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Sesquiterpene Synthesis Essay

1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
2Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
3Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Academic Editors: Y. H. Cheong and H. Verhoeven

Copyright © 2014 Su-Fang Ee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

1. Introduction

Plants have developed a range of strategies to survive and adapt to their environment. One such strategy is to produce a large variety of secondary metabolites [1]. To date, there are an estimated 200,000 secondary metabolites that are produced by plants [2]. Polygonum minus, an aromatic plant that is indigenous to Malaysia, produces large number of secondary metabolites. Traditionally, P. minus is used to treat indigestion and dandruff problems and as a postnatal tonic [3]. This plant has a unique sweet and pleasant flavour and aroma and thus is commonly used in local cuisine. Its unique flavour is mainly due to the secondary metabolites present in the plant [4]. The secondary metabolites present in P. minus are also responsible for its useful biological properties, such as its antioxidant, antiulcer, antiviral, antimicrobial and antifungal activities [5, 6].

Secondary metabolites are divided into three major groups: terpenoids, alkaloids, and phenylpropanoids [7]. Terpenoids are the largest and most structurally diverse class. Different terpenoids are distributed unevenly within the plant kingdom [8]. Some terpenoids are restricted to one species or one genus [8]. The essential oils of P. minus have been shown to contain a high abundance of terpenoids especially the sesquiterpenes [4]. Twenty-four different types of sesquiterpenes including α-humulene, α-farnesene, β-farnesene, valencene, α-panasinsene, α-bergamotene, β-caryophyllene, δ-cadinene, and α-curcumene, have been identified, to date [4]. This diversity suggests that P. minus is a good source of sesquiterpenes for research on secondary metabolites, particularly the sesquiterpenes. Previously, most studies focused on a metabolomic approach to examine this plant, whereas those adopting a molecular approach for characterizing and understanding the biosynthetic regulation of sesquiterpene related genes are still lacking.

Based on our previously constructed cDNA-AFLP transcriptome profiles, a sesquiterpene synthase gene (GenBank: HO079100 and HO079108) was shown to be highly upregulated upon stress induced by salicylic acid [9]. Although transcriptomic data are available, the major challenge for studies of P. minus is the lack of a transformation and regeneration system for the functional study of its genes. To address this concern, the model plant A. thaliana was used in this work. A. thaliana is the first angiosperm to have had its complete genome sequenced. Since then, it has been well studied. Its short life cycle of only 3 months and its ability to produce a large number of progeny seeds have encouraged many researchers to use A. thaliana for functional studies [10]. The well-established floral-dip transformation system also provides a fast and efficient method to transfer genes from Agrobacterium into A. thaliana [11]. This method involves only a simple immersion of the floral buds in an Agrobacterium suspension.

PmSTS was previously cloned and expressed in Lactococcus lactis [12]. That study was aimed solely at maximizing metabolite production in a bacterial system and did not study how the gene is regulated in plants. In addition, the PmSTS gene introduced into L. lactis contained two incorrect nucleotides that resulted in a single amino acid change [12], calling into question the true function of this protein.

In this study, we expressed the correct sequence of PmSTS to determine the function of its protein product in a model plant system. As there is no established transformation and regeneration system available in P. minus, the model plant A. thaliana was used to investigate the role of PmSTS in plants. The overexpression of PmSTS in A. thaliana provides a better understanding of the gene function. The results of this study further enhance our understanding of the biosynthesis and regulation of secondary metabolites in P. minus, particularly the sesquiterpenes.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

A. thaliana ecotype Columbia-0 was grown in a growth chamber (Conviron) at a temperature of 22°C day/20°C night and relative humidity of 50–70%. The photoperiod was set at 16 h day/8 h night, with a light intensity of 100–150 μmoles m−2 s−1 using fluorescent bulbs. The plants were ready for floral-dip transformation one week after the primary inflorescences were clipped. Watering was stopped three days prior to transformation to increase the transformation efficiency.

2.2. In Silico Analysis of PmSTS

The nucleotide sequence of PmSTS was retrieved from the NCBI database with GenBank ID of JX025008. The physiochemical properties of the PmSTS were determined using PROTPARAM software (http://web.expasy.org/protparam/). The presence of signal peptide was predicted using SignalP 4.1 software (http://www.cbs.dtu.dk/services/SignalP/) [13]. Comparative sequence analysis of PmSTS was performed using NCBI BLAST against the protein database (http://blast.ncbi.nlm.nih.gov/). Multiple sequence alignment was done with BIOEDIT software using the default parameters (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The three-dimensional (3D) protein structure homology-modelling of the PmSTS was generated using I-TASSER software [14]. The stereochemical quality of the predicted 3D protein structure was examined through PROCHECK analysis (http://www.ebi.ac.uk/thornton-srv/software/PROCHECK/). Phylogenetic tree was built using MEGA5 software with neighbour joining method. Bootstrap of 1000 replicates was done. Terpene synthases from seven previously recognized TPS subfamilies Tps-a to Tps-g were retrieved from the NCBI GenBank database according to Bohlmann et al. [15] and Danner et al. [16]. The Tps-c and Tps-e subfamilies, which are composed of the copalyl diphosphate (cdp) synthases and kaurene synthases and are involved in primary metabolism, were chosen as outgroups.

2.3. Gene Amplification and Construction of the pCAMSS overexpression Vector

PmSTS (GenBank: JX025008) was amplified by standard PCR methods using the PmSTS specific forward primer 5′-GGGCAGATCTTATGTATTCCATGATC-3′ and reverse primer 5′-GGCTGGTGACCTTATATCAGTATGGG-3′. To facilitate the cloning process, restriction enzymes (RE) sites for BglII and BstEII (underlined) were attached to the 5′ ends of the forward and reverse primers, respectively. The nucleotides in bold type are the start (ATG) and termination (TTA) codons in the open reading frame of the PmSTS gene.

The vector pCAMBIA1301 (Centre for the Application of Molecular Biology of International Agriculture, Black Mountain, Australia) was used as the backbone for the construction of the plant transformation vector, pCAMSS, which harboured the PmSTS gene. To construct the pCAMSS vector, the β-glucuronidase (GUS) reporter gene was first excised from the pCAMBIA1301 vector and then replaced with the PCR-amplified PmSTS gene. Both the pCAMBIA1301 vector and the PmSTS gene were digested with the BglII and BstEII restriction enzymes to generate complementary sticky ends for ligation. The digested fragment of the PmSTS gene was ligated into the corresponding sites of pCAMBIA1301, yielding the pCAMSS vector (Figure 1). The pCAMSS vector was then transformed into Agrobacterium tumefaciens strain GV3101. The cloned pCAMSS vector was sent for sequencing and RE digestion to confirm the integration of the PmSTS gene in the correct orientation.

Figure 1: Schematic diagram of the T-DNA region of pCAMSS vector. The pCAMSS recombinant vector contains the full length PmSTS gene, which is expressed under the control of the constitutive CaMV35S promoter.

2.4. Agrobacterium-Mediated Floral-Dip Plant Transformation

A. thaliana was transformed using the Agrobacterium-mediated floral dip method [11]. Agrobacterium cells were grown to an OD600 of 0.7. The floral-dip inoculation medium contained harvested cells that were resuspended in 5% sucrose and 0.05% Silwet. The secondary inflorescences were immersed in the inoculation medium and swirled gently to allow the intake of Agrobacterium harbouring the pCAMSS vector into the flower gynoecium. The transformed plants were kept in the dark and wrapped with plastic overnight to maintain humidity. The next day, the plants were returned to their normal growth conditions. The transformation was repeated after a week to increase the transformation efficiency. Plants were grown for additional 4-5 weeks, until all of the siliques became brown and dry. The seeds were harvested and stored at 4°C under desiccation.

2.5. Selection of Transgenic A. thaliana

Seeds were surface sterilized with 50% Clorox containing 0.05% Tween-20 for 10 min, followed by 80% ethanol for 2 min, and the seeds were then rinsed three times with distilled water before plating. To select the transformed plants, approximately 100 sterilized seeds were screened on Murashige and Skoog (MS) solid media containing 25 mg L−1 hygromycin. The seeds were cultivated following the standard method of Harrison et al. [17]. The plated seeds were stratified at 4°C for 2 days. The seeds were then placed under light for 6 h to induce germination, followed by 2 days of incubation in the dark, and then returned to normal growth conditions. The putative transgenic plants were selected by two weeks of growth on hygromycin plates. The putative transformants grown on selection media had long hypocotyls, green leaves and long roots. These putative transformants were transferred to pots with soil and grown under normal growth conditions. The seeds from the mature plants were harvested after one month.

To verify the presence of the PmSTS gene, DNA from the putative transformants and wild type A. thaliana was extracted using the standard CTAB extraction method [18]. Wild type A. thaliana were used as the negative control for the PCR amplification. Genomic PCR was performed using a forward primer containing a region of the CaMV35S promoter (5′-TCCCACTATCCTTCGCAAGACCC-) and a reverse primer containing a PmSTS gene-specific sequence (5′-AGTGATAGGCAACTCCAAGC-3′).

2.6. Semiquantitative RT-PCR Analysis of the Transgenic A. thaliana

Semiquantitative RT-PCR was conducted to compare the expression of the PmSTS gene in the T2 transgenic A. thaliana and wild type A. thaliana. Total RNA was extracted from the leaves using the TRI Reagent (Molecular Research Centre, Inc. Cincinnati, OH, USA), according to the manufacturer’s instructions. First strand cDNA was synthesized with the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) using total RNA as the template. Semiquantitative RT-PCR analysis was performed using standard PCR methods with 500 ng of cDNA template. The forward primer (5′-CCATGATGCAGCCAACCGAGAT-3′) corresponded to nucleotide 1523–1544 of the PmSTS gene, while the reverse primer (5′-AATCCATCCTCTCCGGCGTCAT-3′) corresponded to nucleotide 1622–1643. As a control, a PCR with the housekeeping gene 4HPPD (GenBank: AT1G06570.1/NM 100536) was performed in parallel using 5′-GCGCTTCCATCACATCGAGTTC-3′ and 5′-AATCCAATGGGAACGACGACGC-3′ as the forward and reverse primers, respectively.

2.7. GC-MS Analysis of The Transgenic A. thaliana

The volatiles emitted from the leaf samples were extracted using the headspace solid-phase microextraction (HS-SPME) method. A polydimethylsiloxane-(PDMS-) coated fibre was exposed to the headspace of the sample vial containing 2 g of leaves for 30 min at 55–60°C before injection into the gas chromatograph mass spectrometer (GC-MS). The GC-MS analysis was performed on an Agilent 7890A gas chromatograph (GC) that was directly coupled to the mass spectrometer system (MS) of an Agilent 5975C inert MSD with a triple-axis detector. Separation was achieved with a 5% phenyl methylpolysiloxane column (model AB-5MS; Abel Industries) that was 30 m long and 0.25 mm in diameter and had a film thickness of 0.25 μm. Helium was used as the carrier gas, with a flow rate of 1.3 mL min−1. A splitless injection was set at 50°C hold for 3 min, increased to 250°C at a rate of 6°C min−1, and hold at 250°C for 5 min. The peaks were identified by searching the NIST/EPA/NIH mass spectral library (version 2.0), and the results were combined in a GC-MS chromatogram.

3. Results and Discussion

Sesquiterpene synthases broadly refer to enzymes that convert farnesyl diphosphate (FPP) into various sesquiterpenes. Previous studies of sesquiterpenes often comment on the structural complexity and diversity of sesquiterpene metabolism. The main causes of sesquiterpene diversity are the large number of different sesquiterpene synthases that are expressed in plants and the ability of some sesquiterpene synthases to form multiple products from a single FPP substrate [19]. The PmSTS gene has an open reading frame of 1689 bp, which encodes a 562 amino acid protein with a calculated molecular mass of 65.1 kDa and a theoretical isoelectric point (pI) of 5.29. The deduced amino acid sequence of PmSTS showed no signal peptide, which is consistent with other sesquiterpene synthases of 550–580 amino acids, and is shorter than monoterpene synthases (600–650 amino acids). The absence of an N-terminal plastid targeting signal peptide suggests that PmSTS is localized to the cytosol where FPP is found and where sesquiterpene biosynthesis takes place.

Based on the BLASTX analysis (Table 1), the closest homologue to PmSTS is the sesquiterpene synthase from Toona sinensis, with which it shares 43% identity. Although the level of amino acid sequence similarity between PmSTS and the other homologues was relatively low (≤43%), multiple sequence alignment identified several conserved motifs that are found in typical terpene synthases (Figure 2). The two highly conserved aspartate-rich motifs DDXXD (residues 314–318) and NSE/DTE (residues 465–473), which are found in most of the sesquiterpene synthases were highlighted in Figure 2, together with the other commonly conserved RXR motif (residues 277–279) region. The DDXXD and NSE/DTE motifs have been reported to flank the entrance of the active site [20]. They are involved in binding a trinuclear magnesium cluster, with DDXXD binding two magnesium ions and NSE/DTE binding one magnesium ion [21]. Catalysis of the FPP substrate occurs when it reaches the hydrophobic active site, where the diphosphate moiety of FPP interacts with the magnesium ions [22]. Thus, this magnesium cluster is important for the positioning of FPP in the hydrophobic substrate binding pocket of PmSTS [23].

Table 1: BLASTX analysis. PmSTS was compared with the NCBI protein database for gene identification purposes.

Figure 2: Multiple sequence alignment. The deduced amino acid sequence of PmSTS was aligned with homologues identified from the BLASTX analysis. Sequences highlighted in black indicate identical residues, while those in grey indicate similar residues. The conserved motifs RXR, DDXXD, and NSE/DTE are marked. PmSTS: sesquiterpene synthase [P. minus]; TsSTS: sesquiterpene synthase [Toona sinensis]; CsTPS1: terpene synthase 1 [Citrus sinensis]; CsVS: valencene synthase [Citrus sinensis]; VvGAS: germacrene A synthase [Vitis vinifera]; VvBCS: (E-) beta-caryophyllene synthase [Vitis vinifera]; EtACS: alpha-copaene synthase [Eleutherococcus trifoliatus].

Similar to other sesquiterpene synthases, PmSTS contains two large conserved domains, which were identified in a PFAM search; PF01397 corresponds to the terpene synthase family N-terminal domain, and PF03936 corresponds to the terpene synthase family C-terminal metal-binding domain. These domains are shown in the 3D model built using I-TASSER in Figure 3. The 3D protein model was constructed using 5-epi-aristolochene synthase (TEAS) [PDB accession: 1HXG] as a template. The quality of the PmSTS 3D protein model was checked using a Ramachandran plot analysis [24]. The PmSTS 3D protein model exhibited a good fit with the reference geometry with 90.4% of nonglycine and nonproline residues in the most favoured regions. From the 3D model, PmSTS was shown to consist entirely of α-helices and short connecting loops and turns. Both of the conserved aspartate-rich regions, the DDXXD, and NSE/DTE motifs were found in the C-terminal domain demonstrating the importance of the C-terminal domain, which contains the active site, for the catalysis of the substrate FPP [25]. While the actual function of the N-terminal domain remains unknown, it has been suggested to be involved in facilitating the proper folding of the catalytically active C-terminal domain [26]. Phylogenetic analysis of the deduced amino acid sequence of PmSTS showed that it belongs to the Tps-a subfamily of angiosperm sesquiterpene synthases (Figure 4) [15, 16].

Figure 3: Predicted 3D model of PmSTS generated by the I-TASSER server. The two conserved domains are highlighted. The N-terminal domain is in blue and the C-terminal domain is in green. Both aspartate-rich motifs are displayed using ball and stick representation with CPK colour. The wire bonds represent the rest of the protein.

Figure 4: Phylogenetic tree of PmSTS with selected terpene synthases from other plants. Seven previously identified TPS subfamilies (Tps-a to Tps-g) were chosen based on Bohlmann et al. [15] and Danner et al. [16]. The Tps-c and Tps-e subfamilies, which are composed of the copalyl diphosphate (cdp) synthases and kaurene synthases and are involved in primary metabolism, were chosen as outgroups. The alignment was performed using the Clustal Omega algorithm. The tree was built using the neighbour joining method and 1000 replicates for bootstrapping. The numbers indicated are the actual bootstrap values of the branches.

The role and product specificity of PmSTS were determined by generating transgenic A. thaliana. Overexpression of PmSTS in A. thaliana was accomplished using Agrobacterium harbouring the transformation vector pCAMSS. Using the Agrobacterium-mediated floral-dip transformation method, ten hygromycin-resistant transgenic A. thaliana were successfully generated. These plants had long hypocotyls, green leaves, and long main roots with the formation of lateral roots (Figure 5). In contrast, the nontransformants showed short hypocotyls, bleached out leaves, and no lateral root formation (Figure 5). The putative transformants were further verified using PCR amplification of the plant genomic DNA. Fully mature leaves from ten 60-day-old putative transgenic plants and one wild type plant were collected for DNA extraction. All the putative transformants gave rise to a band of the expected size of 365 bp, while the wild type plant showed no amplification. This result confirmed the presence of the PmSTS gene in the genomes of the transgenic plants.

Figure 5: Comparison of hygromycin-sensitive and hygromycin-resistant (shown in arrow) seedlings. The seedlings were screened for 20 days on selection plate containing MS media supplemented with 25 mg L−1 hygromycin.

Two of the transgenic plants, designated as OE3 and OE7, were selected for further analysis. Semiquantitative RT-PCR analysis was performed. Both the OE3 and OE7 plants showed high expression of the PmSTS gene by the amplification of a distinct band at 100 bp, which was absent in the wild type plants (Figure 6). Meanwhile, from the morphological analysis, both plants showed delayed growth compared to the wild type plants (Figure 7). The OE3 and OE7 plants took an additional month to reach the seed maturation step. This phenotype was inheritable, as the T2 plants of these two lines also showed similar growth retardation. The plants also demonstrated smaller basal rosette leaves and shorter and fewer flowering stems. Although flowers and viable seeds were still produced from these plants, the number of seeds obtained was halved compared to the wild type plants.

Figure 6: Semiquantitative RT-PCR for sesquiterpene synthase (PmSTS). PCR for the 4HPPD gene was performed in parallel as a positive control. M: 100 bp DNA ladder; WT1 and WT2: wild type A. thaliana plants; OE3: OE3 transgenic plant; OE7: OE7 transgenic plant; N: no template control.

Figure 7: Comparison of the phenotypes of the transgenic A. thaliana and wild type A. thaliana. (a) Plants (2-month-old) were grown on soil for 1 month. Upper panel: OE3 transgenic plant and wild type plant. Lower panel: OE7 transgenic plant and wild type plant. (b) Two 4-month-old OE3 and OE7 T2 plants compared with a 3-month-old wild type plant.

It has been suggested that the overexpression of the PmSTS gene in transgenic plants interferes with the IPP substrate pool in the cytosol (Figure 8). This overexpression causes the channelling of more isopentyl pyrophosphate (IPP), the building block for terpenes, and FPP to the overexpressed PmSTS. This certainly lowers the flux of IPP to the plastids for the synthesis of other essential and larger isoprene products that are important for the plant growth, such as gibberellins (GA) [27, 28]. This model was further supported when many of the gene modifications involving terpenoid biosynthesis, such as the overexpression of the strawberry linalool/nerolidol synthase (monoterpene) and taxadiene synthase in A. thaliana, also resulted in a dwarf phenotype due to a decrease in the level of GA [27, 29]. The GA-deficient A. thaliana mutants designated as dwarf and delayed-flowering 1 (ddf1) also demonstrated a similar reduction in plant size and other similar phenotypes [30].

Figure 8: A simplified version of the sesquiterpene biosynthetic pathway. This pathway shows the predicted flux of substrate (IPP and FPP) to produce more sesquiterpenes (C15) in transgenic plants overexpressing the PmSTS gene. (+) indicates the increased flux of substrate and (−) indicates the decreased flux of substrate. GA3P: glyceraldehyde 3-phosphate. HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA. DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase. MVA: mevalonate acid. MEP: methyl erythritol phosphate. IPP: isopentyl pyrophosphate and its isomer DMAPP: dimethylallyl diphosphate. FPP: farnesyl diphosphate. GGPP: geranylgeranyl diphosphate (modified from Okada et al. [31] and Vickers et al. [32]).

A GC-MS analysis was performed to identify the specific product produced by transformation with the PmSTS gene. In this analysis, material extracted from the A. thaliana leaf samples was examined using the HS-SPME method. By using leaf samples, we were able to reduce the detection of background terpenes, as A. thaliana leaves were previously reported to not emit or to only emit traces of terpene volatiles [33]. In addition, GC-MS analysis was performed with wild type plants as a control. The GC-MS analysis yielded two chromatograms with similar patterns (Figure 9). However, a very clear difference was observed for the transgenic plant OE3, as an additional peak was present at the retention time of 20.834. This peak was identified as β-sesquiphellandrene, based on the closest hit from a search of the NIST/EPA/NIH library (version 2.0). The mass spectrum of the β-sesquiphellandrene peak compared with that of the highest hit from the library was shown in Figure 10. The production of β-sesquiphellandrene by PmSTS was in agreement with the findings from Song et al. [12]. This result also showed that the point mutation introduced by Song et al. [12] at K266E does not affect the product specificity of PmSTS in producing β-sesquiphellandrene. Having obtained the same product in both L. lactis and A. thaliana, we have showed that PmSTS was responsible for the production of β-sesquiphellandrene through the common mevalonate pathway in sesquiterpene biosynthesis.

Figure 9: GC-MS chromatograms of the transgenic OE3 (b) and a wild type plant (a). The arrow () indicates the peak at the retention time of 20.834 that shows high match with β-sesquiphellandrene in the NIST/EPA/NIH library.

Figure 10: Mass spectra of β-sesquiphellandrene. (a) shows the mass spectrum for the sample compound, while (b) shows the mass spectrum of the highest hit in the NIST/EPA/NIH library.

4. Conclusion

The diversity of the sesquiterpenes found in P. minus renders this plant a major resource for research related to sesquiterpene biosynthesis. In this study, we applied transgenic technology by overexpressing PmSTS in A. thaliana. Growth retardation was clearly observed in the transgenic lines OE3 and OE7. These two plants showed a high expression of the PmSTS gene, which resulted in the production of β-sesquiphellandrene. The β-sesquiphellandrene produced in these A. thaliana transgenic plants indicated the effectiveness of A. thaliana in synthesizing the same product as in previously expressed L. lactis through the common mevalonate pathway of sesquiterpene biosynthesis. This research strongly suggests the potential of the A. thaliana plant system for the study of the sesquiterpene synthase genes in P. minus.

Conflict of Interests

All authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was funded by Universiti Kebangsaan Malaysia through University Research Grants (UKM-GUP-KPB-08-33-135) awarded to Professor Dr. Zamri Zainal. The preparation of this paper was supported by Grant UKM-DLP-2013-004 funded by Universiti Kebangsaan Malaysia. The authors thank Ministry of Science, Technology and Innovation in Malaysia for the National Science Fellowship (NSF) awarded to Su-Fang Ee. They also thank Dr. Md. Imtiaz Uddin and Dr. Nurul Hikma Md Isa for proofreading this paper.

Introduction

ATP-binding-cassette (ABC) proteins are one of the biggest protein families in plants, which function as channels, molecular switches, and transporters (Sugiyama et al., 2006). ABC transporters are divided into different subfamilies depending on the combination of the structural elements (Verrier et al., 2008). One family of these, pleiotropic drug resistance (PDR) transporters, the full size ABCG subfamily, consist of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). The NBDs contain Walker A motifs, Walker B motifs, and the ABC signature motifs (Biemansoldehinkel et al., 2006). In plants, PDR transporters are reported to be involved in varieties of biological functions, including terpenoids and phytohormone transport, cuticular formation, defense against pathogens, and resistance to cadmium and lead (Jasiński et al., 2001; Lee et al., 2005; Stukkens et al., 2005; Ito and Gray, 2006; Kobae et al., 2006; Strader and Bartel, 2009; Kang et al., 2010; Kim et al., 2010; Bessire et al., 2011). The first plant PDR transporter, SpTUR2, was cloned from Spirodela polyrrhiza, which might play a role in response to conditions inhibiting plant growth (Smart and Fleming, 1996). Then SpTUR2 was conferred on the resistance to the antifungal diterpene sclareol (Van Den Brûle et al., 2002). The work on ABC transporters in Nicotiana plumbaginifolia showed that NpABC1 was regulated by the antifungal diterpenes sclareol and sclareolide in cell cultures (Jasiński et al., 2001). Subsequently NpPDR1 was reported to be involved in the secretion of defense-related metabolites (Stukkens et al., 2005). And the expression of NtPDR1 in Nicotiana tabacum BY2 cells and transport tests suggested that NtPDR1 was involved in diterpene transport to defend against biotic threats (Crouzet et al., 2013). Besides, it has been reported that (AtPDR12)/ABCG40 mediates cellular uptake of the phytohormone abscisic acid (Kang et al., 2010). Furthermore, some PDR transporters were reported to contribute to heavy metals resistance, such as cadmium (Cd2+) and lead (Pb2+) (Lee et al., 2005; Kim et al., 2007). Cadmium and lead are common pollutants in soil, which are dangerous to plants growth (Raskin et al., 1997; Lanphear, 1998). In plants, AtPDR8-overexpressing plants showed stronger Cd2+ or Pb2+ resistance, and AtPDR8 RNAi transgenic plants and T-DNA insertion lines were more sensitive to Cd2+ or Pb2+ compared to wild-type plants (Kim et al., 2007). AtPDR12, an ABC transporter, was reported to contribute to Pb2+ resistance in Arabidopsis (Lee et al., 2005).

Artemisia annua L., a traditional Chinese medicinal plant, is famous for producing the sesquiterpenoid endoperoxide artemisinin. Artemisinin-based combination therapies (ACTs) are a recommended treatment against the cerebral and chloroquine-resistant malaria by the World Health Organization (WHO; White, 2008). In addition to artemisinin, a large number of monoterpenes, sesquiterpenes, and triterpenes are presented in A. annua with functions in growth, development and defense in plants (Wei et al., 1992; Fulzele et al., 1995; Holm et al., 1997; Tellez et al., 1999; Bhakuni et al., 2001; Goel et al., 2007). In fact, the monoterpenes from A. annua contain the regular monoterpenes, the rearranged monoterpenes, and the irregular monoterpenes (Charles et al., 1991; Woerdenbag et al., 1994; Jia et al., 1999). The sesquiterpenes β-caryophyllene, β-farnesene, germacrene D, germacrene A, amorphadiene, and epi-cedrol were isolated from A. annua (Fulzele et al., 1995; Bouwmeester et al., 1999; Juteau et al., 2002). Monoterpenes and sesquiterpenes as the major volatile compounds of plants are usually emitted to defend against biotic threats (Degenhardt et al., 2003). For example, (E)-β-farnesene (EβF) is an important volatile compound of plants, which functions as the main component of the aphid alarm pheromones (Bowers et al., 1972; Pickett and Griffiths, 1980; Francis et al., 2004). A sesquiterpene, β-caryophyllene, is distributed in essential oils of plants with the anti-inflammatory, antibiotic, antioxidant, anticarcinogenic, and local anesthetic activities (Legault and Pichette, 2007). The triterpenoids include sterols, steroids, and saponins, are a large and structurally diverse group of natural products, derived from squalene (Xu et al., 2004).

With so many varieties, the sesquiterpene biosynthesis network is quite complicated in A. annua (Figure S1). Fortunately, several sesquiterpene synthases have been reported from A. annua. Sesquiterpenes, like artemisinin, are synthesized via the direct precursor farnesyl diphosphate (FPP) in plants. In sesquiterpene biosynthesis, FPP is converted to an array of cyclization products, such as amorpha-4,11-diene, β-caryophyllene, β-farnesene, germacrene A, and epi-cedrol, by amorpha-4,11-diene synthase (AaADS; Bouwmeester et al., 1999), β-caryophyllene synthase (AaCPS; Cai et al., 2002), β-farnesene synthase (AaBFS; Picaud et al., 2005), germacrene A synthase (AaGAS; Bertea et al., 2006), and epi-cedrol synthase (AaECS; Mercke et al., 1999) respectively in A. annua. In addition, it is well-known that geranyl diphosphate (GPP) is the precursor of monoterpenes. The formation of monoterpene linalool is catalyzed by linalool synthase (AaLAS; Jia et al., 1999). Squalene synthase (AaSQS) is a key enzyme of sterol and triterpene pathway (Liu et al., 2003). The synthesis of triterpene β-Amyrin is catalyzed by β-Amyrin synthase (AaBAS).

There are two kinds of trichomes in A. annua, glandular trichomes and T-shaped trichomes, in which large quantities of secondary metabolites are synthetized, stored and volatilized to protect plants against plant pathogens, neighboring plants, insects, and herbivores (Wagner, 1991; Duke and Paul, 1993; Pichersky and Gershenzon, 2002). The glandular trichomes where artemisinin biosynthesis occurs, contains two stalk cells, two basal cells, and three pairs of secretory cells (Duke and Paul, 1993; Olsson et al., 2009). By contrast, the research on T-shaped trichomes is still largely unknown. Previous studies demonstrated that AaCPS was primarily located in T-shaped trichomes, roots, buds, and flowers, while AaBFS was expressed in T-shaped trichomes, glandular trichomes, and roots (Wang et al., 2013, 2014). The transcriptome of T-shaped trichomes was sequenced using Illumina RNA-Seq. The result showed that the specific terpene metabolic pathways were also existed in the T-shaped trichome (Soetaert et al., 2013). In one publication, the authors cloned PDR1 and PDR2 transporters from A. annua and suggested that PDR2 was related to artemisinin biosynthesis in tobacco, although the substrate was not verified (Wang et al., 2016).

Therefore, these findings indicate that the multicellular T-shaped trichomes have the capacity to synthesize and store large quantities of sesquiterpenes in A. annua. Numerous studies have identified genes related to sesquiterpenes biosynthesis in A. annua, but little is known about the sesquiterpenes transport. Hence, it will be interesting to investigate sesquiterpenes transporters in the biofactories. Here, we identified a PDR transporter PDR3 (AaPDR3) from the T-shaped trichomes RNAseq databases, which is specifically expressed and developmentally regulated in A. annua. The decrease and increase in the transcript levels of AaPDR3 in the RNAi and overexpression plants resulted in the decrease and increase of β-caryophyllene contents, respectively. Besides, when AaPDR3 was expressed in yeast, β-caryophyllene was accumulated faster than the control. From these results, we identified a PDR transporter involved in β-caryophyllene transport in A. annua.

Experimental Procedures

Plant Material and Growth Conditions

A. annua named as “Huhao 1,” originated from Chongqing, was developed in Shanghai after selection for several years. Plants were grown in the greenhouse with a 16/8 h light/dark photoperiod at 25°C.

Isolation and Characterization of AaPDR3

T-shape trichomes were collected from the capitulum of A. annua with laser capture microdissection. The RNA from T-shape trichomes was extracted and sequenced (Soetaert et al., 2013). Arabidopsis ABC protein sequences were obtained from the Arabidopsis Information Resource (TAIR) database. A. annua putative ABC transporters were searched performing a BLASTP analysis against the transcriptome database using Arabidopsis ABC transporter protein sequences as queries with an “E” value over e−120. Then the sequences of polypeptides corresponding to A. annua ABC transporters were analyzed in the Conserved Domain Database (CDD) at NCBI (Cakir and Kilickaya, 2013). The ABC transporters protein sequences from A. annua and PDR protein sequences from Arabidopsis were aligned with ClustalX. The phylogenetic tree was constructed by MEGA software (Tamura et al., 2011). Based on the RNAseq databases, we predicted the full-length AaPDR3 sequence. To obtain the open reading frame (ORF) of AaPDR3, the cDNA was synthesized with 0.5 μg total RNA isolated from leaves of A. annua, and the ORF was amplified using the gene-specific primers (Table S1). The phylogenetic tree analysis was performed with MEGA software version 5 via the neighbor-joining method based on amino acid sequence alignment, and the bootstrap analysis was performed using 1,000 replicates. Roots, stems, young leaves (the two youngest leaves), old leaves (from the 15th to the 16th leaf), buds and flowers of the A. annua plants were collected for RNA extraction using plant RNA isolation reagent (Tiangen, Beijing, China) following the manufacturer's instructions. The leaves from the Leaf0 (meristem), Leaf1, Leaf2, Leaf3, Leaf4, Leaf5, and Leaf6 counted from the apical top of the main stem were collected from 5-month-old A. annua. The total RNA was used to synthesize the first-strand cDNA. All the tissues and leaves collected from three plants were separately pooled for each determination. For hormone treatment, 2-month-old A. annua plants were treated with 100 μM MeJA (Sigma-Aldrich, USA), and then sampled at 0, 0.5, 1.5, 3, 6, 9, 12, 24 h, water with 1% concentration of DMSO as a mock treatment. The fifth leaves collected from three plants were separately pooled for each determination for RNA isolation. Real-time qPCR was carried out using the SuperReal PreMix Plus (SYBR Green) kit (Tiangen, Beijing, China) on lightcycle®96 (Roche, Mannheim, Germany). Three biological repeats were measured for each sample.

Subcellular Localization of AaPDR3

The full-length ORF of AaPDR3 was cloned into BamHI and XbaI sites of pHB-GFP vector. The recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 for A. tumefasciens-based Nicotiana benthamiana leaves transient expression (Voinnet et al., 2003). To confirm the localization of AaPDR3, we co-expressed the fusion protein GFP-AaPDR3 and the plasma membrane protein PIP1-mCherry in tobacco leaf epidermal cells. The GFP signal was observed after 2–3 days by Leica TCS SP5-II confocal laser microscopy (Leica, Wetzlar, Germany).

Molecular Cloning of AaPDR3 Promoter and Promoter-GUS Fusions in Transgenic A. annua

Genomic DNA was extracted from fresh young leaves of A. annua using the CTAB method. The upstream region 2,059 bp of AaPDR3 was obtained from the genome database of A. annua, amplified from genomic DNA with primers containing PstI and BamHI restriction sites and inserted into pCAMBIA1391Z vector. The resulting construct was transformed into A. annua plants, as described previously (Zhang et al., 2009).

Construction of Plant Expression Vector and Transformation of A. annua

The 346 bp fragment of AaPDR3 was amplified, cloned into gateway cloning vector pENTR vector using pENTR™/SD/D-TOPO® Cloning Kit (Invitrogen, Carlsbad, CA, USA), and then transferred to the destination vector pHELLSGATE12 via the LR recombination reaction (Invitrogen). The recombination plasmids (pHB-GFP-AaPDR3 and pHELLSGATE12-iAaDPR3) were introduced into A. tumefaciens strain EHA105 and transformed into A. annua plants, as described previously (Zhang et al., 2009).

Histochemical GUS Staining and Western Blot Analysis

The leaves were sampled from non-transgenic plants and transgenic plants for the histochemical GUS staining (Jefferson, 1987). The photographs were taken using an optical microscope (OLYMPUS, Japan). Two hundred milligrams of young leaves were powdered in liquid nitrogen, solubilized in the 2 volumes of buffer (100 mm Tris-HCl [pH 8], 50 mm KCl, 10 mm MgCl2, 20 mm DTT, and 2% Trixon-100) containing the protease inhibitors Cocktail and 1 mM phenylmethylsulfonylfluoride for 20 min on ice and centrifuged at 10,000 g for 10 min at 4°C twice. The supernatant was denatured by 2x sample buffer (125 mm TrisHCl [pH 6.8], 20% glycerol, 4% SDS, 200 mm DTT, and 0.05% bromophenol blue), incubated at 60°C for 15 min and clarified by centrifugation at 10,000 g for 1 min. The protein samples were separated on 8% SDS-PAGE gels and transferred onto nitrocellulose filters (0.45 μm pore size) (Millipore, USA). The membranes were blocked in 5% (w/v) non-fat milk powder for 2 h, and incubated with a 1:20,000 dilution of the primary antibody (Abmart, China) at 4°C overnight. The membranes were washed, incubated with a 1:10,000 dilution of goat anti-mouse alkaline phosphatase-conjugated secondary antibody (Sigma, USA), and detected using eECL Western Blot Kit (Kangwei Bio Inc., China).

GC-MS Analysis

The fresh samples were ground into fine powder in liquid nitrogen and freeze-dried for 72 h at −50°C. Fifty milligrams powder was suspended in 4 mL chromatographic-grade hexane in 10 mL glass tube with 100 μL trans-farnesol (77.6 μg/mL) as the internal standards, vigorously vortexed for 1 min and extracted for 40 min in an ultrasonic processor (JYD-650; Shanghai Zhisun Instrument Co. Ltd, China). The samples were centrifuged at 4,000 g for 10 min. The supernatants were filtered through 0.25-μm-pore-size filters, then concentrated and redissolved in 200 μL chloroform. GC-MS analysis was performed according to the methods described previously (Zhang et al., 2009). Three biological repeats were measured for each sample. Germacrene D was purchased from ChemFaces. B-caryophyllene and β-farnesene were purchased from Sigma-Aldrich.

Quantification of Artemisinin by HPLC-ELSD

The leaves of A. annua were collected, dried for 48 h at 50°C and ground into powder. One hundred milligrams of powder was extracted with 1 mL methanol for 30 min in an ultrasonic processor twice. The samples were centrifuged at 10,000 g for 10 min. The supernatants were filtered through 0.25-μm-pore-size filters and analyzed by the Waters Alliance 2695 HPLC system coupled with a Waters 2420 ELSD detector (Milford, USA) (Zhang et al., 2009). Three biological repeats were measured for each sample.

Functional Analysis of AaPDR3 in Yeast Cells

AaPDR3 was cloned into the SpelI and PstI sites of pDR196 by In-Fusion PCR cloning kits (Clontech, Palo Alto, CA, USA). The recombinant plasmid was transformed the strain AD1234567833 by the lithium acetate method. The yeast transformant was incubated in 50 mL SD medium (-uracil) at 29°C with shaking at 180 rpm, harvested at A600 = 1.0, and suspended by 50 mL half-strength SD medium (-uracil) containing β-caryophyllene, β-farnesene, and germacrene D, respectively. The cells were cultivated at 29°C with shaking at 180 rpm, harvested at the indicated times by centrifugation, washed twice with sterile water. The cells were disrupted with acid-washed glass beads in methanol for 15 min at 30 Hz (Yu and De Luca, 2013). Yeast cells were incubated in the culture media in the range of 0–1,200 μM β-caryophyllene for 1.5 h at pH 5.9. The cells harvested at the indicated times by centrifugation, washed twice with sterile water. Samples were centrifuged and filtered for GC-MS analysis.

Results

Isolation and Characterization of AaPDR3

Several studies have confirmed that many sesquiterpenes, with important biological functions, are produced in plant flower, leaf, secretory organ and root under constitutive, and stress conditions (Tholl, 2006). So we isolated T-shape trichomes from the capitulum of A. annua with laser capture microdissection and generated an RNA-Seq data based on RNA isolated from T-shape trichomes. Based on the T-shape trichomes transcriptome databases, we identified the 88 putative ABC transporters by performing a BLASTP analysis using Arabidopsis ABC transporter protein sequences as queries (Supplementary Information Data 1). We performed the phylogenetic analysis between PDR subfamily transporters found in Arabidopsis thaliana and the putative ABC transporters from A. annua. The result showed that four proteins were clustered with PDR transporters from Arabidopsis thaliana, and four PDR proteins (contig012562, contig001446, contig009129, and contig004541) were clustered with PDR transporters from Arabidopsis (Figure 1). Subsequently, we performed a phylogenetic tree analysis with the predicted amino acid sequences of four candidate PDR proteins and some PDR transporters containing Arabidopsis PDR transporters, NpPDR1, NtPDR1 and SpTUR2, showing that contig004541 protein sequence was similar to that of PDR proteins (AtPDR12, NpPDR1, NtPDR1, and SpTUR2) involved in terpene transport (Figure 2A). Therefore, this gene, named AaPDR3, was further examined as the candidate gene. AaPDR3, which is 4,278 bp in length, encodes a protein of 1,425 amino acids. This protein, belonging to the full-length size PDR subfamily, contains two nucleotide-binding domains (NBD) and two transmembrane domains (TMD; Figure 2B). Compare the conserved domain of known PDR transporters involved in terpene transport exhibited the high conservation in plants (Figure 2C). Besides, we analyzed the expression of AaPDR3 after the treatment with 100 μM MeJA, showing that MeJA induced the expression of AaPDR3 in A. annua (Figure S2).

Figure 1. Phylogenetic tree showing the relationship ABC transporters expressed in T-shape trichomes of A. annua compared with some PDR transporters from Arabidopsis. The tree presented here is a neighbor-joining tree based on amino acid sequence alignment.

Figure 2. Sequence analysis of AaPDR3. (A) Phylogenetic analysis of PDR proteins from A. annua and some known PDR transporters from Arabidopsis, N. plumbaginifolia NpPDR1, N. tabacum NtPDR1, and S. polyrrhiza SpTUR2. The tree presented here is a neighbor-joining tree based on amino acid sequence alignment. (B) The structure of AaPDR3 was predicted by scanning the deduced amino acid sequence. NBD and TMD indicate the predicted location of NBDs and TMDs, respectively. (C) Multiple alignment of the conserved domain of known PDR transporters involved in terpene transport has the high conservation in plants. The Walker A, Walker B, and ABC signature motifs are shown with shading. The identical amino acid residues in are marked by asterisks.

Expression of AaPDR3 is Tissue-Specifically and Developmentally Regulated in A. annua

Previous studies with CPS and BFS showed that the biosynthesis of related sesquiterpenes took place in roots, stems, leaves, and flower buds where they may play roles in defending the plant against fungal and worm attack (Lv et al., 2016). Consistent with these findings, investigation of AaPDR3 transcript level by RT-qPCR revealed that AaPDR3 expression level was the highest in T-shaped trichomes (Figure 3A). AaPDR3 is also determined in roots, stems, leaves, and flower buds (Figure 3A). Moreover, we analyzed the expression of AaPDR3 in leaves at different developmental stages. The expression level is the lowest in the youngest leaf (leaf0) and increased gradually with the leaves aging (Figure 3B).

Figure 3. Real-time PCR analysis for AaPDR3 expression. (A,B) Relative expression of AaPDR3 in (A) TST (T-shaped trichomes), roots, stems, old leaves, young leaves, buds, flowers, and (B) leaves of different developmental ages of A. annua. ACTIN was used as internal control. The error bars represent the means ± SD (standard deviation) from three technical replicates.

AaPDR3 Is Located to the Plasma Membrane

Analysis of the encoded AaPDR3 protein by the subcellular prediction programs (Predotar: https://urgi.versailles.inra.fr/predotar/predotar.html; WoLF PSORT: http://www.genscript.com/psort/wolf_psort.html) predicted that this protein has no N-terminal signal peptide and is located to the plasma membrane. To examine the subcellular localization of AaPDR3 protein, the green fluorescent protein (GFP) fused to the N-terminal domain of AaPDR3 under CaMV35S promoter was transiently expressed in tobacco leaves. Results showed that GFP fluorescence of leaves expressing GFP-AaPDR3 was only observed in the plasma membrane (Figure 4A). The GFP fused to the N-terminal domain of AaPDR3 together with the established plasma membrane marker PIP1 (Siefritz et al., 2002) fused to mCherry were transiently co-expressed in tobacco leaves. The GFP-AaPDR3 green fluorescent signal was colocalized to the plasma membrane with PIP1-mCherry (Figure 4B). The results were consistent with those from prediction programs, indicating that AaPDR3 was localized in the plasma membrane and might function as a transporter.

Figure 4. The subcellular localization of AaPDR3. (A) Localization of AaPDR3 in tobacco leaves. Bars = 10 μm. (B) AaPDR3 protein co-localized with plasma membrane integral protein PIP1 on the plasma membrane of tobacco leaves determined through confocal microscopy. Bars = 40 μm.

AaPDR3 Is Expressed in T-Shaped Trichomes and Roots of A. annua

To further investigate the tissue-specific expression pattern of AaPDR3 in A. annua, a 2,059-bp genomic fragment corresponding to the predicted AaPDR3 promoter sequence in our genome database was cloned from A. annua and then fused to GUS reporter gene. The recombinant plasmid was introduced into A. annua plants. GUS activity was analyzed in different tissues in A. annua. The result showed that GUS-staining was primarily restricted to T-shaped trichomes of old leaves in transgenic plants (Figures 5A–C). And GUS activity was also observed in roots in transgenic plants (Figure 5D), where a large number of sesquiterpenes are synthesized and stored.

Figure 5. AaPDR3 is mainly expressed in T-shape trichomes and roots. The expression of the proAaPDR3-GUS was observed in (A) the first leaf, (B) the fifth leaf, (C) the sixth leaf, and (D) the root.

AaPDR3 Affects Sesquiterpenes β-Caryophyllene Biosynthesis in A. annua

To explore the function of AaPDR3 in A. annua, we generated 34 AaPDR3-RNAi transgenic plants using an RNAi strategy under the control of the CaMV35S promoter. In the RNAi transgenic plants, four independent lines with 14–34% observably downregulated AaPDR3 expression (Figure 6A) were selected for the detailed metabolic profiling analysis by gas chromatography-mass spectrometry (GC-MS) analysis (Figure S3). In contrast with the wild type, the suppression of AaPDR3 led to a 32–86% reduction of β-caryophyllene content (Figure 6B), while germacrene D and β-farnesene levels remained unchanged in AaPDR3-RNAi lines compared with wild type (Figure 6B). These data indicate that the repression of AaPDR3 markedly results in the suppression of sesquiterpene β-caryophyllene biosynthesis in A. annua.

Figure 6. Comparative analyses of AaPDR3 gene expression and sesquiterpene analyses in wild type (WT), in four AaPDR3-RNAi and four AaPDR3-overexpression plants. (A) Relative expression of AaPDR3 in AaPDR3-RNAi transgenic A. annua lines. (B) The contents of β-farnesene, β-caryophyllene, and germacrene D in AaPDR3-RNAi transgenic A. annua lines. (C) Relative expression of AaPDR3 in AaPDR3-overexpression transgenic A. annua lines. (D) The contents of β-farnesene, β-caryophyllene, and germacrene D in AaPDR3-overexpression transgenic A. annua lines. The error bars represent the means ± SD from three biological replicates, and asterisks indicate statistically significant differences compared with WT. *P < 0.05, **P < 0.01.

AaPDR3 under the control of CaMV35S promoter was overexpressed in A. annua. We obtained 28 AaPDR3-overexpressing transgenic plants. Investigation of AaPDR3 transcript levels by qRT-PCR showed that the AaPDR3 expression was significantly increased in four AaPDR3-overexpression lines (Figure 6C). The four independent transgenic lines were identified by Western blot (Figure S4). Consistent with a role involved in sesquiterpenes biosynthesis transport in planta, the level of β-caryophyllene was increased to 0.48 mg/g FW in AaPDR3-overexpression lines compared to control (0.28 mg/g FW; Figure 6D). Little increases of β-farnesene and germacrene D were observed in AaPDR3-overexpression plants compared to wild type (Figure 6D). Taken together, AaPDR3 is involved in the sesquiterpene β-caryophyllene biosynthesis in A. annua. Moreover, the repression of AaPDR3 observably increased artemisinin contents in the RNAi plants (Figure S5).

AaPDR3 Functions as β-Caryophyllene Transporter in Yeast Strain AD1-8

A heterologous yeast expression system is an informative approach for elucidating the function of transporters (Morita et al., 2009; Shitan et al., 2013; Yu and De Luca, 2013). To investigate the function of AaPDR3 transporter, we expressed the AaPDR3 cDNA in the yeast strain AD12345678 lacking eight major ABC transporters and one transcription factor (Decottignies et al., 1998). Then we selected β-caryophyllene, β-farnesene, and germacrene D as the candidate substrates, respectively. The yeast cells of AaPDR3 transformant and the control (transformed with the empty vector PDR196) were incubated in half-strength Synthetic Dextrose (SD) medium contained 100 μM of each substrates, and the intracellular contents were quantitatively analyzed by LC-MS. Yeast cells expressing AaPDR3 accumulated more β-caryophyllene than the control along the same time course (Figure 7). The AaPDR3 transformants accumulated >44 nmol of β-caryophyllene per gram of cells compared with the control cells the contained almost 27 nmol at 9 h treated by 100 μM β-caryophyllene (Figure 7). The result demonstrated that expression of AaPDR3 increased β-caryophyllene influx. Both AaPDR3 transformants and the control were incubated in the culture media in the range of 0–1,200 μM β-caryophyllene. β-caryophyllene uptake by AaPDR3 followed Michaelis-Menten kinetics with Km of 63.47 ± 8.81 pmol β-caryophyllene and a maximum transport rate Vmax of 80.89 ± 2.46 pmol/g fresh yeast cells/min (Figure S6). No significant differences in the β-farnesene contents accumulated in AaPDR3 expressing yeast cells compared to that in the control group, as well as germacrene D (Figure S7). These results indicated that AaPDR3 was highly specific for the β-caryophyllene transport compared with β-farnesene and germacrene D in yeast.

Figure 7. Time-dependent uptake of β-caryophyllene by AD1-8 yeast cells expressing AaPDR3 and transformed with the empty vector (EV). Yeast was incubated in half-strength SD medium containing 100 μM β-caryophyllene at pH 5.9. The error bars represent the means ± SD from three biological replicates.

Discussion

AaPDR3 Mediates the β-Caryophyllene Biosynthesis in A. annua

The terpenoids are one of the largest groups of plant secondary metabolites (Croteau et al., 2000). Interestingly, the terpenoids are often transported from the cells where these compounds or metabolic intermediates are synthesized to neighboring cells, or even to other tissues or remote organs to be stored. Therefore, many transporter proteins participate in this biology process (Yazaki, 2005). Recently, the ATP-binding cassette (ABC) transporters have been reported to largely contribute to membrane transport of terpenoids in plants, especially for PDR subfamily (Jasiński et al., 2001; Van Den Brûle et al., 2002; Crouzet et al., 2013). Most of identified PDR transporters were expressed in specific tissues. For example, AtPDR8 is predominately expressed in roots and leaves. AtPDR2, AtPDR6, AtPDR9, and AtPDR13 are exclusively expressed in roots but not in shoots in Arabidopsis, while AtPDR14 is expressed only in shoots (Den Brule and Smart, 2002). The expression of AtPDR5 is mainly detected in roots and stems (Bienert et al., 2012). NtABCG5/PDR5, from N. tabacum, was highly expressed in the petals, stem and roots (Bienert et al., 2012). NtPDR1 was detected in stem and leaf tissues (Crouzet et al., 2013). These findings show that PDR genes are predominately expressed in roots and leaves. In plants, roots and leaves are the important tissues connected with environment. In this study, we characterized a PDR transporter AaPDR3 in A. annua. In our investigation, AaPDR3 was mainly active in old leaves, flowers, buds, and roots (Figure 3A). Notably, the GUS analysis exhibited that AaPDR3 was specifically expressed in T-shaped trichomes of old leaves and roots (Figure 5C). Likewise, AaPDR3 exhibited the tissues-specific expression pattern, suggesting that AaPDR3 plays an important role in the defensive compounds biosynthesis in T-shaped trichomes, flowers, buds, and roots. Moreover, the transcript level of CPS was also detected in leaves, flowers, buds, and roots (Lv et al., 2016), which is in accord with that of AaPDR3 in A. annua. Besides, the expression level of AaPDR3 was barely detected in the youngest leaf (leaf0), and increased gradually with the leaves aging (Figure 3B). Although the expression of CPS was highest in youngest leaf (leaf0), the CPS transcript level was also detected with the leaves aging (Lv et al., 2016). It means that β-caryophyllene is synthesized in young leaves and old leaves. From these results, we propose that AaPDR3 as a plasma membrane β-caryophyllene cellular uptake for gathering β-caryophyllene. Then the gathered β-caryophyllene is stored in the some cells of T-shape trichomes to reduce the cell damaged.

To identify the function of AaPDR3 in A. annua, we used RNAi to knock down the expression of AaPDR3. The repression of AaPDR3 resulted in an 86% reduction of β-caryophyllene content in AaPDR3-RNAi-20 transgenic A. annua line (Figure 6B), suggesting that AaPDR3 is essential for β-caryophyllene biosynthesis in A. annua. Transporters are the integral parts in metabolic networks, because they mediate multiple metabolic pathways. We speculated that the AaPDR3 repression would result in β-caryophyllene accumulated in the cells of T-shape trichomes in A. annua, which would prevent the β-caryophyllene biosynthesis. Our results, together with previous findings, indicated that AaPDR3 is involved in β-caryophyllene transport and plays an indispensable role in β-caryophyllene biosynthesis. AaPDR3 transporter reported here is the first transporter related to sesquiterpenes in A. annua, even in family Asteraceae.

AaPDR3 Was Involved in β-Caryophyllene Transport in Yeast

Plant ABC transporters is a large and diverse group of proteins involved in the pathogen response, lipid deposition, and the transport of the phytohormones (Kretzschmar et al., 2011). Therefore, ABC transporters play an important part in plant growth, nutrition, development, and the interaction with the environment (Bird et al., 2007; Kuromori et al., 2010; Ding et al., 2011). Our results preferentially suggest that AaPDR3 is likely to be involved in the sesquiterpene β-caryophyllene transport based on four findings: (i) like other sesquiterpene transporters; the amino acid sequence of AaPDR3 is similar to that of PDR transporters involved in terpene transport (Figure 2A), (ii) the plasma membrane protein AaPDR3 is expressed in the tissues, including the T-shaped trichomes, buds, flowers, and roots, where the sesquiterpenes are synthesized (Figure 3A), (iii) increase and decrease in the AaPDR3 transcript level influence the sesquiterpene β-caryophyllene biosynthesis (Figure 6), and (iv) when AaPDR3 was expressed in yeast mutant AD1-8, yeast expressing AaPDR3 only took up β-caryophyllene faster than controls containing the empty vector (Figure 7). In fact, some ABC transporters are reported to have broad substrate specificity (Kolaczkowski et al., 1998). For example, PDR5 transporter from yeast was confirmed to export some compounds which had different structure and function (Wolfger et al., 2001; Lamping et al., 2010). In Arabidopsis, AtPDR12 is an ABA-uptake transporter in the guard cells and other cells (Kang et al., 2010). The plasma membrane transporter, AtPDR12, also contributes to the resistance of lead (Lee et al., 2005). When the yeast cells expressing AaPDR3 was incubated in SD medium contained 100 μM β-caryophyllene, β-farnesene, and germacrene D, respectively, our results showed that AaPDR3 exhibited narrow substrate specificity (Figure 7 and Figure S6).

AaPDR3 Affects the Artemisinin Biosynthesis in A. annua

Amazingly, we found that knockdown of AaPDR3 resulted in an increase in artemisinin content in AaPDR3-RNAi transgenic plants (Figure S5). AaPDR3 is a specific-expressed transporter gene in T-shape trichomes (Figure 4C), whereas artemisinin is specially synthesized in glandular trichomes. As we known, blocking the competitive pathways of artemisinin biosynthesis is very useful to improve the artemisinin content (Zhang et al., 2009; Lv et al., 2016). Both the artemisinin and dihydroartemisinic acid contents were increased, when CPS was suppressed by anti-sense in A. annua (Lv et al., 2016). The β-caryophyllene content in A. annua was up to 5–10% of the total essential oil (Brown, 2010). When AaPDR3 was down-regulated by RNAi in A. annua, the β-caryophyllene content was observably reduced in transgenic plants (Figure 6B), which might lead to the carbon altered through FPP to artemisinin biosynthetic pathway.

Accession Numbers

AaPDR3 (KR153482), AtPDR1 (NM_112505.4), AtPDR2 (NM_117611.5), AtPDR3 (NM_128548.4), AtPDR4 (NM_128248.2), AtPDR5 (NM_001336647.1), AtPDR6 (NM_129195.6), AtPDR7 (NM_101389.3), AtPDR8 (GQ374243.1), AtPDR9 (NM_115208.4), AtPDR10 (NM_001339062.1), AtPDR11 (NM_105366.4), AtPDR12 (NM_001332173.1), AtPDR13 (NM_001341001.1), AtABCG42 (NM_001203808.2), AtABCG43 (NM_148328.3), NpPDR1 (Q949G3.1), NtPDR1 (Q76CU2.1), SpTUR2 (O24367.1).

Author Contributions

XF and KT designed the research and drafted the manuscript. XF and PS performed the experiments. XF, QH, QS, YM, and PL carried out vector construct, expression analysis, transgene plant generation, subcellular localization and yeast assay. YT, QP, TY, MC, XH, LL, YW, and XS revised the manuscript. All authors approved the manuscript.

Funding

This work was supported by the China National Transgenic Plant Research and Commercialization Project (Grant No. 2016ZX08002-001), China National High Technology Research and Development Program (Grant No. 2011AA100605), and Shanghai Jiao Tong University Agri-Engineering Program (Grant No. AF1500028).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Prof. Masakazu Niimi (Otago University, New Zealand), Prof. André Goffeau (Université Catholique de Louvain, Belgium), and Prof. Mohan Gupta (Chicago University, USA) for providing the yeast AD12345678 strain. We thank the Instrumental Analysis Center of the Shanghai Jiao Tong University for assistance with GC-MS.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fpls.2017.00723/full#supplementary-material

Figure S1. The sesquiterpene biosynthesis in Artemisia annua. HMGR, 3-hydroxy-3- methylglutaryl coenzyme A reductase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductase. FPS, farnesyl diphosphate; ADS, amorpha-4,11-diene synthase; CPR, cytochrome P450 reductase; CYP71AV1, cytochrome P450 monooxygenase; DBR2, artemisinic aldehyde D-11(13)-double bond reductase; ALDH1, aldehyde dehydrogenase 1; CYB5 and ADH1, cytochrome b5 monooxygenase and alcohol dehydrogenase. CPS, beta-caryophyllene synthase; BFS, beta-farnesene synthase; GAS, germacrene A synthase; ECS, 8-epicedrol synthase.

Figure S2. The expression of AaPDR3 was induced by MeJA treatment. ACTIN was used as internal control. The error bars represent the means ± SD from three biological replicates.

Figure S3. Total ion chromatographs of metabolites from leaves of A. annua.

Figure S4. Immunoblotting analysis of GFP-AaPDR3 protein levels in AaPDR3-overexpression transgenic A. annua lines.

Figure S5. The content of artemisinin in overexpression and RNAi transgenic A. annua plants, respectively. The error bars represent the means ± SD from three biological replicates, and asterisks indicate statistically significant differences compared with WT. **P < 0.01.

Figure S6. β-caryophyllene uptake analyses by AD1-8 yeast cells expressing AaPDR3 and transformed with the empty vector (EV). Yeast cells were incubated in the culture media in the range of 0–1,200 μM β-caryophyllene at pH 5.9. β-caryophyllene uptake by AaPDR3 followed Michaelis- Menten kinetics with Km of 63.47 ± 8.81 pmol β-caryophyllene and a maximum transport rate Vmax of 80.89 ± 2.46 pmol/g fresh yeast cells/min (R2 = 0.98). The error bars represent the means ± SD from three biological replicates.

Figure S7. Time-dependent uptake of (A) β-farnesene and (B) germacrene D by AD1-8 yeast cells expressing AaPDR3 and transformed with the empty vector (EV). Yeast was incubated in half-strength SD medium containing 100 μm β-farnesene and germacrene D at pH 5.9, respectively. The error bars represent the means ± SD from three biological replicates.

Table S1. Primers used in this study.

Supplementary Information Data 1. Four selected PDR genes in transcriptome expression in glandular and T-shaped trichomes.

References

Bertea, C. M., Voster, A., Verstappen, F. W., Maffei, M., Beekwilder, J., and Bouwmeester, H. J. (2006). Isoprenoid biosynthesis in Artemisia annua: cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library. Arch. Biochem. Biophys. 448, 3–12. doi: 10.1016/j.abb.2006.02.026

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