Zongyou Lv

AaORA, a trichome‐specific AP2/ERF transcription factor of Artemisia annua, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea

· Six transcription factors of APETALA2/ethylene-response factor (AP2/ERF) family were cloned and analyzed in Artemisia annua. Real-time quantitative polymerase chain reaction (RT-Q-PCR) showed that AaORA exhibited similar expression patterns to those of amorpha-4,11-diene synthase gene (ADS), cytochrome P450-dependent hydroxylase gene (CYP71AV1) and double bond reductase 2 gene (DBR2) in different tissues of A. annua. · AaORA is a trichome-specific transcription factor, which is expressed in both glandular secretory trichomes (GSTs) and nonglandular T-shaped trichomes (TSTs) of A. annua. The result of subcellular localization shows that AaORA is targeted to the nuclei and the cytoplasm. · Overexpression and RNA interference (RNAi) of AaORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71AV1, DBR2 and AaERF1. The up-regulated or down-regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that AaORA is a positive regulator in the biosynthesis of artemisinin. · Overexpression of AaORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2), HEVEIN-LIKE PROTEIN (HEL) and BASIC CHITINASE (B-CHI). After inoculation with Botrytis cinerea, the phenotypes of AaORA overexpression in A. thaliana and AaORA RNAi in A. annua demonstrate that AaORA is a positive regulator of disease resistance to B. cinerea.

A Basic Leucine Zipper Transcription Factor, AabZIP1, Connects Abscisic Acid Signaling with Artemisinin Biosynthesis in Artemisia annua

Artemisinin is a sesquiterpenoid especially synthesized in the Chinese herbal plant, Artemisia annua, which is widely used in the treatment of malaria. Artemisinin accumulation can be enhanced by exogenous abscisic acid (ABA) treatment. However, it is not known how ABA signaling regulates artemisinin biosynthesis. A global expression profile and phylogenetic analysis as well as the dual-LUC screening revealed that a basic leucine zipper family transcription factor from A. annua (namely AabZIP1) was involved in ABA signaling to regulate artemisinin biosynthesis. AabZIP1 had a higher expression level in the inflorescences than in other tissues; ABA treatment, drought, and salt stress strongly induced the expression of AabZIP1. Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that AabZIP1 bound to the ABA-responsive elements (ABRE) in the promoter regions of the amorpha-4,11-diene synthase (ADS) gene and CYP71AV1, which are two key structural genes of the artemisinin biosynthetic pathway. A mutagenesis assay showed that the C1 domain in the N-terminus of AabZIP1 was important for its transactivation activity. Furthermore, the activation of ADS and CYP71AV1 promoters by AabZIP1 was enhanced by ABA treatment in transient dual-LUC analysis. The AabZIP1 variant with C1 domain deletion lost the ability to activate ADS and CYP71AV1 promoters regardless of ABA treatment. Notably, overexpression of AabZIP1 in A. annua resulted in significantly increased accumulation of artemisinin. Our results indicate that ABA promotes artemisinin biosynthesis, likely through 1 activation of ADS and CYP71AV1 expression by AabZIP in A. annua. Meanwhile, our findings reveal the potential value of AabZIP1 in genetic engineering of artemisinin production.

Overexpression of the Artemisia Orthologue of ABA Receptor, AaPYL9, Enhances ABA Sensitivity and Improves Artemisinin Content in Artemisia annua L

The phytohormone abscisic acid (ABA) plays an important role in plant development and environmental stress response. In this study, we cloned an ABA receptor orthologue, AaPYL9, from Artemisia annua L. AaPYL9 is expressed highly in leaf and flower. AaPYL9 protein can be localized in both nucleus and cytoplasm. Yeast two-hybrid assay shows AaPYL9 can specifically interact with AtABI1 but not with AtABI2, AtHAB1 or AtHAB2. ABA can enhance the interaction between AaPYL9 and AtABI1 while AaPYL9-89 Pro→Ser and AaPYL9-116 His→Ala point mutations abolishes the interaction. BiFC assay shows that AaPYL9 interacts with AtABI1 in nucleus in planta. Transgenic Arabidopsis plants over-expressing AaPYL9 are more sensitive to ABA in the seed germination and primary root growth than wild type. Consistent with this, ABA report genes have higher expression in AaPYL9 overexpressing plants compared to wild type after ABA treatment. Moreover, overexpression of AaPYL9 in A. annua increases not only drought tolerance, but also artemisinin content after ABA treatment, with significant enhancement of the expression of key genes in artemisinin biosynthesis. This study provides a way to develop A. annua with high-yielding artemisinin and high drought resistance.

Molecular Cloning and Characterization of a Trichome-Specific Promoter of Artemisinic Aldehyde Δ11(13) Reductase (DBR2) in Artemisia annua

Artemisinin is widely used as an antimalarial drug around the world. Artemisinic aldehyde Δ11(13) reductase (DBR2) is a key enzyme which reduces artemisinic aldehyde to dihydroartemisinic aldehyde in the biosynthesis of artemisinin. In this study, two fragments encompassing a putative promoter of DBR2, designated as DBR2pro1 and DBR2pro2, were isolated using genomic DNA walking. The transcription start site and the putative cis-elements of each version of promoter were predicted using bioinformatic analysis. In order to study the function of the cloned promoter, Artemisia annua was transformed with β-glucuronidase (GUS) reporter gene driven by DBR2pro1 and DBR2pro2, respectively. GUS staining results demonstrated that both DBR2pro1 and DBR2pro2 were strongly expressed in glandular secretory trichomes (GSTs) of leaf primordia and flower buds, but were not obviously expressed in roots, stems, old leaves, and fully developed flowers, thus indicating that the two versions of promoter were functional and specifically expressed in GSTs.

Branch Pathway Blocking in Artemisia annua is a Useful Method for Obtaining High Yield Artemisinin.

There are many biosynthetic pathways competing for the metabolic flux with the artemisinin biosynthetic pathway in Artemisia annua L. To study the relationship between genes encoding enzymes at branching points and the artemisinin biosynthetic pathway, β-caryophyllene, β-farnesene and squalene were sprayed on young seedlings of A. annua. Transient expression assays indicated that the transcription levels of β-caryophyllene synthase (CPS), β-farnesene synthase (BFS) and squalene synthase (SQS) were inhibited by β-caryophyllene, β-farnesene and squalene, respectively, while expression of some artemisinin biosynthetic pathway genes increased. Thus, inhibition of these genes encoding enzymes at branching points may be helpful to improve the artemisinin content. For further study, the expression levels of four branch pathway genes CPS, BFS, germacrene A synthase (GAS) and SQS were down-regulated by the antisense method in A. annua. In anti-CPS transgenic plants, mRNA levels of BFS and ADS were increased, and the contents of β-farnesene, artemisinin and dihydroartemisinic acid (DHAA) were increased by 212, 77 and 132%, respectively. The expression levels of CPS, SQS, GAS, amorpha-4,11-diene synthase (ADS), amorphadiene 12-hydroxylase (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1) were increased in anti-BFS transgenic plants and, at the same time, the contents of artemisinin and DHAA were increased by 77% and 54%, respectively, and the content of squalene was increased by 235%. In anti-GAS transgenic plants, mRNA levels of CPS, BFS, ADS and ALDH1 were increased. The contents of artemisinin and DHAA were enhanced by 103% and 130%, respectively. In anti-SQS transgenic plants, the transcription levels of BFS, GAS, CPS, ADS, CYP71AV1 and ALDH1 were all increased. Contents of artemisinin and DHAA were enhanced by 71% and 223%, respectively, while β-farnesene was raised to 123%. The mRNA level of artemisinic aldehyde Δ11(13) reductase (DBR2) had changed little in almost all transgenic plants.

Overexpression of a Novel NAC Domain-Containing Transcription Factor Gene (AaNAC1) Enhances the Content of Artemisinin and Increases Tolerance to Drought and Botrytis cinerea in Artemisia annua.

The NAC (NAM, ATAF and CUC) superfamily is one of the largest plant-specific transcription factor families. NAC transcription factors always play important roles in response to various abiotic stresses. A NAC transcription factor gene AaNAC1 containing a complete open reading frame (ORF) of 864 bp was cloned from Artemisia annua. The expression of AaNAC1 could be induced by dehydration, cold, salicylic acid (SA) and methyl jasmonate (MJ), suggesting that it might be a key regulator of stress signaling pathways in A. annua. AaNAC1 was shown to be localized to the nuclei by transforming tobacco leaf epidermal cells. When AaNAC1 was overexpressed in A. annua, the content of artemisinin and dihydroartemisinic acid was increased by 79% and 150%, respectively. The expression levels of artemisinin biosynthetic pathway genes, i.e. amorpha-4,11-diene synthase (ADS), artemisinic aldehyde Δ11(13) reductase (DBR2) and aldehyde dehydrogenase 1 (ALDH1), were increased. Dual luciferase (dual-LUC) assays showed that AaNAC1 could activate the transcription of ADS in vivo. The transgenic A. annua exhibited increased tolerance to drought and resistance to Botrytis cinerea. When AaNAC1 was overexpressed in Arabidopsis, the transgenic Arabidopsis were markedly more tolerant to drought. The transgenic Arabidopsis showed increased resistance to B. cinerea. These results indicate that AaNAC1 can potentially be used in transgenic breeding for improving the content of artemisinin and drought tolerance in A. annua.

Overexpression of AaWRKY1 Leads to an Enhanced Content of Artemisinin in Artemisia annua

Artemisinin is an effective component of drugs against malaria. The regulation of artemisinin biosynthesis is at the forefront of artemisinin research. Previous studies showed that AaWRKY1 can regulate the expression of ADS, which is the first key enzyme in artemisinin biosynthetic pathway. In this study, AaWRKY1 was cloned, and it activated ADSpro and CYPpro in tobacco using dual-LUC assay. To further study the function of AaWRKY1, pCAMBIA2300-AaWRKY1 construct under 35S promoter was generated. Transgenic plants containing AaWRKY1 were obtained, and four independent lines with high expression of AaWRKY1 were analyzed. The expression of ADS and CYP, the key enzymes in artemisinin biosynthetic pathway, was dramatically increased in AaWRKY1-overexpressing A. annua plants. Furthermore, the artemisinin yield increased significantly in AaWRKY1-overexpressing A. annua plants. These results showed that AaWRKY1 increased the content of artemisinin by regulating the expression of both ADS and CYP. It provides a new insight into the mechanism of regulation on artemisinin biosynthesis via transcription factors in the future.

Type 2C Phosphatase 1 of Artemisia annua L. Is a Negative Regulator of ABA Signaling

The phytohormone abscisic acid (ABA) plays an important role in plant development and environmental stress response. Additionally, ABA also regulates secondary metabolism such as artemisinin in the medicinal plant Artemisia annua L. Although an earlier study showed that ABA receptor, AaPYL9, plays a positive role in ABA-induced artemisinin content improvement, many components in the ABA signaling pathway remain to be elucidated in Artemisia annua L. To get insight of the function of AaPYL9, we isolated and characterized an AaPYL9-interacting partner, AaPP2C1. The coding sequence of AaPP2C1 encodes a deduced protein of 464 amino acids, with all the features of plant type clade A PP2C. Transcriptional analysis showed that the expression level of AaPP2C1 is increased after ABA, salt, and drought treatments. Yeast two-hybrid and bimolecular fluorescence complementation assays (BiFC) showed that AaPYL9 interacted with AaPP2C1. The P89S, H116A substitution in AaPYL9 as well as G199D substitution or deletion of the third phosphorylation site-like motif in AaPP2C1 abolished this interaction. Furthermore, constitutive expression of AaPP2C1 conferred ABA insensitivity compared with the wild type. In summary, our data reveals that AaPP2C1 is an AaPYL9-interacting partner and involved in the negative modulation of the ABA signaling pathway in A. annua L.

Characterization of a novel ERF transcription factor in Artemisia annua and its induction kinetics after hormones and stress treatments

The full-length cDNA sequence of AaERF3 was cloned and characterized from Artemisia annua. The bioinformatic analysis and phylogenetic tree analysis implied that the AaERF3 encoded a putative protein of 193 amino acids which formed a closely related subgroup with AtERF1, ERF1 and ORA59 in Arabidopsis. The result of subcellular localization showed that AaERF3 targeted to both of the nuclei and the cytoplasm. The qRT-PCR analysis showed that Green young alabastrums had the highest expression level of AaERF3 in the 5-months-old plants. The qRT-PCR analysis also revealed that ABA, Wound and Cold treatments significantly enhanced the transcript expression of AaERF3. MeJA and Ethylene treatment could also slightly induce the accumulation of AaERF3 transcription.

Characterization of the Promoter of Artemisia annua Amorpha-4,11-diene Synthase (ADS) Gene Using Homologous and Heterologous Expression as well as Deletion Analysis

Amorpha-4,11-diene synthase (ADS) is the first key enzyme of artemisinin biosynthetic pathway in Artemisia annua L. In this study, the promoter region of the ADS gene has been cloned and used to demonstrate the expression of GUS reporter gene in both glandular trichomes of A. annua and non-glandular trichomes of Arabidopsis thaliana following homologous and heterologous expression of ADS promoter–GUS fusion. Subsequently, 5′ sequential deletion analysis of the ADS promoter revealed that a short sequence, −350 upstream of the transcription start site, was sufficient for trichome-specific expression in A. thaliana and that the region from −350 to −300 contained essential elements for this observed specificity. However, frequencies of transgenic A. thaliana plants displaying trichome-specific expressions varied between different lines, and all the lines with deleted fragments of the ADS promoter showed lower frequencies than the line with full-length ADS promoter. Most lines with deleted ADS promoter–GUS fusions showed GUS expressions in the guard cells of stomata as well, which was not observed in A. thaliana plants transformed with the full-length ADS promoter. GUS activities varied among different transgenic lines as well, both in transiently transformed Nicotiana benthamiana and stably transformed A. thaliana, with promoter–deletion lines exhibiting higher GUS activities than the full-length ADS promoter line.