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Article

Comparative Transcriptome Analysis of High and Low Thujone-Producing Artemisia argyi Reveals Candidate Genes for Thujone Synthetic and Regulatory Pathway

by
Tingting Zhao
†,
Changjie Chen
†,
Jinxin Li
,
Dandan Luo
,
Yuhuan Miao
,
Chun Gui
,
Qi Liu
and
Dahui Liu
*
Key Laboratory of Traditional Chinese Medicine Resources and Chemistry of Hubei Province, Hubei University of Chinese Medicine, Wuhan 430065, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2023, 9(2), 232; https://doi.org/10.3390/horticulturae9020232
Submission received: 5 January 2023 / Revised: 1 February 2023 / Accepted: 7 February 2023 / Published: 9 February 2023
(This article belongs to the Section Medicinals, Herbs, and Specialty Crops)
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Abstract

:
Artemisia argyi Levl. et Van (A. argyi) is a traditional medicinal plant, which is widely used in health, food and medicine. Thujone is an important cyclic monoterpene derivative in the volatile oil of A. argyi leaves with multiple efficacy. Although the thujone synthetic pathway has been preliminarily analyzed in very few species, genes related to the thujone content in A. argyi leaves remain largely unknown. In this study, we identify candidate genes involved in the synthesis and regulation of thujone content in A. argyi leaves by the comparative transcriptome analysis of two group materials with high and low thujone content. A total of 89 candidate genes related to thujone content are identified including one gene involved in the mevalonate pathway, three genes involved in the methylerythritol phosphate pathway, 19 genes involved in the metabolic process from geranyl pyrophosphate to thujone (four b-terpene synthase, five cytochrome P450, five dehydrogenase, and five reductase-encoding genes) and 66 transcription factor-encoding genes. Taken together, our results provide valuable gene resources for further analyzing the synthetic and regulatory pathway of thujone in A. argyi.

1. Introduction

Artemisia argyi (A. argyi) is a perennial herb belonging to Artemisia in the composite family. Due to its rapid growth and strong stress resistance, A. argyi germplasm resources are widely distributed in the world. Previous studies demonstrate that different A. argyi varieties have rich variation in plant height, leaf shape, moxa yield, the content of flavonoids, phenolic acids, and volatile components [1,2]. For example, the representative A. argyi varieties from Qichun County usually contain more thujone than those from other regions [3]. Nevertheless, the content of moxa in the representative A. argyi varieties from Ningbo City is commonly lower than A. argyi varieties from other regions [2]. The genetic diversity among different A. argyi germplasm resources provides rich materials for investigating the causes of specific traits in characteristic varieties.
The leaves of A. argyi have high medicinal and health value. The moxa extracted from A. argyi leaves is the raw material of moxibustion. In addition, A. argyi leaves are rich in volatile oils, flavonoids and phenolic acids, endowing A. argyi with multiple effects including warming the meridian and stopping bleeding, dispelling dampness and cold, relieving cough and asthma, analgesic and anti-inflammatory, etc. [4,5,6,7,8]. The volatile oil from A. argyi leaves is composed of more than 100 components including monoterpenes and their derivatives, sesquiterpenes and their derivatives, aldehyde, ketones, and phenols [9,10]. Among them, monoterpenoids are the most important class of compounds, and thujone is a cyclic monoterpene derivative. Related pharmacological experiments demonstrate that thujone has multiple effects such as anti-tumor, anti-cancer cell metastasis, anti-diabetes, and anti-reproductive toxicity. Thujone injection to the abdominal cavity of tumor-transplanted mice significantly inhibits the formation of tumor nodules in the lung and further improves the survival rate of model mice [11]. In addition, thujone is also considered as a pivotal pharmacodynamic component of anti-diabetes botanical medicines, and it can reduce the occurrence of diabetes by alleviating palmitate-induced muscle insulin resistance [12]. Moreover, low-dose thujone improves the ability of DNA damage repair of in vitro cells. Therefore, thujone is also a promising biological antimutagenic agent [13].
Geranyl pyrophosphate (GPP) is a common precursor for the synthesis of monoterpenes and their derivatives. GPP is mainly produced by cytoplasmic mevalonic acid (MVA) and plastidial methylerythritol phosphate (MEP) pathways [14,15]. In the MVA pathway, two molecules of acetyl-coenzyme A (CoA) are first condensed to acetoacetyl-CoA by acetyl-CoA C-acetyltransferase (AACT) [16]. Thereafter, acetoacetyl-CoA is catalyzed to generate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) under the action of HMG synthase (HMGS). Then, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) reduces HMG-CoA to produce MVA. MVA is subsequently phosphorylated to MVA 5-diphosphate by MVA kinase (MK) and phospho-MVA kinase (PMK). Ultimately, MVA diphosphate decarboxylase (MPDC) takes MVA 5-diphosphate as a substrate to generate isopentenyl diphosphate (IPP) in an ATP-dependent manner [17,18]. IPP can be isomerized to dimethylallyl diphosphate (DMAPP) by IPP Δ-isomerase (IPPI). In the MEP pathway, D-glyceraldehyde 3-phosphate and pyruvate are initially condensed to 1-deoxy-D-xylulose 5-phosphate (DXP) under the catalyzation of 1-deoxy-D-xylulose 5-phosphate synthase (DXS) [19]. DXP is then isomerized and reduced to MEP by DXP reductoisomerase (DXR). Thereafter, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT) converts MEP to 4-(cytidine 5′-diphospho)-2-C-methyl-Derythritol (CDP-ME). Subsequently, CDP-ME is phosphorylated to 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-Derythritol (CDP-ME2P) by 4-(cytidine 5′-diphospho)-2-C-methyl-Derythritol kinase (CMK). Afterwards, 2-C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MDS) catalyzes the conversion from CDP-ME2P to 2-C-methyl-Derythritol 2, 4-cyclodiphosphate (MEcPP). Then, MEcPP is reduced to 4-hydroxy-3-methylbut-2-enyldiphosphate (HMBPP) under the action of HMBPP synthase (HDS), and HMBPP is finally reduced to a mixture of IPP and DMAPP by HMBPP reductase (HDR) [20]. Finally, GPP synthase (GPPS) catalyzes the formation of GPP using IPP and DMAPP as the substrate [21].
The synthetic pathway of thujone has been reported in very few species. In Salvia officinalis Linn., four steps are required from substrate GPP to generate thujone in total. GPP is first catalyzed to form sabinene under the action of b-terpene synthase (b-TPS). Sabinene undergoes hydroxylation to produce sabinol under the catalyzation of cytochrome P450. Sabinol is then oxidized to generate sabinone by dehydrogenase, and sabinone is ultimately reduced to thujone by reductase [22,23]. Moreover, similarly, the thujone synthetic pathway is also reported in Thuja plicata D. Don, indicating that the thujone synthetic pathway may probably be conserved in different species [24].
Although the thujone synthetic pathway has been preliminarily analyzed and there also have been some transcriptome studies on A. argyi related to terpene or terpenoid synthesis, the thujone synthetic and regulatory pathway is unknown due to a lack of molecular research in A. argyi [25,26,27,28,29]. In this study, we determine the thujone content in a series of A. argyi germplasm resources and screen four varieties with high thujone content and three varieties with low thujone content, respectively. Candidate functional genes involved in the thujone metabolic pathway in A. argyi are identified by a comparative transcriptome analysis of materials with different thujone content. The DEGs were then analyzed to identify candidate transcription factors that regulate the metabolic process of thujone. Our results provide valuable gene resources for further analyzing the thujone synthetic and regulatory pathway in A. argyi.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

A total of 90 A. argyi varieties used in this study were collected from different regions of China during 2018 to 2020 and cultivated under natural conditions in Wuhan (30.45° N, 114.27° E) Hubei Province, China. The research was conducted on 8 June 2022. The detailed information about the collection location is shown at Table S1.

2.2. Headspace Solid-Phase Microextraction Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS)

For HS-SPME-GC-MS, five biological repeats were performed for each sample. First, 0.1 g of fresh A. argyi leaves collected from the middle of the plant was accurately weighed, and the sample was placed in a 20 mL headspace bottle equipped with a Teflon rubber pad. Then, the aged extraction head was inserted into the headspace bottle after sealing. Volatile components in the sample were extracted in the headspace at 80 °C for 40 min. Subsequently, volatile components were entered into the GC-MS system for separation and detection after being desorbed at the injection port at 250 °C for 3 min with a helium flow of 1 mL/min onto the GC column in splitless mode.
The chromatographic column used was TG-1701MS with 60 m × 0.53 mm × 1 µm. The GC oven temperature was programmed starting at 40 °C for 2 min; then, it increased at a rate of 10 °C/min to 116 °C and was kept for 2 min, after which we raised the temperature to 130 °C at the rate of 10 °C/min and maintained it at 130 °C for 2 min. Subsequently, we raised the temperature to 140 °C at a rate of 1 °C/min and maintained it at 140 °C for 2 min, after which we raised the temperature to 150 °C at a rate of 3 °C/min and maintained it for 3 min. Last, we increased at a rate of 5 °C/min to 250 °C and maintained it for 5 min. The carrier gas was high-purity helium with a flow rate of 0.7 mL/min. Using the EI ionization source, the column effluent was ionized by electron impact at 70 eV and a temperature of 280 °C. The full scan mode was adopted to collect data with the scanning ranging from 40 to 600 amu, the transmission line temperature was set to 230 °C, and the solvent was delayed 3 min.

2.3. RNA-Seq Analysis

A total of seven A. argyi varieties including four high thujone content and three low thujone content varieties were used for RNA sequencing. The A. argyi leaves located in the middle of the plant were collected and immediately frozen in liquid nitrogen. The total RNA for RNA-seq analysis was extracted using TRIzol reagent. Three biological repeats were performed for each sample. The RNA library construction and sequencing were performed by Illumina sequencing technology. The expression levels of genes were calculated using RSEM (http://deweylab.biostat.wisc.edu/, accessed on 18 September 2022), and the fragments per kilobase million (FPKM) value was obtained. We used DESEQ (2.0) to identify DEGs between these two groups of A. argyi materials with high and low thujone content with a standard of log2FC > 1 and FDR (q value) < 0.01. The heatmaps of gene expression based on RNA-seq analysis were drawn by the R language software.

3. Results

3.1. Thujone Content Has Rich Variation in A. argyi Varieties

In this study, we collected 90 A. argyi varieties in China, and the contents of thujone in A. argyi leaves were determined by headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) (Figure 1a–g). The results showed that the thujone contents were various in different A. argyi varieties, ranging from 0.00% to 1.58% (Figure 1h). We screened seven extreme phenotypic materials in thujone including four varieties with high thujone content and three varieties with low thujone content, respectively. The contents of thujone in the leaves of four high A. argyi varieties were 1.58 mg/g, 1.52 mg/g, 1.08 mg/g, and 0.93 mg/g, respectively (Figure 1a–d). However, the contents of thujone were below the detectable range in the leaves of three low A. argyi varieties (Figure 1e–g).

3.2. Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Classification and Enrichment Analysis of Differentially Expressed Genes (DEGs) between Two Groups of Thujone Content High and Low Thujone-Producing Materials

To explore the mechanisms underlying different thujone content in different A. argyi varieties, we performed RNA-sequencing (RNA-seq) analysis to identify DEGs in the condition of log2FC > 1 and FDR (q value) < 0.01 between these two groups of high and low thujone-producing materials. A total of 3752 DEGs were identified (Figure 2a). Among these 3752 DEGs, 2397 were upregulated and 1355 were downregulated in A. argyi varieties with high thujone content compared to those with low thujone content, respectively. In addition, the A. argyi varieties with high thujone and low thujone content were clustered to two categories based on DEGs between these two groups, suggesting the obvious difference in expression profile between these two groups (Figure 2b). We then conducted the KEGG pathway classification of upregulated DEGs, and there were 29 genes in KEGG pathways related to the terms of terpenoids and polyketides metabolism (Figure S1). Moreover, there were terpenoid backbone and monoterpenoid biosynthesis in the top 20 significantly enriched pathways of upregulated DEGs (Figure S2), suggesting that the metabolism of monoterpenoids was probably different in these two groups of A. argyi leaves.

3.3. AACT, DXS, and DXR Are Three Main DEG Families of MVA and MEP Pathways in High and Low Thujone-Producing A. argyi Varieties with Different Thujone Content

The terpenoid backbone IPP and its isomer DMAPP were mainly synthetized by cytoplasmic MVA and plastidial MEP pathways. We then investigated the expression levels of genes involved in these two pathways. In the MVA pathway, there were seven gene families whose transcripts could be detected by RNA-seq strategy including AACT, HMGS, HMGR, MK, PMK, MPDC, and IPPI. Comparative transcriptome analysis showed that there was only one DEG (AY062935-RA) encoding AACT between these two groups of A. argyi varieties with different thujone content (Figure 3a).
We further detected the expression levels of related genes participating in the MEP pathway. The results showed that the transcription of seven gene families could be detected by RNA-seq analysis, namely DXS, DXR, MCT, CMK, MDS, HDS, and HDR. Moreover, the expression levels of genes in the MEP pathway were universally higher than those in the MVA pathway, suggesting that IPP and DMAPP were mainly synthesized by the MEP pathway in the plastid in A. argyi leaves. By comparison, two genes (AY291248-RA and AY236918-RA) encoding DXS and one gene (AY028159-RA) encoding DXR were significantly downregulated in the varieties with low thujone content compared to those with high thujone content (Figure 3b). In addition, there was only one geranyl diphosphate synthase (GPPS)-encoding gene (AY284062-RA) whose expression could be detected by transcriptome analysis, and the expression level of AY284062-RA had no significant difference between these two groups of A. argyi varieties with high and low thujone content (Figure 3c). Taken together, these results demonstrated that the differential expression of AACT, DXS, and DXR probably mainly gave rise to the different GPP content in these two groups of A. argyi varieties with different thujone content, which was the precursor of monoterpenoids biosynthesis including thujone.

3.4. Identification of Candidate TPS, Cytochrome P450, Dehydrogenase, and Reductase Encoding Genes Involved in the Thujone Synthetic Pathway

To further investigate candidate genes encoding four crucial protein families (b-TPS, cytochrome P450, dehydrogenase, and reductase) involved in the thujone synthetic pathway (Figure 4a), we searched for upregulated DEGs in varieties with high thujone content compared to varieties with low thujone content. A total of 19 genes were obtained, including four (AY071840-RA, AY071839-RA, AY068699-RA, and AY175386-RA), five (AY264028-RA, AY065675-RA, AY265474-RA, AY187501-RA, and AY065674-RA), five (AY183353-RA, AY183352-RA, AY065617-RA, AY186905-RA, and AY238343-RA), and five (AY061574-RA, AY285957-RA, AY061577-RA, AY061580-RA, and AY258844-RA) genes encoding b-TPS, cytochrome P450, dehydrogenase, and reductase, respectively (Figure 4b and Figure S3). The results indicated that these 19 genes might probably play significant roles in the thujone synthetic pathway in A. argyi.

3.5. Identification of Positive and Negative Candidate Transcription Factors Related to Thujone Accumulation

Previous studies indicate that common transcription factors including MYB, basic helix–loop–helix (bHLH), APETALA2/ethylene-responsive factor (AP2/ERF), basic leucine zipper (bZIP), WRKY, and MADS (MADS-box) participate in the metabolic regulation of terpenoids [25,29]. DEGs encoding these several kinds of transcription factors were further searched in these two groups of A. argyi varieties with different thujone content. In the upregulated DEGs in A. argyi varieties with high thujone content compared to varieties with low thujone content, we identified 17 MYB (AY185566-RA, AY070650-RA, AY270249-RA, AY265305-RA, AY028204-RA, AY278948-RA, AY238121-RA, AY258198-RA, AY029488-RA, AY270965-RA, AY281353-RA, AY150873-RA, AY187319-RA, AY269099-RA, AY030312-RA, AY266842-RA, and AY298916-RA), eight bHLH (AY147223-RA, AY294075-RA, AY151622-RA, AY148915-RA, AY266237-RA, AY279810-RA, AY263979-RA, and AY240592-RA), nine AP2/ERF (AY176540-RA, AY067959-RA, AY066733-RA, AY033761-RA, AY181769-RA, AY033207-RA, AY291402-RA, AY146207-RA, and AY027880-RA), two bZIP (AY064754-RA and AY066804-RA), three WRKY (AY062218-RA, AY061543-RA, and AY031762-RA), and four MADS (AY236939-RA, AY062150-RA, AY268749-RA, and AY062149-RA) encoding genes, respectively (Figure 5a). The results suggested that these transcription factors might probably regulate the accumulation of thujone positively in A. argyi leaves.
A total of 23 downregulated DEGs encoding these transcription factors were identified, including nine MYB (AY183373-RA, AY151374-RA, AY162657-RA, AY152647-RA, AY240631-RA, AY269968-RA, AY264455-RA, AY031023-RA, and AY273511-RA), two bHLH (AY182149-RA and AY056710-RA), five AP2/ERF (AY298833-RA, AY058375-RA, AY076875-RA, AY297131-RA, and AY176857-RA), two bZIP (AY274014-RA and AY297062-RA), two WRKY (AY163285-RA and AY069223-RA), and three MADS (AY281356-RA, AY030926-RA, and AY028314-RA) (Figure 5b). Taken together, these results indicated that a series of transcription factors probably participated in regulating the metabolic process of thujone in A. argyi leaves.

4. Discussion

Thujone is an important monoterpene derivative in the volatile oil of A. argyi leaves, which plays significant roles in the pharmacological effects of volatile oil. Therefore, identifying candidate functional genes and related regulatory factors involved in the accumulation of thujone content is crucial to analyze the thujone synthetic pathway and regulate thujone content directionally in A. argyi leaves. With the rapid development of high-throughput sequencing technology, the omics technology is more and more widely used in the study of the synthetic pathway of secondary metabolite in medicinal plants [30,31,32]. For example, candidate genes involved in C-glucosylquinochalcone biosynthesis are identified by integrated transcriptome and metabolomics analysis of safflower varieties with different colors [33]. The comparative metabolome and transcriptome analysis of cucumber with dark green skin and light green skin cucumber provide valuable clues for identifying the related chlorophyll and anthocyanin metabolism pathway participating in the formation of cucumber fruit skin color [34]. There are also some reports revealing candidate genes related to terpenoid biosynthesis in A. argyi by full-length transcriptome and second-generation transcriptome analysis [25,26,27,29]. However, A. argyi has abundant terpenoids, and these studies can only provide very general gene resources in terpenoid biosynthesis. Therefore, genes related to specific terpene components are still unknown. The A. argyi germplasm resources are very rich, and moreover, the thujone content has great variation in different varieties, which provide tremendous potential for analyzing the thujone synthetic pathway and identifying related candidate genes in A. argyi by the strategy of omics.
Combined with the determination of thujone content in A. argyi leaves of different varieties, we select seven thujone content phenotypic materials including four high varieties and three low varieties. A comparative transcriptome analysis of thujone content in high and low thujone-producing materials is then conducted to identify candidate genes involved in the thujone synthetic pathway in A. argyi leaves. A total of 19 functional protein encoding genes are identified by comparative transcriptome analysis of the two groups of materials with high and low thujone content. These results provide valuable gene resources for further functional verification and analyzing the thujone synthetic pathway systematically in A. argyi.
In addition to related functional genes, transcription factors also play crucial roles in the synthetic and metabolic regulation of terpenoids including MYB, bHLH, AP2/ERF, bZIP, WRKY, and MADS [35,36,37,38]. In Glycine max (Linn.) Merr., GaWRKY1 regulates the biosynthetic pathway of sesquiterpene [39]. The Artemisia annua L. AaWRKY1 activates the expression of Amorpha-4, 11-diene synthase (ADS) by binding to the promoter of ADS to promote the synthesis of artemisinin [40]. Moreover, AaERF1 and AaERF2 also positively regulate the metabolism of artemisinin in A. annua [41]. In Phalaenopsis orchids, the overexpression of PbbHLH4 increases the production of monoterpenoid significantly [42]. In addition, the transcription factor CrWRKY1 regulates the biosynthesis of terpenoid indole alkaloid positively in Catharanthus roseus (L.) G. Don [43]. In addition to the above positive regulatory factors, there are also some negative regulatory factors responsible for the metabolism of terpenoids in plants. In C. roseus, bZIP transcription factors CrGBF1 and CrGBF2 negatively regulate the synthesis of the terpenoid indole alkaloid by suppressing the expression of the strictosidine synthase (Str) encoding gene [44]. The R2R3-MYB transcription factor MsMYB represses terpene biosynthesis in spearmint by suppressing the expression of a geranyl diphosphate synthase large subunit-encoding gene [45]. Previous studies reveal that bHLH transcription factors may probably participate in the terpenoid biosynthesis in A. argyi [28]. In this study, we also screen 66 candidate transcription factor-encoding genes including MYB, bHLH, AP2/ERF, bZIP, WRKY, and MADS that probably regulate the thujone content in A. argyi. The results provide valuable information for the metabolic regulation of thujone.
Related studies demonstrate that terpenoids play significant roles in plant stress resistance and the exchange of information [37,38,46,47,48,49]. The member of the Cupressaceae family of conifers T. plicata has high resistance to insect and fungal damage due to its effective chemical defenses [24]. Recent studies in the Chrysanthemum genus indicate that the volatile component thujanol gives rise to the aphid resistance of plants [50]. As a monoterpenoid volatile component, thujone may probably play an important role in stress resistance and the intraspecific information exchange of A. argyi. The analysis of the thujone synthetic pathway and the excavation of excellent gene resources may probably lay the foundation for cultivating superior A. argyi germplasm resources with stronger resistance based on the content of thujone.
In China, there are four famous genuine producing areas of A. argyi, including Anguo City of Hebei Province, Tangyin County of Henan Province, Qichun County of Hubei Province and Ningbo City of Zhejiang Province. Interestingly, the A. argyi varieties from Qichun County (Qi Ai) usually have higher thujone content than those from the other three genuine producing areas, which is a prominent feature of Qi Ai. High thujone content may probably endow Qi Ai with higher medicinal value compared to other A. argyi varieties. We collect these different A. argyi varieties and plant them in the same place to detect the contents of thujone in various varieties. The contents of thujone varied in different varieties, indicating that genetic factors have crucial roles in the accumulation of thujone [1]. Therefore, the identification of candidate genes involved in the thujone metabolic pathway also plays a significant role in analyzing the genetic connotation of the genuine formation of Qi Ai.

5. Conclusions

In this study, we evaluate a series of A. argyi germplasm resources and screened genetically stable materials with high and low thujone content. By comparative transcriptome analysis of thujone content in high and low thujone-producing materials, we identify candidate functional genes in the thujone synthesis pathway including b-TPS, cytochrome P450, dehydrogenase, and reductase. Moreover, candidate transcription factor encoding genes related to thujone content are also detected. These results provide genetic materials and useful gene resources for further analyzing the thujone synthetic and related regulatory pathway in A. argyi. In the future, we will make efforts to perform a functional verification of these candidate genes and apply these genes to A. argyi breeding.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/horticulturae9020232/s1, Figure S1: KEGG pathway classification of upregulated DEGs in high and low thujone-producing materials with high thujone; Figure S2: The top 20 significantly enriched pathways of upregulated DEGs in high and low thujone-producing materials with high thujone; Figure S3: The expression heatmap of the b-TPS gene subfamily in two groups of A. argyi varieties with high and low thujone content; Table S1: Description of A. argyi varieties in this study.

Author Contributions

Conceptualization, D.L. (Dahui Liu) and T.Z.; validation, D.L. (Dahui Liu) and T.Z.; formal analysis, T.Z.; resources, T.Z. and C.C.; writing—original draft preparation, T.Z.; writing—review and editing, T.Z., C.C., J.L., D.L. (Dandan Luo), Y.M., C.G., Q.L. and D.L. (Dahui Liu). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the grants from the Nature Science Foundation of Hubei Province (2021CFB225), China Postdoctoral Science Foundation (2022M711101), the National Natural Science Foundation of China (32270391), Young Qihuang Scholars Project of the State Administration of Traditional Chinese Medicine, the Young and Middle-aged talents project of the Hubei Provincial Department of Education (Q20212001), and Hubei Provincial Department of Education Excellent Young and Middle-aged Science and Technology Innovation Team Project (T2021008).

Data Availability Statement

The raw data of transcriptome in this study were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/) with GenBank accession number PRJNA914508. The BioSample number of each sample ranges from SAMN32339020 to SAMN32339040, and Sequence Read Archive (SRA) accession number ranges from SRX18799987 to SRX18800007.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Gas chromatogram and the thujone content in high and low thujone-producing materials. (a–g) Gas chromatogram of H1 (a), H2 (b), H3 (c), H4 (d), L1 (e), L2 (f), and L3 (g). The arrows indicate the peak of thujone. (h) The thujone content in seven high and low thujone-producing A. argyi varieties. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
Figure 1. Gas chromatogram and the thujone content in high and low thujone-producing materials. (a–g) Gas chromatogram of H1 (a), H2 (b), H3 (c), H4 (d), L1 (e), L2 (f), and L3 (g). The arrows indicate the peak of thujone. (h) The thujone content in seven high and low thujone-producing A. argyi varieties. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
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Figure 2. The volcano and cluster maps of differentially expressed genes (DEGs) between two groups with high and low thujone content. (a) The volcano map of DEGs between two groups with different thujone content. (b) The cluster maps of seven materials based on DEGs between two groups. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
Figure 2. The volcano and cluster maps of differentially expressed genes (DEGs) between two groups with high and low thujone content. (a) The volcano map of DEGs between two groups with different thujone content. (b) The cluster maps of seven materials based on DEGs between two groups. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
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Figure 3. The expression levels of the MVA pathway, MEP pathway, and GPPS-related genes in materials with high and low thujone content. (a) The expression levels of genes involved in the MVA pathway. The AACT-encoding gene AY062935-RA is differentially expressed in these two groups. (b) The expression levels of genes involved in the MEP pathway. The DXS-encoding genes AY291248-RA and AY236918-RA and the DXR-encoding gene AY028159-RA are differentially expressed between these two groups. (c) The expression level of GPPS (AY284062-RA) in these two groups with different thujone content. DEGs are marked red. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
Figure 3. The expression levels of the MVA pathway, MEP pathway, and GPPS-related genes in materials with high and low thujone content. (a) The expression levels of genes involved in the MVA pathway. The AACT-encoding gene AY062935-RA is differentially expressed in these two groups. (b) The expression levels of genes involved in the MEP pathway. The DXS-encoding genes AY291248-RA and AY236918-RA and the DXR-encoding gene AY028159-RA are differentially expressed between these two groups. (c) The expression level of GPPS (AY284062-RA) in these two groups with different thujone content. DEGs are marked red. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
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Figure 4. The identification of DEGs involved in the thujone synthetic pathway. (a) The proposed thujone synthetic pathway. There are a total of four steps in the metabolic pathway from GPP to thujone. (b) Upregulated genes encoding b-TPS, cytochrome P450, dehydrogenase, and reductase in A. argyi varieties with high thujone content compared to A. argyi varieties with low thujone content. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
Figure 4. The identification of DEGs involved in the thujone synthetic pathway. (a) The proposed thujone synthetic pathway. There are a total of four steps in the metabolic pathway from GPP to thujone. (b) Upregulated genes encoding b-TPS, cytochrome P450, dehydrogenase, and reductase in A. argyi varieties with high thujone content compared to A. argyi varieties with low thujone content. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
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Figure 5. The identification of differentially expressed transcription factor-encoding genes between two groups of materials with different thujone content. (a) Upregulated transcription factor-encoding genes including MYB, bHLH, AP2/ERF, bZIP, WRKY, and MADS in A. argyi varieties with high thujone content compared to A. argyi varieties with low thujone content. (b) Downregulated transcription factor-encoding genes including MYB, bHLH, AP2/ERF, bZIP, WRKY, and MADS in A. argyi varieties with high thujone content compared to A. argyi varieties with low thujone content. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
Figure 5. The identification of differentially expressed transcription factor-encoding genes between two groups of materials with different thujone content. (a) Upregulated transcription factor-encoding genes including MYB, bHLH, AP2/ERF, bZIP, WRKY, and MADS in A. argyi varieties with high thujone content compared to A. argyi varieties with low thujone content. (b) Downregulated transcription factor-encoding genes including MYB, bHLH, AP2/ERF, bZIP, WRKY, and MADS in A. argyi varieties with high thujone content compared to A. argyi varieties with low thujone content. H1, H2, H3, and H4 indicate four varieties with high thujone content. L1, L2, and L3 indicate three varieties with low thujone content.
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Zhao, T.; Chen, C.; Li, J.; Luo, D.; Miao, Y.; Gui, C.; Liu, Q.; Liu, D. Comparative Transcriptome Analysis of High and Low Thujone-Producing Artemisia argyi Reveals Candidate Genes for Thujone Synthetic and Regulatory Pathway. Horticulturae 2023, 9, 232. https://doi.org/10.3390/horticulturae9020232

AMA Style

Zhao T, Chen C, Li J, Luo D, Miao Y, Gui C, Liu Q, Liu D. Comparative Transcriptome Analysis of High and Low Thujone-Producing Artemisia argyi Reveals Candidate Genes for Thujone Synthetic and Regulatory Pathway. Horticulturae. 2023; 9(2):232. https://doi.org/10.3390/horticulturae9020232

Chicago/Turabian Style

Zhao, Tingting, Changjie Chen, Jinxin Li, Dandan Luo, Yuhuan Miao, Chun Gui, Qi Liu, and Dahui Liu. 2023. "Comparative Transcriptome Analysis of High and Low Thujone-Producing Artemisia argyi Reveals Candidate Genes for Thujone Synthetic and Regulatory Pathway" Horticulturae 9, no. 2: 232. https://doi.org/10.3390/horticulturae9020232

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