The plant Artemisia annua is well known due to the production of artemisinin, a sesquiterpene lactone that is widely used in malaria treatment. Phytohormones play important roles in plant secondary ...metabolism, such as jasmonic acid (JA), which can induce artemisinin biosynthesis in A. annua. Nevertheless, the JA-inducing mechanism remains poorly understood.
The expression of gene AaMYC2 was rapidly induced by JA and AaMYC2 binds the G-boxlike motifs within the promoters of gene CYP71AV1 and DBR2, which are key structural genes in the artemisinin biosynthetic pathway.
Overexpression of AaMYC2 in A. annua significantly activated the transcript levels of CYP71AV1 and DBR2, which resulted in an increased artemisinin content. By contrast, artemisinin content was reduced in the RNAi transgenic A. annua plants in which the expression of AaMYC2 was suppressed. Meanwhile, the RNAi transgenic A. annua plants showed lower sensitivity to methyl jasmonate treatment than the wild-type plants.
These results demonstrate that AaMYC2 is a positive regulator of artemisinin biosynthesis and is of great value in genetic engineering of A. annua for increased artemisinin production.
Glandular trichomes are generally considered biofactories that produce valuable chemicals. Increasing glandular trichome density is a very suitable way to improve the productivity of these valuable ...metabolites, but little is known about the regulation of glandular trichome formation. Phytohormone jasmonate (JA) promotes glandular trichome initiation in various plants, but its mechanism is also unknown.
By searching transcription factors regulated by JA in Artemisia annua, we identified a novel homeodomain-leucine zipper transcription factor, HOMEODOMAIN PROTEIN 1 (AaHD1), which positively controls both glandular and nonglandular trichome initiations. Overexpression of AaHD1 in A. annua significantly increased glandular trichome density without harming plant growth. Consequently, the artemisinin content was improved.
AaHD1 interacts with A. annua jasmonate ZIM-domain 8 (AaJAZ8), which is a repressor of JA, thereby resulting in decreased transcriptional activity. AaHD1 knockdown lines show decreased sensitivity to JA on glandular trichome initiation, which indicates that AaHD1 plays an important role in JA-mediated glandular trichome initiation.
We identified a new transcription factor that promotes A. annua glandular trichome initiation and revealed a novel molecular mechanism by which a homeodomain protein transduces JA signal to promote glandular trichome initiation. Our results also suggested a connection between glandular and nonglandular trichome formations.
The glandular secretory trichomes (GSTs) on Artemisia annua leaves have the capacity to secrete and store artemisinin, a compound which is the most effective treatment for uncomplicated malaria. An ...effective strategy to improve artemisinin content is therefore to increase the density of GSTs in A. annua. However, the formation mechanism of GSTs remains poorly understood.
To explore the mechanisms of GST initiation in A. annua, we screened myeloblastosis (MYB) transcription factor genes from a GST transcriptome database and identified a MIXTA transcription factor, AaMIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of AaMIXTA1 resulted in an increase and decrease, respectively, in the number of GSTs as well as the artemisinin content in transgenic plants.
Transcriptome analysis and cuticular lipid profiling showed that AaMIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that AaMIXTA1 could directly activate the expression of genes related to cuticle biosynthesis.
Taken together, AaMIXTA1 regulated cuticle biosynthesis and prompted GST initiation without any abnormal impact on the morphological structure of the GSTs and so provides a new way to improve artemisinin content in this important medicinal plant.
Artemisinin is a type of sesquiterpene lactone well known as an antimalarial drug, and is specifically produced in glandular trichomes of Artemisia annua. However, the regulatory network for the ...artemisinin biosynthetic pathway remains poorly understood. Exploration of trichome-specific transcription factors would facilitate the elucidation of regulatory mechanism of artemisinin biosynthesis.
The WRKY transcription factor GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) was cloned and analysed in A. annua. AaGSW1 exhibited similar expression patterns to the trichome-specific genes of the artemisinin biosynthetic pathway and AP2/ERF transcription factor AaORA. A β-glucuronidase (GUS) staining assay further demonstrated that AaGSW1 is a glandular trichome-specific transcription factor.
AaGSW1 positively regulates CYP71AV1 and AaORA expression by directly binding to the W-box motifs in their promoters. Overexpression of AaGSW1 in A. annua significantly improves artemisinin and dihydroartemisinic acid contents; moreover, AaGSW1 can be directly regulated by AaMYC2 and AabZIP1, which are positive regulators of jasmonate (JA)-and abscisic acid (ABA)-mediated artemisinin biosynthetic pathways, respectively.
These results demonstrate that AaGSW1 is a glandular trichome-specific WRKY transcription factor and a positive regulator in the artemisinin biosynthetic pathway. Moreover, we propose that two trifurcate feed-forward pathways involving AaGSW1, CYP71AV1 and AaMYC2/AabZIP1 function in the JA/ABA response in A. annua.
contains a variety of monoterpenoid indole alkaloids (MIAs), among which bisindole alkaloids vinblastine and vincristine are well-known to have antitumor effects and widely used in clinical ...treatment. However, their contents in
is extremely low and difficult to meet market demands. Therefore, it is of great significance to study the transcriptional regulation mechanism of MIAs biosynthesis for high yielding of bisindole alkaloids in
. Studies have shown that MIAs biosynthesis in
has complex temporal and spacial specificity and is under tight transcriptional regulation, especially bisindole alkaloids. In this study, an AP2/ERF transcription factor CrERF5 was selected by RNA-seq of
organs, and its full-length sequence was cloned and characterized. CrERF5 responds to both ethylene and JA signals and is localized in the nucleus. CrERF5 could activate the transcriptional activity of the TDC promoter. Transient overexpressing CrERF5 in
petals caused a significant increase of the expression levels of key genes in both the upstream and downstream pathways of MIAs biosynthesis while silencing
resulted in a decrease of them. Accordingly, the contents of bisindole alkaloids anhydrovinblastine and vinblastine, monoindole alkaloids ajmalicine, vindoline, and catharanthine were strongly enhanced in
-overexpressing petals while their contents decreased in
-silenced plants. These results suggested that CrERF5 is a novel positive ethylene-JA-inducible AP2/ERF transcription factor upregulating the MIAs biosynthetic pathway leading to the bisindole alkaloids accumulation.
In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate ...the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of (1)H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus.
Plants have evolved sophisticated systems for regulating the biosynthesis of specialized phytochemicals. Artemisinin, which is a sesquiterpene lactone widely used in anti-malaria treatment, is ...produced by the
Artemisia annua
L. plant. However, the artemisinin content in
A. annua
is low and difficult to meet market demands. Studies have shown that artemisinin biosynthesis in
A. annua
has complex temporal and spatial specificity and is under tightly transcriptional regulation. However, the mechanism of transcriptional regulation of artemisinin biosynthesis remains unclear. In this study, we identified two MYC-type bHLH transcription factors (AabHLH2 and AabHLH3) as novel regulators of artemisinin biosynthesis. These bHLH TFs act as transcription repressors and function redundantly to negatively regulate artemisinin biosynthesis. Furthermore, AabHLH2 and AabHLH3 are nuclear proteins that bind to DNA elements with similar specificity to that of AaMYC2, but lack the conserved activation domain, suggesting that repression is achieved by competition for the same
cis
-regulatory elements. Together, our findings reveal a novel artemisinin biosynthesis regulatory network, provide new insight into how specialized metabolites are modulated in plants, and propose a model in which different bHLH TFs coordinated in regulating artemisinin production in the plant. Finally, this study provides some useful target genes for metabolic engineering of artemisinin production via CRISPR/Cas9 gene editing.
Artemisinin is an effective antimalarial sesquiterpene lactone synthesized in
Artemisia annua
. Various transcription factors have been previously reported that can influence the biosynthesis of ...artemisinin; however, the effect of YABBY family transcription factors on artemisinin biosynthesis was unknown. In the present study, we cloned and characterized AaYABBY5: a homolog of MsYABBY5 in
Mentha spicata
which is involved in modulating the monoterpenes, as a positive regulator of artemisinin biosynthesis in
A. annua
. AaYABBY5 was found localized to the nucleus, and its expression was found to be induced by exogenous methyl jasmonic acid (MeJA) treatment. In the dual-luciferase reporter assay, it was found that AaYABBY5 significantly increased the activities of promoters of amorpha-4,11-diene synthase (
ADS
), cytochrome P450 monooxygenase (
CYP71AV1
), double-bond reductase 2 (
DBR2
), and aldehyde dehydrogenase 1 (
ALDH1
) genes. Yeast one hybrid assay showed that AaYABBY5 directly bonds to the promoters of
CYP71AV1
and
DBR2
genes. Quantitative real-time polymerase chain reaction (qPCR) of
AaYABBY5
overexpression and
AaYABBY5
antisense plants revealed a significant increase in the expression of
ADS
,
CYP71AV1
,
DBR2
, and
ALDH1
in
AaYABBY5
overexpression plants and a significant decrease in the expression of these genes in
AaYABBY5
antisense
A. annua
, respectively. Furthermore, the results of high-performance liquid chromatography (HPLC) showed that the artemisinin and its precursor dihydroartemisinic acid were significantly increased in the
AaYABBY5
overexpression plants while
AaYABBY5
downregulation resulted in a significant decrease in the concentration of artemisinin. Taken together, these results explicitly represent that AaYABBY5 is a positive regulator of artemisinin biosynthesis in
A. annua
.
Artemisinin, a sesquiterpenoid endoperoxide, isolated from the plant
L., is widely used in the treatment of malaria. Another sesquiterpenoid, β-caryophyllene having antibiotic, antioxidant, ...anticarcinogenic and local anesthetic activities, is also presented in
. The role played by sesquiterpene transporters in trichomes and accumulation of these metabolites is poorly understood in
and in trichomes of other plant species. We identified
, encoding a pleiotropic drug resistance (PDR) transporter located to the plasma membrane from
. Expression of
is tissue-specifically and developmentally regulated in
. GUS activity is primarily restricted to T-shaped trichomes of old leaves and roots of transgenic
plants expressing
:
. The level of β-caryophyllene was decreased in transgenic
plants expressing
-RNAi while transgenic
plants expressing increased levels of
accumulated higher levels of β-caryophyllene. When AaPDR3 was expressed in transformed yeast, yeasts expressing
accumulated more β-caryophyllene, rather than germacrene D and β-farnesene, compared to the non-expressing control.
Artemisinin is well known for its irreplaceable curative effect on the devastating parasitic disease, Malaria. This sesquiterpenoid is specifically produced in Chinese traditional herbal plant
...Earlier studies have shown that phytohormone abscisic acid (ABA) plays an important role in increasing the artemisinin content, but how ABA regulates artemisinin biosynthesis is still poorly understood. In this study, we identified that AaABF3 encoded an ABRE (ABA-responsive elements) binding factor. qRT-PCR analysis showed that
was induced by ABA and expressed much higher in trichomes where artemisinin is synthesized and accumulated. To further investigate the mechanism of AaABF3 regulating the artemisinin biosynthesis, we carried out dual-luciferase analysis, yeast one-hybrid assay and electrophoretic mobility shift assay. The results revealed that AaABF3 could directly bind to the promoter of
gene, which is a key gene in artemisinin biosynthesis, and activate the expression of
. Functional analysis revealed that overexpression of AaABF3 in
enhanced the production of artemisinin, while RNA interference of AaABF3 resulted in decreased artemisinin content. Taken together, our results demonstrated that AaABF3 played an important role in ABA-regulated artemisinin biosynthesis through direct regulation of artemisinin biosynthesis gene,
.
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