Ecotoxicology and Environmental Safety 170 (2019) 627–634

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Development of bisphenol A (BPA)-sensing indicator Arabidopsis thaliana T

which synthesizes anthocyanin in response to BPA in leaves
⁎
DongGwan Kima,1, Ramin Bahmania,1, Jae-Heung Kob, Seongbin Hwanga,
a
Dept. of Bioindustry and Bioresource Engineering, Dept. of Molecular Biology and Plant Engineering Research Institute, Sejong University, 209, Neungdong-ro, Gwangjin-
gu, Seoul, Republic of Korea
b
Department of Plant & Environmental New Resources, Kyung Hee University, Yongin 446-701, Gyeonggi-do, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Bisphenol A (BPA) is an estrogenic endocrine disruptor which disturbs a normal animal development. We
Endocrine disruptor generated an indicator plant that senses and provides a clear visual indicator of an estrogen-like compound BPA
Bisphenol A in the environment. We developed transgenic Arabidopsis lines expressing a construct designed to synthesize
Anthocyanin anthocyanin (thus showing a red color) in response to BPA. We transformed Arabidopsis with a recombinant
PtrMYB119
vector containing the chimeric estrogen receptor (XVE region), LAP and coding region of PtrMYB119 (tran-
Arabidopsis thaliana
Estrogen receptor
scription factor involved in anthocyanin biosynthesis in poplar and Arabidopsis). Upon binding of the estrogen
compound to the ligand-binding domain of E (estrogen receptor) in XVE, the XV domain binds to LAP promoter
and triggering the transcription of PtrMYB119 with a subsequent enhancement of anthocyanin biosynthetic gene
expression, resulting in anthocyanin synthesis. The leaves of the transgenic Arabidopsis line XVE-PtrMYB119
turned red in the presence of 10 ppm BPA. The transcript level of PtrMYB119 peaked at day 3 of BPA exposure,
then decreased to its minimal level at day 5. Similar expression patterns to that of PtrMYB119 were detected for
genes encoding the anthocyanin biosynthetic enzymes chalcone synthase, chalcone flavanone isomerase, fla-
vanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and UFGT (UGT78D2). The leaves
of transgenic plants did not turn red in response to BPA at concentrations below 10 ppm, but PtrMYB119 ex-
pression was induced by BPA at concentrations as low as 1 ppt BPA. Since this transgenic plant turns red in the
presence of BPA without any experimental procedures, this line can be easily used by non-scientists.

1. Introduction activities, where it contaminates the atmosphere, agricultural soils,

Endocrine-disrupting compounds (EDCs) are chemical compounds
that disrupt the normal physiological functions of endocrine hormones
by competing with them or functioning as hormone analogs. Bisphenol
A (BPA; 2,2-bis(4-hydroxyphenyl) propane) is an estrogenic EDC that is
widely used for making plastic products derived from polycarbonate
and epoxy resins, such as water and infant bottles, medical devices,
coatings for cans, and additives in thermal papers (Cooper et al., 2011;
Goodson et al., 2002; Russo et al., 2017; Wee and Aris, 2017). The
annual production of BPA is approximately 8 million tons in 2016 and
expected to increase to 10.6 million tons in 2022 (Bisphenol, 2016).
Moreover, the production of BPA in the USA and Europe in a period
between 1995 and 2014 has been enhanced to 1000 and 950 kt per year
respectively (IHS Chemical, 2016). It can be released into the en-
vironment through industrial, agricultural, and general human
wastewater effluents, and sewage sludge (Fu and Kawamura, 2010;
Furhacker et al., 2000; Meesters and Schroder, 2002). Concentrations of
BPA vastly varied in the contaminated environments: 17.2 mg/mL
(17,200 ppm) in hazardous landfill leachates (Yamamoto et al., 2001),
21 ppm in freshwater (Crain et al., 2007), 4.4 ng/mL (0.0044 ppm) ~
19 mg/mL (19,000 ppm) in American rivers, and 21.3 μg/g (21.3 ppm)
in soil of hygienic landfill in Brazil (Lovatel et al., 2011), 410 ng/L
(0.00041 ppm) in surface water in Germany (Qiu et al., 2013) and
17.2 mg/L (17.2 ppm) in garbage leachate in Japan (Sun et al., 2013).
To date, most detection methods to identify and quantify chemical
pollutants are based on gas chromatography and mass spectrometry
(Berlioz-Barbier et al., 2014; Magi et al., 2010; Ribeiro et al., 2007;
Selvaraj et al., 2014). Arabidopsis, a herbaceous model plant, has been
modified to detect estrogenic compounds in the environment. Tojo
et al. (2006) generated transgenic Arabidopsis overexpressing a

⁎
Corresponding author.
E-mail addresses: kimdg@sejong.ac.kr (D. Kim), rbahmani@sejong.ac.kr (R. Bahmani), jhko@khu.ac.kr (J.-H. Ko), sbhwang@sejong.ac.kr (S. Hwang).
1
Equal contribution.

https://doi.org/10.1016/j.ecoenv.2018.12.029
Received 19 June 2018; Received in revised form 7 December 2018; Accepted 11 December 2018
Available online 19 December 2018
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
D. Kim et al.
chimeric estrogen receptor consisting of hER–LBD (the human estrogen
receptor ligand-binding domain), LexA–DBD (the LexA DNA-binding
domain from E. coli), and a chimeric nuclear receptor coactivator in-
cluding hTIF2 NID (the nuclear receptor interaction domain of human
transcriptional intermediary factor 2) and VP16 AD (the transactivation
domain of VP16 from the herpes simplex virus) (Tojo et al., 2006). In
this system, upon binding of estrogen to the hER–LBD, the chimeric
receptor interacts with a chimeric coactivator, thereby stimulating the
transcription of the GUS reporter. This line was able to detect estrogen
(17β-estradiol) at concentrations as low as 50 pM (13 ppt), BPA at >
100 nM, and nonyl phenol (NP) at 1 µM. A different transgenic Arabi-
dopsis line expressing LexA DBD, hER–LBD, and VP16 AD displayed GFP
mRNA expression in response to 17ß-estradiol at 1 ppt, BPA at 100 ppb,
and NP at 1 ppm (Inui et al., 2009). Transgenic Arabidopsis plants that
show inducible expression of the Cre recombinase gene in response to
17ß-estradiol have also been generated (Zuo et al., 2006).
Anthocyanins are a group of colorful bioactive natural pigments
that have significant physiological and ecological functions in plants.
They provide protection from ultraviolet light, participate in the
plant–microbe interaction, and play roles in male fertility and in the
response to nutrient availability (Landi et al., 2015; Winkel-Shirley,
2001). One of the most important secondary metabolic pathways stu-
died in plants so far is that of anthocyanins, which represent a special
branch of the flavonoid pathway (Grotewold, 2006; Petroni and Tonelli,
2011). Anthocyanin biosynthesis is regulated by a complex of different
transcription factors (TFs) including R2R3-MYB, basic helix-loop-helix
(bHLH) proteins and WD40 proteins (MYB–bHLH–WD40 TF complex,
MBW) that promotes transcriptions of anthocyanin biosynthetic genes
(Albert et al., 2014; Gonzalez et al., 2008; Petroni and Tonelli, 2011). In
Arabidopsis, different combinations of PRODUCTION OF ANTHOCYA-
NIN PIGMENT1 (PAP1), PAP2, MYB113 and MYB114 (MYB TFs), TT8/
GL3/ EGL3 (bHLH TFs), and TRANSPARENT TESTA GLABRA1 (TTG1)
(WD repeat protein) formulate several different ternary protein com-
plexes (MBW) and activate transcriptions of anthocyanin biosynthesis
genes (Baudry et al., 2004; Shi and Xie, 2014; Zhang et al., 2003;
Zimmermann et al., 2004). By contrast, members of the 1R-MYB tran-
scription factor CPC and MYBL2 act as negative regulators of antho-
cyanin biosynthesis genes by competing with PAP1/PAP2 (positive
regulators) to bind bHLH proteins and disturbing the formation of the
active transcription complex (Matsui et al., 2008; Zhu et al., 2009;
Zimmermann et al., 2004). In addition, PtrMYB119, an R2R3-MYB
transcription factor from poplar (Populus trichocarpa) is homologous to
Arabidopsis PAP1 (production of anthocyanin pigment 1), which is a
transcriptional activator of anthocyanin biosynthesis. Recently, it was
found that when over-expressed in Arabidopsis and poplar, PtrMYB119
promoted anthocyanin (mainly cyanindin-3-O-glucoside) biosynthesis
through enhancing the expression of anthocyanin biosynthetic genes
(Cho et al., 2016). Although several attempts have been carried out to
detect the endocrine-disrupting compounds using XVE inducible system
in plants, they employed reporter genes such as GUS and GFP that show
strong expressions in roots, therefore required a microscopy or staining
technique which are not applicable to non-scientist community (Inui
et al., 2009; Tojo et al., 2006). Therefore, to improve the drawbacks of
the previous detection systems, we generated transgenic Arabidopsis
lines that produce a red color in leaf as a result of anthocyanin bio-
synthesis in response to BPA at concentrations as low as 10 ppm, en-
abling the observation of color change without any additional experi-
mental equipments and processes.
2. Materials and methods
2.1. Plant materials and growth conditions
Arabidopsis thaliana, ecotype Columbia (Col-0), was obtained from
ABRC (Arabidopsis Research Center, USA) and used as the wild-type
and to generate transgenic plants. Arabidopsis seeds were surface-
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Ecotoxicology and Environmental Safety 170 (2019) 627–634
sterilized to avoid bacterial and fungal contaminations with 70%
ethanol for 5 min then in 1.2% bleach solution containing 0.025% (v/v)
Triton X-100. The seeds were incubated at 4 °C for 3 d for vernalization.
Plants were grown under sterile conditions in a growth chamber under
a 16-h light/8-h dark photoperiod with cool white fluorescent light at
150 μmol m−2 s−1 and day/night temperatures of 23/21 °C. The seed-
lings were grown in Petri dishes on half-strength Murashige and Skoog
(MS) medium (pH=5.7, Duchefa Biochemie, Haarlem, Netherlands)
containing 1% (w/v) sucrose and 0.7% (w/v) plant agar (Duchefa
Biochemie, Haarlem, Netherlands). For the BPA chemical treatments,
100 mM stock solution of BPA (Sigma-Aldrich, Cat# 133027) was made
in DMSO, and aliquots were added to 1/2 MS media to produce in-
dicated concentrations (1 ppt ~ 10 ppm).
2.2. Plasmid vector construction and production of transgenic Arabidopsis
To create the estrogen-inducible anthocyanin synthesis constructs,
we used the two Gateway vectors pMDC7 (Curtis and Grossniklaus,
2003) and pB2GW7 (Laboratory of Plant Systems Biology, Ghent Uni-
versity, Belgium). The 5′ FLAG T sequence (encoding the polypeptide
protein tag DYKDDDDK) and the PtrMYB119 cDNA sequence were
amplified using Ex-Taq DNA polymerase (Takara, Japan) and sub-
cloned downstream of LAP (eight copies of the lexA operator with a
−46 minimal promoter) in the pMDC7 vector. The fragment including
LAP, T, and PtrMYB119 was again amplified by PCR to obtain the
LAP_Flag T_PtrMYB119 (LTM) fragment. This fragment was sub-cloned
into the pB2GW7 vector (pB2GW7_LTM). Finally, the XVE region (X,
DNA-binding domain of LexA; V, transcriptional activation domain of
VP16; and E, ligand-binding domain of the human estrogen receptor) of
pMDC7 was amplified using forward and reverse primers linked to an
Spe1 restriction sequence, and then was sub-cloned into pB2GW7_LTM.
Gateway cloning procedure was performed according to the manufac-
turer's instructions (Invitrogen, USA). All vector construction processes
are illustrated in Fig. 1. The resulting constructs were confirmed by
DNA sequencing. The primers used in this study are listed in Supporting
information Table S1. Vectors were introduced into Agrobacterium tu-
mefaciens strain GV3101, and transformed to Arabidopsis by the floral-
dip method (Clough and Bent, 1998). Transformed seeds were selected
and maintained on MS medium containing 5 µg/mL BASTA.
2.3. RNA extraction and qRT-PCR analyses
Total RNA (three independent biological samples) was isolated
using Trizol (Life Technologies, Gaithersburg, MD, USA) according to
the manufacturer's instructions. Then, 2 µg total RNA was reverse-
transcribed to produce first-strand cDNA using a PrimeScript™ RT re-
agent kit (Takara, Japan) according to the manufacturer's instructions.
Quantitative real-time PCR (qRT-PCR) was performed using the CFX
Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA,
USA) with iQ™ SYBR® Supermix (Bio-Rad, Hercules, CA, USA) fol-
lowing manufacturer's manual. The Arabidopsis ACTIN2 gene was used
as the internal quantitative control, and the relative expression level of
each gene was calculated according to the 2-ΔΔC
t method (Pfaffl, 2001).
Each qRT-PCR experiment was carried out three times with different
cDNA sets obtained from three independent biological replicates.
Primer3 software was used for primer design (http:// primer3.ut.ee/).
All primer sequences are shown in Table S1.
2.4. Anthocyanin measurement
Total anthocyanin content was determined as described by Nakata
et al. (2013). Surface-sterilized seeds of wild-type Col-0 and transgenic
Arabidopsis were germinated on 1/2 MS medium containing BPA at
indicated concentrations, and grown under long-day conditions (16-h
light/8-h dark) in a growth chamber. Samples were harvested at in-
dicated times, immediately frozen in liquid nitrogen, and kept at
D. Kim et al.
− 80 °C until analysis. Samples were ground in a mortar and pestle with
liquid nitrogen, then resuspended in five volumes (based on fresh
weight) 45% (v/v) cold methanol and 5% (v/v) acetic acid solution.
After removing cell debris by centrifugation (10,000 g), the clear su-
pernatant was used to determine anthocyanin content. The absorbance
of the solution at 530 nm by anthocyanins and at 657 nm by chlor-
ophylls was measured spectrophotometrically, and then the antho-
cyanin content was calculated as follows to correct the absorbance of
530 nm by chlorophylls: [OD530-(0.25 × OD657)]×extraction volume
(mL) × 1/weight of a tissue sample (g).
2.5. Microscopic analyses
The color change on the abaxial side of Arabidopsis leaves was ob-
served under a Leica Stereoscopic microscope (MZ10F, Wetzlar,
Germany) using Leica Application Suite version 4.6.0. Images were
taken using a Leica DFC450 camera at 10 × to 20 × magnification.
2.6. Statistical analysis
Values are means ± SE of three independent experiments. All data
were subjected to analysis of variance (ANOVA) using SAS software
(version 9.1), and comparisons of the means were performed according
to Tukey's test at P ≤ 0.05 or P ≤ 0.01.
3. Results and discussion
3.1. Generation of BPA-sensing transgenic Arabidopsis
To generate BPA-sensing transgenic plants, Arabidopsis was trans-
formed with a recombinant vector containing the XVE region, LAP
(OLexA, eight copies of the LexA operator sequence; −46, the −46 35S
minimal promoter) and PtrMYB119, a R2R3-MYB transcription factor
from P. trichocarpa (Cho et al., 2016) (Fig. 1). If estrogen compounds
bind to the ligand-binding domain of E (estrogen receptor) in XVE, the
XV domain binds to LAP, inducing the transcription of PtrMYB119 with
a subsequent enhancement of anthocyanin biosynthetic gene expres-
sion, resulting in anthocyanin synthesis (red color production) (Fig. 1).
Using BASTA selection, we obtained a total of 23 transgenic Arabidopsis
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Ecotoxicology and Environmental Safety 170 (2019) 627–634
Fig. 1. Schematic representation of
generating a BPA-inducible vector.
PG10−90, synthetic promoter control-
ling XVE; T3A, rbcsS3A poly(A) addi-
tion sequence; XVE, DNA sequences
encoding a chimeric transcription
factor containing DNA-binding domain
of LexA (residues 1–87), transcriptional
activation domain of VP16 (residues
403–479), and ligand-binding domain
of human estrogen receptor (residues
282–595); LAP (OLexA-46), eight copies
of LexA operator sequence and −46
35S minimal promoter; T, polypeptide
tag; PtrMYB119, R2R3-MYB transcrip-
tion factor from poplar (Populus tricho-
carpa); Hygr, hygromycin B resistance;
attL1 and attR1, GateWay att re-
combination sites; BAR, basta re-
sistance gene; ccdB, toxicity gene for
bacterial selection. Arrows indicate di-
rection of transcription. Diagram
adapted from Zuo et al. (2000). Vector
construction procedure is explained in
detail in Materials and Methods.
.
lines. Two lines (XVE-1 and XVE-2) were selected for further experi-
ments based on their intense red color change in response to 10 ppm
BPA.
3.2. Anthocyanin synthesis in transgenic Arabidopsis in response to BPA is
determined by BPA concentration and transcript levels of PtrMYB119 and
anthocyanin biosynthesis genes
To examine the production of the red color (anthocyanin) by
transgenic Arabidopsis XVE-PtrMYB119 in response to estrogenic com-
pounds, plants (homozygous T3) were grown for 15 days on 1/2 MS
agar medium supplemented with BPA at concentrations ranging from 1
ppt to 10 ppm. The transgenic plants showed that leaves turned red in
response to BPA at concentrations as low as 10 ppm, while control
plants (Col-0) did not display red colors in leaves. Correspondingly, the
anthocyanin content increased in response to BPA at concentrations of
10 ppm or higher (Fig. 2A). By contrast, the transcription of PtrMYB119
was induced by BPA at very low concentrations (as low as 1 ppt), and
reached a 3-fold increase at 10 ppb BPA (Fig. 2B). To explore the reason
for the difference between the concentrations inducing PtrMYB119
expression (1 ppt ~10 ppb) and those inducing anthocyanin accumu-
lation (10 ppm), we quantified the transcript levels of AtCHS (encoding
chalcone synthase, AT5G13930), AtANS (encoding anthocyanidin syn-
thase, AT4G22880) and AtUFGT (encoding UGT78D2, AT5G17050),
which function in the first and last two steps in anthocyanin bio-
synthesis. As shown in Fig. 2C–E, their transcript levels only increased
in response to higher concentrations of BPA (1–10 ppm), even though
that of the transcription factor PtrMYB119, which promotes antho-
cyanin biosynthetic gene expression, was elevated by BPA at much
lower concentrations (1 ppt ~ 10 ppb). By contrast, control plants did
not show any induction in these gene expressions in response to BPA.
To understand why anthocyanin was not synthesized in roots in
response to BPA, we analyzed gene transcript levels separately in roots
and shoots. The transcript level of PtrMYB119 in the root increased in
response to 1 ppt BPA, and reached a 2-fold increase at concentrations
of 10 ppb to 10 ppm BPA although it was lower than the 5-fold increase
in whole seedlings (Fig. 2B and F). However, the transcript levels of the
anthocyanin biosynthesis genes AtCHS and AtUFGT were not enhanced
significantly in the root in response to BPA at concentrations up to
D. Kim et al. Ecotoxicology and Environmental Safety 170 (2019) 627–634

Fig. 2. Anthocyanin contents and transcript levels of PtrMYB119 and anthocyanin biosynthetic genes in control and transgenic Arabidopsis in response to different
concentrations of BPA. (A) Anthocyanin contents in Col-0 and transgenic Arabidopsis plants. Plants were germinated and grown on 1/2 MS medium supplemented
with BPA at different concentrations (from 0 to 10 ppm). Anthocyanin levels were determined on day 11, when red color started to appear. Data are mean values of
three independent experiments, ± SE. (B) Transcript pattern of PtrMYB119 in plants grown in the same conditions as in (A). Transcript levels of PtrMYB119 were
determined on day 11 by qRT-PCR and normalized to that of AtActin. (C), (D), (E) Transcript levels of AtCHS, AtANS, and AtUFGT on day 11 in plants grown in the
same conditions as in (A). (F), (H), (J) Transcript levels of PtrMYB119, AtCHS, and AtUFGT on day 11 in roots of plants grown in the same conditions as in (A). (G),
(I), (K) Transcript levels of PtrMYB119, AtCHS and AtUFGT on day 11 in shoots of plants grown in the same conditions as in (A). All the transcript expression levels
were measured by qRT-PCR and normalized to that of AtActin. Values are means ± SE of three independent experiments.

10 ppm (Fig. 2F, H, J). Thus, anthocyanin was probably not synthesized
in the root in response to BPA even at 10 ppm. In contrast, the transcript
level of PtrMYB119 in the shoot was enhanced by 1 ppt BPA, and
reached a 5-fold increase by 10 ppm BPA which is similar to that of
whole seedlings (Fig. 2B and G). Owing to the 5-fold increase in
PtrMYB119 expression, AtCHS, and AtUFGT transcripts in shoots ele-
vated significantly in response to BPA at 1–10 ppm (Fig. 2G, I, K), and
finally anthocyanin was probably synthesized at 1–10 ppm BPA in
shoots, corresponding to the pattern detected in whole seedlings
(Fig. 2A, C, E). Taken together, these results showed that anthocyanin
synthesis in leaves in response to 10 ppm BPA was attributed to
PtrMYB119 expression in the shoots, while anthocyanin was not syn-
thesized in roots because anthocyanin biosynthesis genes were not ex-
pressed in the roots despite the 2-fold increase of PtrMYB119 expres-
sion. These results suggest that some factor required for PtrMYB119-
driven anthocyanin biosynthetic gene expression is present in the
leaves, but is scarce in the roots.
The lower transcript level of PtrMYB119 in the root (2-fold increase)

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Fig. 3. Color change of transgenic XVE-PtrMYB119 Arabidopsis grown on 1/2 MS medium containing 10 ppm BPA. (A) Color change of leaf abaxial surface in
Arabidopsis expressing BPA-inducible expression vector. Col-0 and transgenic Arabidopsis plants (XVE-1 and −2) were germinated and grown on 1/2 MS medium
containing 10 ppm BPA. Change in leaf color was monitored during 10–15 days after sowing seeds on plates (D: days). (B) Anthocyanin contents in Col-0 and
transgenic Arabidopsis grown in the same conditions as in (A). (C) Expression pattern of PtrMYB119 in plants grown in the same conditions as in (A). PtrMYB119
transcript levels were measured by qRT-PCR and normalized to that of AtActin. Data are mean values of three independent experiments ± SE.

in response to 10 ppm BPA compared to that of shoot (5-fold increase)
may be responsible for no expression of anthocyanin biosynthetic genes
in roots. This suggests that PtrMYB119 should be expressed to some
level (probably 5-fold increase) to make a functional transcriptional
complex (MBW) for promoting transcriptions of anthocyanin biosyn-
thetic genes. This may explain why anthocyanin was not synthesized in
response to BPA at below 10 ppm at which PtrMYB119 expression was
not reached to 5-fold increase. Regarding the requirement of 5-fold
PtrMYB119 expression for anthocyanin biosynthesis, the poplar
PtrMYB119 may not be efficient in producing a functional transcrip-
tional complex (MBW) for anthocyanin biosynthesis in Arabidopsis.
In addition, we have examined the effect of estrogen 17β-estradiol
(EST) and estrogen-like compound nonyl phenol (NP), but our trans-
genic plants did not show color changes (Fig. S1). To understand the
reason, we examined expression levels of MYB119 and CHS. As shown
in Fig. S2, MYB119 level was induced up to 2.5-fold by EST and NP, but
it was far below than 5-fold increase by BPA. Likewise, in roots,
MYB119 expression was increased to 2.5-fold by BPA but roots did not
display a red color (Fig. 3F). Due to the insufficient induction of
MYB119 by EST and NP, CHS level was not enhanced compared to that
of MS (no treatment) (Fig. S2), resulting in no synthesis of anthocyanin
(red color). This result indicates that our transgenic plants display color
changes specifically in response to BPA only.
3.3. Time-dependent anthocyanin synthesis in response BPA in transgenic
Arabidopsis
When transgenic Arabidopsis XVE-PtrMYB119 plants were grown for
15 days on 1/2 MS agar medium supplemented with 10 ppm BPA, their
leaves turned red on day 11 (D11), and the red color peaked on day 12
(D12) before declining and disappearing by day 14 (D14) (Fig. 3A). The
anthocyanin content exactly matched the pattern of red coloration
(Fig. 3B), but the peak in PtrMYB119 expression occurred 1 day before
the peak in red coloration (anthocyanin content), reaching its max-
imum level at D11 and then decreasing to its minimal level at D12 or
D13 (Fig. 3C). By contrast, red colors, anthocyanin and PtrMYB119
expression were not induced by BPA in control plants.
The transcript patterns of anthocyanin biosynthetic genes, AtCHS,
AtCHI (encoding chalcone flavanone isomerase, AT3G55120), AtF3H
(encoding flavanone 3-hydroxylase, AT3G51240), AtDFR (encoding
dihydroflavonol 4-reductase, AT5G42800), AtANS, and AtUFGT were
the same as that of PtrMYB119, showing peak levels at D11 and then
declining to minimum levels at D12 (Fig. 4). In contrast, the transcript
level of AtFLS (encoding flavonol synthase 1, AT5G08640), which is
involved in the branching step of flavonol synthesis, was only slightly
elevated at D11 in response to 10 ppm BPA (Fig. 4). On the basis of
these results, we concluded that the appearance and disappearance of
the red color on leaves of transgenic plants were due to the synthesis
and degradation of anthocyanin, mediated by the expression of an-
thocyanin biosynthetic genes via regulation of the transcription factor
PtrMYB119. The expression pattern of PtrMYB119 which peaked on
D11 and declined to the minimum at D12 or D13 suggests that its
transcription was turned off after D11 by an unknown mechanism. It is
also possible that BPA is degraded or chemically converted into other
compounds (Tojo et al., 2006). However, control plants did not display
enhancements in these gene expressions in response to BPA.
When transgenic Arabidopsis XVE-PtrMYB119 plants were grown for
21 days on 1/2 MS agar medium and then dipped in 1/2 MS liquid
medium containing 10 ppm BPA, the leaves turned red on day 3 and
this coloration was maintained until day 5 (D5) (Fig. 5A). During this

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Fig. 4. Transcript levels of anthocyanin biosynthetic genes in control and transgenic Arabidopsis in response to 10 ppm BPA. Col-0 and transgenic Arabidopsis plants
(XVE-1 and −2) were germinated and grown on 1/2 MS medium in the presence of 10 ppm BPA, and transcript levels of anthocyanin biosynthetic genes were
determined by qRT-PCR during 10–15 days after sowing seeds on plates (D: days). Data are mean values of three independent experiments, ± SE. Transcript levels
were normalized to that of AtActin. Genes and their encoded enzymes are as follows: AtCHS, chalcone synthase (AT5G13930); AtCHI, chalcone flavanone isomerase
(AT3G55120); AtF3H, flavanone 3-hydroxylase (AT3G51240); AtDFR, dihydroflavonol 4-reductase (AT5G42800); AtANS, anthocyanidin synthase (AT4G22880);
AtFLS, flavonol synthase 1 (AT5G08640); AtUFGT, UGT78D2 (AT5G17050).

period, the anthocyanin content increased from day 2, peaked on day 4,
and decreased slightly on day 5 (Fig. 5B), consistent with the pattern of
red coloration. The increase in PtrMYB119 transcript level occurred
1 day before the accumulation of anythocyanin; the PtrMYB119 tran-
script level peaked on day 3 and then decreased sharply to reach the
minimal level on day 5 (Fig. 5C). The transcription patterns of AtCHS,
AtANS, and AtUFGT were the same as that of PtrMYB119 (Fig. 5D, E, F).
These suggest that the anthocyanin pigment remained after the tran-
scription of PtrMYB119, AtCHS, AtANS, and AtUFGT had ceased in the
dipping experiment, while the anthocyanin pigment disappeared more
rapidly in the plating experiment (Fig. 3). Although small induction was
observed after D3 in anthocyanin level, expressions of AtCHS, AtANS,
and AtUFGT, this may be attributed to water stress (Fig. 5).
During the last two decades, various EDC detection systems were
developed using human and yeast cells (Mueller, 2004), and fish (Van
den Belt et al., 2003) which take advantages of reporter genes. How-
ever, these systems have limitations and difficulties such as aseptic
manipulations (for yeast and human cells) and life preservation (for
fish). By contrast, transgenic Arabidopsis plants to detect EDC have
advantages over the above-mentioned systems: (1) Plants can be tested
on both agar and liquid media. (2) Convenient storage of the seeds (up
to several years) in appropriate conditions. (3) Sufficient seeds can be
harvested from each plant and stored (Tojo et al., 2006).
The designed construct for generating transgenic plants in our study
is similar to that of Zuo et al. (2000). Using similar constructs, two
research groups also established transgenic Arabidopsis expressing a
chimeric estrogen receptor linked to the GUS or GFP reporter genes
which are able to detect EST, BPA and NP (Inui et al., 2009; Tojo et al.,

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Fig. 5. Leaf color change of transgenic Arabidopsis during a 5-day exposure to 10 ppm BPA. (A) Color change of leaf abaxial surface in Arabidopsis plants expressing
estrogen-inducible expression vector during a 5-day BPA treatment. Col-0 and transgenic Arabidopsis plants (XVE-1 and −2) were germinated and grown on 1/2 MS
medium for 3 weeks and then transferred to 10 ppm BPA solution. Changes in leaf color were monitored for 5 days (D, day). (B) Anthocyanin contents of Col-0 and
transgenic Arabidopsis plants grown in the same conditions as in (A). (C) Transcription pattern of PtrMYB119 in plants grown in the same conditions as in (A).
PtrMYB119 transcript levels were measured by qRT-PCR and normalized against that of AtActin. (D), (E), (F) Transcript levels of AtCHS, AtANS, and AtUFGT in plants
grown in the same conditions as in (A). AtCHS, AtANS, and AtUFGT transcript levels were measured by qRT-PCR and normalized to that of AtActin. Values are
means ± SE of three independent experiments.

2006). However, these transgenic plants have some weak points; the
expression of the GUS/GFP reporter genes mostly in underground roots,
and the requirement of microscopic and staining skills that cannot be
performed by non-scientist persons. By contrast, our transgenic plants
provide a convenient and cost-effective system to identify the presence
of BPA because the red color appears in the leaves enabling the ob-
servation by naked eye, and additional staining or microscopic analyses
are not required.
4. Conclusions
to detect the presence of BPA because no additional experimental
procedures are required, unlike the previously developed lines expres-
sing GUS or GFP. Therefore, even non-scientists are able to detect BPA
contaminated in soils, water and plastic food cases etc.
Author contribution
S.H. designed and supervised whole research, and wrote the article
with contributions of all the authors; D.K. and R.B. equally performed
most of the experiments; J.K. cloned PtrMYB119.

Using human estrogen receptor and poplar MYB119 genes, we Funding

generated transgenic Arabidopsis lines that produce a red color on
leaves in response to the estrogen-like pollutant, BPA. This response is
caused by the expression of PtrMYB119, which is induced by binding of
BPA to the estrogen receptor, leading to anthocyanin biosynthesis.
These plants can be used as a fast, convenient and cost-effective system
This work was supported by the Civil Research Projects for Solving
Social Problems through the National Research Foundation of Korea
(NRF) funded by the Ministry of Science, ICT & Future Planning [NRF-
2015M3C8A6A06014500], Korea.

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Conflict of interest Magi, E., Di Carro, M., Liscio, C., 2010. Passive sampling and stir bar sorptive extraction
for the determination of endocrine-disrupting compounds in water by GC-MS. Anal.
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The authors have no conflicts of interest to declare. Matsui, K., Umemura, Y., Ohme-Takagi, M., 2008. AtMYBL2, a protein with a single MYB
domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis.
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