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Olofsson, Linda
Publications (6 of 6) Show all publications
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
Olofsson, L., Lundgren, A. & Brodelius, P. E. (2012). Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.. Plant Science, 183, 9-13
Open this publication in new window or tab >>Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.
2012 (English)In: Plant Science, ISSN 0168-9452, E-ISSN 1873-2259, Vol. 183, p. 9-13Article in journal (Refereed) Published
Abstract [en]

The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.

Keywords
LMPC; Artemisia annua; Trichomes; qPCR; Gene expression; Artemisinin biosynthesis
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-18066 (URN)10.1016/j.plantsci.2011.10.019 (DOI)2-s2.0-82155203073 (Scopus ID)
Available from: 2012-03-20 Created: 2012-03-20 Last updated: 2017-12-07Bibliographically approved
Olofsson, L., Engström, A., Lundgren, A. & Brodelius, P. E. (2011). Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L.. BMC Plant Biology, 11, Article ID 45.
Open this publication in new window or tab >>Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L.
2011 (English)In: BMC Plant Biology, ISSN 1471-2229, E-ISSN 1471-2229, Vol. 11, article id 45Article in journal (Refereed) Published
Abstract [en]

BackgroundRecently, Artemisia annua L. (annual or sweet wormwood) has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for treatment of malaria. The plant produces relatively small amounts of artemisinin and a worldwide shortage of the drug has led to intense research in order to increase the yield of artemisinin. In order to improve our understanding of terpene metabolism in the plant and to evaluate the competition for precursors, which may influence the yield of artemisinin, we have used qPCR to estimate the expression of 14 genes of terpene metabolism in different tissues.

ResultsThe four genes of the artemisinin biosynthetic pathway (amorpha-4,11-diene synthase, amorphadiene-12-hydroxylase, artemisinic aldehyde ∆11(13) reductase and aldehyde dehydrogenase 1) showed remarkably higher expression (between ~40- to ~500-fold) in flower buds and young leaves compared to other tissues (old leaves, stems, roots, hairy root cultures). Further, dihydroartemisinic aldehyde reductase showed a very high expression only in hairy root cultures. Germacrene A and caryophyllene synthase were mostly expressed in young leaves and flower buds while epi-cedrol synthase was highly expressed in old leaves. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase exhibited lower expression in old leaves compared to other tissues. Farnesyldiphosphate synthase, squalene synthase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase showed only modest variation in expression in the different tissues, while expression of 1-deoxy-D-xylulose-5-phosphate synthase was 7-8-fold higher in flower buds and young leaves compared to old leaves.

ConclusionsFour genes of artemisinin biosynthesis were highly expressed in flower buds and young leaves (tissues showing a high density of glandular trichomes). The expression of dihydroartemisinic aldehyde reductase has been suggested to have a negative effect on artemisinin production through reduction of dihydroartemisinic aldehyde to dihydroartemisinic alcohol. However, our results show that this enzyme is expressed only at low levels in tissues producing artemisinin and consequently its effect on artemisinin production may be limited. Finally, squalene synthase but not other sesquiterpene synthases appears to be a significant competitor for farnesyl diphosphate in artemisinin-producing tissues.

National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-18063 (URN)10.1186/1471-2229-11-45 (DOI)2-s2.0-79952328863 (Scopus ID)
Available from: 2012-03-20 Created: 2012-03-20 Last updated: 2017-12-07Bibliographically approved
Wang, H., Olofsson, L., Lundgren, A. & Brodelius, P. E. (2011). Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in Artemisia annua L., as Reported by a Promoter-GUS Fusion. American Journal of Plant Sciences, 2(4), 619-628
Open this publication in new window or tab >>Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in Artemisia annua L., as Reported by a Promoter-GUS Fusion
2011 (English)In: American Journal of Plant Sciences, ISSN 2158-2742, E-ISSN 2158-2750, Vol. 2, no 4, p. 619-628Article in journal (Refereed) Published
Abstract [en]

Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the ␣-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activ- ity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.

Keywords
Agrobacterium Tumefaciens, Amorpha-4, 11-Diene Synthase, Artemisia annua, Artemisinin Biosynthesis, ␣-Glucuronidase, Gene Regulation, Promoter Activity, Stable Transformation
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-18065 (URN)10.4236/ajps.2011.24073 (DOI)
Available from: 2012-03-20 Created: 2012-03-20 Last updated: 2017-12-07Bibliographically approved
Olsson, M. E., Olofsson, L., Lindahl, A.-L., Lundgren, A., Brodelius, M. & Brodelius, P. E. (2009). Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L. Phytochemistry, 70(9), 1123-1128
Open this publication in new window or tab >>Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L
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2009 (English)In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Phytochemistry, Vol. 70, no 9, p. 1123-1128Article in journal (Refereed) Published
Abstract [en]

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta 11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1 alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.

National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-2133 (URN)10.1016/j.phytochem.2009.07.009 (DOI)
Available from: 2010-04-06 Created: 2010-04-06 Last updated: 2017-12-12Bibliographically approved
Picaud, S., Olofsson, L., Brodelius, M. & Brodelius, P. (2005). Expression, purification and characterization of amorpha-4,11-diene synthase from Artemisia annua L. Archives of Biochemistry and Biophysics, 436 (2), 215-226
Open this publication in new window or tab >>Expression, purification and characterization of amorpha-4,11-diene synthase from Artemisia annua L
2005 (English)In: Archives of Biochemistry and Biophysics, Vol. 436 (2), p. 215-226Article in journal (Refereed) Published
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-566 (URN)
Available from: 2010-04-01 Created: 2010-04-01 Last updated: 2016-10-25Bibliographically approved
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