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ORIGINAL RESEARCH
published: 30 November 2016
doi: 10.3389/fpls.2016.01819
Edited by:
Jian-Guo Huang,
University of Chinese Academy
of Sciences, China
Reviewed by:
Anitha Kunhikrishnan,
National Institute of Agricultural
Science, South Korea
Lei Chen,
Hokkaido University, Japan
*Correspondence:
Jiasheng Wu
wujs@zafu.edu.cn
†These authors have contributed
equally to this work.
Specialty section:
This article was submitted to
Functional Plant Ecology,
a section of the journal
Frontiers in Plant Science
Received: 30 September 2016
Accepted: 18 November 2016
Published: 30 November 2016
Citation:
Shen J, Song L, Müller K, Hu Y,
Song Y, Yu W, Wang H and Wu J
(2016) Magnesium Alleviates Adverse
Effects of Lead on Growth,
Photosynthesis, and Ultrastructural
Alterations of Torreya grandis
Seedlings. Front. Plant Sci. 7:1819.
doi: 10.3389/fpls.2016.01819
Magnesium Alleviates Adverse
Effects of Lead on Growth,
Photosynthesis, and Ultrastructural
Alterations of Torreya grandis
Seedlings
Jie Shen1†, Lili Song1†, Karin Müller2, Yuanyuan Hu1, Yang Song1, Weiwu Yu1,
Hailong Wang3and Jiasheng Wu1*
1The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Zhejiang, China,
2New Zealand Institute for Plant and Food Research Limited, Ruakura Research Centre, Hamilton, New Zealand, 3Key
Laboratory of Soil Contamination Bioremediation of Zhejiang Province, Zhejiang A & F University, Zhejiang, China
Magnesium (Mg2+) has been shown to reduce the physiological and biochemical
stress in plants caused by heavy metals. To date our understanding of how Mg2+
ameliorates the adverse effects of heavy metals in plants is scarce. The potential effect
of Mg2+on lead (Pb2+) toxicity in plants has not yet been studied. This study was
designed to clarify the mechanism of Mg2+-induced alleviation of lead (Pb2+) toxicity.
Torreya grandis (T. grandis) seedlings were grown in substrate contaminated with 0,
700 and 1400 mg Pb2+per kg−1and with or without the addition of 1040 mg
kg−1Mg2+. Growth parameters, concentrations of Pb2+and Mg2+in the plants’
shoots and roots, photosynthetic pigment, gas exchange parameters, the maximum
quantum efficiency (Fv/Fm), root oxidative activity, ultrastructure of chloroplasts and
root growth were determined to analyze the effect of different Pb2+concentrations
on the seedlings as well as the potential ameliorating effect of Mg2+on the Pb2+
induced toxicity. All measurements were tested by a one-way ANOVA for the effects of
treatments. The growth of T. grandis seedlings cultivated in soils treated with 1400 mg
kg−1Pb2+was significantly reduced compared with that of plants cultivated in soils
treated with 0 or 700 mg kg−1Pb2+. The addition of 1040 mg kg−1Mg2+improved
the growth of the Pb2+-stressed seedlings, which was accompanied by increased
chlorophyll content, the net photosynthetic rate and Fv/Fm, and enhanced chloroplasts
development. In addition, the application of Mg2+induced plants to accumulate five
times higher concentrations of Pb2+in the roots and to absorb and translocate four
times higher concentrations of Mg2+to the shoots than those without Mg2+application.
Furthermore, Mg2+addition increased root growth and oxidative activity, and protected
the root ultrastructure. To the best of our knowledge, our study is the first report on
the mechanism of Mg2+-induced alleviation of Pb2+toxicity. The generated results may
have important implications for understanding the physiological interactions between
heavy metals and plants, and for successful management of T. grandis plantations
grown on soils contaminated with Pb2+.
Keywords: Torreya grandis, lead toxicity, magnesium, heavy metal phytotoxicity, phytoremediation
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
INTRODUCTION
Heavy metal pollution has become a global environmental
threat (Krabbenhoft and Sunderland, 2013;Chen et al., 2015).
Among metal contaminants, lead (Pb2+) is a major concern
because of its extensive distribution in the environment and
the substantial environmental and human health problems it
can cause. Major sources of Pb2+pollution include mining and
smelting activities as well as Pb2+-containing paints, gasoline,
explosives, sewage sludge and fertilizers (Sharma and Dubey,
2005;Buekers et al., 2009). When plants are exposed to Pb2+,
even at micromolar levels, adverse effects can occur on plant
growth (Hadi et al., 2010), root elongation (Liu et al., 2000),
seed germination (Lamhamdi et al., 2011), seedling development
(Kaur et al., 2015), chlorophyll production (Rashid and Popovic,
1990), chloroplast lamellar organization (Hu et al., 2007), and
antioxidant enzymes system (Gupta et al., 2009, 2010). However,
the toxicological response to Pb2+varies depending on the plant
species and tissues analyzed (Pourrut et al., 2011). Huang and
Cunningham (1996) showed significant differences in the uptake
and translocation of Pb2+among Triticum aestivum,Thlaspi
rotundifolium, and Thlaspi caerulescens. Mimosa caesalpiniaefolia
was more tolerant to high Pb2+concentrations in soil than
Erythrina speciosa and Schizolobium parahyba (de Souza et al.,
2012). Huang and Cunningham (1996) found that some dicot
species can accumulate significantly higher concentrations of
Pb2+in the roots than some monocot species.
Heavy metals could be taken up by cation transporters
such as members of the ZIP (Zn-regulated transporter/Fe-
regulated transporter-like protein) and natural resistance-
associated macrophage protein families (Eide et al., 1996;
Korshunova et al., 1999;Guerinot, 2000;Thomine et al., 2000).
These bivalent cation transporters are also important uptake
systems for essential elements. Therefore, nutrients such as
magnesium (Mg2+) are considered to contribute to plants’
tolerance to heavy metal exposure owing to their chemical
similarity as well as sharing common transporters with heavy
metals (Guerinot, 2000;Pittman, 2005). Over the last decade,
studies have revealed the ability of Mg2+to mitigate heavy
metal toxicity caused by aluminum (Al3+) and cadmium (Cd2+)
(Kashem and Kawai, 2007;Bose et al., 2011). Kashem and Kawai
(2007) reported that adding Mg2+to nutrient solutions reduced
Cd concentrations in plants and enhanced the growth of plants
suffering from Cd toxicity. Hermans et al. (2011) indicated that
the protective effect of Mg2+against Cd toxicity may be at
least partly attributed to the protection of the photosynthetic
apparatus. However, only few studies have investigated the effect
of Mg2+on Pb2+toxicity. Therefore, we explored the effect of
Mg2+on Pb2+toxicity and the possible mechanism of Mg2+-
induced alleviation of Pb2+toxicity using a local species Torreya
grandis (T. grandis).
Torreya grandis is a gymnosperm tree species belonging
to the Taxaceae family, mainly grown in eastern China with
significant economic value because of its valuable drupe-like
fruits with medicinal effects from its anthelmintic, antitussive,
carminative, laxative, antifungal, antibacterial, and antitumor
properties (Huang et al., 2001). As the demand for the fruit
increased, the acreage of T. grandis has rapidly expanded, and
the management intensity has increased with higher inputs of
fertilizers and pesticides, such as lead arsenate. In addition, soils
near highways, which are usually polluted by exhaust emissions,
have also been used for growing T. grandis. Therefore, T. grandis
is likely to face with Pb2+stress. It remains unclear whether
T. grandis can be tolerant to high level of Pb2+stress. Thus, in
this study, we performed a pot experiment to test the following
hypotheses: (1) High level Pb2+stress inhibits the growth of
T. grandis seedlings; (2) Mg2+can effectively ameliorate the
negative effects of lead stress on the growth of T. grandis
seedlings. The information obtained in this study is valuable for
the propagation and cultivation of T. grandis under Pb2+stress
conditions.
MATERIALS AND METHODS
Plant Material and Growth Conditions
During All Experiments
All experiments were conducted on the Zhejiang A & F
University campus, Lin’an City, Zhejiang province (330◦230N,
119◦720E), China. Two-year-old uniform and healthy T. grandis
seedlings (mean ground diameter 5 ±0.5 mm and seedling
height 35 ±2 cm) were transplanted into plastic pots (16.5 cm
inner diameter, 18 cm height, with holes in the bottom, one
seedling per pot) filled with 2.5 kg of sterilized substrate mixture
of perlite and quartz sand (1:1, v/v). All pots were irrigated
with 200 ml of Hoagland’s nutrient solution (3.0 mM KNO3,
2.5 mM Ca(NO3)2, 1.0 mM MgSO4.7H2O, 1.2 µM FeEDTA,
4.0 µM MnCl2, 22.0 µM H3BO3, 0.4 µM ZnSO4, 0.05 µM
Na2MoO4, 1.6 µM CuSO4, and 1.0 µM KH2PO4) every three
days and maintained at 75% field capacity of the growth substrate
to keep the plants well watered. Day and night temperature was
kept between 18.0 and 32.0◦C and the relative humidity ranged
between 50 and 80%. The light intensity in the greenhouse was
monitored daily with an external quantum sensor attached to
LI-6400 (Li-COR, Lincoln, NE, USA) and kept within the range
of 500–800 µmol m−2s−1photosynthetically active radiation
(PAR) above the plants.
First Experiment: Exposure of Seedlings
to Pb2+
The first experiment to determine the lead concentration that
adversely affected T. grandis seedlings was carried out between
1 May and 31 June 2014. One month after transplantation of
the seedlings, the height and ground diameter of each seedling
were measured as reference values. A completely randomized
design with three replications per treatment and five plants per
replication was chosen. Total of 45 seedlings were exposed to
Pb2+supplied as Pb (NO3)2at concentrations of 0 (control), 700
and 1400 mg Pb2+kg−1growth substrate. These concentrations
were selected based on a report by Huang et al. (2006), who found
that Pb2+-concentrations in soil exceeding 1000 mg kg−1affected
the growth of Pinus rigida. After 60 days, the height and ground
diameters of the seedlings were recorded.
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
Second Experiment: Exposure of
Seedlings to Pb2+and Mg2+
The above experiment showed that 1400 mg kg−1Pb2+caused
Pb2+toxicity in T. grandis seedlings. Hence, further studies
on the effect of Mg2+on Pb2+toxicity were restricted to the
control and the 1400 mg kg−1Pb2+treatments. This second
experiment was conducted during 1 May to 31 June 2015.
Magnesium (with the irrigation water) was supplied as MgCl2at
1040 mg kg−1. In total, the following four treatments (total of 60
seedlings) were established: T1 (control without Mg2+addition);
T2 (control with 1040 mg kg−1Mg2+); T3 (1400 mg kg−1Pb2+
without 1040 mg kg−1Mg2+) and T4 (1400 mg kg−1Pb2+with
1040 mg kg−1Mg2+). A completely randomized design with
three replications per treatment and five plants per replication
was set up.
Plant Harvest
After 60 days of the second experiment, the third and fourth
leaves from the plant top, which had been completely developed
when Pb2+treatment started, were collected from all plants,
cleaned with tissue paper to remove any surface contamination,
immediately frozen in liquid nitrogen and stored at −70◦C. Plant
growth, concentrations of Pb2+and Mg2+in shoots and roots,
chlorophyll concentration, root oxidative activity, photosynthesis
and ultrastructure of chloroplasts and roots were determined for
all samples.
Growth and Morphology Analysis
After 60 days of the two experiments, all seedlings were harvested
and separated into shoots and roots for growth and morphology
analyses. Shoot biomass and total biomass were measured after
drying of the shoots and roots at 80◦C for 4 days. Seedling height
was defined as the height of the plant from the top of the growth
medium to the tip of the uppermost shoot.
Determination of Pb2+and Mg2+
Concentrations in Plant Shoots and
Roots
To determine the concentrations of Pb2+and Mg2+in the
shoots and roots, the dried plant materials were grounded with
a stainless steel mill and passed through a 0.25 mm sieve for
analysis of Pb2+and Mg2+. An aliquot of 0.1 g of the dried
plant materials of each treatment was digested with HNO3–
HClO4(4:1, v/v), and the digest was diluted with deionized water
(DW) to a final volume of 50 mL. Concentrations of Pb2+and
Mg2+in the filtrates were analyzed by flame atomic absorption
spectroscopy (Perkin Elmer Analyser 300, England). The Pb2+
and Mg2+concentrations in the entire plant were calculated
following Zhang et al. (2011) and expressed in mg kg−1DW and
mg g−1DW, respectively.
Pigment Concentration in Leaves
Approximately 0.1 g of finely cut and well-mixed fresh plant
sample, which was taken from healthy and fully developed
leaves at the same position in each treatment, was repeatedly
extracted with 8 mL of 95% ethanol (100%, Sinopharm Chemical
Reagent Company, Shanghai, China). Pigment was extracted
at 4◦C for 24 h in darkness and shaken three or four
times until the leaf samples blanched (no green color in the
leaf tissue). The absorbance was measured with a Shimadzu
UV-2550 spectrophotometer (Kyoto, Japan) at 664, 649, and
470 nm after centrifugation of the mixture. The chlorophyll a
(Chla), chlorophyll b (Chlb), total chlorophyll (Chl(a+b)), and
carotenoid (Car) contents were calculated using the following
formulas (Lichtenthaler, 1987). Results are expressed in mg g−1
fresh weight (FW).
CagL−1=13.36A664 −5.19A649 (1)
CbgL−1=27.43A649 −5.10A664 (2)
Ca+bgL−1=5.24A664 −22.24A649 (3)
Cx +cgL−1=1000A470 −2.13Ca−97.64Cb
209 (4)
Where, Ca, Cb, Ca+b,and Cx+cwere the concentrations of Chla,
Chlb, Chl (a+b), and Car, respectively. A664,A649, and A470 were
the absorbances of pigment extract solution at 664, 649, and
470 nm wavelengths, respectively.
Photosynthetic Parameters and the
Maximum Quantum Efficiency of Psii
Photochemistry (Fv/Fm)
The youngest healthy and fully developed leaves randomly
selected from the first branch were chosen for gas exchange
measurements. Field gas exchange measurements were
conducted with a LI-6400 portable photosynthesis system
(Li-COR, Inc. Lincoln, NE, USA) with a standard leaf chamber
equipped with a 6400-02B LED light source (LI-6400, Li-COR,
Lincoln, NE, USA). Measurements were conducted at an air
concentration of 21% O2, 400 µmol mol−1carbon dioxide
(CO2), 800 µmol m−2s−1PAR, 50% relative humidity and a
temperature of 20 ±2◦C. The gas exchange measurements were
performed on sunny days from 8:30 to 11:30 am.
Chlorophyll fluorescence (Fv/Fm) was determined in the
morning (08:00 am–11:00 am) on the healthy and fully developed
leaves with a pulse modulation fluorometer (PAM-2500, Walz,
Effeltrich, Germany). After 30 min of adaptation to the dark
(Tang et al., 2015), the minimum fluorescence (Fo) was
determined in a measuring light of approximately 0.5 µmol
photon m−2s−1, and the maximum fluorescence (Fm) was
determined under a 0.8-s saturating flash of 10,000 µmol photon
m−2s−1. The Fv/Fm value was calculated as (Fm−Fo)/Fm
(Maxwell and Johnson, 2000).
Determination of Root Morphological
Traits
After gently washing the roots with deionized water, the total
length, volume, and surface area of the root samples were
determined by image analysis. The roots were photographed
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
FIGURE 1 | Effect of Pb2+addition to the growth medium on T. grandis seedlings. T1, control, T2, 700 mg kg−1Pb2+, T3, 1400 mg kg−1Pb2+.
and then the images were analyzed with the root image analysis
system software WinRHIZO1.
Root Oxidative Activity
The root oxidative activity was measured according to the
method of Mishra (2012) with a slight modification. About
3 g fresh root were immersed in 300 ml of 20 ppm á-
naphthylamine solution for 10 minutes to exclude any initial
rapid absorption of á-naphthylamine by roots. The intact
roots were then transferred to another vial with 300 ml of
20 ppm of á-naphthylamine solution and incubated for four
hours at 25 ±1◦C. Then, 2 ml of the incubated solution
were mixed with 10 ml of 0.1% sulfanilic acid (in 3%
acetic acid) and 2 ml of 50 ppm NaNO2, and diluted to
25 ml using distilled water. The absorbance of the colored
solution was determined at 530 nm using spectrophotometry.
Root oxidative activity was expressed as µg á-naphthylamine
h−1g−1FW.
Ultrastructure of Chloroplast and Root
To examine the chloroplast ultrastructure of mesophyll cells,
fresh leaves were immediately fixed in 2.5% (v/v) glutaraldehyde
(0.1 mol L−1phosphate buffer, pH 7.2) for at least 48 h after
detachment from the plants. The samples were immersed in
1% (v/v) osmium acid for post-fixation, embedded in resin, and
ultrathin sectioned for transmission electron microscopy (H7650,
Hitachi, Tokyo, Japan).
Data Analysis
Because the Pb2+and Mg2+treatments were not applied
independently to each seedling, the plants in each treatment
combination were not true replicates (Hurlbert, 1984;Maherali
1www.regentinstruments.com
and DeLucia, 2000). Therefore, averages of subsamples (five
seedlings per replicate) were used for the analysis of variance.
All measurements were tested by a one-way ANOVA for
the effects of treatments (combinations of Pb2+and Mg2+
concentration). The effects were considered significant at
P<0.05. Before ANOVA, data were checked for normality
and homogeneity of variances, and log-transformed to correct
deviations from these assumptions when needed. Significant
differences among treatment means were analyzed using Tukey’s
multiple comparison post hoc tests. The used statistical software
package was SPSS 16 for Windows (SPSS Inc., Chicago, IL, USA).
RESULTS
Effect of Lead on Plant Growth and
Development
Plants grown for 60 days at 0, 700, and 1400 mg Pb2+per kg−1
soil could be visually differentiated. Plants grown at 700 mg
kg−1were larger than those of other treatments (Figure 1).
Compared with the control, soil contamination of 700 mg Pb2+
kg−1significantly increased the growth of T. grandis seedlings
(P=0.0001, Figure 2). However, the 1400 mg kg−1Pb2+
treatment inhibited plant growth and ground diameter by 60.5%
(P=0.0001) and 83.0% (P=0.0001), respectively (Figure 2).
Effect of Mg2+on Dry Biomass, Plant
Growth, and Morphological Traits of
Roots Under Lead Toxicity
Compared with the control seedlings, exposure of plants to
1400 mg kg−1Pb2+in a growth medium for 60 days
significantly decreased the dry mass of shoots and roots by
19.6% (P=0.0002) and 24.1% (P=0.0038), respectively
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
FIGURE 2 | Effect of Pb2+addition to growth medium on the seedling height (A) and ground diameter (B) of T. grandis seedlings. T1, control, T2, 700 mg
kg−1Pb2+, T3, 1400 mg kg−1Pb2+. Data points and error bars represent mean ±standard deviation (n=3). Different lower case letters above the columns
indicate significant (P<0.05) difference between treatments.
(Table 1). The Mg2+-treated plants had significantly higher
shoot (P=0.0172) and root (P=0.0118) dry mass than
plants under Pb2+toxicity without Mg2+application (Table 1).
However, Mg2+had no significant effect on the dry mass
of shoots (P=0.5937) and roots (P=0.9235) of the
non-Pb2+-stressed plants. The leaf area (P=0.0001) and
seedling height (P=0.0001) of plants under Pb2+toxicity
were significantly lower than those of the control plants.
Application of Mg2+increased the leaf area (P=0.0004) and
seedling height (P=0.0001) of plants under Pb2+toxicity
(P<0.05, Table 1). However, there were no significant
differences in leaf area (P=0.4141) and seedling height
(P=0.1411) in the non-lead-stressed plants treated with or
without Mg2+.
Total length, surface area and volume of plant roots under
Pb2+toxicity decreased significantly by 26.9% (P=0.0004),
28.8% (P=0.0001), and 33.5% (P=0.0001), respectively,
compared with the control plants (Table 1). Treatment
of Pb2+-stressed plants with Mg2+significantly increased
total length, surface area and volume of roots by 27.7%
(P=0.0029), 24.3% (P=0.0113), and 24.0% (P=0.0001),
respectively, compared with plants treated only with Pb2+for
60 days.
Effect of Mg2+on Photosynthetic
Pigments and Gas Exchange Parameters
of Plants Under Lead Toxicity
Variations in the levels of photosynthetic pigments, including
chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids
(Car), were evaluated in T. grandis seedlings under lead
toxicity (Table 2). The Chla (P=0.0001) concentrations, Chlb
(P=0.0001) concentrations, Car (P=0.0004) concentrations
and Chla/Chlb (P=0.0003) ratios were lower but the
Car/Chl(a+b) (P=0.0003) ratios were higher in the Pb2+-
treated plants than in the control plants. Application of Mg2+
resulted in higher Chla (P=0.0001), Chlb (P=0.0001),
and Car (P=0.0019) concentrations, and also increased the
Chla/b (P=0.0012) ratios but lowered the Car/Chl(a+b)
TABLE 1 | Effects of Mg2+on the dry biomass of shoots and roots, leaf area (LA), seedling height, and root morphological traits of T. grandis seedlings
grown under Pb2+toxicity.
Treatment Shoot
Biomass (g)
Root
Biomass (g)
LA (cm2) Seedling
height (cm)
Total root
length (cm)
Root surface
area (cm2)
Root volume
(cm3)
T1 26.0 ±1.20a11.6 ±0.64a0.7 ±0.04ab 14.3 ±0.17a1614.6 ±70.54a865.2 ±7.42a38.8 ±0.41a
T2 26.7 ±0.35a11.9 ±0.21a0.8 ±0.02a14.8 ±0.15a1682.1 ±7.78a919.7 ±5.45a41.9 ±0.39a
T3 20.9 ±0.35c8.8 ±0.49b0.5 ±0.02c7.3 ±0.10c1179.7 ±128.0b616.4 ±8.48c25.8 ±2.34c
T4 23.4 ±0.78b11.1 ±1.48a0.7 ±0.01b10.8 ±0.15b1506.8 ±23.74a766.8 ±14.90b32.0 ±2.50b
Data points and error bars represent mean ±standard deviation of three replicates. Different letters indicate significant differences (P <0.05); n =3. The same letter
in the same column denotes no significant difference among treatments. Treatments: T1, control; T2, control +1040 mg kg−1Mg2+; T3, 1400 mg kg−1Pb2+; T4,
1400 mg kg−1Pb2++1040 mg kg−1Mg2+.
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
TABLE 2 | Effects of Mg2+on chlorophyll a (Chla), chlorophyll b (Chlb), carotenoids (Car), Chl(a+b), chlorophyll a:b ratio (Chla/Chlb), and Car/ Chl(a+b) of
T. grandis seedling leaves under Pb2+toxicity.
Treatment Chla (mg/g) Chlb (mg/g) Car (mg/g) Chl (a+b) (mg/g) Chla/Chlb Car/Chl(a+b)
T1 1.0 ±0.01a0.5 ±0.01a0.3 ±0.01a1.5 ±0.01a2.1 ±0.02a0.2 ±0.01b
T2 1.0 ±0.03a0.5 ±0.02a0.3 ±0.01a1.5 ±0.04a2.1 ±0.07a0.2 ±0.02b
T3 0.5 ±0.02c0.3 ±0.01b0.2 ±0.02b0.8 ±0.02c1.8 ±0.02c0.3 ±0.03a
T4 0.9 ±0.04b0.5 ±0.02a0.3 ±0.02a1.4 ±0.03b2.0 ±0.04b0.2 ±0.01b
Data points and error bars represent mean ±standard deviation of three replicates. Different letters indicate significant differences (P <0.05); n =3. The same letter
in the same column denotes no significant difference among treatments. Treatments: T1, control; T2, control +1040 mg kg−1Mg2+; T3, 1400 mg kg−1Pb2+; T4,
1400 mg kg−1Pb2++1040 mg kg−1Mg2+.
FIGURE 3 | The net photosynthetic rate (Pn) (A), stomatal conductance (Gs) (B), internal carbon dioxide concentration (Ci) (C), and transpiration rate (Tr) (D) of
T. grandis seedlings grown in media amended with various amounts of Pb2+and Mg2+. Treatments: T1, control; T2, control +1040 mg kg−1Mg2+; T3, 1400 mg
kg−1Pb2+; T4, 1400 mg kg−1Pb2++1040 mg kg−1Mg2+. Error bars are standard deviation (n=3). Different lower case letters above the columns indicate
significant (P<0.05) difference between treatments.
(P=0.0005) ratios in the leaves of plants exposed to
1400 mg kg−1Pb2+(Table 2). However, significant difference
in chlorophyll and carotenoid concentrations between seedlings
treated with and without Mg2+under no Pb2+toxicity was not
found.
Compared with the control, Pb2+toxicity significantly
decreased the photosynthetic rate (Pn), stomatal conductance
(Gs) and transpiration (Tr) by 54.6% (P=0.0001), 39.8%
(P=0.0001), and 58.4% (P=0.0001), respectively, while it
increased intercellular CO2(Ci) by 49.1% (P=0.0001) (Figure 3;
P<0.05). In leaves of plants under Pb2+toxicity, Mg2+
treatment significantly increased the levels of Pn, Gs, and Tr by
91.8% (P=0.0001), 21.0% (P=0.0001), and 86.4% (P=0.0001),
respectively, whereas it decreased Ci levels by 21.4% (P=0.0001)
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
compared with non-Mg2+-treated plants under Pb2+toxicity
(Figure 3).
Effect of Mg2+on Chlorophyll
Fluorescence and Oxidative Activity of
Roots in Plants Under Lead Toxicity
The Fv/Fm value was significantly decreased by 21.6%
(P=0.0001) in plants under Pb2+toxicity compared with
the control (Figure 4B). Application of Mg2+significantly
increased the Fv/Fm value by 23.7% (P=0.0001)in leaves of
seedlings exposed to 1400 mg kg−1Pb2+(Figure 4A).
The root oxidative activity in plants under Pb2+toxicity
decreased compared with the control (P=0.0001, Figure 4B).
Application of Mg2+to Pb2+-stressed plants significantly
increased root oxidative activity by 102.9% (P=0.0001)
compared with plants only treated with Pb2+(Figure 4A).
However, significant difference in oxidative activity between
plants treated with and without Mg under no Pb2+toxicity was
not found (P=0.4361, Figure 4B).
Effect of Mg2+on Pb2+and Mg2+
Accumulation in Plant Tissues Under
Lead Toxicity
Under Pb2+toxicity, Pb2+contents in the roots were three times
higher than in the shoots, indicating that the roots accumulated
the majority of the absorbed Pb2+. After application of Mg2+,
the Pb2+concentrations in the roots were higher than in roots of
the Pb2+-stressed plants, whereas the Pb2+uptake in the above-
ground parts was lower compared to the plants without Mg2+
application. Interestingly, the distribution of Mg2+in roots and
shoots of the T. grandis seedlings differed significantly. The Mg2+
concentration in the shoots of the control plants was higher than
in the roots. However, Pb2+toxicity had no significant effect
on the distribution of Mg2+between roots (P=0.6412) and
shoots (P=0.8785) compared with the control. Application of
Mg2+significantly increased Mg2+accumulation in the shoots
(P=0.0001) and roots (P=0.0002), and Mg2+concentration
was four times higher in the shoots than in the roots (Table 3).
Effect of Mg2+on Ultrastructural
Modifications of Leaves and Roots in
Plants Under Lead Toxicity
Application of Mg2+caused significant differences in the
ultrastructure of the chloroplasts of the T. grandis seedlings
grown under Pb2+toxicity (Figure 5). Elliptical-shaped
chloroplasts with thylakoids were found in the control plants.
However, the integrity of the ultrastructure was severely affected
by Pb2+toxicity. Chloroplasts were swollen and had irregularly
shaped grana, decreased lamellae, and increased osmiophilic
granule numbers, and the thylakoid membrane system in plants
was in disorder. Interestingly, application of Mg2+promoted the
development of chloroplasts, grana and stroma lamellae as well
as reduced the osmiophilic granule numbers.
Lead toxicity had a marked influence on the ultrastructure of
the seedlings’ roots (Figure 6). Compared with the control, the
root cell structure under lead toxicity was completely destroyed.
The nucleus was almost invisible and the mitochondria appeared
as hollow bubbles. Application of Mg2+protected the integrity
of the root cells as evidenced by visible nuclei, slightly condensed
chromatin and irregularly swollen mitochondria with fractured
and fuzzy cristae.
DISCUSSION
The growth of T. grandis seedlings was highest at 700 mg kg−1
Pb2+in soil, whereas the lowest growth of plants was found at
1400 mg kg−1Pb2+(Figures 1 and 2), indicating that lead stress
toxicity in T. grandis seedlings did not occur at 700 mg kg−1
Pb2+in soil. Meanwhile, visible toxic symptoms, such as old
leaves yellowing and chlorosis, were observed in plants exposed to
1400 mg kg−1Pb2+(Figure 1). The treatment with 1400 mg kg−1
Pb2+significantly decreased plant growth and the development
of the T. grandis seedlings, as indicated by the decreased shoot
dry mass, root dry mass, seedling height and leaf area (Table 1).
Similarly, Hadi et al. (2010) found that 500 mg kg−1Pb2+in soil
did not affect the germination rate of maize (Zea mays) seeds and
that the young maize seedlings did not exhibit any visible toxic
symptoms.
Chlorophyll fluorescence is the focus in studies of
photosynthetic regulation and plant responses to the
environment due to its sensitivity, convenience and non-
destructive characteristics (Dai et al., 2009). Generally, plants
subjected to heavy metal stress typically have lower Fv/Fm
values than non-stressed plants, which is associated with
photoinhibition of PSII (Krause and Weis, 1991;Wu et al., 2014).
In the present study, the Fv/Fm ratio was significantly reduced
in the plants treated with 1400 mg kg−1Pb2+(Figure 4A).
Indeed, this result was consistent with the gas exchange results,
in which lead toxicity decreased Pn, Tr and Gs compared
with the control (Figure 3), indicating photoinhibition of the
photosynthetic capacity in T. grandis seedlings under lead
stress conditions. A similar result was also described by Rashid
and Popovic (1990) for spinach leaves treated with 2 mM
Pb2+. Interestingly, in this study the Ci value was higher in
Pb2+-treated plants than in the control plants, indicating that
the reduction of photosynthesis under lead stress conditions
primarily resulted from non-stomatal limitations. Additionally,
photoinhibition and reduction of the photosynthetic capacity
under lead stress conditions were manifested by changes of
leaf chlorophyll contents. This was explained by its important
role in photosynthesis and plant growth. In the present study,
we found a strong reduction in the levels of Chla, Chlb, total
Chl content and carotenoids in plants treated with 1400 mg
kg−1Pb2+(Table 2), which was consistent with the results of
de Souza et al. (2012), who reported that Pb2+exposure led
to a reduction of Chla and Chlb contents in leaves as well as a
reduction of Car levels. Application of Mg2+to Pb2+-stressed
plants improved plant growth, which was accompanied with
increased chlorophyll contents, Pn levels and Fv/Fm ratios
(Tables 1 and 2;Figures 3 and 4A). Thus, the positive and
beneficial effects of Mg2+on the growth of T. grandis seedlings
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
FIGURE 4 | The maximum quantum efficiency (Fv/Fm) (A) and root oxidative activity (B) of T. grandis seedlings grown in media amended with various amounts
of Pb2+and Mg2+. Treatments: T1, control; T2, control +1040 mg kg−1Mg2+; T3, 1400 mg kg−1Pb2+; T4, 1400 mg kg−1Pb2++1040 mg kg−1Mg2+. Error
bars are standard deviation (n=3). Different lower case letters above the columns indicate significant (P<0.05) difference between treatments.
might be associated with improving the photosynthetic capacity
and alleviating photoinhibition. A similar result was reported by
Hermans et al. (2011), who indicated that the protective effect
of Mg2+against Cd toxicity may be at least partly attributed to
the protection of the photosynthetic apparatus. It is well known
that photoinhibition primarily results from overproduction
of reactive oxygen species (ROS) through the photosynthetic
electron transport chain under stress circumstances (Critchley,
1981). However, it needs to be further elucidated if Mg2+
protects the photosynthetic membrane from photo-oxidation by
effectively scavenging ROS under lead stress conditions.
The influence of heavy metal on cellular organization is
important for understanding physiological alterations under
stress conditions (Souza et al., 2005). In the present study,
chloroplasts were highly susceptible to stress induced by high
lead conditions, as indicated by decreased lamellae, increased
numbers of osmiophilic granules and disrupted thylakoid
membranes (Figure 5). Damage to chloroplasts and thylakoid
membranes in plants treated with heavy metals has been reported
TABLE 3 | Effects of Mg2+on concentrations of Pb2+and Mg2+in shoots
and roots of T. grandis seedlings grown under Pb2+toxicity.
Treatment Pb2+content
(mg kg−1) Root
Shoot Mg2+content
(mg g−1) Root
Shoot
T1 n.d. n.d. 0.39 ±0.03b0.74 ±0.03b
T2 n.d. n.d. 0.73 ±0.08a3.27 ±0.05a
T3 689.1 ±30.5b231.4 ±12.3a0.44 ±0.05b0.79 ±0.02b
T4 876.1 ±20.2a156.2 ±3.6b0.76 ±0.01a3.37 ±0.2a
Data points and error bars represent mean ±standard deviation of three replicates.
Different letters indicate significant differences (P <0.05); n =3. The same letter in
the same column denotes no significant difference among treatments, n.d. =not
determined. Treatments: T1, control; T2, control +1040 mg kg−1Mg2+; T3,
1400 mg kg−1Pb2+; T4, 1400 mg kg−1Pb2++1040 mg kg−1Mg2+.
by Wu et al. (2014). We found that Mg2+ameliorated the
chloroplast ultrastructural disorders caused by lead (Figure 5D),
which might explain the improved photosynthesis of the Mg-
treated plants. Meanwhile, as also suggested by our study,
the main processes underlying the improvement in plant
photosynthesis induced by Mg2+treatment are the enhanced
light-use-efficiency and the protection of the chloroplast
structures.
Many scientists have reported that Mg2+supplementation
enhances the tolerance to toxic metals by reducing the uptake
and translocation of metals, including Cd2+and Al3+(Kashem
and Kawai, 2007;Bose et al., 2011). We found that the
accumulation of lead was greater in the roots than in the shoots
of T. grandis seedlings (Table 3), indicating that the plants
translocated lower concentrations of metals into the shoots (Yang
et al., 1998). Higher Pb2+concentrations in roots than shoots
were observed in the Mg2+-alleviated plants, whereas the shoot
Mg2+concentrations were four-fold higher than the root Mg2+
concentrations in the Mg2+-alleviated plants (Table 3). These
results suggest that Mg2+application through the watering
solution helped decrease the Pb2+accumulation in the shoots.
Similar findings have been reported by Kashem and Kawai (2007),
who found that magnesium-alleviated plants showed decreased
shoot Cd2+concentration in Japanese mustard spinach (Brassica
rapa L. var. pervirdis).
Furthermore, lead uptake significantly reduced total root
length, surface area and volume compared with the control
plants (Table 1). However, the application of Mg2+increased
the indices of root morphological traits of T. grandis seedlings
under lead toxicity. Root oxidative activity implies the degree
of root development and metabolic status (Liu et al., 2008). In
the present study, lower root oxidative activity was found in
T. grandis seedlings under Pb2+toxicity than in the non-Pb2+-
treated control plants, whereas higher root oxidative activity was
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
FIGURE 5 | The transmission electron micrographs of chloroplasts in T. grandis seedlings grown in media amended with various amounts of Pb2+
and Mg2+. (A) Control; (B) control +1040 mg kg−1Mg2+;(C) 1400 mg kg−1Pb2+;(D) 1400 mg kg−1Pb2++1040 mg kg−1Mg2+.
FIGURE 6 | The transmission electron micrographs of root of T. grandis seedlings grown in media amended with various amounts of Pb2+and Mg2+.
(A) Control; (B) control +1040 mg kg−1Mg2+;(C) 1400 mg kg−1Pb2+;(D) 1400 mg kg−1Pb2++1040 mg kg−1Mg2+.
observed in the Mg2+-alleviated plants than in the Pb2+-toxic
plants (Figure 4B). These findings indicate that additional Mg2+
might increase the absorptive area of roots and, hence, increase
the uptake of water and nutrients to improve plant growth
(Glinka, 1980). This finding was consistent with the observed
ultrastructure of the T. grandis seedlings roots. The Mg2+
application protected the integrity of the root cells, resulting in
a visible nucleus, slightly condensed chromatin and irregularly
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Shen et al. Mg2+Alleviates Lead Toxicity in T. grandis Seedlings
swollen mitochondria with fractured and fuzzy cristae (Figure 6).
Thus, Mg2+application is an effective method to alleviate Pb2+
toxicity in T. grandis seedlings by improving root growth and root
oxidative activity and protecting root ultrastructure.
CONCLUSION
Torreya grandis seedlings exposed to 1400 mg kg−1Pb2+
exhibited stress toxicity as indicated by reduced shoot growth.
Mg2+addition under Pb2+stress conditions might have
beneficial effects on the growth of T. grandis seedlings, as
evidenced by increased shoot dry biomass, root dry biomass,
chlorophyll contents, and photosynthesis as well as improved
chloroplast ultrastructure. Moreover, additional Mg2+in the
solution containing Pb2+decreased the Pb2+concentration
in the shoots and increased the Mg2+concentration in the
shoots. Furthermore, we showed that the positive effects of Mg2+
on the growth of T. grandis were triggered by protecting the
morphology, activity and ultrastructure of the roots. To our
knowledge, this study is the first study to show Mg2+-induced
alleviation of lead toxicity in T. grandis seedlings and is of great
importance for the cultivation of T. grandis seedlings in China,
where soils are often contaminated with lead.
AUTHOR CONTRIBUTIONS
All authors listed, have made substantial, direct and intellectual
contribution to the work, and approved it for publication.
ACKNOWLEDGMENT
This work was financially supported by the Special Fund for
Forest Scientific Research in the Public Welfare (201504708).
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2016 Shen, Song, Müller, Hu, Song, Yu, Wang and Wu. This is an
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