Positive Correlation Between PSI Response and Oxidative
Pentose Phosphate Pathway Activity During Salt Stress in
an Intertidal Macroalga
1
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
College of Earth Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
3
Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science and
Technology, Tianjin 300457, China
*Corresponding author: E-mail, [email protected]; Fax, +86-532-82880645.
(Received March 28, 2014; Accepted April 28, 2014)
2
Studies have demonstrated that photosynthetic limitations
and starch degradation are responses to stress; however, the
relationship between the two is seldom described in detail.
In this article, the effects of salt stress on photosynthesis, the
levels of NADPH and total RNA, the starch content and the
activities of glucose-6-phosphate dehydrogenase (G6PDH)
and ribulose-5-phosphate kinase (RPK) were evaluated.
In thalli that underwent salt treatments, the cyclic electron
flow through PSI showed greater stress tolerance than the
flow through PSII. Even though the linear electron flow was
suppressed by DCMU, the cyclic electron flow still operated.
The electron transport rate I (ETRI) increased as the salinity
increased when the thalli recovered in seawater containing
DCMU. These results suggested that PSI receives electrons
from a source other than PSII. Furthermore, the starch content and RPK activity decreased, while the content of
NADPH and total RNA, and the activity of G6PDH increased
under salt stress. Soluble sugar from starch degradation may
enter the oxidative pentose phosphate pathway (OPPP) to
produce NADPH and ribose 5-phosphate. Data analysis suggests that NADPH provides electrons for PSI in Ulva prolifera
during salt stress, the OPPP participates in the stress
response and total RNA is synthesized in excess to assist
recovery.
Keywords: NADPH OPPP Photosynthesis Salt stress Starch.
Abbreviations: ETR, electron transport rate; Fv/Fm, maximum quantum yield of PSII; G6P, glucose 6-phosphate;
G6PDH, glucose 6-phosphate dehydrogenase; MTT, 3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; OPPP,
oxidative pentose phosphate pathway; P700, primary electron donor of PSI; PES, phenazine ethosulfate; R5P, ribose
5-phosphate; RPK, ribulose-5-phosphate kinase.
Regular Paper
Li Huan1,2, Xiujun Xie1, Zhenbing Zheng1,2, Feifei Sun1, Songcui Wu1,2, Moyang Li3, Shan Gao1,2,
Wenhui Gu1,2 and Guangce Wang1,*
Introduction
Many marine macroalgae inhabit the intertidal zone, where
they are submerged in seawater at high tide and exposed to
air at low tide (Smith and Berry 1986). When at low tide, the
thalli are severely challenged by environmental changes, such as
high salinity and temperature (Davison and Pearson 1996).
However, they demonstrate an extreme tolerance to these
stresses. Although they lose 80–95% of their original weight
during the diurnal low tides, they can still survive and recover
physiological activity when rehydrated (Dring and Brown 1982,
Blouin et al. 2011, Gao and Wang 2012). Moreover, the intertidal zone is the transition region where organisms evolved
from living in the sea to living on land. Thus, the tolerance to
extreme stresses exhibited by macroalgae suggests that they are
ideal subjects for studying stress-resistant mechanisms not only
in marine algae but also in terrestrial higher plants.
Among marine algae, the Chlorophyta are the most closely
related to higher plants (Lewis and McCourt 2004, Aquino et al.
2011). In a previous study of Ulva sp. (Chlorophyta), it was
found that electron transport rate II (ETRII) decreased to zero
when the absolute water content of the thalli was close to 35%,
but ETRI still had a value (7 mmol m2 s1). After 5 min of
rehydration, the PSI activity increased instantly and was fully
restored, whereas restoration of the PSII activity was relatively
slow. Cyclic electron flow around PSI plays a crucial physiological role in Ulva sp. responses to stress (Gao et al. 2011).
This phenomenon was also found in other marine macroalgae,
such as Porphyra yezoensis (Gao and Wang 2012), Porphyra
haitanensis (Gao et al. 2013), and even in the higher plants
Hordeum vulgare (Golding and Johnson 2003) and Paraboea
rufescens (Huang et al. 2012). In addtion, PSII activity appeared
highly salt sensitive compared with PSI, as reflected by a
continuous decrease over time in Anabaena (Rai et al. 2014).
Plant Cell Physiol. 55(8): 1395–1403 (2014) doi:10.1093/pcp/pcu063, available online at www.pcp.oxfordjournals.org
! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 55(8): 1395–1403 (2014) doi:10.1093/pcp/pcu063 ! The Author 2014.
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This raises the following question: when the linear electron transport is weakened by stresses, what is the electron
source?
It was suggested that stromal reductants, including
NAD(P)H, which are mostly produced by starch breakdown,
play an important role in the donation of electrons to PSI
(Bukhov et al. 2002). Hibino et al. (1996) reported that the
PSI content and cyclic electron flow were enhanced by salt
via NAD(P)H dehydrogenase in the Cyanobacterium
Aphanothece halophytica. Therefore, it seems that NAD(P)H
is related to the maintenance of PSI activity when the cells
are under stress. In addition, when responding to stresses,
starch degradation was found in many organisms, such as the
alga Ulva lactuca (Murthy et al. 1988) and the higher plant
Arabidopsis (Sun et al. 2008). The soluble sugar degraded by
starch plays an important role in maintaining osmotic pressure
to reduce the damage to the cells caused by stress (Watanabe
et al. 2000). Apart from this, is there a relationship between
starch degradation and the generation of NAD(P)H when cells
respond to stress?
In this study, U. prolifera was chosen as the representative
species to study the salt stress response. We investigated the
photosynthetic performance of PSI and PSII in the thalli when
exposed to a range of solutions of different salinity. In addition,
changes in the levels of NAD(P)H, starch and soluble sugar were
detected and analyzed. Related enzyme activities and total RNA
levels were also determined. The results show that the NADPH
produced by starch degradation provides an electron source for
PSI in U. prolifera under salt stress. Additionally, total RNA was
synthesized in excess to help the thalli to get over stress.
Results
Photosynthetic performance of U. prolifera in
response to salt stress
After being incubated in different salt concentrations for 6 h,
the maximum quantum yield of PSII (Fv/Fm) of the thalli changed as the salinity increased. No significant differences were
found between the 3% and 5% salt treatments (P > 0.05).
After that, Fv/Fm declined as the salinity increased, with the
lowest ratio occurring in the 18% treatment group. However,
after recovering in natural seawater for 30 min, Fv/Fm increased
significantly (P < 0.05) (Fig. 1A). In addition, as the salinity
increased, ETRII declined and dropped almost to zero in the
18% treatment group. ETRI also decreased as the salinity
increased, although when ETRII dropped almost to zero, ETRI
retained a high value (10 mmol m2 s1). During the recovery
period, both ETRI and ETRII increased significantly (P < 0.05)
relative to their values in the 18% treatment group (Fig. 1B).
In the control group (3% salinity), when the linear electron
flow was inhibited by DCMU, ETRII decreased to zero while
ETRI had a low value (6 mmol m2 s1). As the salinity
increased, the value of ETRI increased. ETRI was highest in the
18% treatment group, with a value as high as that of the normal
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Fig. 1 Variations of Ulva prolifera thalli parameters after being cultured under different salinity conditions for 6 h. Recovery denotes
thalli that were placed at 18% salinity for 6 h and then transferred
to natural seawater (3% salinity) to recover for 30 min. Data are the
mean of five independent experiments (±SD). (A) Fv/Fm. (B) ETR
(electron transport rate). (C) ETRI (the linear electron flow was
inhibited when the thalli recovered in seawater containing DCMU).
state of the thalli. After the 30 min recovery period, the value
decreased (Fig. 1C).
The levels of NAD(P)H and NAD(P)+
When responding to salt stress, the NADPH level increased as
the salinity increased, while the NADP+ level decreased.
However, when the thalli recovered, the NADPH level
decreased and the NADP+ level showed a slight increase
(Fig. 2A, B). There were no significant changes in the levels
of NADH and NAD+ when the thalli were under salt stress
(Fig. 2C, D).
The degradation of starch during the salt stress
response
Fig. 3A shows that starch granules became lighter in color and
smaller in size as the thalli were subjected to increasing salt
concentrations for 6 h, which suggests that starch was being
degraded. Additionally, the starch content declined as the
Plant Cell Physiol. 55(8): 1395–1403 (2014) doi:10.1093/pcp/pcu063 ! The Author 2014.
PSI and OPPP correlation under stress
Fig. 2 Effects of salt stress on NAD(P)H and NAD(P)+ levels in thalli of U. prolifera. Recovery denotes thalli that were placed at 18% salinity for 6 h
and then transferred to natural seawater (3% salinity) to recover for 30 min. Data are the mean of four independent experiments (±SD). (A)
NADPH, (B) NADP+, (C) NADH, (D) NAD+.
salinity increases. When the thalli recovered, the starch content
increased significantly (Fig. 3B). The soluble sugar content
increased slightly as the salinity increased, except in the 18%
treatment group where it declined. The soluble sugar content
increased after recovery to a level similar to that of the control
group (Fig. 3C).
Changes in enzyme activities and total RNA
content
The salt treatment significantly increased the activity of
glucose-6-phosphate dehydrogenase (G6PDH), which is the
enzyme that catalyzes the first step of the oxidative pentose
phosphate pathway (OPPP) to produce NADPH. Compared
with the control group, the activity of G6PDH in the salt
treatment groups increased significantly (Fig. 4A). Ribulose-5phosphate kinase (RPK), an enzyme involved in the Calvin
cycle, decreased when the thalli responded to high salinity.
After recovery, its activity showed a small increase although
this was not significant (Fig. 4B). In addition, the salt treatment
significantly increased the total RNA content (Fig. 4C).
Discussion
PSI is restored quickly after recovery from
salt stress
Fv/Fm is a sensitive indicator of the photosynthetic activity
of plants and decreases when plants are subjected to stresses,
such as high temperature, dehydration and high salinity
(Maxwell and Johnson 2000, Gao et al. 2011). In this study,
Fv/Fm decreased significantly (Fig. 1A), showing that the
U. prolifera thalli were affected by the environmental factor.
In the 18% treatment group, ETRII decreased almost to zero
whereas ETRI remained high (10 mmol m2 s1) (Fig. 1B),
indicating that the cyclic electron flow around PSI showed
greater tolerance to high salinity than the electron flow
around PSII. Similar phenomena have been reported for other
species, such as P. yezoensis (Gao and Wang 2012). In addition, it
was reported that PSII was more sensitive to cadmium treatment than PSI in the green alga Chlorella pyrenoidosa (Wang
et al. 2013). When U. prolifera thalli recovered in natural seawater, both ETRI and ETRII increased, but ETRI increased more
rapidly (Fig. 1B). This suggested that there are more electron
transport pathways for PSI than for PSII.
When the thalli from different salt treatments recovered in
natural seawater containing DCMU, although the linear electron
flow was inhibited, ETRI could still be restored. More interestingly, it increased significantly as the salinity increased. When the
18% treatment group recovered with DCMU, ETRI was restored
to its original level. However, when the thalli from the 18% salinity treatment group recovered for 30 min and then DCMU was
added, ETRI did not increase to the same level, indicating that
the recovery has a negative impact on the restoration of ETRI
(Fig. 1C). Therefore, in addition to the electrons from water
oxidation during photosynthesis, there may be other sources
providing electrons to PSI under salt stress.
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Fig. 3 Effects of salt stress on starch and soluble sugar contents in thalli of U. prolifera. Recovery denotes thalli that were placed at 18% salinity for
6 h and then transferred to natural seawater (3% salinity) to recover for 30 min. (A) Staining of starch granules; scale bar = 100 mm. (B) Starch
content. (C) Soluble sugar content.
The starch content decreases significantly and the
soluble sugar content increases slightly as the
salinity of the treatments increases
The staining results and the starch content analysis showed
that starch degradation in U. prolifera thalli increased as the
salinity of the treatments increased. The soluble sugar content
increased slightly as the salt concentration increased (Fig. 3).
1398
This suggests that the salt treatment accelerated starch degradation and soluble sugar synthesis. The same phenomenon was
also reported in other studies. Yang et al. (2001) suggested that
in rice stems, water stress treatment accelerated the reduction
of starch, while more soluble sugars, including sucrose, accumulated. Temperature stress also leads to the induction of the
transcripts and/or the activity of b-amylase. It was also found
that this increase has been linked to an increased maltose
Plant Cell Physiol. 55(8): 1395–1403 (2014) doi:10.1093/pcp/pcu063 ! The Author 2014.
PSI and OPPP correlation under stress
Fig. 4 Effects of salt stress on glucose-6-phosphate dehydrogenase (G6PDH) and ribulose-5-phosphate kinase (RPK) activities and total RNA
content in thalli of U. prolifera. Recovery denotes thalli that were placed at 18% salinity for 6 h and then transferred to natural seawater
(3% salinity) to recover for 30 min. (A) G6PDH activity. (B) RPK activity. (C) Total RNA content.
content (Kaplan and Guy 2004, Kaplan et al. 2006). In addition,
Rizhsky et al. (2004) reported that since photosynthesis was
suppressed in Arabidopsis cells subjected to a combination of
drought and heat stress, sucrose may have been synthesized
following starch degradation. They identified that pathways for
starch degradation and sucrose biosynthesis were specifically
elevated in plants during a combination of drought and heat
stress (Watanabe et al. 2000). When U. prolifera thalli were
under salt stress, photosynthesis was suppressed. Therefore,
the increase in the soluble sugar content was mainly from
starch degradation.
It has been suggested that during stress soluble sugar can
function in two ways: as an agent to maintain osmotic pressure,
which protects the cell from stress, and as an osmoprotector,
Plant Cell Physiol. 55(8): 1395–1403 (2014) doi:10.1093/pcp/pcu063 ! The Author 2014.
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stabilizing proteins and membranes (Bohnert et al. 1995,
Sánchez et al. 1998). Additionally, the biological process of
starch breakdown plays an important role in the donation
of electrons to the photosynthetic cyclic electron flow in
Chlamydomonas reinhardtii (Johnson and Alric 2012). It will
be of interest to study the mechanism behind this role.
normally even though transcription is inhibited. Until the
late cleavage period, a large number of mRNA transcripts are
synthesized in the early embryo.
OPPP participates in the salt response
Preparation of U. prolifera
In this study, we found that when the thalli responded to different salt concentrations, the content of NADPH increased
significantly, while NADH did not change significantly
(Fig. 2). This suggested that NADPH may be more important
than NADH in the salt stress response. NADPH is produced by
the first two steps of the OPPP, the major source of NADPH in
the cytoplasm of plant cells (Schnarrenberger et al. 1973), which
are catalyzed by G6PDH and 6-phosphogluconate dehydrogenase. G6PDH, the rate-limiting and key regulatory enzyme of the
OPPP, controls the flow of carbon through this pathway and
produces reductant to meet the cellular needs for reductive
biosynthesis (Kletzien et al. 1994). The activity of G6PDH
increased when the thalli were under salt stress (Fig. 4A),
which is consistent with the increased NADPH level. Liu et al.
(2007) reported that G6PDH activity was enhanced rapidly in
the presence of NaCl in red kidney bean roots. In Anabaena, the
level of NADPH was induced in cells under salt stress; the induction of NADPH and transketolase suggested the operation
of the OPPP (Rai et al. 2014). Reports suggest that NAD(P)H can
donate electrons to intersystem electron carriers, which may
have significant effects on the cyclic electron flow (Bukhov et al.
2002, Bukhov and Carpentier 2004, Johnson and Alric 2012, Gao
et al. 2013). According to these results and the literature, we
propose that the NADPH derived from starch degradation provides electrons for the operation of cyclic electron flow around
PSI in thalli under salt stress.
In addition to being the major provider of reducing equivalents, OPPP also provides ribose 5-phosphate (R5P) (Williams
et al. 1987, Porter et al. 2000). R5P not only plays a part in the
Calvin cycle, but it can also be utilized for nucleotide synthesis
(Debnam and Emes 1999). When cells are placed under stress,
the activities of Calvin cycle-related enzymes weaken (Dias and
Brüggemann 2010). In this study, the activity of RPK, which
catalyzes conversion of R5P to ribulose 1,5-bisphosphate,
decreased under salt stress (Fig. 4B). Due to the increase of
G6PDH activity and the decrease of RPK, there may be an accumulation of pentoses when the cell is under salt stress. This
increase of pentoses is likely to initiate the synthesis of RNA.
This hypothesis was corroborated by the increased total RNA
content of thalli under salt stress (Fig. 4C). It is speculated that
the purpose of the excess synthesis of total RNA is in order to
help the cells get over the stress and ready for the non-stressed
condition. This is similar to what occurs in most animal species,
where the oocyte contains abundant mRNAs and proteins,
which control the rate of early embryo cell division after fertilization. In the early embryo cleavage stage, the genome of the
zygotic embryo is not functional. The early embryo can develop
Samples of U. prolifera were collected from the intertidal zone
at Zhanshan (36 30 1400 N, 120 220 1700 E), Qingdao, China. The
thalli were rinsed gently in sterile seawater to remove any sediment, small grazers or epiphytes. Thalli in a similar physiological
state were chosen for use in the experiments.
Materials and Methods
Photosynthetic performance of U. prolifera in
response to salt stress
To investigate the response of U. prolifera to salt stress, thalli
were incubated under different concentrations of salt (3, 5, 9, 12
and 18%) for 6 h. In addition, some thalli were transferred to
natural seawater (3%) for a 30 min recovery period after being
incubated at 18% salinity for 6 h. The photosynthetic properties
of the thalli, including the Chl fluorescence of PSII and the
absorbent changes of PSI, were measured using a Dual-PAM100 (Walz) connected to a PC equipped with WinControl software. Two parameters, Fv/Fm and ETR, were used in this study.
Fv/Fm was calculated according to the formula: (Fm – F0)/Fm
(Kramer et al. 2004, Schreiber 2004), in which F0 and Fm were
determined after 10 min dark adaptation. ETRI and ETRII were
calculated by the Dual-PAM software.
In addition, after being treated in the different situations
shown above, the thalli were transferred to natural seawater
containing 10 mM DCMU. DCMU is known to block electron
transport after the primary acceptor in PSII (Joët et al. 2002).
The photosynthetic properties of the thalli were measured after
5 min.
NAD(P)H and NAD(P)+ assays
An equal fresh weight of each differently treated U. prolifera
thallus was transferred to 0.1 M pre-heated NaOH or HCl
solution. The mixtures were heated in a boiling water bath
for 5 min, cooled in an ice bath and then centrifuged at
10,000g at 4 C for 10 min (Zhao et al. 1987, Gibon
and Larher 1997). NADPH and NADH are specifically extracted in the supernatant obtained after the alkaline treatment, while NADP+ and NAD+ are extracted after an acidic
treatment.
Enzyme cycling assays were employed using 3-(4,5dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide (MTT)
as the terminal electron acceptor (Nisselbaum and Green 1969).
Procedures for the assays were described by Matsumura and
Miyachi (1980). Reactions were carried out under low lighting
due to the light sensitivity of phenazine ethosulfate (PES) and
MTT. The acidic, as well as the alkaline, extract was placed in a
1.5 ml microtube and neutralized by adding an equivalent
amount of NaOH or HCl, followed by the addition of equal
Plant Cell Physiol. 55(8): 1395–1403 (2014) doi:10.1093/pcp/pcu063 ! The Author 2014.
PSI and OPPP correlation under stress
volumes of 1 M Bicine-NaOH buffer pH 8.0, 40 mM EDTA,
4.2 mM MTT and 16.6 mM PES. Then, 0.05 ml of 50 mM glucose
6-phosphate (G6P) was added for the determination of
NADP(H), and 0.1 ml of 5 M ethanol for the determination of
NAD(H). The total volume of the assay medium was adjusted to
1 ml by adding H2O, and the tubes were incubated for 5 min in a
37 C water bath. Enzyme cycling was started by adding 0.02 ml
of 35 U ml1 G6PDH [for NADP(H)] or 500 U ml1 alcohol dehydrogenase [for NAD(H)]. The cycling time was 40 min, and
the absorbancy at 570 nm was determined. The rate of reduction of MTT was directly proportional to the concentration of
the coenzyme.
In order to eliminate the effect of salt stress on the moisture
content of the cells, the thalli in different treatments were
dried at 80 C to a constant weight. The percentage moisture
content was calculated as moisture lost during drying/
FW 100%. The other part of the portion (represent as DW)
was used for the assay, so did the enzymes activities and total
RNA content in the following.
Staining of starch granules
Ulva prolifera thalli were incubated in 3, 9 and 18% saline seawater for 6 h. The starch samples were fixed in 4% paraformaldehyde for 24 h, decolorized in 70% ethanol for another 24 h
and stained with a solution of KI–I2 (2.5% KI and 1% I2) for
30 min (Seguchi et al. 2000, Seguchi et al. 2001). Starch granules
were observed through a Leica DM 2500 microscope, and
microphotographs were taken.
Assays determining G6PDH and RPK activities,
and total RNA content
Ulva prolifera thalli that had undergone different salt treatments were frozen in liquid nitrogen and ground into
powder with a mortar and pestle. For the determination of
G6PDH activity, extraction buffer (50 mM Tris–HCl pH 8.0,
300 mM NaCl, 0.1 mM benzamidine and 0.1 mM phenylmethylsulfonyl fluoride) was added. The extracts were centrifuged at
10,000g for 10 min at 4 C, and the supernatants were retained
for enzyme assays. G6PDH activity was determined by measuring the change of the 340 nm absorbance at 30 C in 0.1 M Tris–
HCl pH 8.0, 0.4 mM NADP+ and 2 mM G6P (Graeve et al. 1994,
Fahrendorf et al. 1995). For the determination of RPK activity,
extraction buffer (100 mM HEPES-NaOH pH 8.0, 10 mM MgCl2,
0.4 mM EDTA, 1% polyvinylpyrrolidinone, 100 mM Naascorbate and 0.1% bovine serum albumin at 4 C) was added.
Its initial activity was measured in 30 mM HEPES-NaOH pH 8.0,
10 mM MgCl2, 2 mM ATP, 2 mM Phosphoenolpyruvate, 1 mM
R5P, 0.3 mM NADH, 2 U ml1 of lactate dehydrogenase, pyruvate kinase and ribose phosphate isomerase. The reaction was
initiated by adding the extract supernatant (Rao and Terry
1989).
The RNAs of the thalli that had undergone different salt
treatments were extracted using a Total RNA Kit (Omega)
and the concentrations were measured by a NanoDrop 1000
Spectrophotometer (Thermo).
Funding
Starch and soluble sugar analyses
In this study, the method described by Sánchez et al. (1998),
and by Brányiková et al. (2011), was used with minor modifications. Ulva prolifera thalli that underwent different salt treatments (3, 5, 9, 12 and 18%, and recovery) were frozen in liquid
nitrogen and ground into powder with a mortar and pestle. The
powder was dried at 105 C to a constant weight. Soluble sugar
was extracted using 8 ml of 80% ethanol at 68 C for 15 min and
centrifuged. This procedure was repeated three times. The
supernatants were merged and volatilized in an 85 C water
bath to a volume of 2–3 ml. Distilled water was then added
to a final total volume of 10 ml. For the total hydrolysis of
starch, 3.3 ml of 30% perchloric acid was added to the sediment,
stirred for 15 min and centrifuged. This procedure was also repeated three times. The extracts were combined and perchloric
acid was then added to a final volume of 10 ml.
Samples of 0.1 ml of the soluble sugar and of the starch
extract were cooled to 0 C, and 1 ml of anthrone solution
[2 g of anthrone in 1 liter of 72% (v/v) H2SO4] was added and
blended quickly. The mixtures were kept in a water bath at
100 C for 8 min, cooled to 20 C, and the 625 nm absorbance
levels were measured. The standard curve was carried out simultaneously using glucose. The soluble sugar contents were
determined according to the calibration curve, while the
starch contents were obtained by multiplying the measured
values by 0.9.
This work was supported by the National Natural
Science Foundation of China [41176137; 41376164]; 863 project
[2012AA100811-5]; 863 key project [2012AA100806]; the
Innovative Foundation of the Chinese Academy of Sciences
[XDA05030401].
Disclosures
The authors have no conflicts of interest to declare.
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