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Abstract Background As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. Results To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants. Conclusions In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.

Summary: Glandular trichomes and cuticles are both specialized structures that cover the epidermis of aerial plant organs. The former are commonly regarded as ‘biofactories’ for producing valuable natural products. The latter are generally considered as natural barriers for defending plants against abiotic and biotic stresses. However, the regulatory network for their formation and relationship remains largely elusive. Here we identify a homeodomain‐leucine zipper (HD‐ZIP) IV transcription factor, AaHD8, directly promoting the expression of AaHD1 for glandular trichome initiation in Artemisia annua. We found that AaHD8 positively regulated leaf cuticle development in A. annua via controlling the expression of cuticle‐related enzyme genes. Furthermore, AaHD8 interacted with a MIXTA‐like protein AaMIXTA1, a positive regulator of trichome initiation and cuticle development, forming a regulatory complex and leading to enhanced transcriptional activity in regulating the expression of AaHD1 and cuticle development genes. Our results reveal a molecular mechanism by which a novel HD‐ZIP IV/MIXTA complex plays a significant role in regulating epidermal development, including glandular trichome initiation and cuticle formation. [ABSTRACT FROM AUTHOR]

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, Aa MIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of Aa MIXTA1 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 Aa MIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that Aa MIXTA1 could directly activate the expression of genes related to cuticle biosynthesis., Taken together, Aa MIXTA1 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. [ABSTRACT FROM AUTHOR]

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 ( Aa GSW1) was cloned and analysed in A. annua. Aa GSW1 exhibited similar expression patterns to the trichome-specific genes of the artemisinin biosynthetic pathway and AP2/ ERF transcription factor Aa ORA. A β-glucuronidase (GUS) staining assay further demonstrated that Aa GSW1 is a glandular trichome-specific transcription factor., Aa GSW1 positively regulates CYP71 AV1 and Aa ORA expression by directly binding to the W-box motifs in their promoters. Overexpression of Aa GSW1 in A. annua significantly improves artemisinin and dihydroartemisinic acid contents; moreover, Aa GSW1 can be directly regulated by Aa MYC2 and Aab ZIP1, which are positive regulators of jasmonate ( JA)- and abscisic acid ( ABA)-mediated artemisinin biosynthetic pathways, respectively., These results demonstrate that Aa GSW1 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 Aa GSW1, CYP71 AV1 and Aa MYC2/ Aab ZIP1 function in the JA/ ABA response in A. annua. [ABSTRACT FROM AUTHOR]

• 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. [ABSTRACT FROM AUTHOR]

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 Aa MYC2 was rapidly induced by JA and Aa MYC2 binds the G-box-like motifs within the promoters of gene CYP71 AV1 and DBR2, which are key structural genes in the artemisinin biosynthetic pathway., Overexpression of Aa MYC2 in A. annua significantly activated the transcript levels of CYP71 AV1 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 Aa MYC2 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 Aa MYC2 is a positive regulator of artemisinin biosynthesis and is of great value in genetic engineering of A. annua for increased artemisinin production. [ABSTRACT FROM AUTHOR]

Jasmonates (JAs) are important signaling molecules in plants and play crucial roles in stress responses, secondary metabolites' regulation, plant growth and development. In this study, the promoter of AaAOC, which was the key gene of jasmonate biosynthetic pathway, had been cloned. GUS staining showed that AaAOC was expressed ubiquitiously in A. annua. AaAOC gene was overexpressed under control of 35S promoter. RT-Q-PCR showed that the expression levels of AaAOC were increased from 1.6- to 5.2-fold in AaAOC-overexpression transgenic A. annua. The results of GC-MS showed that the content of endogenous jasmonic acid (JA) was 2- to 4.7-fold of the control level in AaAOC-overexpression plants. HPLC showed that the contents of artemisinin, dihydroartemisinic acid and artemisinic acid were increased significantly in AaAOC-overexpression plants. RT-Q-PCR showed that the expression levels of FPS (farnesyl diphosphate synthase), CYP71AV1 (cytochrome P450 dependent hydroxylase) and DBR2 (double bond reductase 2) were increased significantly in AaAOC-overexpression plants. All data demonstrated that increased endogenous JA could significantly promote the biosynthesis of artemisinin in AaAOC-overexpression transgenic A.annua. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Flower colour is a complex phenomenon that involves a wide range of secondary metabolites of flowers, for example phenolics and carotenoids as well as co-pigments. Biosynthesis of these metabolites, though, occurs through complicated pathways in many other plant organs. The analysis of the metabolic profile of leaves, stems and roots, for example, therefore may allow the identification of chemomarkers related to the final expression of flower colour. Objective To investigate the metabolic profile of leaves, stems, roots and flowers of Catharanthus roseus and the possible correlation with four flower colours (orange, pink, purple and red). Methods 1H-NMR and multivariate data analysis were used to characterise the metabolites in the organs. Results The results showed that flower colour is characterised by a special pattern of metabolites such as anthocyanins, flavonoids, organic acids and sugars. The leaves, stems and roots also exhibit differences in their metabolic profiles according to the flower colour. Plants with orange flower s featured a relatively high level of kaempferol analogues in all organs except roots. Red-flowered plants showed a high level of malic acid, fumaric acid and asparagine in both flowers and leaves, and purple and pink flowering plants exhibited high levels of sucrose, glucose and 2,3-dihydroxy benzoic acid. High concentrations of quercetin analogues were detected in flowers and leaves of purple-flowered plants. Conclusions There is a correlation between the metabolites specifically associated to the expression of different flower colours and the metabolite profile of other plant organs and it is therefore possible to predict the flower colours by detecting specific metabolites in leaves, stems or roots. This may have interesting application in the plant breeding industry. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.