The cold-induced transcription factor bHLH112 promotes artemisinin biosynthesis indirectly via ERF1 in Artemisia annua.

Basic helix-loop-helix (bHLH) proteins are the second largest family of transcription factors (TFs) involved in developmental and physiological processes in plants. In this study, 205 putative bHLH TF genes were identified in the genome of Artemisia annua and expression of 122 of these was determined from transcriptomes used to construct the genetic map of A. annua. Analysis of gene expression association allowed division of the 122 bHLH TFs into five groups. Group V, containing 15 members, was tightly associated with artemisinin biosynthesis genes. Phylogenetic analysis indicated that two bHLH TFs, AabHLH106 and AabHLH112, were clustered with Arabidopsis ICE proteins. AabHLH112 was induced by low temperature, while AabHLH106 was not. We therefore chose AabHLH112 for further examination. AabHLH112 was highly expressed in glandular secretory trichomes, flower buds, and leaves. Dual-luciferase assays demonstrated that AabHLH112 enhanced the promoter activity of artemisinin biosynthesis genes and AaERF1, an AP2/ERF TF that directly and positively regulates artemisinin biosynthesis genes. Yeast one-hybrid assays indicated that AabHLH112 could bind to the AaERF1 promoter, but not to the promoters of artemisinin biosynthesis genes. Overexpression of AabHLH112 significantly up-regulated the expression levels of AaERF1 and artemisinin biosynthesis genes and consequently promoted artemisinin production.

Introduction

The gene family of basic helix-loop-helix (bHLH) transcription factors (TFs) is one of the largest TF families in plants. bHLH proteins have a typical bHLH signature domain composed of about 60 amino acids. The bHLH domain contains two functionally distinctive regions, the basic region and the HLH region. The basic region, composed of ~15 residues rich in basic amino acids, is involved in DNA binding (Li ; Carretero-Paulet ). The bHLH domain, containing two amphipathic α-helices linked by a variable loop, participates in protein interactions to form homomeric or heteromeric complexes (Heim ). More than 100 bHLH TFs have been identified in Arabidopsis and Oryza sativa (rice). As one of the largest TF families in plants, bHLH TFs play important roles in developmental and physiological processes, such as the development of trichomes and floral organs, hormone and light signaling, diverse stress responses, and regulation of secondary metabolism (Elomaa ; Payne ; Vom Endt ; Morohashi ; Lau and Deng, 2010; Zhou and Memelink, 2016; Wang ). The Lc gene was the first plant bHLH gene identified and is involved in regulating maize tissue-specific anthocyanin biosynthesis (Ludwig ). Since then, many bHLH TFs have been shown to play critical roles in regulating the biosynthesis of anthocyanins in diverse plant species. It is well known that the MYB-bHLH-WD40 complex controls the biosynthesis of anthocyanins and proanthocyanidins, with bHLH being an essential component of the interaction with MYB and WDR (Xie ; Schaart ; Schulz ). Besides regulating anthocyanin biosynthesis, bHLH TFs also control the production of different types of secondary metabolites, including alkaloids and terpenoids (Yamada ; Fabian ). BIS1 is a bHLH TF that is a master regulator that activates the biosynthesis of terpenoid indole alkaloids in Catharanthus roseus (Van Moerkercke ). The biosynthesis of isoquinoline alkaloids is positively regulated by CjbHLH1, a bHLH TF in Coptis japonica (Yamada ), and MYC2 plays a key role in regulating terpenoid biosynthesis in plants such as Arabidopsis and Artemisia annua (Hong ; Shen ).

Artemisia annua, belonging to the family Asteraceae, is a well-known medicinal plant that has been used in traditional Chinese medicine for nearly 2000 years (Klayman, 1993). This important plant is the only known source for the commercial production of artemisinin, a first-line drug used in the treatment of malaria (Nosten and White, 2007; White, 2008; Tu, 2011). Artemisinin production can be promoted by diverse factors such as plant growth regulators and environmental factors. The biosynthesis of artemisinin is tightly regulated due to spatial and temporal expression of artemisinin biosynthesis genes that are genetically controlled by different types of TFs. Plant growth regulators such as methyl jasmonate (MeJA) and abscisic acid (ABA) also control artemisinin biosynthesis (Jing ; Wang ; Caretto ; Xiang ). With the discovery of a series of TFs, molecular mechanisms for the regulation of artemisinin biosynthesis by plant growth regulators have been extensively studied. AaERF1 and AaMYC2, responsive to MeJA, positively regulate artemisinin biosynthesis genes by directly binding to their promoter regions (Yu ; Shen ). AabZIP1 plays a crucial role in regulating artemisinin biosynthesis via ABA signaling, and transactivates ADS and CYP71AV1 by binding to the ABRE motifs in their promoter regions (Zhang ). Meanwhile, AabZIP1 is phosphorylated by AaAPK1 (Zhang ). AaGSW1, a WRKY family TF, integrates JA and ABA signals to positively regulate the biosynthesis of artemisinin (Chen ). Overexpressing genes encoding each of these TFs significantly promotes artemisinin biosynthesis in A. annua. These in-depth studies have facilitated the development of new A. annua varieties that yield higher levels of artemisinin, and they have also suggested that artemisinin biosynthesis may be regulated by different types of TFs under different conditions.

Besides plant growth regulators, environmental factors such as temperature, water, and light also significantly affect the biosynthesis of secondary metabolites including artemisinin (Wang ; Pan ; Soni and Abdin, 2017). Artemisia annua plants are cultivated commercially in warm subtropical and hot tropical regions. Interestingly, the biosynthesis and production of artemisinin can be significantly enhanced when plants are exposed to low temperature (Wallaart ; Yang ; Liu ). For example, artemisinin production was found to be elevated after a night-frost period (Wallaart ). This might be explained by higher levels of 1O2 during a night-frost period, which may trigger the conversion of dihydroartemisinic acid to artemisinin (Wallaart ). When plants are exposed to low temperature conditions, the transcript levels of artemisinin biosynthesis genes are markedly increased (Yang ; Liu ). Coordinated expression of artemisinin biosynthesis genes in response to low temperature strongly suggests the involvement of specific TFs in the regulatory networks underlying artemisinin biosynthesis under such conditions. Cold-induced bHLH TFs, especially inducers of CBF expression (ICEs), play crucial roles in tolerance to low temperature or cold stress (Chinnusamy ; Fursova ; Kurbidaeva ). As key regulators in cold signaling, ICE TFs might be involved in regulating the biosynthesis of secondary metabolites in plants. However, the roles of ICE TFs in regulating terpenoid biosynthesis are unknown. In A. annua, artemisinin biosynthesis is positively regulated by two bHLH TFs, AaMYC2 (Shen ) and AabHLH1 (Ji ). AaMYC2 and AabHLH1, responsive to MeJA and ABA, respectively, activate expression of artemisinin biosynthesis genes by binding to their promoters, suggesting that different members of the bHLH TF family might distinctly regulate biosynthesis under different conditions. It is reasonable to predict that certain cold-induced bHLH TFs might regulate biosynthesis under low temperature conditions.

The genetic map of A. annua was released in 2010, and was constructed using RNA-seq technology (Graham ). On World Malaria Day (April 25th) in 2018, our group released the whole genome of A. annua (Shen ). These data opened the door for genome-wide analyses of the bHLH TF family. In order to determine whether bHLH TFs regulate artemisinin biosynthesis at low temperature, such an analysis was performed to find candidate bHLH members in A. annua. Using comprehensive molecular, biochemical, biotechnological, and phytochemical technologies, the present study revealed that a low-temperature-induced bHLH member (AabHLH112) regulates artemisinin biosynthesis. The discovery of AabHLH112 establishes an improved understanding of the regulation of artemisinin biosynthesis under low temperature conditions.

Material and methods

Plant materials and growth conditions

Different organs were harvested from Artemisia annua plants grown in the field at the botanical garden of Southwest University (Chongqing, China) in September 2015, and immediately stored in liquid nitrogen. Seeds were also harvested from the same botanical garden. Bacteria-free seedlings for genetic transformation were developed according to the methods of Zhang , 2018). Seeds were planted in perlite:vermiculite:pindstrap moss, (1:6:3), irrigated with liquid compound fertilizer, and cultivated in a growth chamber under a 16/8 h light/dark photoperiod at 25±1 °C. When the plantlets reached ~10 cm in height, they were used for low temperature (4 °C) treatment (see below). Nicotiana benthamiana plants were grown under the same conditions as those of A. annua. When the N. benthamiana plants had five pairs of leaves, they were used for subcellular localization and dual-luciferase assays (Zhang , 2018; Lv ).

Bioinformatic analysis

For identification of A. annua bHLH family members, the hidden Markov model (HMM) file of the HLH domain (PF00010) was downloaded (Pfam database, http://pfam.xfam.org/) and used as a query to scan the translated coding sequences from the genome of A. annua using the HMMER program (version 3.1, http://hmmer.org/). Sequences of candidate bHLH genes were retrieved and used for the following analyses. To analyse the expression of A. annua bHLH genes in different tissues, RNA-seq data were obtained from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (Graham ) (SRR019547 for mature leaf trichome; SRR019254 for meristem; SRR019548 for bud trichome; SRR019549 for cotyledon; SRR019546 for young leaf trichome). These data were converted to FASTA format using the SRA toolkit (http://www.ncbi.nlm.nih.gov/Traces/sra/?view=software). The expression values of candidate bHLH genes in different tissues of A. annua were converted to transcripts per kilobase per million using the Salmon program, in which the candidate bHLH genes were used as the query against the RNA-seq data of different tissues of A. annua. Hierarchical cluster analysis was performed using the R package pheatmap, v1.0.10 (https://rdrr.io/cran/pheatmap/). Putative bHLH transcription factor sequences of A. annua and Arabidopsis were aligned using CLUSTALX. A maximum-likelihood phylogenetic tree was reconstructed using the FastTree software (Price ). Protein sequences of bHLH transcription factors from Arabidopsis were obtained from the TAIR website (https://www.arabidopsis.org/).

Low temperature treatment

Plants of A. annua were incubated at 4 °C for the low temperature treatment with untreated plants as the control. Leaves were gathered after 0, 3, 6, 12, or 24 h of treatment. Three biological replicates were used for each experiment. Leaf samples were used for RNA isolation and gene expression analysis of the 15 bHLH TFs for which tissue patterns were tightly associated with those of artemisinin biosynthesis genes, namely AabHLH38, AabHLH62, AabHLH63, AabHLH74, AabHLH77, AabHLH79, AabHLH81, AabHLH93, AabHLH103, AabHLH106, AabHLH112, AabHLH114, AabHLH135, AabHLH145, and AabHLH169.

Gene cloning and sequence analysis

The core sequences of AabHLH106 and AabHLH112 were obtained according to transcriptome and whole-genome data (Shen ), after which the two fragments were subjected to rapid amplification of cDNA ends PCR (RACE-PCR) to obtain the full-length sequence. For AabHLH106 and AabHLH112 cDNA isolation, 3′-RACE/5′-RACE was performed according to the protocol of the RACE System (Clontech, CA, USA). The primers used are listed in Supplementary Table S2 at JXB online. The promoter of AaERF1 was cloned using a pair of primers, pAaERF1F and pAaERF1R, according to the released sequence (JQ513909). The promoters of ADS, CYP71AV1, DBR2, and ALDH1 were isolated according to previous studies (Wang , 2013; Jiang ; Liu ) using primers listed in Supplementary Table S2. The BLASTP algorithm (Johnson ) was used to analyse the similarity between AabHLH112 and ICE proteins from other plant species. Multiple alignments were performed using CLUSTAL W (Thompson ). Promoters were analysed in silica using PLACE (Higo ).

Gene expression analysis

To detect the expression levels of AabHLH106 and AabHLH112 under the low-temperature treatments, total RNA was extracted using an RNA Simple Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. The concentration of total RNA was measured using a NanoDrop spectrophotometer (NanoDrop, Wilmington, NC) and checked by agarose gel electrophoresis. First-strand synthesis of cDNA was carried out using a TIANScript cDNA Kit (TIANGEN) according to the manufacturer’s instructions. RNA (2 μg) was reverse-transcribed with 2 μl reverse primers mix. The first-strand cDNA was used as a template for quantitative real-time PCR (qPCR). The expression levels of AabHLH106 and AabHLH112 were determined by qPCR using an IQ5 thermocycler (Bio-Rad, CA). First single-stranded cDNA was used as the template in 20 μl reaction mixture including 10 μl SYBR Premix ExTaq and 10 pmol of each primer. The primers used are listed in Supplementary Table S2. The qPCR conditions were 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s, 58 °C for 20 s, and 72 °C for 20 s. The β-actin gene was used as a reference for normalization (Wang ; Fu ). To analyse the tissue profile of AabHLH112 expression, total RNAs were isolated from different organs, namely glandular secretory trichomes (GSTs), flower buds, leaves, stems, and roots using an RNA Prep Pure Plant Kit (TIANGEN). The GSTs were collected as described by Teoh . All experiments were performed using three biological replicates. Relative expression levels were calculated using the 2−ΔΔ method (Livak and Schmittgen, 2001). The primers used for qPCR are listed in Supplementary Table S2.

Subcellular localization of AabHLH112

The coding sequence of AabHLH112 was amplified by PCR with primers adding the SalI and BamHI restriction sites and inserted into the pCAMBIA1300-GFP vector in frame with green fluorescent protein (GFP), generating the pCAMBIA-AaBHLH112-GFP plasmid. This plasmid was then introduced into Agrobacterium tumefaciens strain GV3101 and infiltrated into N. benthamiana leaves according to methods described previously (Zhang ). The empty pCAMBIA1300-GFP vector was used as a control. DAPI (4’,6-diamidino-2-phenylindole) was used to specifically stain nuclei (Hu ; Tan ). Fluorescent signals were observed using a Leica TCS SP5 laser confocal scanning microscope.

Dual-luciferase assays

Dual-luciferase assays were performed using methods reported previously (Zhang , 2018). The promoters of ADS (AY528931), CYP71AV1 (FJ870128), DBR2 (KC118523), ALDH1 (KU214890), and AaERF1 (JQ513909) were cloned and inserted into the pGreenII0800-LUC vector upstream of the LUC gene. The generated plasmids, pADS::LUC, pCYP71AV1::LUC, pDBR2::LUC, pALDH1::LUC, and pAaERF1::LUC, were used as reporters. Meanwhile, the coding sequence of AabHLH112 was inserted into the plasmid pHB under control of a double cauliflower mosaic virus (CaMV) 35S promoter to generate the pHB-AabHLH112 plasmid, which was used as an effector. Plasmid pHB-YFP, in which the coding sequence of yellow fluorescent protein (YFP) was under the control of a double CaMV 35S promoter, was used as a negative control. The reporter and effector constructs were introduced into A. tumefaciens strain GV3101. Each reporter strain was co-infiltrated into leaves of N. benthamiana with the effector strain or a negative control. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System (Promega, WI). Each sample was collected from at least five individual plants from the same growth conditions.

Yeast one-hybrid assay

Yeast one-hybrid assays were performed as described previously (Zhang ). AabHLH112 was inserted into the pB42AD vector fused with the GAL4 AD sequence to generate the plasmid pB42AD-AabHLH112. The fragments of each promoter of artemisinin biosynthesis genes were individually inserted into the p178 vector upstream of the lacZ reporter gene. The p178 constructs harboring the promoter fragments were as follows: pADS (–1718~–1127), pADS (–1144~–539), pADS (–562~–1); pCYP71AV1 (–1150~–840), pCYP71AV1 (–844~–560), pCYP71AV1 (–286~–1); pDBR2 (–1651~–1128), pDBR2 (–1651~–1128), pDBR2 (–1219~–448), pDBR2 (–467~–1); pALDH1 (–2004~–1406), pALDH1 (–1490~–934), pALDH1 (–1015~–437), pALDH1 (–538~–1); pAaERF1 (–1384~–922), pAaERF1 (–994~–448), pAaERF1 (–532~–1). The pB42AD-AabHLH112 plasmid was co-transformed into yeast strain EGY48 with each of these constructs. Yeast cells were grown on selective medium deficient in Trp and Ura for 3 d. Five independent yeast cells were transferred into liquid selective medium for 24 h. The yeast cells were then collected by centrifugation and resuspended in sterile water, then dropped onto an X-gal medium agar plate and cultivated at 30 °C for 3 d. Empty pB42AD or p178 plasmids were used as negative controls.

Establishment of transgenic Artemisia annua plants

Plasmid pHB-AabHLH112 was introduced into A. tumefaciens strain EHA105 to generate the engineered bacterial strain. Transgenic plants of A. annua were developed as described previously (Lu ; Zhang , 2018). Micropropagated plants were transferred into pots with substrate as described above and cultured in a growth chamber under a 16/8 h light/dark photoperiod at 25±1 °C. When plants reached 70–80 cm in height, leaves were harvested for molecular and chemical analyses.

Molecular analysis of transgenic plants

To confirm positive transgenic A. annua plants, genomic DNA was isolated from the leaves and used as a template for detecting the DNA fragments of interest by PCR, including the hygromycin resistance gene and the coding sequence of AabHLH112 with partial RUBISCO (rbc) terminator. Three independent transgenic lines were randomly selected and used for further analysis. Total RNA was also extracted from leaves to analyse expression levels of AabHLH112, AaERF1, and four artemisinin biosynthesis genes (ADS, CYP71AV1, DBR2, and ALDH1) by qPCR. The leaves of wild-type A. annua plants were used as a control because the empty pHB vector did not alter artemisinin production (Zhang ). At least three replicates were used in these experiments. The primers are listed in Supplementary Table S2.

Detection of artemisinin and dihydroartemisic acid

Leaves were collected from transgenic and wild-type A. annua plants and ground into a fine powder after drying at 50 °C. Leaf powder (0.2 g) was used to extract artemisinin and dihydroartemisic acid in 25 ml petroleum ether under ultrasound for 30 min in a 50 °C water bath. Leaf debris was removed by filtration, and the extraction solution was evaporated to dryness under vacuum at 50 °C. Finally, the residue was dissolved in 1 ml of methanol. All samples were centrifuged at 12 000 rpm for 10 min, and the supernatants were passed through nitrocellulose filters for HPLC determination as described previously (Xiang ). At least three replications were performed. Standard samples of artemisinin and dihydroartemisic acid were purchased from Sigma-Aldrich.

Statistical analysis

Statistical significance was analysed by Student’s t-test or Duncan’s multiple-range test using SPSS.

Results

Genome-wide analysis of the bHLH transcription factor family in A. annua

As one of the biggest TF families in plants, the bHLH family has 133 members in Arabidopsis (Heim ) and 167 members in rice (Li ). To examine the mechanisms involved in increases in artemisinin biosynthesis under low temperature, we performed a genome-wide analysis to identify bHLH genes using the publicly sequenced genome of A. annua and putative full-length protein sequences. We identified 205 bHLH TFs based on the HMMER scanning program using the hidden Markov model and named these as AabHLH1 to AabHLH222 (Supplementary Table S1). Compared with Arabidopsis and rice, A. annua had many more bHLH TF members. Among the 205 bHLH TFs, 122 were also identified from transcriptome data used to construct the genetic map of A. annua (Graham ). In the bHLH TF family of A. annua, only two members, AabHLH1 and AabHLH76, have been functionally characterized. One is a MYC2 gene (AaMYC2 or AabHLH76) and the other is a bHLH1 gene (AabHLH1), both of which positively regulate artemisinin biosynthesis (Ji ; Shen ). AabHLH1 and AaMYC2 were identified in the genome and transcriptomes of A. annua. To identify candidate bHLH TFs that might regulate artemisinin biosynthesis, expression relationships between the 122 bHLH TFs and artemisinin biosynthesis genes were analysed using the released transcriptome data of A. annua. In the resulting heatmap, these bHLH TFs clustered into five groups with distinct expression patterns (Fig. 1). Group V, composed of 15 bHLH TFs, was tightly associated with artemisinin biosynthesis genes. Expression analysis suggested that these 15 bHLH TFs could be selected as candidates that regulate artemisinin biosynthesis. To further find candidates that might be involved in cold signaling, a phylogenetic tree was constructed using the 15 bHLH TFs of A. annua and all the bHLH TFs of Arabidopsis. Notably, two bHLH TFs of A. annua, AabHLH106 and AabHLH112, were clustered with the well-known ICE TFs of Arabidopsis (AT3G26744/AtICE1 and AT1G12860/AtICE2) in the same clade (Fig. 2), suggesting that the two bHLH TFs might play similar functions to the Arabidopsis ICE TFs that are known to be crucial in cold signaling (Chinnusamy ; Fursova ; Kurbidaeva ). Given the results of the expression and phylogenetic analyses, AabHLH106 and AabHLH112 were selected for further study and we cloned their full-length cDNAs. Both AabHLH106 and AabHLH112 contained one basic helix-loop-helix domain (Supplementary Fig. S1), and they shared 23.2% and 43.7% identity with Arabidopsis ICE1, respectively (Supplementary Fig. S2).

Fig. 1.

Heatmap of gene expression levels of the 122 bHLH transcription factors and artemisinin biosynthesis genes in mature leaf trichomes (MLT), meristem, cotyledon, young leaf trichomes (YLT), and flower bud trichomes (FT) of Artemisia annua.

Fig. 2.

Phylogenetic analysis of all bHLH transcription factors of Arabidopsis and 15 bHLH transcription factors of Artemisia annua. The Arabidopsis bHLH transcription factors are shown in black, while those of A. annua are in red. AabHLH106 and AabHLH112 are marked with stars. The clade including AtICE1, AtICE2, AabHLH106, and AabHLH112 is highlighted with a green background. The numbers on branches represent the bootstrap values, and the scale bar indicates the genetic distance.

Nuclear-localized AabHLH112 has similar expression patterns to artemisinin biosynthesis genes

To identify which of the two bHLH TFs might be involved in regulating artemisinin biosynthesis under low temperature, expression analysis of AabHLH106 and AabHLH112 was carried out. When plants were exposed to 4 °C, AabHLH106 was not responsive to the low-temperature treatment (Fig. 3A). In contrast, expression of AabHLH112 was quickly up-regulated, peaking at 6 h of low-temperature treatment, and was maintained at high levels for up to 24 h of treatment (Fig. 3B). The 13 other bHLH genes in Group V were not up-regulated by low temperature (Supplementary Fig. S3). According to a previous study, transcript levels of artemisinin biosynthesis genes and AaERF1 are increased under low temperature conditions (Liu ). Since the expression patterns of AabHLH112 and artemisinin biosynthesis genes were similar under low-temperature conditions, it was deduced that AabHLH112 might be the TF of interest. To investigate potential similarities in tissue expression patterns between AabHLH112 and artemisinin biosynthesis genes, relative expression levels of AabHLH112 in different tissues were examined. AabHLH112 was expressed at variable levels in different organs including GSTs, flower buds, leaves, stems, and roots (Fig. 3C). The transcript level was very high in GSTs, high in flower buds and leaves, moderate in roots, and very low in stems. The tissue expression profile of AabHLH112 was similar to that of artemisinin biosynthesis genes (Teoh , 2009; Zhang ). These results suggested that AabHLH112 might be the candidate bHLH gene that regulates artemisinin biosynthesis under low-temperature conditions.

Fig. 3.

Expression analysis of AabHLH106 and AabHLH112 in Artemisia annua plants exposed to low temperature. (A) Expression levels of AabHLH106 in plants subjected to 4 °C for 0–24 h. (B) Expression levels of AabHLH112 in plants subjected to 4 °C for 0–24 h. (C) Tissue profile of AabHLH112 expression. R, roots; S, stems; L, leaves; F, flower buds; GST, glandular secretory trichomes. Data are means (±SE), n=3. Significant differences between the treated plants and the mock controls in (A, B) were determined using Student’s t-test (**P<0.01). Different letters in (C) indicate significant differences as determined by Duncan’s test (P<0.01). (This figure is available in colour at JXB online.)

TFs are usually localized in the nucleus where they regulate target genes at the transcriptional level. To investigate the subcellular localization of AabHLH112, the coding sequence of AabHLH112 was fused in-frame to GFP. The nucleus was specifically stained by DAPI. When AabHLH112-GFP was transiently expressed in tobacco cells, strong fluorescence was clearly and exclusively observed in the nucleus (Fig. 4). All the fluorescence signal from AabHLH112-GFP was co-localized with that from DAPI, whilst strong fluorescence was diffusely observed throughout tobacco cells expressing GFP alone. These data indicated that AabHLH112 was specifically localized to the nucleus, in accordance with its potential role as a TF.

Fig. 4.

Subcellular localization of AabHLH112 in leaves of Nicotiana benthamiana. AabHLH112 fused in-frame with green fluorescent protein (GFP) was transiently expressed in tobacco leaf cells. YFP was used as a control. Nuclei were stained by DAPI. Scale bars are 20 μm. (This figure is available in colour at JXB online.)

AabHLH112 transactivates artemisinin biosynthesis genes and AaERF1

The expression levels of artemisinin biosynthesis genes as well as AaERF1, which positively regulates artemisinin biosynthesis through binding to the promoters of ADS and CYP71AV1 (Yu ), are significantly elevated under low-temperature conditions (Liu ). Both artemisinin biosynthesis genes and AaERF1 could thus be target genes of AabHLH112. To test this hypothesis, we performed dual-luciferase assays in tobacco leaves. Previously, we had cloned the promoters of four artemisinin biosynthesis genes and used them as reporters in dual-luciferase assays (Zhang , 2018). In this present study, we isolated the 1384-bp promoter region of AaERF1 based on the publicly available sequence (JQ513909), namely pAaERF1. The promoters of artemisinin biosynthesis genes and AaERF1 were fused to FIREFLY LUCIFERASE (LUC) to generate the reporter constructs pAaERF1::LUC, pADS::LUC, pCYP71AV1::LUC, pDBR2::LUC, and pALDH1::LUC. Meanwhile, AabHLH112 driven by the cauliflower mosaic virus (CaMV) 35S promoter was used as an effector construct (Fig. 5A). Pairs of effector and reporter constructs were co-infiltrated into tobacco leaves using A. tumefaciens GV3101. When AabHLH112 was expressed, LUC/REN values were significantly increased by 2.19-fold for pAaERF1::LUC, 9.71-fold for pADS::LUC, 2.49-fold for pCYP71AV1::LUC, 6.46-fold for pDBR2::LUC, and 8.37-fold for pALDH1::LUC compared with LUC/REN values in tobacco leaves co-transfected with a YFP control (Fig. 5B). These results suggested that AabHLH112 up-regulated AaERF1 and artemisinin biosynthesis genes at the transcriptional level.

Fig. 5.

Dual-luciferase assays in leaves of Nicotiana benthamiana to study the transactivation of AabHLH112 on AaERF1 and artemisinin biosynthesis genes. (A) Diagrams of effector and reporter constructs. (B) Results of AabHLH112 dual-luciferase assays using the promoters of artemisinin biosynthesis genes and AaERF1. Data are means (±SE), n=5. Significant difference between means were determined using Student’s t-test (**P<0.01). (This figure is available in colour at JXB online.)

AabHLH112 was able to bind to the promoter of AaERF1 that directly transactivates artemisinin biosynthesis genes

Many studies have shown that bHLHs, such as AaMYC2 and AabHLH1, act through recognition of, and binding to, a G-box hexanucleotide sequence in the promoter regions of artemisinin biosynthesis genes (Ji ; Shen ). Bioinformatic analysis showed that pAaERF1 also contained these G-box motifs (Supplementary Fig. S4). Although the results of the dual-LUC assays had indicated that the cold-induced AabHLH112 gene positively regulated artemisinin biosynthesis through elevation of the expression levels of AaERF1 and artemisinin biosynthesis genes, it was not known whether this was due to direct or indirect regulation at the transcriptional level. To determine whether AabHLH112 binds directly to the G-box in the promoters of artemisinin biosynthesis genes or AaERF1, yeast one-hybrid (Y1H) assays were performed. The promoter of each artemisinin biosynthesis gene was divided into overlapping fragments and inserted into the lexA Y1H vector p178, which contained the lacZ reporter gene, to generate reporter constructs. AabHLH112 was fused to the B42 activation domain to generate the effector construct pB42AD-AabHLH112, which was co-transformed into yeast strain EGY48 with the respective reporter construct (Fig. 6A). As shown in Fig. 6B–E, none of the yeast clones on medium with X-gal were stained blue, suggesting that AabHLH112 did not bind to the promoters of artemisinin biosynthesis genes.

Fig. 6.

Yeast one-hybrid (Y1H) assays between AabHLH112 and promoter fragments of artemisinin biosynthesis genes and AaERF1. (A) Diagrams of constructs used in the assays. (B–F) Y1H assay between (B) AabHLH112 and pADS, (C) AabHLH112 and pCYP71AV1, (D) AabHLH112 and pDBR2, (E) AabHLH112 and pALDH1, and (F) AabHLH112 and pAaERF1.

To investigate the interaction between AabHLH112 and the AaERF1 promoter, the 1384-bp promoter region of AaERF1 was divided into three fragments (see Methods). These fragments were inserted upstream of the lacZ reporter gene to generate reporter constructs. The pB42AD-AabHLH112 effector construct was co-transformed into yeast strain EGY48 with each of the reporter constructs containing an AaERF1 promoter fragment. As shown in Fig. 6F, yeast cells harboring pB42AD-AabHLH112 and pAaERF1(–1384~–922)::lacZ were clearly stained. This Y1H analysis therefore indicated that AabHLH112 could bind to the AaERF1 promoter region from –1384 to –922 bp, suggesting that it is a direct target gene. AaERF1 has previously been shown to directly bind to and regulate the promoters of ADS and CYP71AV1 (Ji ). However, the transactivation of AaERF1 on the promoters of DBR2 and ALDH1 has not been characterized. To assess whether AaERF1 is able to transactivate the promoters of the four artemisinin biosynthesis genes that we selected, we also performed dual-LUC assays in tobacco leaves. As described above, AaERF1 was used as an effector and co-infiltrated into leaves with the respective reporters, which were constructed using the promoters of the artemisinin biosynthesis genes (Fig. 7A). As shown in Fig. 7B, AaERF1 significantly induced the activities of the promoters of the four artemisinin biosynthesis genes.

Fig. 7.

Dual-luciferase assays in leaves of Nicotiana benthamiana to study transactivation of AaERF1 on artemisinin biosynthesis genes. (A) Diagrams of effector and reporter constructs. (B) Results of AaERF1 dual-luciferase assays using promoters of artemisinin biosynthesis genes. Data are means (±SE), n=5. Significant differences between means were determined using Student’s t-test (**P<0.01). (This figure is available in colour at JXB online.)

Molecular analysis and detection of metabolite of transgenic plants of A. annua

Since the results above suggested that AabHLH112 could positively regulate AaERF1 and artemisinin biosynthesis genes, we were interested to study its role in regulating biosynthesis in transgenic plants of A. annua over-expressing AabHLH112 (Supplementary Fig. S5). Fragments specific to the marker gene (hygromycin resistance) and the 557-bp fragment from AabHLH112 to the transcriptional termination sequence of the Rubisco gene (rbc) were respectively amplified from the AabHLH112-over-expressing (-OE) plants and the plant expression vector pHB-AabHLH112 (positive control), but were not obtained from control plants (Supplementary Fig. S6). In addition, expression levels of AabHLH112 were detected using qPCR and were significantly higher in AabHLH112-OE plants than in controls (Fig. 8A). Based on the PCR and qPCR results, authentic transgenic plants of A. annua were confirmed in which AabHLH112 was overexpressed. Expression levels of AaERF1 and the artemisinin biosynthesis genes ADS, CYP71AV1, DBR2, and ALDH1 were also analysed. Compared with the expression levels in control plants, the transcript levels of AaERF1 (Fig. 8A) and the biosynthesis genes were markedly elevated in AabHLH112-OE plants (Fig. 8B). These data were consistent with the results of the dual-luciferase assays, suggesting that over-expression of AabHLH112 significantly up-regulated the expression levels of AaERF1 and artemisinin biosynthesis genes.

Fig. 8.

Molecular analysis and detection of metabolites in leaves of wild-type and transgenic Artemisia annua plants. (A) Expression levels of AabHLH112 and AaERF1. (B) Expression levels of artemisinin biosynthesis genes. (C) Contents of artemisinin and dihydroartemisinic acid. WT, wild-type; OX28, OX33, and OX37 are independent lines of AabHLH112-overexpressing plants. Data are means (±SE), n=3. Significant differences between means were determined using Student’s t-test (**P<0.01). (This figure is available in colour at JXB online.)

Artemisinin and dihydroartemisinic acid content were then analysed in control and transgenic plants of A. annua. Control plants produced 8.42 mg g−1 DW of artemisinin and 0.86 mg g−1 DW of dihydroartemisinic acid, while the three independent AabHLH112-OE lines (OX28, OX33, and OX37) exhibited variable but significant increases in production of both (Fig. 8C). OX28, OX33, and OX37 contained artemisinin at levels of 14.35 mg g−1 DW, 13.82 mg g−1 DW, and 12.44 mg g−1 DW, respectively, representing 70.42%, 64.13%, and 47.62% increases in content compared with control plants. Contents of dihydroartemisinic acid were increased by 104%, 498%, and 269% in the AabHLH112-OE lines. These data indicated that overexpression of AabHLH112 elevated the expression levels of AaERF1 and artemisinin biosynthesis genes, and consequently enhanced the production of artemisinin and dihydroartemisinic acid.

Discussion

The bHLH TFs comprise the second largest TF family in plants. There are 133 and bHLH TFs in Arabidopsis (Heim ) and 167 in rice (Li ). We identified more than 200 bHLH TFs in the genome of A. annua (Supplementary Table S1), i.e. exceeding the number of genes in both Arabidopsis and rice. The 1.74-gigabase genome of A. annua has 62 336 protein-coding genes, with 2717 TF genes found among them, and these numbers are among the largest for any sequenced plant genome (Shen ). The large number of genes might be due to gene family expansion in A. annua. As a terpenoid-rich plant species, it might require more bHLH TFs to regulate the biosynthesis of terpenoids, including artemisinin, under different conditions. Among the 205 bHLH TFs, only 122 were expressed in the five high-quality transcriptomes used to construct the genetic map of A. annua (Graham ). TFs that positively regulate artemisinin biosynthesis are often positively correlated with artemisinin biosynthesis genes at the transcriptional level (Shen ). Analysis of gene expression relationships revealed that only 15 bHLH TFs among the 122 identified were clustered with artemisinin biosynthesis genes in the same clade with similar tissue profiles (Fig. 1). It was therefore reasonable to deduce that novel bHLH candidates regulating artemisinin biosynthesis might be present among these 15 members.

We focused on bHLH TFs that regulate artemisinin biosynthesis under low temperature, and specifically sought to find putative regulators involved in cold signaling among the 15 bHLH TFs. Many previous studies have reported that ICE TFs, belonging to the bHLH TF family, play crucial roles in cold signaling (Chinnusamy ; Badawi ; Fursova ; Kurbidaeva ). A phylogenetic analysis of the 15 bHLH TFs of A. annua and all of the Arabidopsis bHLH TFs revealed that two from A. annua, AabHLH106 and AabHLH112, were grouped into the same clade with the Arabidopsis ICE proteins AtICEl and AtICE2 (Fig. 2). It is well known that both AtICE1 and AtICE2 regulate cold signaling in Arabidopsis (Chinnusamy ; Fursova ; Kurbidaeva ), so our results suggested that AabHLH106 and AabHLH112 might be candidates for regulating artemisinin biosynthesis under low-temperature conditions. Phytohormones and environmental factors significantly affect artemisinin biosynthesis. Among phytohormones, MeJA is often used to promote artemisinin production. MeJA-responsive TFs, especially AaERF1 (Yu ) and AaMYC2 (Shen ), may activate artemisinin biosynthesis genes by directly binding to their promoters. It is notable that MeJA can also markedly enhance tolerance to cold stress in A. annua (Liu ). The production of jasmonate and artemisinin can be promoted when plants are exposed to low temperature, due to the elevated expression levels of biosynthesis genes involved in JA and artemisinin production (Liu ). Although AaMYC2 and AaERF1 positively regulate artemisinin biosynthesis via JA signaling, neither of these compounds are directly involved in cold signaling. ICE TFs play crucial roles in cold signaling and regulate cold tolerance in plants; however, it is unknown whether they regulate the biosynthesis of secondary metabolites. Artemisinin biosynthesis genes usually exhibit coordinated expression patterns. They have high expression levels in organs such as flower buds and young leaves that are rich in glandular trichomes, and they are also responsive to phytohormones including MeJA or ABA (Maes ; Zhang ; Shen ). In addition, TFs that positively regulate artemisinin biosynthesis usually have similar expression patterns to artemisinin biosynthesis genes. Thus, similar expression patterns allow convenient screening of candidate regulatory genes involved in controlling artemisinin biosynthesis. AabHLH112 was highly expressed in glandular trichomes, flower buds, and leaves (Fig. 3C), and it could also be induced by low temperature (Fig. 3B). The similarities in expression patterns of AabHLH112, artemisinin biosynthesis genes, and AaERF1, especially their expression induced by low temperature, suggested that these genes might be involved in the same regulatory network.

AabHLH112 belongs to the family of bHLH TFs containing the conserved HLH domain, which can bind to G-box motifs of target gene promoters (Jakoby ; Chinnusamy ; Badawi ). The G-box motifs in the promoters of AaERF1 and artemisinin biosynthesis genes thus provide putative binding sites for AabHLH112 (Ji ; Shen ). The results of Y1H assays indicated that AabHLH112 directly bound to the AaERF1 promoter (Fig. 6F), but it could not bind to the promoters of artemisinin biosynthesis genes (Fig. 6B–E). These results suggested that AaERF1 was a direct target gene of AabHLH112, whereas artemisinin biosynthesis genes were not. However, TFs can regulate biosynthesis genes directly or indirectly. According to previous studies, the TFs AaERF1, AaMYC2, AabZIP1, and AaGSW1 positively regulate artemisinin biosynthesis genes by binding to their promoters (Yu ; Zhang ; Shen ; Chen ). In contrast, another ERF TF, AaORA, indirectly regulates the genes, but its overexpression greatly promotes artemisinin biosynthesis in transgenic plants of A. annua (Lu ). Recently, it was found that a complex composed of AaTCP14 and AaORA regulates artemisinin biosynthesis genes, and AaTCP14 was shown to bind to their promoters (Ma ). Our dual-luciferase assays provided evidence to support AabHLH112 playing a positive role in regulating artemisinin biosynthesis. When AabHLH112 was expressed the promoter activities of AaERF1 and artemisinin biosynthesis genes were significantly increased (Fig. 5). These results suggested that AabHLH112 may positively regulate the activity of AaERF1, because it could bind to the promoter of AaERF1. Although the selected artemisinin biosynthesis genes were not direct targets of AabHLH112, the activities of their promoters were also enhanced by AabHLH112 in dual-luciferase assays performed in tobacco cells. Some unknown TFs in tobacco might be directly transactivated by AabHLH112, and they might further up-regulate the promoters of the artemisinin biosynthesis genes in the dual-luciferase assays. In fact, the tobacco ERF TF NtERF32 positively regulates nicotine biosynthesis by directly binding to G-box motifs of the PMT promoter (Sears ). In our recent study, the SnRK2-type kinase (AaAPK1) could also enhance the activities of the promoters of artemisinin biosynthesis genes in dual-luciferase assays, despite the observation that they were not direct target genes of AaAPK1 (Zhang ). It should be noted that AaERF1 positively and directly regulates artemisinin biosynthesis genes through binding to the promoters of ADS and CYP71AV1 (Yu ). Taken together, we conclude that AabHLH112 positively regulates artemisinin biosynthesis genes through direct transactivation of AaERF1, which further positively regulates artemisinin biosynthesis genes by binding to their promoters.

The results of the Y1H and dual-luciferase assays strongly suggested that AabHLH112 positively regulated artemisinin biosynthesis through AaERF1. As expected, overexpression of AabHLH112 markedly elevated the expression level of AaERF1, which was the direct target TF of AabHLH112 (Fig. 8A). Because AaERF1 positively regulates artemisinin biosynthesis genes by binding to their promoters (Yu ), the elevated expression of AaERF1 subsequently up-regulated these genes at the transcriptional level (Fig. 8B). With increased expression of the genes, the biosynthesis of artemisinin and dihydroartemisinic acid were eventually enhanced in AabHLH112-overexpressing plants of A. annua (Fig. 8C). In summary, we present a simplified model to illustrate AabHLH112 regulation of artemisinin biosynthesis (Fig. 9). When plants of A. annua are exposed to low temperature, AabHLH112 is induced and it can transactivate AaERF1 through binding to its promoter; artemisinin biosynthesis genes, as the direct target genes of AaERF1, are then up-regulated to enhance biosynthesis of artemisinin.

Fig. 9.

A simplified model illustrating regulation of artemisinin biosynthesis by AabHLH112 under low temperatures. Exposure to cold induces AabHLH112, which transactivates AaERF1 through binding to its promoter. Artemisinin biosynthesis genes are the direct targets of AaERF1, and their subsequent up-regulation thus enhances production of artemisinin. (This figure is available in colour at JXB online.)

Supplementary data

Supplementary data are available at JXB online.

Table S1. The 205 bHLH transcription factors of Artemisia annua.

Table S2. Primers used in this study.

Fig. S1. Conserved structures of AabHLH106 and AabHLH112 protein as predicted in NCBI.

Fig. S2. Protein alignment analysis of AabHLH106 and AabHLH112 with AtbHLH116.

Fig. S3. Expression levels of 13 bHLH TF genes in A. annua in response to low temperature.

Fig. S4. Nucleotide sequence of the cloned AaERF1 promoter with putative cis-elements indicated.

Fig. S5. Illustration of how of AabHLH112-overexpression transgenic plants of A. annua were obtained.

Fig. S6. PCR analysis of AabHLH112-overexpression transgenic plants of A. annua.

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