Dynamic Metabonomic Responses of Tobacco (Nicotiana tabacum

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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 1904

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Journal of Proteome Research 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 1905

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Journal of Proteome Research 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 1906

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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 1907

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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

metabolite quantity (mean ( SD, mg/g freeze-dried plants)

coefficient(r)c

ppm

B/A

C/B

D/C

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

0.84

0.95

-0.84

-0.86

0.4 ( 0.1

17.59 ( 3.7d,e,f,g

14.89 ( 3.8

Carbohydrates Suc 5.42

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

0.77

0.08 ( 0.0

0.08 ( 0.0

0.05 ( 0.0

0.09 ( 0.0

0.19 ( 0.0d,e,f,g

0.1 ( 0.0

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

Uri 5.91

-0.70

-0.96

-0.88

0.71

0.17 ( 0.0

0.15 ( 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

a

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.

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 1908

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Journal of Proteome Research 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.

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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. 1909

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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 1910

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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 1911

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Journal of Proteome Research 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

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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 1912

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Journal of Proteome Research 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. ’ REFERENCES (1) FAO, Global network on integrated soil management for sustainable use of salt affected soils; FAO: Rome, 2005. (2) Zhu, J. K. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 2002, 53, 247–273. (3) Zhu, J. K. Cell signaling under salt, water and cold stresses. Curr. Opin. Plant Biol. 2001, 4, 401–406.

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