Open Journal of Forestry
2012. Vol.2, No.2, 71-76
Published Online April 2012 in SciRes (
Copyright © 2012 SciRes. 71
Traffic Pollution Influences Leaf Biochemistries of
Broussonetia papyrifera
Yuanwen Kuang1, Dan Xi1,2, Jiong Li1, Xiaomin Zhu1,2, Lingling Zhang1
1Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China
Botanical Garden, Chinese Academy of Sciences, Guangzhou, China
2Graduate University of the Chinese Academy of Sciences, Beijing, China
Received January 12th, 2012; revised February 20th, 2012; accepted February 28th, 2012
Paper mulberry (Broussonetia papyrifera) is one of multifunctional species in agroforestry systems as
well as one of traditional forages in many countries of Asia. Fully expanded tender leaves of B. papyrifera
wildly growing under two traffic densities (a high traffic loads bearing more than 1000 vehicles per hour,
HT; and a relatively clear section with almost no traffic loads, NT) were collected for carbohydrates,
amino acids and phytohormones analysis. Leaves exposed to traffic pollutants were revealed to have sig-
nificant lower amounts of carbohydrates and total amino acids than those growing at relatively clear en-
vironment. The levels of abscisic acid in the leaves significantly increased, while gibberellin acid, in-
doleaetic acid, and zeatin riboside in the leaves significantly decreased, with the traffic densities. The re-
sults indicated that the contents of carbohydrates, amino acids and phytohormones in the leaves of B. pa-
pyrifera could be adversely affected by traffic pollution. Variations of the leaf biochemistries of B. pa-
pyrifera exposed to traffic pollutants implied that B. papyrifera could physiologically regulate itself to
adapt or resist traffic stress.
Keywords: Amino Acids; Broussonetia papyrifera; Carbohydrates; Phytohormounes; Traffic Pollutants
Rapid development of livestock is bringing huge demands
for forages globally. During the last few years, public concerns
regarding food safety have intensively increased as a conse-
quence of the increasing prevalence of some fatal diseases (e.g.
Salmonella enteritidis in meat products and Escherichia coli
0157: H7 in beef) endangering human health. Frequent misuse
of antibiotics, antibacterial, vitamins, hormones and the addi-
tive of some trace metals in animal feedstuffs also brought po-
tential risks on human being. Controlling of hazard materials
into livestock forages is one of internationally important issues
for public health. Development of high quality plant forages has
been prompted to avoid infectious agents into the animal feed-
stuffs and thus to strengthen the bio-security of humans
(Martínez et al., 2005).
Paper mulberry (Broussonetia papyrifera) is a fast growing
tree or shrub of the Moraceae family. This species commonly-
naturally grows in various environments in Asia and Pacific
countries (Malik & Husain, 2007) with large biomass and rapid
propagation by shoot regeneration either from root or stem
cuttings or seeding. It usually takes only 12 - 18 months to
reach the harvest size of 3 - 4 m height (http://www.agrofor- Particularly, once have
been harvested, the species could present faster growth rate and
larger biomass than newly planted ones. Since the ancient time,
B. papyrifera was widely used as multifunctional species in
agroforestry ecosystems, e.g. manufacturing high-quality pa-
pers, cloths, and ropes (Liao et al., 2006), treating diseases as
one of traditional Chinese medicines (Lee et al., 2001).
Differing from a variety of woody species used for furniture
and manufacturing, Paper mulberry has been traditionally used
as forage of livestock in many mountainous regions in China
for long history when their tender leaves and twigs were har-
vested from natural stands. With the globally rapid develop-
ment of domestic livestock and the huge demands for forages,
values of wild plant resources such as mulberry have been in-
tensively concerned (Hibib, 2004). Recently, farmers in moun-
tainous provinces of China (e.g. Hubei, Guangxi and Jiangxi)
have been encouraged to grow large area of Paper mulberry as
a cash crop. Based on the literature survey, research on this
species mainly focused on medicinal properties (Kwak et al.,
2003), bark yield (Saito et al., 2009), tissue culture and rapid
propagation (Li et al., 2008), influence on native scrub forest
(Malik & Husain, 2007) and efficiency of heavy metals re-
moval (Nagpal et al., 2011). Being one of traditional fodder
shrubs with high levels of crude protein, minerals and digesti-
bility in the leaves and twigs, B. papyrifera has been less inves-
tigated on the changes in biochemistries under the exposure of
traffic exhaust. Researchers have demonstrated that pollution
did depress the properties of fodder trees (shrubs) (Sanz et al.,
2011). Automobile exhaust gas, a dominant cause of atmos-
pheric pollution in urban and rural areas owing to the increasing
number of vehicles, could be transported from urban to remote
mountain areas (Sakugawa & Cape, 2007). In the present study,
we detected the variations of carbohydrates, amino acids as
well as phytohormones in the leaves of B. papyrifera exposed
to traffic exhaust. The objectives were to examine the potential
impacts of traffic pollution on the fodder properties, and to
elucidate the mechanism by which this species responded to
traffic stress.
Materials and Methods
Plant Sampling and Processing
Naturally-growing Paper mulberry shrubs were sampled from
two environments with different traffic densities in Guangzhou
city, southern China. The first one was selected along two
freeways running along South China Botanical Garden with
mean traffic loads of more than 1000 vehicles per hour. This
section stood for the environment receiving high levels of
automobile exhaust from point sources (high traffic loads, HT).
The reference environment was selected in the botanical garden
where vehicles were forbidden to enter. This section stood for
the relatively clear environment without direct point source of
traffic exhaust (NT). The two sampling sections, partitioned by
the bounding wall of the garden, had similar soil property and
climate condition.
Fifteen Paper mulberry shrubs with similar appearance and
without visible injury in leaves were randomly selected from
the different environment in November, respectively. All Paper
mulberry shrubs were selected at least 500 m away from each
other. The shrubs were all annual with about 9-month old. For
each selected shrub, a composite sample with between 15 and
20 fully expanded tender leaves was taken from the outer can-
opy, stored in an icebox and carried back to the laboratory im-
In the laboratory, all the leaf samples from each section were
divided into two parts. One part was freshly weighted and fro-
zen in liquid nitrogen and stored at –20˚C for plant hormones
analysis. The other part was washed thoroughly with distilled
water, and dried at 60˚C for at least 48 hrs and ground using a
mortar and pestle for later analysis.
Air Quality Monitoring
Ambient air quality was monitored from early-November to
mid-December for total suspended particulates (TSP), particu-
late matter less than 2.5 microns in diameter (PM2.5), sulphur
dioxide (SO2) and nitrogen oxides (NOx including NO and NO2)
with moderate-volume sampler (TH-150CIII, China) located at
1.5 m above ground level. Total suspended particulates and
PM2.5 were trapped on quartz fibre filters (tare weighted before
sampling) attached to the hopper of the samplers operated con-
tinuously for 24 hrs. After sampling, the filters were stored in a
desiccator with constant temperature for at least 24 hrs and
re-weighted with a precision balance. The concentrations of
SO2 were determined by absorbing air (0.5 L·min–1 for 45 - 60
mins, 5 replicates) into a buffering solution of formaldehyde,
which was later analyzed through a pararosaniline spectropho-
tometry (SEP-HJ482, 2009). Nitrogen oxides were absorbed
(0.4 L·min–1 for 45 - 60 mins, 5 replicates) by N-ethylene dia-
mine dihydrochloride and then were determined spectropho-
tometrically (SEP-HJ479, 2009). The data were presented as 24
hrs average concentrations, and expressed as μg·m–3.
Carbohydrate Analysis
Approximately 50 mg of the oven-dried leaf powder of each
sample was extracted with 80% ethanol (v/v) at 85˚C for 1 h.
The solutions were then centrifuged at 12000 g for 10 mins.
The ethanol extraction step was repeated three times. The three
resulting supernatants were combined, treated with activated
charcoal, and evaporated to dryness in a vacuum evaporator.
The residues were redissolved in distilled water, and subjected
to soluble sugar analysis using the anthrone-sulfuric acid
method (Ebell, 1969). Following removal of soluble sugars, the
remaining pellets were oven-dried overnight at 60˚C and re-
tained for starch analysis according to the procedures described
in previous publications (Vu et al., 2002). Total nonstructural
carbohydrates (TNC) were calculated as the sum of soluble
sugar and starch. Cellulose content was determined by the
method of Updegraff (1969). All the analyses were repeated
five times.
Determination of Amino Acids
An amount (100.0 - 200.0 mg) of leaf powder was placed in
a hydrolysis tube. Each sample had 3 parallel repetitions. The
hydrolysis tube was added in 15 mL of 6 M hydrochloric acid
(HCl) and 1.0 mL of 1% mercaptoacetic acid, then sealed and
heated in vacuum at 110˚C for 22 hrs. After hydrolysis, the
hydrolysis solution was filtered into a 50.0 mL volumetric flask.
The dilute solution (1.0 mL) was transferred into a 25.0 mL
beaker in vacuum, and vaporized. Repeat this process once
again by adding 1.0 - 2.0 mL of distilled water. Finally, the
residual was dissolved in 1.0 mL of 0.02 M HCl, and filtered
through a membrane (0.22 μm). The solution was used to de-
termine the contents of aspartic acid (Asp), threonine (Thr),
serine(Ser), glutamic acid (Glu), glycine (Gly), alanine (Ala),
cystine (Cys), valine (Val), methionine (Met), isoleucine (Ile),
leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys),
histidine (His), arginine (Arg) and proline (Pro) by Amino Acid
Analyzer (L-8800, Hitachi, Japan). The analysis was carried out
according to the standard analytical procedures proposed by
Chen et al. (2008).
Determination of Leaf Hormones
The extraction, purification and determination of endogenous
levels of indoleaetic acid (IAA), gibberellin acid (GA), abscisic
acid (ABA) and zeatin riboside (ZR) by an indirect enzyme-
linked immunosorbent assay (ELISA) technique were per-
formed as described by Zhao et al. (2006). The samples were
homogenized in liquid nitrogen and extracted in cold 80% (v/v)
methanol with butylated hydroxytoluene (1 mmol·L–1) over-
night at 4˚C. The extracts were collected after centrifugation at
10000 × g (4˚C) for 20 mins, the extracts were passed through a
C18 Sep-Pakcatridge (Waters, Milford, MA) and dried in N2.
The residues were dissolved in PBS (0.01 mol·L–1, pH 7.4) in
order to determine the levels of IAA, GA, ABA and ZR. Mi-
crotitration plates (Nunc) were coated with synthetic IAA, GA,
ABA or ZR ovalbumin conjugates in NaHCO3 buffer (50
mmol·L–1, pH 9.6) and left overnight at 37˚C. Ovalbumin solu-
tion (10 mg·mL–1) was added to each well in order to block
nonspecific binding. After incubation for 30 min at 37˚C, stan-
dard IAA, GA, ABA, ZR, samples and antibodies were added
and incubated for a further 45 min at 37˚C. The antibodies
against IAA, GA, ABA and ZR were obtained as described by
Yang et al. (2001). Then horseradish peroxidase-labelled goat
antirabbit immunoglobulin was added to each well and incu-
bated for 1 h at 37˚C. Finally, the buffered enzyme substrate
(orthophenylenediamino) was added, and the enzyme reaction
was carried out in the dark at 37˚C for 15 min, then terminated
using 3 mol·L–1 H
2SO4. The absorbance was recorded at 490
nm. Calculations of the enzyme-immunoassay data were per-
Copyright © 2012 SciRes.
formed as described by Yang et al. (2001). In this study the
percentage recovery of each hormone was calculated by adding
known amounts of standard hormone to a split extract. Per-
centage recoveries were all above 90%, and all sample extract
dilution curves paralleled the standard curves, indicating the
absence of nonspecific inhibitors in the extracts. All the hor-
mones were analyzed at College of Crop Science, China Agri-
cultural University.
Statistical Analysis
The data were shown as mean ± standard deviation. Mean
comparison was performed to test the differences between the
two traffic loads at the confidence level of 95% by paired-
samples T-test using software SPSS 10.0 (SPSS Inc., Chicago,
Ambient Pollutants
Considering the main pollutants at the different environments,
traffic exposure brought significantly higher concentrations of
TSP, PM2.5, NOx, and SO2 (Table 1). The ambient mean con-
centrations of gas pollutants at HT site were nearly 10 and 4
times higher than those at NT for NOx and SO2, respectively.
Traffic emission deteriorated the ambient air quality at HT, thus
gave the opportunity to compare the biochemistries in the leaf
of B. papyrifera grown under the traffic exposure.
Carbohydrates Content
Exposure to traffic loads not only caused significant decrease
of soluble sugars and total nonstructural carbohydrates (TNC),
but also caused dramatic decrease of cellulose content in the
leaves of B. papyrifera (Table 2). Total soluble sugars in leaves
of B. papyrifera exposed to traffic pollutants decreased by
approx 50% (P < 0.01) relative to those growing under rela-
tively clear environment (NT). Considering leaf soluble sugars
and starch together, the TNC content decreased by c. 46% (P <
0.01) in leaves at HT, despite starch contents were not signifi-
cantly different between the sites. Noticeably, traffic exposure
statistically decreased the content of some structural carbohy-
drates in B. papyrifera leaves (P < 0.01), e.g. cellulose de-
creased more than 26%, compared to those at NT. However, the
decreased magnitude in cellulose contents was not as high as
Table 1.
Comparison of the main pollutants between the environments with high
traffic loads of more than 1000 vehicles per hour (HT) and with not
traffics (NT). Data of total suspended particulates (TSP) and particulate
matter less than 2.5 microns in diameter (PM2.5) was 24-hour average
value. Nitrogen oxides including NO and NO2 (NOx) and sulphur diox-
ide (SO2) were shown as the mean and standard variation of 5 replica-
tion measurements. Data was presented as μg·m–3.
Pollutants HT NT
TSP 252.93 ± 80.40** 94.88 ± 12.00
PM2.5 131.18 ± 41.35** 63.31 ± 18.75
NOx 76.83 ± 20.74** 7.71 ± 2.59
SO2 158.90 ± 23.34** 44.56 ± 9.09
**Extremely statistical difference between the environments with the values of P <
the ones in soluble sugars and TNC.
Leaf Amino Aci d s
The individual and total amino acids in the leaves of B. pa-
pyrifera grown under different traffic densities were shown in
Table 3. Among the detected 17 individual amino acids, Glu,
Asp, Les and Lys were the most abundant amino acids while
His, Cys and Met were the lowest ones accounting for about
40% and only 5% of total amino acids, respectively, in the
leaves at both environments. Traffic exposure significantly
Table 2.
Carbohydrate contents (mg·g–1 of dry weight) in the leaves of B. pa-
pyrifera growing at the environments with high traffic loads of more
than 1000 vehicles per hour (HT) and with not traffics (NT). Values
given were mean ± standard deviation. Mean values (n = 15 samples)
were compared by paired-samples T-test at the significant level of P <
Contents HT NT
Soluble sugar 98.10 ± 24.12 205.07 ± 57.26**
Starch 12.64 ± 2.42 13.22 ± 1.44
Cellulose 169.05 ± 47.74 229.91 ± 28.99**
TNC 110.74 ± 24.11 217.52 ± 61.89**
**Extremely statistical difference between the environments with the values of P <
Table 3.
Comparison of amino acids (% of dry weight) in the leaves of B. pa-
pyrifera growing at the environments with high traffic loads of more
than 1000 vehicles per hour (HT) and with not traffics (NT). Values
given were mean ± standard deviation. Mean values (n = 15 samples)
were compared by paired-samples T-test at the significant level of P <
Species HT NT
Glu 1.87 ± 0.10 2.09 ± 0.12**
Asp 1.74 ± 0.08 1.83 ± 0.09
Leu 1.33 ± 0.09 1.69 ± 0.07**
Lys 1.30 ± 0.08 1.50 ± 0.11**
Phe 1.17 ± 0.02 1.28 ± 0.11
Gly 0.98 ± 0.05 1.09 ± 0.06
Ala 0.96 ± 0.09 1.23 ± 0.10
Pro 0.90 ± 0.12 0.82 ± 0.16
Val 0.82 ± 0.01 0.98 ± 0.07**
Ile 0.81 ± 0.03 0.98 ± 0.04**
Tyr 0.80 ± 0.04 0.82 ± 0.03
Thr 0.79 ± 0.05 0.88 ± 0.06**
Ser 0.78 ± 0.02 0.87 ± 0.06
Arg 0.76 ± 0.14 1.06 ± 0.17**
His 0.47 ± 0.05 0.53 ± 0.03
Cys 0.22 ± 0.04 0.15 ± 0.02
Met 0.19 ± 0.04 0.23 ± 0.04
Total 15.89 ± 0.50 18.05 ± 0.89**
**Extremely statistical difference between the environments with the values of P <
Copyright © 2012 SciRes. 73
decreased the leaf total amino acids, with expectedly highest
contents in the leaves from the relatively clear environment
(NT). However, concentrations of Asp, Phe, Gly, Ala, Pro, Tyr,
Ser, His, Cys, and Met did not respond to the presence of traf-
Leaf Phytohormones
Facing to traffic exhausts, paper mulberry patterned distin-
guishingly for certain hormones in the leaves (Table 4). It
could be easily observed that the plant hormones varied spe-
cies-specifically in the leaves between the cases. There were
significant increase in ABA and significant decrease in GA,
IAA, and ZR in leaves exposed to traffic exhausts (HT) com-
pared with those grown at the relatively clear environment (NT).
Levels of IAA were revealed particularly affected by the traffic
pollutants, with almost 3 times higher in the leaves at HT than
at and NT.
Partitioned only by the bounding wall of the botanical garden,
the two sampling locations were considered with no significant
difference in soil and climate properties. The influence of traf-
fic pollutants was mainly discussed in this study. It’s well
known that vehicles could directly emit a large amount of TSP
and PM2.5, which could have significant effects on ambient
quality (Kunzli et al., 2006). As revealed by Sakugawa et al.
(2011), nitrite was a dominant source of photochemical forma-
tion of OH radical in both gasoline and diesel car exhausts. The
atmospheric NO2 concentration at the roadside in the forest was
highly correlated with the traffic density of buses (Kume et al.,
2009). In this study, the significantly high concentrations of
TSP, PM2.5, NOx and SO2 at HT indicated that automobile ex-
haust might have harmful effects on plant species (Shigihara et
al., 2008). At the same time, high SO2 in NT suggested that
wind transported a considerable amount of pollutants from
traffic site to the botanical garden.
As revealed by researchers that environmental stresses like
heavy metal and air pollution could lead to major alterations in
carbohydrate metabolism of plants by decreasing of maximum
photosynthetic rate and stomata conductance in plant leaves,
increasing ethylene emission, and reducing leaf longevity
(Thomas et al., 2006; Devi et al., 2007; Kume et al., 2009; Sa-
kugawa et al., 2011). Total content of carbohydrates (in par-
ticular soluble sugar and TNC) in leaves of forages added nutri-
tive value to animals (Shewmaker et al., 2006). In this study,
the significant decrease in soluble sugar, TNC and cellulose
contents in the leaves of B. papyrifera affected by traffic expo-
sure agreed with Tripathi and Gautam (2007) who found that
even short duration of air pollution significantly decreased the
soluble sugar contents. Traffic pollutants could adversely affect
plant physiological and morphological characteristic directly or
indirectly. Various physiological deteriorations of plant leaves
were correlated with the NO2 concentration (Kume et al., 2000).
We speculated that the noticeable decrease of soluble sugar,
NTC in the leave of B. papyrifera might be due to: 1) the inhi-
bition of RUBP carboxylase activity caused by traffic exhausts,
because RUBP carboxylase was a most abundant key enzyme
in photosynthesis for carbohydrates assimilation in plants (Tri-
pathi & Gautam, 2007). The decrease of RUBP carboxylase
activity thereby resulted in reduced levels of carbohydrates; 2)
Table 4.
The levels of phytohormones (mg·g–1 of fresh weight) in the leaves of B.
papyrifera growing at the environments with high traffic loads of more
than 1000 vehicles per hour (HT) and with not traffics (NT). Values
given were mean ± standard deviation. Mean values (n = 15 samples)
were compared by paired-samples T-test at the significant level of P <
Contents HT NT
ABA 68.86 ± 1.26** 61.25 ± 1.09
GA 19.79 ± 0.42 24.47 ± 0.61**
IAA 37.33 ± 0.81 117.03 ± 2.18**
ZR 15.66 ± 0.26 26.77 ± 0.61**
**Extremely statistical difference between the environments with the values of P <
the increased respiration and decreased CO2 fixation because of
chlorophyll deterioration caused by the traffic exhausts. Usually,
plants could increase soluble sugar in leaves when assimilation
rates were in excess of carbohydrate consumption rates; 3) the
lower allocation of carbohydrates to cell walls or a decrease in
the activity of cellulose synthase leading to the reduction of
cellulose in the leaves of B. papyrifera exposed under traffic
loads. We suggested that the leaf carbohydrate contents of B.
papyrifera be indicators of traffic pollution for early nutritive
diagnosis or as markers for physiological damage to forage
prior to the onset of visible injury symptoms.
Amino acids were known to play a vital role in the osmotic
adjustment and in the tolerance and detoxification of plants
(Hall, 2002). For instance, Pro was revealed to be very impor-
tant in ameliorating environmental stress in many higher plants
(Wang et al., 2009). Another amino acid, His, also typically
involved in metal stress tolerance (Sharma & Dietz, 2006). In
this study, however, both of the two amino acids did not in-
crease from NT to HT (Table 3). The similar levels of Pro and
His found in the leaves of B. papyrifera collected from the two
environments compared to other amino acids indicated that this
species might be able to actively accumulate some amino acids,
other than Pro and His, depending on the type of environmental
stress. This finding was in agreement with Hussein and Terry
(2002) who reported similar observations in some plants grow-
ing at crude oil contaminated saline environment, but in dis-
agreement with other studies in which the concentration of Pro
was found markedly increased under environmental stress de-
spite of the decrease of total amino acids (Balestrasse et al.,
2005). Cysteine, a SH containing amino acid, was a key con-
stituent of phytochelatins and played an important role in metal
detoxification. An increase in Cys content was recorded in
leaves of plants irrigated with effluents (Chandra et al., 2009).
However, Cys did not increase or decrease in the leaves of B.
papyrifera between the traffic loads, implying that this species
might detoxify by accumulating other amino acids. In the pre-
sent study, B. papyrifera was found to biochemically-physio-
logically respond to the traffic pollution. Traffic exposure did
decrease some individual amino acid (Glu, Leu, Lys, Val, Ile,
Thr, Arg) as well as the total amino acids levels. This result was
well consistent with numerous findings concerning plants fac-
ing to environmental stresses (Wang et al., 2009).
Plants could respond both physiologically and anatomically
to environmental stresses usually under the regulations of plant
hormones including ABA, GA, IAA, and ZR (Li et al., 2002).
Copyright © 2012 SciRes.
Plant ABA was considered as an inhibitor of leaf growth and
was suggested to be a regulator of leaf stomatal aperture (Jiang
et al., 2003). In the present study, the significantly higher levels
of ABA in leaves exposed to traffic exhaust implied that B.
papyrifera might resist the traffic pollution by decreasing the
leaf stomatal aperture and increasing leaf hydraulic conductiv-
ity. It was reported that high ABA levels found in leaves were
generally consistent with low stomatal aperture and high hy-
draulic conductivity (Jiang et al., 2003). The patterns of leaf
ABA in the present study were also revealed by Monni et al.
(2001) who found plants growing near pollution source had
higher contents of ABA in their stems compared to those
growing farther.
Unlike the significant increase in IAA, GA, ZR and signifi-
cant decrease in ABA in the leaves of Arabidopsis thaliana
grown under the elevated CO2 (Teng et al., 2006), plant hor-
mones in this study were observed noticeably reduced in IAA,
GA and ZR in the leaves grown at the traffic environment. GA
and IAA could enhance plant growth and development by
stimulating cell division, cell elongation and protein synthesis
(Yong et al., 2000). The noticeable reduce of GA and IAA in
the leaves affected by traffic exhausts implied that the growth
of B. papyrifera might be adversely affected. We proposed that
change in the levels of plant hormones probably was one of
physiological responses regulating the adaptability or resistance
of B. papyrifera growing under traffic pollution.
Traffic pollution significantly decreased the carbohydrates
and total amino acids in the leaves of B. papyrifera, which
might adversely decrease the nutritive values of this species.
Concerns on this decrease should be arisen when the wild shrub
was frequently and increasingly used as one of important plant
forages. The variations of plant hormones in the leaves exposed
to traffic pollutants implied that this species could physiologi-
cally and bio-protectively regulate itself to adapt or resist traffic
pollution. The biochemistries could be used as indicators for
early properties diagnosis.
This research was jointly supported by the Knowledge Inno-
vation Program of the Chinese Academy of Sciences (No.
KSCX2-EW-J-28), Guangdong Natural Science Foundation (No.
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