAO declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600223 Project supported by the National High-Tech R&D Program (863) of China (No. 22011AA100605) 2016-05-17 2016-09-10 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 18