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. 2017 Jun 8;12(7):e1335845. doi: 10.1080/15592324.2017.1335845

Metabolomics analysis of rice responses to salinity stress revealed elevation of serotonin, and gentisic acid levels in leaves of tolerant varieties

Poulami Gupta 1, Bratati De 1,✉
PMCID: PMC5586353  PMID: 28594277

ABSTRACT

A GC-MS based analytical approach was undertaken to understand the metabolomic responses of seedlings of 2 salt sensitive (Sujala and MTU 7029) and 2 tolerant varieties (Bhutnath, and Nonabokra) of indica rice (Oryza sativa L.) to NaCl induced stress. The 4 varieties responded differently to NaCl treatment with respect to the conserved primary metabolites (sugars, polyols, amino acids, organic acids and certain purine derivatives) of the leaf of rice seedlings. However, there were significant differences in salt induced production of chorismic acid derivatives. Serotonin level was increased in both the salt tolerant varieties in response to NaCl induced stress. In both the salt tolerant varieties, increased production of the signaling molecule gentisic acid in response to NaCl treatment was noticed. Salt tolerant varieties also produced increased level of ferulic acid and vanillic acid. In the salt sensitive varieties, cinnamic acid derivatives, 4-hydroxycinnamic acid (in Sujala) and 4-hydroxybenzoic acid (in MTU 7029), were elevated in the leaves. So increased production of the 2 signaling molecules serotonin and gentisic acid may be considered as 2 important biomarker compounds produced in tolerant varieties contributing toward NaCl tolerance.

KEYWORDS: GC-MS, gentisic acid, leaf, metabolomics, rice, salt stress, serotonin

Introduction

High salinity stress is the most severe environmental stress which impairs crop production on at least 20% of irrigated land worldwide.1 More than 30% of world food is estimated to be produced in irrigated land.2 Nearly 10% of the land surface (9.5 × 108 ha) and 50% of irrigated land (2.3 × 108 ha) are salt affected. It is important to improve the salt tolerance of crops in a salinized world with situations of increasing populations, declining crop yields, and a decrease in agricultural lands.3 For this it is necessary to understand the biochemical and metabolic responses of plants to salt stress to exploit genetic resources to develop tolerant varieties.

Rice (Oryza sativa L.), the staple food for more than half of the world's population is the most salt sensitive cereal.4 Seedling emergence and early seedling growth stages are most sensitive to salinity.5-7 Salinity stress response is multigenic, as several processes involved in the tolerance mechanism are affected, such as various compatible solutes/osmolytes, polyamines, reactive oxygen species and antioxidant defense mechanism, ion transport and compartmentalization of injurious ions.8 It has been shown that transcriptional and translational machineries are important determinants in controlling salt stress response.9 Salt stress responses using transcriptomic approaches have been described for rice.10-12 Analysis of changes of the proteome following salt stress in rice has also been reported.13-14 Specific proteins expressed in specific regions of rice show a coordinated response to salt stress.15 A 31 kDa polypeptide was identified as a salt induced protein in rice leaf sheaths of Thai rice cv. Leaung Anan.16 The QTL mapping approach has culminated in finding of the SKC1 protein, and its function in rice salinity tolerance as Na+-selective transporter.17 Plant proteins may have important roles in rice roots.18 Rice roots rapidly changed broad spectrum of energy metabolism upon challenging salt stress, and suppression of GA signaling by salt stress may be responsible for the rapid arrest of root growth and development.19 In spite of several good reviews, an integrated vision of current information on rice tolerance to salt stress is lacking.4 Recent insights point toward involvement of both ion transport and metabolic aspects in rice performance under elevated soil salinity.20 Metabolomics provides a better understanding of the changes in cellular metabolism induced by salt stress.3 A metabolic depletion syndrome was discovered at early vegetative stages in roots of salt sensitive rice cultivars. Depletion of at least 30 primary metabolites including sucrose, glucose, fructose, glucose-6-P, fructose-6-P, organic acid, and amino acids has been reported.20 But any biomarker of salt tolerance is yet unknown. During the present study, we compared metabolic changes in the leaves of the seedlings of 4 varieties of rice, differing in their responses to NaCl stress, following GC-MS based metabolomics approach, to search for biomarkers of salt tolerance.

Results

Metabolic changes in the 4 rice varieties consisting of 2 tolerant varieties (Nonabokra and Bhutnath) and 2 sensitive varieties (Sujala and MTU7029) to short-term NaCl stress were different from each other. In total 91 metabolites could be identified from the leaf part. They included 29 amino acids, 14 organic acids, 3 fatty acids, 10 phenols, 6 xanthine derivatives, 22 sugars, polyols and derivatives, and 7 other metabolites (Table 1). The data (relative response ratio of the metabolites) (Table S1) were analyzed by multivariate statistical analysis to understand the metabolic responses of the 4 varieties of rice seedlings to NaCl induced stress.

Table 1.

Metabolites identified from rice leaves.

ORGANIC ACIDS Identification PHENOLS Identification
trans-Aconitic acid A O-Acetylsalicylic acid A, B
DL-3-Aminoisobutyric acid A Ferulic acid A, B
Citric acid A, B Gentisic acid A, B
Fumaric acid A Gallic acid A, B
3-Hydroxy-3-methylglutaric acid A 4-Hydroxybenzoic acid A, B
Gluconic acid lactone A, B 4-Hydroxycinnamic acid A, B
Glyceric acid A 4-Hydroxy-3-methoxybenzoic acid A, B
Glycolic acid A Kaempferol A, B
4-Guanidinobutyric acid A Shikimic acid A
L-(+) Lactic acid A Quinic acid A, B
Lactobionic acid A SUGARS AND DERIVATIVES  
D-Malic acid A, B Fructose A
Malonic acid A Galactinol A
Methylmalonic acid A D(+)Galactose A
Mucic acid A Methyl-β-D-galactopyranoside A
Oxalic acid A, B D-Glucose A
Pantothenic acid A D-Glucose-6-phosphate A
Pyruvic acid A Glycerol A
Succinic acid A, B Glycerol-1-phosphate A
Phosphoric acid A Guanosine A
AMINO ACIDS   D-Lyxose A
β-alanine A D-Lysosylamine A
L-Alanine A, B Maltose A
Aspartic acid A, B D-Mannitol A
L-Asparagine A, B D-Sorbitol A
β-cyano-L-Alanine A Sucrose A, B
Citrulline A Talose A
L-Glutamic acid A, B Tagatose A
L-Glutamine A, B D-(+) Trehalose A, B
Glycine A, B Raffinose A, B
L-Histidine A, B FATTY ACIDS  
L-Homoserine A Lauric acid A, B
DL-Isoleucine A, B Palmitic acid A, B
L-Leucine A, B Stearic acid A, B
L-Lysine A, B OTHERS  
L-Methionine A, B Adenine A
L-Ornithine A, B Adenosine A
Phenylalanine A, B Xanthine A
L-Proline A, B Porphine A
trans-4-Hydroxy-L-proline A, B Allantoin A
Putrescine A Phenylethylamine A
L-Pyroglutamic acid A urea A
L-Serine A, B Uracil A
Serotonin A, B 1,3-Diaminopropane A
Spermidine A O-Phosphocolamine A
L-Threonine A, B    
L-Tryptophan A, B    
Tyramine A    
L-Tyrosine A, B    
L-Valine A, B    
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A: RT, RI, MS of Fiehn Library

B: RT, RI, MS of authentic compound

Hierarchical cluster analysis showed that the 4 varieties segregated separately (Fig. 1) on the basis of metabolomic responses. The metabolite profiles in different varieties in response to different NaCl treatment were also analyzed by Heatmap (Fig. 2). It could be visualized that the varieties responded differently to the NaCl induced stresses. It is also clear that the salt tolerant variety Nonabokra biosynthesized least level of metabolites, even after stress responses, compared with the other 3 varieties. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal PLS-DA also segregated the tolerant varieties from the sensitive varieties on the basis of metabolite levels in each variety (Fig. 3). But Nonabokra and Bhutnath were different metabolically in all cases. As Nonabokra produced small amount of metabolites on the basis of relative quantity, metabolic responses to stress could not be explained from the important variables generated from the above models.

Figure 1.

Figure 1.

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Hierarchical cluster analysis of metabolites in 4 rice varieties.

Figure 2.

Figure 2.

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Heat map of normalized response ratios of leaf metabolite areas in 4 varieties of rice seedlings in response to different concentrations of NaCl treatment.

Figure 3.

Figure 3.

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Separation of salt tolerant and sensitive varieties (a) PCA; (b) PLS-DA; (c) OPLS-DA.

So the responses of individual plants to stress were studied. PCA and PLS-DA showed that, for each variety, the control and different salt treated plants separated very distinctly from each other on the basis of metabolite profile (Figs. 4–7). The findings suggested that the plants responded differently to different treatment concentrations of NaCl. To find out the metabolites responsible for such variation, biplots (Fig. S1) were generated from PCA model and VIP scores (Fig. 8) were found from PLS-DA model. In sujala, a salt sensitive variety, the responsible metabolites identified by VIP scores were glucose-6-P, lyxose, adenosine, spermidine, alanine, trans-aconitic acid, adenine, 4-hydroxycinnamic acid, homoserine, palmitic acid, stearic acid, lauric acid, glycolic acid, tryptophan, and talose. A few more metabolites such as gallic acid, citrulline, histidine, shikimic acid, sorbitol, trehalose, lysoxamine, and gentisic acid were detected from the biplot (Fig. S1). In another salt sensitive variety MTU 7029, the metabolites identified from VIP scores (Fig. 8) and biplot (Fig. S1) for the separation of clusters were stearic acid, methyl β-D-galactopyranoside, sorbitol, lyxose, homoserine, histidine, glucose-6-P, palmitic acid, serotonin, adenine, lactobionic acid, uracil, pyruvic acid, gallic acid, spermidine, porphine, oxalic acid, galactinol, 4-hydroxybenzoic acid, ferulic acid, and quinic acid. In Bhutnath, a salt tolerant variety, the identified metabolites for separation of treated plants into different clusters were gluconic acid, phosphocolamine, gentisic acid, histidine, quinic acid, 4-hydroxy-3-methoxybenzoic acid, adenine, uracil, trans-aconitic acid, lactobionic acid, guanosine, methylmalonic acid, raffinose, malonic acid, gallic acid, galactose, pyruvic acid, porphine, and spermidine (Fig. 8). The metabolites responding to salt stress were identified from VIP scores (Fig. 8) and biplot (Fig. S1) in the other salt tolerant variety Nonabokra. They were trans-4-hydroxyproline, pyruvic acid, glucose, trans-aconitic acid, ferulic acid, serotonin, gluconic acid, 4-hydroxybenzoic acid, oxalic acid, talose, phenylethylamine, gentisic acid, palmitic acid, spermidine, 4 guanidinobutyric acid, lysoxamine, porphine, glucose-6-P, tagatose, proline, sorbitol, tyramine, and adenine. The trend of increase or decrease of these metabolites in response to NaCl, as analyzed by ANOVA, were compared in the Table 2. However, decreases or increases of most of the metabolites could not be correlated to distinguish the tolerant and sensitive varieties, except trehalose, trans-4-hydroxy-proline, serotonin, 2 bases (adenine, and adenosine), and 4 phenolic metabolites (quinic acid, ferulic acid, gentisic acid, and 4-hydroxy-3-methoxybenzoic acid or vanillic acid).

Figure 4.

Figure 4.

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Segregation of treated leaves from control on the basis of metabolite changes in Sujala (a) 3-D PCA score plot; (b) 3-D PLS-DA score plot (R2 0.89022, 0.99146, 0.99647; Q2 0.79366, 0.97352, 0.99167).

Figure 5.

Figure 5.

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Segregation of treated leaves from control on the basis of metabolite changes in MTU 7029 (a) 3-D PCA score plot; (b) 3-D PLS-DA score plot (R2 0.81814, 0.98408, 0.99322; Q2 0.75205, 0.96716, 0.98196).

Figure 6.

Figure 6.

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Segregation of treated leaves from control on the basis of metabolite changes in Bhutnath (a) 3-D PCA score plot; (b) 3-D PLS-DA score plot (R2 0.85868, 0.97674, 0.99067; Q2 0.70661, 0.95471, 0.98165).

Figure 7.

Figure 7.

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Segregation of treated leaves from control on the basis of metabolite changes in Nonabokra (a) 3-D PCA score plot; (b) 3-D PLS-DA score plot (R2 0.95674, 0.99052, 0.99434; Q2 0.91835, 0.98112, 0.9865).

Figure 8.

Figure 8.

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VIP scores for metabolites responsible for separation of clusters in PLS-DA (a) Sujala; (b) MTU 7029; (c) Bhutnath; (d) Nonabokra.

Table 2.

Changes in metabolite level in the leaves of 4 varieties of rice seedlings.

  SUGARS AND POLYOLS
Varieties
 Galactinol
 D-Galactose
 D-Glucose
 D-Glucose-6-p
 Lactobionic acid
 D-Lyxose
 D-Lysosylamine
 Methyl-b-D-galactopyranoside
 Raffinose
 D-Sorbitol
 Tagatose
 D-Trehalose
 Talose
Sujala ⇓ − ⇔ ⇑ − ⇔ ⇓ − − ⇔ ⇓ ⇓ ⇓
MTU 7029 ⇔ − ⇔ ⇓ ⇑ ⇔ − ⇔ − ⇑ ⇔ ⇔ −
Bhutnath ⇔ ⇑ ⇔ ⇑ ⇑ ⇔ − ⇔ ⇑ − ⇔ ⇑ −
Nonabokra ⇔ − ⇔ ⇓ ⇑ ⇔ ⇔ ⇔ − ⇑ ⇔ ⇑ ⇔
 
  AMINO ACID
               BASES
 
 L-Alanine
 Citrulline
 L-Histidine
 L-Homoserine
 4-Hydroxyproline
 L-Proline
 Serotonin
 Spermidine
 Tryptophan
 Tyramine
 Lauric acid
 Palmitic acid
 Stearic acid
Sujala ⇓ ⇔ − ⇑ − ⇔ − ⇓ ⇓ − ⇑ ⇓ ⇓
MTU 7029 ⇔ ⇔ ⇓ ⇓ − ⇓ ⇓ ⇔ − − ⇔ ⇓ ⇓
Bhutnath   ⇔ ⇑ ⇔ ⇑ ⇔ ⇑   − − ⇔ − −
Nonabokra ⇔ ⇔ − ⇔ ⇑ ⇔ ⇑ ⇓ − ⇑ ⇔ ⇓ ⇓
 
  PHENOLS
                 FATTY ACIDS
 
 Ferulic acid
 Gallic acid
 Gentisic acid
 4-Hydroxybenzoic acid
 Vanillic acid
 4-Hydroxy- Cinnamic acid
 Kaempferol
 Shikimic acid
 Quinic acid
  
 Lauric acid
 Palmitic acid
 Stearic acid
Sujala − ⇔ ⇔ ⇓ ⇓ ⇑ ⇔ ⇓ ⇔   ⇑ ⇓ ⇓
MTU 7029 ⇔ ⇓ − ⇑ − − − ⇓ ⇔   ⇔ ⇓ ⇓
Bhutnath ⇑ ⇔ ⇑ ⇓ ⇑ − − ⇓ ⇑   ⇔ − −
Nonabokra ⇑ − ⇑ ⇓ ⇑ − − ⇓ ⇑   ⇔ ⇓ ⇓
 
  ORGANIC ACIDS
           BASES
      
 
 Fumaric acid
 Gluconic acid
 Glycolic acid
 4-Guanidinobutyric acid
 Malonic acid
 Oxalic acid
 Pyruvic acid
  
 Guanosine
 Adenine
 Adenosine
 Uracil
  
Sujala ⇓ − ⇓ ⇓ − − −   − ⇓ − −  
MTU 7029 ⇔ − ⇔ ⇔ ⇑ − ⇑   ⇑ ⇔ ⇔ ⇑  
Bhutnath ⇑ ⇓ ⇔ ⇔ − − ⇑   ⇑ ⇑ ⇓ ⇑  
Nonabokra ⇔ − ⇔ ⇔ ⇔ ⇓ ⇑   ⇔ ⇑ ⇓ −  
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⇑ Increase; ⇓ Decrease; ⇔ No change; − Absaent; Tolerant; Sensitive

Discussion

From the metabolomic responses of 2 tolerant and 2 sensitive varieties of rice to salinity, it can be said that the 4 varieties of rice responded differently to salt stress. The conserved metabolites e.g. amino acids, organic acids, sugars, polyols and the xanthin derivatives increased or decreased without showing specific relation to known salt tolerance level of the varieties.

Sugars act as osmoprotectants, help to maintain osmotic balance, stabilize macromolecules, provide immediate energy source to plants restarting growth.21 Sugars commonly accumulate in response to salt stress as previously reported in barley (Hordeum vulgare L.),22 Lotus japonicas,23 Limonium latifolium,24 Thellungiella halophila, Arabidopsis thaliana,25 and Populus euphratica.26 However, previous study reported no increase in sugars following salt stress in different rice cultivars and fructose and sucrose levels decreased.27 During the present study, it was revealed that the leaves of both salt tolerant and salt susceptible varieties responded differently to salt stress with accumulation of different levels of sugars. So it is difficult to conclude universally their role to salinity tolerance as pointed out previously.28 But there were increases in trehalose in leaves of tolerant varieties. Trehalose, a nonreducing disaccharide of glucose, is an osmoprotectant, stabilize biomolecules (dehydrated enzymes, proteins lipid membranes) under stress in some cases.21,29 Polyols act as Reactive Oxygen Species (ROS) scavengers.30 But the polyols were not found to respond positively in the rice varieties.

Proline, an osmoprotectant, is accumulated in many plant species in response to environmental stress.31 Increased levels of proline content have previously been reported in rice seedlings.32-33 Increased accumulation of proline, has been reported in rice seedlings in response to salt stress.34-36 Salt tolerant Nonabokra accumulated less proline than the salt sensitive cultivars.33 Present study showed that there was either decrease or no significant changes in proline content in the leaves of both tolerant and susceptible varieties of rice. However, in Bhutnath and Nonabokra trans-4-hydroxy-L-proline level increased. Presence of trans-4-hydroxy-L-proline in stress tolerant rice seedlings under control condition and its induced assimilation in response to stress is noteworthy. To the best of our knowledge putative identification of this metabolite is reported for the first time from rice seedlings. Enhanced biosynthesis of trans-4-hydroxy-L-proline in response to NaCl stress was reported in Vicia faba L. leaf. But the role of this metabolite during salinity stress is unknown.37

The signaling molecule serotonin (5-hydroxytryptamin), a pineal hormone in mammals, also increased significantly in the 2 tolerant varieties Nonabokra and Bhutnath in response to NaCl induced stress. Serotonin, a conserved signaling molecule, has previously been reported from a wide range of plants species.38,39 Serotonin has also been reported from rice leaves and seeds.40 Accumulation of serotonin is suggested to play a positive role against reactive oxygen species (ROS) delaying senescence in rice leaves. Serotonin synthesis could be associated with accumulation of ammonia which occurs during senescence.39 Serotonin and its derivative metabolite melatonin regulate the modulation in the signaling events of abiotic stress in plants.41 Increased production of serotonin in salt tolerant varieties suggests their positive role in combating stress and other negative responses to NaCl treatment. The salt sensitive variety Sujala failed to produce this metabolite. In MTU 7029, serotonin level decreased after NaCl treatment.

Salicylic acid (2-hydroxybenzoic acid), a shikimic acid pathway metabolite and benzoic acid derivative, is considered to be mediators of plant responses to biotic and abiotic stresses.42 It has been found that environmental stress factors like salinity also induces salicylic acid production43,44 in leaves of rice seedlings. However, during the present GC-MS based analysis, salicylic acid could not be detected in the leaves of any of the varieties considered for the experiment. The tolerant varieties of rice Bhutnath and Nonabokra, on the other hand, produced gentisic acid (2,5-dihydroxybenzoic acid) in response to salinity stress. This metabolite could not be detected in any of the varieties when grown without salt stress. Sujala produced small amount of gentisic acid in a few replica after treatment with NaCl. MTU 7029, considered to be a salt susceptible variety did not produce detectable amount of gentisic acid. Interestingly, in this variety 4-hydroxybenzoic acid production was increased in response to salt stress. In the tolerant varieties Bhutnath and Nonabokra, the level of 4-hydroxybenzoic acid decreased significantly. Previously gentisic acid was reported to be a pathogen induced signal in tomato (Lycopersicon esculentum cv Rutgers),45 Cucumis sativus L. cv Wisconsin SMR-58, Gynura aurantiaca DC.,46 and potato (Solanum tuberosum L.).47 Gentisic acid has been proposed as a signal molecule for plant defense response.45 To our knowledge, no report is available regarding the induced production of the signaling molecule gentisic acid in response to abiotic stresses. Gentisic acid has shown a differential activity with respect to salicylic acid for inducing defense proteins and the 2 signals can act in a complementary manner.46 In rice, gentisic acid could not be detected in the tolerant varieties without salt stress. Its production was induced after NaCl treatment in the salt tolerant varieties. Therefore, the metabolite is plausibly acting as a signaling molecule in response to salinity stress also in these stress tolerant varieties.

In addition to the above mentioned signaling molecules, the salt tolerant varieties also produced increased (although not statistically significant) level of ferulic acid and vanillic acid, the other 2 biosynthetically related metabolites. These 2 phenolic compounds increased in the tolerant varieties and decreased in the susceptible varieties. Such type of increased production of phenolic acids in response to salinity stress has recently been reported in rice.48 The well- known antioxidant ferulic acid possibly acts as reductants to scavenge ROS in tolerant varieties of rice through its increased production.

In Vietnamese cultivars, GC-TOF-MS based metabolomics, failed to identify single marker metabolite with unequivocal distinction between tolerant cultivars from the remaining. Drastic increase in amino acids, e.g., glycine, phenylalanine, glutamate and glutamine in 2 out of 14 cultivars were reported.27 Single unambiguous marker molecule will be rare in primary metabolism and perhaps more frequent in secondary metabolism.27 Conservation of metabolic responses to environmental stress should be observable within pathways of primary metabolism. Secondary metabolism, not conserved among plants, reflects successful adaptation of species through acquisition of novel biosynthetic capacities.23 During the present study, conserved decrease or increase of sugars, polyols, organic acids and amino acids were variety specific. Thus the salt tolerant variety Bhutnath showed increased level of many sugars and amino acids, whereas, in other varieties these components were either depleted or showed no significant changes. Increased production of the signaling molecule gentisic acid, in response to NaCl stress in the tolerant varieties Bhutnath and Nonabokra, suggests that it is a biomarker compound in response to salinity also. Gentisic acid is a catabolite of aspirin (o-acetylsalicylic acid).49 In this regard, putative identification of o-acetylsalicylic acid in the leaves of all the experimental rice varieties is of significant importance. In the salt sensitive MTU 7029, gentisic acid could not be detected but there was significant increase in o-acetylsalicylic acid. Gentisic acid could not be detected in seedling leaves without NaCl treatment. De novo production of this secondary metabolite in salt tolerant varieties, thus, may be considered as an important biomarker of salt tolerance. This signaling molecule45 is probably produced contributing to salt tolerance in rice seedlings in tolerant varieties. Fig. 9 clearly shows that chorismic acid derived metabolites play important role in salt tolerant varieties with increase in the level of serotonin and gentisic acid. There was a shift in biosynthetic pathway from cinnamic acid toward formation of the signaling molecule gentisic acid in Bhutnath and Nonabokra. In the sensitive varieties the biosynthetic pathway was diverted toward formation of 4-hydroxycinnamic acid (Sujala) and 4-hydroxybenzoic acid (MTU 7029). Changes in the level of shikimic acid derived metabolites including the chorismic acid derived ones in the 4 varieties are compared in Fig. 10. Thus the production of metabolites derived from chorismic acid are significant in rice in determining their sensitivity to NaCl induced salt stress.

Figure 9.

Figure 9.

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Mapping shikimate derived metabolites on the metabolic pathway: drawn following KEGG after simplification. The colored squares depict significance level of changes in metabolites calculated from the differences among treatments applying one way ANOVA.

Figure 10.

Figure 10.

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Shikimic acid derived metabolites: Changes due to effect of NaCl treatment *Significant increase; #Significant decrease.

Materials and methods

Plant materials

Four indica varieties of rice grains (Oryza sativa L.) e.g., 2 salt sensitive varieties (Sujala, and MTU 7029) and 2 tolerant varieties (Bhutnath, and Nonabokra) were collected from Rice Research Station, Chinsurah, Directorate of Agriculture, Government of West Bengal, India. Nonabokra is one of the most salt-tolerant varieties50 and a salt tolerance donor in classical breeding.51

Chemicals

Fatty acid methyl esters (FAME), methoxyamine hydrochloride, and ribitol were purchased from Sigma (USA). Authentic amino acids alanine, aspartic acid, asparagines, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, trans-4 hydroxy-L-proline, serine, serotonin, threonine, tryptophan, tyrosine and valine, collected from SRL (Sisco Research Laboratory, India); authentic organic acids like citric acid, gluconic acid lactone, malic acid, oxalic acid, and succinic acid from SRL; authentic phenols O-acetylsalicylic acid (Sigma), ferulic acid (SRL), gentisic acid (Aldrich), gallic acid (SRL), 4-hydroxybenzoic acid (SRL), 4-hydroxycinnamic acid (Himedia), 4-hydroxy-3-methoxybenzoic acid (Sigma-Aldrich), kaempferol (SRL), and quinic acid (SRL); authentic sugars sucrose, trehalose, and raffinose; and fatty acids like lauric acid, palmitic acid, and stearic acid collected from SRL, were injected for further authentication of the identified peaks. Methanol used was of HPLC grade, purchased from Sigma (USA). All other chemicals were purchased from Merck Specialties Pvt. Ltd., India.

Plant growth and treatment conditions

The seeds were surface sterilized by incubating in 5% sodium hypochlorite solution for 15 min and then rinsed thoroughly with distilled water. The seeds were germinated in the dark on filter paper moistened with distilled water in petridishes. Three days after germination, the seedlings were transferred near the rim of culture tubes (inside layered with blotting paper and bottom filled with hydroponic solution without NaCl) to allow root growth. Two seedlings were grown in one culture tube. The nutrient solution22 consisted of Ca(NO3)2 (2 mM), KNO3 (5 mM), NH4NO3 (5 mM), MgSO4 (2 mM), KH2PO4 (0.1 mM), Na2Si3 (0.5 mM), NaFe(III)EDTA (0.05 mM), MnCl2 (5 µM), ZnSO4 (5 µM), CuSO4 (0.5 µM), NaMoO3 (0.1 µM), and H3BO3 (5 µM). The seedlings were allowed to grow at 32 ± 2°C and photoperiodic condition 12 h light (photon flux intensity 135 µmol m−2 s−1) and 12 h darkness. After 7 days, the nutrient solution was replaced by the same solution supplemented with 0, 25, 50, 100 and 150 mM NaCl. Seedlings were harvested within 6 to 7 h of light period on day 15 (after 5 d of salt stress). The leaf tissues were snap frozen in liquid nitrogen.

Extraction and derivatization of tissue metabolites

Leaf tissue (120 ± 20 mg) homogenized in liquid nitrogen were extracted with 1 ml 50% methanol after addition of internal standard ribitol (20 μl of 0.2% aqueous solution) for 30 min at 60°C temperature. The process was repeated 5 times for each sample (biologic replicates). The extracts were centrifuged. Aliquots of leaf (150 µl), after evaporation, were considered for derivatization. Each sample was derivatized with methoxyamine hydrochloride and MSTFA. FAME markers (methyl esters of C8, C10, C12, C14, C16, C18, C20, C20, C24 and C26 linear chain fatty acids) were used as internal Retention Index (RI) markers.52

Gas chromatography – mass spectrometry (GC-MS) analysis

GC-MS analysis was performed using Agilent 7890 A GC (software driver version 4.01(054) equipped with 5795C inert MSD with Triple Axis Detector. The HP-5MS capillary column [Agilent J & W; GC column (USA)] of dimensions 30 m X 0.25 mm X 0.25 μm was used. The analysis was performed52 with oven temperature program (oven ramp 1 min hold at 60°C, to 325°C at 10°C /min, held for 10 min before cool-down producing a run time of 37.5 minutes). Injection temperature was set at 250°C, the MSD transfer line at 290°C, ion source at 230°C. Helium was used as carrier gas with flow rate 0.723 ml/min (carrier linear velocity 31.141 cm/sec). Derivatized samples were injected onto the column via split mode (split ratio 10:1). Mass spectra from 50 to 500 m/z were recorded. Following users' guide, the method was calibrated with the FAME standards available on the Fiehn GC-MS Metabolomics RTL Library (Agilent Technologies, USA, 2008) by RT locking method.53 The analysis for identification of metabolites was performed using automated mass spectral deconvolution and identification system (AMDIS) to deconvolute resulting chromatogramme. Fragmentation pattern of the mass spectra, and RT in Agilent Fiehn GC/MS Metabolomics Library were compared for identification of the peaks. For many of the metabolites RT, RI, and MS were also compared with that of authentic samples.

Statistical analysis

The relative response ratios of all the identified metabolites were obtained by normalizing the compound peak area by dividing with that of ribitol and fresh weight of sample. Missing values were replaced by half of the detection limit52 before normalization. The differences among treatments were also tested by applying one way analysis of variance (ANOVA). The differences in values of control and each treatment were compared by t-test, considering p < 0.05 as statistically significant. Data were also subjected to Hierarchical Cluster Analysis, Principal Component Analysis (PCA), Partial Least Squares - Discriminant Analysis (PLS-DA), Orthogonal Partial Least Squares - Discriminant Analysis (OPLS-DA) using MetaboAnalyst 3.0: a comprehensive tool suite for metabolomic data analysis. Heatmap was also generated following this tool.

Supplementary Material

Supplemental_Material.zip

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgements

The work was supported by the Department of Science and Technology, Government of West Bengal under grant 104/WBSCST/F/0520/14 sanctioned to PG. We also acknowledge Department of Science and Technology, Government of India (FIST program) for Instrumental facility. Dr. Santanu Aich, Rice Research Station, Chinsurah is gratefully acknowledged for his help in collection of plant materials.

References

  • 1.Tuteja N. Mechanisms of high salinity tolerance in plants. Methods Enzymol 2007; 428:419-38; PMID:17875432; https://doi.org/ 10.1016/S0076-6879(07)28024-3 [DOI] [PubMed] [Google Scholar]
  • 2.Munns R. Comparative physiology of salt and water stress. Plant Cell Environ 2002; 25:239-50; PMID:11841667; https://doi.org/ 10.1046/j.0016-8025.2001.00808.x [DOI] [PubMed] [Google Scholar]
  • 3.Ruan CJ, Da Silva JAT, Mopper S, Qin P, Lutts S. Halophyte improvement for a salinized world. Crit Rev Plant Sci 2010; 29:329-59; https://doi.org/ 10.1080/07352689.2010.524517 [DOI] [Google Scholar]
  • 4.Negrao S, Courtois B, Ahmadi N, Abreu I, Saibo N, Oliviera MM. Recent updates on salinity stress in rice: From physiological to molecular responses. Crit Rev Plant Sci 2011; 30:329-77; https://doi.org/ 10.1080/07352689.2011.587725 [DOI] [Google Scholar]
  • 5.Flowers TJ, Yeo AR. Variability in the resistance to sodium chloride salinity within rice (Oryza sativa L.) varieties. New Phytol 1981; 88:363-73; https://doi.org/ 10.1111/j.1469-8137.1981.tb01731.x [DOI] [Google Scholar]
  • 6.Lutts S, Kinet JM, Bouharmont J. Changes in plant responses to NaCl during development of rice (Oryza sativa L.) varieties differing in salinity resistance. J Exp Bot 1995; 46:1843-52; https://doi.org/ 10.1093/jxb/46.12.1843 [DOI] [Google Scholar]
  • 7.Lauchli A, Grattan SR. Plant growth and development under salinity stress In Jenks MA et al. (Eds), Advances in molecular breeding toward drought and salinity tolerant crops Chapter 1 2007; 1:1-32 [Google Scholar]
  • 8.Sairam RK, Tagi A. Physiology and molecular biology of salinity stress tolerance in plants. Curr Sci 2004; 86:407-21 [Google Scholar]
  • 9.Sahi C, Singh A, Kumar K, Blumwald E, Grover A. Salt stress response in rice: Genetics, molecular biology, and comparative genomics. Funct Integr Genomics 2006; 6:263-84; https://doi.org/ 10.1007/s10142-006-0032-5 [DOI] [PubMed] [Google Scholar]
  • 10.Walia H, Wilson C, Zeng L, Ismail AM, Condamine P, Close TJ. Genome-wide transcriptional analysis of salinity-stressed japonica and indica rice genotypes during panicle initiation stage. Plant Mol Biol 2007; 63:609-23; PMID:17160619; https://doi.org/ 10.1007/s11103-006-9112-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Kumari S, Sabharwal VP, Kushwaha HR, Sopory SK, Singla-Pareek SL, Pareek A. Transcriptome map for seedling stage specific salinity stress response indicates a specific set of genes as candidate for saline tolerance in Oryza sativa L. Funct Integr Genomics 2009; 9:109-23; PMID:18594887; https://doi.org/ 10.1007/s10142-008-0088-5 [DOI] [PubMed] [Google Scholar]
  • 12.Schmidt R, Schippers Jos HM, Welker A, Mieulet D, Guiderdoni E, Mueller-Robber B. Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. Japonica. AoB Plants 2012; 2012:pls 011; PMID:22616023; https://doi.org/ 10.1093/aobpla/pls011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Nohzadeh MS, Habibi RM, Heidari M, Salekdeh GH. Proteomics reveals new salt responsive proteins associated with rice plasma membrane. Biosci Biotechnol Biochem 2007; 71:2144-54; PMID:17827676; https://doi.org/ 10.1271/bbb.70027 [DOI] [PubMed] [Google Scholar]
  • 14.Zang X, Komatsu S. A proteomics approach for identifying osmotic stress –related proteins in rice. Phytochemistry 2007; 68:426-37; PMID:17169384; https://doi.org/ 10.1016/j.phytochem.2006.11.005 [DOI] [PubMed] [Google Scholar]
  • 15.Abbasi FM, Komatsu SA. Proteomic approach to analyze salt-responsive proteins in rice leaf sheath. Proteomics 2004; 4:2072-81; PMID:15221768; https://doi.org/ 10.1002/pmic.200300741 [DOI] [PubMed] [Google Scholar]
  • 16.Kong-ngern K, Daduang S, Wongkham C, Bunnang S, Kosittrakun M, Theerakulpisu P. Protein profiles in response to salt stress in leaf sheaths of rice seedlings. Sci Asia 2005; 31:403-8; https://doi.org/ 10.2306/scienceasia1513-1874.2005.31.403 [DOI] [Google Scholar]
  • 17.Ren ZH, Gao JP, Li LG, Cai XL, Huang W, Chao DY, Zhu MZ, Wang ZY, Luan S, Lin HX. A rice quantitative train locus for salt tolerance encodes a sodium transporter. Nat Genet 2005; 37:1141-6; PMID:16155566; https://doi.org/ 10.1038/ng1643 [DOI] [PubMed] [Google Scholar]
  • 18.Zhang L, Tian L, Zhao J, Song Y, Zhang C, Guo Y. Identification of an apoplastic protein involved in the initial phase of salt stress response in rice root by two-dimensional electrophoresis. Plant Physiol 2009; 149:916-28; PMID:19036832; https://doi.org/ 10.1104/pp.108.131144 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Nam MH, Bang E, Kwon TY, Kim Y, Kim EH, Cho K, Park W, Kim B, Yoon IS. Metabolite profiling of diverse rice germplasm and identification of conserved metabolic markers of rice roots in response to long-term mild salinity stress. Int J Mol Sci 2015; 16:21959-74; PMID:26378525; https://doi.org/ 10.3390/ijms160921959 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Siapoosh MR, Sanchez DH, Schlereth A, Acofield GN, Furbank RT, Van Dongen JT, Moons A, Bauw G, Prinsen E, Montagu MV, et al.. Molecular and physiological responses to abscicic acid and salts in roots of salt-sensitive and salt tolerant Indica rice varieties. Plant Physiol 1995; 107:177-86; PMID:7870812; https://doi.org/ 10.1104/pp.107.1.177 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yancey PH. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J Exp Biol 2005; 208:2819-30; PMID:16043587; https://doi.org/ 10.1242/jeb.01730 [DOI] [PubMed] [Google Scholar]
  • 22.Widodo Patterson JH, Neubegin ED, Tester M, Bacic A, Roessner U. Metabolic responses to salt stress of Barley (Hordeum vulgare L.) cultivars, Sahara and Clipper, Which differ in salinity tolerance. J Exp Bot 2009; 60:4089-103; PMID:19666960; https://doi.org/ 10.1093/jxb/erp243 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sanchez DH, Siahpoosh MR, Roessner U, Udvardi M, Kopka J. Plant metabolomics reveals conserved and divergent metabolic responses to salinity. Physiol Plant 2008; 132:209-19; PMID:18251862; https://doi.org/ 10.1111/j.1399-3054.2007.00993.x [DOI] [PubMed] [Google Scholar]
  • 24.Gagneul D, Ainouche A, Duhaze C, Lugan R, Larher FR, Bouchereau AA. Reassessment of the function of the so-called compatible solutes in the halophytic plumbaginaceae Limomium latifolium. Plant Physiol 2007; 144:1598-611; PMID:17468212; https://doi.org/ 10.1104/pp.107.099820 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gong Q, Li P, Ma S, Rupassara SI, Bohnert H. Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana. Plant J 2005; 44:826-39; PMID:16297073; https://doi.org/ 10.1111/j.1365-313X.2005.02587.x [DOI] [PubMed] [Google Scholar]
  • 26.Brosche M, Vinocur B, Alatalo ER, Lamminmaki A, Teichmann T, Ottow EA, Djllianov D, Afif D, Bogeat-triboulot B, Altman A, et al.. Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert. Genome Biol 2005; 6:R101; PMID:16356264; https://doi.org/ 10.1186/gb-2005-6-12-r101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zuther E, Koehl K, Kopka J. Comparative metabolome analysis of the salt response in breeding cultivars of rice In Jenks MA et al. (Eds.), Advances in molecular breeding toward drought and salt tolerant crops Chapter 12 2007; 12:285-315 [Google Scholar]
  • 28.Ashraf M, Harris PJC. Potential biochemical indicators of salinity tolerance in plants. Plant Sci 2004; 166:3-16; https://doi.org/ 10.1016/j.plantsci.2003.10.024 [DOI] [Google Scholar]
  • 29.Garg AK, Kim J, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ. Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci U S A 2002; 99:15898-903; PMID:12456878; https://doi.org/ 10.1073/pnas.252637799 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Noiraud N, Maurousset L, Lemoine R. Transport of polyols in higher plants. Plant Physiol Biochem 2001; 39:717-28; https://doi.org/ 10.1016/S0981-9428(01)01292-X [DOI] [Google Scholar]
  • 31.Szabados L, Savoure A. Proline: A multifunctional amino acid. Trends Plant Sci 2009; 15:89-97; PMID:20036181; https://doi.org/ 10.1016/j.tplants.2009.11.009 [DOI] [PubMed] [Google Scholar]
  • 32.Demiral T, Turkan I. Comparative lipid peroxidation, antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance. Environ Exp Bot 2005; 53:247-57; https://doi.org/ 10.1016/j.envexpbot.2004.03.017 [DOI] [Google Scholar]
  • 33.Lutts S, Kinet JM, Bouharmont J. Effects of salt stress on growth, mineral nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) cultivars differing in salinity resistance. Plant Growth Regul 1996; 19:207-18; https://doi.org/ 10.1007/BF00037793 [DOI] [Google Scholar]
  • 34.Chunthaburee S, Dongsansuk A, Sanitchon J, Pattanagul W, Theerakulpisut P. Physiological and biochemical parameters for evaluation and clustering of rice cultivars differing in salt tolerance at seedling stage. Saudi J Biol Sci 2016; 23:467-77; PMID:27298579; https://doi.org/ 10.1016/j.sjbs.2015.05.013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Kanawapee N, Sanitchon J, Lontom W, Threerakulpisut P. Evaluation of salt tolerance at the seedling stage in rice genotypes by growth performance, ion accumulation, proline and chlorophyll content. Plant Soil 2012; 358:235-49; https://doi.org/ 10.1007/s11104-012-1179-6 [DOI] [Google Scholar]
  • 36.Igarashi Y, Yoshiba Y, Sananda Y, Yamaguchi-Shinozaki K, Shinozaki K. Characterization of the gene for delta1-pyrroline-5carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L. Plant Mol Biol 1997; 33:857-65; PMID:9106509; https://doi.org/ 10.1023/A:1005702408601 [DOI] [PubMed] [Google Scholar]
  • 37.Geilfus C, Niehaus K, Godde V, Hasler M, Zorb C, Gorzolka K, jezek M, Senbayram M, Ludwig-Muller J, Muhling KH. Fast responses of metabolites in Vicia faba L. to moderate NaCl stress. Plant Physiol Biochem 2015; 92:19-29; PMID:25900421; https://doi.org/ 10.1016/j.plaphy.2015.04.008 [DOI] [PubMed] [Google Scholar]
  • 38.Badria FAI. Melatonin, serotonin, and tryptamine in some Egyptian food and medicinal plants. J Med Food 2002; 5:153-7; PMID:12495587; https://doi.org/ 10.1089/10966200260398189 [DOI] [PubMed] [Google Scholar]
  • 39.Kang K, Park S, Kim YS, Lee S, Back K. Biosynthesis and biotechnological production of serotonin derivatives. Appl Microbiol Biotechnol 2009; 83:27-34; PMID:19308403; https://doi.org/ 10.1007/s00253-009-1956-1 [DOI] [PubMed] [Google Scholar]
  • 40.Kang S, Kang K, Lee K, Back K. Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice. Planta 2007; 227:263-72; PMID:17763868; https://doi.org/ 10.1007/s00425-007-0614-z [DOI] [PubMed] [Google Scholar]
  • 41.Kaur H, Mukherjee S, Baluska F, Bhatla SC. Regulatory roles of serotonin and melatonin in abiotic stress tolerance in plants. Plant Signal Behav 2015; 10(11):e1049788; PMID:26633566; https://doi.org/ 10.1080/15592324.2015.1049788 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Wildermuth MC. Variations on a theme: Synthesis and modification of plant benzoic acids. Curr Opin Plant Biol 2006; 9:288-96; PMID:16600669; https://doi.org/ 10.1016/j.pbi.2006.03.006 [DOI] [PubMed] [Google Scholar]
  • 43.Sawada H, Shim I, Usui K. Induction of benzoic acid 2-hydroxylase and salicylic acid biosynthesis – modulation by salt stress in rice seedlings. Plant Sci 2006; 171:263-70; https://doi.org/ 10.1016/j.plantsci.2006.03.020 [DOI] [Google Scholar]
  • 44.Jayakannan M, Bose J, Babourina O, Rengel Z, Shabala S. Salicylic acid in plant salinity stress signalling and tolerance. Plant Growth Regul 2015; 76:25-40; https://doi.org/ 10.1007/s10725-015-0028-z [DOI] [Google Scholar]
  • 45.Belles JM, Garro R, Fayos J, Navarro P, Primo J, Conejero V. Gentisic acid as a pathogen-inducible signal, additional to salicylic acid for activation of plant defences in tomato. Mol Plant-Microbe Interact 1999; 12:227-35; https://doi.org/ 10.1094/MPMI.1999.12.3.227 [DOI] [Google Scholar]
  • 46.Belles JM, Garro R, Pallas V, Fayos J, Rodrigo I, Conejero V. Accumulationofgentisic acid as associated with systemic infections but not with the hypersensitive response in plant pathogen interactions. Planta 2006; 223:500-11; PMID:16331468; https://doi.org/ 10.1007/s00425-005-0109-8 [DOI] [PubMed] [Google Scholar]
  • 47.Kresic-Stres H, Vucak C, Ravnikar M, Kovac M. Systemic potato virus infection and levels of salicylic acid and gentisic acids in different potato genotypes. Plant Pathol 2005; 54:441-7; https://doi.org/ 10.1111/j.1365-3059.2005.01209.x [DOI] [Google Scholar]
  • 48.Minh LT, Khang DT, Ha PTT, Tuyen PT, Minh TN, Quan NV, Xuan TD. Effects of salinity stress on growth and phenolics of rice (Oryza sativa L.). Int Lett Nat Sci 2016; 57:1-10; https://doi.org/ 10.18052/www.scipress.com/ILNS.57.1 [DOI] [Google Scholar]
  • 49.Fernandez IS, Cuevas P, Angulo J, Lopez-navajas P, Canales-Mayordomo A, Gonzalez-Corrochano R, Lozano RM, Valverde S, Jimenez-Barbero J, Romero A, et al.. Gentisic acid, a compound associated with plant defense and a metabolite of aspirin, heads a new class of in vivo fibroblast growth factor inhibitors. J Biol Chem 2010; 285:11714-29; PMID:20145243; https://doi.org/ 10.1074/jbc.M109.064618 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Kurotani K, Yamanaka K, Toda Y, Ogawa D, Tanaka M, Kozawa H, Nakamura H, Hakata M, Ichikawa H, Hattori T, et al.. Stress tolerance profiling of a collection of extant salt-tolerant rice varieties and transgenic plants overexpressing abiotic stress tolerance genes. Plant Cell Physiol 2015; 56:1867-76; PMID:26329877; https://doi.org/ 10.1093/pcp/pcv106 [DOI] [PubMed] [Google Scholar]
  • 51.Moons A, Bauw G, Prinsen E, Montagu MV, Straeten DVD. Molecular and physiological responses to abscisic acid and salts in roots of salt-sensitive and salt-tolerant indica rice varieties. Plant Physiol 1995; 107:177-86; PMID:7870812; https://doi.org/ 10.1104/pp.107.1.177 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Fiehn O. Metabolite profiling in Arabidopsis. Hum Press 2006; 323:439-47; PMID:16739598; https://doi.org/ 10.1385/1-59745-003-0:439 [DOI] [PubMed] [Google Scholar]
  • 53.Das S, Dutta M, Chaudhury K, De B. Metabolomic and chemometric study of Achras sapota L. fruit extracts for identification of metabolites contributing to the inhibition of α-amylase and α-glucosidase. Eur Food Res Technol 2016; 242:733-43; https://doi.org/ 10.1007/s00217-015-2581-0 [DOI] [Google Scholar]

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