In A. annua FPP is a precursor for not only artemisinin and other sequiterpenes but also squalene and sterols and these could all therefore be additional sinks for FPP that does not flux into the artemisinin pathway via AMS (**Figure 1**). To determine if the silencing of AMS led to a redirection of FPP flux, squalene and sterol levels were quantified from dried leaf material by GC-MS. Squalene, stigmasterol, β-sitosterol and campesterol were identified in both the NTC and transformed lines (**Figure 7**). Stigmasterol and β-sitosterol were present at higher levels in comparison to squalene and campesterol but overall no significant

differences were found between the NTC and the AMS silenced lines.

To determine if AMS silencing led to an increase in sesquiterpenes other than artemisinin, GC- and UPLC-MS analysis was carried out on fresh, young, and mature leaf material, and pooled dried leaf material. From the GC-MS analysis of NTC and transformed lines, 105 compounds were identified, 30 of which were sesquiterpenes. Comparisons between the leaf material sampled (young/mature/dried) revealed the level of sesquiterpenes to be higher in the young leaf samples in comparison to the mature and dried leaf material (Supplemental Table 1). Further statistical analysis to investigate differences

FIGURE 6 | Expression of functionally characterized sesquiterpene synthase (AMS, GAS, ECS, CPS, and FS) and SQUALENE SYNTHASE (SQS) genes in young leaf tissue from NTC and AMS silenced lines. Error bars ±SD (NTC—n = 6, AMS\_RNAi lines—n = 3).

between the 3 AMS silenced lines and the NTC found that for 17 of the sesquiterpene compounds levels were significantly higher in the NTC. Significant increases in the AMS silenced lines were found for only 6 sesquiterpene compounds (Supplemental Table 1). In young leaf material these were: beta-farnescene and germacrene; in mature leaf there were increases in 2 unknown compounds with putative C15H24 formulae, and in dried leaf material germacrene D and ledene oxide were significantly higher (**Figure 8**). For the other 7 sesquiterpene compounds no differences were observed.

As well as artemisinin and its associated compounds derived from the artemisinin pathway, the UPLC-MS analysis also identified four putative novel oxygenated sesquiterpene (C15H24O) compounds in the AMS silenced lines. These

FIGURE 9 | The concentration (µg/mg extracted dry weight) of putative novel sesquiterpene compounds in young and mature leaves and dried pooled leaf material for the AMS silenced lines and NTCs. (A) sesquiterpene M255.1946T53, (B) sesquiterpene M345.1205T24, (C) —M239.2007T65, and (D) —sesquiterpene M239.2005T78 Error bars ±SD (NTC—n = 6, AMS\_RNAi lines—n = 3). Letters represent Bonferroni test results after ANOVA, groups not sharing letters indicate statistically significant differences (p ≤ 0.05).

compounds were identified as being significantly increased although the levels at which they were present was very low, ranging from 0.03- to 0.4 µg/mg DW which is 100–10 times (respectively) lower than artemisinin levels (**Figure 9**). Putative sesquiterpenes: M255.1946T53 M239.2007T65 and M239.2005T78 were all found to be significantly increased in young leaf tissue in the transformed lines in comparison to the NTC. M345.1205T24 was found to be significantly increased in only the dried leaf tissue of the AMS silenced lines in comparison to the NTC.

#### DISCUSSION

#### Silencing AMS Leads to Accumulation of the Sesquiterpene Precursor FPP in *A. annua* GSTs

In A. anuua the first committed step in the artemisinin pathway converting FPP to amorpha-4,11-diene is AMS. It was hypothesized that by blocking this step FPP would either accumulate or be channeled into the production of known or novel sesquiterpenes. Previous work had shown that AMS was not only highly expressed in the Artemis cultivar, but that recovered AMS gene sequences were polymorphic (Graham et al., 2010). This indicated that multiple copies of the gene could exist which we confirmed by qPCR (**Figure 2**). The high copy numbers for AMS present in the A. annua cv. Artemis could be linked to its high artemisinin yield, and its success as an elite hybrid for commercial production of artemisinin (Delabays et al., 2001). To effectively silence all the AMS copies two separate sections of the sequence were selected and joined to create an AMS specific sequence, this was driven by the cyp71av1 trichome specific promoter (Wang et al., 2011). Stably transformed lines expressing the construct were achieved, with AMS expression reduced to less than 4% of the NTC. Artemisinin content was reduced by 95% alongside a reduction in all artemisinin-related compounds downstream of the AMS-catalyzed step.

One exception was amorpha-4,11-diene where levels were found to be significantly higher in young leaf tissue of the AMS silenced lines compared to the NTC (**Figure 4A**). No such increase was present in mature or dry leaves. The levels of amorpha-4,11-diene in A. annua are reported to be low as a consequence of artemisinin biosynthesis (Bouwmeester and Wallaart, 1999). Detection of this early step precursor in young leaves of NTC is consistent with previous findings (Czechowski et al., 2016) which suggest the pathway to artemisinin only becomes active as leaves mature. The increase in the AMS silenced lines is unexpected and the reason not obvious but could relate to the metabolic sink being somehow further compromised as a result of the decreased AMS.

To establish the impact of silencing AMS on FPP levels we adapted a protocol from Nagel et al. (2014) that allowed us to quantify this important precursor for the first time in A. annua. We found that silencing of AMS led to a significant accumulation of FPP in young leaf tissue and this increase was also confirmed as being trichome specific by carrying out the same extraction on young leaf isolated GSTs (**Figures 5A,B**).

### The Downstream Effect of Increased FPP Levels on Sterol and Sesquiterpene Synthesis in Artemis as Quantified by UPLC-MS and GC-MS

Increasing FPP by knocking down AMS had no effect on the expression of any of the other known sesquiterpene synthases genes or squalene synthase known to be expressed in either the GSTs or young leaf tissue (**Figure 6**). UPLC-MS and GC-MS analysis of leaf material was carried out to determine if the FPP accumulating in the AMS silenced lines was being redirected into sterol or sesquiterpene production. GC-MS analysis found no significant differences in squalene and sterol levels between the NTC and the AMS silenced lines (**Figure 7**) despite this pathway being considered the main competitor for FPP after artemisinin (Zhang et al., 2009). The GC-MS analysis also revealed very few changes in volatiles in the AMS silenced lines (Supplemental Table 1). Where significant differences were observed the magnitude, changes were very low (**Figure 8**). These results differ to the findings of Ma et al. (2015) who silenced AMS in a low artemisinin background (artemisinin yields of 0.025 µg/mg). Alongside reporting a decrease in artemisinin, they also found a significant increase in the levels of caryophyllene and copaene in their AMS silenced plants. The increase in these sesquiterpene compounds could be linked to the low artemisinin cultivar used for transformation having other active endogenous sesquiterpene synthases. In Artemis a high yielding cultivar the endogenous synthases would appear not to be as active in their ability to utilize the FPP made available away from artemisinin being silenced.

Although no differences were seen in known sesquiterpene levels in the AMS silenced lines, putative novel sesquiterpene compounds were detected and characterized by UPLC-MS. Significant differences were seen between Artemis NTCs and the AMS silenced lines for 4 putative sesquiterpene compounds although the concentrations were around 30 times lower than artemisinin. The compounds were also mainly identified in young leaf tissue suggesting that these compounds are not end products but rather are further converted as the leaf matures. Compound M345.1205T24 was an exception to the other 3 as it was found in dried leaf only. The very low concentrations of these novel compounds in available plant material ruled out structural determination attempts by NMR.

The lack of diversion of the accumulated FPP to other sesquiterpenes or sterols in the AMS silenced lines is somewhat surprising. One possible explanation for this is that the Artemis hybrid has been selected for high yield artemisinin and the flux of FPP may already be optimized to flow toward artemisinin production.

#### Trichomes With Elevated FPP as a Potential Production Platform for High Value Sesquiterpenes

A. annua is already established as a very efficient crop plant for artemisinin production, with the potential to produce this high value chemical at a relatively low cost of less than \$250 per kilogram. Disruption of cyp71av1, leading to novel arteannuin X accumulation demonstrated the plasticity of GST metabolism in A. annua, suggesting their potential as factories for new compound production (Czechowski et al., 2016). The GSTs provide an optimal environment for the synthesis of many natural products based on the availability of precursors, coenzymes, mRNA and protein processing. In A. annua the problem of toxicity of some of these compounds is overcome as the GSTs can sequester them in the extracellular cavities of the trichome secretory cells. This coupled with the location of the GSTs on the surface of leaves is advantageous as the compounds are both contained and readily extractable.

By silencing AMS in a high artemisinin yielding A. annua cultivar we have significantly decreased the amount of artemisinin and related compounds produced in the GSTs. As a further result of the silencing we also show that the precursor FPP is accumulated in the GSTs and not catalyzed by endogenous synthases. The lack of production of novel compounds at significant amounts suggests that the elevated pool of GST localized FPP is either not available to or not utilized by other sesquiterpene, squalene synthase enzymes. Consequently, the AMS silenced lines may represent a platform for production of other high value compounds that require FPP as a precursor and for which genes encoding biosynthetic enzymes are known.

### AUTHOR CONTRIBUTIONS

TC, TMC, and IG designed the experiments; TC, TMC, CB, NS, JH, and DH performed the experiments; TMC, TC, CB, DH, and TL analyzed the data; TMC, TC, TL, and IAG wrote

#### REFERENCES

the manuscript; and all authors revised and approved the manuscript.

#### FUNDING

We acknowledge financial support for this project from The Bill and Melinda Gates Foundation as well as from The Garfield Weston Foundation.

#### ACKNOWLEDGMENTS

We thank the University of York horticulture team for horticultural assistance; C. Calvert, W. Lawley, for project management; D. Rathbone and S. Heywood for preliminary experimental involvement. We thank X. Simonnet and Médiplant for access to the Artemis pedigree. Mass spectrometry analysis was in part supported by instrumentation within the York Centre of Excellence in Mass Spectrometry (CoEMS). The CoEMS was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1; EP/M02 8127/1).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 00547/full#supplementary-material

in terpenoid metabolism. Proc. Natl. Acad. Sci. U.S.A. 113, 15150–15155. doi: 10.1073/pnas.1611567113

**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.

Copyright © 2018 Catania, Branigan, Stawniak, Hodson, Harvey, Larson, Czechowski and Graham. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Overexpression of Artemisia annua Cinnamyl Alcohol Dehydrogenase Increases Lignin and Coumarin and Reduces Artemisinin and Other Sesquiterpenes

Dongming Ma1,2† , Chong Xu1† , Fatima Alejos-Gonzalez2 , Hong Wang3 , Jinfen Yang1 , Rika Judd2 and De-Yu Xie2 \*

1 Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine, Guangzhou, China, 2 Department of Plant & Microbial Biology, North Carolina State University, Raleigh, NC, United States, 3 Graduate University of Chinese Academy of Sciences, Beijing, China

#### Edited by:

Tomasz Czechowski, University of York, United Kingdom

#### Reviewed by:

Patrick Smithers Covello, Biotechnology Research Institute (NRC-CNRC), Canada Yongzhen Pang, Institute of Botany (CAS), China

\*Correspondence: De-Yu Xie<EMAIL_ADDRESS>†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science

Received: 23 January 2018 Accepted: 28 May 2018 Published: 19 June 2018

#### Citation:

Ma D, Xu C, Alejos-Gonzalez F, Wang H, Yang J, Judd R and Xie D-Y (2018) Overexpression of Artemisia annua Cinnamyl Alcohol Dehydrogenase Increases Lignin and Coumarin and Reduces Artemisinin and Other Sesquiterpenes. Front. Plant Sci. 9:828. doi: 10.3389/fpls.2018.00828 Artemisia annua is the only medicinal crop that produces artemisinin for malarial treatment. Herein, we describe the cloning of a cinnamyl alcohol dehydrogenase (AaCAD) from an inbred self-pollinating (SP) A. annua cultivar and its effects on lignin and artemisinin production. A recombinant AaCAD was purified via heterogeneous expression. Enzyme assays showed that the recombinant AaCAD converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates involved in the biosynthesis of lignin. Km, Vmax, and Vmax/Km values were calculated for all three substrates. To characterize its function in planta, AaCAD was overexpressed in SP plants. Quantification using acetyl bromide (AcBr) showed significantly higher lignin contents in transgenics compared with wildtype (WT) plants. Moreover, GC-MS-based profiling revealed a significant increase in coumarin contents in transgenic plants. By contrast, HPLC-MS analysis showed significantly reduced artemisinin contents in transgenics compared with WT plants. Furthermore, GC-MS analysis revealed a decrease in the contents of arteannuin B and six other sesquiterpenes in transgenic plants. Confocal microscopy analysis showed the cytosolic localization of AaCAD. These data demonstrate that AaCAD plays a dual pathway function in the cytosol, in which it positively enhances lignin formation but negatively controls artemisinin formation. Based on these data, crosstalk between these two pathways mediated by AaCAD catalysis is discussed to understand the metabolic control of artemisinin biosynthesis in plants for high production.

Keywords: Artemisia annua, artemisinin, arteannuin B, cinnamyl alcohol dehydrogenases, coumarin, lignin, sesquiterpenes

#### INTRODUCTION

Artemisinin-based combination therapy (ACT) is the first-line treatment for malaria (WHO, 2006, 2013; Maude et al., 2009). Artemisia annua L. (sweet wormwood), an effective antimalarial plant, is the only natural resource to produce artemisinin, an endoperoxide sesquiterpene lactone. Due to the global demand for ACT, understanding artemisinin biosynthesis in this medicinal

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plant is critical for metabolic engineering purposes for high production of this sesquiterpene. To date, numerous studies have molecularly and biochemically characterized the main steps of the artemisinin biosynthetic pathway, starting with amorpha-4,11-diene (amorphadiene) localized in the cytosol of glandular trichomes (**Figure 1**) (Bouwmeester et al., 1999; Teoh et al., 2006; Covello et al., 2007; Zhang et al., 2008; Czechowski et al., 2016). A recent review summarized enzyme assay, synthetic biology, and multiple transgenic studies that solidly demonstrate the first step catalyzed by amorpha-4, 11 diene synthase (ADS) s (Xie et al., 2016). The second step is catalyzed by a cytochrome P450 mono-oxygenase (CYP71AV1) coupled with a cytochrome P450 reductase 1 (CPR1). These two enzymes were originally demonstrated to convert amorpha-4, 11 diene to artemisinic alcohol and then to artemisinic aldehyde (Ro et al., 2006;Teoh et al., 2006). Recently, a new gene encoding an A. annua alcohol dehydrogenase 1 (AaADH1) was cloned from glandular trichomes of A. annua and demonstrated to catalyze the conversion of artemisinic alcohol to artemisinic aldehyde (Paddon et al., 2013). This discovery has greatly enhanced our understanding of the second step.  The spontaneous oxidation of DHAA finally produces artemisinin. In addition, arteannuin B is derived from artemisinic aldehyde (**Figure 1**). Recently, a cyp71av1 mutant of A. annua was generated to provide fundamental genetic evidence demonstrating the essential role of CYP71AV1 in controlling the artemisinin biosynthetic pathway in planta (Czechowski et al., 2016). This mutant further provided solid evidence to demonstrate the formation of artemisinin through spontaneous oxidation. In addition to research conducted in plants, fundamental successes in synthetic biology have further demonstrated the steps catalyzed by ADS, CYP71AV1, CPR1, and AaADH1 (Ro et al., 2006; Paddon et al., 2013; Turconi et al., 2014). Although these previous achievements have demonstrated fundamental promising methods to improve the artemisinin supply, global production from current sweet wormwood crops still lacks stability and is unable to meet the increase in medicinal demands (Paddon et al., 2013; Ma et al., 2017a). Therefore, continuous research efforts are urgently necessary to elucidate the regulatory mechanisms of artemisinin biosynthesis to improve the global yield.

Cinnamyl alcohol dehydrogenase (CAD) is categorized in a group of short-chain oxidoreductases in the family of nicotinamide adenine dinucleotide phosphate (reduced form) dependent enzymes. It has been appropriately characterized to catalyze the conversion of phenylpropenyl aldehydes to alcohols in the late steps of lignin biosynthesis (**Figure 1**) (Sarni et al., 1984; Somers et al., 1995; Baucher et al., 1999; Chabannes et al., 2001). A large number of studies have succeeded in manipulating CAD expression in potent biotechnological efforts to reduce lignin in trees and crops for different economic applications (Baucher et al., 1999; MacKay et al., 1999; Chabannes et al., 2001; Fu et al., 2011; d'Yvoire et al., 2013; Trabucco et al., 2013; Anderson et al., 2015; Ozparpucu et al., 2017). The main

successes associated with using CAD downregulation include improved digestibility of forage crops, reduction of lignin in trees for pulping and biofuel, and different renewable plants for biofuel feedstock (Lapierre et al., 2000; Fu et al., 2011; Wang Y.H. et al., 2015; Ozparpucu et al., 2017; Ponniah et al., 2017). For example, downregulation of CAD in alfalfa has been shown to lead to a decrease in lignin and improved digestibility of this forage crop (Baucher et al., 1999). Suppression of CAD in rice has demonstrated the potential to facilitate cellulose production for biofuel feedstock (Ponniah et al., 2017). A downregulation of CAD has also been reported to improve saccharification in switchgrass for biofuel conversion (Fu et al., 2011).

arrow for AaCAD in the artemisinin pathway denotes a partial reverse. The long arrow for CYP71AV1 and CPR1 denotes the main direction toward

artemisinin.

In our previous study, we reported the cloning of a CAD homolog from glandular trichomes of an heterozygous (crosspollination) A. annua cultivar and in vitro enzyme assays to show that it used cinnamyl aldehyde, coniferyl aldehyde, sinapyl aldehyde, and artemisinic aldehyde as substrates (Li et al., 2012). To date, whether this CAD can affect lignin and other metabolic pathways in A. annua remains to be investigated. Herein, we report the cloning of a new CAD homolog from a novel self-pollinating (SP) A. annua and characterize its enzyme kinetics and overexpression in plants. Phytochemical analysis, GC-MS-based metabolic profiling, and LC-MS analysis were conducted to characterize phenylpropanoid and terpenoid metabolism in transgenic plants. The resulting data showed that not only was this new AaCAD involved in lignin biosynthesis, but it was also associated with coumarin formation.

By contrast, artemisinin and other sesquiterpenes contents were significantly decreased in transgenic plants. These data show that AaCAD can play a dual function by establishing a crosstalk between two distinct cytosolic pathways, the phenylpropanoid and sesquiterpene pathways. Although overexpression of AaCAD leads to a reduction of artemisinin contents, this research is very instructional for future metabolic engineering designs to improve artemisinin production in A. annua.

### MATERIALS AND METHODS

#### Plant Materials and Growth Conditions

The progeny of a self-pollinating (SP) A. annua variety were grown in the phytotron for seeds as described previously (Alejos-Gonzalez et al., 2011). The photoperiod and temperature in the phytotron was 16/8 h (light/dark) and 25/22◦C (day and night). Seedlings of the F3 progeny were used for gene cloning, genetic transformation, and metabolite analysis.

### Cloning of AaCAD cDNA

DNA-free total RNA was isolated from 2-month-old seedlings of SP A. annua using the RNeasy Plant Mini Kit (Qiagen, United States) as described previously (Ma et al., 2015). The first-strand cDNA was synthesized with 1.0 µg of total RNA and Powerscript reverse transcriptase (Clontech). The resulting cDNA was used as template to clone AaCAD. A pair of primers consisting of CAD-F (50 - ATG GGA AGC ATG AAA GAA GAA AG-30 ) and CAD-R (50 -ATT TGT TGT TTC CTC TTC CAA A-30 ) was designed for RT-PCR, which was carried out to obtain the open reading frame (ORF) fragment of AaCAD. The PCR product was further sequenced to analyze its nucleotides. The resulting ORF was deduced to determine the amino acid sequence, which was aligned with a reported CAD sequence obtained from GenBank. The sequence alignment was completed using an online Cluster Omega program1 .

### Heterogeneous Expression of Recombinant AaCAD

The ORF of AaCAD containing its stop codon TAA was cloned into the pENTR/D-TOPO vector (Gateway, Invitrogen) to obtain a recombinant pENTR-AaCAD plasmid. The LR Clonase II enzyme mix (Invitrogen) was used to digest the pENTR-AaCAD plasmid and the destination vector pDEST17 (6xHis tag). As a result, the AaCAD ORF was cloned into pDEST17 to obtain a new pDEST17-AaCAD plasmid for protein expression. All cloning steps followed the manufacturer's protocol. The pDEST17-AaCAD plasmid was further introduced into competent E. coli strain BL21 cells to induce recombinant protein.

For protein induction, a single colony was selected and then cultured in 200 ml liquid LB medium supplied with 50 mg/L ampicillin at 37◦C in 500-ml E-flasks. When the OD value of the suspension culture was approximately 0.6 at 600 nm, 0.1% L-arabinose and 0.1 mM IPTG were added the flask. The

1http://www.ebi.ac.uk/Tools/msa/clustalo/

suspension culture was continuously incubated for an additional 20 h at 16◦C. As described in our recent report (Ma et al., 2017a), the recombinant AaCAD was purified using Ni-NTA Superflow Columns (Qiagen, 1.5 ml) according to the manufacturer's protocol. The resulting purified recombinant AaCAD was loaded onto PD-10 columns (Amersham Pharmacia Biotech, now GE Healthcare Life Sciences, http://www.gelifesciences. com) to remove salts. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to examine the purification of the recombinant AaCAD. The resulting desalted protein was used for the enzyme assay immediately or stored in a −20◦C freezer for late use as described below.

#### Enzyme Assay and Kinetics Analysis

Three lignin substrates, coniferyl aldehyde, p-coumaryl aldehyde, and sinapyl aldehyde (Sigma-Aldrich), were used to examine the catalytic activity of the recombinant AaCAD. Three substrates were dissolved in methanol. Enzymatic reactions were carried out in a 200-µl volume composed of 0.1 mM substrate, 50 mM Tris–HCl (pH 7.5), 0.5 mM NADPH, 2.0 mM dithiothreitol, and 1.3 µg purified recombinant AaCAD. All reactions were initiated by addition of AaCAD to the reaction mixture at 30◦C. After 30 min, all reactions were stopped by addition of 15 µl of glacial acetic acid, followed by centrifugation at 10,000 × g for 10 min. The resulting supernatant was pipetted into a 200-µl glass insert, which was further placed into a 2-ml glass vial for high-performance liquid chromatograph (HPLC) analysis. All experiments were repeated three times, each with three replicates.

HPLC analysis was performed on a Waters 2695 instrument. Metabolites were separated on an Agilent ZORBAX Eclipe XDB-C18 column (4.6 × 150 mm, 5 µm). Two HPLC grade solvents, methanol (solvent A) and water (solvent B), were used as the mobile phase. A gradient solvent program, which was composed of A:B from 5:95 to 95:5 from 0 to 40 min, was developed to elute the metabolites. The flow rate was 1.0 ml/min, and the injection volume was 20 µl. The wavelengths for detection of the metabolites were 260 nm and 340 nm.

To characterize the kinetics of the recombinant AaCAD, eight concentrations (6, 8, 10, 12, 14, 20, and 30 µM) of each substrate were tested. Except for the different concentrations of the substrates used, other components in the reaction mixture were the same as described above. All reactions were performed in 200 µl in 96-well microplates. Measurements of enzymatic products were performed using an Epoch Microplate reader (BioTek instruments Inc., United States). All reactions were initiated by the addition of enzyme and then maintained at 30◦C for 10 min. All tubes were placed on a microplate for 40 min of declination recorded at 340 nm, which is classically used to quantify aldehyde and NADPH because they have maximum absorbance values at this wavelength (Goffner et al., 1992; Ma, 2010; Saathoff et al., 2011; Rong et al., 2016). This method uses extinction coefficients (14.7 × 10−3 to 19.45 × 10−3 M−1 cm−1 ) for both aldehyde and NADPH to calculate the relative contribution of each to the 340-nm signal (Hawkins and Boudet, 1994; Saathoff et al., 2011), which allows the elimination of potential spectrophotometric interference. We used this method to record absorbance values at one-min

intervals for each tube (reaction). Each reaction was continuously recorded 40 times to obtain 40 values. The resulting data were analyzed using GraphPad Prism 6 software to determine the Km, Vmax, and Kcat values. All experiments were performed three times, each with three replicates.

### Development of Binary Vector, Genetic Transformation of A. annua, and Genotyping

The AaCAD ORF, including its stop codon, was cloned into the pENTR/D-TOPO vector (Gateway, Invitrogen) to obtain a new recombinant pENTR-AaCAD plasmid, which was introduced into competent E. coli DH5α cells. The destination vector used for the development of binary vectors was PMDC-84 (Xi et al., 2016). The pENTR-AaCAD and PMDC-84 plasmids were digested and interchanged using the LR Clonase II enzyme mixture (Invitrogen) following the manufacturer's protocol. This cloning step resulted in a binary vector, namely, PMDC-84-AaCAD, in its T-DNA cassette of which AaCAD was cloned at the immediate downstream of a 2× 35S promoter, and a hygromycin gene was used for selection (**Figure 3A**). This binary vector was introduced into competent A. tumefaciens strain LBA4404 cells, from which one positive colony was selected for genetic transformation of A. annua, as described previously (Ma et al., 2015). Multiple transgenic shoots were generated on the hygromycin selection medium and rooted to obtain plantlets. More than 10 plantlets were planted in pot soil and placed in the phytotron for continuous growth to flower and then seed production. In addition, transgenic plants for other genes reported previously (Ma et al., 2015) were used as vector controls in this study. We further used a Leica MZ FLIII fluorescence stereomicroscope to examine trichomes including glandular and other shaped trichomes on the surfaces of leaves and stems of transgenic vs. wild-type plants.

Genomic DNA was extracted from leaf tissues of transformed plants using the DNeasy Plant Mini Kit (Qiagen, United States). A total of 50 ng of genomic DNA was used as template for PCR using a pair of primers consisting of a forward primer (50 -TCT AGA ACT AGT TAA TTA AGA AT-30 ) designed based on the 35S promoter and a reverse primer (50 -ATT TGT TGT TTC CTC TTC CAA A-30 ) designed based on AaCAD. The thermal program for PCR was composed of 94◦C for 5 min, followed by 35 cycles at 94◦C for 1 min, annealing for 1 min at 5◦C, and extension of 1 min at 72◦C, and a final extension at 72◦C for 5 min. All PCR experiments were performed with three biological replicates, each repeated at least three times.

#### Subcellular Localization Analysis

Subcellular localization of AaCAD was carried out as described in our recent reports (Ma et al., 2017a,b). In brief, a gateway technique was used to insert the AaCAD ORF without its stop codon into the pENTR/D-TOPO vector (Gateway, Invitrogen, United States) following the manufacturer's protocol. This cloning generated a new recombinant plasmid, namely, pENTR/D-TOPO-CAD. The destination vector used was pSITEII-N1-enhanced green fluorescent protein (EGFP) (with the EGFP epitopic tag in the C-terminus). Then, AaCAD in the pENTR/D-TOPO-CAD vector was cloned into pSITEII-N1 by LR reactions following the manufacturer's protocol. This cloning step generated a new pSITEII-N1-CAD/EGFP plasmid, in which AaCAD (without its stop codon) was directly ligated at the 50 -end of EGFP. The new plasmid was then introduced into Agrobacterium tumefaciens strain GV3101. A positive colony was selected and then activated for leaf agroinfiltration of N. benthamiana to transiently analyze protein expression. After 30 h of infection, leaf tissues were examined using a confocal microscope (Carl Zeiss). GFP fluorescence was excited at 488 nm and observed between 495 and 550 nm according to a method reported previously (Wang G.F. et al., 2015). All analyses were performed with three groups of biological replicates, each with at least three replicates.

### Reverse Transcription-Polymerase Chain Reaction and Western Blot Analysis

Total RNA was isolated from the young leaves of transgenic candidates and WT plants using an RNeasy Plant Mini Kit (Qiagen, CA, United States). Samples were then treated with DNase to obtain DNA-free RNA. The first-strand cDNA was synthesized using 1.0 µg of DNA-free RNA and Powerscript reverse transcriptase (RT, Clontech, United States). One microliter of cDNA was used as template for PCR using Taq polymerase (Promega, United States). The steps of these three types of experiments followed the manufacturers' protocols, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out for AaCAD transgenic and WT plants. The housekeeping gene β-actin was used as a reference control. The gene-specific primer pair for PCR was 5-ATGGGAAGCATGAAAGAAGAAAG-3 (forward primer) and 50 -ATTTGTTGTTTCCTCTTCCAAA-30 (reverse primer). The thermal program was as described above for genotyping. RT-PCR experiments were performed with three groups of biological replicates, each with three replicates.

A polypeptide consisting of MGSMKEERKITGWAC selected from the AaCAD amino acid sequence was used for antibody development. This peptide was synthesized and then used to develop a polyclonal antibody in rabbit at Genscript Company (NJ 08854, United States) for western blot analysis. Western blot was performed as described in our recent reports (Ma et al., 2017a,b). In brief, total proteins were extracted from leaves of transgenic vs. wild-type plants and separated by SDS-PAGE. Separated proteins were transferred to a nitrocellulose membrane and then probed with anti-AaCAD antibody. An anti-rabbit IgG HPR conjugate was used as a second antibody (Promega). An enhanced chemiluminescence system (Thermo Scientific, IL, United States) was used to detect the hybridization signal for immunoblot analysis. Western blot analysis was repeated three times.

#### Lignin Measurement

The lignin content was measured using the acetyl bromide (AcBr) method (Fukushima and Kerley, 2011; Moreira-Vilar et al., 2014). Small revisions were developed to obtain the protein-free cell wall

fraction. All dried stems and branches of each plant were ground into powder and filtered using a sieve (120 µm). One gram of fine powder was suspended in 20 ml sodium phosphate buffer (0.1 M, pH 7.2) in a 50-ml capped polyethylene tube at 37◦C for 30 min, followed by centrifugation at 4000 rpm for 10 min. The remaining pellet was suspended in 10 ml 70% ethanol, placed in an 80◦C water bath for 5 min, and then centrifuged for 10 min to remove the supernatant. This step was repeated five times. The remaining pellet in the tube was suspended in 10 ml acetone at room temperature for 5 min and centrifuged for 5 min to remove the supernatant. These treatments obtained protein-free cell wall fraction. The pellet was completely dried in a 37◦C oven until there was no weight change. Five milligrams of the dry residue sample was dissolved in 2.5 ml of acetyl bromide:acetic acid (1:3, v/v) solution in a glass tube. This treatment was maintained 24 h at room temperature. The mixture was completely transferred to a 10-ml volumetric flask, followed by addition of 0.35 ml of 0.5 M hydroxylamine hydrochloride. Glacial acetic acid was added to the volumetric flask to 10 ml. The flask was gently shaken in the upside-down direction to thoroughly mix the sample. The absorbance of the resulting mixture was recorded at 280 nm using an ultra-visible spectrometer. An extinction coefficient of 23.077 g−1 l cm−1 was used to calculate the AcBr concentration. The resulting data were used to calculate the contents of lignin extracted from the samples. Three biological replicates were analyzed for each plant. Each biological replicate was repeated three times.

### Extraction of Non-polar Metabolites and Gas Chromatograph-Mass Spectrometry Analysis

A protocol was developed to extract and profile nonpolar metabolites from tissues of A. annua (Ma et al., 2015). In brief, hexane was used to extract nonpolar metabolites from the leaves of 8–12 nodes of two-month-old plants grown in the phytotron (**Figure 3B**), and gas chromatographmass spectrometry (GC-MS) was performed using a gas chromatograph 6890 coupled with 5975C MSD (Agilent Technologies, United States). A RTX-5 capillary column (30 m × 0.25 mm × 0.25 µm) was used to separate the metabolites. Splitless mode was used in the inlet. The injection temperature was set at 250◦C. The temperature was initially set at 60◦C, then ramped to 260◦C at a constant rate of 10◦C/min, and held at 260◦C for 25 min. Pure helium was used as the carrier gas, with a flow rate of 1 ml/min. A positive electron impact ion source (70 eV) was used to ionize compounds, and mass fragments were scanned in the range from 40 to 800 (m/z), with 4 min of solvent delay. Three biological replicates were analyzed for each genotypic plant. Each biological replicate was repeated three times.

#### High-Performance Liquid Chromatography-Mass Spectrometry Analysis of Artemisinin

Leaf samples used for nonpolar metabolite analysis were also used for artemisinin measurements. Extraction of artemisinin and high-performance liquid chromatographymass spectrometry (HPLC-MS) analysis were performed using a 2010 eV LC/UV/ESI/MS instrument (Shimadzu) following our protocols reported previously (Alejos-Gonzalez et al., 2011, 2013). Three biological replicates were analyzed for each genotypic plant, with each biological replicate repeated three times.

#### Statistical Analyses

Experimental data were analyzed by one-way ANOVA. Subsequent multiple comparisons were performed using Duncan's multiple range test. All statistical analyses were performed using IBM SPSS Statistics Professional Edition, and the statistical significance was set at P < 0.01.

## RESULTS

### Kinetics of Recombinant AaCAD for Three Lignin Substrates

According to a CAD homolog sequence (ACB54931) that was cloned from a cross-pollinating cultivar (Li et al., 2012) and curated in the GenBank at NCBI database, we used RT-PCR to clone an AaCAD open reading frame (ORF) (GenBank accession ID#: MH017050) from the leaf tissues of self-pollinating (SP) A. annua. The full-length ORF is composed of 1086 base pairs of nucleotides that are deduced to encode 362 amino acids. An alignment between the deduced amino acid sequence and the homologous ACB54931 sequence only revealed three different amino acids, showing the high identity of the sequence and predicted structure (Supplementary Figures S2, S3).

The ORF was cloned into pDEST17 (6xHis tag) to induce the recombinant AaCAD. The purified recombinant enzyme was further obtained using Ni-NTA Superflow Columns (Supplementary Figure S1). Three substrates, coniferyl aldehyde, sinapyl aldehyde, and p-coumaryl aldehyde, were used to evaluate the enzymatic activity. After the recombinant AaCAD was incubated with the three substrates, HPLC analysis showed that the recombinant enzyme efficiently converted the three substrates to their corresponding alcohol products, coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol, respectively (**Figures 2A–C**). These results demonstrated the biochemical involvement of AaCAD in the lignin biosynthetic pathway (**Figure 1**).

Kinetic analysis was carried out to characterize Km, Vmax, Kcat, and Kcat/Km values for the three substrates. Given that coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol produced from AaCAD catalysis have maximum absorbance values at 340 nm, the use of a microplate reader is a highly efficient way to measure their concentrations in the same reaction time frame. The resulting data showed that the Km value of sinapyl aldehyde was lower than those of coniferyl aldehyde and p-coumaryl aldehyde, which had similar values (**Figure 2D**). This result supports a catalytic preference for sinapyl aldehyde. The Vmax values for the three substrates were similar. The Kcat/Km value for sinapyl aldehyde was higher than those of coniferyl

in D were carried out with three replicates to calculate the values.

aldehyde and p-coumaryl aldehyde, which had similar values (**Figure 2D**).

#### Overexpression of AaCAD in SP A. annua

The ORF of AaCAD was cloned into the PMDC-84 vector, and its expression was driven by a 2 × 35S promoter (**Figure 3A**). This construct was introduced into SP A. annua via A. tumefaciens-mediated transformation of leaf explants. Numerous hygromycin-resistant shoots were regenerated from infected explants and further rooted to obtain multiple transgenic plantlets. More than 10 plantlets were planted in soil contained in pots and grown in the phytotron as reported previously (Ma et al., 2017a). As shown in the photograph of line OE3 and a wild-type plant in **Figure 3B**, the transgenic candidates grew similarly. In addition, vector control transgenic plants such as ADS transgenic plants (Ma et al., 2015) were used as a vector control, and no difference in plant growth was observed between ADS and AaCAD transgenic plants.

Based on the 35S promoter and AaCAD sequences, primers were designed for PCR-based genotyping. The resulting PCR

and a reverse CAD primer. (D) Gel images show the increased expression of AaCAD in transgenic plants. (E) Gel images show the increased AaCAD protein in transgenic lines. Abbreviations in T-DNA: AaCAD, Artemisia annua cinnamyl alcohol dehydrogenase; GFP, green florescent protein; Hyg, hygromycin; Tnos, Nos terminator. Plant name abbreviation: OE2 and OE3, two transgenic lines; WT, wild-type control. PCR and RT-PCR experiments were performed with three biological replicates, each with at least three replicates. Western blotting was repeated three times.

data, as shown for transgenic examples OE2 and OE3 in **Figure 3C**, demonstrated the integration of the 35S and AaCAD transgenes into the genome of transgenic candidates. These two candidate lines were further selected for RT-PCR and western blot analysis. RT-PCR using gene-specific primers showed higher expression levels of AaCAD in these two transgenic plants compared with the wild-type control (**Figure 3D**). Western blot analysis demonstrated an increased protein level of AaCAD in the two transgenic plants (**Figure 3E**). These data demonstrate that the two candidates are transgenic plants.

To characterize the subcellular localization of AaCAD, confocal microscopy analysis was performed. The stop codon TAA was removed from the AaCAD ORF. The resulting TAA-eliminated sequence was fused to the N-end of an EGFP for protein localization. As reported previously for testing transgene functions in SP A. annua (Ma et al., 2017a,b), transient expression of AaCAD-EGFP was carried out in Nicotiana benthamiana. Both green and red channels were used to localize the proteins. The resulting data from two-channel observations showed that epidermal cells exhibited strong green fluorescence in the cytosol (**Figure 4**), indicating that AaCAD catalyzes reactions in the cytosol.

### Lignin and Coumarin Contents Are Increased in Transgenic Plants

Lignin and other phenylpropanoid metabolites were analyzed in the transgenic and wild-type plants. Given that T0 transgenic

FIGURE 4 | Subcellular localization of AaCAD-EGFP. (A) The green fluorescence signal of GFP in cells is shown in images photographed using green and bright channels. (B) The green fluorescence signal of GFP in cells is shown in images photographed using green and red channels. Green, green fluorescence of GFP; red, autofluorescence of chloroplasts; merged, green fluorescence of GFP and red autofluorescence of chloroplasts. This experiment was repeated three times, each with at least three biological replicates.

plants were regenerated at different time points, in this study, we mainly focused on OE2 and OE3, which were regenerated at the same time and grew in a synchronous manner. The main

contents in transgenic plants. (A) AcBr analysis-based calculation showing the lignin content in leaves of two transgenic lines and wild-type plants. (B) Overlay of total ion chromatographs showing increases in coumarin abundance in leaves of the two transgenic lines. (C) Peak area values show significant increases in coumarin in leaves of the two transgenic lines. OE2 and OE3, two transgenic lines overexpressing the AaCAD transgene; WT, wild-type. ∗ in (A) and ∗∗ in (C) denotes significant differences between transgenic lines and wild-type plants (N = 3, p < 0.01). All analyses were conducted with three biological replicates, each repeated three times.

stem and all branches of OE2 and OE3 were harvested for lignin analysis after the seeds were collected. Acetyl bromide (AcBr) analysis was completed to measure the lignin contents. The resulting data showed that lignin was significantly increased in the stems and branches of these two transgenic lines (**Figure 5A**).

Coumarin (2H-chromen-2-one) was annotated by GC-MS analysis. The resulting data showed a significant increase in coumarin content in the leaves of these two lines compared with the leaves of the wild-type plants (**Figures 5B,C**), demonstrating that AaCAD is associated with coumarin formation in plants.

#### Artemisinin, Arteannuin B, and Other Sesquiterpenes Are Decreased in Transgenic Plants

Artemisinin, arteannuin B, and other sesquiterpenes were profiled in the leaves of AaCAD transgenic vs. wild-type plants. As reported previously (Ma et al., 2015), artemisinin was measured using HPLC-MS. The resulting data showed that the contents of artemisinin were reduced significantly in two transgenic lines compared with wild-type plants (**Figure 6A**). In addition, arteannuin B and other sesquiterpenes were analyzed using GC-MS as reported previously (Ma et al., 2015). The resulting total ion chromatographs showed that the abundance of many non-polar metabolites was reduced in the two transgenic lines compared with wild-type plants (**Figure 6B**). Peak deconvolution allowed annotation of arteannuin B and other sesquiterpenes (**Figure 6B**). Peak values were recorded for arteannuin B and six other main sesquiterpenes. The resulting data showed that the peak values of arteannuin B and six sesquiterpenes were significantly reduced in the transgenic plants compared with wild-type controls (**Figures 6C,D**). These data indicate that the biosynthetic activity of sesquiterpenes is reduced in AaCAD transgenic plants. To determine whether the reduction of these metabolites was associated with the density of glandular trichomes, leaves and stems of transgenic vs. wild-type plants were examined using a Leica MZ FLIII fluorescence stereomicroscope and photographed (Supplementary Figures S4, S5). The resulting data showed that although many transparent or semi-transparent trichomes were observed in both abaxial and adaxial surfaces, most of them are T-shaped trichomes (Supplementary Figure S5) or other sharp stick trichomes. The density of glandular trichomes was low and similar on those leaves between transgenic and wild-type plants (Supplementary Figure S4).

### DISCUSSION

The present study shows that understanding the function of AaCAD, a short chain oxidoreductase in A. annua, is fundamental for the metabolic engineering of this medicinal crop to achieve high production levels of artemisinin. Numerous studies have reported that CAD is a key enzyme in the biosynthesis of lignin and plays an essential role in plant development associated with plant biomass (Mitchell et al., 1994; Feuillet et al., 1995; Halpin et al., 1998; Bagniewska-Zadworna et al., 2014; Pan et al., 2014; Choi et al., 2016). Downregulation of CAD expression or its knockout causes a reduction of lignin, which leads to severe plant dwarfism and a decreased biomass (Sirisha et al., 2012; Anderson et al., 2015; Ozparpucu et al., 2017; Ponniah et al., 2017) as well as decreased plant resistance to pathogens (Bagniewska-Zadworna et al., 2014;

Preisner et al., 2014; Rong et al., 2016). However, whether an increase in total lignin via CAD overexpression can affect the biosynthesis of terpenoids, such as artemisinin biosynthesis in A. annua, remains uninvestigated. We recently reported the cloning of a CAD homolog from a cross-pollinating heterozygous cultivar and its biochemical analysis. Our previous in vitro enzyme assays showed that the recombinant CAD used coumaryl, coniferyl, and sinapyl aldehydes as substrates in the presence of NADPH (Li et al., 2012). Our previous enzyme assays also showed that this CAD could use artemisinic aldehyde, a sesquiterpenoid metabolite, as a substrate to produce to artemisinic alcohol. Although the Km value of CAD for artemisinic aldehyde is higher than those of coumaryl, coniferyl, and sinapyl aldehydes, the activity using this sesquiterpenoid substrate indicates its catalytic promiscuity. Accordingly, we hypothesize that CAD not only essentially involves lignin formation, but it can also control artemisinin accumulation in A. annua. However, a functional analysis of CAD in planta has not been performed, given that the crosspollinating cultivar used to clone CAD is a heterozygous species that causes progeny segregation. The heterozygous cultivar is not appropriate material to generate transgenic plants to understand this gene function in planta. Herein, we re-cloned a new homolog from our novel inbred SP A. annua (F3 progeny) and designated it AaCAD to further characterize its functions in vitro and in vivo. Our enzyme assay showed that this new recombinant AaCAD efficiently converted coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols (Supplementary Figure S2). As expected, the overexpression of AaCAD significantly increased the lignin content in SP A. annua plants (**Figure 5A**), demonstrating its involvement in lignin biosynthesis. In addition, overexpression increased the content of coumarin (**Figures 5B–C**), indicating its additional function in the phenylpropanoid pathway. Given that amino acid sequence analysis showed that this AaCAD and the previous one from a cross pollinating cultivar had only three amino acid

differences (Supplementary Figure S2) and structural modeling showed the same structure conformation (Supplementary Figure S3), two homologs were anticipated to have similar enzymatic activity to convert artemisinic aldehyde to alcohol. As anticipated, the overexpression of this new AaCAD led to significant decreases in the contents of artemisinin, arteannuin B, and other sesquiterpenes in leaves (**Figures 6A,C**). Although these results were not a goal of our metabolic engineering strategy, they supported the findings of our previous in vitro enzyme assay showing that recombinant CAD from a cross-pollinating A. annua cultivar converted artemisinic aldehyde to artemisinic alcohol (Li et al., 2012), a late step in the reverse direction of the artemisinin pathway (**Figure 1**). To understand whether this reaction could occur in the cytosol of transgenic plant cells, we used GFP fusion to characterize the subcellular localization. Our confocal microscopy analysis revealed the cytosolic localization of AaCAD (**Figure 4**). These data further revealed that the cytosolic localization of AaCAD could reverse the step toward the formation of artemisinin catalyzed by CYP71AV1, CPR1, and AaADH1 and thus catalytically reduce the efficacy of three enzymes in the cytosol. These data indicate that overexpression of AaCAD negatively controls the formation of artemisinin in A. annua.

Glandular trichomes are the localization of the artemisinin biosynthesis (Duke et al., 1994; Xiao et al., 2016). A field study reported that application of salicylic acid or chitosan oligosaccharidea (out of different stress treatments) could significantly decrease 4–9 glandular trichomes per square of millimeter only on upper leaves from mature plants grown in the field (Kjaer et al., 2012). In the same study, three plants selected from the field were propagated via cutting and the resulting clones were grown in the greenhouse. Kjaer et al. (2012) observed that application of nine stress treatments did not affect the density of glandular trichomes on upper leaves but two stress conditions could significantly reduce about two trichomes per square of millimeter on lower leaves of clones. These results indicate that the density of glandular trichomes can be affected by external stress conditions. In our study, although whether CAD can alter glandular and other trichome development remains unknown, we examine glandular trichomes on leaves from 8–12 nodes and those nodes. We did not observe significant density difference of glandular trichomes between wild-type and transgenic plants. It was interesting that certain visual alterations in T-shaped trichomes were observed under microscope (Supplementary Figure S4). Given that glandular trichomes are the tissue of the artemisinin biosynthesis, our herein observation indicates a valuable interest in future studies to thoroughly examine glandular trichomes on all leaves and their effects on artemisinin contents in CAD transgenic progeny.

The increase in lignin and the reduction of artemisinin and other sesquiterpenes reveal an interesting crosstalk between two completely distinct pathways via AaCAD catalysis in transgenic plants. Lignin biosynthesis is mainly specialized in vascular tissues in all land plants (Weng and Chapple, 2010; Espineira et al., 2011). By contrast, artemisinin biosynthesis is mainly observed in A. annua and potentially other Artemisia species. Furthermore, this unique pathway is considered to be limited to a type of specialized glandular trichomes on leaves and flowers (Covello et al., 2007), although its cellular specialization remains controversial (Xie et al., 2016). This type of crosstalk in transgenic plants is most likely associated with two pathway intermediates and co-localization in the cytosol, despite their metabolic distinction (**Figure 1**). In the present transgenic plants, the AaCAD transgene was driven by two 35S promoters (**Figure 3A**), leading to constitutive overexpression of the transgene in the tissues, including trichomes. Our previous in vitro assay also showed that the recombinant AaCAD catalyzed the conversion of artemisinic aldehyde to alcohol (Li et al., 2012) (**Figure 1**). Accordingly, it is likely that overexpression of AaCAD (**Figure 3E**) enhances the catalytic conversion of artemisinin aldehyde to alcohol in the presence of NADPH in trichomes. In addition to our observations, CAD or CAD-like enzymes have been reported to be involved in the formation of monoterpenoid indole alkaloids in Rauvolfia serpentine (Geissler et al., 2016). Moreover, additional biochemical studies have shown that CAD is characterized by a high promiscuity level of different substrates (Chao et al., 2014; Geissler et al., 2016). Based on our and other reports, one hypothesis is that CAD-mediated crosstalk can occur in other metabolic pathways. As more studies are performed, it is anticipated that this type of enzyme-based crosstalk will be observed in different plants. In summary, the observed AaCADmediated crosstalk leading to reduced artemisinin contents is instructional for the engineering of A. annua for high production.

In conclusion, a new AaCAD was cloned from and overexpressed in inbred SP A. annua. The enzyme is localized in the cytosol. Overexpression of AaCAD in A. annua increased lignin and coumarin contents, but it decreased artemisinin, arteannuin B, and other sesquiterpene contents. These data revealed an AaCAD-mediated metabolic crosstalk between the phenylpropanoid (lignin and coumarin) and the sesquiterpene (artemisinin) pathways in the cytosol.

### AUTHOR CONTRIBUTIONS

DM and D-YX conceived and designed the research. DM, CX, FA-G, JY, HW, RJ, and D-YX performed the experiments and analyzed the data. DM and D-YX wrote the manuscript. D-YX supervised the entire project. All authors read and approved the manuscript.

### FUNDING

This research was supported by the North Carolina Biotechnology Center (grant #: 550031 and reference #: 2009-MRG-1117) and the National Natural Science Foundation of China (81303163).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00828/ full#supplementary-material

#### REFERENCES

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improved saccharification efficiency in switchgrass. BioEnergy Res. 4, 153–164. doi: 10.1007/s12155-010-9109-z

cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis. Plant Cell 26, 3709–3727. doi: 10.1105/tpc.114.127399

**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.

Copyright © 2018 Ma, Xu, Alejos-Gonzalez, Wang, Yang, Judd and Xie. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fpls-09-00828 June 17, 2018 Time: 12:20 # 12

# Molecular Characterization of the 1-Deoxy-D-Xylulose 5-Phosphate Synthase Gene Family in Artemisia annua

Fangyuan Zhang1† , Wanhong Liu2† , Jing Xia1 , Junlan Zeng1 , Lien Xiang1 , Shunqin Zhu1 , Qiumin Zheng1 , He Xie3 , Chunxian Yang1 , Min Chen4 and Zhihua Liao1 \*

1 Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), Chongqing Key Laboratory of Plant Ecology and Resources Research in Three Gorges Reservoir Region, SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing, China, 2 School of Chemistry and Chemical Engineering, Chongqing University of Science and Technology, Chongqing, China, 3 Tobacco Breeding and Biotechnology Research Center, Yunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, China, 4 SWU-TAAHC Medicinal Plant Joint R&D Centre, College of Pharmaceutical Sciences, Southwest University, Chongqing, China

#### Edited by:

Henrik T. Simonsen, Technical University of Denmark, Denmark

#### Reviewed by:

Ian A. Graham, University of York, United Kingdom Mehar H. Asif, National Botanical Research Institute (CSIR), India

\*Correspondence:

Zhihua Liao<EMAIL_ADDRESS><EMAIL_ADDRESS>
†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science

Received: 15 January 2018 Accepted: 13 June 2018 Published: 02 August 2018

#### Citation:

Zhang F, Liu W, Xia J, Zeng J, Xiang L, Zhu S, Zheng Q, Xie H, Yang C, Chen M and Liao Z (2018) Molecular Characterization of the 1-Deoxy-D-Xylulose 5-Phosphate Synthase Gene Family in Artemisia annua. Front. Plant Sci. 9:952. doi: 10.3389/fpls.2018.00952 Artemisia annua produces artemisinin, an effective antimalarial drug. In recent

decades, the later steps of artemisinin biosynthesis have been thoroughly investigated; however, little is known about the early steps of artemisinin biosynthesis. Comparative transcriptomics of glandular and filamentous trichomes and 13CO2 radioisotope study have shown that the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, rather than the mevalonate pathway, plays an important role in artemisinin biosynthesis. In this study, we have cloned three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) genes from A. annua (AaDXS1, AaDXS2, and AaDXS3); the DXS enzyme catalyzes the first and rate-limiting enzyme of the MEP pathway. We analyzed the expression of these three genes in different tissues in response to multiple treatments. Phylogenetic analysis revealed that each of the three DXS genes belonged to a distinct clade. Subcellular localization analysis indicated that all three AaDXS proteins are targeted to chloroplasts, which is consistent with the presence of plastid transit peptides in their N-terminal regions. Expression analyses revealed that the expression pattern of AaDXS2 in specific tissues and in response to different treatments, including methyl jasmonate, light, and low temperature, was similar to that of artemisinin biosynthesis genes. To further investigate the tissue-specific expression pattern of AaDXS2, the promoter of AaDXS2 was cloned upstream of the β-glucuronidase gene and was introduced in arabidopsis. Histochemical staining assays demonstrated that AaDXS2 was mainly expressed in the trichomes of Arabidopsis leaves. Together, these results suggest that AaDXS2 might be the only member of the DXS family in A. annua that is involved in artemisinin biosynthesis.

Keywords: Artemisia annua, artemisinin, 1-deoxy-D-xylulose 5-phosphate synthase, gene expression, MEP pathway

## INTRODUCTION

fpls-09-00952 August 2, 2018 Time: 15:3 # 2

Terpenoids, also known as isoprenoids, play several important roles in several plant processes. Despite their diverse structures and functions, all terpenoids are derived from the common five-carbon (C5) building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, the C5 building blocks are biosynthesized via twoindependent pathways: cytosolic mevalonate (MVA) pathway that is found in most eukaryotes and 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway that is found in the chloroplasts of photosynthetic eukaryotes and in eubacteria (Querol et al., 2002). Both these pathways are thought to be largely independent. The MVA pathway is primarily responsible for the biosynthesis of sesquiterpenes and triterpenes, whereas the MEP pathway produces precursors for the biosynthesis of major photosynthetic pigments, hormones, and monoterpenes and diterpenes (Dudareva et al., 2005). However, crosstalk between the MVA and MEP pathways occurs during the biosynthesis of some sesquiterpenes, such as artemisinin (Schramek et al., 2009).

The first reaction of the MEP pathway is the condensation of pyruvate with glyceraldehyde-3-phosphate to produce 1-deoxy-D-xylulose 5-phosphate (DXS), which is catalyzed by DXS (Querol et al., 2002). Subsequently, MEP is converted into a 5:1 mixture of IPP and DMAPP via six enzymatic reactions (Rodriguez-Concepcion and Boronat, 2002). Various studies suggest that DXS is a rate-limiting enzyme in the biosynthesis of terpenoids. The expression of DXS increases in plant tissues that require high levels of isoprenoids, as exemplified by maize (Zea mays; Cordoba et al., 2011), tomato (Solanum lycopersicum; Paetzold et al., 2010), glandular trichomes of peppermint (Mentha piperita; Lange et al., 1998), and young seedlings of arapdiopsis (Arabidopsis thaliana; Estévez et al., 2001). Overexpression or suppression of DXS alters the levels of specific isoprenoids in arapdiopsis (Estévez et al., 2001), tomato (Enfissi et al., 2005), and potato (Solanum tuberosum; Morris et al., 2006). Thus, DXS is an important target for the manipulation of isoprenoid biosynthesis.

Although most of the enzymes in the MEP pathway are encoded by single copy genes, the DXS enzymes are usually encoded by a small gene family (Rodriguez-Concepcion and Boronat, 2002; Cordoba et al., 2009), which is divided into three distinct phylogenetic clades (Carretero-Paulet et al., 2013). The expression of genes in the different clades varies with development, tissue type, and environmental conditions. The DXS proteins in clade 1, such as DXS1 in arabidopsis (Cloroplastos alterados 1, CLA1), primarily perform housekeeping functions (Estévez et al., 2000). In contrast, the expression of DXS genes in clade 2 is associated with isoprenoid accumulation. For instance, the expression of MtDXS2 in barrelclover (Medicago truncatula) and ZmDXS2 in maize is correlated with the production of certain apocarotenoids during mycorrhization (Walter et al., 2002; Cordoba et al., 2011). The suppression of these MtDXS2 results in reduced apocarotenoid accumulation, whereas the accumulation of MtDXS1 does not affect apocarotenoid production (Walter et al., 2002). Similarly, the expression of SlDXS2 in tomato exhibits a positive correlation with colonization by mycorrhizal fungi and the accumulation of apocarotenoids (Paetzold et al., 2010). Furthermore, the suppression of SlDXS2 in tomato leads to a decrease in the accumulation of the monoterpene, β-phellandrene, and an increase in the levels of two sesquiterpenes in leaf trichomes (Paetzold et al., 2010). Likewise, OsDXS3 in rice (Oryza sativa) has been suggested to be involved in defense responses and secondary metabolism (Okada et al., 2007). Together, these findings suggest that the DXS proteins in clade 2 are dedicated to secondary metabolism.

Artemisinin is a sesquiterpene endoperoxide that has been isolated from sweet wormwood (Artemisia annua) and extensively used in the treatment of malaria. Artemisinin has received tremendous interest in recent years because of its potential to treat cancer, diabetes, and tuberculosis (Zheng et al., 2017). Remarkable advances have been made in understanding the artemisinin biosynthetic pathway in recent decades. The first committed step in artemisinin biosynthesis is the cyclization of farnesyl diphosphate (FPP) to amorpha-4, 11-diene by amorpha-4, 11-diene synthase (ADS). Through three additional consecutive enzymatic reactions, amorpha-4, 11-diene is converted into dihydroartemisinic acid (DHAA), which is subsequently converted into artemisinin in an enzyme-independent reaction. Additionally, all four artemisinin biosynthesis genes (ADS, CYP71AV1, DBR2, and ALDH1) are specifically expressed in glandular secretory trichomes (GSTs), which are 10-cell structures located primarily on the surface of leaves and flower buds in A. annua (Chen et al., 2017).

While substantial progress has been made in understanding the later steps of artemisinin biosynthesis, little is known about the MVA and MEP pathways that supply the precursors for artemisinin biosynthesis. It has long been assumed that terpenoid/isoprenoid precursors provided by the MVA pathway are predominantly responsible for artemisinin biosynthesis; however, more recent studies suggest that the MEP pathway also supplies precursors for artemisinin biosynthesis. Either mevinolin (MVA pathway-specific inhibitor) or fosmidomycin (MEP pathway-specific inhibitor) decreases the artemisinin production in treated plants of A. annua (Towler and Weathers, 2007). Furthermore, the 13CO2 study demonstrates that the MEP pathway provides the central isoprenoid unit for the biosynthesis of FPP, which is the substrate for ADS (Schramek et al., 2009). Thus, both pathways are involved in artemisinin biosynthesis; however, genes that encode the enzymes of the MEP and MVA pathways, and the functions of these genes, remain elusive.

Given that the MEP pathway supplies precursors for artemisinin biosynthesis, and the DXS is the rate-limiting enzyme in the MEP pathway, a deeper understanding of the individual DXS gene family members will further help in understanding artemisinin biosynthesis and provide new target(s) for manipulating this metabolic pathway. Previously, Graham et al. (2010) have reported two DXS genes in A. annua with differential expression patterns, based on transcriptome sequencing. In the present work, we performed a detailed analysis of AaDXS gene family. Three AaDXS genes were cloned, each of which represented a distinct phylogenetic clade. Additionally, comprehensive expression analyses showed that the expression

pattern of AaDXS2 was highly similar to that of artemisinin biosynthesis genes, suggesting that AaDXS2 is the primary member of the AaDXS gene family that is involved in artemisinin biosynthesis.

### MATERIALS AND METHODS

#### Plant Materials and Treatments

Seeds of A. annua were collected from the botanical garden of Southwest University, Chongqing, China and stored at 4◦C. Seedlings were grown in the greenhouse at 25 ± 1 ◦C under 16 h light/8 h dark photoperiod. For light induction analysis of DXS genes, one-month-old seedlings were grown in the dark for 24 h and then shifted to light (Cordoba et al., 2011). Gene expression was examined in the leaves collected at different time points, ranging from 5 min to 12 h; leaves collected at 0 min were used as a control. In order to determine the expression pattern of DXS genes during development, the apical bud and the top seven leaves (leaf 1–8, except leaf 3) on the main stem of two-monthold plants were subjected to quantitative real-time polymerase chain reaction (qPCR) analysis (Lu et al., 2013; Czechowski et al., 2016). To analyze the expression of DXS genes in response to methyl jasmonate (MeJA), leaves of 2-month-old plants were treated with 300 µM MeJA and harvested at the indicated time points; leaves harvested from plants treated with 0.8% alcohol were used as a control. To study the effect of cold temperature on DXS gene expression, A. annua seedlings were transferred to the illumination incubator at 4◦C (Liu et al., 2017). Subsequently, leaves collected at the indicated time points were subjected to qPCR analysis; leaves harvested at 0 h were used as a control. For analyzing tissue-specific expression profiles of DXS genes,

five-month-old plants were transferred to 8 h light/16 h dark photoperiod to promote flowering. Flowers, leaves, stems, and roots of A. annua plants were collected and used for analyzing tissue expression profiles of DXS genes. All treatments performed in this study were replicated three times.

#### Cloning of DXS Genes and Sequence Analysis

Total RNA was isolated from plant materials using RNAsimple Kit (No. DP419; Tiangen Biotech, Beijing, China) according to the manufacturer's protocol. Promega M-MLV Kit (Promega, United States) and SMART rapid amplification of cDNA ends (RACE) cDNA Amplification Kit (Clontech, United States) were used for cloning the 30 - and 50 -end of DXS cDNAs, respectively. The first-strand 30 - and 50 -RACE-Ready cDNAs were prepared and used as templates for 30 - and 50 -RACE, respectively, according to the manufacturer's protocol. For sequence analysis, DXS amino acid sequences of A. thaliana were used as queries to search for AaDXS nucleotide sequences in the expressed sequence tag (EST) database of A. annua (taxid: 35608) using tBLASTn program. Touchdown PCR was carried out to clone the 30 - and 50 -ends of AaDXS genes. Each PCR product was cloned into the pMD-18T vector (Takara, Japan) and sequenced. Subsequently, full-length cDNAs of AaDXS genes were amplified by PCR using gene-specific primers (**Supplementary Table S1**). Multiple sequence alignments of DXS proteins were performed using Vector-NTI Advance 11.5 software package (Invitrogen, Carlsbad, CA, United States). Phylogenetic tree of DXS proteins was constructed with MEGA version 3.0 (Kumar et al., 2004) using the neighbor-joining method with a bootstrap of 1,000 replicates (Saitou and Nei, 1987).

#### Subcellular Localization

The putative plastid transit peptides of DXS isoforms were predicted using TargetP (Emanuelsson et al., 2000). Fragments of AaDXS genes encoding transit peptides were amplified by PCR using KOD plus (TOYOBO, Japan). Subsequently, all fragments harboring SacI and SalI restriction sites were inserted into the multiple cloning site of pCAMBIA1300-green fluorescent protein (GFP) in-frame with the coding sequence of the GFP gene. Approximately, 20 µg of each plasmid was introduced into mesophyll protoplasts of tobacco (Nicotiana tabacum) using polyethylene glycol-mediated transformation (Yoo et al., 2007). GFP fluorescence and chlorophyll autofluorescence were observed using ZEISS LSM700 laser confocal microscope (ZEISS, Germany) at excitation wavelengths of 488 and 555 nm, respectively.

#### qPCR Analysis

To investigate the expression of AaDXS genes in different tissues as well as under various conditions, qPCR was performed using iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, United States). The total RNA (2 µg) of A. annua was used for first-strand cDNA synthesis using GoScriptTM Reverse Transcription System (Promega, United States). The qPCR reaction mixtures were prepared with GoTaq qPCR Master

constructed with the neighbor-joining method using MEGA program 3.0 with 1,000 bootstrap values. The TAIR or Genebank accession numbers of DXS amino acid sequences used for phylogenetic analysis are as follows: Arabidopsis thaliana (DXS1 = At4g15560, DXS2 = At3g21500, and DXS3 = At5G11380); Ginkgo biloba (DXS1 = AAS89341.1 and DXS2 = AAR95699.1); Hevea brasiliensis (DXS1 = AAS94123.1 and DXS2 = ABF18929.1); Medicago truncatula (DXS1 = CAD22530.1 and i = CAN89181.1); Nicotiana tabacum (CBA12009.1); Oryza sativa (DXS1 = NP\_001055524, DXS2 = NP\_001059086, and DXS3 = BAA83576); Pinus densiflora (DXS1 = ACC54557.1 and DXS2 = ACC54554.1); Pinus taeda (DXS1 = ACJ67021.1 and DXS2 = ACJ67020.1); Populus trichocarpa (DXS1 = XP\_002312717.1, DXS2-1 = XP\_002303416.1, DXS2-2 = XP\_002331678.1, and DXS3 = XP\_002308644.1); Ricinus communis (DXS1 = XP\_002516843.1, DXS2 = XP\_002533688.1, and DXS3 = XP\_002514364.1); and Salvia miltiorrhiza (DXS1 = ACF21004.1, DXS2 = ACQ66107.1).

Mix (Promega, United States), according to the manufacturer's protocol. PCR amplifications were performed using the following conditions: denaturation at 95◦C for 30 s, followed by 40 cycles at denaturation at 95◦C for 5 s and annealing and extension at 60◦C for 30 s, and a final extension at 72◦C for 20 s. Melting curve was used to determine the specificity of amplifications. The ACTIN gene was used as reference for normalization of qPCR CT values. Gene-specific primers used for qPCRs were designed using Primer Premier 6 (**Supplementary Table S1**). The 2−11CT method was used to calculate the relative fold-change in gene expression (Livak and Schmittgen, 2001).

### Promoter Cloning and β-Glucuronidas (GUS) Histochemical Staining

Genomic DNA of A. annua was isolated using the CTAB method. A genome walking method, that is, fusion primer and nested integrated PCR (Wang et al., 2011) was carried out to amplify the promoter of AaDXS2 (pAaDXS2). Primers used to amplify pAaDXS2 are listed in **Supplementary Table S1**. The TSSP software was used to determine the transcription start site of AaDXS2 (Solovyev and Shahmuradov, 2003). The cis-elements in pAaDXS2 were analyzed using PlantCARE website1 and PLACE website2 .

To investigate the expression pattern of AaDXS2 in plants, the pAaDXS2 was cloned into pCAMBIA1391.Z to drive the expression of GUS gene. The pAaDXS2::GUS construct was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into A. thaliana by the floral dip method (Zhang et al., 2006). Mature leaves and flowers of 45-day-old arabidopsis seedlings as well as siliques from 2-month-old transgenic A. thaliana were used for GUS histochemical staining as described previously (Jefferson et al., 1987). GUS stained tissues were observed under Olympus SZX16 microscope, and pictures were taken using Olympus DP73 digital camera.

#### RESULTS

### The A. annua Genome Harbors Three DXS Genes

To investigate the DXS genes in A. annua, tBLASTn program3 was used. Amino acid sequences of AtDXS proteins (At4G15560, At3G21500, and At5G11380) were used as queries against the EST database of A. annua (taxid: 35608). Three AaDXS partial coding sequences (contig16978, contig19217, and contig6280) were obtained. Subsequently, primers for RACE PCRs were designed based on the longest EST sequence. The resulting fulllength cDNAs of three AaDXS genes were named as AaDXS1, AaDXS2, and AaDXS3.

The full-length AaDXS1 cDNA was 2,529 bp in length and contained 126 bp 50 untranslated region (50UTR), 2,142 bp open reading frame (ORF), and 261 bp 30UTR (**Supplementary Figure S1**). The AaDXS2 cDNA consisted of 52-bp 50UTR, 2,187 bp ORF, and 214 bp 30UTR (**Supplementary Figure S2**). The AaDXS3 cDNA was the longest among the three AaDXS cDNAs (2,761 bp) and comprised 248 bp 50UTR and 374 bp 3 0UTR (**Supplementary Figure S3**). All three AaDXSs showed features similar to those of known DXS from other plant species, including the presence of an N-terminal targeting sequence, a conserved thiamine diphosphate binding site, and pyridine binding DRAG domain (**Figure 1**).

A phylogenetic tree was constructed using neighbor-joining method to reveal the evolutionary relationship among the DXS proteins of thirteen plant species, including A. annua (**Figure 2**). Phylogenetic analysis showed three clusters of DXS proteins. AaDXS1 grouped in clade 1, which contained well-characterized DXS1 proteins of arabidopsis and M. truncatula. AaDXS2 grouped in clade 2 with the well-characterized DXS2 proteins of M. truncatula and Salvia miltiorrhiza. Partial members within clade 2 play important roles in plant secondary metabolism, especially isoprenoid biosynthesis (Floss et al., 2008; Kai et al., 2012). Compared with clades 1 and 2, clade 3 contained five DXS proteins from A. annua, A. thaliana, and O. sativa. The biological functions of DXS proteins in clade 3 are unclear; however, the DXS proteins in clade 3 are contained at lower expression levels than those in clades 1 and 2 in maize (Cordoba et al., 2011). Studies in arabidopsis and tomato have shown that the DXS proteins in different clades are involved in different biological processes (Paetzold et al., 2010; Carretero-Paulet et al., 2013). Overall, phylogenetic analysis showed that the three AaDXS proteins clustered into three distinct clades, suggesting that these three proteins are involved in different biological processes.

#### AaDXS Proteins Localize to the Chloroplast

In silico analysis with TargetP 1.1 showed that all three AaDXS proteins carried a plastid transit peptide. To further validate the in silico results, nucleotide sequences encoding the putative transit peptides were amplified by PCR and cloned into pCAMBIA1300-GFP vector. These constructs containing the putative transit peptides fused with GFP were then transformed into tobacco mesophyll protoplasts. The GFP signal resulting from the transformation of each vector localized together with the autofluorescence signal of chloroplasts (**Figure 3**). These results demonstrated

1http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

2http://www.dna.affrc.go.jp/PLACE/

3https://blast.ncbi.nlm.nih.gov/Blast.cgi

that all three AaDXS proteins localized to the chloroplast; this is consistent with the plastid localization of the MEP pathway.

### AaDXS2 and Artemisinin Biosynthesis Genes Show Similar Tissue-Specific Expression

The expression profiles of AaDXS genes were analyzed in different tissues, including flowers, leaves, stems, and roots. Many studies have shown that the artemisinin biosynthesis genes have the highest expression levels in flower buds, followed by leaves, and finally, the roots (Zhang et al., 2015). As shown in **Figure 4**, expression levels of AaDXS1 and AaDXS3 were significantly lower in flowers than in other tissues of A. annua. However, the expression level of AaDXS2 in flowers was 3 fold higher than that in leaves and 10-fold higher than that in stems and roots (**Figure 4**). Additionally, the expression of AaDXSs was also detected in leaves at different positions on the stem, where the density of glandular trichomes and expression of artemisinin biosynthetic genes show significant difference (Lu et al., 2013). Furthermore, the level of artemisinin precursor, DHAA, progressively declines during leaf maturation (Czechowski et al., 2016). Results of qPCR analysis showed that the expression of AaDXS2 was high in apical buds, whereas they declined sharply during leaf development (**Figure 5B**). The expression pattern of AaDXS2 was highly similar to that of the ADS gene, which encodes the first enzyme involved in artemisinin biosynthesis (**Figure 5D**). By contrast, the expression of AaDXS1 was relatively constant in leaves at different positions (**Figure 5A**), and the expression of AaDXS3 was higher in leaf 8 than in other leaves (**Figure 5C**). Overall, the tissue-specific expression profiles of AaDXS genes showed that AaDXS2 was the only gene whose expression pattern was similar to that of the artemisinin biosynthesis genes, which further indicated that AaDXS2 was probably highly expressed in glandular trichomes, where artemisinin is biosynthesized.

### AaDXS2 and Artemisinin Biosynthesis Genes Exhibit Similar Expression Patterns Under Multiple Treatments

Many elicitors and environmental factors, such as MeJA, low temperature, and light, regulate artemisinin biosynthesis (Hao et al., 2017; Liu et al., 2017). The expression of artemisinin biosynthesis genes increases in response to MeJA, cold, and light. To determine which of the three AaDXS genes was more important for artemisinin biosynthesis, we analyzed the expression of all three AaDXS genes under MeJA, cold, and light treatment. As shown in **Figure 6**, the expression of AaDXS1 and AaDXS3 was mildly induced by MeJA treatment at 3 and 9 h (**Figures 6A,C**), whereas that of AaDXS2 was strongly induced from 1 to 12 h (**Figure 6B**). Under cold treatment, the expression of AaDXS1 and AaDXS3 decreased through the time course (**Figures 7A,C**). Although the expression of AaDXS2 was downregulated at 1 and 3 h under cold stress, its expression was significantly upregulated at 6 and 12 h compared with the control (**Figure 7B**). Additionally, the expression of all three AaDXS genes in cold-treated plants was lower than that in the control from 1 to 3 h.

To investigate the effect of light on the expression of AaDXS genes, A. annua seedlings were placed in a dark room for 24 h and then transferred to the illumination incubator. Leaves were harvested for qPCR analysis at different time points, ranging from 5 min to 12 h. Results of qPCR analysis showed that the expression of AaDXS1 was slightly induced after 1 h of light exposure (**Figure 8A**); however, no significant differences were detected in the expression of AaDXS3 throughout the experiment (**Figure 8C**). In contrast, the expression of AaDXS2 rapidly reached a peak at 5 min and then gradually declined, returning to the control level (**Figure 8B**). Furthermore, expression patterns of ADS and CYP71AV1 under light treatment were similar to that of AaDXS2 (**Figures 8D,E**).

Overall, the expression analysis of AaDSX genes under MeJA, cold, and light treatment demonstrated that among the three AaDXS genes, the expression pattern of only AaDXS2 was similar

to that of the artemisinin biosynthesis genes. These data suggest that AaDXS2 is more important than AaDXS1 and AaDXS3 in artemisinin biosynthesis.

### Analysis of AaDXS2 Promoter Activity in Transgenic A. thaliana

Results of qPCR analysis showed that the expression of AaDXS2 in different tissues and under different conditions was similar to that of the artemisinin biosynthesis genes. Subsequently, we cloned the 1,494-bp promoter of AaDXS2 (pAaDXS2; **Supplementary Figure S4**). Sequence analysis of pAaDXS2 using PLACE and PlantCARE revealed the presence of several lightresponsive elements, such as Box I and GATA-motifs; MeJAresponsive elements, such as CGTCA- and TGACG-motifs; and stress-responsive elements, such as Box-W1 that responds to fungal elicitors, HSE involved in high temperature stress, and TC-rich repeats associated with plant defense and stress. To investigate the cellular compartmentalization of AaDXS2, we cloned pAaDXS2 upstream of the GUS reporter gene and transformed the pAaDXS2::GUS construct in A. thaliana. Different organs of 45-day-old transgenic arabidopsis plants, including mature leaves, flowers, and siliques, were used for

FIGURE 7 | Relative expression levels of AaDXS1–3 under low temperature (4◦C). (A) AaDXS1; (B) AaDXS2; and (C) AaDXS3. Statistically significant differences are indicated using asterisks (Student's t-test, ∗p < 0.1; ∗∗p < 0.05; and ∗∗∗p < 0.01). Data represent mean ± SD of three biological replicates.

GUS histochemical staining. GUS staining was observed in the stigma, stamen, and pedicel but not in petals and young carpel (**Figures 9A–C**). More importantly, strong GUS staining was observed in the trichomes of mature leaves (**Figure 9B**).

#### DISCUSSION

The MEP pathway provides structural molecules for the synthesis of numerous key metabolites, such as terpenoids, phytohormones, chlorophyll, and carotene. Understanding this biosynthetic route is important for modulating the production of key isoprenoids. Although most of the enzymes in the MEP pathway are encoded by single-copy genes (Rodriguez-Concepcion and Boronat, 2002; Cordoba et al., 2009), the first key enzyme, DXS, is encoded by a small gene family composed of 2–4 genes in plants, such as arabidopsis (Carretero-Paulet et al., 2013), alfalfa (Medicago sativa; Walter et al., 2002), and maize (Cordoba et al., 2011). Souret et al. (2002) reported a DXS gene, named DXSPS, in A. annua; the nucleotide sequence of AaDXSPS was 98.7% similar to that of AaDXS1 cloned in this study (**Supplementary Figure S5**). Thus, we propose that DXPS and AaDXS1 represent the same gene. However, transcript levels of AaDXS1 only showed a slight increase under light treatment, whereas those of DXPS significantly increased in root cultures grown under continuous light compared with those grown in the dark (Souret et al., 2002). This discrepancy might be due to the different plant material used in these experiments; Souret et al. (2002) performed expression analysis in A. annua root cultures,

whereas we used leaves in this study. Previously, 454 sequencing of EST libraries in A. annua have revealed two DXS contigs (EZ216572 and EZ167196; Graham et al., 2010). Aligning these two contigs with the AaDXS genes cloned in this study showed that EZ216572 and EZ167196 represent partial coding sequences of AaDXS1 and AaDXS2, respectively. Moreover, transcriptome data showed that the expression of EZ167196 was significantly higher in the GSTs of flower buds and young leaves than in those of mature leaves and young leaf meristem. In contrast, EZ216572 showed higher expression levels in the GSTs of young leaves and in cotyledons. These data are consistent with the expression analysis results of this study, showing that AaDXS2 was mainly expressed in young leaves and flower buds.

Several studies suggest that different genes in the DXS family are responsible for the biosynthesis of different isoprenoids (Enfissi et al., 2005; Paetzold et al., 2010; Cordoba et al., 2011). Henceforth, it is important to investigate the roles of different DXS isoforms in the biosynthesis of specific metabolites. Although enzymes involved in artemisinin biosynthesis have been determined, little is known about the enzymes involved in the MEP pathway. In the present study, we cloned three AaDXS genes, each of which encoded plastid-localized proteins. Expression analysis of AaDXS genes indicated that the expression pattern of AaDXS2 was similar to that of the artemisinin biosynthesis genes. The analysis of AaDXS2 promoter in transgenic arabidopsis demonstrated that AaDXS2 was mainly expressed in trichomes.

Amino acid sequence alignment revealed that the three AaDXS proteins shared 42.3% identity. Moreover, low conservation in the TPP and GAP binding sites of AaDXS3 suggested that AaDXS3 diverged from AaDXS1 and AaDXS2. The relatively low similarity in DXS protein sequences implies the functional divergence of DXS family members that have been shown previously (Cordoba et al., 2011). Phylogenetic analysis showed that AaDXS1 clustered with AtDXS1 and soybean (Glycine max) DXS1 in clade 1; it has been shown that the function of AtDXS1 and GmDXS1 is related to chloroplast development and chlorophyll synthesis (Mandel et al., 1996; Zhang et al., 2009). Most of the DXS enzymes involved in the biosynthesis of secondary metabolites in plants belonged to clade 2. For example, MtDXS2, which plays an important role in apocarotenoid biosynthesis, groups in clade 2 (Floss et al., 2008). The expression of DXS2 in hairy roots of red sage (Salvia miltiorrhiza) is positively correlated with the accumulation of tanshinones (Kai et al., 2012). Although the biological functions of DXS proteins in clades 1 and 2 in plants are relatively clear, those of DXS proteins in clade 3 are still unclear. The expression of ZmDXS3 is lower than that of the other DXS isogenes in maize, by which we predicted that ZmDXS3 might be involved in the biosynthesis of derivatives from the MEP pathway at a lower level, such as plant hormones (Cordoba et al., 2009).

Because artemisinin biosynthesis genes are specifically expressed in glandular trichomes, transcripts of artemisinin biosynthesis genes are accumulated to the highest levels in flowers followed by young leaves, as these tissues have more glandular trichomes than the other plant tissues (Lu et al., 2013). In this study, we showed that the expression of both AaDXS1 and AaDXS3 was lower in flowers than in other tissues, whereas that of AaDXS2 was the highest in flowers. Additionally, the expression level of AaDXS2 in leaves at different positions on the stem was similar to that of the artemisinin biosynthesis genes. This differential expression of AaDXS2 is consistent with the density of glandular trichomes, which is the highest in the apical bud and decreases in leaves with an increasing developmental age (Lu et al., 2013). Results showed that AaDXS2 was mainly expressed in the glandular trichomes of leaves, which was consistent with the GUS staining patterns observed in transgenic A. thaliana (**Figure 9B**). In addition to trichomes of mature leaves, GUS staining of transgenic arabidopsis showed that AaDXS2 was also expressed in the stigma, stamen, and silique. This result was similar to the expression analysis of AtDXS2 in arabidopsis in which transcripts were detected at highest levels in siliques and inflorescences (Carretero-Paulet et al., 2013). We hypothesized that this might be due to DXS, which is a rate-limiting enzyme in MEP pathway, and which is responsible for biosynthesis of many isoprenoids. SlDXS2 plays an important role in isoprenoid biosynthesis and trichome development in tomato. The suppression of SlDXS2 via RNAi in tomato results in a decrease in the level of the monoterpene, β-phellandrene, and an increase in trichome density (Paetzold et al., 2010). AaDXS2 might be responsible for the biosynthesis of other isoprenoids excepted to artemisinin. Notably, among the three DXS genes of A. annua, only AaDXS2 showed tissue-specific expression patterns that were similar to those of artemisinin biosynthesis genes, suggesting that AaDXS2 might play a more important role in biosynthesis of artemisinin.

Generally, the DXS genes belonging to clade 1 exhibit constitutive expression in plants. For example, PaDXS1 is ubiquitously expressed in various tissues and is not induced by elicitors (Phillips et al., 2007). Nevertheless, many DXS genes in clade 2 exhibit higher expression in response to various elicitors, such as MeJA and light (Cordoba et al., 2011; Kai et al., 2012). Previous studies have shown that MeJA is a powerful elicitor of artemisinin biosynthesis genes; its application induces the expression of artemisinin biosynthesis genes, resulting in a greater accumulation of artemisinin in MeJA-treated plants (Wang et al., 2010; Caretto et al., 2011; Xiang et al., 2015). The strong induction of AaDXS2 expression following MeJA treatment observed in this study is consistent with the expression of artemisinin biosynthesis genes under MeJA treatment shown previously (Gong et al., 2006; Phillips et al., 2007). Cold stress is effective in enhancing artemisinin biosynthesis via two-independent pathways: converting DHAA into artemisinin (Wallaart et al., 2000) and promoting endogenous MeJA biosyntheis (Liu et al., 2017). Among the three AaDXS genes, the expression of AaDXS1 and AaDXS3 was inhibited under cold stress, whereas that of AaDXS2 was significantly increased.

In A. thaliana, the MEP pathway genes, except HDR, are upregulated by light, suggesting that light is an important regulator of the MEP pathway in plants (Hsieh and Goodman, 2005). Overexpression of AtCRY1, a blue light receptor, in A. annua promotes artemisinin accumulation, leading to the conclusion that light is a vital factor in regulating artemisinin biosynthesis (Hong et al., 2009). This conclusion is corroborated by a recent study (Hao et al., 2017). The expression of artemisinin biosynthesis genes (ADS, CYP71AV1, DBR2, and ALDH1) is upregulated under light. Furthermore, JA-induced artemisinin biosynthesis is dependent on light. Consistent with the study of Hao et al. (2017), we showed that the expression of ADS and CYP71AV1 increased under light. More importantly, only AaDXS2 was induced rapidly and dramatically after light exposure; this expression pattern of AaDXS2 was similar to that of the artemisinin biosynthesis genes. Similarly in maize, the expression pattern of ZmDXS1 and ZmDXS2 were increased in plants exposed to light treatment (Cordoba et al., 2011). Additionally, the expression pattern of AaDXS2 was similar with ADS and CYP71AV1 in A. annua, indicating that AaDXS2 is involved in the light-mediated regulation of artemisinin biosynthesis.

#### CONCLUSION

In conclusion, this study reports a detailed analysis of the expression profiles of three DXS genes in different tissues of A. annua. Additionally, we have shown that elicitors, such

#### REFERENCES

Caretto, S., Quarta, A., Durante, M., Nisi, R., De Paolis, A., Blando, F., et al. (2011). Methyl jasmonate and miconazole differently affect arteminisin production and gene expression in Artemisia annua as MeJA, and various environmental factors modulate the expression of AaDXS genes. The expression of AaDXS2 was very similar to that of the artemisinin biosynthesis genes, suggesting that AaDXS2 might be the only DXS isoform involved in the biosynthesis of artemisinin. This work extends the analyses of DXS genes in other plants and provides a new potential target for the modulation of artemisinin production.

#### AUTHOR CONTRIBUTIONS

FZ and ZL conceived and designed the study. FZ, WL, and JX performed the RNA isolation, qRT-PCR analysis, and plant transformation. JZ, HX, and LX performed the subcellular localization and GUS staining. SZ, QZ, and CY managed Artemisia annua and Arabidopsis. MC analyzed the data. ZL prepared the manuscript. All authors have read and approved the manuscript.

#### FUNDING

This work was financially supported by the NSFC project (31770335 and 31300333), Fundamental Research Funds for the Central Universities (XDJK2016C114), China Postdoctoral Science Foundation (2015M582497), Foundation of YNTC (2016YN22), the National Undergraduate Training Programs for Innovation and Entrepreneurship of China<PHONE_NUMBER>75), the Scientific and Technological Research Program of Chongqing Municipal Education Commission (KJ1501321), Chongqing Science and Technology Commission (cstc2016shmszx80101), and the open fund of Chongqing Key Laboratory of Industrial Fermentation Microorganism (LIFM201714).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00952/ full#supplementary-material

FIGURE S5 | Amino acidsequence alignment of AaDXS1 and DXPS (AF182286.2).

TABLE S1 | The primers used in this study.

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**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.

Copyright © 2018 Zhang, Liu, Xia, Zeng, Xiang, Zhu, Zheng, Xie, Yang, Chen and Liao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Seasonal and Differential Sesquiterpene Accumulation in *Artemisia annua* Suggest Selection Based on Both Artemisinin and Dihydroartemisinic Acid may Increase Artemisinin *in planta*

#### Jorge F. S. Ferreira1 \*, Vagner A. Benedito2 , Devinder Sandhu1 , José A. Marchese3 and Shuoqian Liu4

1 US Salinity Laboratory, Riverside, CA, United States, 2 Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, United States, 3 Biochemistry and Plant Molecular Physiology Laboratory, Agronomy Department, Federal University of Technology–Paraná, Pato Branco, Brazil, 4 Department of Tea Science, College of Horticulture and Hardening, Hunan Agricultural University, Changsha, China

#### *Edited by:*

Ian A. Graham, University of York, United Kingdom

#### *Reviewed by:*

Kexuan Tang, Shanghai Jiao Tong University, China Tony Larson, University of York, United Kingdom

> *\*Correspondence:* Jorge F. S. Ferreira<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science

*Received:* 23 December 2017 *Accepted:* 06 July 2018 *Published:* 13 August 2018

#### *Citation:*

Ferreira JFS, Benedito VA, Sandhu D, Marchese JA and Liu S (2018) Seasonal and Differential Sesquiterpene Accumulation in Artemisia annua Suggest Selection Based on Both Artemisinin and Dihydroartemisinic Acid may Increase Artemisinin in planta. Front. Plant Sci. 9:1096. doi: 10.3389/fpls.2018.01096 Commercial Artemisia annua crops are the sole source of artemisinin (ART) worldwide. Data on seasonal accumulation and peak of sesquiterpenes, especially ART in commercial A. annua, is lacking while current breeding programs focus only on ART and plant biomass, but ignores dihydroartemisinic acid (DHAA) and artemisinic acid (AA). Despite past breeding successes, plants richer in ART are needed to decrease prices of artemisinin-combination therapy (ACT). Our results show that sesquiterpene concentrations vary greatly along the growing season and that sesquiterpene profiles differ widely among chemotypes. Field studies with elite Brazilian, Chinese, and Swiss germplasms established that ART peaked in vegetative plants from late August to early September, suggesting that ART is related to the photoperiod, not flowering. DHAA peaks with ART in Chinese and Swiss plants, but decreases, as ART increases, in Brazilian plants, while AA remained stable through the season in these genotypes. Chinese plants peaked at 0.9% ART, 1.6% DHAA; Brazilian plants at 0.9% ART, with less than 0.4% DHAA; Swiss plants at 0.8% ART and 1% DHAA. At single-date harvests, seeded Swiss plants produced 0.55–1.2% ART, with plants being higher in DHAA than ART; Brazilian plants produced 0.33–1.5% ART, with most having higher ART than DHAA. Elite germplasms produced from 0.02–0.43% AA, except Sandeman-UK (0.4– 1.1% AA). Our data suggest that different chemotypes, high in ART and DHAA, have complementary pathways, while competing with AA. Crossing plants high in ART and DHAA may generate hybrids with higher ART than currently available in commercial germplasms. Selecting for high ART and DHAA (and low AA) can be a valuable approach for future selection and breeding to produce plants more efficient in transforming DHAA into ART in planta and during post-harvest. This novel approach could change the breeding focus of A. annua and other pharmaceutical species that produce more than one desired metabolite in the same pathway. Obtaining natural variants with high ART content will empower countries and farmers who select, improve, and cultivate A. annua as a commercial pharmaceutical crop. This selection approach could enable ART to be produced locally where it is most needed to fight malaria and other parasitic neglected diseases.

Keywords: seasonal sesquiterpene accumulation, sesquiterpene-based selection, sesquiterpene seasonal peak, high-DHAA germplasm selection, different chemotypes

#### INTRODUCTION

Active pharmaceutical ingredients (APIs) based on artemisinin (ART), such as artemether and artesunate, are key components of the most effective antimalarial drugs. The Nobel Assembly at Karolinska Institute awarded the Nobel Prize in Physiology or Medicine to Prof. Youyou Tu in 2015 in recognition of her early work in the late 1960s and 70s that culminated with the discovery of ART, the most effective natural anti-malarial medicine after quinine. Currently, all ART that is used as raw material for the production of artemisinin-combination therapy (ACT) is obtained from the plant Artemisia annua (Family: Asteraceae). Although other plants and microorganisms have been engineered with ART-pathway genes, tobacco was only able to produce artemisinic acid (AA) at 0.12% of leaf dry weight (DW) (Fuentes et al., 2016), and ART at less than 0.0007% of leaf DW (Farhi et al., 2011); transgenic moss produced ART at 0.021% of leaf DW (Khairul Ikram et al., 2017). These yields are 47 (moss) to 1,400 times (tobacco) less than an A. annua plant that produces 1% ART. Also, moss is not a feasible alternative considering its low biomass yield. Although baker's yeast produced 25 g of AA per liter of culture (Paddon et al., 2013), derivatization of AA to ART is further needed, making the process economically unfeasible compared to plant-based ART (Peplow, 2016). The low yields of ART in tobacco may be in part due to the fact that ART is phytotoxic (Duke et al., 1987) and must be stored extracellularly, inside epicuticular spaces of glandular trichomes (Duke et al., 1994; Ferreira and Janick, 1995b). Although other Artemisia species can produce ART (Mannan et al., 2010), their ART shoot concentrations are not large enough to justify commercialization. The no-cost/no-profit price of semi-synthetic ART by Sanofi is estimated to range from US\$350 to 400 kg−1 , which is well above the US\$250 kg−1 for the naturally-produced ART (Peplow, 2016). Although heterologous systems are valuable to improve the knowledge of the ART pathway, more efficient ways to stabilize market prices and reduce ACT costs are urgently needed. Rather than pursuing ART production in heterologous systems, more viable approaches to increase plant-based ART supply should focus on: (1) breeding plants that are higher in ART than the currently reported average of approximately 1.5% ART, (2) producing cultivars that are less variable in plant-toplant ART concentrations, (3) producing cultivars that are higher in DHAA and able to convert DHAA into ART more efficiently, (4) improving ART commercial extraction to over 70% efficiency, and (5) recycling DHAA from ART commercial waste to produce more ART.

Since the initial plant screenings by the Chinese government in the late 1960s, plants have been selected only for their high biomass and ART leaf content. In 1985, Chinese accessions were reported to range from 0.01 to 0.5% ART, with plants from the Sichuan and Chongqing provinces being the highest in ART (Klayman, 1985), and those north of the Huaihe river bank reported to have 0.1% ART or less (Li et al., 2017). Only a few breeding programs managed to increase ART shoot concentration from 0.5 to 1.5% or higher. Because A. annua has a high degree of self-incompatibility (allogamy) that precludes selfpollination, and the word "hybrid" often used does not denote true hybrids generated from homozygous parents, but rather the F1 progeny of two distinct, highly heterozygous, parents. The oldest breeding program with published reports in English and French is from the company Mediplant (Conthey, Switzerland). In the early 1990s this program produced several crossings that generated plants with over 1% ART (Debrunner et al., 1996; Magalhães et al., 1999). Later, Mediplant reported a new line named "Hybrid 1" with up to 1.8% ART and 2.9 tons of dry leaves ha−1 (Simonnet et al., 2008), but we are not sure whether "Hybrid 1" was ever available commercially or to breeding programs destined to generate high-ART plants for humanitarian purposes. The Brazilian breeding program, in collaboration with Mediplant, developed hybrids named "CPQBA," the acronym (in Portuguese) for the Multidisciplinary Center for Chemical and Biological Research (Campinas, Brazil) with seeds that sold for US\$40 g−1 , with 12–15 thousand seeds g−1 . Despite the fact that plants from both Mediplant and CPQBA selections were late flowering in the environment where they were selected (Nicolas Delabays, personal communication), they flowered prematurely and produced little biomass when planted close to the equator (5–7◦ latitude) due to a short day length that inhibit vegetative development and accelerated the reproductive stage (Ferreira et al., 2005). Selected plants from this Swiss (Mediplant) cultivar named Artemis R produced from 0.65 to 1.9% ART when cultivated in an Appalachian Gilpin soil (Beaver, WV, 37◦ 44 ′ N 81◦ 8 ′W) from May to August in a Quonset greenhouse under a mild potassium stress (Ferreira, 2007). Most recently, the cross "Hyb8001r" was developed and introduced by the Centre for Novel Agricultural Products (CNAP) in the UK, which is now commercialized by East-West Seed International. In field trials worldwide, "Hyb8001r" produced shoot ART up to 1.44% (g/100 g DW) and up to 4.4 tons/ha of dry leaf biomass, with a theoretical ART yield of 54 kg/ha (Suberu et al., 2016). In their work, "Hyb8001r" is referred to as CNAP8001, with seeds available to growers linked to the ACT raw material supply chain (https://www.artemisiaf1seed.org/hyb8001r/).

The literature provides evidence that A. annua plants accumulate not only ART, but also its biosynthetic precursor DHAA and AA (Wallaart et al., 2000; Ferreira, 2007; Ferreira and Luthria, 2010). After artemisinic aldehyde (AO), the ART pathway diverts into either AA, arteannuin B, and artemisitene (AT) or into DHAA, dihydroartemisinic hydroperoxide (DHAHP), and ART (Kjær et al., 2013; Bryant et al., 2015) (**Figure 1**). Despite the fact that AA can be the main sesquiterpene produced (over 1% of leaf DW) by certain chemotypes of A. annua (Ro et al., 2006), the chemotype used for commercial purposes produce mainly ART, with DHAA as its main precursor (Wallaart et al., 1999b; Brown and Sy, 2004; Ferreira and Luthria, 2010). Contrary to previous reports of that in vitro photooxidative conversion of DHAA into ART could occur with almost 27% efficiency in organic solvents and with the presence of chlorophyll a (Acton and Roth, 1992; Wallaart et al., 1999a). Recent work suggests enzymatic action in the final stages of the pathway (Zhu et al., 2014; Bryant et al., 2015).

It was originally postulated that ART was produced and sequestered in glandular trichomes of leaves (Duke et al., 1994) and flowers (Ferreira and Janick, 1995a,b); and that the isolation of enzymes from these trichomes would confirm this hypothesis (Ferreira and Janick, 1995a). Since then, ART biosynthetic enzymes have been isolated from glandular trichomes (Olofsson et al., 2012; Tan et al., 2015; Chen et al., 2017). However, although the pathway produces AA, DHAA, and ART simultaneously, the two main chemotypes found in the literature either accumulate mainly AA (Elhag et al., 1992; Ro et al., 2006) or mainly DHAA and ART (Wallaart et al., 1999b, 2000; Ferreira, 2008). Detailed experiments based on the use of synthetically-produced radiolabeled precursors fed to plants also concluded that, compared to AA, DHAA is more cost-effective to produce ART in geneticallyengineered yeast (Brown, 2010). A two-step auto-photooxidation step is postulated to occur in plants to convert DHAA into ART while plants senesce, or are dried under oven, shade, or sun (Brown and Sy, 2004; Ferreira and Luthria, 2010). Sun drying for 1–3 weeks was the most efficient way to convert DHAA into ART (over 90%), whereas forced-air oven drying (45◦C from 12 to 16 h) only achieved a 40% conversion (Ferreira and Luthria, 2010). These findings suggest that drying plants in a forced-air oven illuminated with UV light may convert DHAA into ART more efficiently than in a dark oven. The average content of ART in plants used for industrial extraction has been reported to be 0.7% based on leaf dry weight (Malcolm Cutler, personal communication). However, ART yields can be maximized if the plants are harvested at the time ART reaches its seasonal peak, which is prior to flowering (Delabays et al., 2001; Ferreira, 2008). Swiss plants field-cultivated in West Virginia and harvested toward the end of their vegetative stage (no flowers), in early September, produced 0.7% ART and an average of 450 g dry leaves plant−1 , or 4.5 tons ha−1 for a plant density of 1 plant m−2 (Ferreira, 2007).

A. annua plants increased ART production in response to abiotic stresses, such as potassium deficiency (Ferreira, 2007), drought (Marchese et al., 2010), post-harvest drying (Ferreira and Luthria, 2010), senescence (Lommen et al., 2007), and salinity (Qureshi et al., 2005; Qian et al., 2007; Yadav et al., 2017). Interestingly, environmental stresses such as drought, wound, and cadmium (Xiao et al., 2016), and application of the hormones JA and cytokinin (Maes et al., 2011) increased

FIGURE 1 | Biosynthetic pathway of artemisinin starting from farnesyl diphosphate (FPP). 1. Amorpha-4,11-diene synthase (ADS) synthesizes the first committed product of the artemisinin pathway, AD. 2. Cytochrome P450 monooxygenase CYP71AV1 oxidizes AD to AA in three successive reactions (2, 3, and 8). CYP71AV1 is the main rate-limiting enzyme of the committed artemisinin pathway. Cytochrome P450 reductase (CPR) restores the active state of CYP71AV1. 3. AO can be produced by CYP71AV1/CRP enzymatic system, and alcohol dehydrogenase 1 (ADH1) can also make AO using AOH as a substrate. 4. AO is utilized by the enzyme artemisinic aldehyde 111(13) reductase (DBR2), which generates DHAO, as well as the third reaction of CYP71AV1 (7), which generates AA. 5. Aldehyde dehydrogenase 1 (ALDH1) converts DHAO to DHAA. 6–7. The conversion of DHAA into ART through the intermediate DHAAHP is postulated to be non-enzymatic, but induced only by ultraviolet light and oxygen. 8. The third reaction of CYP71AV1 converts AO into AA, diverting the pathway from producing ART and DHAA. 9. AA can be converted non-enzymatically into AB. 10. AB can be converted into AT. 11. DHAAHP may also generate DART. Once extracted from plants and purified, ART is derivatized into dihydroartemisinin (dART), then into other antimalarial drugs (e.g., artemether and artesunate), which the body metabolizes to the bioactive DART.

trichome density in A. annua. Trichome development and ART biosynthesis have been linked through the transcription factor TAR1 and its role in upregulating ADS, CYP71AV1, and ART biosynthesis (Tan et al., 2015) and several other genes, reviewed elsewhere (Xiao et al., 2016). The literature also suggests that reactive oxygen species (ROS) triggered by stress may lead to the transformation of DHAA into ART, although that has not yet been shown in planta through direct correlation between quantified ROS build-up and the increased conversion of DHAA into ART. Also, although potassium deficiency stress increased ART leaf concentration by 75%, it had no apparent effect on the concentrations of either DHAA and AA (Ferreira, 2007). Thus, the simple effect of photooxidation seems more plausible at this point, although it is unknown how DHAA is converted into ART as the plant metabolism shuts down during senescence and drying. For instance, compared to freeze dried sub-samples, shade, oven, and sun dried shoots had significantly higher concentrations of ART and decreased concentrations of DHAA and AA, and sun drying was more efficient in converting DHAA into ART (Ferreira and Luthria, 2010). Although it is currently debatable whether the last steps of the pathway (leading from DHAA to ART and deoxyartemisinin (dART) through dihydroartemisinic acid hydroperoxide (DHAAHP) are enzymatic or not, if selection and breeding focus exclusively on optimizing ART yields (and neglects DHAA), the potential to increase ART production in planta through the conversion of DHAA into ART during post-harvest drying will be wasted.

In order to validate their production potential, promising elite germplasm should be tested in areas with similar edaphoclimatic conditions of potential commercial production. A new cultivar ("Artemis R ") developed in Conthey, Switzerland (46◦ 13 ′ N 7 ◦ 17 ′ E, elevation 485 m above sea level - asl), and another ("A3") developed by the University of Campinas, Brazil (22◦ 48′ S, 47◦ 07 ′W, 749 m asl) have been tested in Kenya, Tanzania, and Nigeria, and produced from 0.7 to over 1% ART DW. However, a Chinese genotype from Chongqing, China (29.43◦N, 106.91◦E, 238 m asl) was grown only in China and Vietnam until its field trials in West Virginia (37◦ 45′N 80◦ 50′W, 890 m asl) reported here. Further selections of the Brazilian cultivar better adapted to lowland humid tropics resulted in plants with higher ART (1% w/w) and leaf dry biomass (3 ton/ha) than the Chinese, Indian, and U.S. clones (0.4–0.5% ART, 1.5–2 ton/ha of dry leaf biomass) in Calabar, Nigeria (4.96◦N 8.3◦E, 50 m asl) (Brisibe et al., 2012). To our knowledge, there are no current breeding programs engaged in producing A. annua genotypes that are rich in both ART and DHAA, or with a less variable ART yield from plant to plant.

The aims of this work are to: (1) show the seasonal and differential accumulation of ART, DHAA, and AA of elite Brazilian, Chinese, and Swiss cultivars cultivated in a West Virginian field of similar latitude and altitude to Chongqing (China), where approximately 90% of the world's A. annua is cultivated for ART extraction; (2) provide evidence that a high-ART genotype can also be high in DHAA, which should be considered as an important sesquiterpene and biochemical marker that can be used in the selection and breeding to produce new high-ART A. annua lines.

### MATERIALS AND METHODS

#### Plant Material and Field Cultivation

The three main high-ART cultivars used in this study to evaluate seasonal and individual accumulation of sesquiterpenes, were donated by Mediplant (Switzerland, cv. Artemis R ), Centro Pluridisciplinar de Pesquisas (CPQBA-Sao Paulo, Brazil, cv. 3M), and Holley Pharma (Chongqing, China, cultivar not identified). Another cultivar (Sandeman Seeds, UK) with high concentration of AA, but low ART and DHAA, was donated by a colleague (Dr. Dae-Kyun Ro). Plants from Brazilian, Chinese, and Sandeman cultivars were started from seeds, while a Swiss selection was cloned in a Quonset greenhouse under long-day photoperiod before transferring to the field. From here on, these genotypes will be mainly referred to as Brazilian, Chinese, Sandeman, and Swiss. For all field experiments reported here, all genotypes were transferred to the field in the first week of June 2006, 2007, or 2009 on an Appalachian soil (Gilpin silt loam—fine-loamy, mixed, mesic Typic Hapludults) at the Richmond School Farm, Beaver, WV (37◦ 45′N 80◦ 50′W, 890 m asl) and provided twice with 45 kg N, 20 kg P, and 37 kg K per hectare during the five-six months of cultivation (June to October/November). Soil pH was 5.8 and plants were irrigated for the first month, until established, with further irrigation only provided by rain. Soil analysis is provided elsewhere (Ferreira, 2007). To determine the seasonal accumulations of ART, DHAA, and AA, Chinese and Brazilian plants were field cultivated in 2006 and 2007, respectively, with three seed-generated plants of each genotype sampled bi-weekly (non-destructively) throughout the season. Seeds of the Swiss genotype (Artemis R ), were cultivated in West Virginia and quantified for ART, DHAA, and AA. One selection (named MDP-11) was cloned in 2006 to generate enough plants for the determination of seasonal peak ART. Thus, for the Swiss genotype, three plants of the cloned MDP-11 were harvested at each collection date, the whole plant was oven dried, and a dry sample from the bottom, middle, and top part of each plant was pooled for HPLC-UV analysis. To evaluate the natural segregation of ART, DHAA, and AA in both Swiss and the Brazilian genotypes, approximately 100 plants generated from seeds were transferred to the field, and 55–65 plants were harvested at random on August 24, 2007 (Brazilian) and August 18/19, 2008 (Swiss). Dried leaves were separated from stems, ground to 0.5 mm particle size in a Wiley mill, and saved in a −20◦C freezer until extraction for HPLC-UV analysis of underivatized ART and its precursors (Ferreira and Gonzalez, 2009).

#### Extraction of ART, its Precursors, and HPLC Analysis

ART, deoxyartemisinin (co-synthesized with ART), DHAA, and AA were extracted from 500 mg of A. annua dry leaf samples, refluxed with 50 mL of petroleum ether (45◦C) for 1 h, transferred to beakers and left to dry overnight in a fume hood. Next day, samples were reconstituted in 20 mL of acetonitrile (two washes of 10 mL each), filtered through a 0.45µm nylon filter attached to a 10-mL luer-lock syringe and transferred to a 20-mL scintillation vial. Samples were transferred to 1.8 mL HPLC vials and 10 µL were injected by an HPLC auto-sampler into the system (Agilent 1100 series). ART, DHAA, and AA were quantified by HPLC-UV (Ferreira and Gonzalez, 2009). Standards of ART were purchased from Sigma/Aldrich (sigmaaldrich.com) and standards of DHAA and AA were donated by Amyris (Amyris.com). To better follow results and discussion, and the roles of ART, DHAA, and AA and their competing pathways, see **Figure 1**.

#### Relationship Among Sesquiterpenes in *A. annua*

The mathematical model formula used to evaluate ART leaf concentration (ART%) in relation to shoot concentration of AA (AA%) in **Figures 4**, **5** is shown in Equation 1, and is equivalent to a log-normal distribution fit, where ln = Napierian logarithm and e is Euler number =2.1718.

$$ART(\%) = \frac{0.36 \pm 0.06}{AA(\%)} \times e^{\left(-0.5 \times \left(\frac{\ln\left(\frac{AA(\%)}{0.34 \pm 0.03}\right)}{\ln(0.19 \pm 0.01)}\right)^2\right)} \tag{1}$$

#### RESULTS

#### Differential Seasonal Sesquiterpene Accumulation in *A. annua*

In West Virginia, the seasonal peak of ART concentration for the Brazilian, Chinese, and Swiss cultivars occurred between the end of August and the first week of September, declining steadily thereafter (**Figure 2**). All Plants of these three cultivars were high in ART and DHAA, but low in AA. The Brazilian cultivar had lower DHAA than ART, the Swiss cultivar had DHAA in similar or slightly higher concentrations than ART, and the Chinese cultivar had higher DHAA than ART. The Chinese cultivar displayed the highest concentrations of DHAA, reaching 1.6% DHAA and 0.95% ART at its seasonal peak (Sept 1). The Swiss plants that were asexually propagated as clones, expectedly showed a small plant-to-plant variation in sesquiterpene content than the Brazilian or Chinese plants generated from seeds (**Figure 2**). At the peak, the Brazilian and Swiss plants produced an average ART concentration of 0.75%, but a few plants reached 1.5% ART (data not shown). The peak for DHAA concentration coincided with ART in the Chinese and Swiss plants, whereas DHAA was lower than ART during most of the season in Brazilian plants. Plants peaked in their DHAA concentration at 0.6% (July 23, first harvest), 1.58% (Sept 1), and 1.25% (Sept 08) for the Brazilian, Chinese and Swiss plants, respectively. The Brazilian, Chinese, and Swiss genotypes were all low in AA, ranging from 0.1-0.2% and remained fairly constant throughout the whole experiment (**Figure 2**).

#### Sesquiterpene Profiling of Different *A. annua* Chemotypes

Due to the phenotypical segregation observed for DHAA accumulation in 2006, when over 50 seed-generated plants of

the Brazilian cultivar were field grown and analyzed (data not shown), three segregating individual plants were cloned and harvested weekly to originate the data in **Figure 2**. Data for each segregating clone (3M-8, 3M-43, and 3M-49) are shown in **Figure 3** regarding the seasonal differences in ART, DHAA, and AA concentrations. Relative concentrations of ART, DHAA, and AA were clearly different among the three genotypes throughout the growing season (**Figure 3**). ART concentration ranged from 0.2 to 0.9% with maximum and minimum concentrations on August 28th and October 30th, respectively. All three plants peaked in ART concentration on August 28th. Opposite to the Chinese and Swiss genotypes, DHAA content starts high and decreases throughout the growing season in all three Brazilian (3M) clones (**Figure 3**). AA content was largely unchanged in each of the genotypes throughout the growing season. Genotypes 3M-8 and 3M-43 were most similar in their chemical profiles for

all three sesquiterpenes, whereas the 3M-49 displayed lower ART and DHAA content and higher AA content as compared to the other two genotypes for most of the growing season (**Figure 3**).

In 2008, 50 seed-originated Brazilian and Swiss plants were cultivated in a West Virginia field and in 2009, some seedoriginated Sandeman plants were cultivated in the same field. The data for the three elite cultivars and the Sandeman cultivar were grouped for shoot artemisinin concentration (ART %) and artemisinic acid concentrations (AA%) and evaluated through a three-parameter non-linear fit (**Eq. 1**).

The data were also grouped for leaf ART% and leaf DHAA% and presented as both Pearson (r, n = 95) and linear coefficient of determination (R 2 , n = 95) (**Figure 4**). When these 95 sexually propagated (37 Brazilian, 48 Swiss, and 10 Sandeman) plants were randomly collected on Aug 18–19, 2008 (Brazilian and Swiss) and 2009 (Sandeman), the Brazilian plants ranged from 0.25 to 1.3% ART, 0.2–1.5% DHAA, and 0.02–0.12 AA, whereas the Swiss plants ranged from 0.58–1% ART, 0.9–1.6% DHAA, and 0.1–0.43 AA, and the Sandeman plants from 0.07–0.2% ART, 0.01–0.12% DHAA, and 0.41–1.18% AA (**Figure 4**). Data modeling analysis of these 95 plants of four different genotypes (based on leaf sesquiterpene concentrations) resulted in highly significant Pearson (r = −0.56∗∗∗ , n = 95) and a linear coefficient of determination (or fit) of R 2 = 0.31∗∗∗. However, a higher, and highly significant, non-linear fit of R 2 = 0.57∗∗∗ was obtained for the observed vs. predicted relationship between ART% vs. AA% (**Figure 4A**) according to the model in **Eq. 1**. The root mean square error for the ART% predictions, or the diversion of the observed data from the prediction by the model presented in **Eq. 1** was only 0.18%. Interestingly, the range of AA concentration in the sexually propagated progenies was 0.02– 0.12% for Brazilian, 0.1–0.43% for Swiss, and 0.41–1.18% for Sandeman plants (**Figure 4A**).

When correlating ART to DHAA in the three elite cultivars plus the Sandeman plants, both Pearson (r = 0.74∗∗∗ , n = 95) and the linear fit (R 2 = 0.54∗∗∗ , n = 95) were highly significant (**Figure 4B**). There was no significant correlation between ART and either AA or DHAA, or between AA and DHAA, for the sexually-propagated Sandeman plants (correlations not shown). The implications of these comparisons across germplasms is explored in the Discussion section.

The several years of HPLC-UV (Ferreira and Gonzalez, 2009) quantification of ART, DHAA, and AA in greenhouse and field-grown plants allowed for a selection of several Brazilian and Swiss clones with contrasting concentrations of all three sesquiterpenes, including a genotype from Sandeman Seeds (UK) that produces over 1% AA and 0.2% or less of either ART or DHAA (**Figure 5**). The Swiss clones were relatively high in DHAA and ART as compared to the Brazilian and Sandeman plants (**Figure 5**). For these selected genotypes, DHAA presented significant and high correlation with ART, while AA did not show a significant correlation with ART (**Figures 5B,C**). Although the Brazilian clones were lower in DHAA than the Swiss clones, three clones that were high in DHAA (3M13, 3M39, and 3M85) were also high in ART (**Figure 5A**).

#### DISCUSSION

Our field data revealed that, regardless of genotype, plants from different origins planted in the same location will reach their ART and DHAA peaks at the same time in the season (**Figure 2**). The ART peak was not related to flowering and agrees with reports that ART in Swiss plants peaked at the end of August also in Conthey, Switzerland, independently of the physiological state for the hybrid cultivar Artemis R (Delabays et al., 2001). Our results suggest that plants of elite germplasms, regardless of their origins, allocated more resources toward DHAA and ART accumulation, while AA remained below 0.43%, and stable, during the whole season. DHAA biosynthesis was favored in Chinese, similar to ART in Swiss, and lower than ART in Brazilian plants, while AA biosynthesis (opposite to ART or DHAA) was favored in Sandeman plants. Results