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Wang, Hongzhen
Publications (6 of 6) Show all publications
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

Correction published in: http://dx.doi.org/10.1007/s11103-015-0332-z

Available from: 2016-07-22 Created: 2016-07-22 Last updated: 2018-08-30Bibliographically 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
Wang, H., Han, J., Kanagarajan, S., Lundgren, A. & Brodelius, P. E. (2013). Studies on the Expression of Sesquiterpene Synthases Using Promoter-b-Glucuronidase Fusions in Transgenic Artemisia annua L. PLoS ONE, 8(11), Article ID e80643.
Open this publication in new window or tab >>Studies on the Expression of Sesquiterpene Synthases Using Promoter-b-Glucuronidase Fusions in Transgenic Artemisia annua L
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, article id e80643Article in journal (Refereed) Published
Abstract [en]

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for beta-caryophyllene (CPS), epi-cedrol (ECS) and beta-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-31304 (URN)10.1371/journal.pone.0080643 (DOI)000327541700031 ()2-s2.0-84894239991 (Scopus ID)
Available from: 2013-12-18 Created: 2013-12-18 Last updated: 2017-12-06Bibliographically approved
Wang, H., Han, J., Kanagarajan, S., Lundgren, A. & Brodelius, P. E. (2013). Trichome-specific expression of the amorpha-4,11-diene 12-hydroxylase (cyp71av1) gene, encoding a key enzyme of artemisinin biosynthesis in Artemisia annua, as reported by a promoter-GUS fusion. Plant Molecular Biology, 81(1-2), 119-138
Open this publication in new window or tab >>Trichome-specific expression of the amorpha-4,11-diene 12-hydroxylase (cyp71av1) gene, encoding a key enzyme of artemisinin biosynthesis in Artemisia annua, as reported by a promoter-GUS fusion
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2013 (English)In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 81, no 1-2, p. 119-138Article in journal (Refereed) Published
Abstract [en]

Artemisinin derivatives are effective anti-malarial drugs. In order to design transgenic plants of Artemisia annua with enhanced biosynthesis of artemisinin, we are studying the promoters of genes encoding enzymes involved in artemisinin biosynthesis. A 1,151 bp promoter region of the cyp71av1 gene, encoding amorpha-4,11-diene 12-hydroxylase, was cloned. Alignment of the cloned promoter and other cyp71av1 promoter sequences indicated that the cyp71av1 promoter may be different in different A. annua varieties. Comparison to the promoter of amorpha-4,11-diene synthase gene showed a number of putative cis-acting regulatory elements in common, suggesting a co-regulation of the two genes. The cyp71av1 promoter sequence was fused to the beta-glucuronidase (GUS) reporter gene and two varieties of A. annua and Nicotiana tabacum were transformed. In A. annua, GUS expression was exclusively localized to glandular secretory trichomes (GSTs) of leaf primordia and top expanded leaves. In older leaves, there is a shift of expression to T-shaped trichomes (TSTs). Only TSTs showed GUS staining in lower leaves and there is no GUS staining in old leaves. GUS expression in flower buds was specifically localized to GSTs. The recombinant promoter carries the cis-acting regulatory elements required for GST-specific expression. The cyp71av1 promoter shows activity in young tissues. The recombinant promoter was up to 200 times more active than the wild type promoter. GUS expression in transgenic N. tabacum was localized to glandular heads. Transcript levels were up-regulated by MeJA. Wound responsiveness experiment showed that the cyp71av1 promoter does not appear to play any role in the response of A. annua to mechanical stress.

Keywords
Agrobacterium tumefaciens, Amorpha-4, 11-diene 12-hydroxylase, Artemisia annua, Artemisinin biosynthesis, beta-glucuronidase, Gene regulation, Promoter activity, Stable transformation
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-24509 (URN)10.1007/s11103-012-9986-y (DOI)000312872900009 ()2-s2.0-84871460414 (Scopus ID)
Available from: 2013-02-22 Created: 2013-02-22 Last updated: 2017-12-06Bibliographically 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
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