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pubs.acs.org/jpr
Dynamic Metabonomic Responses of Tobacco (Nicotiana tabacum)
Plants to Salt Stress
Jingtao Zhang,†,‡,§ Yong Zhang,†,§,|| Yuanyuan Du,‡,§ Shiyun Chen,*,|| and Huiru Tang*,‡
‡
)
State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance,
Wuhan Institute of Physics and Mathematics, the Chinese Academy of Sciences, Wuhan 430071, P. R. China
Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, The Chinese Academy of Sciences,
Wuhan 430071, P. R. China
§
Graduate School of the Chinese Academy of Sciences, P. R. China
bS Supporting Information
ABSTRACT: Metabolic responses are important for plant
adaptation to osmotic stresses. To understand the dosage and
duration dependence of salinity effects on plant metabolisms,
we analyzed the metabonome of tobacco plants and its dynamic
responses to salt treatments using NMR spectroscopy in
combination with multivariate data analysis. Our results showed
that the tobacco metabonome was dominated by 40 metabolites
including organic acids/bases, amino acids, carbohydrates and
choline, pyrimidine, and purine metabolites. A dynamic trajectory was clearly observable for the tobacco metabonomic
responses to the dosage of salinity. Short-term low-dose salt
stress (50 mM NaCl, 1 day) caused metabolic shifts toward
gluconeogenesis with depletion of pyrimidine and purine
metabolites. Prolonged salinity with high-dose salt (500 mM NaCl) induced progressive accumulation of osmolytes, such as
proline and myo-inositol, and changes in GABA shunt. Such treatments also promoted the shikimate-mediated secondary
metabolisms with enhanced biosynthesis of aromatic amino acids. Therefore, salinity caused systems alterations in widespread
metabolic networks involving transamination, TCA cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis,
shikimate-mediated secondary metabolisms, and the metabolisms of choline, pyrimidine, and purine. These findings provided new
insights for the tobacco metabolic adaptation to salinity and demonstrated the NMR-based metabonomics as a powerful approach
for understanding the osmotic effects on plant biochemistry.
KEYWORDS: metabonomics, salinity, tobacco plants, NMR, multivariate data analysis
1. INTRODUCTION
Salinity is a major adverse environmental factor for plant
growth limiting the utilization of about 830 million ha of
agriculture land globally, among which about 80 million ha of
irrigated and dryland agriculture are seriously affected worldwide.1 This is one of the most important land resource problems
for the global food production especially with respect to the
rapidly increasing demands due to global population growth. The
ultimate solution to such problems is probably the development
of salt-tolerant plants based on comprehensive understandings of
the salinity effects on plant biochemistry and plant adaptation
mechanisms in systems level.
Salinity has detrimental effects on almost all aspects of plants
including seed germination, plant development and growth.
These effects are related to activation of salinity-induced molecular networks involved in stress perception, signal transduction,
regulations of stress-related genes, protein expressions and
subsequently metabolisms. For example, salt stress alters many
kinase-based signal transduction pathways of plant cells such as
r 2011 American Chemical Society
the mitogen-activated and calcium-dependent protein kinases,
glycogen synthase kinase and histidine kinase signaling.2 It is also
known that salinity disturbs the ion and osmotic homeostasis,
induces oxidative stresses, affects plant hormone biosynthesis
and alters metabolisms such as photosynthesis.2
More recently, transcriptomic and proteomic analyses indicated
that salt stress induced complex plant biological changes in the
systems level. It is particularly interesting to note that there are
some common responses of gene expressions and protein regulations for both the model and crop plants which are associated
not only with functions in terms of cell development and selfprotection, signal transduction and material transportation but also
with metabolisms.3-5 For example, salt stress induced more than
2-fold expression changes in more than two thousand Arabidopsis
genes, which accounted for about 30% of its genome although
the functions of many such genes remained unknown.6 Salt stress
Received: November 15, 2010
Published: February 16, 2011
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also resulted in significant changes for 65 protein spots of Physcomitrella patens with functions related to plant cell development, selfprotection, signal transduction, ion homeostasis and metabolisms.7
Salt sensitive and tolerant wheat cultivars showed significant
differences in more than 100 root protein spots8 and about half of
them were associated with signal transduction, transportation,
chaperone functions and plant cell metabolisms.8 Nevertheless,
the differences in salinity responses for different species or cultivars
imply species/cultivar-dependent salinity adaptation strategies
although such dependence remains to be thoroughly investigated.
Metabonomic analysis ought to have an important role to play
in understanding the molecular responses to salinity since
metabonomics is the branch of science concerned with the
metabolite complement (metabonome) of an integrated biological system and its holistic responses to both endogenous and
exogenous factors.9-11 Practically, metabonomics involves detecting and quantifying the metabolic changes with techniques
such as NMR spectroscopy and mass spectrometry and mining
the resultant data with multivariate statistical techniques such as
principal component analysis (PCA) and orthogonal signal
correction projection to latent structure discriminant analysis
(OPLS-DA).12,13 Being able to simultaneously detect all 1H
containing metabolites with concentrations above tens of micromolar level, the NMR-based metabonomic analysis has already
been successfully applied in studies of the stress effects on both
mammals13-15 and plants16-18 and become a powerful tool in
plant pathophysiological studies.11,19,20
A number of previous studies indicated that the plant metabolic responses were critically important for plant adaptation and
salt-tolerance. Five terrestrial plant species were examined using
1
H NMR combined with gas-chromatographic methods with 41
metabolites confidently determined under different salinity
treatments.21 It was found that salt stress led to significant
alterations in glucose, malate and proline levels in grapevines
implying disturbed energy metabolism, photosynthesis and
osmolyte biosynthesis.22 Short-term salt stress to Arabidopsis
thaliana cell cultures induced changes in the methylation cycle,
the phenylpropanoid pathway for lignin production and glycinebetaine (GB) biosynthesis whereas the long-term stress induced changes in glycolysis and sucrose metabolism.23 Recent
results also showed that the effects of drought and salt stresses on
shoots and roots of two rice cultivars were highlighted by a
significant accumulation of amino acids and sugars and clear
differences were present between two rice cultivars.24
The accumulation of so-called “compatible” osmolytes, such
as glucose, fructose, myo-inositol, proline, GB and γ-amino-butyrate
(GABA), appears to be a common plant metabolic response to
salinity25 to maintain osmotic balance and protect protein structures. However, interspecies even intercultivar differences are apparently present in terms of producing the combination of such
“compatible” osmolytes under salinity.26-28 Consequently, it remains to be clarified whether the pathways producing particular
osmolytes or the osmolytes themselves are more important to plant
tolerances to osmotic stresses.29 Further holistic metabonomic
studies are clearly warranted on different species in terms of their
global and dynamic metabolic responses to salinity.
Tobacco (Nicotiana tabacum) has been employed as one of the
most common models in developing salt tolerant plants by introducing osmoprotectant genes coding biosyntheses of GB,30 proline31
and mannitol.32 Consequently, there were a number of classical
metabolism studies carried out on the cultured tobacco cells in the
context of salinity or its resistances33,34 and some preliminary
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metabonomics studies on tobacco plants have also been reported.35
However, there has been no work, for the time being, published on
comprehensive investigations on the whole-plant metabonomic
responses of tobacco to salinity in terms of salt dosages and durations.
In this work, therefore, we systematically analyzed the metabonomic features of tobacco plants and their dynamic responses
to salt stresses using 1H NMR spectroscopy in conjunction with
multivariate data analysis. The objectives of such study are to
further define the metabonome of tobacco plants and its dynamic
changes associated with salt stress as a function of stress dosages
(salt concentrations) and durations.
2. MATERIALS AND METHODS
2.1. Chemicals
Sodium chloride, K2HPO4 3 3H2O and NaH2PO4 3 2H2O (all
in analytical grade) were purchased from Guoyao Chemical Co.
Ltd. (Shanghai, China) and used without further treatments.
D2O (99.9% in D) and sodium 3-trimethlysilyl [2,2,3,3-D4]
propionate (TSP) were purchased from Cambridge Isotope
Laboratories (Miami, FL). Phosphate buffer (100 mM, pH 7.4)
was prepared in H2O containing 10% D2O to provide an NMR
field lock and 0.02 mM TSP as an internal reference, where
K2HPO4/NaH2PO4 were employed for their good solubility and
storage stability.36
2.2. Plant Growth Conditions and Salt Stress Treatments
Seeds of a salt susceptible tobacco cultivar, Nicotiana tabacum
L. cv Xanthi, were sterilized in 0.5% hypochlorite solution for
5 min in an eppendorf (EP) tube. Following 3 times rinsing with
sterilized distilled water, the seeds were germinated on Murashige and Skoog (MS) basal agar medium.37 Three weeks after
germination, the seedlings were transferred into sterilized sands
and grown in a growth chamber at 26 °C with 70% relative
humidity and light (150 μmol photons m-2 s-1) on a cycle of
16 h light (08:00-24:00) and 8 h dark (0.00-8:00). When grew
to the 4-5 leave stage (∼2 months postgermination), the plants
were transferred into 15-ml glass centrifuge tube containing
15 mL of autoclaved 1/10 MS basal solution (i.e., one-tenth
of the original concentration of MS) in a growth chamber for
3 days. A stepwise increase of external NaCl was applied to
tobacco plants to induce different severities of salt stress. In
details, the hydroponic solution was changed to fresh 1/10 MS
solution with 50 mM NaCl for one day, then changed to 1/10 MS solution with 500 mM NaCl for one week. The aerial parts of
treated plants were respectively collected (at around 11:00 a.m.)
for each biological replicate one day after 50 mM NaCl treatment,
followed with 500 mM NaCl treatment for 1 day, 3 and
7 days. Plants without salt treatment were used as controls. Each
group has 10 independent plants as replicates. Samples were snapfrozen in liquid nitrogen and stored at -80 °C until further processing.
2.3. Sample Extraction Procedures
To observe possible differences resulting from different extraction solvents, plant samples were extracted with CH3OH/
H2O (1:1) and with phosphate buffer directly38 without addition
of EDTA. In both cases, each sample was ground in liquid
nitrogen with a mortar and a pestle followed by lyophilization for
about 24 h. About 25 mg of freeze-dried materials was added with
1 mL aqueous methanol (50%) or precooled phosphate buffer
(4 °C, 0.1 M, pH7.4) and agitated in a 2-mL EP tube with a vortex
at room temperature for 30 s followed with 3 min intermittent
sonication (1 min sonication and 1 min break, repeated for
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3 times) in an ice bath. After 5 min centrifugation (16 100 g) at
4 °C, 600 μL of the supernatant from buffer extracts was
transferred into a 5 mm NMR tube for NMR analysis; whole
process took less than 1 h. For aqueous methanol extraction,
insoluble residues were further extracted twice using the same
procedure and supernatants from three extractions were combined. Following removal of methanol under vacuum for 20 h,
the supernatants were lyophilized in a freeze-drier for at least
24 h.18 Then the powder (about 2 mg) were added with 500 μL
100% D2O together with 100 μL phosphate buffer (PB) containing 10% D2O and 0.02 mM TSP. After 5 min centrifugation
(16 100 g) at 4 °C, 550 μL of the supernatant was transferred
into a 5 mm NMR tube for NMR analysis. No obvious insoluble
matters can be observed in the EP tubes. Two extraction blanks
were always added in parallel during extraction.
Some fresh plant samples were also extracted with buffer without
pre- or postextraction lyophilization treatments to assess possible
presence of volatile metabolites. In such case, extraction procedures
were similar to the above but with supernatants directly transferred
into NMR tubes followed with immediate data acquisition (taking
less than 20 min) without any drying treatment.
2.4. NMR Measurements
All 1H NMR spectra were recorded at 298 K on a Bruker AVIII
600 NMR spectrometer (600.13 MHz for 1H) equipped with a
5 mm inverse cryogenic probe (Bruker Biospin, Germany). A
standard one-dimensional pulse sequence noesypr1d (recycle delay-90°-t1-90°-tm-90°-acquisition) was used to obtain metabolic
profiles of plant extracts with the 90° pulse length of about 10 μs
and t1 of 3 μs. Water suppression was achieved with a weak
irradiation during the recycle delay (RD, 2 s) and mixing time
(tm, 100 ms). 64 transients were collected into 32 768 data points for
each spectrum with a spectral width of 12 kHz. An exponential
window function with line-broadening factor of 0.5 Hz was applied to
free induction decays (FIDs) prior to Fourier transformation (FT).
For resonance assignment purposes, 1H-1H TOCSY,
1
H-1H COSY, 1H-13C HSQC and 1H-13C HMBC
2D NMR spectra were acquired as previously reported16,43 for
selected samples. In COSY and TOCSY experiments, 48 transients were collected into 2048 data points for each of 256
increments with the spectral width of 10 ppm for both dimensions. Phase insensitive mode was used with gradient selection
for the COSY experiments whereas the well-known MLEV-17
was employed as the spin-lock scheme in the phase sensitive
TOCSY experiment (TPPI) with the mixing time of 100 ms.
1
H-13C HSQC and HMBC NMR spectra were recorded using
the gradient selected sequences with 200 transients and 2048
data points for each of 128 increments. The spectral widths were
6313 Hz for 1H and 26 410 Hz for 13C in HSQC (33 202 Hz in
HMBC) experiments. The data were Fourier transformed into a
4 2k matrix with appropriate apodization functions.
To ensure no changes during the extraction and data acquisition processes, we also investigated the sample stability of
extracts (within 24 h) as a function of time at room temperature
by continuously acquiring 1H NMR spectra of two samples from
both solvents (i.e., phosphate buffer and aqueous methanol). It
took about 5 min to acquire each spectrum. So obtained spectra
were compared directly by scaling to TSP peak assuming no
enzymatic effects on TSP. In this paper, only the data from
aqueous methanol extractions were discussed since the results
from buffer extraction might be potentially affected by enzymic
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activities even though broadly similar conclusions were reached
in terms of salinity-induced changes in metabolic pathways.
2.5. Data Reduction and Multivariate Data Analysis
1
H NMR spectra were manually corrected for phase and
baseline distortions using TOPSPIN (V2.0, Bruker Biospin)
and the spectral region of δ0.5-9.5 was uniformly integrated
into 3166 buckets with width of 0.003 ppm (1.8 Hz) using the
AMIX package (v3.8.3, Bruker Biospin). The region δ4.67-5.15
was discarded to eliminate the effects of imperfect water presaturation. The spectral areas of all buckets were normalized to
the weight of extracts employed for measurements. Principal
component analysis (PCA) was carried out on the meancentered NMR data with the software package SIMCA-Pþ
(v11.0, Umetrics, Sweden). The orthogonal projection to latent
structure with discriminant analysis (OPLS-DA)39 was carried out
using the NMR data as X-matrix and class information as Y-matrix
with unit variance scaling and 7-fold cross-validation. The model
qualities were assessed with the total explained variables (R2X
values) and the model predictability (Q2 values) followed with
rigorous permutation tests40 with the permutation number of 200.
In both cases, the results were visualized with scores plots
where each point represented a sample’s metabonome. Loadings
plots from OPLS-DA results showed variables (i.e., metabolites)
contributing to the group differences. The loadings obtained
from OPLS-DA were back-transformated12 and color-coded
with the absolute values of coefficients (|r|) using an in-house
developed Matlab script. In such a coefficient plot, the observed
phase (positive or negative) of the resonance signals represents
the relative changes (rise or decline) in the concentration of
metabolites. The color indicated the significances of variables
contributing to the intergroup discrimination with hot colored
(e.g., red) variables showing more significant contribution than
the cold colored (e.g., blue) ones. The statistically significant
changes of metabolites were obtained with the coefficient values
based on the discrimination significance at the level of p < 0.05,
which was determined according to the discriminating significance of the Pearson’s product-moment correlation coefficient.39
Absolute levels of metabolites were calculated, as milligram
per gram freeze-dried plant extracts, from the least overlapping
NMR signals of metabolites and TSP with known concentration
assuming little intersample variations of spin-lattice relaxation
time for the same protons.16 These semiquantitative data were
expressed in the form of mean ( standard deviation and were
also subjected to classical one-way ANOVA analysis using SPSS
13.0 software with a Turkey post-test (p < 0.05). The ratios of
metabolite changes during the entire salt stress process were also
calculated against the controls, i.e., [Ci - CA]/CA, where Ci and
CA stand for the concentration in the salt stress sample i (Group B,
C, D and E) and in the control tobacco (Group A), respectively.
3. RESULTS
3.1. Assignments for the Metabolites of Tobacco Plants
Using NMR Spectroscopy
Figure 1 shows 1H NMR spectra of the aqueous methanol
extracts from the tobacco plants treated with no salt (A), 50 mM
NaCl for 1 day (B), 50 mM NaCl for 1 day followed with
500 mM NaCl for 1 day(C), 3 days (D) and 7 days (E),
respectively. The metabolite resonances were assigned for both
1
H and 13C data (Table S1) based on the literature data,41,42 our
in-house databases and publicly available databases.43 These
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Figure 1. Typical 600 MHz 1H NMR spectra of aqueous methanol extracts from tobacco plants treated with (A) no salt, (B) 50 mM for 1 day,
(C) 50 mM for 1 day followed with 500 mM for 1 day, (D) for 3 days, and (E) for 7 days. Keys: 1, Leucine; 2, Valine; 3, Isoleucine; 4, Lactate; 5, Alanine;
6, γ-Amino-butyrate; 7, Acetate; 8, Glutamate; 9, Glutamine; 10, Proline; 11, Dimethylamine; 12, Aspartate; 13, Asparagine; 14, Ethanolamine;
15, Choline; 16, Methanol; 17, Threonine; 18, Fructose; 19, Malate; 20, Galactose; 21, β-Glucose; 22, R-Glucose; 23, Tyrosine; 24, Sucrose; 25, Uridine;
26, Fumarate; 27, Phenylalanine; 28, Formate; 29, N-Methylnicotinamide; 30, Nicotine; 31, Histidine; 32, Tryptophan; 33, Allantoin; 34,
R-Ketoglutarate; 35, Succinate; 36, Uracil; 37, myo-Inositol; 38, Dimethylglycine; 39, Methylamine; 40, Hypoxanthine; 41, Arginine 42, Unknown.
assignments were further confirmed with extensive 2D NMR
data from COSY, TOCSY, HSQC and HMBC spectra. It is
apparent that the tobacco metabonome is dominated by 16
amino acids, 5 carbohydrates, 15 organic acids/amines, 5 nucleotide derivatives and 1 unknown metabolite (Figure 1 and Table S1,
Supporting Information). Among them, the contents of sucrose,
glucose (Glc), fructose (Fruc), myo-inositol, proline (Pro), asparagine (Asn), aspartate (Asp), glutamine (Gln), γ-aminobutyrate
(GABA), malate and nicotine in some samples were above 1 mg per
gram freeze-dried plant samples (Table 1). We did not detect
chlorogenic acid in our extracts as reported in a previous work35
probably due to different cultivars employed.
It is also important to note that within 8 h at ambient, no
significant metabolite level changes are observable for the
tobacco extracts from both solvents employed here (data not
shown). However, obvious changes can be observed for the
buffer extracts when standing for 12 h or longer at room
temperature though only slight such changes can be observed
for aqueous methanol extracts after 24 h at room temperature.
This means that the durations of extraction and data acquisition
have to be well controlled for plant studies. Furthermore, even
with good control of such durations, levels of some metabolites
(e.g, proline and sucrose) in the phosphate-buffer extracts
differed, to some extent, from these in the aqueous methanol
extracts. This is probably due to solubility-limited extraction efficiency rather than enzymatic effects being in good agreement with
previous findings from other plants.17,44 Moreover, a singlet of
methanol (3.36 ppm) was clearly visible in the extracts of fresh
samples from buffer extracts without pre- or postextraction lyophilization (data not shown) whereas such a signal was much less
intense in aqueous methanol extracts probably due to freeze-drying.
This implies that cares have to be taken for the sample extractions
if methanol (or ethanol) related metabolisms are important. This
is particularly important in the case of LC-MS analysis even
without lyophilization steps.
Nevertheless, Figure 1 showed clear metabolic changes for
tobacco plants under both short-term low-dose sodium chloride
(50 mM for 1 day) and the prolonged stress with high-dose NaCl
(500 mM for 1-7 day). It is worth noting that treatments with
50 mM salt cause little phenotypic changes, in the morphological
level, for the tobacco plants whereas treatments with 500 mM salt
result in withering and growth retardance (Figure S1 in Supporting Information). Direct visual inspection can readily reveal that
the most obviously changed metabolites included sucrose and
proline in samples treated with 500 mM salt for 3-7 days
(Figure 1D and E) compared with control sample (Figure 1A).
In contrast, the low-dose salt stress led to level increases for
sucrose and glucose with little changes for proline (Figure 1B).
To obtain the detailed metabonomic changes caused by salt
stresses, multivariate data analyses were applied to the NMR data.
3.2. Metabonomic Trajectory for the Salinity-Induced Responses of Tobacco Plants
The scores plot from principal component analysis (PCA)
(Figure 2) shows that more than 85% variables can be explained
with two principal components for all five groups of samples (AE). A clear stress-induced trajectory for tobacco metabonomic
changes is evident indicating the dosage dependence and dynamic responses of tobacco plants to salt stress (Figure 2). This
further implies that 1H NMR-based metabonomic method is
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Table 1. Correlation Coefficients from OPLS-DA and the Metabolite Content in Extracts of Tobacco Plants under Different Salt
Stressa
metabolitesb
coefficient(r)c
ppm
B/A
C/B
D/C
metabolite quantity (mean ( SD, mg/g freeze-dried plants)
E/D
A
B
C
D
E
Amino acids
Pro 4.14
0.21
0.84
0.84
0.40
1.83 ( 0.5
2.28 ( 0.7
3.39 ( 0.9
10.01 ( 3.1d,e,f,g
Asn 2.87
Asp 2.82
0.52
0.44
0.82
-0.88
0.78
-0.37
-0.35
0.46
1.26 ( 0.3
1.09 ( 0.2
1.64 ( 0.5
1.17 ( 0.2
1.91 ( 0.5
0.8 ( 0.2d,e,f,g
11.71 ( 3.4
3.41 ( 1.0d,e,f,g
0.78 ( 0.2
3.05 ( 1.1
0.86 ( 0.2
Gln 2.45
0.67
-0.90
-0.82
-0.48
6.16 ( 1.5
7.28 ( 1.7
4.33 ( 1.5d,e,f,g
3.6 ( 1.0
2.46 ( 0.4
Ala 1.48
-0.11
-0.88
0.56
0.60
0.52 ( 0.1
0.58 ( 0.1
0.56 ( 0.1
0.74 ( 0.2
0.92 ( 0.2
Val 1.04
0.12
0.93
0.67
-0.55
0.1 ( 0.0
0.13 ( 0.0
0.25 ( 0.1d,e,f,g
0.33 ( 0.1d,e,f,g
0.33 ( 0.1
Ile 1.01
0.08
0.92
0.62
0.54
0.11 ( 0.0
0.14 ( 0.0
0.34 ( 0.1d,e,f,g
0.37 ( 0.1
0.37 ( 0.1
Phe 7.42
0.23
0.84
0.50
-0.56
0.31 ( 0.0
0.34 ( 0.1
0.53 ( 0.2d,e,f,g
0.63 ( 0.2
0.58 ( 0.1
His 7.09
0.30
0.14
0.38
0.62
0.2 ( 0.1
0.22 ( 0.1
0.23 ( 0.1
0.32 ( 0.1
0.39 ( 0.1
Trp 7.74
GABA 3.02
0.35
0.47
0.90
-0.93
0.76
-0.87
-0.38
0.61
0.13 ( 0.0
3.86 ( 1.2
0.15 ( 0.0
4.03 ( 1.3
0.22 ( 0.1
3.54 ( 1.1
0.42 ( 0.1d,e,f,g
2.65 ( 0.9
0.37 ( 0.2
4.7 ( 1.0
Tyr 7.19
0.44
0.92
-0.49
-0.40
0.12 ( 0.0
0.14 ( 0.0
0.24 ( 0.1d,e,f,g
0.28 ( 0.1
0.24 ( 0.1
Suc 5.42
0.84
0.95
-0.84
-0.86
0.4 ( 0.1
17.59 ( 3.7d,e,f,g
14.89 ( 3.8
Carbohydrates
4.7 ( 1.8d,e,f,g
3.47 ( 2.4d,e,f,g
Glc 5.24
0.64
-0.78
-0.68
0.49
3.65 ( 1.5
4.48 ( 1.4
2.33 ( 1.0
Fruc 3.81
0.68
-0.71
-0.60
-0.52
19.2 ( 6.4
24.08 ( 5.8
21.26 ( 6.1
mIno3.29
0.33
0.88
0.75
-0.10
1.85 ( 0.3
2.29 ( 0.5
2.76 ( 0.7
3.78 ( 0.9d,e,f,g
3.56 ( 0.6
Fum 6.52
Mal 4.32
0.58
-0.66
-0.60
-0.39
-0.77
-0.68
-0.48
0.61
0.04 ( 0.0
3.73 ( 0.8
0.04 ( 0.0
2.28 ( 0.7d,e,f,g
0.03 ( 0.0d,e,f,g
1.95 ( 0.6
0.02 ( 0.0
1.48 ( 0.2
0.01 ( 0.0
1.79 ( 0.3
Succ2.42
-0.20
0.65
-0.56
0.44
0.39 ( 0.1
0.37 ( 0.1
0.45 ( 0.1
0.33 ( 0.1
0.37 ( 0.1
0.09 ( 0.0
0.19 ( 0.0d,e,f,g
d,e,f,g
1.63 ( 0.5
2.17 ( 0.7
17.74 ( 4.3
15.08 ( 3.2
TCA cycle
Nucleotide derivatives
Ura 5.80
-0.46
-0.79
0.69
0.77
0.08 ( 0.0
0.08 ( 0.0
0.05 ( 0.0
Uri 5.91
-0.70
-0.96
-0.88
0.71
0.17 ( 0.0
0.15 ( 0.0
0.1 ( 0.0
hXan 8.22
-0.62
-0.93
-0.66
0.63
0.16 ( 0.0
0.14 ( 0.0d,e,f,g
Nico7.60
NMNN 9.27
Allan 5.39
d,e,f,g
0.12 ( 0.0
0.07 ( 0.0
0.14 ( 0.0
0.09 ( 0.0
0.11 ( 0.0
0.39
0.80
-0.72
0.35
1.07 ( 0.2
1.13 ( 0.3
1.31 ( 0.8
0.91 ( 0.7
1.08 ( 0.4
-0.39
0.53
-0.72
0.83
-0.71
0.76
-0.10
0.42
0.11 ( 0.0
0.16 ( 0.0
0.1 ( 0.0
0.23 ( 0.1
0.08 ( 0.0
0.35 ( 0.1
0.07 ( 0.0
0.53 ( 0.1d,e,f,g
0.05 ( 0.0
0.58 ( 0.2
Others
MA 2.62
-0.12
-0.25
-0.33
0.36
0.04 ( 0.0
0.04 ( 0.0
0.03 ( 0.0
0.02 ( 0.0
0.02 ( 0.0
DMA 2.74
-0.57
-0.73
-0.36
0.64
0.09 ( 0.0
0.08 ( 0.0
0.07 ( 0.0
0.05 ( 0.0
0.06 ( 0.0
0.16 ( 0.0
EA 3.15
-0.50
-0.83
-0.74
-0.21
0.19 ( 0.0
0.18 ( 0.0
0.18 ( 0.0
0.18 ( 0.0
Cho 3.20
-0.47
-0.99
-0.70
-0.26
0.88 ( 0.1
0.79 ( 0.1
0.47 ( 0.1d,e,f,g
0.19 ( 0.0d,e,f,g
0.12 ( 0.0
Form 8.46
-0.55
0.76
-0.61
0.58
0.01 ( 0.0
0.01 ( 0.0
0.02 ( 0.0
0.01 ( 0.0
0.05 ( 0.0
Tobacco plants were stressed by 0 mM (A), 50 mM NaCl for 1 day (B), 50 mM NaCl for 1 day followed with 500 mM NaCl for 1 day(C), for 3 days (D)
and for 7 days (E). b Consult abbreviations for these metabolites. c Coefficients from OPLS-DA results, positive and negative signs indicate positive and
negative correlation, respectively. The cutoff value 0.60 was used for the significant difference (p < 0.05). d,e,f,g significant differences from one-way
ANOVA (p < 0.05) between B and A, C and B, D and C, E and D respectively . ND: not determined due to small quantity or peak overlapping.
a
powerful and efficient for depicting the physiological states of
salt-stressed tobacco plants. It is also interesting to notice the
close intragroup sample clusters suggesting good reproducibility
in the extraction procedures and NMR measurements.
3.3. Tobacco Metabolic Responses to Short-Term Salt Stress
To obtain the detailed information on salt-induced metabolic
alterations and the significance of metabolites contributing to the
alterations, pairwise comparative OPLS-DA was conducted with
one orthogonal and one predictive component calculated for all
models derived from two classes of samples. The metabolites
showing significant level changes were tabulated in Table 1
together with the metabolites’ concentration data expressed as
mg/g freeze-dried powder. In this study, a correlation coefficient
of |r| > 0.60 (i.e., r > 0.60 or r < -0.60) was used as the cutoff
value for the statistical significance based on the discrimination
significance at the level of p < 0.05.39
The scores plot of OPLS-DA results showed clear separation
between the tobacco plants stressed with 50 mM salt for 1 day
and controls (Figure 3a) with good model quality (R2X = 0.76,
Q2 = 0.91). Such differences were also evident (Figure 3b)
between the 50 mM salt prestressed for 1 day and followed with
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500 mM for another day (R2X = 0.68, Q2 = 0.91). The validities
of these models were further confirmed by rigorous permutation
tests with 200 permutations (Figure S2, Supporting Information). The coefficient-coded loadings plots indicated that
50 mM salt treatments (for 1 day) induced marked metabonomic
alterations for tobacco plants; such changes were highlighted
with the significant elevation of sucrose, glucose, fructose and
glutamine accompanied with decreases of malate, hypoxanthine
and uridine. Proline level was increased although not statistically
significant with p < 0.05 (Figure 3a, Table 1).
Following the prestress, further stress with 500 mM NaCl (for
1 day) led to level increase for sucrose, myo-inositol, Pro, Asn,
Val, Ile, Phe, Trp, Tyr, succinate, nicotine, formate and allantoin
together with decrease of Asp, Ala, GABA, choline, EA, DMA, Nmethylnicotinamide (NMNN), hypoxanthine, uracil and uridine. However, this additional stress also led to reverse changes
in the levels of Glc, Fruc, Gln and fumarate whereas the malate
level was not significantly changed (Figure 3b, Table 1).
3.4. Tobacco Metabolic Responses to Long-Term High Dose
Salt Stress
The salinity induced progressive metabonomic alterations
showed duration dependence (from 1 to 7 days) for the group
Figure 2. PCA scores plot showing the metabonomic trajectory for
tobacco plants treated with (A) no salt, (B) 50 mM for 1 day, (C) 50 mM
for 1 day followed with 500 mM for 1 day, (D) for 3 days, and (E) for
7 days.
ARTICLE
treated with 500 mM NaCl. Significant metabonomic differences
were evident for tobacco stressed for 1 (Group C) and 3 days
(Group D), and for 3 (Group D) and 7 days (Group E) with
good model qualities in both cases (Figure 4) which were further
confirmed with permutation tests (with 200 permutations)
(Figure S2, Supporting Information). Compared with one-day
treatment, three-day treatment with 500 mM salt caused significant elevation of Pro, Asn, Val, Ile, Trp, myo-inositol, uracil
and allantoin together with reduction of sucrose, glucose,
fructose, Gln, GABA, malate, fumarate, choline, EA, uridine,
hypoxanthine, nicotine, NMNN and formate (Figure 4a,
Table 1). Such salt treatments for further 4 days led to almost
two-thirds level decline for sucrose and significant elevation of
Ala, GABA, malate, hypoxanthine, uracil and uridine (Figure 4b,
Table 1).
Semiquantitative data (Table 1) showed that the progressive
elevation of proline was associated with the increased duration of
salt stress together with the decrease of choline, EA and NMNN.
Sucrose responded more drastically than any other metabolites
by showing rise of more than 40 folds under persistent salinity
(than controls) even though prolonged high-dose salt actually
suppressed its level to large extent (about 4-folds from day 3 to
day 7 under 500 mM NaCl) (Figure 4b). Such dynamic
metabonomic changes induced by salt were reported for the first
time, to the best of our knowledge, and clearly involved a
complex network. On the basis of the quantification results
(Table 1), the above changes were more clearly illustrated with
the ratios of metabolite-concentration changes (against controls)
(Figure 5) for the transamination-related metabolites (Asn, Gln
and GABA), cell membrane-related metabolites (choline and
EA), sugars (sucrose and Glc), osmolytes (proline and myoinositol) and shikimate-mediated metabolites (Phe and Trp).
4. DISCUSSIONS
The above results suggest that the NMR-based metabonomic
analysis is an excellent information-rich approach for understanding the stress-induced dynamic plant biochemical changes
in the systems level and for pinning down the important metabolic
pathways which may play vital roles in plant adaptation to salinity.
Our results have shown the presence of a salinity-induced plant
Figure 3. OPLS-DA scores and loadings plots showing dose-dependence of salinity effects on tobacco metabolism. (a) No salt treatment (A) vs 50 mM
for 1 day (B); (b) 50 mM for 1 day followed with 500 mM for 1 day (C) vs 50 mM 1 day (B). Metabolite keys are the same as in the legend of Figure 1.
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ARTICLE
Figure 4. OPLS-DA scores and loadings plots showing time-dependence of salinity effects on tobacco metabolism; (a) 50 mM for 1 day followed with
500 mM for 1 day (C) vs for 3 days (D); (b) 500 mM treated for 3 days (D) vs 500 mM treated for 7 days (E). Metabolite keys are the same as in the
legend of Figure 1.
and adaptation to the salinity stress. To the best of our knowledge, this is the first report of the study on the dynamic
metabonomic responses to salt stress for tobacco plants in terms
of dosages and durations stress. Based on the metabolites
showing significant changes, the metabolic pathways of tobacco
plants responding to salt stress were highlighted (Figure 6) and
further discussed.
4.1. Short-Term Salinity Induced Metabonomic Alterations
in Tobacco Plants
Figure 5. Salinity-induced metabolite concentration changes against
these in controls for tobacco plants treated with (A) no salt, (B) 50 mM
NaCl for 1 day, (C) 50 mM for 1 day followed with 500 mM NaCl for
1 day, (D) for 3 days and (E) for 7 days respectively. The ratios of
metabolite changes were calculated against the controls, that is, [Ci CA]/CA, where Ci and CA stand for the concentration in the salt stress
sample i (Group B, C, D and E) and in the control tobacco (Group A),
respectively.
metabonomic trajectory from 50 to 500 mM salt stresses and
further to the increased stress duration (Figure 2). Such doseand time-dependences probably indicate the presence of a
progressive development axis for the plant metabolic responses
to the salinity severity. The results have also indicated that
salinity causes profound biochemical alterations to many metabolic processes of the salt-susceptible tobacco plants, including
transamination, glycolysis/gluconeogenesis, TCA cycle and
photosynthesis, glutamate-mediated proline biosynthesis, choline metabolism, shikimate-mediated metabolisms, and pyrimidine and purine metabolisms (Figure 6). This revealed greater
details on the salinity induced metabonomic changes than the
literature reported for tobacco cells and seedlings.45,46 Moreover,
this progressive development of metabolic responses was probably related to the stress management of plants to osmotic shocks
Salinity often induces generation of reactive oxygen species
(ROS), such as H2O2 and O2 3 -, and causes protease activation
and intracellular hyperammonia.47 To avoid the hyperammonia
caused cytotoxicity, plant cells normally react by either transforming ammonium ions into transamination metabolites (e.g.,
Asn, Asp, Glu and Gln) with asparagine synthetase (AS),
aspartate aminotransferase, glutamine synthetase/synthase
(GS/GOGAT) and glutamate dehydrogenase (GDH), which
is abundant in plant tissues,48 involving TCA cycle intermediates,
2-oxoglutarate and oxaloacetate. Glutamate can further be converted into proline with Δ1-pyrroline-5-carboxylate synthetase
(P5CS).
Our results demonstrated that short-term 50 mM salt treatment (for 1 day) induced preferential accumulation of Gln, Glc,
Fruc and especially sucrose with an increase of more than an
order of magnitude (Table 1 and Figure 5) together with
significant decreases of malate, uridine and hypoxanthine. Proline showed elevation to some extent though without statistical
significance. Such accumulation of sucrose, glucose and fructose
was also observed in Actinidia seedlings49 and tomato50 under
short-term salt stresses. In plant cells, sugars such as glucose,
fructose and sucrose were derived from photosynthesis, gluconeogenesis and degradation of polysaccharides. Since the apparent photosynthetic rate was reported to be similar in both control
and 50 mM NaCl treated tobacco during the first 3 days,51 our
results implied that gluconeogenesis was promoted under such lowdose and short-term salinity with the transamination products fed
into TCA cycle. The short-term salinity induced decreases of TCA
cycle intermediate, malate, also supports the notion of gluconeogenesis promotion probably as salt-shock effects. Such responses
not only efficiently relieved the salt-induced hyperammonia but
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ARTICLE
Figure 6. Metabolic changes for tobacco plants upon salt stress obtained from OPLS-DA analysis. The proposed metabolic pathways were based on
web-based metabolic pathway database MetaCyc (http://www.metacyc.org) and literatures.59,60 Metabolites with red boxes denote significant increases
while with green ones denote significant decreases. The bold-lettered metabolites were detected in this study. The level of significance was set at p < 0.05.
Metabolite identities are listed in the abbreviation list.
also generated sucrose, glucose and fructose, which probably
function as carbon store, ROS scavengers and compatible
osmolytes to maintain osmotic balance. The depletion of uridine
here may also result from the salinity-induced gluconeogenesis
since uridine is an intermediate in pyrimidine catabolism linked
with UDP-glucose, glutamine and pentose phosphate pathway
(via 5-phosphoribosyl 1-pyrophosphate) through UMP. Hypoxanthine in purine metabolism is more distantly linked to glutamine and pentose phosphate pathway via IMP. Nevertheless, the
decreases of uridine and hypoxanthine further suggest that shortterm salinity probably also causes alterations of DNA and RNA
biosynthesis/degradation since they are catabolic intermediates
of pyrimidines and purines, respectively.
Following one-day treatment with 50 mM NaCl, further stress
to tobacco with 500 mM salt for another day led to significant
elevation of proline with about 50% concentration increase implying
that such treatment probably enhances the 1-pyrroline-5-carboxylate (P5C) mediated biosyntheses of proline. Such enhancement of
proline biosynthesis is a typical response of plant cells to osmotic
stress to provide an extra compatible osmolyte, storage for carbon
and nitrogen, scavenger for ROS and regulator for intracellular
pH.52,53 Further elevation of sucrose and decrease of transamination-related metabolites (Asp, Gln and GABA) (Table 1, Figure 5)
induced by such treatment indicates that both sugars and glutamatemediated proline biosynthesis (Figure 6) are important for controlling salinity induced osmotic pressure.
However, this additional stress with 500 mM (for one day) led
to clear elevation of myo-inositol and reverse changes in the levels
of glucose and fructose. Similar observation of changes for myoinositol has been made for the salt-stressed Actinidia (kiwifruit)
leaves under high-dose salinity conditions.49 myo-Inositol is
normally synthesized from glucose-6-phosphate via myo-inositol-1-phosphate54 and, as one of the polyols, this metabolite may
be employed by plants for the osmotic stress management
purposes especially under salinity. myo-Inositol also has important functions in membrane biosynthesis, membrane protection
as free-radical scavengers, plant cell signaling and the biosynthesis of cell wall components.49 Furthermore, plant cells require
relatively constant amounts of compensating osmolytes as the
amount of Naþ in the plant fluctuates little during light and
dark.49 With the high diurnal dependence for the levels of sugars
including glucose and fructose, the above observation suggests
that myo-inositol may also function as a better carbon storage and
osmolyte than sugars. The level decline of Asp, Gln and GABA is
indicative that the biosynthesis of these osmolytes is probably
also associated with transformation of transamination products
through GABA shunt as well. The changes of Ala, Val and Ile are
probably related to gluconeogenesis as a way of relief of
transamination products since they are glucogenic amino acids
linked to pyruvate metabolism.
Salinity-induced elevation of Tyr, Trp and Phe under such
stress may indicate stress-promoted enhancement for the shikimate-mediated plant secondary metabolisms since these aromatic amino acids are intermediates for biosynthesis of (4hydroxy) cinnamic acid which is the key precursor for secondary
metabolites (e.g., polyphenols) through shikimate pathway.
The level changes for choline and ethanolamine (EA) are
probably related to salinity-induced alterations in membrane synthesis since both EA and choline are intermediates for biosynthesis of
the cell membrane components. The depletion of these metabolites
and the choline-degradation product (dimethylamine) here suggests that salinity may induce inhibition of membrane synthesis or
enhanced membrane degradation. Such changes may also result
from the salinity-induced promotion of gluconeogenesis via glycine
and 3-phosphorylglycerate mediation.
The decrease of uracil, uridine and hypoxanthine suggest that
further salinity with high concentration of salt probably further
causes alterations of DNA and RNA biosynthesis/degradation as
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they are catabolic intermediates of pyrimidines and purines. The
elevation of allantoin further supports such notion since it is a
metabolite of hypoxanthine and xanthine in purine metabolism.
Elevation of nicotine and decrease of N-methyl-nicotinamide
(NMNN) indicate the salinity-induced alterations in nicotianamide metabolism in which nicotine can be generated from
NMNN via nicotinamide and nicotinate. Furthermore, as discussed earlier, the depletion of uracil and uridine and hypoxanthine may also be associated with the salinity-induced
gluconeogenesis since these metabolites are linked with UDPglucose, glutamine and pentose phosphate pathway through
UMP and IMP, respectively.
4.2. Dynamic Metabonomic Responses of Tobacco Plants to
High-Dose Salinity
Metabonomic changes caused by high-dose (500 mM) salt
showed clear duration dependence. It was particularly interesting
to note that proline level was not significantly affected by shortterm 50 mM salt stress but increased consistently upon 500 mM
salt treatment with an increase of more than four folds from oneday to seven-day high-dose (500 mM) salt treatment (Table 1,
Figure 5). Such increase was accompanied with significant level
alterations of transamination related metabolites such as Asn, Gln,
and GABA. Similar results were reported for tobacco suspension
cells that little proline increases were observed under low-dose
short-term external NaCl treatment whereas its level rose sharply
when stressed with salt solution containing above 100 mM NaCl.34
This implies that the glutamate-mediated proline biosynthesis is a
dominant metabolic response to prolonged salt stress since severe
salt stress has been found to promote the expression of glutamate
dehydrogenases in tobacco.48
Proline accumulation is considered as a common metabolic
response of higher plants to water deficits16,17 and salinity stress
by protecting plant cell membranes and proteins and functioning
as a ROS scavenger. In our study, proline was one of the most
significantly changed metabolites for long-term high-dose salt
stress and accumulated even with 500 mM salt treated for 7 days.
Such observations were consistent with findings from tobacco
cell cultures adapted to 428 mM NaCl,33 where proline accounted for more than 80% of free amino acids and its accumulation was mainly due to increased glutamate-mediated biosynthesis.55 Another study showed that the proline level in tobacco
leaves was increased when treated with up to 300 mM NaCl but
decreased when treated with 400 mM NaCl.56 In our study,
however, proline accumulation was consistently noticeable for
tobacco plants when treated with 500 mM salt for 7 days
(Figure 5). Since proline accumulation was observable upon
ionic but not nonionic hyperosmotic stresses in Arabidopsis,57
the differences for proline changes under short-term low-dose
and long-term high-dose treatments suggested the proline accumulation as a metabolic response to long-term ion cytotoxicity
rather than early stage osmotic stress.
The decrease of transamination related metabolites (e.g., Asp,
Gln and GABA) with prolonged high-dose salt stress were
consistent with the diversion of metabolic activities toward
proline biosynthesis. The decrease in the levels of uridine and
hypoxanthine is also supportive to the demands of proline
biosynthesis probably through glutamate/glutamine mediated
routes although these changes may also be related to their
catabolism which is supported with the elevation of the degradation product (allantoin). The elevation of uracil, uridine and
hypoxanthine following seven days treatment with 500 mM salt
ARTICLE
probably indicates that such long-term salinity with high salt
concentration promotes severe degradation of DNA/RNA. The
decrease of N-methylnicotinamide and nicotine suggests that the
prolonged salinity causes alterations in nicotinamide metabolism. Furthermore, the elevation of Ala, Val, Ile and Phe were
probably associated with inhibition of protein biosynthesis or
enhanced protein degradation since the plant growth was clearly
inhibited with prolonged salinity especially after seven days
(Figure S1, Supporting Information). The elevation of aromatic
amino acids (Tyr, Trp and Phe) is probably also related to the
shikimate-mediated secondary metabolism since they are all
precursors for biosynthesis of polyphenols, which function as
plant endogenous antioxidants.
Choline and EA are plant metabolites contributing to the
synthesis of membrane phospholipids, phosphatidylcholines and
phosphatidylethanolamines, which account for more than half of
the lipids in nonplastid plant membranes. Suppression of EA and
choline metabolism might cause restriction of cell membrane
elongations leading to the restriction of tobacco growth under
high salinity stress conditions. Choline is also a precursor for an
effective compatible osmolyte, glycine-betaine (GB). However,
characteristic NMR signal for GB was not observable in our
NMR spectra probably due to the lack of appropriate enzymes
responsible for GB synthesis in tobacco (Nicotiana tabacum) as
reported previously.27 Under such situation, tobacco cells can
only utilize Pro, sucrose and myo-inositol as osmolytes. This also
explains the sustained high sucrose level during salinity although
with some decline around day 7 of treatments.
Moreover, glutamate/glutamine conversion seems to be a
critical check point in osmotic crisis management for plant cells
in terms of both transamination and proline biosynthesis. Gln
showed an obvious level changes in both short-term low-dose salt
stress and long-term high-dose salinity indicating continued
demands for transamination during the salinity to reduce the
risk of hyperammonia-induced toxicity to plant cells. Under such
salt stress, tobacco cells seem to adopt consistent metabolic
responses to prevent hyperammonia by rapidly converting the
transamination products to compatible osmolytes through networks involving extensive and multiple metabolic pathways.
GABA shunt appears to function as one of these pathways. In
this case, GABA was probably not functioning as an osmoregulator and cytosolic pH regulator58 but as an intermediate contributed to carbon-nitrogen balance to relieve hyperammonia
and assist biosynthesis of osmolytes such as sucrose, myo-inositol
and proline. Similarly, the immediate products of transamination,
such as Asn, Asp, Gln and Glu, were all functioning as intermediates of relieving hyperammonia.
To sum up, salinity-induced metabonomic responses for
tobacco plants showed clear severity dependences with completely different responses to different salt stresses in terms of salt
concentrations and durations. Short-term low-dose salt stress
caused preferentially accumulation of sucrose, glucose, fructose
and myo-inositol with more than 10 folds concentration increase
for sucrose (Figure 5). Such changes indicated a possible shift
from nitrogen to carbon flux through transamination, TCA cycle
and further to gluconeogenesis. In contrast, lengthy stresses with
high-dose salt solution led to outstanding proline accumulation
(Figure 5), indicating the importance of gluconeogenesis and
proline biosynthesis for relieving hyperammonia and regulating
osmotic stress. In both cases, nevertheless, relieving hyperammonia via transamination and generating effective compatible
osmolytes seem to be the most important strategies for tobacco
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plants to adapt the salinity conditions. These findings have
revealed systems and dynamic responses of tobacco plants to
various salinity conditions and demonstrated that the NMRbased metabonomics may provide useful information for the
development of salt tolerant plants.
’ ASSOCIATED CONTENT
bS
Supporting Information
Supplemental figures and information on NMR data for the
metabolites of tobacco extracts, representative photographs of
the tobacco plants treated with different salt treatments and
results of permutation tests. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Huiru Tang, e-mail: [email protected]. Tel: þ86-(0)2787198430. Fax: þ86-(0)27-87199291. Shiyun Chen, e-mail:
[email protected]. Tel: þ86-(0)27-87199354. Fax: þ86-(0)2787199354.
Author Contributions
†
These authors equally contributed to this manuscript.
’ ACKNOWLEDGMENT
We acknowledge the financial supports from the Ministry of
Agriculture of China (2009ZX08012-023B) and the National
Natural Science Foundation of China (20825520, 20921004).
We also thank Dr. Hang Zhu of Wuhan Institute of Physics and
Mathematics for developing MATLAB scripts used for color-coded
OPLS-DA coefficient plots, which was originally downloaded from
http://www.mathworks.com/matlabcentral/fileexchange.
’ ABBREVIATION LIST
FID, free induction decay; FT, Fourier transformation; NMR,
nuclear magnetic resonance; OPLS-DA, orthogonal partial leastsquares discriminant analysis; PCA, principal components analysis; 3-PGA, 3-phosphoglycerate; R-KG, R-ketoglutarate; Ade,
adenosine; Ala, alanine; Allan, allantoin; Asn, asparagine; Asp,
aspartate; Cho, choline; Cit, citrate; DMA, dimethylamine;
EA, ethanolamine; Fruc, fructose; F-6-P, fructose-6-phosphate;
Form, formate; Fum, fumarate; GABA, γ-amino-n-butyrate;
G-6-P, glucose-6-phosphate; Gal, galactose; Glc, glucose; Gln,
glutamine; Glu, glutamate; Gly, glycine; His, histidine; Ile, isoleucine; mIno, myo-inositol; Thr, threonine; Leu, leucine; MA,
methylamine; Mal, malate; NMNN, N-methylnicotianamide;
Nic, nicotine; OAA, Oxalacetic acid; P5C, 1-pyrroline-5-carboxylate; PB, phosphate buffer; Phe, phenylalanine; Pro, proline;
Pyr, pyruvate; Shik, shikimate; Suc, sucrose; Succ, succinate; Trp,
tryptophan; Tyr, tyrosine; Ura, uracil; Uri, uridine; Val, valine.
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