American Journal of Plant Sciences, 2011, 2, 619-628
doi:10.4236/ajps.2011.24073 Published Online October 2011 (
Copyright © 2011 SciRes. AJPS
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
Hongzhen Wang, Linda Olofsson, Anneli Lundgren, Peter E. Brodelius
School of Natural Sciences, Linnaeus University, Kalmar, Sweden.
Received August 16th, 2011; revised September 7th, 2011; accepted September 20th, 2011.
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 o rder
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 β-glucuronidase (GUS) reporter gene. Transgenic
plants of A. annua expressing this fusion were g enerated 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 ac tivity 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 enha ncing 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, Artemisi nin Biosynthesi s,
β-Glucuronidase, Gene Regulation, Promoter Activity, Stable Transformation
1. Introduction
Artemisinin is an effective anti-malarial drug, which has
become an important component of artemisinin-based
combination therapies (ACTs) [1]. The content of ar-
temisinin in Artemisia annua is, however, very low and
ranges between 0.1% and 0.8% of dry weight [2]. Selec-
tion of high-producing varieties of A. annua has met lim-
ited success but hybrids producing increased amounts of
artemisinin have been produced [3]. However, there is
still a shortage in the supply o f artemisinin [4]. Different
methods have been tried to improve the artemisinin pro-
duction such as treatment with abscisic acid [5], gibbere-
lic acid [6-8] and elicitors [9-11]. Various transgenic A.
annua plants have been produced to increase the yield of
artemisinin. These attempts include down-regulation of
squalene synthase [12,13] or up-regulation of farnesyl
diphosphate synthase (FDS) [14].
Cyclization of farnesyl diphosphate (FDP) to amor-
pha-4,11-diene by amorpha-4,11-diene synthase (ADS)
is the initial step of the artemisinin biosynthetic pathway
[15] and amorpha-4,11-diene is the committed precursor
[16]. Tissue specificity of ADS expression has been
shown by GUS expression in Arabidopsis thaliana using
a fusion of the ADS promoter and the reporter gene [17].
In the following step of the artemisinin biosynthesis,
amorpha-4,11-diene is hydroxylated to yield artemisinic
alcohol. This reaction is catalyzed by a cytochrome P450
dependent amorpha-4,11-diene 12-hydroxylase (CYP71AV1)
Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis
620 in Artemisia annua L., as Reported by a Promoter-GUS Fusion
[18]. This enzyme is multifunctional and can also oxidize
the alcohol to artemisinic aldehyde and then further on to
artemisinic acid [19]. However, artemisinic acid is a di-
rect precursor of arteannuin B biosynthesis. For artemisi-
nin biosynthesis the artemisinic aldehyde must be re-
duced to dihydroartemisinic aldehyde, which subse-
quently is oxidized to dihydroartemisinic acid and then
converted to artemisinin [20]. These two steps are cata-
lyzed by artemisinic aldehyde 11 (13) reductase [21]
and aldehyde dehydrogenase 1 [22], respectively. Glan-
dular trichome specific expression of ADS and CYP71-
AV1 [17,18] strongly supported the notion that artemisi-
nin is sequestered and localized to glandular trich omes of
A. annua [23,24].
In order to understand the mechanisms behind the in-
crease in artemisinin biosynthesis after treatment with
hormones [5-8] or biotic elicitors [9-11], a detailed
knowledge about th e regulation of expr ession of differ ent
enzymes involved in artemisinin biosynthesis will be
essential. To our knowledge, there is no data available on
the regulation of expression of biosynthetic genes in A.
annua using promoter-reporter gene fusions in transgenic
plants of A. annua. We have initiated studies on the
regulation of terpene metabolism in different tissues of A.
annua [25] and here we report on results from transgenic
A. annua expressing a fusion of the reporter gene (GUS)
and the promoter of the key enzyme ADS.
2. Materials and Methods
2.1. Plant Materials
Plants of A. annua were grown under 16 h days and 8 h
nights at 22˚C to a height of approximately 1 m followed
by induction of flowering at 8 h days and 16 h nights at
22˚C. Flower buds, young leaves, old leaves, stems and
roots were collected for GUS staining. Samples of these
tissues were also frozen in liquid nitrogen, ground and
used for RNA extraction.
2.2. Promoter Cloning
Genomic DNA was extracted from fresh young leaves of
A. annua using the CTAB method [26]. The promoter
sequence of ADS was available in the GenBank (acces-
sion number AY528931). The sequence was used to de-
sign primers for PCR amplification (primers 1 and 2 in
Table 1) of a 1929 bp fragment of the ADS promoter
using Pfu polymerase (Fermentas). Primers 1 and 2 car-
ried Xm a I and NcoI restriction site, respectiv ely, used for
cloning the fragment into the plant transformation vector.
2.3. Construction of Transformation Vector
The pCAMBIA 1381Z vector (CambiaLabs, Brisbane,
Table 1. Nucleotide sequence of primers used. Restriction
sites are underlined; f = forward; r = reverse.
PrimerNamePrimer Sequence
Australia) carrying the GUS-gene was used for the trans-
formation of A. annua. However, the vector was modi-
fied in that the plant resistance gene was changed from
hygromycin to kanamycin. An XhoI fragment (0.88 kb)
carrying the NPTII gene was cut out from pCAMBIA
2301 and ligated into pCAMBIA 1381Z digested with
XhoI. Restriction analysis was used to determine that the
XhoI fragment had been ligated in the right direction.
The ADS promoter was double-digested with NcoI/
XmaI and inserted into the modified pCAMBIA 1381Z
vector digested with the same restriction enzymes. The
plant transformation vector obtained (pCAMBIA1381Z-
pADS-GUS) was introduced into E. coli BL21, which
was grown on LB medium containing kanamycin (50
mg/L). The vector was purified using the GeneJet Plas-
mid Miniprep Kit (Fermentas) and used for transforma-
tion of Agrobacetrium tumefaciens EHA105.
The plant transformation vector was introduced into A.
tumefaciens EHA105 by the freeze and thaw method. A.
tumefaciens EHA105 carrying the plant transformation
vector was grown on YEP medium containing kanamy-
cin (100 mg/L), rifampicin (40 mg/L) and streptomycin
(25 mg/L) at 28˚C to an OD600 = 0.8 – 1. The cells were
collected by centrifugation and resuspended in MSMO
liquid medium to an OD600 = 0.3 – 0.5 .
2.4. Plant Transformation
A. annua seeds (variety Chongqin) were immersed in
75% ethanol for 1 min, surfaced sterilized with 5% so-
dium hypochlorite (NaOCl) for 20 min, washed 3 - 4
times with sterile distilled water. The sterilized seeds
were germinated on solid MSMO medium (Sigma)
(MSMO powder 4.4 g/L, sucrose 30 g/L, agar 8 - 9 g/L,
pH = 5.8) at 28˚C for 20 - 25 days. The leaflets of 20 - 25
days old seedlings were used as explants for transforma-
tion. The explants were immersed into a suspension of A.
tumefaciens EHA105 carrying the pCAMBIA1381Z-
pADS-GUS transformation vector. After 30 min the ex-
plants were transferred to co-cultivation medium (MSMO
Copyright © 2011 SciRes. AJPS
Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis 621
in Artemisia annua L., as Reported by a Promoter-GUS Fusion
medium containing 6-BA (0.5 mg/L), NAA (0.05 mg/L))
and the A. tumefaciens EHA105. Co-cultivation was car-
ried out in darkness at 28˚C for 48 h. Subsequently, the
explants were transferred to selection and germination
medium (MSMO medium containing 6-BA (0.5 mg/L),
NAA (0.05 mg/L), kanamycin (100 mg/L) and carbeni-
cillin (250 mg/L)). The explants were transferred to new
medium every second week. Shoots started to grow out
after around two weeks. The shoots were transferred to
root inducing medium (half strength MSMO medium
containing kanamycin (100 mg/L) and carbenicillin (100
mg/L)). After rooting the plantlets were left 4 weeks be-
fore they were transferred to soil.
2.5. Scanning Electron Microscopy
Scanning electron microscopy (SEM) was carried out on
unfixed tissues from wild-type and transgenic A. annua
plants using a relatively low energy beam (5 kV) on a
LEO 435VP scanning electron microscope.
2.6. GUS Assay
Leaf primordia at the apex and expanded leaves at dif-
ferent nodes, stems, roots from shoots and flowering
plants, and flower buds were sampled from transgenic A.
annua plants to perform GUS analysis. GUS histo-
chemical staining of the various plant tissues was carried
out as previously described [27]. GUS stained tissues
were studied under a microscope (Nikon ECLIPSE E400)
and photographs were taken using a digital camera
(Nikon DP11).
2.7. qPCR
Different tissues of transgenic A. annua plants were
sampled to analyze the relative expression pattern of
pADS-GUS and pADS-ADS using β-actin as the reference
gene. RNA was extracted using PurelinkTM Plant RNA
Reagent (Invitrogen, Carlsbad, CA, USA) according to
the manufacturer’s instruction. Genomic DNA was re-
moved by treatment with DNase I (Fermentas, St Leo-
Roth, Germany). RNA (1 μg) was reverse transcribed
using RevertAidTM H Minus-MuLV reverse transcriptase
(Fermentas) primed with 0.5 μg oligo (dT)18 primer.
RNA was removed from the cDNA obtained by treat-
ment with RNas e H (Fermentas).
The qPCR was performed on a 7500 qPCR thermocy-
cler (Applied Biosystems, Foster City, USA) using prim-
ers listed in Table 1 for GUS (primers 3 and 4), ADS
(primers 5 and 6) or actin (primers 7 an d 8). First single-
stranded cDNA was used as template in a 20 μl reaction
mixture containing 10 μl Power SYBR® Green PCR
Master Mix (Applied Biosystems) and 2 pmol of each
primer. The qPCR thermal cycling was performed at
50˚C (2 min), 95˚C (10 min), 40 cycles at 95˚C (15 sec),
60˚C (1 min) and finally a dissociation state at 95˚C (15
sec), 60˚C (1 min) and 95˚C (15 sec). Triplet samples
were run for each cDNA sample.
2.8. Statistical Methods
Grubbs test (G = |Suspect value-xmean|/s) was used to test
for outliers. The outliers were rejected if Gcalculated >
Gcritical at P = 0.05. The critical value of G is 1.155 when
the sample size is three [19].
3. Results and Discussion
3.1. Cloning of Promoter of ADS
Based on the sequence of the promoter region of ADS
(GenBank accession number AY528931), a promoter
region of 1929 bp upstream of the translation initial
codon (ATG) as shown in Figure 1 was isolated from A.
annua genomic DNA by PCR using high fidelity poly-
merase and primers 1 and 2 as listed in Table 1.
3.2. Prediction of Transcription Start Site and
Core Promoter Elements
The transcription start site (TSS) of the cloned promoter
was predicted using the TSSP software (http://linux1. A putative TSS of ADS (la-
beled +1 in Figure 1) was predicted 51 bp upstream of
the translation initiation ATG-codon.
The TATA-box is a cis-regulatory element found in
the promoter region of many genes in eukaryotes with
the core sequence or a variant, which is usually follo wed
by three or more adenine bases. It is considered to be the
core promoter sequence and the binding site of either
general tran scription factors or histones (the binding of a
transcription factor blocks the binding of a histone and
vice versa). It is involved in the process of transcription
by RNA polymerase and is usually located around 25 bp
upstream of the TSS. A putative TATA-box was found at
positions –29 to –24 (TATAAA) in the ADS promoter
(Figure 1) upstream of the putative TSS.
The CAAT-box signal is the binding site for the RNA
transcription factor, and is typically accompanied by a
conserved consensus sequence (GGCCAATCT). Genes
that have this element seem to require it for the gene to
be transcribed in sufficient quantities. This box along
with the GC-box is known for binding general transcrip-
tion factors. CAAT- and GC-boxes are primarily located
in the region from 100 - 150 bp upstream from the
TATA-box. Binding of specific protein is required for
the CAAT-box activation. These proteins are known as
CAAT-box binding proteins/CAAT-box binding factors.
e could not find any CAAT-box with the consensus W
Copyright © 2011 SciRes. AJPS
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
Copyright © 2011 SciRes. AJPS
Figure 1. Nucleotide sequence of the cloned ADS promoter with putative cis-elements shown.
sequence GGCCAATCT. However, CAAT-box sequ-
ences may differ considerably from the consensus se-
quence as exemplified by the promoters of the EAS3 and
EAS4 genes encoding the sesquiterpene synthase epi-
aristolochene synth ase in Nicotiana tabacum [28], which
were determined to be GATCAATTA for both promoters.
Two putative CAAT- boxes were identified within 150
bp upstream of the TATA-box of the ADS promoter as
shown in Figure 1 (e.g. AATCAATTG orATTCAATTG
at positions –145 to –137 and –77 to –69 upstream of the
TSS, respectively). It may be pointed out that by in-
specting the promoter sequences for TATA- and CAAT-
Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis 623
in Artemisia annua L., as Reported by a Promoter-GUS Fusion
boxes a number of alternative putative TSSs may be
3.3. Prediction of Cis-Elements of Promoter
Region of ADS
Putative cis-elements of the promoter were predicted by
using the PLANTCARE software (http://bioinformatics.
The py-rich sequence TTTCTTCTC is a cis-acting
element conferring high transcription levels. Such an
element was found at position –1204 to –1212 as shown
in Figure 1.
Yang and Poovaiah (2000) reported that the tobacco
ethylene-responsive gene NtER1 encodes a calmodulin-
binding protein [29] and demonstrated later that there is
one NtER1 homolog as well as five related genes in
Arabidopsis [30]. These six Arabidopsis genes are rap-
idly and differentially induced by environmental signals
such as extreme temperatures, UV-B, salt, wounding,
hormones such as ethylene and abscisic acid (ABA), and
signal molecules such as methyl jasmonate (MeJA),
H2O2, and salicylic acid (SA). Hence, they were design-
nated as AtSR1 to 6 (Arabidopsis thaliana signal-re-
sponsive genes). AtSR1 targets the nucleus and specifi-
cally recognizes a 6-bp CGCG-box (A/C/G)CGCG(G/T/
C). Multiple CGCG cis-elements are found in promoters
of genes involved in ethylene, ABA and SA signaling,
and light signal perception. A putative CGCG-box at
positions –240 to –235 was found in the ADS promoter
(Figure 1).
The TCA-element with the seq u ence GAG AA GAATA
is involved in responsiveness of genes to SA [31]. One
such element with the sequence GAGAAGAAAA (–1212
to –1203) was foun d in the ADS promoter (Figure 1). A
putative ethylene responsiveness ERE-element (ATTTC-
AAA) was localized to –256 to –268. Furthermore, a
putative gibberellin (GA)-responsive element (the
GARE-motif AAACAGA) is present in the ADS pro-
moter at position –1354 to –1348 and a modified GARE-
motif (TAACAAG) at position –348 to –342. A TC-rich
repeat, which is involved in defense and stress response,
was localized to position –387 to –396 (ATTGTCTTCA).
Finally, an AU-RR core sequence (GGTCCAT), which is
involved in auxin responsiveness, was found at position
–1116 to –1122.
The elements described above may be involved in the
response of A. annua to plant hormones (ABA, SA, eth-
ylene, MeJA and GA). In fact, treatment of A. annua
plants with ABA (10 μM) resulted in the induction of a
number of genes encoding enzymes involved in artemis-
inin biosynthesis [5]. The expression levels of 3-hy-
droxy-3-methylglutaryl coenzyme A reductase (HMGR),
farnesyl diphosphate synthase (FDS), CYP71AV1 and
cytochrome P450 reductase (CPR) were significantly
induced while ADS only showed a slight increase. The
treatment with ABA resulted in a 65% increase in ar-
temisinin content in A. annua plants [5].
Treatment of A. annua plants with 1 mM SA resulted
in a gradual increase in the expression of the HMGR
gene and a temporary peak in the expression of the ADS
gene [32]. The expression of the FDS and CYP71AV1
genes showed little change. At 96 h after SA treatment,
the concentration of artemisinin, artemisinic acid and
dihydroartemisinic acid were 54%, 127% and 72%
higher than th at of the control, respectively [32].
In a study on the production of artemisinin in cell sus-
pension cultures of A. annua , acetyl-SA (20 mg/L), JA (5
mg/L) or GA (10 mg/L) were efficient elicitors and gave
around 2-, 4- and 2.5-fold increased yield of artemisinin,
respectively [10], indicating an increased expression of
the key regulatory enzyme ADS. The beneficial effect of
GA3 on artemisinin accumulation is also supported by
the findings that treatment of A. annua plants with half-
strength Hoagland’s solution containing 14 mM GA3
increased artemisinin content from 0.14% to 0.64% (w/w)
when applied to 74-day-old plants [8]. No studies on the
effects of GA on the expression level of enzymes in-
volved in artemisinin biosynthesis have been reported.
Two W1-boxes ((C/T)TGAC(C/T)), defined as elicit-
tor-responsive elements, have been iden tified at positions
–255 to –250 and –926 to –931 (Figure 1). The tran-
scription factor WRKY binds to these elements trigger-
ing the transcription of the gene. Transient expression of
the A. annua transcription factor AaWRKY in agroinfil-
trated leaves of A. annua resulted in increased levels of
HMGR, ADS, CYP71AV1 and DBR2 indicating that
several of the genes encoding enzymes involved in ar-
temisinin biosynthesis are induced by binding of
AaWRKY to the W-boxes [33]. In cotton, the GaWRKY1
is regulating the activity of cadinene synthase 1 (CAD1)
involved in the biosynthesis of sesquiterpene phytoalex-
ins [34]. The CAD1 promoter carries two W-boxes to
which the GaWRKY 1 binds. A recen t study showed that
foliar application of an elicitor (chitosan) resulted in in-
crease of dihydroartemisinic acid and artemisinin by 72%
and 53%, respectively, in A. annua [11]. Furthermore,
semi-quantitative RT-PCR showed an increased level of
ADS transcripts 2 h after application of chitosan.
GATA-factors, involved in light-mediated regulation,
are a class of transcriptional regulators present in plants
that normally recognize the consensus sequence (T/A)
GATA(G/A) [35]. Twelve putative GATA-boxes were
found in the promoter r egion of ADS as shown in Figure
1. Other elements putatively involved in light-mediated
Copyright © 2011 SciRes. AJPS
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
Copyright © 2011 SciRes. AJPS
3.4. Activity of the ADS Promoter regulation are the ACE-motif (AAACCGGTTA; position
–982 to –973), the AE-box (AGAAACAA; position
–1350 to –1343), the Box 1 (TTTCAAA; position –257
to –263 and +47 to +41), the Box 4 (ATTAAT; positio ns
–784 to –779 and –1455 to –1450), the G-box (CACGT
(T/C); position –1529 to –1524, –1418 to – 1413 and
–472 to –467), the GT1-motif (AATCCACA; position
–1266 to –1259), the Sp1-motif (CC(G/A)CCC; position
–1063 to –1068) and the TCT-motif (TCTTAC; position
–274 to –279).
In order to study the temporal and spatial expression of
ADS, the cloned ADS promoter was inserted into the
modified (kanamycin) pCAMBIA 1381Z plant transfor-
mation vector carrying the GUS reporter gene. Trans-
genic A. annua plants carrying the fusion of the ADS
promoter and the GUS gene were produced using Agro-
bacterium tumefaciens. Twenty-seven positive T0 trans-
genic lines were selected by PCR. Fifteen of these T0
transgenic lines showed similar GUS staining pattern
while the other twelve lines did not show any obvious
GUS staining, which may be due to silencing of the in-
troduced GUS gene .
Two putative MYB binding sites (MBS) (TAACNG)
at positions –697 to –692 and –1499 to –1504 were
found in the ADS promoter. In Arabidopsis, they are the
binding site for the myb proteins ATMYB1 and AT-
MYB2 involved in regulation of genes that are respon-
sive to water stress [36] and in Petunia hybrida for
MYB.Ph3 involved in regulation of flavonoid biosynthe-
sis [37]. Twenty-eight MYB transcription factors have
been identified in A. annua according to the plant tran-
scription factor database (
index.php? sp=Aan). To our knowledge, no study on the
effects of MYB transcription factors on metabolism in A.
annua has been reported.
There are two kinds of trichomes on aerial organs of A.
annua, i.e. glandular secretory trichomes (GSTs) and
non-glandular T-shaped trichomes (TST) (Figure 2). GUS
staining was observed in GSTs of leaves (Figure 3(a)),
stems of one-week old plantlets after root initiation (Fig-
ure 3(b)), leaf primordia (Figure 3(d)) and flower buds
(Figure 3(e) and (f)). No GUS staining was observed in
roots of plantlets (Figure 3(c)) but GUS staining was ob-
served in roots at the reproductive stage. Furthermore, no
GUS staining was observed in TSTs on leaves (Figure
3(g)). Based on data from the promoter/reporter fusion
studies, it appears that ADS, the key enzyme of artemisi-
nin biosynthesis, is almost exclusively expressed in
GSTs and consequently the biosynthesis of artemisinin
precursors is localized to this type of trichomes.We have
previously reported that ADS only is expressed in apical
cells of glandular trichomes [40]. However, the results
obtained here clearly shows that the ADS pr o mo t e r also i s
active in sub-apical cells (Figure 3(h)). This difference
may be due to fixation of the tissue with formaldehyde
before isolation of the apical cells by laser microdissec-
tion [40]. In a recent study, we have shown that isolation
of RNA from formaldehyde-fixed cells is problematic
and difficult to do in a reproducible way [L. Olofsson et
al., unpublished]. However, isolation of RNA from un-
fixed cells with subsequent amplification is a good
A number of amorphane sesquiterpenes have been iso-
lated from seeds of A. annua including artemisinic acid,
dihydroartemisinic acid and arteannuin B indicating that
enzymes of artemisinin biosynthesis are expressed in
seeds [38]. These results have been questioned since
there are no trichomes on seeds [39]. However, the ADS
promoter contains a number of putative cis-elements in-
volved in endosperm expression. These include eight
Skn-1 (GTCAT) and two GCN4-motifs (TG(T/A)GTCA).
Consequently, ADS may be expressed and amorphane
sesquiterpenes produced in seeds of A. annua even
though no trichomes are present. Finally, two HSE ele-
ments (AAAAAATTTC), which are involved in response
to heat, were localized to positions –698 to –706 and –83
to –74 and one CBFHV element (GTCGAC), involved in
salt and dehydration stress, at pos ition –1341 to –1336.
Figure 2. Scanning electron microscopy of A. annua. (a) close-up of young leaf; (b) young leaf; (c) old leaf (leaf before senes-
cence). GST: glandular secretory trichome; TST: T-shaped trichome.
Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis 625
in Artemisia annua L., as Reported by a Promoter-GUS Fusion
Figure 3. Gus-staining of transgenic A. annua transformed using the pADS-GUS plasmid. (a) leaf; (b) stem; (c) root of plant-
lets; (d) leaf primordial; (e) and (f) flower bud; (g) close-up of leaf; (h) glandular trichome. GST: glandular secretory
trichome; TST: T-shaped trichome.
method to study transcripts in trichome cells by quantita-
tive real time PCR (qPCR). Using this method, ADS
transcripts were detected in both apical and sub-apical
cells of glandular trichomes from A. annua [L. Olofsson
et al., unpublished].
The number of GUS-stained GSTs decreased with age
of the leaves (Figure 4). A high GST density is observed
in young leaves at the apex (Figure 4(a)) while essen-
tially no GSTs are present on old leaves at lowest node
(Figure 4(c)). It is quite obvious that the biosynthesis of
artemisinin precursors take place in young tissues of A.
The activity of the ADS promoter varies temporally
and in old tissues essentially no GUS staining could be
observed (Figures 3 and 4). It is difficult to quantita-
tively analyze the expression levels of ADS and GUS
controlled by endog enous promoter (pADS-ADS) and the
recombinant ADS promoter (pADS-GUS), respectively,
in transgenic plants. In order to establish the expression
pattern of the two promoters, qPCR was used to analyze
the relative expression levels of pADS-GUS and pADS-
ADS in different tissues of transgenic plants setting the
expression in stem to 1.0 (Figure 5). The expression
pattern of GUS and ADS in aerial parts of the transgen ic
plant, i.e. flower buds at early or late stages of develop-
ment and leaf primordia, was essentially the same indi-
cating that the cis-elements controlling glandular tricho-
me-specific expression are included in the cloned pro-
moter. The differences in relative expression levels in
these tissues reflect the density of GSTs.
In old leaves and roots at the reproductive stage, the
cloned promoter shows considerably higher activity than
Figure 4. Gus-staining of transgenic A. annua transformed using the pADS-GUS plasmid. a: leaf at apex; b: leaf at node 2; c:
ld leaf at node 6.
Copyright © 2011 SciRes. AJPS
Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis
626 in Artemisia annua L., as Reported by a Promoter-GUS Fusion
Figure 5. Expression pattern of pADS-ADS (a) and pADS-
GUS (b) of different tissues. All activities relative to the
activity in stem, which was set to 1. 0.
the wild-type promoter (Figure 5). A possible explana-
tion for this observation is that a silencing cis-element
controlling ADS expression in these tissues has been
deleted in the cloned ADS promoter.
Relative expression levels of pADS-GUS and pADS-
ADS were estimated using the 2∆∆Ct method using β-
actin as reference gene [41]. The wild-type promoter
showed a considerably higher activity (up to 250-fold)
than the recombinant promoter (Figure 6). Apparently,
one or more enhancer cis-elements are not included in
the cloned ADS promoter. It is possible that such enhan-
cers are located downstream of the ADS start codon, e.g.
in an intron as has been re po rt e d for ot her plant genes [42,
43]. In addition to this, the stability of the two mRNAs
may differ considerably, i.e. the GUS mRNA is broken
down much faster than the ADS mRNA.
The results presented are consistent with ADS being
Figure 6. Ratio of expression level of pADS-ADS to pADS-
GUS in aerial tissues of transgenic A. annua. For each tissue
the expression level for pADS-GUS was set to 1.0.
specifically located to glandular trichomes and the high-
est concentration of artemisinin precursors being in upper
leaves of A. annua plants [44].
4. Acknowledgements
We wish to thank Professor K. Tang , Shanghai Jiao Tong
University, for hosting HW during a visit to his labora-
tory to carry out initial experiments on the genetic trans-
formation of A. annua. We also wish to acknowledge the
financial support from the Faculty of Natural Sciences
and Engineering, Linnaeus University.
[1] D. Rathore, T. F. McCutchan, M. Sullivan and S. Kumar,
“Antimalarial Drugs: Current Status and New Develop-
ments,” Expert Opinion on Investigational Drugs, Vol. 14,
No. 7, 2005, pp. 871-883.
[2] T. E. Wallart, N. Pras and W. J. Ouax, “Seasonal Varia-
tions of Artemisinin and Its Biosynthetic Precursors in
Tetraploid Artemisia annua Plants Compared with the
Diploid Wild-Type,” Planta Medica, Vol. 65, No. 8, 1999,
pp. 723-728. doi:10.1055/s-1999-14094
[3] N. Delabays, X. Simonnet and M. Gaudin, “The Genetics
Copyright © 2011 SciRes. AJPS
Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis 627
in Artemisia annua L., as Reported by a Promoter-GUS Fusion
of Artemisinin Content in Artemisia annua L. and the
Breeding of High Yielding Cultivars,” Current Medicinal
Chemistry, Vol. 8, No. 15, 2001, pp. 795-1801.
[4] M. Hommel, “The Future of Artemisinins: Natural, Syn-
thetic or Recombinant?” Journal of Biology, Vol. 7, No.
10, 2008, p. 38. doi:10.1186/jbiol101
[5] F. Jing, L. Zhang, M. Li, Y. Tang, Y. Wang, Y. Wang, Q.
Wang, Q. Pan, G. Wang and K. Tang, “Abscisic Acid
(ABA) Treatment Increases Artemisinin Content in Ar-
temisia annua by Enhancing the Expression of Genes in
Artemisinin Biosynthetic Pathway,” Biologia, Vol. 64,
No. 2, 2009, pp. 319-323.
[6] T. C. Smith, P. J. Weathers and R. D. Cheetham, “Effects
of Gibberellic Acid on Hairy Root Cultures of Artemisia
annua: Growth and Artemisinin Production,” In Vitro
Cellular & Developmental Biology-Plant, Vol. 33, No. 1,
1997, pp. 75-79. doi:10.1007/s11627-997-0044-4
[7] P. J. Weathers, G. Bunk and M. C. McCoy, “The Effect
of Phytohormones on Growth and Artemisinin Production
in Artemisia annua Hairy Root,” In Vitro Cellular & De-
velopmental Biology-Plant, Vol. 41, No. 1, 2005, pp.
47-53. doi:10.1079/IVP2004604
[8] Y. S. Zhang, H. C. Ye, B. Y. Liu, H. Wang and G. F. Li,
“Exogenous GA3 and Flowering Induce the Conversion
of Artemisinic Acid to Artemisinin in Artemisia annua
Plants,” Russian Journal of Plant Physiology, Vol. 52, No.
1, 2005, pp. 58-62. doi:10.1007/s11183-005-0009-6
[9] W. Putalun, W. Luealon, W. De-Eknamkul, H. Tanaka
and Y. Shoyama, “Improvement of Artemisinin Produc-
tion by Chitosan in Hairy Root Cultures of Artemisia an-
nua L.,” Biotechnology Letters, Vol. 29. No. 7, 2007, pp.
1143-1146. doi:10.1007/s10529-007-9368-8
[10] A. Baldi and V. K. Dixit, “Yield Enhancement Strategies
for Artemisinin Production by Suspension Cultures of Ar-
temisia annua,” Bioresource Technology, Vol. 99, No. 11,
2008, pp. 4609-4614. doi:10.1016/j.biortech.2007.06.061
[11] C. Lei , D. Ma, G. Pu, X. Qiu, Z. Du, H. Wang, G. Li, H.
Ye and B. Liu, “Foliar Application of Chitosan Activates
Artemisinin Biosynthesis in Artemisia annua L.,” Indus-
trial Crops and Products, Vol. 33, No. 1, 2011, pp.
176-182. doi:10.1016/j.indcrop.2010.10.001
[12] L. Zhang, F. Jing, F. Li, M. Li, Y. Wang, G. Wang, X.
Sun and K. Tang, “Development of Transgenic Artemisia
annua (Chinese Wormwood) Plants with an Enhanced
Content of Artemisinin, an Effective Anti-Malarial Drug,
by Hairpin-RNA-Mediated Gene Silencing,” Biote-
chnology and Applied Biochemistry, Vol. 52, No. 3, 2009,
pp. 199-207. doi:10.1042/BA20080068
[13] L. -L. Feng, R. -Y. Yang, X. -Q. Yang, X. -M. Zeng. W.
-J. Lu and Q. -P. Zeng, “Synergistic Re-Channeling of
Mevalonate Pathway for Enhanced Artemisinin Produc-
tion in Transgenic Artemisia annua,” Plant Science, Vol.
177, No. 1, 2009, pp. 57-67.
[14] J. -L. Han, B. -Y. Liu, H. -C. Ye, H. Wang, Z. -Q. Li and
G. -F. Li, “Effects of Overexpression of the Endogenouse
Farnesyl Diphosphate Synthase on the Artemisinin Con-
tent in Artemisia annua L.,” Journal of Integrative Plant
Biology, Vol. 48, No. 4, 2006, pp. 482-487.
[15] P. Mercke, M. Bengtsson, H. J. Bouwmeester, M. A.
Posthumus and P. E. Brodelius, “Molecular Cloning, Ex-
pression, and Characterization of Amorpha-4,11-Diene
Synthase, a Key Enzyme of Artemisinin Biosynthesis in
Artemisia annua L.,” Archives of Biochemistry and Bio-
physics, Vol. 381, No. 2, 2000, pp. 173-180.
[16] H. J. Bouwmeester, T. E. Wallaart, M. H. Janssen, B. va n
Loo, B. J. Jansen, M. A. Posthumus, C. O. Schmidt, J. W.
de Kraker, W. A. Konig and M. C. Franssen, Amor-
pha-4,11-Diene Synthase Catalyses the First Probable
Step in Artemisinin Biosynthesis,” Phytochemistry, Vol.
52, No. 5, 1999, pp. 843-854.
[17] S. H. Kim, Y. J. Chang and S. U. Kim, “Tissue Specific-
ity and Developmental Pattern of Amorpha-4,11-Diene
Synthase (ADS) Proved by ADS Promoter-Driven GUS
Expression in the Heterologous Plant, Arabidopsis
thaliana,” Planta Medica, Vol. 74, No. 2, 2008, pp.
188-193. doi:10.1055/s-2008-1034276
[18] K. H. Teoh, D. R. Polichuk, D. W. Reed, G. Nowak and P.
S. Covello, “Artemisia annua L. (Asteraceae) Trichome-
Specific cDNAs Reveal CYP71AV1, a Cytochrome P450
with a Key Role in the Biosynthesis of the Antimalarial
Sesquiterpene Lactone Artemisinin,” FEBS Letters, Vol.
580, No. 5, 2006, pp. 1411-1416.
[19] D. -K. Ro, E. M. Paradise, M. Ouel let, K. J. Fisher, K. L.
Newman, J. M. Ndungu, K. A. Ho, R. A. Eachus, T. S.
Ham, J. Kirby, M. C. Y. Chang, S. T. Withers, Y. Shiba,
R. Sarpong and J. D. Keasling, “Production of the Anti-
malarial Drug Precursor Artemisinic Acid in Engineered
Yeast,” Nature, Vol. 440, No. 7, 2006, pp. 940-943.
[20] G. D. Brown and L. K. Sy, In Vivo Transformations of
Dihydroartemisinic Acid in Artemisia annua Plants,”
Tetrahedron, Vol. 60, No. 5, 2004, pp. 1139-1159.
[21] Y. Zhang, K. H. Teoh, D. W. Reed, L. Maes, A.
Goossens, D. J. Olson, A. R. Ross and P. S. Covello,
“The Molecular Cloning of Artemisinic Aldehy de 11(13)
Reductase and Its Role in Glandular Trichome-Dependent
Biosynthesis of Artemisinin in Artemisia annua,” The
Journal of Biological Chemistry, Vol. 283, No. 31, 2008,
pp. 21501-21508. doi:10.1074/jbc.M803090200
[22] K. H. Teoh, D. R. Polichuk, D. W. Reed and P. S.
Covello, “Molecular Cloning of an Aldehyde Dehydro-
genase Implicated in Artemisinin Biosynthesis in Ar-
temisia annua,” Botany, Vol. 87, No. 6, 2009, pp.
635-642. doi:10.1139/B09-032
[23] M. V. Duke, R. N. Paul, H. N. Elsohly, G. Sturtz and S. O.
Duke, “Localization of Artemisinin and Artemisitene in
Copyright © 2011 SciRes. AJPS
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
Copyright © 2011 SciRes. AJPS
Foliar Tissues of Glanded and Glandless Biotypes of Ar-
temisia annua L.,” International Journal of Plant Sci-
ences, Vol. 155, No. 3, 1994, pp. 365-372.
[24] P. S. Covello, K. H. Teoh, R. Devin, D. R. Polichuk,
“Function Genomic and Biosynthesis of Artemisinin,”
Phytochemistry, Vol. 68, No. 14, 2007, pp. 1864-1871.
[25] L. Olofsson, A. Engström, A. Lundgren and P. E. Brode-
lius, “Relative Expression of Genes of Terpene Metabo-
lism in Different Tissues of Artemisia annua L.,” BMC
Plant Biology, Vol. 11, No. 45, 2011, pp. 1-12.
[26] J. Fütterer, A. Gisel, V. Iglesias, A. Klöti, B. Kost, O.
Mittelsten Scheid, G. Neuhaus, G. Neuhaus-Url, M.
Schrott, R. Shillito, G. Spangenberg and Z. Y. Wang,
“Standard Molecular Techniques for the Analysis of
Transgenic Plants,” In: I. Potrykus and G. Spangenberg,
Eds., Gene Transfer to Plants, Springer Verlag, Berlin,
1995, pp. 215-263.
[27] R. A. Jefferson, T. A. Kavanagh and M. W. Bevan, “GUS
Fusions: Beta-Glucuronidase as a Sensitive and Versatile
Gene Fusion Marker in Higher Plants,” The EMBO
Journal, Vol. 6, No. 13, 1987, pp. 3901-3907.
[28] S. Yin, L. Mei, J. Newman, K. Back and J. Chappell,
“Regulation of Sesquiterpene Cyclase Gene Expression;
Characterization of an Elicitor- and Pathogen-Inducible
Promoter,” Plant Physiology, Vol. 115, No. 2, 1997, pp.
437-451. doi:10.1104/pp.115.2.437
[29] T. Yang and B. W. Poovaiah, “An Early Ethylene
up-Regulated Gene Encoding a Calmodulin-Binding Pro-
tein Involved in Plant Senescence and Death,” The Jour-
nal of Biological Chemistry, Vol. 275, No. 49, 2000, pp.
38467-38473. doi:10.1074/jbc.M003566200
[30] T. Yang and B. W. Poovaiah, “A Calmodulin-Binding/
CGCG-Box DNA-Binding Protein Family Involved in
Multiple Signaling Pathways in Plants,” The Journal of
Biological Chemistry, Vol. 277, No. 47, 2002, pp.
45049-45058. doi:10.1074/jbc.M207941200
[31] L. D. Zhang, K. J. Zuo, F. Zhang, Y. F. Cao, J. Wang, Y. D.
Zhang, X. F. Sun and K. X. Tang, “Conservation of
Non-Coding Microsatellites in Plants: Implication for Gene
Regulation,” BMC Genomics, Vol. 7, No. 323, 2006 , pp. 1-14.
[32] G. -B. Pu, D. -M. Ma, J. -L. Chen, L. -Q. Ma, H. Wang,
G. -F. Li, H. -C. Ye and B. -Y. Liu, “Salicylic Acid Acti-
vates Artemisinin Biosynthesis in Artemisia annua L.,”
Plant Cell Reports, Vol. 28, No. 7, 2009, pp. 1127-1135.
[33] D. Ma, G. Pu, C. Lei, L. Ma, H. Wang, Y. Guo, J. Chen,
Z. Du, H. Wang, G. Li, H. Ye and B. Liu; “Isolation and
Characterization of AaWRKY1, an Artemisia annua
Transcription Factor that Regulates the Amorpha-4,11-
Diene Synthase Gene, a Key Gene of Artemisinin Bio-
synthesis,” Plant and Cell Physiology, Vol. 50, No. 12,
2009, pp. 2146-2161. doi:10.1093/pcp/pcp149
[34] Y. -H. Xu, J. -W. Wang, S. Wang, J. -Y. Wang and X. -Y.
Chen, “Characterization of GaWRKY1, a Cotton Tran-
scription Factor that Regulates the Sesquiterpene Syn-
thase Gene (+)-δ-Cadinene Synthase-A,” Plant Physiol-
ogy, Vol. 135, No. 1, 2004, pp. 507-515.
[35] J. C. Reyes, M. I. Muro-Pastor and F. J. Florencio, “The
GATA Family of Transcription Factors in Arabidopsis
and Rice,” Plant Physiology, Vol. 134, No. 4, 2004, pp.
1718-1732. doi:10.1104/pp.103.037788
[36] T. Urao, K. Yamaguchi-Shinozaki, S. Urao and K. Shi-
nozaki, “An Arabidopsis myb Homolog Is Induced by
Dehydration Stress and Its Gene Product Binds to the
Conserved MYB Recognition Sequence,” Plant Cell, Vol.
5, No. 11, 1993, pp. 1529-1539.
[37] R. Solano, C. Nieto, J. Avila, L. Cañas, I. Diaz and J.
Paz-Ares, “Dual DNA Binding Specificity of a Petal
Epidermis-Specific MYB Transcription Factor (MYB.
Ph3) from Petuni a hybrida,” The EMBO Journal, Vol. 14,
No. 8, 1995, pp. 1773-1784.
[38] G. D. Brown, G. -Y. Liang and L. -K. Sy, “Terpenoids
from the Seeds of Artemisia annua,” Phytochemistry, Vol.
64, No. 1, 2003, pp. 303-323.
[39] J. F. S. Ferre ira, J. E. Simon and J. Janick, “Developmen-
tal Studies of Artemisia annua: Flowering and Artemisi-
nin Production under Greenhouse and Field Conditions,”
Planta Medica, Vol. 61, No. 2, 1995, pp. 167-170.
[40] M. E. Olsson, L. M. Olofsson, A. L. Lindahl, A.
Lundgren, M. Brodelius and P. E. Brodelius, “Localiza-
tion of Enzymes of Artemisinin Biosynthesis to the Api-
cal Cells of Glandular Secretory Trichomes of Artemisia
annua L.,” Phytochemistry, Vol. 70, No. 9, 2009, pp.
[41] K. J. Livak and T. D. Schmittgen, “Analysis of Relative
Gene Expression Data using Real-Time Quantitative PCR
and the 2CT Method,” Met h od s, Vol. 25, No. 4, 2001,
pp. 402-408. doi:10.1006/meth.2001.1262
[42] D. Mascarenhas, I. J. Mettler, D. A. Pierce and H. W.
Lowe, “Intron-Mediated Enhancement of Heterologous
Gene Expression in Maize,” Plant Molecular Biology,
Vol. 15, No. 6, 1990, pp. 913-920.
[43] M. K. Deyholos and L. E. Sieburth, “Separable Whorl-
Specific Expression and Negative Regulation by Enhan-
cer Elements within the AGAMOUS Second Intron,”
Plant Cell, Vol. 12, No. 10, 2000, pp. 1799-1810.
[44] S. O. Duke and R. N. Paul, “Development an Fine Struc-
ture of the Glandular Trichomes of Artemisia annua L.,”
International Journal of Plant Sciences, Vol. 154, No. 1,
1993, pp. 107-118. doi:10.1086/297096