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Plant Cell, Tissue and Organ Culture (PCTOC)
https://doi.org/10.1007/s11240-020-01914-2
ORIGINAL ARTICLE
Overexpression ofthemetallothionein gene PaMT3‑1 fromPhytolacca
americana enhances plant tolerance tocadmium
JunkaiZhi1,2· XiaoLiu1,2· PengYin1,2· RuixiaYang2· JiafuLiu2· JichenXu1,2
Received: 18 May 2020 / Accepted: 11 August 2020
© Springer Nature B.V. 2020
Abstract
Metallothionein (MT) is a cysteine-rich, low-molecular-weight protein that can bind to cadmium ions and reduce their toxic-
ity to plants. In this study, we cloned the PaMT3-1 gene encoding an unstable protein of 63 amino acids from the cadmium
hyperaccumulator Phytolacca americana. The gene was inserted into a plant expression vector and introduced into tobacco
plants. The cadmium content of the transgenic plants was measured after treatment with 100 mM CdCl2 for 7 days. Transgenic
and wild-type roots had similar cadmium contents, whereas the cadmium content of transgenic leaves was 66.28–78.70%
of the wild type. The transport coefficient of cadmium in transgenic plants was decreased by 23.31–35.52% relative to the
wild type. According to various physiological indexes, including malondialdehyde content, relative electrolyte leakage,
root activity, and soluble sugar content, the transgenic plants performed better than the wild type. The PaMT3-1 gene can
significantly improve plant resistance to cadmium and has potential as an important gene resource in phytoremediation. Our
findings could also contribute to an understanding of complex processes and mechanisms involved in phytoremediation.
Key message
Over-expressing a metallothionein gene PaMT3-1 from Phytolacca americana intobacco plants showed enhanced cadmium
tolerance that would be important gene resource forphytoremediation.
Keywords Metallothionein· Phytolacca americana L· Cadmium· Transformation· Resistance
Introduction
Phytoremediation, which exploits absorption, transpor-
tation, and accumulation processes in plants, is the most
effective way to treat soils contaminated with heavy metals
(Stephenson etal. 2014). Numerous relevant genes, such as
those encoding metallothionein (MT) (Cobbett etal. 2002),
phytochelatin (PCs) (Chen etal. 2015), Natural resistance
associated macrophage protein (NRAMP) (Nakanishi-
Masuno etal. 2018), Yellow stripe-like protein (YSL) (Curie
etal. 2009), and heavy metal ATPase (HMA) (Wang etal.
2018), have currently been identified.
Metallothionein is a cysteine-rich, low-molecular-weight
protein. The sulfhydryl group of cysteine can bind cadmium,
zinc, and other heavy metal ions to form a barely or non-
toxic complex that enhances plant tolerance to metal toxicity
and facilitates their accumulation. Several gene transforma-
tion experiments have definitively confirmed that metal-
lothionein can improve plant heavy-metal resistance. For
example, Turchi etal. (2012) transformed a pea MTA1 gene
into poplar, and the transgenic plants showed more resist-
ance to zinc and copper stress. Arabidopsis plants with a
knocked-out MT1 gene were found to be more sensitive to
cadmium and had significantly lower arsenic, cadmium, and
zinc leaf contents (Zimeri etal. 2005). Suh etal. (1998)
transformed a mouse metallothionein gene into tobacco. The
transgenic plants grew normally under cadmium stress (200
μm), whereas wild-type plants were strongly affected. Zhang
Communicated by Mohammad Faisal.
Zhi Junkai and Liu Xiao contributed equally to this work
* Jichen Xu
jcxu282@sina.com
1 Beijing Advanced Innovation Center forTree Breeding
byMolecular Design, Beijing Forestry University,
Beijing100083, China
2 National Engineering Laboratory forTree Breeding, Beijing
Forestry University, Beijing100083, China
Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
etal. (2003) transformed the TyMT gene from cattail into
Arabidopsis, and the cadmium tolerance of the transgenic
plants was significantly increased. Gorinova etal. (2007)
transferred a MT gene from Silene conoidea to tobacco,
which increased root and leaf cadmium accumulations in
the transgenic plants. On the basis of these experiments,
metallothionein is considered to be an important component
in the phytoremediation of heavy-metal-contaminated soil.
In a previous study, Phytolacca americana was confirmed
to be a cadmium-hyperaccumulator species. According to
a transcriptome analysis by Chen (2017), metallothionein
genes in P. americana, especially type-III genes, are sig-
nificantly regulated by cadmium. In a prokaryotic expres-
sion test, PaMT3-1 was found to be more resistant to cad-
mium than two other MT genes. The report here supposed
to explore the role of this gene in phytoremediation. We
transformed the PaMT3-1 gene into tobacco and measured
cadmium resistance and accumulation levels in the resulting
transgenic plants. Our findings should aid understanding of
the mechanism of the action of this gene in phytoremedia-
tion and possibly provide an important gene resource for
future molecular breeding programs.
Materials andmethods
Materials
The PaMT31 was cloned previously (Chen etal. 2018) in
our lab. The pEZR(K)-LC was used as the plant expression
vector, and the Agrobacterium tumefaciens strain LBA4404
and Nicotiana tabacum L. were used for gene transforma-
tion experiment.
Characteristics andevolutionary analysis
ofPaMT3‑1 fromPhytolacca americana
Based on the PaMT3-1 gene sequence reported earlier, its
encoding peptide was refereed via DNAMAN software.
ProtParam was used to characterize the protein properties
(https ://web.expas y.org/protp aram/). The metallothionein
type III gene sequences from 15 plant species were down-
loaded in NCBI (National Center for Biotechnology Infor-
mation). The phylogenetic tree was constructed using the
MEGA software based on their amino acids.
Construction ofplant expression vector
withPaMT3‑1
The specific primers of PaMT3-1 were designed at both
side of the gene with HindIII and XbaI restriction site
(PaMT31HindIII, 5’-CCC AAG CTT ATG TCG GAC A AG
TGT GGA AACT-3’, HindIII site underlined; PaMT31XbaI,
5’-TGC TCT AGA TTA GTG ACA TCC ACA TCC GCAG-3’,
XbaI site underlined). The full-length candidate gene frag-
ment was obtained by PCR amplification with the procedure:
94°C for 5 min followed by 35 cycles of 94 °C for 30 s, 55
°C for 40 s, 72 °C for 1 min, and finally extension at 72 °C
for 10 min. The recovered fragment and the plant expression
vector pEZR(K)-LC were digested separately with Hind III
and XbaI. The recovered DNA fragments were ligated by
replacing GFP with PaMT3-1 gene. The recombinant plas-
mid was introduced into Agrobacterium tumefaciens strain
LBA4404 by electroporation (Eppendorf eporator, USA).
The positive clones were identified by PCR test.
PaMT31 gene transformation totobacco plants
The positive LBA4404clone with the recombinant vector
pEZR(K)-LC-PaMT31 was cultivatedinYEB medium con-
taining 50 mg/L kanamycin and 50 mg/L rifampicin at 28 °C
overnight. The activated bacteria were transferred to antibi-
otic-free YEB liquid medium for 4–5 h until OD600 of 0.5.
Tobacco leaves were cut in small pieces (1.5 × 1.5 cm) and
immersed in Agrobacterium solution for 10 minutes, then
transferred to pre-culture medium [Murashige and Skoog
Basal Medium (MS) +2mg/L 6-benzylamino-purine (6-BA)
+ 0.1mg/L naphthlcetic acid (NAA) + 3 % (w/v) sucrose +
0.2 % (w/v) phytagel] in dark for 3 days. They were then cul-
tured in differentiation medium [MS + 2 mg/L 6-BA + 0.1
mg/L NAA +50 mg/L Kanamycin +200 mg/L Cefotaxime
+ 3 % (w/v) sucrose + 0.2 % (w/v) phytagel] for 4 weeks.
The regenerated buds in height of 1–2cm were transferred
on root-inducing medium [MS + 0.1 mg/L NAA +50 mg/L
Kanamycin + 200 mg/L Cefotaxime + 3 % (w/v) sucrose
+ 0.2 % (w/v) phytagel] for another 2 weeks. The pieces of
rooted tobacco leaves were harvested for DNA and RNA
extraction. The positive transgenic plants were determined
by PCR with the specific primer of PaMT31 gene.
Physiological test ofthetransgenic lines inresponse
tocadmium treatment
The leaves of the positive transgenic tobacco lines were
harvested for regeneration experiment again to obtain mul-
tiple clonal plants. The seedlings were transplanted in the
plastic pots (10 × 10 cm) with vermiculite and grew in a
growth chamber (25°C, 8h dark/16h light) for a month. The
well-growing and identical plants were selected for cad-
mium treatment (20ml of 100mM CdCl2 for each pot) for
7 days. The leaves and roots were then harvested, washed
with distilled water, and used for physiological indexes test
and cadmium content measurement.
Relative electrolyte leakage (REL) was estimated based
on the method by Liu (2016), 0.1 g fresh sampled leaves
were cut in pieces of 0.5 cm2, soaked in 30 ml of deionized
Plant Cell, Tissue and Organ Culture (PCTOC)
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water, and shaken overnight. The electrical conductivity
of the solution was measured as R1. After boiling for 15
min and shaken overnight, the electrical conductivity was
measured as R2. REL was calculated by R1/R2×100%
Malondialdehyde (MDA) content was measured accord-
ing to the method by Liu (2016). 0.1 g fresh leaves were
ground in 1 ml of 10% trichloroacetic acid (TCA) solu-
tion. After centrifugation, the supernatant was mixed with
0.6% thiobarbituric acid (TBA) in equal volume and kept
in boiling water bath for 15 min. The absorbance values
of the supernatant were measured at wavelength of 450,
532, and 600 nm by using UV-754N Spectrophotometer.
The MDA content (μmol/g) was calculated as: [6.459 ×
(OD532−OD600)−0.56×OD450] × V/W. (V, extraction vol-
ume; W, fresh sample weight).
Soluble sugar content (SSC) was measured based on
the method by Zhang (2019). 0.1 g fresh leaves in 2 ml
deionized water was boiled for 20 min. 50 μL of the super-
natant was mixed with 450 μL distilled water and 2.5ml
anthrone reagent, then boiled for 10min. The absorbance
value of the solution was measured at wavelength of 620
nm by using UV-754N Spectrophotometer. The soluble
sugar content (μg/g) was calculated as: W1×V1×dilution
factor/(V2×W2×106). (W1, soluble sugar content from the
standard curve; V1, extraction volume; V2, test sample vol-
ume; W2, fresh sample weight).
Root activity was determined by TTC (2,3,5-triphe-
nyltetrazolium chloride) method. 0.1 g cleansed roots was
incubated in 2.5 mL of 0.4% TTC for 24 h in the dark.
After rinsing with distilled water and drying out with fil-
ter paper, the samples were put into 5 mL 95% ethanol
at 60°C for 4 h. Absorbance at 490 nm was measured by
using UV-754N Spectrophotometer. Based on the standard
curve, the tripheny formazan (TTF) content (μg/g/h) was
inferred and computed for the sample root vitality by TTF
content/root dry weight (g)
Cadmium content measurement
Plant root and leave samples were harvested individually
and dried in oven at 80°C for 48 h, then ground to pow-
der. 0.05g sample powder of each was digested in HNO3/
HClO4 (4:1, v/v) at 100°C for 20 min, heated at 190°C for
60 min until the liquid evaporated, then dissolved in 100
mL deionized water overnight. The plant Cd content (mg/
kg) was measured by an Agilent 7500 ICP-MS instrument
(Agilent Technologies Inc, USA), converted based on cad-
mium standard curve, and calculated as (C × 0.1)/W (C,
the cadmium concentration detected by the instrument; W,
sample dry weight). The transfer coefficient was calculated
as cadmium content in leaves/cadmium content in roots.
Statistics analysis
Each line was tested with three propagated plants for physi-
ological test and cadmium content measurement. The mean
of the repliacates and the standard error were calculated.
The significant difference between the transgenic plants and
wild type was analyzed according to LSD test (SPSS 19.0
ANOVA) at 0.05 (marked as single asterisk) or 0.01 (maked
as doble asterisk) probability levels. The physiological index
response (IR) for each transgenic plant under stress was cal-
culated by the formula of (transgenic plant–wild type)/wild
type.
Results
Characteristics ofthePaMT3‑1 protein
ofP. americana
The PaMT3-1 gene from P. americana was found to be 192-
bp long and predicted to encode 63 amino acids. The small
peptide had a theoretical isoelectric point of 4.77, a molecu-
lar weight of 6.76 kDa, and a protein instability index of
44.49. Thus, PaMT3-1 is a slightly acidic, unstable protein.
A phylogenetic tree of amino acid sequences encoded by
metallothionein type-III genes from P. americana and 16
other plant species was constructed using MEGA6 software
(Fig.1). In this tree, PaMT3-1 and proteins from Theobroma
cacao, Gossypium raimondii, Ricinus communis, Eucalyp-
tus grandis, Nicotiana sylvestris, Arabidopsis thaliana, and
Brassica napus clustered in a group heavily represented by
cadmium hyperaccumulator plants, while sequences from
Carica papaya, Solanum nigrum, Coffea arabica, Jatropha
curcas, and Citrus sinensis were classified into another
group. The closest homologs of PaMT3-1 were proteins
from cruciferous plants. The topology of the tree based on
metallothionein sequences was consistent with traditional
evolutionary species relationships.
Construction ofanexpression vector harboring
PaMT3‑1 andtransformation intotobacco plants
Primers with restriction enzyme sites (Hind III/XbaI) on
both sides of the PaMT3-1 gene sequence were designed and
used to amplify the complete gene sequence with a plasmid
template. The pEZR (K)-LC vector and the recovered gene
fragment were cleaved by HindIII and XbaI, respectively,
and ligated to form the recombinant plant expression vector
pEZR(K)-LC-PaMT3-1 (Fig.2).
The recombinant plasmid was transformed into Agrobac-
terium tumefaciens strain LBA4404 and then introduced into
tobacco plants. Four positive transgenic lines were identified
by PCR using the PaMT3-1 specific primer (Fig.3).
Plant Cell, Tissue and Organ Culture (PCTOC)
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Cadmium content oftransgenic plants harboring
PaMT3‑1
The positive transgenic lines were individually propagated.
At maturity, the transgenic tobacco plants were subjected to
cadmium treatment for 7 days. Measured cadmium contents
of aboveground parts of transgenic lines were significantly
lower than in the wild type, with an average decrease of
25.69% (Fig.4). Cadmium contents of underground portions
of transgenic plants were similar to those of the wild type.
The transport coefficient of cadmium from underground to
aboveground portions of the transgenic tobacco lines was
Fig. 1 The phylogenetic tree of
type III metallothionein genes
from Phytolacca americana and
15 other species based on amino
acids
Brassica juncea(BAB85601. 1)
Brassica napus(XP 013681151.1)
Arabis alpina(KFK38876.1)
Arabidopsis thaliana(NP 566509.1)
Nicotiana sylvestris(XP 009757034.1
)
Eucalyptus grandis(KCW67342.1)
Ricinus communis(XP 002525821.1)
Phytolacca americana(PaMT3-1)
Gossypium raimondii(KJB61887.1)
Theobroma cacao(XP 007021480.1)
Jatropha curcas(XP 012084959.1)
Citrus sinensis(K DO62487.1)
Sesamum indicum(XP 011098316. 1)
Coffea arabica(AGL34968.1)
Carica papaya(Q96386.1)
Solanum nigrum(ACL80668. 1)
100
76
93
49
78
93
33
48
66
87
77
99
75
npt II PaMT3-1OCS-ter
Hind IIIXba I
pEZR(K)-LC-PaMT3-135S
npt II GFPOCS-ter
Hind IIIXba I
pEZR(K)-LC 35S
Fig. 2 Construction of the recombinant expression vector pEZR(K)-
LC-PaMT3-1
Fig. 3 Regenerated seedling (a)
and RNA detection of PaMT3-1
gene (b) 1, 3, 4, and 7, trans-
genic tobacco lines; 9, a positive
control of the cloned gene; 10,
a negative control of wild-type
tobacco plant; M, DNA Marker
(A)
(B)
1 3 4 7 9 10 M
Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
64.48–76.69% of that of wild-type plants. Obviously, the
binding of cadmium ions to PaMT3-1 proteins in root cells
was not conducive to their secretion into stem microtubule
tissue, thus limiting the transport of the ions to leaves.
Cadmium resistance ofPaMT3‑1 transgenic plants
The tabacco plants were growing well before cadmium treat-
ment, and no phenotype difference was revealed between the
transgenic and wild plants. When the cadmium applied for
7 days, the leaves of all tobacco lines tended to be slightly
wilted, yellow, and withered from the bottom to top. Signifi-
cantly, the transgenic tobacco plants performed better than
wild-type plants (Fig.5), thus indicating that PaMT3-1 might
play a role in resistance to cadmium.
Measurements of physiological indexes yielded similar
results that well responded to the phenotype performance
under cadmium treatment (Fig.6). No significant differences
were revealed between WT and transgenic plants without
cadmium treatment, while each physiological index was
really changed when processing cadmium treatment. In
cadmium treatment for 7 days, the relative conductivity of
transgenic tobacco plants was approximately 29.44% lower
than that of wild-type plants, whereas the malondialdehyde
content of wild-type tobacco plants was 1.67 times that of
Fig. 4 Cadmium content in
leaves and roots of transgenic
(T1-T7) and wild-type tobacco
plants (WT) (a), and transport
coefficient (b). The experiment
was replicated for three times.
Each column represents an
average of three replicates, and
bars indicate SD. “*” and “**”
Indicate significant differences
in comparison with relative WT
performance at P < 0.05 and P
< 0.01.
**
**
**
**
0
100
200
300
400
500
600
700
800
900
1000
WT T1 T3 T4 T7 WT T1 T3 T4 T7
tooRfaeL
Tobacco lines
Cadmium content(µg/g)
(A)
***
*
0
0.5
1
1.5
2
WT T1 T3 T4 T7
Tobacco lines
Transfer coefficien
(B)
(A)
WT T1 T3 T4 T7
(B)
WT T1 T3 T4 T7
Fig. 5 Phenotype of tobacco lines before (a) and after (b) cadmium
treatment. T1, T3, T4, and T7, transgenic tobacco lines; WT, wild
type tobacco line.
Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
Fig. 6 The physiological index
test of the transgenic (T1-T7)
and wild type line (WT) with
or without 100mM Cd treat-
ment for 7 d. The experiment
was replicated for three times.
Each column represents an
average of three replicates, and
bars indicate SD. “*” and “**”
Indicate significant differences
in comparison with relative WT
performance at P < 0.05 and P
< 0.01. a Relative electrolyte
leakage (REL); b Malondialde-
hyde (MDA) content; c Soluble
sugar content (SSC); d Root
activity
(A)
(B)
(C)
(D)
Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
transgenic plants, which indicates that wild-type tobacco
plants experienced serious membrane damage. The soluble
sugar content of the transgenic lines was 1.4 times higher
than that of wild-type tobacco. This accordingly resulted
in free water content decreased while bound water con-
tent increased in transgenic plant cells, thereby increased
plant resistance. Furthermore, the root activity of wild-type
tobacco plants was only half that of the transgenic lines,
which reflects the robustness of transgenic plant cells able
to actively defend against heavy metal ion toxicity.
Discussion
In this study, we transformed PaMT3-1 from P. americana
into tobacco plants. As expected, the resulting transgenic
lines showed better tolerance to cadmium whatever phe-
notype or physiological performance, which indicates that
the PaMT3-1 gene was able to confer stronger resistance to
heavy metal ion stress on tobacco. Some other MT genes
have given similar results, such as ThMT3 from Tamarix
hispida (Yang etal. 2015), SpMTL from Sedum plumbizin-
cicola (Peng etal. 2017), and SaMT2 from Sedum alfredii
(Zhang etal. 2014).
In regards to a possible mechanism, cysteines in metal-
lothioneins undoubtedly play a key role by chelating heavy
metal ions to weaken the toxicity of ions toward cells. Thus,
the number of cysteines in the peptide is an important deter-
minant of the strength of resistance to cadmium stress. In a
previous prokaryotic expression analysis (Chen etal. 2018),
we analyzed three MT3 proteins and found that the one with
the most cysteines, PaMT3-1 with 12, conferred the highest
cadmium resistance. The well performance of the gene in
tobacco plants confirmed that PaMT3-1 could be an effi-
cient gene resource in phytoremediation. This observation is
consistent with other strongly performing proteins, such as
BjMT3 from Brassica juncea (Mobin etal. 2007), BnMT3
from Brassica napus (Wang etal. 2015), AaMT3 from Ara-
bis alpina (Willing etal. 2015), and AtMT3 from Arabidop-
sis thaliana (Lee etal. 2004), all of which have 12 cysteines.
In contrast, most species that are generally used in phytore-
mediation have a MT3 protein with only 10 cysteines; exam-
ples include CsMT3 from Citrus sinensis (Wu etal. 2018),
GrMT3 from Gossypium raimondii (Paterson etal. 2012),
NsMT3 from Nicotiana sylvestris (Sierro etal. 2013), and
SiMT3 from Sesamum indicum. (Wang etal. 2016). There-
fore, the gene replacement could be expectant to enhance the
efficiency of phytoremediation.
Unfortunately, metallothioneins differ greatly in their ability
to enhance the enrichment and transport of heavy metal ions.
Some proteins can promote metal ion accumulation; for exam-
ple, significantly increased zinc accumulation, as high as 48%
in roots compared with the wild type, has been been observed
in transgenic plants carrying the SaMT2 gene from Sedum
alfredii (Zhang etal. 2014). Some metallothionein performed
differently; for instance, copper accumulation in roots, stems,
and leaves of ThMT3-expressing transgenic plants under cop-
per stress was similar to that of wild-type plants (Yang etal.
2015). Cadmium concentrations in leaves and roots of trans-
genic Arabidopsis plants harboring SpMTL have been found
to be significantly reduced relative to the wild type (Peng etal.
2017), with the cadmium transport coefficient of transgenic
lines (0.1) significantly lower than that of the wild type (0.16)
under cadmium stress. In a study by Li (2001), the lead (Pb)
content in leaves of MT-expressing transgenic plants was sig-
nificantly lower than that of the control, while Pb contents
in stems and roots were much higher under Pb stress. In the
present study, cadmium content in roots of PaMT3-1 trans-
genic tobacco plants was not significantly decreased compared
with wild-type plants, whereas shoot cadmium content was
significantly lower, and the transport coefficient was reduced
by an average of 30.41%. For the ambiguous data, we pro-
pose the following mechanism to explain these observations.
First, metallothionein genes exhibit high sequence variation;
for example, amino acid similarities between PtMT3-1 and
MT3 proteins from 16 other species range from 42.86–59.68%,
great variation that can seriously affect the protein’s spatial
structure and ultimately its ion-binding ability. Second, several
types of genes are involved in heavy metal ion detoxification
in plants; an example is phytochelatins (PCs), whose main
function is ion enrichment and transport. Similar to metal-
lothioneins, cysteine is the key functional group in PCs for
chelation of heavy metal ions, which may lead to biosynthetic
competition between PCs and MT in plants used for heavy
metal detoxification. Several reports contributed the evidences
that over-expression of MT in transgenic plants may reduce
PC synthesis and ultimately reduce heavy metal ion transport
(Schat etal. 2002; Zhao etal. 2003; Hernandez etal. 2006;
Peng etal. 2017). Finally, metallothionein stability may affect
the transport and accumulation of heavy metal ions. As men-
tioned above, PtMT3-1 is an unstable protein with an instabil-
ity index of 44.49 (In comparison, the other two MT3 proteins
from P. americana are stable proteins with instability indexes
of 25.31 and 38.65). We speculate that its protein stability
can accordingly be increased by binding metal ions. And, an
increase in protein stability may limit the release and dispersal
of binding ions and lead to decreased transport and accumula-
tion. Further exploration of MT proteins would thus be useful
and help elucidate their roles and mechanisms of action in
phytoremediation.
Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
Conclusions
PtMT3-1 from Phytolacca americana displays high
sequence variation relative to proteins encoded by homol-
ogous genes in other species. PtMT3-1 can significantly
improve plant resistance to cadmium but limits cadmium
transport and enrichment. The pyramiding of PtMT3-1 with
other related genes may be a suitable strategy for cadmium
phytoremediation.
Acknowledgements The research was granted by Beijing Natural Sci-
ence Foundation (#5122019) and the Fundamental Research Funds for
the Central Universities Grants (No. 2015ZCQ-SW-01)
Author Contributions All authors contributed to the study conception
and design. ZJ and LX performed material preparation and research;
YP, YR and LJ analyzed data; ZJ and XJ wrote the paper. All authors
read and approved the final manuscript.
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