Introduction
The heavy metal cadmium (Cd) is widely distributed in the natural
environment (Lalor et al. 2004). Although Cd is not a
necessary element for plants, can readily absorb by plants because of the
similarity with many divalent metal ions such as zinc, iron, and calcium (Pottier et al.
2015). Once it enters the plant, Cd may depress the growth and
yield of plants (Wang et al. 2013; Naggar et al.
2014). Additionally, Cd was able to be accumulated in the edible
portions of plants, especially in crops. Because Cd is migratory along the food
chain, it may pose potential risks to humans and other animals (Clemens et al.
2013).
Over the last decade, Cd-contaminated soil has
been increased dramatically (Chibuike and Obiora
2014). Hence, it is essential to conduct Cd-contaminated soil restoration.
Phytoremediation, as an environment-friendly and sustainable remediation method
for contaminated soils, has attracted wide attention (Pilon-Smits and Freeman 2006; Rosa
et al. 2014). However, the uptake, transport, accumulation and redistribution
of Cd from soil to other tissues of plants require different transporters, for
example the ZIP family (ZRT: zinc regulated transporters, IRT-like protein:
iron-regulated transporter-like protein); the HMA family (heavy metal ATPase
transporters); the MATE family (multidrug and toxic compound extrusion protein
transporters); the NRAMP family (natural resistance-associated macrophage
protein); the ABC family (ATP-binding cassette proteins); and the oligopeptide transporters family and so on (Nevo and Nelson 2006; Sasaki et al. 2016; Wu et al. 2016).
Transporters are essential for Cd absorption, translocation and distribution in
plants. To under the molecular mechanisms of Cd transport process and the role
of transporters is helpful to the development of hyperaccumulators.
The ideal plant for using in phytoremediation should grow fast, high biomass,
preferably in the aboveground parts, ability to absorb and tolerate high metal
concentrations (Baker and Whiting 2002; Whiting
2010; Goswami and Das 2015). At present, the heavy metal hyperaccumulators such as the Noccaea caerulescens and the Arabidopsis halleri
screened have the capacity for Cd accumulation, but most of them are
slow-growing, the aboveground biomass is low, which is not conducive to
large-scale mechanization. It is common knowledge that Brassicaceae are the heavy metal
accumulators, the accumulation ranges of heavy metals in shoots have been
studied for many years (Ghosh and Singh 2005; Flores-Caceres et al. 2015). Indian mustard (B. juncea) and
B. napus
belong to Brassicaceae
family and the former is considered to be one of the most potential phytoremediation species, because of
their higher biomass and higher heavy metal accumulating capacity in the shoot (Eapen and D'Souza 2005; Goswami and Das 2015).
However, the oil production of B. juncea is very low and its growth area is very limited.
It is difficult to perform large-scale planting of B. juncea in China. B. napus, as an important oil crop, has
larger biomass and higher oil production compared to B. juncea. It is popularly planted in the
middle and lower reaches of the Yangtze River in China. Because of its prolific
growth, B.napus can be grown advantageously for
phytoremediation (Meng et al. 2008). Hence, researching the physiological and
genetic processes of Cd uptake, transport and accumulation in B. juncea and B. napus, are beneficial to the improvement of Cd tolerance and
resistance in plant, further controlling the migration process of Cd in food
chain.
In this study, one potential Cd accumulation cultivar of B. napus and
the normal species of B. juncea were selected and compared under different Cd
stress conditions. A comprehensive investigate on the biomass, physiological characteristics, Cd and mineral
element enrichment ability and grain quality, subsequent the genetic processes
of Cd uptake and transport at different Cd concentrations were studied. The
current research is very important to find out the differences between the two Brassica species in response to Cd
stress, obtaining an oilseed rape species that can enrich Cd without affecting
its economic value, finally providing a theoretical basis for the screening of practical
Cd hyperaccumulators in response to agricultural
Cd-contaminated soil.
Materials
and Methods
Plant materials
and treatment
The
pot experiment was conducted at Southwest University, Beibei
District, Chongqing, China (106.4 N, 29.8E). The tested soil in the
experimental consisted of a typical peat (Klasmann-Deilmann,
pH6.0, no Cd, Germany) and vermiculite (diameter 3–6 mm). Seeds of B. napus (Zhongyou 821) and B. juncea (Ping shan
qing cai) were obtained
from the College of Agronomy and Biotechnology of the Southwest university.
Seeds of B. napus
and B. juncea
were surface sterilized using 6% (w/v) NaClO
(including 0.05% Twain20) for 3 min, rinsed completely in sterile water and
incubated in petri dishes. The seeds were then vernalized at 4°C for 14 days in
the darkness and then grown in an culture room with 16 h light (25°C, 5000Lux)
and 8 h dark (20°C) photoperiod. After 8 days for germination, uniform
seedlings of Brassica were transferred into plastic pots (25×33 cm) containing
2 kg of tested soil, one plant per pot and watered every three days. The
experiments were carried out in the greenhouse of Southwest University under
normal illumination and temperature conditions (the winter temperature ranges
from 0 to 10°C necessary for vernalization).
After approrimately
100 days growth, one week before the initial flowering stage, the plants were
exposed to the soils with 0, 50 and 200 mg kg-1 Cd stress
for 30 days, eight repetitions per treatment.
The first sampling was carried out after 30 days
of Cd treatment. Four repetitions of different Cd treatments were harvested. At
harvest, plants were divided into roots (R), lower stems (LS), lower leaves
(LL), upper stems (US), upper leaves (UL) and siliques
(S). The leaves of some samples were taken out, immediately frozen in liquid nitrogen and then stored at -80°C for
RNA extraction. All other tissues were dried in oven at 70℃ until they
reached constant mass and weighed for biomass determination. And the rest of
plants were harvested when the siliquesss had fully
matured and the leaves had senesced. Corning peeled to leave rapeseed for Cd
content and determination of fatty acid components.
Mineral nutrients
contents
The sample of oven-dried was
crushed and sieved (60 mesh) for mineral nutrients detection. The contents of
Cadmium (Cd), Calcium (Ca), Magnesium (Mg), Copper
(Cu), Zinc (Zn), Iron (Fe) and Manganese (Mn) in
plant tissues were measured via Flame Atomic Absorption Spectrometry (FAAS)
after digestion of the plant samples with diacid (3
HNO3: 1 HClO4; v/v).
Fatty acid components
Fatty acid components of seeds,
removed from the siliques, was detected by GC
analysis (GC-2010, Shimadzu, Japan) as mentioned by Lian
(Lian et
al. 2017). In all of 200 mg dried rapeseed were crushed and then transferred
to 5 mL glass tube and added 2 mL petroleum ether and ether solution (1:1),
then the sample was shaken gently. After dissolving (about 40 min), 1 mL
KOH-methanol solution (0.4 mol/L) was added to the
sample, and then the mixture was mixed to carry out methyl esterification
(about 30 min at the room temperature). Added 2 mL distilled water, mixed and
shaken. After demixed, taking 1 mL of supernatant to
the GC tube for detected.
The chromatograph column type is a DB-WAX (30 m × 0.246 mm × 0.25 ppm).
The stationary phase was polyethylene glycol. The column temperature was 185°C,
the vaporization temperature was 250°C, and the detector temperature also was
250°C. The gas flow rate of nitrogen (carrier gas) was 60 mL/min, hydrogen was
40 mL/min and air was 400 mL/min. 2 uL sample was
injected and the peak retention time was 13 min. According to the
meteorological chromatogram of each sample to be measured, the peak area normalization
method is used to calculate the percentage of each fatty acid component.
Photosynthetic efficiency
After 30 days of Cd treatment, the shoot of plant was divided into two
layers with the third long-handled leaves at the under of lowest effective branch.
Photosystems activity parameters were all detected in Portable photosynthesis
systems (CIRAS2) between 10:00 to 11:00 at Sunny morning.
RNA isolation, complementary
DNA (cDNA) synthesis and real-time quantitative-PCR
The EZ-10 DNAaway RNA Mini-prep Kit (Sangon, Shanghai) was used to extract the
total RNA from frozen upper leaves of B. napus and B. juncea
according to the manufacturer’s protocol. The concentration and
quality of RNA samples have been tested by NanoDrop 2000 spectrophotometer (Thermo
Scientific, USA). PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Tapan) was used for the cDNA synthesis.
Real-time quantitative-PCR was used to identify the expression level of Cd
transport related genes for which homologous to A. thaliana had been reported. All genome sequences were blast and
downloaded from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All primers
(Additional file 2: Table S2) for RT-qPCR were
designed based on tool Primer3 (http://bioinfo.ut.ee/primer3–0.4.0/primer3/) and synthesized commercially
(BGI, Beijing, China). All the expression data were obtained from three
individual biological replicates and three independent technical replications
for each cDNA template. The samples were standardized
as the selected internal reference gene and the expression levels of related
genes were calculated by the 2_DDCt method.
Statistical analysis
The SPSS 18.0 and GraphPad Prism 5 were used to statistically analyze all
data. The data sets were analyzed by variance analysis to calculate the mean
values and SD of each treatment. The Duncan’s multiple range test was used for significant analysis of differences
between groups of samples (p < 0.05). Student's t test was
used to compares two means and the significant difference between two groups (p < 0.05).
Results
The effects of Cd stress on the biomass in the two brassica species
The dry weight of roots and stems of B. napus and B. juncea were
decreased remarkably under Cd treatment (Fig.
1A and B). The 50 mg kg-1 Cd treatment
significantly affected the dry weight of leaves of B. napus and B. juncea. The dry weight of B. napus
leaves was slightly reduced, while that of B.
juncea was increased, under
200 mg kg-1 Cd treatment condition (Fig. 1C). On the contrary, with
the increase of Cd concentration, the dry weights of siliques
of B. napus
and B. juncea
were also increased, respectively. When the Cd concentration was up to
200 mg kg-1, the dry weight of siliques
of B. napus
was up to 2.46 g twice as much as that under normal conditions (Fig. 1D).
However, the biomass of B. napus was weakly decreased under 50 mg kg-1 Cd treatment condition (Fig. 1E). The
root/shoot ratio of B. napus was almost unaffected by Cd treatments. However,
the root/shoot ratio of B. juncea was significantly decreased under Cd treatment
condition. The results suggested that the Cd stress had no significant effect
on the biomass of both B. napus and B. juncea after 30 days of Cd treatment at flowering
stage. Comparably, B. juncea
was more sensitive to Cd treatment when comparing with B. napus.
The effect of Cd stress on photosynthetic efficiency
The Cd treatment weakly decreased
the Ci in UL of B.
napus. The Ci in LL of B. napus and B. juncea were
not affected by Cd treatment, respectively (Fig. 2A). The Tr in UL of B. napus was improved by Cd treatments, while the Tr in LL was weakly reduced under 50 mg kg-1 Cd treatment condition. The effect of
Cd stress on Tr of B. juncea was consistent with that of B. napus (Fig.
2B). The Gs in both ULand
LL of B. napus were
reduced under 50 mg kg-1 Cd
treatment condition, in contrast, the Gs in both ULand LL of B.napus were increased under 200 mg kg-1 Cd treatment condition. Moreover, the Gs of UL and LL of B.
napus were 699ppmol/m2/s and
623ppmol/m2/s, which was 1.4 times and 1.5 times higher than that in
the normal condition, respectively. The Gs in UL and
LL of B. juncea
were weakly increased (Fig. 2C). The Pn in both UL
and LL of B. napus
were reduced under 50 mg kg-1 Cd
treatment condition, and then were increased under 200 mg kg-1 Cd treatment condition. The
effect of Cd treatment on the Pn in UL of B. juncea was
consistent with that of B. napus, but had no obviously effect on the Pn in LL of B. juncea (Fig. 2D). The results revealed that the
photosynthesis of lower leaves (LL) which were also older leaves, in both Brasscia species, especially in B. juncea, was more significantly
affected by Cd stress compared with the upper leaves (UL), which were also
younger leaves.
The effects of different Cd concentrations on the Cd contents of different
organs of the two brassica species
%20829-837_files/image002.jpg)
Fig. 1: Effects of different Cd
concentrations on dry weights of root (A), stem (B), leaf
(C), silique (D), whole plant (E) and the root/shoot
ratio (F) of two Brassica species
Data were statistically evaluated using Duncan’s
test (P < 0.05). At least four biological replicates were
performed for each treatments
%20829-837_files/image004.jpg)
Fig. 2: The effect of Cd stress on Ci: Intercellular CO2 concentration (A), Tr:Transpiration (B), Gs: stomatal conductance (C) and Pn: Variation of net CO2 assimilation (D) of the
two Brassica species
Data presented are the means (n=4), and error bars denote the standard
deviations. Data were statistically evaluated using Duncan’s test (P < 0.05)
The result showed that higher Cd stress could increase Cd uptake and
accumulation activities in B. napus and B. juncea (Fig. 3). Interestingly, the Cd contents in B. napus were
mostly higher than that in B. juncea under different Cd treatments (Fig. 3A–F), except for the Cd contents in
the seeds under 200 mg kg-1 Cd treatment
condition (Fig. 3E). The accumulation of shoot in B. napus was higher than that in B. juncea
(Fig. 3F). Under 50 mg kg-1 Cd stress, the accumulation of shoot in
B. napus
were up to 1532.30 µg/plant, were 6.13 times to that of B. juncea,
respectively. Under 200 mg kg-1 Cd stress, the accumulation of shoot in B. napus were up to2219.27 µg/plant,
which were 2.80 times to that of B. juncea. The result revealed that B. napus can significantly accumulate
more Cd comparing with B. juncea under same Cd treatment conditions. Therefore,
the cultivar of B. napus chosen in the present study is an potential species for agricultural soil heavy metal
remediation in the future.
%20829-837_files/image006.jpg)
Fig. 3: Effects of different Cd
concentrations on the Cd contents ofroots (A), stems
(B), leaves (C), siliques (D), seeds (E) and Cd
accumulation of shoot (F) of the two Brassica species
Data were statistically evaluated using Student's t test (P < 0.05).
At least four biological replicates were performed for each treatments
The effects of Cd stress on the
uptake, transport and accumulation
The BCF is the ratio of Cd concentration in plant tissues to exogenous Cd.
TF is the ratio of Cd concentration in shoot to that in root. Therefore, higher
the BCF value, the stronger was cadmium accumulation ability. Likewise, higher the
TF value, the higher was cadmium transfer ability from root to shoot. Table 1
showed that the BCFroot/soil,
BCFlower stem/soil, BCFlower leaves/soil, BCFupper stem/soil, BCFupper leaves/soil, BCFsiliques/soil of B. napus were
decreased when the Cd concentration increased from 50 to 200 mg kg-1
and the BCFroot/soil, BCFlower stem/soil, BCFlower leaves/soil, BCFupper stem/soil, BCFupper leaves/soil, BCFsiliques/soil were reduced by 38.81, 58.88, 69.90, 57.14, 69.70
and 72%, respectively. The BCFroot/soil, BCFlower
leaves/soil and BCFupper stem/soil of
B. juncea were
decreased when the Cd concentration increased from 50 to 200 mg kg-1, while the BCFlower
stem/soil, BCFupper leaves/soil
were increased, the BCFsiliques/soil had
no differences. However, all the BCF showed significantly differences between
the two Brassica species at 50 mg kg-1 Cd concentration condition (p<0.05),
the BCF of B. napus
was higher than that of B. juncea, expect for the BCFlower
stem/soil. The TF values of B. napus were 2.52 and 1.35 (≥1) under 50 and 200 mg
kg-1 Cd treatments condition, respectively. The TF values of B. juncea were 1.73 and 5.49 (≥1) under 50 and
200 mg kg-1 Cd treatments condition, respectively (Table 1). The
results revealed that the accumulation and transformation capacity pfCd in B. napus were higher than that in B.
juncea at comparably lower (50 mg kg-1)
Cd stress condition.
Table 1: Biological enrichment factor (BCF)
and Translocation factor (TF) values of different Cd concentration of the two
oilseed rape cultivars
|
Cd ( mg kg-1) |
Cultivars |
BCFroot/Soil |
BCFlowerstem/Soil |
BCFlowerleaves/Soil |
BCFupperstem/Soil |
BCFupperleave/Soil |
BCFSilique/Soil |
TF |
|
50 |
B.napus |
2.19±0.15 ** |
1.07±0.05 ** |
1.96±0.39 * |
0.70±0.05 |
1.32±0.32 * |
0.25±0.05 * |
2.52±0.22 ** |
|
|
B.juncea |
1.11±0.12 |
0.51±0.06 |
0.54±0.17 |
0.73±0.24 |
0.40±0.16 |
0.05±0.00 |
1.73±0.04 |
|
200 |
B.napus |
1.34±0.17 ** |
0.44±0.03 |
0.59±0.07 |
0.30±0.04 |
0.40±0.04 |
0.07±0.01 |
1.35±0.09 |
|
|
B.juncea |
0.45±0.20 |
0.56±0.06 * |
0.49±0.08 |
0.40±0.09 |
0.48±0.14 |
0.05±0.00 |
5.49±0.64 ** |
Data were the means±standard deviation (n = 4). Values followed by different asterisk indicate significant difference (P≤0.05) among
different Cd levels in B.napus
and B.juncea
(*: P < 0.05, **: P < 0.01)
The effect of Cd stress on mineral
nutrients in the two brassica species
%20829-837_files/image008.jpg)
Fig. 4: The effect of Cd stress on Ca contents (A), Mg contents (B), Fe contents (C), Zn
contents (D), Cu contents (E) and Mn contents (F) of
different organs in the two Brassica species
Data were statistically evaluated using Duncan’s test
(P < 0.05). At least four biological replicates were performed
for each treatment
There were obvious differences
between the two Brassica species for the six mineral nutrients in different
parts of B. napus
and B. juncea (Fig.
4). The Ca contents of B. napus were not affected by the Cd
stress, only the Ca content was increased under 50 mg kg-1 Cd treatment condition. The Ca content in US of B.
juncea was increased under 50 mg kg-1
Cd treatment condition, while in LL was decreased under 200 mg kg-1
Cd treatment condition (Fig. 4A). With the increase of Cd concentration, Mg
content in LL, UL and S of B. napus was decreased. The Mg contents in LS, US, LL and
UL of B. juncea
were improved by the 50 mg kg-1 Cd treatment (Fig. 4B). The Cd stress had obviously effect on the Fe content in
all parts of B. napus
and B. juncea.
The Fe content in roots of B. apus was increased under 50 mg kg-1 Cd
treatment condition, which was 2.42
times higher than that in the normal condition. With the increase of Cd
concentration, the content of Fe in LS, UL, LL and S of B. napus decreased, but the Fe content in
UL was increased. The Fe contents in R, LS, UL and S of B. juncea were decreased with the
increase of Cd concentrations. The Fe content in US of B. juncea was obviously increased under 200 mg kg-1 Cd treatment condition, while
that in LL was also obviously increased at 50 mg kg-1 Cd
concentration (Fig. 4C). The Zn contents in R, LS, LL and UL of B. napus were
increased under 50 mg kg-1 Cd treatment, then decreased under 200 mg
kg-1 Cd treatment, while the Zn content in S was decreased with the
increase of the Cd concentrations. The Zn contents in LS, US, LL and UL of B. juncea were
increased under 200 mg kg-1 Cd treatment
condition, while other tissues of B.
juncea were not affected by the Cd stress (Fig.
4D). The Cu contents in LS, US, LL and UL of B. napus were
increased under 200 mg kg-1 Cd treatment condition, the Cu contents in R and S were no affected by the Cd treatments. The Cu contents in R,
US, LL and S of B. juncea
were affected by the Cd stress.
%20829-837_files/image010.jpg)
Fig. 5: The expression levels of CET2 (A),
CET3 (B), CET4 (C), HMA3;1 (D), OPT3 (E), HMA4;1 (F) NRAMPs in B. napus (G)
and B. juncea
(H) in leaf of the two Brassica species in response to Cd stress
TData were statistically evaluated using Student's t test
(P < 0.05). At least three biological replicates were performed
for each treatment
The 50 mg kg-1 Cd treatment
had improved the Cu contents in R and LL up to 11.20 and 105.81 mg
kg−1 of B. juncea, which was 1.82 and 4.23 times higher than that
in the normal condition (Fig. 4E). The Cd stress had significantly effect on
the Mn contents in LL and UL of B. napus. The Mn
contents in R, LS and US were weakly decreased with the increase of Cd
concentrations of B. napus.
The Mn contents in R, US and LL of B. juncea were
increased under 50 mg kg-1 Cd treatment condition,
while the Mn content in LS was decreased with the
increase of Cd concentrations (Fig. 4F).
The results indicated that the uptaken and
translocation of micronutrients in the two Brassica species were affected in
different levels under gradient increased Cd treatment. Interestingly, Zn
contents were significantly affected in B.
napus, which imply that Cd and Zn may compete the transport pathways in B. napus, which may not happen in B. juncea.
The effects of Cd stress on seeds
characteristic of two brassica species
It can be seen from Table 2 that the fatty
acid component of seeds were quite different under the
Cd stress. In normal condition, the percentage of palmitic
acid, stearic acid, oleic acid, linolenic acid and arachidonic acid were higher in seeds of B. napus than
that of B. juncea,
while the percentage of linolenic
acid and erucic acid was lower in seeds of B. napus.
Under 50 mg kg-1 Cd treatment, the percentage of palmitic acid, stearic acid, linoleic acid and arachidonic acid were obviously higher in seeds of B. napus than
that of B. juncea,
the percentage of oleic acid, linolenic acid and erucic acid were lower in seeds of B. napus. Under 200 mg kg-1 Cd treatment, the percentage of palmitic
acid, linoleic acid and arachidonic acid were higher
in seeds of B. napus
(Table 2). The results suggested that the oil contents in both B. napus and B. juncea were
almost = affected with = Cd treatment.
Gene expression
analysis of Cd transport-related genes in leaves of two brassica species
In order to understand the relationship between Cd transport and the
phenotypes of oilseed rape under Cd stress, 12 genes belonging to four gene
families were selected. The 4 gene families were CET, OPT, HMA and NRAMP. It was shown that all the candidate genes from B. napus and B. juncea were
significantly different in the expression levels at different Cd concentrations for 30 days exposure to Cd (Fig. 5). Compared with normal conditions, the expression levels of CET2 gene in B. napus and B. juncea was up-regulated in Cd
treatments, especially the expression level of CET2 in B. juncea was up-regulated 2.23 times in 200 mg kg-1
Cd treatment condition (Fig. 5A). The expression levels of CET3 gene was significant differences between B. napus and B. Table 2: Effect of Cd stress on seeds fatty acid composition of B.napus and B.juncea
|
0mg kg-1 |
50 mg kg-1 |
200 mg kg-1 |
|||||
|
Brassica napus |
Brassica juncea |
Brassica napus |
Brassica juncea |
Brassica napus |
Brassica juncea |
||
|
Total fatty acid (%) |
Palmitic acid C16:0 |
5.138±0.431*** |
2.530±0.185 |
3.906±0.211 |
2.772±0.775 |
3.916±0.019* |
2.730±0.481 |
|
Stearic acid C18:0 |
2.246±0.176** |
1.517±0.083 |
1.718±0.264 |
1.588±0.234 |
1.827±0.139 |
1.960±0.228 |
|
|
Oleic acid C18:1(9) |
30.055±1.399 |
27.569±0.603 |
26.706±0.395 |
29.395±1.515 |
29.210±0.728 |
31.177±1.074 |
|
|
Linoleic acid C18:2(9,12) |
17.813±0.593*** |
7.994±0.379 |
15.536±0.243*** |
7.276±0.158 |
13.630±0.159** |
8.248±1.538 |
|
|
Linolenic acid C18:3(9,12,15) |
4.444±0.029 |
5.134±0.116** |
3.839±0.577 |
5.354±0.055* |
3.349±0.144 |
4.019±0.375 |
|
|
Arachidonic acid C20:1(11) |
10.355±0.339* |
8.682±0.984 |
9.473±0.586 |
9.291±0.854 |
9.901±0.164 |
9.686±0.150 |
|
|
Erucic acid C22:1(13) |
32.662±1.309 |
46.070±0.531*** |
38.426±0.667 |
45.736±0.963*** |
38.168±0.482 |
42.180±1.397* |
|
Data were the means±standard deviation (n = 4). Means followed by different asterisk indicate significant difference (P≤0.05) among different Cd
levels in B. napus
and B. juncea
(*: P < 0.05, **: P < 0.01, ***: P< 0.001)
juncea. Compared with the normal condition, the expression levels of CET3 gene in B. napus was down-regulated in the Cd
treatments, then the expression levels of CET3
gene in B. juncea
was not affected by the Cd treatments (Fig. 5B). Under the 50 mg kg-1
Cd treatment condition, the expression levels of CET4 gene from B. napus was down-regulated compared with the normal
condition, then obvious up-regulated under the 200 mg kg-1 Cd
treatment condition. The expression levels of CET4 gene from B. juncea were down-regulated under the Cd treatments
condition (Fig. 5C). The genes HMA3;1
and HMA4;1 in B. napus and B. juncea was lower expressed under the
Cd treatments condition, while HMA4;1
gene in B. juncea
expressed highly when the Cd concentration was up to 200 mg kg-1
(Fig. 5D and F). The expression levels of OPT3 gene from B. napus and B. juncea were up-regulated compared with
the normal condition (Fig. 5E). Normally, the expression levels of NRAMP genes family were down-regulated
under the 50 mg kg-1 Cd treatment compared with the normal
condition. The BnNRAMPs
were highly up-regulated under the 200 mg kg-1 Cd treatment
condition. The Cd concentration up to 200 mg kg-1 didn,t change the
expression of BjNRAMPs (Fig. 5G and 5H). The result revealed
that BnCET4, BnOPT3, BnHMA4;1 in B. napus, and BjCET2,
BjOPT3 in B. juncea may play important roles in
response to Cd stress.
Discussion
In soil, high Cd stress interferes with
the physiological metabolism, inhibited the growth and development, reduced the
biomass, and even lead to plant death. Some researchs
have indicated that Cd can obviously inhibit plants growth (Li et al. 2013;
Ehsan et al. 2014; Irfan et al. 2014; Hassan et al. 2016). In addition，the application of Cd had
suppressed the Pn, Gs and Tr (Ehsan et al. 2014; Kaur et al. 2017). In this study, dry weights of roots, stems
and leaves of the two Brassica species were reduced under Cd treatments, were
consistent with that in B .napus L. (Ehsan et al. 2014). However, the
biomass of both B. napus
and B. juncea were
not significant affected under the Cd stress. These results were similar to Wu,s
study (Wu
et al. 2015). It is possible for plants to use
the ability to accumulate Cd of their shoot to mitigate this danger in the soil
(Rome et
al. 2016). For example, Thlaspi caerulescens
and T. goesingense
can grow on the soil which containing Cd2+ or Ni2+
without any damage (Persans et al. 2001; Rigola et al. 2006). Furthermore, in the present study, according to the
changes of biomass and the root/shoot ratio in response to Cd treatment, B. juncea was
more sensitive to Cd treatment when comparing with B. napus.
Many studies have indicated that increasing Cd
concentration can lead to the accumulation of Cd in plants (D'Alessandro et
al. 2013; Wang et al. 2013; Pietrini et al. 2016). In this study, the
Cd contents in roots, stem, leaves and seeds of the two Brassica species were
significantly increased with the increasing of exogenous Cd concentration. While, B. napus
accumulated a great deal of Cd in the shoots compared with B. juncea,
which is in consistent with the study of Nouairi (Nouairi et al.
2006). Different plant varieties lead to significant differences
in Cd uptake of B. napus
and B. juncea.
Generally speaking, the high Cd uptake capacity of B. napus may be related to its biomass
and regulatory mechanisms (Fig. 1 and Table 1; Gallego et al. 2012).
It is also meaningful to under the influence of cadmium on plants and the
characteristics of cadmium accumulation in plants. It was reported that Cd in
soil was absorbed by plant roots, most of which remain in roots, some were
transported to shoots and then only a small was transferred to grains (Xue et al.
2013; Sterckeman et al. 2015; Wu et al. 2015). In the current
study, B. napus
can significantly accumulate more Cd in the shoot comparing with B. juncea
under same Cd treatment conditions. It is likely that the difference of Cd
accumulation between B. napus and B. juncea may be mainly affected by root-shoot process
rather than soil-root process (Xue et al. 2013). Therefore, six Cd
transport-related genes were expressed at different levels in both B. napus and B. juncea
under Cd treatment. CET is one Cation-efflux
transporters, which plays an important role in the detoxification of high
concentration Cd. The expression levels of CET2 in B. juncea were higher than that in B. napus, which
may be the reason why B. juncea accumulated less Cd. This may be due to reports
that CET2 enhances heavy metal efflux in plants. Overexpression of BjCET2 in B. juncea
increases the tolerance of heavy metal and accumulated a large number of Cd in
leaves (Xu
et al. 2009). The expression levels of CET3 in B. napus were higher than that in B. juncea.
This may be due to the different role of BjCET3 under some stress conditions,
which seems to be different from the cation-efflux
transporters such as AtMTP1 and BjCET2 (Lang et al. 2011).
Additionally, OPT is another important oligopeptide transporter. In this study, Cd stress
increased the expression of OPT3 gene to some extent under 200 mg kg-1
Cd treatment. Under high Cd treatment, plant tolerance to Cd increased, which
increased the accumulation of Cd. This may be due to the significant expression
of TcOPT3 on the ground. In addition, in situ hybridization analyses indicated
that TcOPT3 is expressed in plant vascular systems, especially in the pericycle, which might be involved in the long-distance
transport. When the expression of OPT3 is impaired, incorrect gene regulation
mediates uptake and mobilization of trace metals, leading to excessive cadmium
in seeds, not other metals (Hu et al. 2012; Mendoza-Cozatl et al. 2014). Recently, many
studies have shown that the expression of HMA3 in the roots of A. thaliana, limiting the long-distance
migration of Cd from root to shoot. So, the high expression of HMA3 in B. juncea
limited the transport of Cd from root to shoot. But, over-expression of HMA3 is
also responsible for high Cd accumulation and tolerance in other plants (Ueno et al.
2011; Zhang et al. 2016). In
addition, the overexpression of AtHMA4 enhanced the
transport of Cd and Zn from root to shoot, thus enhancing the tolerance of
plant to the stress of heavy metal (Verret et al. 2004; Wu et al. 2015). The combination of mutations in both homeologs of HMA4 was proposed as a strategy to limit the
accumulation of Cd in leaves without affects the development (Liedschulte et
al. 2017). In the present study, B.
juncea demonstrated stronger ability to express
HMA3;1, B. napus
demonstrated stronger ability to express HMA4;1 under 200 mg kg-1 Cd
treatment condition, especially for HMA4;1, leading to Cd accumulation in shoot
increased.
NRAMP genes family plays a key role in absorption
and transport of divalent transition metals. Researches have shown that the
mutations selectively modifies Cd2+ and Zn2+ accumulation without affecting Fe2+
transport which was mediated by NRAMP4 in plants (Pottier et al. 2015).
AtNRAMP6 is localized in a vesicular-shaped endomembrane and as a Cd
intracellular transporter contributes to the detoxification of Cd (Cailliatte et
al. 2009). Furthermore, OsNRAMP1 and OsNRAMP5 genes are expressed in
roots have affinity with the cadmium, iron and manganese,
participate in Cd absorption and transport (Ishimaru et al. 2012; Takahashi et al. 2014). The expression levels of NRAMP genes family in both B.
napus and B.
juncea were differ. The elevated expression of NRAMPs
may also be a consequence rather than a cause of the increased Cd levels (Oomen et al.
2009). These results showed that genes related to Cd transport were significantly
up-regulated or down-regulated under Cd treatment and interactived.
Conclusion
The B. napus cultivar used in the present study accumulated more Cd in the shoot
compared to B. juncea
under Cd treatment condition. According to the dry weight, biomass,
root/shoot ration and photosynthesis related elements analysis,
B. napus was
more tolerant to Cd stress when comparing with B. juncea. The micronutrient detection
analysis and the qRT-PCR assays revealed that there
were different Cd uptake and translocation pathways in B. napus and B. juncea in response to Cd stress.
Acknowledgements
This work was supported by National Key R & D Program of China
(2018YFD0200903), National Natural Science Foundation of China (31870587；31400063; 31500038) and Fundamental
Research Funds for the Central Universities (XDJK2017B030; SWU116021;
XDJK2018C095; SWU118114; SWU115018), Research Funds of Scientific Platform and
Base Construction (cstc2014pt-sy0017) and The Recruitment Program for Foreign
Experts (No. WQ20125500073).
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