Ecotoxicology and Environmental Safety 240 (2022) 113685

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Ecotoxicology and Environmental Safety

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

Distribution of phenanthrene in the ospho2 reveals the involvement of

phosphate on phenanthrene translocation and accumulation in rice
Xiaowen Wang a, b, Ajay Jain c, Mengyuan Cui a, Siwen Hu a, Gengmao Zhao a, Yue Cao d, *,
Feng Hu a, *
a
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China
b
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
c
Amity Institute of Biotechnology, Amity University Rajasthan, Jaipur, India
d
School of Environmental Science and Engineering, Guangdong Provincial Key Lab for Environmental Pollution Control and Remediation Technology, Sun Yat-sen
University, Guangzhou 510006, China

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

Edited by Dr R Pereira The intricate mechanisms involved in the acquisition and translocation of polycyclic aromatic hydrocarbons
(PAHs) in plants have not been elucidated. Phosphate (Pi) is the bioavailable form of essential macronutrient
Keywords: phosphorus, which is acquired and subsequently assimilated for plant optimal growth and development. Rice
Polycyclic aromatic hydrocarbons (PAHs) phosphate overaccumulator 2 (OsPHO2) is a central constituent of the regulation of Pi homeostasis in rice. In the
Nutrient
present study, the role of OsPHO2 in regulating the translocation and accumulation of phenanthrene (Phe) and
Phosphate (Pi)
the involvement of Pi in this process were investigated. The temporal study (1 d-35 d) revealed a significant and
ospho2
Rice (Oryza sativa) gradual increase of Phe accumulation in Pi-deprived roots of wild-type (WT) seedlings. Compared with the WT,
the concentrations of Phe were significantly higher in the shoots of ospho2 (OsPHO2 mutant) grown hydro­
ponically with Phe (1.5 mg/L) under +Pi (200 μM) and -Pi (10 μM) conditions. The sap experiment clearly
showed the significant increases in levels of Phe in the xylem sap of ospho2 than the WT grown hydroponically
with Phe and +Pi. Further, the concentrations of both Phe and P were coordinately higher in the culms and flag
leaves of the mutants than WT at maturity in potting soil with LPhe (6 mg/kg) and HPhe (60 mg/kg). However,
the concentrations of Phe in the seeds were comparable in the WT and mutants, suggesting a pivotal of OsPHO2
in attenuating Phe toxicity in the seed. In +Phe WT, the relative expression level of OsPHO2 in the shoots was
significantly lower, while those of Pi transporters (PTs) OsPT4 and OsPT8 were significantly higher in the roots
compared with -Phe. Together, the results provided evidence towards the involvement of Pi in OsPHO2-regulated
translocation and accumulation of Phe in rice.

1. Introduction excitation microscopy was employed for tracking the acquisition of

phenanthrene (Phe) from the polluted growth medium into the roots of
Polycyclic aromatic hydrocarbons (PAHs), generally produced by the maize (Zea mays) and wheat (Triticum aestivum) for two months (Wild
partial burning of organic substances, resistant to degradation, ubiqui­ et al., 2005). The study revealed the binding of Phe to the epidermis in
tously present in the environment, pose a concern due to the toxicity and the elongation zone, then traversed the epidermal cells radially to reach
are potentially carcinogenic to humans (Alshaarawy et al., 2016; Meng the cortex of the root hair and branching zones of the root, and subse­
et al., 2019; Ruby et al., 2016). Soils contaminated by PAHs are mainly quently mobilized slowly and laterally to the shoot.
responsible for the continuous formation of environmentally persistent Rice (Oryza sativa) is a staple food and the main source of dietary
and relatively stable free radicals (Jia et al., 2017). It has been reported energy for more than 50% of the population in the world (www.irri.org/
that these chemicals could enter the plant roots from the polluted soils rice-today). In China, people’s exposure to PAHs and the related cancer
and be transferred to the aerial parts subsequently (Alagić et al., 2015; risk due to the consumption of rice is much higher compared to that in
Liu et al., 2021; Shen et al., 2020; Wang et al., 2021). Two-photon other countries (Ding et al., 2012). Cultivation of rice augmented the

* Corresponding author.
E-mail addresses: caoy85@mail.sysu.edu.cn (Y. Cao), fenghu@njau.edu.cn (F. Hu).

https://doi.org/10.1016/j.ecoenv.2022.113685
Received 10 December 2021; Received in revised form 9 May 2022; Accepted 20 May 2022
0147-6513/© 2022 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Wang et al. Ecotoxicology and Environmental Safety 240 (2022) 113685

dissipation and transport of PAHs (Ma et al., 2012; Wang et al., 2015). In 42-D-old as per the requirement of the experiment) were then transferred
rice, PAHs were more readily absorbed by the root than adsorbed on the to an aerated hydroponic system with a relative stable at pH 5.5 buffered
surface, and their level was relatively higher in the lateral root than in by 2-(N-morpholino) ethanesulfonic acid (MES). The culture solution
the nodal root (Jiao et al., 2007). The analysis for a sampling of various was supplemented with different concentrations of Phe (+Phe [1.5
rice tissues revealed a higher accumulation in the roots compared with mg/L] and -Phe [0 mg/L]) and Pi (+Pi [200 μM] and -Pi [10 μM]) for
the aerial tissues of rice cultivated in the PAHs-contaminated field (Su different time intervals (1, 8, 16, and 35 d) (Wang et al., 2021; Zhang
et al., 2008; Tao et al., 2006). et al., 2015). Rice seedlings were kept in a greenhouse with a light/dark
Among the macronutrients, phosphorus (P) is a vital component of photoperiod of 14-h/10-h, and a day/night temperature of 30 ◦ C/24 ◦ C,
various organic molecules (ATP, nucleic acids, and phospholipids), plays and the humidity was maintained at about 60%. The medium in the
a key role in signal transduction and metabolic pathways, and is thus hydroponic system was changed every 3 d. For the pot experiments, the
vital for the growth and development of plants (Gutiérrez-Alanís et al., soil was obtained from the top surface (~20 cm) of a field at a research
2018; Crombez et al., 2019; Paz-Ares et al., 2022). Membrane-localized base of Nanjing Agricultural University. The soil was air-dried, then the
phosphate (Pi) transporters (PTs) belonging to the PHOSPHATE plant debris in it was manually removed, and the soil was sieved through
TRANSPORTER1 (Pht1) family facilitate the acquisition of Pi, which is a mesh (pore size 2 mm). The soil (12.5 kg) was supplemented with low
the available form of P in the rhizosphere (Raghothama, 2000; Nus­ Phe (LPhe, 6 mg Phe/kg soil), high Phe (HPhe, 60 mg Phe/kg soil),
saume et al., 2011; Gu et al., 2016). Intricate crosstalk was demonstrated K2SO4 (200 mg/kg soil), KH2PO4 (20 mg/kg soil), and urea (CH4N2O) as
between the essential macronutrients (nitrogen [N], P, and potassium described (Wang et al., 2021). The pot experiment was carried out in
[K]) and the uptake and translocation of Phe in wheat (Zhan et al., 2013, triplicate.
2015; Yang et al., 2016). The crosstalk between P and PAHs was also
evident in rice as indicated by an adverse effect of PAHs on the phos­ 2.3. Collection of xylem sap
pholipid metabolism, which triggered damage to the cell membrane and
consequent growth inhibition (Liu et al., 2021). The activities of the The WT and ospho2 plants were grown for 6 weeks in the Hoagland
PAH-biodegrading bacteria are also influenced by the carbon (C)/P/N solution in a growth room. The seedlings (42-D-old) were transplanted to
ratio in the contaminated soils (Leys et al., 2005). These studies high­ a hydroponic system with 8 L Hoagland solution, and the pH was buff­
lighted the prevalence of crosstalk between P and PAHs in taxonomically ered to 5.5 by MES solution. The Hoagland solution was supplemented
diverse bacteria and crop species. Therefore, it was intriguing to assume with Phe (1.5 mg/L) for 3 d. Seedlings were cut 5 cm above the root to
a likely role of the molecular mechanism involved in the acquisition and shoot junction, and the xylem sap exudates were sampled at the cutting
mobilization of Pi from the roots to shoots, and its crosstalk effect on the culm base after discarding the first drop as described (Yang et al., 2014).
tissue-specific accumulation of Phe in rice grown in the
Phe-contaminated rhizosphere. 2.4. Quantification of Phe concentration
In model plants (Arabidopsis thaliana and rice), phosphate over­
accumulator2 (PHO2) is a ubiquitin-conjugating (UBC) E2 enzyme that Quantification of Phe concentration was performed as described
has been implicated in regulating the Pi transporters during the mobi­ (Wang et al., 2021). Phe was extracted thrice from rice (~0.2 g) samples
lization of Pi from the roots to shoots (Aung et al., 2006; Bari et al., 2006; with 10 mL of hexane: dichloromethane solution (1:1, v/v) for 30 min in
Dong et al., 1998; Hu et al., 2011). The concentration of Pi in the leaves an ultrasonic bath. The solvent fractions were combined and passed
of OsPHO2 knockout mutants (ospho2) was significantly higher through a silica gel column (top layer anhydrous Na2SO4 [2 g] and
compared with the wild-type (WT), which provided evidence of sub-layer silica gel [2 g]), and then eluted with 10 mL of hexane:
OsPHO2-mediated mobilization of Pi from the roots to shoots (Cao et al., dichloromethane solution (1:1, v/v). The collected elutes were then
2014). Therefore, the likely role of OsPHO2 in the translocation and evaporated, suspended in methanol (2 mL), and filtered through a filter
accumulation of Phe under different Pi regimes was assumed. For unit (0.22 µm). The samples were then analyzed by UHPLC (UltiMate
empirical evidence, the effects of mutation in OsPHO2 on the temporal 3000, Thermo Fisher Scientific) with a fluorescence detector (UltiMate
translocation and accumulation of Phe were investigated during growth 3000). The UHPLC was fitted with a reverse phase C18 column (Φ 4.6 ×
under different conditions (hydroponics and potting soil). By comparing 150 mm, 2.6 µm particle) and methanol and ultrapure water were used
the Phe and P distribution in the WT and the mutants, the results pro­ as the mobile phase at a flow rate of 1 mL/min. Chromatography was
vided evidence that Phe translocated and redistributed through the performed at 40 ◦ C, and injection volume was 20 µL. The external
OsPHO2-regulated P translocation pathway in rice. standard method was used for all the samples to ensure the accuracy of
the Phe detection.
2. Materials and methods
2.5. Quantification of total P concentration
2.1. Plant materials and chemicals
Total P concentration was quantified as described (Nagarajan et al.,
WT rice and OsPHO2 Tos17 insertion mutants (ospho2-1 and ospho2- 2011). Briefly, the oven-dried tissue sample (~20–30 mg) was digested
2) in Nipponbare background were procured from the National Institute in glass tubes with H2SO4 (5 mL) for 2 h. The digested sample (10 µL)
of Agrobiological Sciences, Functional Genomics Laboratory (Japan). was diluted with deionized water (790 µL) and added 200 µL of the
OsPHO2 mutant lines were used in our earlier study to demonstrate its molybdenum blue reagent (5N H2SO4, 4% [w/v] ammonium molybdate,
pivotal role in the utilization of Pi in rice (Cao et al., 2014). In this study, and 10% [w/v] ascorbic acid). The reaction mixture was incubated at
all the organic solvents used were ultra-high performance liquid chro­ 45 ◦ C for 20 min and the total P concentration was quantified by spec­
matography (UHPLC) grade. trophotometric assay at 880 nm.

2.2. Treatment with Phe and Pi 2.6. RT-qPCR

For hydroponic experiments, the seeds of the WT and OsPHO2 mu­ TRIzol reagent (Invitrogen) was used for total RNA extraction from
tants (ospho2-1 and ospho2-2) were first surface-sterilized for 30 min the rice tissue samples. The quality and integrity of the total RNA
with 30% (v/v) sodium hypochlorite solution, washed, and then extracted were determined by running an aliquot (~1000 ng) of the
germinated on 1/2 MS medium (Murashige and Skoog, 1962) for 4 d at sample on a 1.5% (w/v) denaturing agarose gel stained with ethidium
25 ◦ C in dark. The uniformly grown germinated seedlings (14-D-old or bromide. Detection of clear and sharp 18 S and 28 S rRNA bands

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X. Wang et al. Ecotoxicology and Environmental Safety 240 (2022) 113685

suggested the intact total RNA. Further, the purity of the total RNA was 3.2. Mutation of OsPHO2 enhanced Phe translocation under the different
assayed spectroscopically by computing the ratio of absorbance at 260 Pi regimes
nm and 280 nm, which was 1.8–2.0. First-strand cDNA was synthesized
by HiScript II Q RT SuperMix (+gDNA wiper) kit. RT-qPCR assays were OsPHO2 played a role in the mobilization of Pi from the roots to the
performed using the AceQ qPCR SYBR Green Master Mix on the shoots and its mutation resulted in aberrated translocation and remo­
QuantStudio™ 6 Pro Real-Time PCR System (Thermo Fisher Scientific) bilization of Pi in rice shoots causing Pi toxicity (Cao et al., 2014; Hu
as described (https://www.vazymebiotech.com). The relative expres­ et al., 2011). Since the present work (Fig. 1) and earlier studies (Leys
sion levels of OsPHO2, OsPTs (OsPT1, OsPT2, OsPT4, and OsPT8) were et al., 2005; Yang et al., 2016) suggested the crosstalk between Pi and
computed using the 2-ΔC
T method of relative quantification, and OsActin PAHs, the potential role of OsPHO2 in the acquisition and accumulation
was used as an internal control (Livak and Schmittgen, 2001). The of Phe in rice was assumed. Therefore, to determine the effect of the
primers used for RT-qPCR are listed in Table S1. mutation in OsPHO2 on the translocation of Phe under different Pi re­
gimes, the WT and OsPHO2 mutant (ospho2-1 and ospho2-2) seedlings
were grown hydroponically in Hoagland solution supplemented with
2.7. Statistical analysis Phe under +Pi and -Pi conditions. The harvested roots and shoots were
assayed for concentration and translocation factor (TF) of Phe (Fig. 2).
IBM SPSS 20.0 software package (https://www.ibm.com) was Although -Pi triggered a significant increase in the concentration of
employed for the data analysis by using Duncan’s test for the multiple Phe in the roots of both the WT (3.75 folds higher) and ospho2 (6.18
comparisons of two-way and/or one-way ANOVA. Letters on the bars folds higher) compared with +Pi, the values were comparable between
indicated statistically different (P < 0.05). the WT and the mutants irrespective of the Pi regime (Fig. 2A). The
concentration of Phe in the shoots of both the WT and ospho2 was
3. Results and discussion significantly lower compared with the roots under different Pi regimes
(Fig. 2A). The results suggested that only some part of the acquired Phe
3.1. Pi deficiency triggers the accumulation of Phe in roots by the roots was subsequently translocated to the shoot. Contrary to the
roots, no significant effect of Pi deprivation was observed on the con­
To determine the temporal effect of Pi on the acquisition of Phe in centration of Phe in the shoots of the WT and the mutants (Fig. 2A).
rice, the WT seedlings (14-D-old) were hydroponically grown in Hoag­ Notably, the concentrations of Phe were significantly augmented in the
land solution with Phe under Pi replete (+Pi) and Pi deprivation (-Pi) shoots of the mutants than the WT under +Pi (1.73 folds higher) and -Pi
conditions. Roots were harvested and assayed for Phe concentrations at (1.93 folds higher) (Fig. 2A). The TF of Phe was significantly higher in
the different treatment time points (after 1 d, 8 d, 16 d, and 35 d). As both the WT and ospho2 under +Pi compared with -Pi, being 6.69 and
shown in Fig. 1, after 1 d treatment, there was no significant difference 8.63 folds, respectively (Fig. 2B). Furthermore, the TF in ospho2 was
of Phe concentration between +Pi and -Pi. However, compared with 2.88 folds higher than the WT under +Pi and 2.10 folds higher under -Pi,
+Pi, Phe concentrations under -Pi were significantly increased with the which indicated the TF got further accentuated in the mutants than the
increasing treatment time by 0.56, 1.07, and 3.10 folds at 8, 16, and 35 WT relatively more under +Pi than under -Pi (Fig. 2 B). These results
d, respectively (Fig. 1). The result revealed the stimulatory effect of Pi showed that OsPHO2 mutation increased the translocation of Phe but
deficiency treatment for ~ a week or more on the accumulation of Phe in had no effect on its uptake. This regulation pattern of OsPHO2 mutation
the roots and was consistent with an earlier study demonstrating sig­ on Phe was similar to Pi, as the OsPHO2 mutation only augmented the P
nificant acquisition and accumulation of Phe in the roots of Pi-deprived level in the shoots but not in the roots (Cao et al., 2014; Hu et al., 2011).
wheat seedlings (Yang et al., 2016), suggesting that Pi is involved in the Interestingly, Pi-deficiency and PAHs exposure also disturbed phos­
accumulation of Phe in certain ways in plants. P content of the soil also pholipid (PL) metabolism in rice in the same pattern, which was
exerted a significant influence on the survival and activity of the decreasing the contents of the same typical PLs (Liu et al., 2021; Sun
PAH-degrading bacteria Mycobacterium and Sphingomonas (Leys et al., et al., 2020). Thus, it is reasonable to presume a similar pathway is
2005). Therefore, Pi may play pivotal role in the acquisition, accumu­ involved in the OsPHO2-mediated translocation of Phe and Pi.
lation, and biodegradation of PAHs in diverse microorganisms and plant
species. 3.3. Mutation of OsPHO2 significantly increased Phe concentration in
xylem sap

The acropetal translocation of Phe was mediated by both phloem and

xylem pathways and the Phe concentration was found to be relatively
higher in the xylem sap than in phloem sap (Shen et al., 2020). There­
fore, to further understand the higher Phe translocation in OsPHO2
mutants, the effect of the mutation in OsPHO2 on the concentration of
Phe in the xylem sap was investigated. The WT and the mutants seed­
lings were grown hydroponically in a Hoagland solution supplemented
with Phe and Pi. Their culms were cut, then the xylem sap exudate was
collected from the cut ends and assayed for the concentration of Phe
(Fig. 3). The results showed a significant increase in the level of Phe in
the xylem sap of ospho2, being 3.64 folds of the WT. The translocation of
P from the nodal root was influenced by the xylem vascular architecture
(Hay and Hamilton, 1996). In rice, the efficacy of Pi mobilization from
Fig. 1. Temporal effect of different Pi regimes on the accumulation of Phe. The
the roots to shoots was positively correlated with the Pi concentration in
WT seedlings (14-D-old) were grown hydroponically supplemented with Phe
the xylem sap (Zhang et al., 2015; Pei et al., 2017; Ai et al., 2020).
(1.5 mg/L) under +Pi (200 μM) and -Pi (10 μM) conditions. Roots were har­
vested after 1 d, 8 d, 16 d, and 35 d, and assayed for Phe concentration. Values Furthermore, it had demonstrated that the mutation of OsPHO2
are means ± SE (n = 5). Uppercase letter above bars indicates the significant augmented the Pi concentration in the xylem sap (Cao et al., 2014).
difference between Pi regimes at each time point and lowercase letter indicates Taken together, it was speculated that OsPHO2 regulated the concomi­
the significant difference between the time points under each Pi regime tant translocation of Phe with Pi from the roots to aerial tissues via
(P < 0.05, one-way ANOVA). xylem.

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Fig. 2. Mutation in OsPHO2 affects the trans­

location of Phe under different Pi regimes. The
WT and ospho2 seedlings (14-D-old) were grown
hydroponically supplemented with Phe
(1.5 mg/L) under +Pi (200 μM) and -Pi (10 μM)
conditions for 35 d. The root and shoot were
harvested and assayed for Phe concentration.
Data are presented for (A) Phe concentration in
the root and shoot and (B) Translocation factor
of Phe. Values are means ± SE (n = 5). Upper­
case letter above bars indicates the significant
difference between plant lines (WT, ospho2-1
and ospho2-2) under each Pi regime and
lowercase letter indicates the significant dif­
ference between Pi regimes in each line
(P < 0.05, one-way ANOVA).

Fig. 3. Mutation of OsPHO2 affects the concentration of Phe in the xylem sap.
The WT and ospho2 seedlings (42-D-old) were grown hydroponically supple­
Fig. 4. Mutation in OsPHO2 affects the concentrations of Phe and total P in the
mented with Phe (1.5 mg/L) and Pi (200 μM) for 3 d. The xylem sap was
culm and flag leaves at maturity. The WT and ospho2 seedlings (21-D-old) were
collected above a cut root-shoot junction and assayed for Phe concentration.
grown in a potting soil with supplemented LPhe (6 mg/kg) and HPhe (60 mg/
Values are means ± SE (n = 4). Uppercase letter on the bars indicates the sig­
kg) for 20 weeks. The culm and flag leaf were harvested and assayed for the
nificant differences (P < 0.05, one-way ANOVA).
concentrations of Phe and total P. Data are presented for the concentrations of
(A) Phe, and (B) Total P in the culm and flag leaf. Values are means ± SE
3.4. Mutation of OsPHO2 enhanced the concentration of Phe and total P (n = 4). Uppercase letter above bars indicates the significant difference be­
in the culms and flag leaves tween plant lines (WT, ospho2-1 and ospho2-2) under each Phe regime and
lowercase letter indicates the significant difference between Phe regimes in
The mutation of OsPHO2 increased Phe accumulation in the shoots each line (P < 0.05, one-way ANOVA).
(Fig. 2) and xylem sap (Fig. 3) when rice was grown in the hydroponic
system. To determine whether OsPHO2 regulates the translocation of (Fig. 4A). The results indicated that the mutation of OsPHO2 triggered
Phe during the whole growth period, the WT and ospho2 seedlings were the significant Phe accumulation in the shoots, xylem sap, culms, and
grown in potting soil with LPhe (6 mg/kg) and HPhe (60 mg/kg) for 20 flag leaves during growth under the different conditions (hydroponics
weeks and the culms and flag leaves were assayed for the concentrations and potting soil) and the regimes of Phe (Figs. 2, 3, 4A). On the contrary,
of Phe and total P (Fig. 4). Consistent with the hydroponic experiments Phe concentration in the roots of the WT and ospho2 were comparable
(Figs. 2, 3), OsPHO2 mutants (ospho2-1 and ospho2-2) translocated more under both LPhe and HPhe conditions (Fig. S1). These results further
Phe to the culms and flag leaves compared with WT under LPhe con­ corroborated the effect of mutation of OsPHO2 on the translocation of
dition and the effect was significantly augmented under HPhe condition Phe from the roots to aerial parts (culms and flag leaf). Furthermore,
(Fig. 4A). Under HPhe, Phe concentrations in the culms and flag leaves total P concentration also revealed a trend similar to that of Phe with
of the mutants were 9.44 and 1.68 folds higher than the WT, respectively higher values in culms and flag leaves of the mutants than WT under

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both LPhe (6.50 and 10.7 folds) and HPhe (3.79 and 23.2 folds) condi­ in the mutants was also relatively lower in the seeds (Fig. 5B) than culms
tions (Fig. 4B). This phenomenon exhibited the pivotal role of OsPHO2 and flag leaves (Fig. 4B). Together, it was logical to speculate that
in regulating the concomitant translocation of Pi and Phe from the roots OsPHO2 coordinated greatly the allocation of Phe and Pi from the shoots
to aerial tissues. to the seeds in the maturity rice but with different regulation patterns for
N and K also play important roles in the acquisition and translocation them. Compared to traditional flooding irrigation, water-saving irriga­
of Phe in gramineous species (Zhan et al., 2013, 2015; Wang et al., tion has shown to be a relatively effective strategy for reducing the
2021). In our previous study, the overexpression of a nitrate transporter concentration of PAHs in rice grain by up to 50% (Zhao et al., 2018).
(OsNRT2.3b) in rice triggered the reduction of Phe uptake and trans­ Therefore, it would be interesting to investigate the efficacy of OsPHO2
location, and there was a reverse association between N and Phe in the mutation under water-saving irrigation in mitigating the concentration
OsNRT2.3b-overexpressing plants at maturity (Wang et al., 2021). On of Phe in the seed, which merits further studies.
the contrary, in the present study, P and Phe showed a synergic asso­
ciation in the OsPHO2 mutants at maturity (Fig. 4). Moreover, the effect
of OsPHO2 mutation on the Phe translocation was also significant under 3.6. OsPHO2 regulated the acquisition and translocation of Phe through
LPhe (Fig. 4A) but overexpression of OsNRT2.3b only functioned under OsPTs in rice
HPhe (Wang et al., 2021). It demonstrated OsPHO2 played a stronger
role in regulating the translocation of Phe than OsNRT2.3b in rice. The effect of Phe on the tissue-specific relative expression levels of
Therefore, it is logical to assume intricate crosstalk between the mac­ OsPHO2 and Pi transporters OsPT1, OsPT2, OsPT4, and OsPT8 was
ronutrients (N, P, and K) and the acquisition and mobilization of Phe in investigated. The WT seedlings were grown hydroponically in Hoagland
different plant species. solution under +Phe and -Phe and the tissues harvested were then
determined for the relative expression levels using qRT-PCR (Fig. 6). The
3.5. Increased concentration of Pi with the unchanged concentration of results showed that the expression levels of OsPHO2 were higher in the
Phe in the seeds of ospho2 roots compared with the shoots under both Phe conditions. Although the
relative expression of OsPHO2 was comparable in the roots under
Since the accumulation of PAHs in rice and its consumption by the different Phe regimes, it was significantly reduced by 85.6% in the
Chinese population has been considered one of the factors for devel­ shoots under +Phe condition (Fig. 6). The results suggested the differ­
oping cancer (Ding et al., 2012), a maintainable mitigation strategy is ential effects of Phe on the relative expression of OsPHO2 in a tissue-
needed urgently. Our studies revealed that the mutation of OsPHO2 led specific manner.
to elevated concentration of Phe in different young and mature tissues Pi acquisition and translocation in plants are directly regulated by Pi
and triggered its translocation from the roots to shoots during growth for transporters (Harrison et al., 2002; Seo et al., 2008). In rice, there are 13
variable time intervals under different growth conditions and Phe re­ Pi transporters (OsPT1-OsPT13) in the PHT1 family (Paszkowski et al.,
gimes (Figs. 2, 3, 4A). It would be of attraction and significance to 2002), and the functions for the Pi uptake and translocation of the
identify the Phe and P levels in the seeds. Therefore, the concentrations higher expression abundance members have been investigated in rice
of Phe and total P were assayed in the WT and ospho2 seedlings grown in (Ai et al., 2009; Jia et al., 2011; Sun et al., 2012; Zhang et al., 2015).
a potting soil supplemented with HPhe for 20 weeks (Fig. 5). Under Pi replete condition, the relative expression levels of OsPT1,
Despite the higher accumulation of Phe in the culms and flag leaves OsPT2, OsPT4, and OsPT8 increased significantly in the roots of OsPHO2
of the mutants than the WT during growth in potting soil (Fig. 4A), the mutant, which suggested the likely role of OsPHO2 in mobilizing Pi by
concentration of Phe in the seeds was observed to be comparable be­ regulating the tissue-specific expression of OsPTs (Hu et al., 2011; Cao
tween the WT and ospho2 (Fig. 5A). The results thus suggested that the et al., 2014). In the present study, the mutation of OsPHO2 enhanced
mutation of OsPHO2 attenuated the allocation of Phe from the shoots to
the seeds and thereby mitigated the Phe toxicity in the seed. As the
shown in Fig. 5B, the concentration of total P in the seeds was 44.8%
higher in ospho2 than in the WT. Noticeably, the concentration of total P

Fig. 6. Phe differentially affects the relative expression levels of OsPHO2 and
OsPTs. The WT seedlings (14-D-old) were grown hydroponically supplemented
with +Phe (1.5 mg/L) and -Phe (0 mg/L) for 8 d. Tissues (root and/or shoot)
Fig. 5. Mutation in OsPHO2 differentially affects the concentrations of Phe and were harvested and assayed for the relative expression levels of OsPHO2, OsPTs
total P in the seeds. The WT and ospho2 seedlings (21-D-old) were grown in a (OsPT1, OsPT2, OsPT4, and OsPT8) by qRT-PCR. Actin (OsRac1;
potting soil with Phe (60 mg/kg) for 20 weeks. Seeds were harvested and LOC_Os03g50885) was used as an internal control. Values are means ± SE
assayed for the concentrations of Phe and total P. Data presented for the con­ (n = 3). Uppercase letter above bars indicates the significant difference be­
centrations of (A) Phe and (B) Total P. Values are means ± SE (n = 3). Up­ tween Phe regimes of each gene in the root or shoot and lowercase letter in­
percase letter above bars indicates the significant difference between plant lines dicates the significant difference between the root and shoot of OsPHO2 under
(WT, ospho2-1 and ospho2-2) (P < 0.05, one-way ANOVA). each Phe regime. (P < 0.05, one-way ANOVA).

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X. Wang et al. Ecotoxicology and Environmental Safety 240 (2022) 113685

Phe translocation and accumulation in rice significantly (Figs. 2–4). Alagić, S.C., Maluckov, B.S., Radojičić, V.B., 2015. How can plants manage polycyclic
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https://doi.org/10.1016/j.envpol.2006.10.025.
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The authors declare that they have no known competing financial nitrogen/phosphorus ratio on polycyclic aromatic hydrocarbon degradation by
interests or personal relationships that could have appeared to influence Mycobacterium and Sphingomonas in soil. Appl. Microbiol. Biotechnol. 66, 726–736.
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This work was supported by the National Natural Science Foundation https://doi.org/10.1016/j.envpol.2021.117073.
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