Introduction
The current large-scale use of artificial nanomaterials has released more
quantum dots into the environment and has an unpredictable impact on the
environment and human health (Wiesner et
al. 2006). Recent studies showed that the water environment provides a way
for these nanomaterials (NPs) to enter the environment (Kim et al. 2010) and will eventually sink and accumulate in the soil (Navarro
et al. 2010). Plants are one of the
many organisms that are directly affected by NPs since these are grown in soil.
It is also confirmed that NPs can penetrate different biological barriers, from
insect to plant cells (Lin et al.
2009).
The QDs are crystalline NPs, first synthesized in
the early 1980s and used in the electronics industry (Brus 1984). They are a type
of nanocrystalline semiconductor material. The core and shell of a
semiconductor nanocrystal are composed of elements of groups III to Ⅴ or
II to VI, and their sizes range from 1 to 10 nm. At such a size, these NPs exhibit unique
photoluminescence (Chandan et al. 2018). The widely studied QDs are
CdTe QDs, cadmium selenide (CdSe) QDs and cadmium sulfide (CdS) QDs (Ensafi et
al. 2017).
The CdTe is one of the most
important II-VI semiconductors and has been extensively applied in the fields
of biomedicine, optoelectronic devices and photonic crystals (Wang et al.
2018). As a new type of nanomaterial, CdTe QDs showed toxicity in vitro and in vivo (Schneider et al. 2018). Liver cancer cells showed reduced metabolic activity,
increased apoptotic cells, increased intracellular reactive oxygen species
(ROS) contents along with reduced glutathione (GSH) and catalase (CAT) contents
and glutathione thiol transferase (GST) activity after exposure to CdTe QDs (Nguyen et
al. 2013). After treating with CdTe
QDs and daunorubicin in HepG2 cells, the proportion of apoptosis after synergistic
action was at the highest level (Zhang et al.
2011). The CdTe QDs and CdTe/CdS QDs decreased the survival rate of
human epithelial cells in 24 h, and most of the cells died in 48 h (Su et al. 2009). While in plants, CdTe-QDs and enhanced
UV-B radiation triggered antioxidant enzyme metabolism and programmed cell
death in wheat (Triticum aestivum L.)
seedlings (Chen et al. 2014).
In addition to studying the toxic effects of CdTe
QDs on cells, researchers have also conducted preliminary investigations into
the mechanism of toxic effects (Whiteside et al. 2009). The CdTe QDs exposure is found to be associated
with ROS production, mitochondrial damage, protein and DNA damage, and
apoptosis (Wang et al. 2017). Comet tests
showed that CdTe QDs damaged cellular DNA in a dose-dependent manner (Zhang et
al. 2015) and it affected some
processes of mitochondrial biosynthesis (Li et al.
2014). Genetic research showed that the tail DNA content of the CdTe QDs
treatment was significantly higher than the control. The possible reason is the
released content of Cd2+ (Nguyen et al.
2015).
Although previous studies confirmed that Cd2 +
released from the core of CdTe QDs are transported into the cells causes
cytotoxicity (Wang et al. 2010). Fewer studies
have evaluated the toxicity of CdTe QDs in plants (Chen et al. 2014), especially in food crops such as
wheat, rice (Oryza sativa L.) and
corn (Zea mays L.). Therefore, this
study was designed to evaluate the effects of CdTe QDs on wheat seedlings and
mesophyll protoplasts. Moreover, the results of this study will provide a
foundation to further study the toxicity and biological safety of CdTe QDs in
plants.
Materials and Methods
Plant materials
The wheat variety Lin Y8198 donated by Shanxi Wheat Research Institute was
used as experimental material.
Preparation of CdTe QDs
The CdTe QDs were configured with distilled water (DW), M standard
solution (5 mmol/L CaCl2·2H2O, 0.5 mol/L mannitol, 0.5
mmol/L KH2PO4, 2 mmol/L MgSO4, 3 mmol/L
2-(N-Morpholino) ethanesulfonic acid 4-Morpholineethanesulfonic acid, MES, pH=
5.6), Phosphate Buffered Saline (PBS) (NaCl 137 mmol/L, KCl 2.7 mmol/L, Na2HPO4
4.3 mmol/L, KH2PO4 1.4 mmol/L, pH= 7.2), and PEM (50
mmol/L PIPES, 5 mmol/L EGTA, 5 mmol/L MgSO4, 0.225 mol/L Sorbitol,
pH= 6.9). The CdTe QDs had different stability and fluorescence intensity in
different solvents (Fig. 1a) and the CdTe QDs configured with PBS were more
effective. The PBS configured CDTe QDs were suitable for living environment of
leaf protoplasts and alkaline environment of the solvent. This made the CdTe
QDs more stable and the fluorescence intensity stronger. In order to
investigate the effect of different concentrations of CdTe QDs on protoplasts,
different concentrations of CdTe QDs were configured with PBS as a solvent (Fig.
1b) and then the Image J software was used to calculate the fluorescence
intensity of the same area in different treatment groups.
Six different concentrations of CdTe QDs solutions
configured with PBS solution as 0, 50, 100, 200, 500 and 1000 mg/L were used to
observe the fluorescence intensity in the gel imager system. Compared with control (CK) group, the fluorescence
intensity of the CdTe QDs in the concentrations 50–500 mg/L increased with
increasing concentration, but the fluorescence intensity of the Q5 treatment
group (1000 mg/L) did not increase further (Fig. 2).
Wheat cultivation
Wheat seeds with full and uniform grains without damage and mildew were
selected, washed with tap water 2 to 3 times and then sterilized with 1.5%
NaClO for 10 min. The disinfection solution was washed with distilled water,
and then was added an appropriate amount of distilled water. Thirty seeds were
cultured per Petri dish in a growth chamber at 25°C with 55%
relative humidity and were watered daily. From the day the
seeds germinated, six treatments were performed, each repeated three times. Briefly, seeds were soaked with either CdTe QDs (0, 50,
100, 200, 500 and 1000 mg/L) all day long. After 7 days of growth, the plant
height and root length were measured with a ruler. Eighty seedlings per replicate per treatment were randomly
selected for analysis.
Measurement of MDA and soluble
sugar concentrations
Trichloroacetic acid (TCA) method was used to determine MDA concentration.
Fresh tissues (1.0 g) were ground with SiO2 in 2 mL 10% TCA. After
centrifugation at 4000 rpm for 10 min, the supernatant was removed, 2 mL of
0.6% (w/v) thiobarbituric acid was added, and the mixture was incubated in a
100°C water bath for 15 min. After centrifugation at 4000
rpm for 15 min, the supernatant was measured at 532 and 450 nm, respectively.
The total sugar concentration was determined by anthrone colorimetry. Dry plant
tissues (50 mg) ware triturated with 4 mL of 80% ethanol. The supernatant was
collected after continuous stirring in a water bath at 80°C for 40 min.
Activated carbon (10 mg) was used to decolorize the solution for 30 min, after
which 5 mL anthrone was added and samples were incubated in a water bath at
100°C for 10 min. Samples were then cooled for 5 min before spectrophotometric
absorbance assessment at 625 nm. The concentration was determined using
standard curves.
Isolation and identification of
protoplasts
The 7-day-old leaves of wheat seedlings without being soaked with CdTe QDs
were taken. The wheat mesophyll protoplasts were prepared by enzymatic
hydrolysis, and the centrifuge tubes containing the prepared protoplasts were
numbered 0, 1, 2, 3, 4, 5; then 3 mL of CdTe QDs of different concentrations
were taken and added to the numbered centrifuge tube, finally the centrifuge
tube was covered and shake up and down gently to mix well, and incubated at
37°C in the dark overnight.
Leaf protoplast suspension (0.2 mL) was taken and
FDA staining solution (0.2 mL) was added (Cheng
and Belanger 2000). A small amount
of the mixed solution was taken on a glass slide and fluorescence microscope
was used to take photos of protoplasts under a 565 nm
fluorescence filter to observe and count live (yellow) protoplasts and dead
(red) protoplasts (Silva and Menendezyuffa 2006).
Fig. 1: Comparison
of fluorescence intensity of CdTe QDs in different solvents (a) and with
different concentrations in PBS (b). The red box indicates the area used
to calculate the fluorescence intensity
Here DW= Distilled water; PBS= Phosphate
buffered saline; M= M standard solution; PEM= PIPES, EGTA, MgSO; CK= 0 mg/L;
Q1= 50 mg/L; Q2= 100 mg/L; Q3= 200 mg/L, Q4= 500 mg/L; Q5= 1000 mg/L
Fig. 2: Comparison of fluorescence intensity of CdTe
quantum dots with different concentrations in PBS
Here CK= 0 mg/L; Q1= 50 mg/L; Q2= 100 mg/L; Q3= 200 mg/L, Q4= 500 mg/L; Q5= 1000 mg/L
Extraction of leaf protoplast
protein
The treated leaf protoplasts were centrifuged and the supernatant was
discarded. Aprotinin (50 μL) and cell extract (2 mL) were added to
each centrifuge tube and mixed thoroughly and an appropriate amount of quartz
sand was added to the mortar. The treated leaves in the centrifuge tube were
ground separately. After grinding, aliquot was transferred into a new
centrifuge tube and marked accordingly. Then the supernatant was centrifuged to
obtain the protein extraction solution, stored at-80°C for future use.
Determination of protein contents
The protein was quantified using the Bradford protein assay; bovine serum
albumin at different dilutions was measured using the standard curve method,
and the absorbance was measured colorimetrically at 595 nm, repeated 3 times,
and recorded; using spectrophotometry determine the absorbance of the sample
extract under different processing conditions formed at 595 nm, repeated 3
times, and recorded. The total protein content of leaf protoplasts was measured
according to the formula.
Analysis of leaf protoplast
proteins by SDS-PAGE
After cleaning, Vaseline was used to prepare the rubber sheet. After
checking for leakage with distilled water, it was wiped clean with filter
paper. The glue was made according to the method of separating the glue and
pour the glue. After pouring the glue, N-butanol was used to press the surface
immediately. After the gel is solidified, upper layer of N-butanol was poured
off. The concentrated gel was configured, poured to full. The
comb was inserted into the gel for gelation; the electrophoresis tank was
prepared, the electrode buffer was poured, the bottom rubber strip was remove, the
comb was pulled out, then the gel plate was inserted into the electrophoresis
tank, and air bubbles were eliminated with a syringe. Spot the sample and plug
in the power. Initially, the constant pressure of the concentrated gel is 80 v
and the constant pressure of the separation gel is 120 v. Stain for 3 h, then
decolorizes and observe.
Statistical analysis
Fig. 3: Effects of different concentration of CdTe QDs
on 7-day-old wheat seedlings
Here CK=
0 mg/L; Q1= 50 mg/L; Q2= 100 mg/L; Q3= 200 mg/L, Q4= 500 mg/L; Q5= 1000 mg/L
Fig. 4: Morphology of unpurified (upper) and purified
(lower) wheat leaf protoplasts without FAD staining (a) and purified
leaf protoplasts stained with FAD under 10x magnification (b)
Here CK= 0 mg/L; Q1= 50 mg/L; Q2= 100 mg/L; Q3= 200 mg/L, Q4= 500 mg/L; Q5= 1000 mg/L
Data results are expressed as means
± standard error (SE). Statistical significance of data were assessed using
one-way analysis of variance (ANOVA) tests using General Linear Model and Tukey
test was performed using the S.P.S.S. 21.0 and Sigma-plot 12.5 to compare the
treatments means.
Results
Effects of CdTe-QDs on wheat
seedlings growth
Compared with control the shoot and root length of wheat seedlings
gradually decreased with increasing CdTe QDs concentrations (Fig. 3). Shoot
length of wheat seedlings at 50 (Q1), 100 (Q2), 200 (Q3), 500 (Q4) and 1000
mg/L (Q5) concentration groups decreased by 5.0, 10.1, 20.1, 73.8 and 85.5%,
respectively compared with control (Fig. 3a). The root length at Q1 ~ Q5 treatment
groups was decreased by 13.2, 22.9, 41.1, 71.1 and 85.8%, respectively over
control (Fig. 3d). The soluble sugar contents in shoots and roots of wheat
seedlings were gradually decreased with the increasing concentrations of CdTe
QDs. Compared with control, the Q1 ~ Q5 treatment groups reduced soluble sugar
contents by 7.1, 13.7, 21.8, 77.9 and 88.5% in shoots and by 21.1, 30.1, 53.4,
79.3 and 93.1 in roots (Fig. 3c, f). Moreover, the contents of MDA increased with the
increase of CdTe QDs concentration (Fig. 3b, e). Compared with control group,
Q1 ~ Q5 treatment groups increased MDA contents in shoot by 40.4, 75.7, 111.8, 146.5 and 187.2% (Fig. 3b) while in
root MDA contents the increase was 40.8, 115.6, 229.6, 355.5 and 468.7,
respectively over control (Fig. 3e).
Determination of protoplast
viability
Before staining with FAD, the purified protoplasts were round and almost
free of impurities (Fig. 4a). The protoplasts in the control group showed
bright green fluorescence after FDA staining, indicating that their protoplasts
were more active. Compared with the control group, the number of protoplasts in
the leaves of the Q1 ~ Q5 treatment groups gradually decreased with the
increase of CdTe QDs concentrations, which may be due to membrane rupture and
cells death (Fig. 4b).
Fig. 5: Standard curve: bovine serum albumin content at
different dilutions (a) and total protoplast protein content of wheat
leave protoplasts in different treatments (b)
Here CK=
0 mg/L; Q1= 50 mg/L; Q2= 100 mg/L; Q3= 200 mg/L, Q4= 500 mg/L; Q5= 1000 mg/L
Fig. 6: SDS-PAGE electrophoresis profiles of total
protein in wheat leaf protoplasts treated with different concentrations of CdTe QDs. The red lines in the above figure are
automatically added by the Quantity One software which indicated the protein
bands
Here L =
Leaves of the CK group; CK= 0 mg/L; Q1= 50 mg/L; Q2= 100 mg/L; Q3= 200 mg/L, Q4= 500 mg/L; Q5= 1000 mg/L
Determination of
protein content
As shown in Fig. 5a, the total protein contents of protoplast of wheat
seedlings of each group were calculated according to the standard curve.
Compared with control, the total protein contents of protoplast of wheat
seedlings of the Q1 treatment group was significantly reduced and the total
protein contents in the Q2 group were significantly increased. From the Q2 ~ Q5
treatment groups, the total protein contents of
protoplast of wheat seedlings decreased significantly with increase of CdTe QDs
concentrations. The protein content of the Q3 treatment group and the control
did not change significantly, and the total protein contents of the Q4 and Q5
groups were both lower than the control group (Fig. 5b).
Analysis of leaf protoplast protein
by SDS-PAGE
In order to studying the relationship between the concentration of CdTe
QDs and the total protein content of wheat protoplasts, the effect of QDs
concentration on protein types was further explored. The results showed that
the Mw, CK, Q1, Q2, Q3, Q4, and Q5 treatment groups contained 5, 16, 17, 21,
23, 26 and 31 bands, respectively (Fig. 6), which means the protein types
increased with the increasing concentration of CdTe QDs. From Fig. 5, it could
be seen that these increased protein bands were mainly distributed between 97.4
kD and 66.2 kD.
Discussion
In this study, high-concentration CdTe QDs significantly
inhibited the growth of wheat roots and shoots. The impacts of NPs on crop
plants are unavoidable because plants cannot move but only acclimate to
environmental changes. More and more literature has
confirmed that CdTe QDs have a significant toxic effect on wheat growth and
development, and have been studied in terms of physiology and biochemistry (Su et al. 2010; Chen et al. 2014). When CdTe QDs were
absorbed by plants, they can reduce the amount of antioxidants in cells or
increase the production of reactive oxygen species (ROS) (Santos et al. 2012), increased ROS will inhibit wheat root length
(Marmiroli et al. 2015). It is well reported that
CdTe QDs can reduce the expression of intracellular proteins involved in auxin
transport (Marmiroli et al. 2016), thereby reducing auxin
synthesis, inhibiting cell elongation and shoot length.
Results unveiled that the exogenous CdTe QDs
could be absorbed and gathered in cell vacuoles in wheat and displayed
inhibitory effects on wheat seedlings growth. Since some acid enzymes were in
the vacuole which made CdTe QDs released Cd2+ in the vacuole;
thereby deepening the toxic effects on wheat (Hassan et al. 2016). At the concentration of Q4 (500 mg/L), shoot growth
of wheat plants were significantly inhibited (Fig. 3a). It might be due to the
CdTe QDs absorbed from wheat roots and entered to shoots through vascular
tissue; the CdTe QDs dissociated into more Cd2+ in the plant and
showed more toxicity (Wang et al. 2010). This needs to be taken
seriously, because the effects of CdTe QDs on plants may be twofold, namely the
effects of CdTe QD particles and the toxic effects of Cd2+ on
plants.
Externally added CdTe QD particles caused the decline and
death of wheat mesophyll protoplasts (Fig. 4b); which indicated that plant
cells without cell walls are more susceptible to the toxicity of CdTe QDs.
Since, the plant cells were directly treated with CdTe QDs in vitro, which avoided the degradation effects of plants on the
CdTe QDs during transportation from plant roots to shoots. Therefore, in vitro treatment of mesophyll
protoplasts with CdTe QDs can more accurately reflect the response of mesophyll
cells to CdTe QDs. This system has not been reported yet, but it is necessary
to directly reflect the impact of NPs on plant cells at the cellular level.
From the effect of CdTe QDs
concentration on the protein content of wheat leaf protoplasts (Fig. 5), low
concentration of CdTe QDs can inhibit protein synthesis, but within a certain
concentration range, QDs can promote protein synthesis. The CdTe QDs may
inhibit the activity of proteolytic enzymes and activate the expression of
wheat resistance genes at this concentration. When the
concentration of CdTe QDs continued to increase, its toxicity exceeded
the resistance of wheat itself, destroying wheat genes and some life-active
substances such as enzymes, leading to the death of wheat leaf protoplasts.
Significant changes have taken place in the protein
expression pattern in wheat mesophyll protoplasts treated with CdTe QDs. As the
concentration of treatments increased, more and more different types of
proteins were detected (Fig. 6). Based on previous studies conducted
by Marmiroli et al. (2015) combined with
the growth phenotype analysis of the seedlings, it is speculated that the
increased protein types should be related to oxidative stress response, auxin
synthesis and transport, and cell metabolism. CdTe QDs not only affect the
synthesis of intracellular proteins, but also cause cellular DNA damage and
inhibit DNA repair, but the mechanism of DNA damage is still insufficiently
explored. Therefore, future research directions should focus on the
interconnections between various mechanisms, studying the response mechanism of
crop plants to environmental CdTe QDs at the molecular level, and continue to
explore the potential mechanisms of NPs toxicity in plants.
Conclusion
Results
obtained revealed that CdTe QDs application inhibited wheat seedlings growth
and show toxicity to mesophyll protoplasts. The CdTe QDs application led to a
decrease in soluble sugar concentration along with simultaneous increase in MDA
contents in shoots and roots. Also, CdTe QDs changed the total protein pattern
of the mesophyll protoplasts. More in
vitro experiments are needed to study the effect of CdTe QDs on crop
plants.
Acknowledgments
The first author acknowledges the Natural Science Foundation of China
under Grant No. 31900251, China.
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