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Shen, Q., Zhang, L., Liao, Z., Wang, S., Yan, T., Shi, P., . . . Tang, K. (2018). The Genome of Artemisia annua Provides Insight into the Evolution of Asteraceae Family and Artemisinin Biosynthesis. Molecular Plant, 11(6), 776-788
Open this publication in new window or tab >>The Genome of Artemisia annua Provides Insight into the Evolution of Asteraceae Family and Artemisinin Biosynthesis
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2018 (English)In: Molecular Plant, ISSN 1674-2052, E-ISSN 1752-9867, Vol. 11, no 6, p. 776-788Article in journal (Refereed) Published
Abstract [en]

Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin.

Keywords
Artemisia annua, artemisinin, genome, evolution, transcriptome, metabolic engineering
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-76879 (URN)10.1016/j.molp.2018.03.015 (DOI)000434429000003 ()29703587 (PubMedID)
Available from: 2018-07-17 Created: 2018-07-17 Last updated: 2018-07-17Bibliographically approved
Matias-Hernandez, L., Jiang, W., Yang, K., Tang, K., Brodelius, P. E. & Pelaz, S. (2017). AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana. The Plant Journal, 90(3), 520-534
Open this publication in new window or tab >>AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana
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2017 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 3, p. 520-534Article in journal (Refereed) Published
Abstract [en]

The effective anti-malarial drug artemisinin (AN) isolated from Artemisia annua is relatively expensive due to the low AN content in the plant as AN is only synthesized within the glandular trichomes. Therefore, genetic engineering of A. annua is one of the most promising approaches for improving the yield of AN. In this work, the AaMYB1 transcription factor has been identified and characterized. When AaMYB1 is overexpressed in A. annua, either exclusively in trichomes or in the whole plant, essential AN biosynthetic genes are also overexpressed and consequently the amount of AN is significantly increased. Artemisia AaMYB1 constitutively overexpressing plants displayed a greater number of trichomes. In order to study the role of AaMYB1 on trichome development and other possibly connected biological processes, AaMYB1 was overexpressed in Arabidopsis thaliana. To support our findings in Arabidopsis thaliana, an AaMYB1 orthologue from this model plant, AtMYB61, was identified and atmyb61 mutants characterized. Both AaMYB1 and AtMYB61 affected trichome initiation, root development and stomatal aperture in A. thaliana. Molecular analyses indicated that two crucial trichome activator genes are misexpressed in atmyb61 mutant plants and in plants overexpressing AaMYB1. Furthermore, AaMYB1 and AtMYB61 are also essential for gibberellin (GA) biosynthesis and degradation in both species by positively affecting the expression of the enzymes that convert GA(9) into the bioactive GA(4) as well as the enzymes involved in the degradation of GA(4). Overall, these results identify AaMYB1/AtMYB61 as a key component of the molecular network that connects important biosynthetic processes, and reveal its potential value for AN production through genetic engineering.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2017
Keywords
artemisinin, gibberellin, MYB transcription factors, trichome, Artemisia annua, Arabidopsis thaliana
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-64356 (URN)10.1111/tpj.13509 (DOI)000399732200007 ()28207974 (PubMedID)
Available from: 2017-05-24 Created: 2017-05-24 Last updated: 2017-05-24Bibliographically approved
Guo, M., Lu, X., Wang, Y. & Brodelius, P. E. (2017). Comparison of the interaction between lactoferrin and isomeric drugs. Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy, 173, 593-607
Open this publication in new window or tab >>Comparison of the interaction between lactoferrin and isomeric drugs
2017 (English)In: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy, ISSN 1386-1425, E-ISSN 1873-3557, Vol. 173, p. 593-607Article in journal (Refereed) Published
Abstract [en]

The binding properties of pentacyclic triterpenoid isomeric drugs, i.e. ursolic acid (UA) and oleanolic acid (OA), to bovine lactoferrin (BLF) have been studied by molecule modeling, fluorescence spectroscopy, UV-visible absorbance spectroscopy and infrared spectroscopy (IR). Molecular docking, performed to reveal the possible binding mode or mechanism, suggested that hydrophobic interaction and hydrogen bonding play important roles to stabilize the complex. The results of spectroscopic measurements showed that the two isomeric drugs both strongly quenched the intrinsic fluorescence of BLF through a static quenching procedure although some differences between UM and OA binding strength and non-radiation energy transfer occurred within the molecules. The number of binding sites was 3.44 and 3.10 for UA and OA, respectively, and the efficiency of Forster energy transfer provided a distance of 0.77 and 1.21 nm for UA and OA, respectively. The conformation transformation of BLF affected by the drugs conformed to the "all-or-none" pattern. In addition, the changes of the ratios of alpha-helices, beta-sheets and beta-turns of BLF during the process of the interaction were obtained. The results of the experiments in combination with the calculations showed that there are two modes of pentacyclic triterpenoid binding to BLF instead of one binding mode only governed by the principle of the lowest bonding energy.

Keywords
Ursolic acid, Oleanolic acid, Bovine lactoferrin, Interaction
National Category
Analytical Chemistry Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-60150 (URN)10.1016/j.saa.2016.10.029 (DOI)000390502900082 ()
Available from: 2017-01-24 Created: 2017-01-24 Last updated: 2018-05-31Bibliographically approved
Guo, M., Wang, X., Lu, X., Wang, H. & Brodelius, P. E. (2016). alpha-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of alpha-Mangostin to HSA or TRF. PLoS ONE, 11(9), Article ID e0161566.
Open this publication in new window or tab >>alpha-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of alpha-Mangostin to HSA or TRF
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 9, article id e0161566Article in journal (Refereed) Published
Abstract [en]

In order to obtain the biological active compound, alpha-mangostin, from the traditional native mangosteen (Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of a-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of alpha-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (H-1 NMR and C-13 NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained alpha-mangostin was 95.6% as determined by HPLC analysis. The binding of native alpha-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that amangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to alpha-mangostin is 6.4832x10(5) L/mol and 1.4652x10(5) L/mol at 298 K and 7.8619x10(5) L/mol and 1.1582x10(5) L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between alpha-mangostin and HSA or TRF were also obtained by virtue of the Forster theory of non-radiation energy transfer. The effect of alpha-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between amangostin and HSA is hydrophobic interactions, while the main interaction between alpha-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results.

National Category
Organic Chemistry
Research subject
Chemistry, Organic Chemistry
Identifiers
urn:nbn:se:lnu:diva-57613 (URN)10.1371/journal.pone.0161566 (DOI)000382855600041 ()2-s2.0-84990831662 (Scopus ID)
Available from: 2016-10-26 Created: 2016-10-25 Last updated: 2017-11-29Bibliographically approved
Liu, M., Shi, P., Fu, X., Brodelius, P. E., Shen, Q., Jiang, W., . . . Tang, K. (2016). Characterization of a trichome-specific promoter of the aldehyde dehydrogenase 1 (ALDH1) gene in Artemisia annua. Plant Cell Tissue and Organ Culture, 126(3), 469-480
Open this publication in new window or tab >>Characterization of a trichome-specific promoter of the aldehyde dehydrogenase 1 (ALDH1) gene in Artemisia annua
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2016 (English)In: Plant Cell Tissue and Organ Culture, ISSN 0167-6857, E-ISSN 1573-5044, Vol. 126, no 3, p. 469-480Article in journal (Refereed) Published
Abstract [en]

Artemisinin is a frequently used anti-malaria drug extracted from glandular trichomes (GSTs) in Artemisia annua L. In this study, we report on the characterization of the promoter of aldehyde dehydrogenase 1 (ALDH1) involved in the biosynthesis of artemisinin. A 1620-bp promoter fragment was cloned upstream of the ALDH1 start codon. Putative regulatory cis-acting elements are predicted by software, revealing that this gene is affected by complex factors. The activity of the ALDH1 promoter was analyzed using a reporter gene GUS. GUS expression showed a spatial difference in leaves at different ages. In young leaves, GUS staining was exclusively discovered in GSTs. In older leaves, both GSTs and T-shaped trichomes (TSTs) showed GUS signals. Only TSTs showed GUS staining in lower leaves. No GUS staining was detected in the bottom leaves. The result demonstrates that the ALDH1 promoter is trichome-specific. The RT-Q-PCR analysis revealed that both wild-type and recombinant promoters showed similar activity in A. annua. After application of exogenous 100 μM methyl jasmonate, 100 μM gibberellin and 100 μM salicylic acid separately, the transcript levels were increased significantly, indicating that ALDH1 may play an important role in the response to hormones in A. annua.

Keywords
Aldehyde dehydrogenase 1, Artemisia annua, Hormonal treatment, Promoter, Transgene
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biotechnology
Identifiers
urn:nbn:se:lnu:diva-56071 (URN)10.1007/s11240-016-1015-4 (DOI)000382151200009 ()2-s2.0-84983050078 (Scopus ID)
Available from: 2016-09-16 Created: 2016-08-31 Last updated: 2017-11-21Bibliographically approved
Han, J., Wang, H., Kanagarajan, S., Hao, M., Lundgren, A. & Brodelius, P. E. (2016). Promoting Artemisinin Biosynthesis in Artemisia annua Plants by Substrate Channeling [Letter to the editor]. Molecular Plant, 9(6), 946-948
Open this publication in new window or tab >>Promoting Artemisinin Biosynthesis in Artemisia annua Plants by Substrate Channeling
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2016 (English)In: Molecular Plant, ISSN 1674-2052, E-ISSN 1752-9867, Vol. 9, no 6, p. 946-948Article in journal, Letter (Refereed) Published
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-54682 (URN)10.1016/j.molp.2016.03.004 (DOI)000377531600018 ()26995295 (PubMedID)2-s2.0-84966538972 (Scopus ID)
Available from: 2016-07-22 Created: 2016-07-21 Last updated: 2017-12-07Bibliographically approved
Wang, B., Kashkooli, A. B., Sallets, A., Ting, H.-M., de Ruijter, N. C. A., Olofsson, L., . . . van der Krol, A. R. (2016). Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua. Metabolic engineering, 38, 159-169
Open this publication in new window or tab >>Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua
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2016 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 38, p. 159-169Article in journal (Refereed) Published
Abstract [en]

Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.

Keywords
ABC transporters, Artemisia annua, Artemisinin, Lipid transfer proteins, Nicotiana benthamiana, Pleiotropic Drug Resistance protein
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-56057 (URN)10.1016/j.ymben.2016.07.004 (DOI)000387984600017 ()27421621 (PubMedID)2-s2.0-84982854633 (Scopus ID)
Available from: 2016-09-16 Created: 2016-08-31 Last updated: 2017-11-21Bibliographically approved
Yang, K., Rashidi Monfared, S., Wang, H., Lundgren, A. & Brodelius, P. E. (2015). The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.. Plant Molecular Biology, 88(4-5), 325-340
Open this publication in new window or tab >>The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.
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2015 (English)In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 88, no 4-5, p. 325-340Article in journal (Refereed) Published
Abstract [en]

The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the ‘2/39’, ‘Chongqing’ and ‘Anamed’ varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the ‘Meise’, ‘Iran#8’, ‘Iran#14’, ‘Iran#24’ and ‘Iran#47’ varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties. © 2015, Springer Science+Business Media Dordrecht.

Keywords
Artemisia annua, Artemisinin, Cis-acting regulatory elements, DBR2, GC–MS, Promoter cloning, qPCR
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-54998 (URN)10.1007/s11103-015-0284-3 (DOI)000357052100001 ()2-s2.0-84933675640 (Scopus ID)
Note

Erratum: http://dx.doi.org/10.1007/s11103-015-0332-z

Available from: 2016-07-22 Created: 2016-07-22 Last updated: 2017-11-28Bibliographically approved
Han, J., Wang, H., Lundgren, A. & Brodelius, P. E. (2014). Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants. Phytochemistry, 102, 89-96
Open this publication in new window or tab >>Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants
2014 (English)In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 102, p. 89-96Article in journal (Refereed) Published
Abstract [en]

The effective anti-malarial medicine artemisinin is costly because of the low content in Artemisia annua. Genetic engineering of A. annua is one of the most promising approaches to improve the yield of artemisinin. In this work, the transcription factor AaWRKY1, which is thought to be involved in the regulation of artemisinin biosynthesis, was cloned from A. annua var. Chongqing and overexpressed using the CaMV35S promoter or the trichome-specific CYP71AV1 promoter in stably transformed A. annua plants. The transcript level of AaWRKY1 was increased more than one hundred times under the CaMV35S promoter and about 40 times under the CYP71AV1 promoter. The overexpressed AaWRKY1 activated the transcription of CYP71AV1 and moreover the trichome-specific overexpression of AaWRKY1 improved the transcription of CYP71AV1 much more effectively than the constitutive overexpression of AaWRKY1, i.e. up to 33 times as compared to the wild-type plant. However the transcription levels of FDS, ADS, and DBR2 did not change significantly in transgenic plants. The significantly up-regulated CYP71AV1 promoted artemisinin biosynthesis, i.e. up to about 1.8 times as compared to the wild-type plant. It is demonstrated that trichome-specific overexpression of AaWRKY1 can significantly activate the transcription of CYP71AV1 and the up-regulated CYP71AV1 promotes artemisinin biosynthesis.

Keywords
AaWRKY1; Artemisia annua; Artemisinin biosynthesis; CYP71AV1 promoter; CYP71AV1; Trichome-specific overexpression
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-33362 (URN)10.1016/j.phytochem.2014.02.011 (DOI)000336339700008 ()2-s2.0-84899438852 (Scopus ID)
Available from: 2014-03-27 Created: 2014-03-27 Last updated: 2017-12-05Bibliographically approved
Wang, H., Kanagarajan, S., Han, J., Hao, M., Yang, Y., Lundgren, A. & Brodelius, P. E. (2014). Studies on the expression of linalool synthase using a promoter-beta-glucuronidase fusion in transgenic Artemisia annua. Journal of plant physiology (Print), 171(2), 85-96
Open this publication in new window or tab >>Studies on the expression of linalool synthase using a promoter-beta-glucuronidase fusion in transgenic Artemisia annua
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2014 (English)In: Journal of plant physiology (Print), ISSN 0176-1617, E-ISSN 1618-1328, Vol. 171, no 2, p. 85-96Article in journal (Refereed) Published
Abstract [en]

Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to β-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652 bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.

National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-31306 (URN)10.1016/j.jplph.2013.09.019 (DOI)000331008600011 ()2-s2.0-84890220315 (Scopus ID)
Available from: 2013-12-18 Created: 2013-12-18 Last updated: 2017-12-06Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-8899-5046

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