Plant Biotechnol Rep (2015) 9:259267
DOI 10.1007/s11816-015-0362-7
ORIGINAL ARTICLE
Competitive activity as a constitutive promoter
in the 50 -proximal regulatory region of the Capsicum
capsanthincapsorubin synthase gene
Sun-Hwa Ha1
Received: 23 June 2015 / Accepted: 24 June 2015 / Published online: 10 July 2015
Korean Society for Plant Biotechnology and Springer Japan 2015
Abstract Depending on the nature of the Capsicum
capsanthincapsorubin synthase gene (CaCcs) showing a
chromoplast specificity, the 50 -upstream region of CaCcs
from -2279 to ?30 was isolated from the Korean red
pepper genomic library for promoter analysis. The full-
length 2.3-kb promoter and its deletion mutants similarly
drove GUS expression in chromoplastic tissues such as
anthers and styles in transgenic Arabidopsis flowers.
However, the shortest 397-bp promoter (CaCcs-P1) con-
taining the -367 to ?30 region displayed histochemically
high GUS expression in non-chromoplastic tissues such as
sepals and leaves, as well as in chromoplastic tissues. The
efficient constitutive expression of CaCcs-P1 was higher
than that of the dual 35S promoter (d35S-P) with respect to
mRNA expression (at least threefold) and enzyme activity
(fourfold) and was maintained in the whole body of plants
during all developmental stages. Ubiquitous GUS expres-
sion by CaCcs-P1 was elicited at the highest level in stems,
followed by leaves, flowers, roots, young siliques, and
mature seeds, with respect to both transcript levels and
enzyme activity. More effective activity of CaCcs-P1
compared to d35S-P (1.4-fold higher) was also confirmed
in transgenic tobacco plants. Together with the prediction
of cis-acting motifs that allow diverse spatial specificity,
we report a promoter displaying efficient constitutive
Electronic supplementary material The online version of this
article (doi:10.1007/s11816-015-0362-7) contains supplementary
material, which is available to authorized users.
& Sun-Hwa Ha
sunhwa@khu.ac.kr
1
Graduate School of Biotechnology, Kyung Hee University,
Yongin 446-701, Republic of Korea
Online ISSN 1863-5474
Print ISSN 1863-5466
characteristics with the advantages of a plant origin and
size competitiveness compared with the 35S promoter.
Keywords Arabidopsis  Capsanthincapsorubin
synthase  Capsicum  Chromoplast-specific  Constitutive 
Promoter  Tobacco
Introduction
The capsanthincapsorubin synthase gene (CaCcs) is
specifically expressed during chromoplast development in
fruits that accumulate the Capsicum-specific keto-
carotenoids, capsanthin, and capsorubin (Bouvier et al.
1994). The CaCcs expression is very tightly linked with the
ripening process, showing simultaneous strong induction in
ripe fruits, but not in any other plant organs including
leaves, stems, roots, and green fruits (Ha et al. 1999).
Moreover, transcription of CaCcs is not detected in Cap-
sicum varieties that show yellow ripe colors because of
impairment of the biosynthetic pathway of capsanthin and
capsorubin, the pigment metabolites responsible for the red
color of Capsicum fruits through their de novo biosynthesis
during ripening (Bouvier et al. 1994; Ha et al. 2007;
Ramchiary et al. 2014). This intrinsic feature of CaCcs
expression in Capsicum makes it a good candidate gene for
the development of chromoplast-specific promoters. In
previous studies, a putative promoter region containing a 50
2.3-kb fragment upstream of the ATG initiation codon of
CaCcs was examined for activity by tomato explant
regeneration (Kuntz 2002). Activity of the CaCcs promoter
was compared with that of the fibrillin promoter through a
b-glucuronidase (GUS) assay in diverse organs (leaves,
fruits, and flowers) and during different developmental
stages (fruits and flowers). The fibrillin gene encodes a
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protein that is tightly associated with the Capsicum chro-
moplasts for the packaging and organization of carotenoids
(Derue`re et al. 1994). As expected, both promoters dis-
played chromoplast specificity in fruits and flowers. Their
activity at 4 days after breaker stage was even higher than
that of the known ripening-induced tomato polygalactur-
onase promoter (Bird et al. 1988). In contrast to the fibrillin
promoter, which was also activated in mature green fruits,
the CaCcs promoter showed a large increase in activity
around the mature stages of fruits and anthers of flower
tissues, accompanied by changes to visibly red color phe-
notypes (Kuntz 2002).
In addition to the chromoplast specificity of the CaCcs
promoter described above, oxidative stress-inducible char-
acteristics have also been suggested from experiments
showing that diverse reactive oxygen species (ROS) progen-
itors and pro-oxidants triggered rapid induction of simulta-
neous expression of multiple carotenogenic genes including
CaCcs, even in mature green pericarp disks (Bouvier et al.
1998). A partial CaCcs promoter of 650 bp was confirmed to
elicit high expression of a GUS reporter activity under
oxidative stress conditions in a transient assay in fruit.
On the basis of the above findings, the CaCcs promoter
might be considered a target with great potential to extend
our range of options for the expression of foreign genes in
the production of genetically modified (GM) crops. To
evaluate this possibility, we isolated a CaCcs promoter of
over 2.3 kb by plaque screening of a genomic library
constructed with Korean red pepper. This promoter was
progressively deleted to give a series of six truncated forms
that were transformed in combination with the GUS
reporter gene into Arabidopsis plants. Fortunately, we
discovered efficient constitutive promoter activity in the
shortest promoter of 397 bp including the region from 367
to ?30, named CaCcs1 promoter (CaCcs-P1). The strength
of CaCcs-P1 was compared with that of the dual 35S
promoter (d35S-P) in two transgenic plant systems, Ara-
bidopsis and tobacco, to evaluate its usefulness as an effi-
cient constitutive promoter for use in plant biotechnology.
Materials and methods
Cloning of a 50 -upstream regulatory region
of the Capsicum Ccs promoter
Using a genomic library from Korean red pepper (Capsicum
annuum cv. NocKwang) constructed in the kEMBL3/BamHI
vector (Stratagene, La Jolla, CA), approximately 105 plaques
were screened with 32P-labeled CaCcs full-length cDNA as a
probe (Ha et al. 1999). After EcoRI digestion of putative
CaCcs genomic clones, a positive 2.5-kb signal band detected
on Southern blotting with a 32P-200 bp fragment of the
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Plant Biotechnol Rep (2015) 9:259267
N-terminal region of pepper CaCcs cDNA was subcloned into
pBluescript SK (?) (Stratagene) as shown in supplementary
Fig. S1. A 2.3-kb PCR product (-2279 to ?30) generated by
removal of a 246-bp region in the N-terminal coding region of
CaCcs was finally cloned into the EcoRI and XbaI sites of
pBluescript KS (?) (Stratagene). This 50 2309-kb region
upstream from the ATG start codon of the CaCcs was con-
sidered the full-length CaCcs promoter (referred to as CaCcs-
P6 hereafter) for analysis of promoter activity.
Construction of a deletion series of the 50 -upstream
regulatory region of CaCcs
To prepare the deletion series of the 2.3-kb CaCcs pro-
moter that is mainly composed of multiple direct repeats,
self-ligations after dual restriction enzyme digestion were
designed to give promoter regions of 2.0, 1.4, 1.0, 0.7, and
0.4 kb as follows: EcoRV and ScaI for CaCcs-P5, EcoRV
and klenow fragment-treated AvaII (partial cut) for CaCcs-
P4, ClaI and TaqI for CaCcs-P3, EcoRV and StuI for
CaCcs-P2, EcoRV and klenow fragment-treated AvaII (full
cut) for CaCcs-P1 (Supplementary Fig. S2).
Next, the Gus:Tnos fragment derived from pBI221 was
incorporated into six deletion promoter series vectors using
the same XbaI and SacI sites. Vectors expressing Gus
under the control of the six CaCcs-P deletion series were
named pKS-CaCcs-P1-6::Gus:Tnos.
Binary vector construction and plant transformation
To express the CaCcs-P deletion series in plants, each
cassette containing CaCcs-P1-6::Gus:Tnos was excised by
SalI and EcoRI and ligated into pCAMBIA 2300 (Cambia,
Canberra, Australia). A 2-kb fragment of the Bar expres-
sion cassette was additionally incorporated into each
plasmid to prepare six binary vectors of CaCcs P1-6. These
constructs were then separately transformed into
Agrobacterium strain EHA105 using the freezethaw
method and consecutively introduced into Arabidopsis
ecotype Columbia (Col-0) using the floral dip method. A
construct for CaCcs-P1 was further transformed into
Agrobacterium strain GV3101 and used to generate trans-
genic tobacco plants using the previously reported leaf disk
method (Lim et al. 2012). As reference controls for pro-
moter strength comparison, previously confirmed trans-
genic plants including a single 35S promoter (s35S-P-
transgenic Arabidopsis) and d35S-P-transgenic plants of
Arabidopsis and tobacco were used (Chung et al. 2008;
Liang et al. 2009; Lim et al. 2013). All transgenic plants
were selected using 0.3 % Basta spray and grown in a
chamber under a 16-h photoperiod at 22 C during the day
and 18 C at night.
Plant Biotechnol Rep (2015) 9:259267
Histochemical and fluorometric GUS assays
To perform the GUS histochemical assay, Arabidopsis
plants were stained with 1 % 5-bromo-4-chloro-3-indolyl
b-D-glucuronide (X-Gluc) solution and then observed and
imaged using a light microscope (Olympus, Tokyo, Japan)
equipped with a digital camera (Olympus). To measure
GUS activity fluorometrically, a 4-methyl umbelliferyl
b-D-glucuronide (MUG) assay was carried out using Ara-
bidopsis and tobacco plants as previously reported (Liang
et al. 2011; Lim et al. 2013). A Qubit fluorometer (Invit-
rogen, Carlsbad, CA) and the FluorAceTM b-glucuronidase
Reporter Assay Kit (Bio-Rad, Hercules, CA) were used for
quantification and assaying enzyme activity of GUS pro-
tein, respectively. Values with error bars were calculated
from three experimental replicates.
RT-PCR analysis
Total RNA from various tissues of Arabidopsis prepared
with a previously described procedure (Lim and Ha 2013)
Fig. 1 Schematic illustration of binary vectors used for promoter
deletion analysis and comparison of their promoter activities in
transgenic Arabidopsis plants. a Gus expression vectors driven by the
full-length CaCcs promoter (CaCcs-P6) and its serial truncation
promoters (CaCcs-P5, P4, P3, P2, and P1) were constructed using
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was converted into first-strand cDNAs and subjected to
semi-quantitative PCR using an mRNA Selective PCR Kit
(Takara, Tokyo, Japan). The reaction was carried out for 25
cycles using a Gus-specific primer set (50 -
ACCTGCGTCAATGTAATGTTCTGC-30 /50 -CTCCCTGC
TGCGGTTTTTCA-30 ). A primer set specific for the Ara-
bidopsis elongation factor 1 gene (AtEF1a; 50 -GTTCACA
TTAACATTGTGGTCATT-30 /50 -CAGGTACCAGTGATC
ATGTTCTTG-30 ) was used as a reference to normalize the
amount of RNA. Two PCR products of 478 and 305 bp in
size, respectively, are expected. PCR band intensity was
quantitatively analyzed using a densitometer and Quantity
One software (Bio-Rad, Hercules, CA).
Computational analysis
The 50 -upstream DNA sequence including the -367 to
?30 region for CaCcs1-P was analyzed to search for cis-
acting elements using public web sites including PLACE
(http://www.dna.affrc.go.jp/PLACE/signalscan.html),
PlantCARE (http://bioinformatics.psb.ugent.be/
pCAMBIA as the plasmid backbone. b Histochemical GUS staining
results for leaves (left) and flowers (right) of representative lines for
CaCcs-P6, P5, P4, P2, and P1. Unfortunately, transgenic lines for
CaCcs-P3 have not been obtained. NC indicates non-transgenic
control Arabidopsis Col-0 plants
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webtools/plantcare/html/), and Softberry (http://
linux1.softberry.com/berry.phtml?topic=tssp&group=
programs&subgroup=promoter) databases.
Results and discussion
Activity of the CaCcs promoter and its deletion
series in transgenic Arabidopsis plants
Based on the chromoplast-specific characteristics of the 50
upstream 2.3-kb CaCcs promoter that showed strong
expression in fruits and anthers and weak expression in
petals of transgenic tomato plants (Kuntz 2002), six
deletion vectors driving GUS reporter expression were
constructed with promoter sizes of 2309 bp (P6), 2001 bp
(P5), 1,379 bp (P4), 984 bp (P3), 661 bp (P2), and 397 bp
(P1) to identify the necessary region for regulation of
chromoplast specificity in the flower organs of Arabidopsis
plants via stable transformation (Fig. 1a and supplementary
Fig. S2). More than 10 independent transgenic plants were
generated and stained for GUS expression driven by full-
length CaCcs-P6 and the 50 deletion fragments CaCcs-P5,
P4, P2, and P1; unfortunately transgenic plants were not
generated for CaCcs-P3. Unlike observations in tomato
flowers, GUS expression driven by CaCcs-P6 in Ara-
bidopsis was displayed weakly in anthers but strongly in
sepals. Progressive 50 -deletions from CaCcs-P5 to CaCcs-

Fig. 2 Comparison of activity

between d35S-P and CaCcs-P1
in transgenic Arabidopsis
plants. a Histochemical GUS
staining was performed with
five independent d35S-P:Gus
and CaCcs-P1:Gus transgenic
Arabidopsis lines at the 3-week-
old seedling stage. b Enzymatic
values for GUS activity levels
were quantified using a
fluorometric MUG assay with
the same five independent lines
for d35S-P and CaCcs-P1.
Error bars show the standard
deviations of four technical
replicates. Numbers above the
bars indicate fold changes
relative to the value of the d35S-
P-10 line

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Fig. 3 Maintenance of
constitutive expression by
CaCcs-P1 compared with two
types of 35S-P. Histochemical
GUS staining was performed
with whole plant bodies of
transgenic Arabidopsis plants at
two different developmental
stages of 4 and 5 weeks old
P2 slightly strengthened GUS expression in anthers but
abolished expression in sepals (Fig. 1b). Interestingly, for
the shortest element, CaCcs-P1, high GUS expression in
sepals was revived and higher GUS expression was
observed in the anthers and filaments in stamens and the
styles in pistils. Moreover, CaCcs-P1 showed an unex-
pectedly deep blue signal in leaves, suggesting its ability to
strongly drive GUS expression in transgenic Arabidopsis
leaves in contrast to the other promoters (Fig. 1b). The
observation of GUS staining driven by CaCcs-P1 in non-
chromoplastic tissues (leaves and sepals) as well as chro-
moplastic tissues (anthers and styles among flower organs)
suggests the potential of CaCcs-P1 as a ubiquitously
expressed constitutive promoter.
CaCcs-P1 has more efficient activity than
d35S-P in transgenic Arabidopsis plants
To compare the promoter strength of CaCcs-P1 with that
of d35S-P for constitutive expression, 12 Arabidopsis
plants of CaCcs-P1:Gus and 15 of d35S-P:Gus were
stained with X-Gluc solution at the 3-week-old seedling
stage of T1 generation. All transgenic plants showed a
similar dense blue color due to GUS expression in whole
body regions of transgenic seedlings. Among them, five
representative lines for each promoter were selected based
on their strong blue staining (Fig. 2a) and their T-DNA
insertion was confirmed to be low copy number (\2) by
genomic Southern blot analysis except for the presence of
four copies in the d35S-P-2 line (data not shown). The five
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lines for each promoter were further developed toward T2
generation and their promoter activities were compared by
GUS fluorometric assay (Fig. 2b). In general, CaCcs-P1
resulted in 3- to 31-fold higher expression than d35S-P;
d35S-P-10 could be reasonably considered a representative
line with average activity whereas d35S-P-5 was excluded
as an exceptional line displaying 88-fold higher activities
than the average line of d35S-P-10 and 29-fold higher
activity than the lowest line of CaCcs-P1-2 (Fig. 2b).
To determine whether the efficient constitutive expres-
sion of CaCcs-P1 is maintained during the entire process of
plant development, histochemical GUS staining of two
lines (CaCcs-P1-4 and CaCcs-P1-12) was performed at
further developmental stages of 4 and 5 weeks compared
with a d35S-P-5 as an exceptionally dominant line and an
additional s35S-P-1 line, as previously reported (Chung
et al. 2008). In accordance with high GUS enzyme activ-
ities induced by CaCcs-P1 (Fig. 2b), the GUS staining
signal in two CaCcs-P1 lines was maintained much more
strongly in the whole plant body including leaves, stems,
roots, and flowers during the three stages of plant devel-
opment, with levels similar to those in the d35S-P-5 line
but much higher than in the s35S-P-1 line (Fig. 3).
Ubiquitous expression driven
by CaCcs-P1 in transgenic Arabidopsis plants
To investigate the promoter characteristics on the basis of
organ specificity, the ability of CaCcs-P1 to drive Gus
expression was compared among different organs of rosette
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Fig. 4 Activity of CaCcs-P1 in

driving GUS expression among
different organ types of
transgenic Arabidopsis plants.
a Detection of Gus transcripts
by semi-quantitative RT-PCR
(upper) and relative expression
levels (lower) driven by CaCcs-
P1 in rosette leaves (RL),
cauline leaves (CL), stems (St),
roots (R), flowers (F), siliques
(Si), and seeds (Se). Two
independent lines of d35S-
P were used to compare the
levels of Gus transcripts with
those of CaCcs-P1 in the same
RL organs; Arabidopsis
elongation factor 1-a (AtEF1a)
was used as a normalization
control for RNA quantity. The
graph was drawn with d35S-P-
10 assigned a value of 1.
Additional numbers above bars
indicate fold changes among
diverse organs for the CaCcs-
P1-4 line. Error bars show the
standard deviations (SDs) of
three replicates. b GUS enzyme
activities were measured by
MUG assay using two
independent CaCcs-P1 lines.
Results represent mean SD
for six values, including two
biological and three technical
repeats. Numbers above bars
indicate fold changes among
diverse organs for each CaCcs-
P1 line

leaves (RL), cauline leaves (CL), stems (St), roots (R),
flowers (F), young siliques (Si), and mature seeds (Se) of
CaCcs-P1:Gus transgenic Arabidopsis plants by semi-
quantitative RT-PCR analysis (Fig. 4a). In particular, Gus
transcript levels for CaCcs-P1 in rosette leaves were
compared with those for d35S-P using the representative
d35S-P-10 line and the transnormal d35S-P-5 line. Con-
cordant with the results of GUS enzyme assays showing at
least threefold higher activity (Fig. 2b), the fourfold higher
expression levels of Gus mRNA in CaCcs-P1-4 compared
with d35S-P-10 confirmed the potent activity of CaCcs-P1
compared with d35S-P in rosette leaves (Fig. 4a). Among
organs, the activity of CaCcs-P1 was highest in stems,
followed by leaves of cauline and rosette, flowers, roots,
young siliques, and mature seeds (Fig. 4a). Moreover, Gus
mRNA was more abundant in green organs of leaves and
stems and much less abundant in seeds than in other
organs. Fluorometric GUS assays were also performed with
the same organ samples for CaCcs-P1-4 and CaCcs-P1-12
(Fig. 4b). Except for low activity in seeds, high activity of
GUS enzyme was observed in all organs with a moderate
difference between the two CaCcs-P-transgenic Ara-
bidopsis plants.
Collectively, these data indicate that CaCcs-P1 could
induce exogenous gene expression with higher activity
than d35S-P and in all organs examined, thus supporting its
ubiquitous function (Figs. 2, 3, and 4). These results were
extremely consistent for mRNA level and enzyme activity,

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as well as for histochemical staining for GUS reporter
expression in Arabidopsis plants.
CaCcs-P1 has more efficient activity
than d35S-P in transgenic tobacco plants
To evaluate whether CaCcs-P1 acts as an efficient pro-
moter in other plant systems, ectopic GUS expression was
also examined in tobacco plants via stable transformation.
Among 12 and 17 transformants for d35S-P and CaCcs-P1
respectively, 4 and 8 independent lines showing moderate
expression levels were selected on the basis of histo-
chemical GUS staining signals of leaf disk (data not
shown) and their GUS enzyme activities were measured
and compared by fluorometric assay (Fig. 5). On average,
CaCcs-P1 was a 1.4-fold stronger driver of GUS expres-
sion than d35S-P, suggesting that CaCcs-P1 might have
greater potential as an efficient constitutive promoter than
d35S-P in diverse dicotyledonous plants, as confirmed in
Arabidopsis and tobacco plants.
Putative cis-acting elements conferring organ
specificity of CaCcs-P1
To further investigate the surprising characteristic of con-
stitutive expression of CaCcs-P1, the 50 -proximal DNA
sequences in the -367 to ?30 region were computationally
analyzed using public databases of PLACE, PlantCARE,
and Softberry (Fig. 6 and Supplementary Table S1). The in
silico analysis predicted a total of 46 cis-acting elements
for diverse spatial specificity in green tissues of leaves and
stems (11), roots (5), flowers (20), and seeds (10) in both
DNA orientations. Combinatory positioning of regulatory
cis-acting factors with respect to quality and quantity in

Fig. 5 Comparison of promoter

efficiency of d35S-P and
CaCcs-P1 in transgenic tobacco
plants. Enzymatic values for
GUS activity levels were
quantified using a fluorometric
MUG assay with four and eight
independent lines for d35S-
P and CaCcs-P1, respectively at
the 3-week-old seedling stage.
Error bars show the standard
deviations of three technical
replicates

Fig. 6 Schematic diagram

showing cis-acting motifs
computationally predicted for
diverse organ and tissue
specificity in both orientations
of the CaCcs-P1 region (-367
to ?1)

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diverse organs might affect the constitutive expression of
CaCcs-P1. The additional prediction of 17 cis-acting ele-
ments suggests that CaCcs-P1 might be responsive to light
and related to defense, salt, and water stress as a stress
regulator (Supplementary Table S1). Further promoter
analysis is needed to test this hypothesis.
In this study, we report a novel promoter of plant origin
that displays efficient constitutive characteristics. The most
commonly used promoters for plant biotechnology are the
35S promoter and its derivatives, for example those of
plant pathogens like Cauliflower Mosaic Virus (Odell et al.
1985; Omirulleh et al. 1993). To date, the 35S promoter has
been used in almost all GM crops currently grown or tes-
ted, and is considered the favored choice due to its stably
effective and constitutive expression in almost any type of
plant cell and tissue at any developmental stage, with the
additional merit of its short length for engineering pur-
poses. However, issues regarding its capacity to be active
in gut pathogens and soil bacteria and other potential
hazards have constantly been a subject of debate in terms
of GMO safety assessment (Lewin et al. 1998; Ho et al.
2000; Podevin and du Jardin 2012). Hence, the use of a
plant-derived promoter could be a good alternative to
overcome these issues associated with the poor public
acceptance of GM crops. In addition to the matter of gene
source, DNA size might be also important for extensive use
as a constitutive promoter. Since the full-size 35S promoter
(-941 to ?9) was identified, diverse shorter forms of the
35S promoter including the 35S enhancer fragment region
(-209 to -46) have been developed and incorporated into
plant binary vectors (Odell et al. 1985; Fang et al. 1989).
Among constitutive promoters from dicot plants, some
relatively small promoters have also been reported, such as
the duplicated tobacco iCUP promoter of 633 bp and the
Arabidopsis ubiquitin-10 promoter of 634 bp (Malik et al.
2002 and Grefen et al. 2010). However, a wider choice of
suitable promoters is required in order to introduce multi-
ple transgenes at once and to avoid involuntary gene
silencing (Park et al. 2013).
In addition, efforts to shorten the length of the promoter
by deletion analysis have been reported to increase the
spatial specificity of the Solanum habrochaites CYC-
B (ShCYC-B) promoter and to decrease the strength of the
Medicago truncatula HP (MtHP) promoter (Xiao et al.
2005; Dalal et al. 2010). Through promoter deletion anal-
ysis, the function of the chromoplast-specific ShCYC-
B promoter was enhanced within a short promoter region
(-436 bp to ATG) compared with the full-length promoter
(908 bp), and the strong constitutive expression of the full-
length MtHP promoter (1,548 bp) became weaker as the
promoter became shorter up to a 107-bp length. CaCcs-P1
is the first example of a promoter that is transformed into
an efficient constitutive promoter with loss of chromoplast
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Plant Biotechnol Rep (2015) 9:259267
specificity via deletion. We believe that the 397-bp pro-
moter region of CaCcs-P1 (-367 to ?30) might be the best
choice for driving high levels of constitutive expression of
transgenes in dicot plants for plant engineering because of
its advantages of plant origin and size competitiveness
compared with the 35S promoter.
Acknowledgments This work was supported by Grants from the
Next-Generation BioGreen 21 Program (PJ011286012015 and
PJ011094012015), Rural Development Administration, Republic of
Korea, and by the Basic Science Research Program through the
National Research Foundation of Korea (NRF-2013R1A1A2062998).
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