Ecotoxicology and Environmental Safety 145 (2017) 150–160

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Ecotoxicology and Environmental Safety

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

Stomatal and non-stomatal factors regulated the photosynthesis of soybean MARK

seedlings in the present of exogenous bisphenol A
⁎ ⁎⁎
Liya Jiaoa,c, Lihong Wanga,c, Qing Zhoua,c, , Xiaohua Huangb,
a
State Key Laboratory of Food Science and Technology, School of Environment and Civil Engineering, Jiangsu Key Laboratory of Anaerobic Biotechnology, Jiangnan
University, Wuxi 214122, China
b
Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, School of Chemistry and Materials Science,
Nanjing Normal University, Nanjing 210046, China
c
Jiangsu Cooperative Innovation Center of Water Treatment Technology and Materials, Suzhou University of Science and Technology, Suzhou 212003, China

A R T I C L E I N F O A B S T R A C T

Keywords: Bisphenol A (BPA) is an emerging environmental endocrine disruptor that has toxic effects on plants growth.
BPA Photosynthesis supplies the substances and energy required for plant growth, and regulated by stomatal and non-
Net photosynthetic rate stomatal factors. Therefore, in this study, to reveal how BPA affects photosynthesis in soybean seedlings (Glycine
Stomatal factor max L.) from the perspective of stomatal and non-stomatal factors, the stomatal factors (stomatal conductance
Non-stomatal factor
and behaviours) and non-stomatal factors (Hill reaction, apparent quantum efficiency, Rubisco activity, car-
Soybean seedling
boxylation efficiency, the maximum Rubisco carboxylation velocity, ribulose-1,5-bisphospate regeneration ca-
pacities mediated by maximum electron transport rates, and triose phosphate utilization rate) were investigated
using a portable photosynthesis system. Moreover, the pollution of BPA in the environment was simulated. The
results indicate that low-dose BPA enhanced net photosynthetic rate (Pn) primarily by promoting stomatal
factors, resulting in increased relative growth rates and accelerated soybean seedling growth. High-dose BPA
decreases the Pn by simultaneously inhibiting stomatal and non-stomatal factors, and this inhibition decreases
the relative growth rates further reducing soybean seedling growth. Following the withdrawal of BPA, all of the
indices were restored to varying degrees. In conclusion, low-dose BPA increased the Pn by promoting stomatal
factors while high-dose BPA decreased the Pn by simultaneously inhibiting stomatal and non-stomatal factors.
These findings provide a model (or, hypothesis) for the effects of BPA on plant photosynthesis.

1. Introduction
Bisphenol A [2,2-bis(4-hydroxyphenyl)propane; BPA] is considered
to be an environmental endocrine disruptor, and is a commercially
important monomer used in synthesis of polycarbonate plastics, epoxy
resins, packaging coatings and flame retardants (Goodson et al., 2002).
These products are widely used in common products, including plastic
food containers, medical devices, baby bottles and thermal receipt
paper (Geens et al., 2011). Because of the tremendous demand in
commercial and daily life, BPA has become one of the world's most
frequently produced chemicals, with an annual output of 6.8 million
tons (Jandegian et al., 2015). Due to its widespread use and prevalence
in industrial discharge, BPA is ubiquitous in the environment with an
increasing content. For example, the mean BPA content of soils in sa-
nitary landfills in Brazil has been measured at 21.3 μg/g (Vieceli et al.,
2011). The BPA contents in hazardous landfill leachates and soil in
China are in the ranges of 1–370 μg/L and 0.1–10.5 μg/g, respectively
(Huang et al., 2012). Furthermore, the BPA content in the hazardous
landfill leachates in Japan reaches 1.3–17.2 mg/L (Yamamoto et al.,
2001) and rivers in South Florida are in the range of 4.4–190 mg/L
(Singh et al., 2010). In incidents of pollution by BPA emitted acciden-
tally, the BPA content in the environment can exceed 17.2 mg/L, which

Abbreviations: BPA, bisphenol A; Pn, net photosynthetic rate; CO2, carbon dioxide; Gs, stomatal conductance; AQY, apparent quantum efficiency; Rubisco, ribulose-l,5-bisphosphate
carboxylase oxygenase; CE, carboxylation efficiency; Vc max , maximum Rubisco carboxylation velocity; Jmax, ribulose-1,5-bisphospate regeneration capacities mediated by maximum
electron transport rates; VTPU, triose phosphate utilization rate; A/Ci curve, the corresponding CO2 response curve; PGRs, growth index and plant growth rates; Ci, intercellular CO2
concentration; Ls, the relative stomatal limitation of photosynthesis; Lns, the relative non-stomatal limitation of photosynthesis; Cc, CO2 partial pressure at Rubisco; Rd, dark respiration;
Γ*, photorespiratory compensation point
⁎
Corresponding author at: State Key Laboratory of Food Science and Technology, School of Environment and Civil Engineering, Jiangsu Key Laboratory of Anaerobic Biotechnology,
Jiangnan University, Wuxi 214122, China.
⁎⁎
Corresponding author.
E-mail addresses: qingzhou510@yahoo.com (Q. Zhou), huangxiaohuanjnu@yahoo.com (X. Huang).

http://dx.doi.org/10.1016/j.ecoenv.2017.07.028
Received 25 January 2017; Received in revised form 30 June 2017; Accepted 11 July 2017
0147-6513/ © 2017 Elsevier Inc. All rights reserved.
L. Jiao et al.
has affected the biological activity of living organisms (Crain et al.,
2007). Therefore, the United States Environmental Protection Agency
proposed that 1.5 mg/L BPA is the upper safety limit for drinking water
(Geens et al., 2011).
Numerous studies have documented the negative effects of BPA on
animals and humans, particularly the impact on reproductive health
and immunological functions, as well as its ability to alter genetic
material (Flint et al., 2012; Gies and Soto, 2012; Rahman et al., 2015;
Rogers et al., 2013; Tiwari et al., 2012; Wong and Cheng, 2011). In
comparison, relevant research detailing the effects of BPA on plants,
especially terrestrial plants, is rare. The environmental risk assessment
report on BPA proposed further extended studies to address the po-
tential risks and toxic effects of BPA on plants, especially terrestrial
plants (Commission, 2008). Terrestrial plants can absorb BPA and
translocate it from the soil to aboveground areas (Nakajima et al.,
2002). In turn, BPA can affect plant growth and development (Ferrara
et al., 2006). Present literature has indicated that low doses of BPA
(0.01 and 1.5 mg/L) promote the growth of soybean (Glycine max L.)
and carrot (Daucus carota L.) seedlings (Qiu et al., 2013; Terouchi et al.,
2004), although higher doses (10.0, 17.2 and 50.0 mg/L) have been
found to inhibit the growth of tomato (Solanum lycopersicum Mill.),
durum wheat (Triticum durum Desf.), broad bean (Vicia faba L.) and
lettuce (Lactuca sativa L.) (Ferrara et al., 2006), as well as inhibit the
growth and ammonium assimilation in soybean (Glycine max L.) roots
(Qiu et al., 2013; Sun et al., 2013). Another study has revealed that
10.0 mg/L and higher doses of BPA inhibit the germination and elon-
gation of kiwifruit (Actinidia deliciosa A. Chev.) (Speranza et al., 2011).
Visible plant growth is based on imperceptible physiological activity
at the microscopic level. The basic physiological function of photo-
synthesis is to assimilate CO2 to form organic matter, which can gen-
erate the substances and energy required for plant growth and devel-
opment (Heldt and Piechulla, 2011). Recent studies have demonstrated
that different doses of BPA exert varying effects on plant growth and
development by regulating photosynthesis. For instance, BPA has been
found to regulate net photosynthetic rate (Pn) by controlling chlor-
ophyll synthesis and fluorescence reactions, thus affecting soybean
seedling growth (Jiao et al., 2015; Qiu et al., 2013).
However, photosynthesis is a complex physiological process that
involves primary reaction, electron transport, photophosphorylation
and carbon dioxide (CO2) assimilation. Thus, the mechanisms under-
lying the effects of BPA on plant photosynthesis may also be extremely
complex. Unfortunately, extensive and systematic research on the me-
chanisms underlying the effects of BPA on the Pn is scarce. In the pre-
sent study, the effects of BPA and the potential mechanisms underlying
the influences of BPA on plant photosynthesis, particularly the stomatal
and non-stomatal factors that directly impact plant photosynthesis,
were examined. To achieve this goal, stomatal factors and non-stomatal
factors were examined on soybean seedling leaves. Stomatal factors
included stomatal conductance (Gs) and behaviours. And non-stomatal
factors included Hill reaction activity, apparent quantum efficiency
(AQY), the activity of ribulose-l,5-bisphosphate carboxylase oxygenase
(Rubisco), carboxylation efficiency (CE), the maximum Rubisco car-
boxylation velocity (Vcmax), ribulose-1,5-bisphospate regeneration ca-
pacities mediated by maximum electron transport rates, and triose
phosphate utilization (VTPU) rate. The results may serve as reference for
objectively understanding and evaluating the potential environmental
risks of BPA pollution.
2. Materials and methods
2.1. BPA solution preparation
The selection of BPA doses was based on the following three factors:
(1) BPA concentrations used in previous reports on living organisms
(Ferrara et al., 2006; Mihaich et al., 2009). (2) Current global BPA
pollution levels, especially in relation to BPA pollution trends in
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Ecotoxicology and Environmental Safety 145 (2017) 150–160
developing countries (Saiyood et al., 2013). (3) High environmental
levels of BPA emitted accidentally. Based on these three considerations,
five BPA concentrations (1.5, 7.0, 12.0, 17.2, 50.0 mg/L) were selected.
Of these concentrations, 1.5 mg/L is considered to be a safe dose of BPA
in potable water and has been assigned as the upper safety limit of BPA
for individuals (Geens et al., 2011). Therefore, this level was used to
test the effect of BPA in plants (Adamakis et al., 2013; Li et al., 2008;
Speranza et al., 2011). The concentrations from 7.0 to 17.2 mg/L are
equivalent to the BPA concentrations of hazardous landfill leachates
and dust (Loganathan and Kannan, 2011; Yamada et al., 1999;
Yamamoto et al., 2001; Yasuhara et al., 1997). A 50.0 mg/L BPA level
denotes accidental contamination by high BPA concentrations in soil.
This concentration is often used in international reports to indicate the
toxicity of BPA in plants (Adamakis et al., 2013; Dogan et al., 2012;
Mihaich et al., 2009); thus, it was applied to illustrate the effect of BPA
on photosynthetic mechanisms. The BPA solutions (1.5, 7.0, 12.0, 17.2
and 50.0 mg/L) were prepared by dissolving BPA of a particular quality
(Sinopharm Chemical Reagent Co., Ltd, China) in half-strength Hoa-
gland's solution (pH 7.0), with sonication at 25 °C. At this temperature,
the water solubility of BPA is within the range of 120–300 mg/L and
has low volatility (Ferrara et al., 2006). Before BPA exposure, the
analytic confirmation of actual exposure concentrations was performed
by high performance liquid chromatography based on a previously
described method (Mihaich et al., 2009). The concentrations of BPA
residues in the exposure solution of BPA were 0.00, 1.49, 6.98, 11.97,
17.18 and 49.95 mg/L, corresponding to the nominal concentrations of
0.0, 1.5, 7.0, 12.0, 17.2 and 50.0 mg/L (Table 1).
2.2. Plant culture and treatment
The soybean seeds (Glycine max L.) (Zhonghuang 25, Wuxi seed Co.,
Ltd., China) were sterilized for 5 min using a 0.1% HgCl2 solution and
then washed several times using deionised water. The sterilized seeds
were placed on three layers of gauze in a dish and germinated in an
incubator (25 ± 1 °C). After the radicle lengths of the soybean seedlings
were close to 1 cm, the six plants were uniformly held using foam
boards with a similar size and six holes and transplanted into 10.5 L
plastic pots (35 × 25 × 12 cm, six plants per pot). The pots were filled
with distilled water, which was replaced daily. Soybean seedlings were
cultured in half-strength Hoagland's solution (pH 7.0) in a greenhouse
at a temperature of 20–25 °C, a relative humidity of 70–80% and a 14 h
photoperiod with a light intensity of 300 μmol/m2/s directed from
above (Flexas et al., 2006).
At the stage when the third set of true soybean leaves had developed
(almost 30 d following germination), the plants were transplanted into
the different test doses of BPA solution. The control plants were cul-
tured in half strength Hoagland's solution lacking BPA. All the treat-
ments were carried out in triplicate. To ensure sufficient nutrients for
plant growth and the stabilization of pH, the culture solution was re-
placed every 3 d. Before different doses of BPA exposures, 100 pots of
healthy plants with similar growing situation were selected for the
Table 1
The measured exposure concentrations of BPA in the exposure solution.
Nominal concentrations of BPA (mg/ Actual exposure concentrations of BPA
L) (mg/L)
0.0 0.00 ± 0.00a
1.5 1.49 ± 0.08a
7.0 6.98 ± 0.04a
12.0 11.97 ± 0.56a
17.2 17.18 ± 0.32a
50.0 49.95 ± 2.30a
Each value represents mean ± SD of three replicates. The letter “a” denotes no sig-
nificant differences between nominal concentrations and actual exposure concentrations
of BPA at p < 0.05 in the same row.
L. Jiao et al.
control and BPA treatment. From the 100 pots, three pots of soybean
seedlings (six plants per pot) were selected randomly to be exposed to
one concentration of BPA solution. The plants were exposed to the BPA
solution for 7 d, then cultured in half strength Hoagland's solution (pH
7.0) in the absence of BPA and stored for another 7 d. These conditions
were based on our pre-experiment, in which the results indicated that
after BPA exposure for 7 d and withdrawal of BPA exposure for another
7 d, the effects of BPA on soybean seedlings stabilized. After 7 d of
varying BPA exposure and withdrawal of BPA exposure, one plant per
pot in the same treatment group was selected for the determination of
each index. All measurements were made between 9:00 to 11:00 a.m.
on sunny, cloudless days to reduce experimental error.
2.3. Growth indices and plant relative growth rates (PGRs) determination
The test growth indices included the dry weight (leaves and stems)
and leaf areas of each plant. A CI-203 Laser Area Meter (CID, Inc., USA)
was used to measure leaf area. The third leaves from the same position
of soybean seedlings were sampled for the determination of leaf area.
The soybean materials were collected at 1, 7, 8 and 14 d following BPA
exposure, after which they were dried at 105 °C for 10 min, and were
then further dried to constant weight at 80 °C for 24 h in order to
measure the dry weight. The relative growth rates (PGRs) were calcu-
lated using the following equation: RGR = lnW2 – lnW1/(t2 – t1), where
ln is the natural logarithm (the logarithm to base e), W1 and W2 re-
present the stem and leaf dry weights (g), respectively, and t1 and t2
denote the time (day) (Kingsbury et al., 1984).
2.4. Determination of Pn, Gs, intercellular CO2 concentration (Ci) and
stomatal assay
Gas exchange characteristics were determined using a portable gas
exchange fluorescence system (GFS-3000, WALZ, Effeltrich, Germany).
The measurements were carried out under constant chamber CO2
concentration (385 μmol/mol), photosynthetic quantum flux density
(800 μmol/m2/s), leaf temperature (25 °C), chamber relative humidity
(70%) and air flow rate (750 μmol/s). The third fully expanded leaf was
enclosed in a gas exchange chamber (8 cm2) to perform the gas ex-
change measurements. Three leaves were measured in each treatment,
the Pn, Gs and Ci were recorded until the steady-state values were ob-
tained.
Stomatal assays were performed based on a previously described
method (Chen et al., 2010). Specifically, peeled abaxial epidermal strips
of the third fully expanded soybean leaves were immersed in a buffer
(pH 6.1) containing 10 mM KCl and 5 mM 2-(N-morpholino) propane-
sulfonic acid prior to observation. For each epidermal strip, three
images were captured using a Leica microscope (Leica Microsystems
AG, Solms, Germany) equipped with a Nikon NIS-F1 CCD camera and a
DS-U3 controller (Nikon, Tokyo, Japan). Stomatal aperture was calcu-
lated using a micrometre, and the quantity of open stomata were
counted using 10 fields for each sample (Meckel et al., 2007).
2.5. Determination of A/Ci curve and curve fit estimates of Vcmax, Jmax,
VTPU and CE, the relative stomatal limitation of photosynthesis (Ls) and the
relative non-stomatal limitation of photosynthesis (Lns)
The response relationship between net photosynthesis and internal
leaf CO2 concentration (A/Ci curve) was measured using a portable gas
exchange fluorescence system (GFS-3000, Waltz, Effeltrich, Germany).
Experiments started at a CO2 concentration of 400 μmol/mol and the
chamber temperature, relative humidity and air flow rate were set at
25 °C, 70% and 750 μmol/s, respectively. The light levels were main-
tained at 1400 μmol/m2/s saturating light levels that were determined
for the soybean seedlings grown under the conditions of the experi-
ment. While the steady state was reached, the internal leaf CO2 con-
centration was decreased to 30 μmol/mol and then progressively
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Ecotoxicology and Environmental Safety 145 (2017) 150–160
increased up to 1200 μmol/mol. Fifteen points of net photosynthesis
were used to construct an A/Ci response curve. The varying CO2 con-
centration was achieved by means of an automatically controlled CO2
injector. CE was regarded as the initial slope of the A/Ci (Ci < 200
μmol/mol) line (Wang et al., 2014).
The A/Ci data was used to estimate Vcmax, Jmax and VTPU, using the
Microsoft Excel utility presented in previous studies (Sharkey et al.,
2007). When A is Rubisco-limited, the response of A to CO2 can be
described by the following equation:
Cc − Γ *
A =Vcmax [ O
]−Rd
Cc + K c (1+ K ) (1)
o
where Vcmax is the maximum velocity of Rubisco for carboxylation, Cc is
the CO2 partial pressure at Rubisco, Kc is the Michaelis constant of
Rubisco for CO2, O is the partial pressure of oxygen at Rubisco and Ko is
the inhibition constant (usually taken to be the Michaelis constant) of
Rubisco for oxygen. This equation lends itself to a linear regression
approach to estimating Vcmax as the slope and –Rd as the intercept (Long
and Bernacchi, 2003). The symbol Γ* is the CO2 concentration, where
oxygenation proceeds at twice the carboxylation rate, causing photo-
synthetic uptake of CO2 to be exactly compensated by photorespiratory
CO2 release (Ethier and Livingston, 2004). Thus, Γ* is the photo-
respiratory compensation point, which is slightly lower than the overall
compensation point of the leaf. Rd is respiratory CO2 release other than
by photorespiration (day respiration) and is presumed to be primarily
mitochondrial respiration. Because of non-photorespiratory CO2 losses,
there is a net release of CO2 from leaves when air around the leaf has a
CO2 concentration equal to Γ*.
When A is limited by RuBP regeneration:
Cc − Γ *
A=J – Rd
4Cc +8Γ * (2)
where J is the rate of electron transport. J is sometimes used to estimate
a maximum rate (Jmax) that could be obtained with saturating light. The
J provided here is the rate of electron transport required to support
NADP+ reduction for RuBP regeneration at the measurement light in-
tensity.
When A is limited by TPU:
A = 3TPU – R d (3)
where TPU is the rate of use of triose phosphates, but can also be any
export of carbon from the Calvin cycle including direct use of photo-
respiratory glycine or serine. When significant glycine or serine use
occurs, TPU-limited photosynthesis
can decrease with a decrease in O2 or increase in CO2 (Harley et al.,
2010). The reverse sensitivity to CO2 and O2 can also occur because
during TPU-limited photosynthesis, high phosphoglyceric acid levels
can suppress starch synthesis by inhibiting phosphoglucoisomerase.
Therefore, TPU-limited photosynthesis is seen as no increase in A with
increasing CO2 or a decrease with increasing CO2 (Sharkey et al., 2007).
The relative stomatal limitation of photosynthesis (Ls) were quan-
tified using the A/Ci response curves (Battie-Laclau et al., 2014). The Ls
was calculated according the formula: Ls (%) = 100 × (Aci – Aca)/Aci,
where Aci and Aca represent net photosynthesis at the Ci level (the rate
at which stomatal resistance to CO2 diffusion is assumed to be zero) and
ambient CO2 concentration of 400 μmol/mol, respectively. The relative
non-stomatal limitation of photosynthesis (Lns) levels were estimated as
the decrease of maximum CO2 assimilation rates under saturated light
conditions and CO2 levels under BPA exposure relative to the value of
the control (Lns = 0 in the control group).
2.6. Determination of AQY
AQY was determined by means of a portable gas exchange fluor-
escence system (GFS-3000, WALZ, Effeltrich, Germany). To determine
L. Jiao et al. Ecotoxicology and Environmental Safety 145 (2017) 150–160

the light response curve, the leaf temperatures were set at 25 °C, CO2 Table 2
concentrations were set to 385 μmol/mol and chamber relative hu- Relationships between the Pn and PGRs, growth and the Pn and Hill reaction activity,
AQY, CE, Rubisco activity and Vcmax, Jmax, VTPU, Gs and Ci levels in leaves following BPA
midity at 70%. The following light intensities were used for the AQY
exposure and the withdrawal of BPA exposure.
measurement: 1400, 1200, 1000, 800, 600, 400, 300, 200, 170, 150,
130, 100, 70, 50, 30, 10 and 0 μmol/m2/s. Approximately 5 min was y x Linear regression equation Correlation
allotted for a leaf to achieve steady-state photosynthesis under each coefficient (r)
light intensity. The AQY was calculated according to the slopes of the
After BPA exposure
linear segments (light intensity < 200 μmol/m2/s) of the light-response Leaf DW Pn y = 0.0051x+0.1117 0.931**
curves of photosynthesis (Zhang et al., 2012). Stem DW y = 0.0032x+0.0081 0.946**
Leaf area y = 0.2322x+6.5003 0.957**
2.7. Hill reaction activity determination PGRs y = 0.0145x–0.0460 0.916**
Pn Hill reaction y = 1.3672x+2.0368 0.951**
activity
The chloroplasts were extracted based on previously described AQY y = 268.50x+2.0615 0.933**
methods (Wen et al., 2011). The leaf tissue (2 g) was mechanically CE y = 268.82x–2.1325 0.954**
ground in a frozen extraction solution of 20 mM Tris-HCl buffer (pH Rubisco activity y = 0.4431x+1.9879 0.951**
Vcmax y = 0.2086x+3.0831 0.972**
7.8) containing 5 mM MgCl2, 10 mM NaCl, 300 mM sucrose and 1 mM
Jmax y = 0.1720x–1.0579 0.982**
EDTA. The homogenate was filtered using four layers of cheesecloth, VTPU y = 1.7843x+1.708 0.972**
and then the filtrate was centrifuged at 200 ×g for 2 min (4 °C). After Gs y = 0.0885x–14.836 0.958**
that, the supernatant was centrifuged at 3000 ×g for 5 min (4 °C). The Ci y = –0.1908x+69.868 –0.870**
final pellet containing the chloroplasts was suspended with a minimal Following withdrawal of BPA exposure
Leaf DW Pn y=0.0073x+0.0892 0.888**
amount of extraction solution. The chloroplast suspension was re-
Stem DW y = 0.0046x–0.0028 0.892**
frigerated for analysis. The Hill reaction activities were measured using Leaf area y = 0.3929x+4.7042 0.822**
a spectrophotometer as indicated by the rate of 2,6-di- PGRs y = 0.0023x–0.0103 0.898**
chlorophenolindophenol (2,6-DCPIP) photo-reduction, with water Pn Hill reaction activity y = 1.1299x+2.7636 0.927**
AQY y = 174.46x+5.4460 0.908**
(H2O→DCPIP) or 1,5-diphenyl carbazide (DPC→DCPIP) used as the
CE y = 161.07x+3.9045 0.961**
electron donor. The final 3 mL reaction mixture included chloroplasts Rubisco activity y = 0.3828x+3.2648 0.954**
that contained 15 μg chlorophyll, 15 μM DCPIP, 0.01 M phosphate Vcmax y = 0.1196x+7.2611 0.932**
buffer (pH 6.8), 0.1 M KCl and 100 μM MgSO4. This mixture was ex- Jmax y = 0.0954x+4.7703 0.943**
posed to saturated white light through a 10 cm water filter to reduce VTPU y = 1.4807x+2.8416 0.915**
Gs y = 0.0073x–19.835 0.926**
infrared radiation and heat levels for 30 s. DCPIP bleaching caused by
Ci y = –0.1427x+55.325 –0.780**
photoreduction was measured at 600 nm and 0.5 mM DPC was added.
** Significance at p < 0.01.
2.8. Rubisco activity determination
performed using the software SPSS 17.0.
It was reported that the content of Rubisco protein reaches up to
more than 50% of the contents of total soluble proteins in the normal
leaves (Spreitzer and Salvucci, 2002), and thus the Rubisco activity is 3. Results
more influenced by the degree of activation of Rubisco carboxylase, not
simply related to the content of Rubisco (Manter et al., 2005). There- 3.1. Effects of BPA on the PGRs, growth indices, and Pn in the soybean
fore, in this manuscript, we only determined the Rubisco activity. To seedlings
measure Rubisco activity, two discs (4 cm2) obtained from the same
leaves used for the gas exchange measurements were frozen with liquid The changes in the PGRs, dry weights (leaves and stems) and leaf
nitrogen and stored at −70 °C. The leaves were ground into a powder area in the soybean seedlings exposed to different doses of BPA are
using a mortar pre-chilled with liquid nitrogen, and were homogenized presented in Fig. 1. Following an exposure of 1.5 mg/L of BPA, the
in 1 mL of a cooling extraction medium containing 50 mM Tris, 15 mM PGRs, dry weights and leaf area increased relative to that of the ex-
MgCl2, 0.1 mM EDTA and 10% glycerol (pH 8.0). Extracts were then posure of 0.0 mg/L BPA. Following 7 d of 7.0 mg/L BPA exposure, the
centrifuged at 11,000 ×g for 2 min (4 °C), and the supernatant was PGRs and growth indices indicated no obvious changes relative to that
immediately assayed at 25 °C for Rubisco activity. Rubisco activity was of the exposure of 0.0 mg/L BPA. Following 7 d of exposure with 12.0,
analysed in a reaction mixture containing 50 mM Tris,15 mM MgCl2, 17.2 and 50.0 mg/L of BPA, the PGRs and growth indices decreased and
0.1 mM EDTA, 20 mM bicarbonate, 0.2 mM NADH (Sigma-Aldrich Co., this effect became more evident as the BPA dose increased. Further-
Ltd, USA), 5 mM DTT, 1 mM ATP, five units of glyceraldehyde-3- more, 1.5 mg/L BPA exposure clearly improved the Pn compared with
phosphate dehydrogenase from rabbit muscle (GAPDH; Sigma-Aldrich the exposure of 0.0 mg/L BPA (Fig. 2). Following 7.0 mg/L BPA ex-
Co., Ltd, USA), five units of phosphoglycerate kinase from yeast (PGK; posure, the Pn did not change relative to that of the exposure of 0.0 mg/
Sigma-Aldrich Co., Ltd, USA) and 1 mM ribulose 1,5-bisphosphate L BPA; however, following higher doses of BPA exposure, the Pn de-
(RuBp; Sigma-Aldrich Co., Ltd, USA). The enzyme reaction (3 mL) was creased relative to the exposure of 0.0 mg/L BPA. Greater decreases in
initiated by adding 20 μL extracts to the mixtures. The absorbance at the Pn occurred as the BPA doses increased (Fig. 2). The correlation
340 nm was calculated after 1 min at 25 °C and the Rubisco activity was analysis demonstrated that there was a positive correlation between the
expressed on a per plant area basis (Yang and Liu, 2015). growth patterns and Pn, and the changes in the Pn were found to partly
contribute to the effect of BPA on the growth of soybean plants
2.9. Statistical analysis (Table 2).
Following the withdrawal of 1.5, 7.0 and 12.0 mg/L BPA, the PGRs,
Significant differences between the different treatments were de- growth indices and Pn in the soybean seedlings were restored to the
termined by employing an analysis of variance (ANOVA) using the exposure of 0.0 mg/L BPA groups. Following the withdrawal of the
software SPSS 17.0. A Fisher's least squares difference (LSD) test was higher doses of BPA, the PGRs, growth indices and Pn in the soybean
performed to determine the significance of difference among the seedlings remained lower relative to the exposure of 0.0 mg/L BPA
treatments (p < 0.05 or p < 0.01). Correlation analysis (Table 2) was groups. However, compared to the exposure of 0.0 mg/L BPA group at

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L. Jiao et al. Ecotoxicology and Environmental Safety 145 (2017) 150–160

Fig. 1. Effects of bisphenol A (BPA) on the growth indices and the

relative growth rates (PGRs) of the soybean seedling leaves fol-
lowing BPA exposure (measured after exposure to BPA for 7 d,
empty columns) and the withdrawal of BPA exposure (measured
withdrawal of BPA exposure for another 7 d, shaded columns).
Each value represents mean ± SD of three replicates and error
bars represent SD. Significant differences at p < 0.05 are denoted
with different letters.

Fig. 2. Effects of BPA on the net photosynthetic rate (Pn) (A), stomatal conductance (Gs) (B), intercellular CO2 concentration (Ci) (C), the quantity of open stomata (D) and the stomatal
aperture (length and width) (E, F) in soybean seedling leaves following BPA exposure (measured after exposure to BPA for 7 d, empty columns) and the withdrawal of BPA exposure
(measured withdrawal of BPA exposure for another 7 d, shaded columns). The stomatal aperture and quantity of open stomata was obtained from Fig. 3 using software that comes with
microscope. Each value represents mean ± SD of three replicates and error bars represent SD. Significant differences at p < 0.05 are denoted with different letters.

154
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the same growth period, the decreases in the PGRs, growth indices and
Pn after BPA exposure withdrawal were lower than those after BPA
exposure. This suggests that the changes in the PGRs, growth indices
and Pn resulting from BPA exposure were halted when exposure to BPA
was removed, and indicates that the effect of BPA on soybean seedling
growth is associated with the Pn. Thus, this indicates that the changes in
the Pn and the effects of BPA on growth recovered following the
withdrawal of varying doses of BPA.
3.2. Effects of BPA on the leaf Gs, Ci and stomata behaviours
Fig. 2 indicates the changes in the Gs and Ci in soybean seedling
leaves subjected to different doses of BPA. Following exposure with
1.5 mg/L BPA, the Gs and Ci increased relative to that of the exposure of
0.0 mg/L BPA. However, there was no significant change in Gs or Ci
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Ecotoxicology and Environmental Safety 145 (2017) 150–160
Fig. 3. Representative stomatal images for the mea-
surements of stomatal parameters in the soybean
seedlings following exposure to different concentra-
tions (1.5, 7.0, 12.0, 17.2 and 50.0 mg/L) of BPA and
the withdrawal of BPA exposure. Aperture width
(red solid arrow), aperture length (red dashed arrow)
are indicated by double-headed arrows in the above
image. (For interpretation of the references to color
in this figure legend, the reader is referred to the web
version of this article.)
following exposure with 7.0 mg/L BPA. Following exposures to 12.0,
17.2 and 50.0 mg/L BPA, the Gs clearly decreased while Ci levels in-
creased relative to that of the exposure of 0.0 mg/L BPA. These effects
became more significant with increasing BPA doses. The effects of BPA
on stomatal behaviours (stomatal aperture and the quantity of open
stomata) in the soybean seedling leaves are shown in Figs. 2 and 3,
respectively. Subsequent to exposure with 1.5 mg/L BPA, the stomatal
aperture and the quantity of open stomata increased relative to that of
the exposure of 0.0 mg/L BPA. Following exposure to 7.0 mg/L BPA,
stomatal aperture and the quantity of open stomata were not obviously
different from that of the exposure of 0.0 mg/L BPA. Following ex-
posures to 12.0, 17.2 and 50.0 mg/L BPA, stomatal aperture and the
number of open stomata tended to decrease significantly. These effects
were greater following higher levels of BPA exposure.
Following the withdrawal of the 1.5, 7.0 and 12.0 mg/L BPA
L. Jiao et al. Ecotoxicology and Environmental Safety 145 (2017) 150–160

Fig. 4. Effects of BPA on the corresponding CO2 response

curve (A/Ci curves) of the soybean seedling leaves following
BPA exposure (measured after exposure to BPA for 7 d, empty
columns) (A) and the withdrawal of BPA exposure (measured
withdrawal of BPA exposure for another 7 d, shaded columns)
(B). Effects of BPA on the relative stomatal limitation of
photosynthesis (Ls) (C), the relative non-stomatal limitation of
photosynthesis (Lns) (D), the maximum Rubisco carboxylation
velocity (Vcmax) (E), maximum electron transport rates (Jmax)
(F) and triose phosphate utilization (VTPU) (G) in the soybean
seedlings. Each value represents mean ± SD of three re-
plicates and error bars represent SD. Significant differences at
p < 0.05 are denoted with different letters.

156
L. Jiao et al.
exposure conditions, the Gs and Ci levels did not significantly change
relative to those of the exposure of 0.0 mg/L BPA groups. Following the
withdrawal of the higher BPA exposure levels, the Gs levels were lower
than the exposure of 0.0 mg/L BPA group, while those of Ci were higher
than the exposure of 0.0 mg/L BPA. However, for all exposure groups of
BPA, the Gs and Ci levels recovered relative to those recorded during the
period of BPA exposure. Following the withdrawal of the 1.5, 7.0 and
12.0 mg/L BPA exposure conditions, stomatal aperture and the number
of open stomata became similar to that of the exposure of 0.0 mg/L BPA
groups, and following the withdrawal of the higher doses of BPA, sto-
matal aperture and opening remained lower relative to that of the ex-
posure of 0.0 mg/L BPA groups. However, for all exposure groups of
BPA, the stomata behaviours recovered relative to those recorded
during the exposure period. In conjunction, the data suggest that the Gs,
Ci and stomatal behaviours triggered by exposure to BPA recovered
after the withdrawal of different doses of BPA.
3.3. Effects of BPA on the A/Ci curve of the leaves
Fig. 4 shows the effects of BPA on the A/Ci curve and five indices
(Vcmax, Jmax, VTPU, Ls and Lns) of the soybean seedling leaves. Following
exposures to 1.5 and 7.0 mg/L BPA, the Vcmax, Jmax and VTPU did not
significantly change, although the Ls increased under the exposure of
1.5 mg/L BPA. Following exposure with 12.0, 17.2 and 50.0 mg/L BPA,
the Vcmax, Jmax and VTPU values decreased significantly relative to those
of the exposure of 0.0 mg/L BPA group. These effects became more
significant as the BPA doses increased. The Ls decreased and Lns in-
creased as the BPA doses increased.
Following the withdrawal of the 1.5, 7.0 and 12.0 mg/L BPA ex-
posure conditions, Ls, Vcmax, Jmax and VTPU did not significantly change
except for the Lns that increased significantly. Following the withdrawal
of the higher dose BPA exposure conditions, the Vcmax, Jmax and VTPU
remained lower, but the Lns was higher relative to that of the exposure
of 0.0 mg/L BPA group. However, for all exposure groups of BPA, the
Vcmax, Jmax and VTPU recovered relative to those recorded during the
exposure period. Thus, the data suggest that the impact of BPA on
Vcmax, Jmax and VTPU was relieved following the withdrawal of different
doses of BPA.
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Ecotoxicology and Environmental Safety 145 (2017) 150–160
3.4. Effects of BPA on the Hill reaction activity, AQY, CE and Rubisco
activity in the soybean seedlings
Fig. 5 indicates the changes in the Hill reaction activity, AQY, CE
and Rubisco activity of soybean leaves exposed to varying doses of BPA.
Following 1.5 mg/L BPA exposure, the CE and Rubisco activity in-
creased while both the Hill reaction activity and AQY indicated no
significant changes relative to that of the exposure of 0.0 mg/L BPA
group. The Hill reaction activity, AQY, CE and Rubisco activity in the
leaves following 7 d of 7.0 mg/L BPA exposure revealed no notable
change, which is in accordance with the results for the Pn (Fig. 2).
Following 7 d of exposure to higher dose BPA, the Hill reaction activity,
CE, AQY and Rubisco activity decreased significantly relative to those
of the exposure of 0.0 mg/L BPA group. In addition, the changes were
more obvious at higher BPA dose.
Following the withdrawal of exposure to 1.5, 7.0 and 12.0 mg/L
BPA, the Hill reaction activity, AQY, CE and Rubisco activity were not
significantly different from the exposure of 0.0 mg/L BPA groups.
Following the withdrawal of the higher doses of BPA, the Hill reaction
activity, AQY, CE and Rubisco activity levels were still lower than those
of the exposure of 0.0 mg/L BPA groups. However, for all exposure
groups of BPA, the Hill reaction activity, AQY, CE and Rubisco activity
levels recovered relative to those recorded during the exposure period.
Thus, these data suggest that the changes in the Hill reaction activity,
AQY, CE and Rubisco activity were relieved after the withdrawal of BPA
exposure.
3.5. Correlation analysis
To determine how BPA affects the Pn and produces changes in
soybean seedling growth, correlation analyses among the Pn, PGRs,
growth indices, Hill reaction activity, AQY, CE, Rubisco activity, Vcmax,
Jmax and VTPU in the soybean seedlings were performed. The Pn was
positively correlated with the PGRs, growth indices, Hill reaction ac-
tivity, AQY, CE, Rubisco activity and Vcmax, Jmax and VTPU, but was
negatively correlated with the Ci in the soybean seedlings (p < 0.01;
Table 2).
Fig. 5. Effects of BPA on the Hill reaction activity (A), apparent
quantum efficiency (AQY) (B), carboxylation efficiency (CE) (C)
and Rubisco activity (D) in the soybean seedling leaves following
BPA exposure (measured after exposure to BPA for 7 d, empty
columns) and the withdrawal of BPA exposure (measured with-
drawal of BPA exposure for another 7 d, shaded columns). Each
value represents mean ± SD of three replicates and error bars
represent SD. Significant differences at p < 0.05 are denoted with
different letters.
L. Jiao et al.
4. Discussion
Both dry weight (leaf and stem) as well as leaf area are direct re-
flections of plant growth conditions, and are affected by the PGRs (Long
et al., 2010). Notably, the Pn serves as an important parameter that
determines relative growth rates in plants (Huang et al., 1997), and the
results suggested that low-dose BPA (1.5 mg/L) promoted the Pn, PGRs
and growth, while high doses BPA (12.0, 17.2 and 50.0 mg/L) re-
strained the Pn, PGRs and growth of soybean seedlings (Figs. 1 and 2).
In addition, the Pn, PGRs and growth levels of soybean seedlings were
significantly correlated (Table 2) which is consistent with our previous
studies (Qiu et al., 2013).
Photosynthesis is a complex physiological process, and the main
internal factors that affect plant leaf Pn include stomatal factors that
regulate the stomatal behaviours and non-stomatal factors involved in
the photosynthetic light/dark reactions of mesophyll cells (Ainsworth
and Rogers, 2007; Camejo et al., 2005). Thus, BPA exposure might
affect the Pn in soybean seedling leaves in various ways. Our A/Ci curve
analysis indicated that the effects of BPA on the Pn are related to sto-
matal and non-stomatal factors, although the effects of these two factors
on the Pn at different BPA doses varied (Fig. 4). Our study indicated that
low-dose BPA increased the Gs and extracellular CO2 transport into
mesophyll cells (Fig. 2) and generated sufficient substrates (CO2) for
photosynthetic carbon reduction (Salazar-Parra et al., 2015). These
effects subsequently increased the Pn in the plant leaves and further
increased the PGRs (Figs. 1 and 2). Thus low-dose BPA produced the
same effects by improving stomatal factors. Studies have shown that the
increased Gs induced by low-dose BPA are related to improvements in
the stomatal aperture and quantity of open stomata (Fig. 2). However,
the methods by which BPA affects the stomatal aperture and the
quantity of open stomata require future study. In addition, low-dose
BPA improved the Rubisco activity and photosynthetic carboxylation
efficiency, but they did not change the Vcmax, Jmax and VTPU (Fig. 4).
This result may have been related to the significantly altered negative
free energy levels caused by the carboxylation reaction in the carbox-
ylation phase of the dark reaction and the strong relationship between
Rubisco and CO2 (Galmes et al., 2014). In turn, the increased Rubisco
activity negatively affected the Vcmax. However, the regeneration of
RuBP mediated by the Jmax and VTPU was related to the assimilatory
power levels (ATP and NADPH) driven by electrons and protons, with
electrons and protons restricted by the Hill reaction and AQY (Flexas
et al., 2004). Thus, the lack of any obvious change in the Jmax and VTPU
induced by low-dose BPA might explain the unaltered the Hill reaction
and AQY (Fig. 5). Overall, the impact of low-dose BPA on non-stomatal
factors was limited, and the effect of low doses of BPA on Ls was higher
than that of Lns (Ls > Lns, Fig. 4). This regulation increased the PGRs
and promoted soybean seedling growth (Table 2).
High-dose BPA decreased the Gs (Fig. 2), inhibited extracellular CO2
diffusion to mesophyll cells, depleted the substrates available for pho-
tosynthesis dark reactions (Grassi and Magnani, 2005; Kanechi et al.,
1996), and further diminished the Pn in the soybean seedlings (Fig. 2).
Concurrent observations of stomatal behaviour showed that there was a
decrease in the regulatory action related to the number of open stomata
(Fig. 2). These observations lead to the following questions: How does
BPA affect the stomatal aperture and the number of open stomata?
Reactive oxygen species commonly induce stomatal closure (Kanechi
et al., 1996). Therefore, the opening degree and the number of stomata
may be related to the increased content of reactive oxygen species in-
duced by high doses of BPA (Zhang et al., 2016). These questions must
be answered in future investigations. Additional studies have shown
that high-dose BPA exposure also decreased the Pn by reducing the light
reaction processes (Hill reaction and AQY) and dark reaction processes
(Rubisco activity, CE, Vcmax, Jmax, and VTPU) (Figs. 4 and 5). These
changes may be related to the following factors. First, high-dose BPA
decreased the RuBP carboxylation efficiency and activity as well as the
chloroplast CO2 concentrations through decreases in the Gs (Fig. 2).
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Ecotoxicology and Environmental Safety 145 (2017) 150–160
These trends led to a decline in the Vcmax during the RuBP dark reaction
carboxylation phase (Peñarojas et al., 2004). Second, high-dose BPA
decreased the Hill reaction activity and AQY and ultimately inhibited
the formation of assimilatory power molecules (ATP and NADPH),
which further limited RuBP regeneration controlled by the Jmax and
VTPU (Fig. 4) and decreased the Jmax and VTPU (Campos et al., 2014;
Lawlor and Cornic, 2002). Clearly, high-dose BPA had negatively ef-
fects on stomatal and non-stomatal factors and inhibited the Pn of
soybean seedlings by simultaneously limiting stomatal and non-sto-
matal factors. However, the relative contribution of non-stomatal fac-
tors to the Pn were more significant compared with that of stomatal
factors (Lns > Ls and increased Ci) (Farquhar and Sharkey, 1982). As a
consequence, high-dose BPA decreased the PGRs and inhibited soybean
seedling growth (Fig. 1 and Table 2). Whether high-dose BPA inhibited
stomatal and non-stomatal factors related to reactive oxygen species
increases that can damage structures of the leaf cell, chloroplast thy-
lakoid and photosynthetic pigment (Speranza et al., 2012) requires
further study.
Overall, BPA affected the Pn in the soybean seedlings by affecting
both stomatal and non-stomatal factors. Therefore, these factors may be
regarded as two “metabolic targets” of BPA. Interestingly, we observed
significant differences in the regulatory effects of stomatal and non-
stomatal factors on the net photosynthesis of soybean seedlings under
different BPA exposure levels despite the same metabolic targets were
used.
Following the withdrawal of low-dose BPA exposure conditions, the
promoting effects of BPA on the Gs, Ci, Hill reaction activity, AQY,
Rubisco activity, CE, Vcmax, Jmax and VTPU were diminished and led to
the recovery of the Pn back to the levels of 0.0 mg/L BPA exposure.
These changes may have been related to a decline in the effects of BPA
on stomatal aperture, quantity of open stomata, Rubisco activities, and
CE because of the self-detoxification of BPA or BPA metabolism cata-
lysed by certain enzymes in plants (Nakajima et al., 2002), thus limiting
the role of stomatal aperture regulation and quantity of open stomata.
Following the withdrawal of the high-dose BPA exposure conditions,
the inhibitory effects of BPA on the Gs, Hill reaction activity, AQY,
Rubisco activity, CE, Vcmax, Jmax and VTPU also diminished. The degree
to which the above indices and Pn recovered was related to the BPA
doses. After the withdrawal of high-dose BPA, the BPA content in plants
decreased because BPA had been metabolized in the plants (Nakajima
et al., 2007) and its toxic effects were diminished. Accordingly, the
inhibitory effects of BPA on the stomatal and non-stomatal factors de-
clined. In addition, whether the recovery of the inhibitory effect of BPA
on the stomatal and non-stomatal factors was related to decreased
thylakoid and guard cell damage caused by a decrease in active oxygen
content and recovery of protective enzymes following the removal of
high BPA doses (Wang et al., 2015) requires further study.
5. Conclusions
In conclusion, a significant dose-effect relationship was observed
between BPA and stomatal factors, non-stomatal factors, Pn, PGRs and
growth in soybean seedlings. Thus, BPA affects Pn by affecting the
stomatal (Gs and stomatal behaviour) and non-stomatal factors (AQY,
Hill reaction, Rubisco activity, CE, Vcmax, Jmax and VTPU), further in-
fluencing CO2 absorption and changing photosynthetic substrates;
however, the degree of regulation was dependent on the BPA dosage
levels. Low-dose BPA increased the Pn by improving the stomatal fac-
tors, whereas high-dose BPA decreased the Pn by inhibiting the stomatal
and non-stomatal factors in soybean seedling leaves, with the non-sto-
matal factors providing a greater contribution to the diminishing Pn.
The effects of BPA on the stomatal and non-stomatal factors might re-
present an effect pathway of BPA on plant photosynthesis. Compared
with the low potential environmental risks that occur under low-dose
BPA conditions, the potential negative effects of high-dose BPA on plant
photosynthesis and growth are worthy of concern.
L. Jiao et al.
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgments
The authors are grateful for the financial support provided by the
Natural Science Foundation of China (21371100, 31170477,
21501068), National Water Pollution Control and Management
Technology Major Project (2012ZX07101_013), the Project Funded by
the Priority Academic Program Development of Jiangsu Higher
Education Institutions and Research and Innovation Project for
Postgraduate of Higher Education Institutions of Jiangsu Province in
2015 (KYLX15_1189, KYLX15_1164).
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