Research Article

Morphometric Variability and Biochemical Analysis of Growth Seedlings under Salt Stress in Tomato (Lycopersicon esculentum Mill.) Cultivars.  

Touati Amar , Yahia Nourredine , Fyad-Lamèche  Fatima Zohra
Laboratoire de Biotechnologie des Rhizobiums et Amélioration des Plantes (Equipe 4: Génétique et Amélioration des Plantes), Département de Biologie, Faculté des Sciences de la Nature et de la Vie. Université Ahmed Ben Bella (Oran 1), Oran, Algeria
Author    Correspondence author
Molecular Plant Breeding, 2016, Vol. 7, No. 4   doi: 10.5376/mpb.2016.07.0004
Received: 10 Nov., 2015    Accepted: 26 Dec., 2015    Published: 02 Jan., 2016
© 2016 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Touati Amar, Yahia Nourredine and Fyad-Lamèche Fatima Zohra, 2016, Morphometric Variability and Biochemical analysis of Growth Seedlings under Salt stress in Tomato (Lycopersicon esculentum Mill.) Cultivars., Molecular Plant Breeding, 7(04): 1-9 (doi: 10.5376/mpb.2016.07.0004)

Abstract

Biotic and abiotic stresses impose a major threat to agriculture. Increase crop productivity need to develop stress tolerant plants. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Plants may be exposed to different salt levels at various stages of development; seed germination represents a major bottleneck in the species life cycle. In this context, seeds of eight varieties of tomato were tested under different levels of NaCl. Two approaches were adopted in this investigation: (a) Morphometric variability measurement, (b) biochemical changes of protein profile and iso-enzyme esterase activity. For the morphometric variability, two quantitative components were measured, vigor seed and length of seedling, for each parameter tolerance index (IT = treated / control) was evaluated. Statistical analysis was carried by software STATISTICA (version6. France). One-way ANOVA analysis indicates a significant difference between treatments and between varieties by depending on the increasing salt level for both components, differences between means were determined. Following the biometric analysis two genotypes were selected to complete biochemical analysis under stress conditions, MCH (salt-tolerant) and MRMD (moderately-tolerant). SDS-PAGE electrophoresis was used to determinate the effect of NaCl on protein expression and Native-PAGE electrophoresis to study isozymatic activity of esterase (EC 1.11.2). Results showed that salt-tolerant genotype increase of presently consecutive expression proteins; in contrast high levels of NaCl induce denaturation of proteins isoforms in sensitive genotype. Another interesting finding about antioxidant esterase activities; an important elevate expressed of esterase isoforms was founded in the salt-tolerant than the sensitive one. High levels of salt stress inhibited completely some of them in both genotypes.

Keywords
Salt stress; Seeds vigor; Seedling length; Tolerance Index; SDS-PAGE proteins; Esterase (EC 1.11.2); Lycopersicon esculentum Mill

Background
Plants are immobile and depend on their environment for growth and development. This environment is variable and challenges plants with abiotic stress situations throughout their life cycle: light (quality and quantity), mineral nutrition (toxicity and salinity) (Dolferus, 2014). Plants are typically exposed during their life cycle to conditions of abiotic stress. Most plants have evolved strategies to protect themselves against these conditions. However, if the severity and duration of the stress conditions are too great, the effects on plant development, growth, and yield of most crops plant profound. Continuous exposure to stress causes major alterations in the plant metabolism. These great changes in metabolism ultimately lead to cell death and consequently yield losses. Several lines of evidence have indicated that molecular tailoring of genes has the potential to overcome a number of limitations in creating (Mousumi et al., 2011).
 
Soil salinity is one of the main factors, which reduces crop production in semiarid and arid areas all around the word (Kholova et al., 2009; Li et al., 2011; Carillo et al., 2011). Globally, about 950 million hectares of arable land are affected by salinity, 250 million hectares of which is irrigated land. The result is a $27.3 billion annual loss in revenue.  Consequently, to achieve the challenge of feeding 9.3 billion people by 2050 requires a qualitative breakthrough in attempts to develop salt-tolerant germplasm (Shabala et al., 2015). Investigating germination and seedling emergence percentage at early seedling growth stage of four genotypes of grass pea to salinity stress, Piwowarczyk et al. (2015) highlighted that germination and seedling emergence percentage was not significantly affected by 50 mM and 100 mM of NaCl. However, NaCl in 200 mM concentration lowered these parameters. These authors stippled also that generally, exposure to NaCl stress significantly reduced length of grass pea seedling organs (root and shoot) but did not influence the content of dry weight in shoots and increased it in the roots in two cases.
 
Straight a consequence of salinity is the induction of stress proteins or antioxidant enzymes by exposed plants to minimize damage caused by reactive oxygen species (Jebara et al., 2005; Murhed et al., 2014; Karami et al., 2015 ; Pawar and Panneerselvam, 2015).  The development of salt-tolerant crops that can tolerate high levels of salinity in the soils would be a practical solution of such problem (Yamaguchi and Blumwald, 2005).
 
Tomato (Lycoersicon esculentum Mill.) is a model plant for studying the mechanism of plant salt tolerance (Amini et al., 2007 ; Mittova et al., 2015). In Mediterranean countries, tomatoes have also been studies as a model crop in salinized or dry land agriculture, and a large amount of data about the effects of salinity on the growth (seed germination, vegetative growth) of tomato plants has been produced (Saito and Matsukura, 2015).
 
To protect themselves from the detrimental effects of salt stress, plants have evolved many biochemical and molecular mechanisms. The main biochemical strategies: (i) induction of antioxidative enzymes, (ii) ion homeostasis and (iii) synthesis of compatible solutes Manoj et al., (2011). In the same case, Zhu et al., (2002) indicated that excess salt in soil or in solutions interferes with several physiological and biochemical process, resulting in problems such as ion imbalance, mineral deficiency, osmotic stress, ion toxicity and oxidative stress; these conditions ultimately interact with cellular components, including DNA, proteins, lipids, and pigments in plants. The popularity of the tomato for scientists has increased over the years, until it has become a model organism for research programs, both for applied and theoretical purposes. This is probably due to the possibility of growing in different conditions, allowing an understanding of the adaptability to different abiotic stresses and its relative cycle life (Bergougnoux, 2013).
 
Several studies were conducted to investigate salt-tolerance in tomato, we cited those realized by (Foolad, 2004; Cuartero et al, 2006; Junming et al, 2011).
 
A few studies was related about changes in iso-enzyme of esterase activities to stress responses (Ashraf and Harris, 2004; Joshi et al. 2010). Amini et al., (2007), when they comparing the protein profiles between control plants and those treated with different salt concentrations using SDS-PAGE, showed that NaCl treatment induced only few changes in the pattern of proteins. They found that the intensity of some protein bands was increased in salt-treated plants. In contrast, some other protein in salt-treated plants decreased dramatically. This research was conducted to determine effects of salt concentrations on seedling development and biochemical changes of protein and esterase (EC 3.1.1.2) of tomatoes varieties.
 
1 Results and Discussion
1.1 Effect of NaCl on seed germination
As shown in Table 1, results indicate that under control conditions (0 mM of NaCl) a very significant germination rate was observed for all varieties. Germination had its maximum in MCH variety (97%). For the other varieties, the results indicate a response to germination which varies between 89% and 96%. Rate of germination was affected at 51.33 mM of salt treatment. This trend was more pronounced in the Hybrid AG (53%). However, the maximum germination was reported in PICO with a percentage of 86%. Regarding the variability for this treatment, we find that the standard deviations oscillating between 0.033 and 0.045. AG (F1) indicates the most important value (0.045). RG and PICO recorded the same standard deviation (0.033).

 

 

Table 1 Means of germination rate evaluated under different levels of NaCl. Values are mean ± standard deviation. Data represents the mean of four replicates ± standard deviation

  

Comparing with the control, germination was strongly affected by salt treatment in all varieties. A significant decrease of germination was observed at 128.42 mM NaCl treatment. Indeed, compared with the control, the percentages of germination fall and reached values ranging between 10% and 20% among all genotype tested. MCH, RG and STP showed a highest percentage with 46% ± 0.48 in MCH variety followed by PICO variety. The other varieties have standard deviations oscillating between 0.02 and 0.04. Seed germination was completely inhibited at 256.54 mM Na Cl for all Varieties. This concentration drastically affects seed germination. These results were in accordance with those of Piwowarczyk et al. (2015). These authors reported that germination and seedling emergence percentage was not significantly affected by 50 mM and 100 mM of NaCl. However, NaCl in 200 mM concentration lowered these parameters. Piwowarczyk et al. (2015) highlighted that germination and seedling emergence percentage was not significantly affected by 50 mM and 100 mM of NaCl. However, NaCl in 200 mM concentration lowered these parameters. Results obtained by Kandil et al. (2015) showed that increasing salinity levels from 0 to 15 dSm-1significantly decreased germination percentage, germination index and seedling vigor index.
 
High value of the tolerance index indicates important tolerance. Tolerance index T2/T0 appears to be the best indicator to discriminate and classify genotypes according to their degrees of tolerances. Reading the values of the three index of tolerance allows identifying three categories of varieties: one type of variety that is more tolerant concentration of 51.33 mM and becomes sensitive to a concentration of 128.42 mM, this type includes PICO, RG and HZ varieties. Other varieties have a stackable ranking at both concentrations (ELG, MMD and AG); these varieties behave in the same way as and as the concentration increases. Both varieties MCH and STP, behave better because of a high concentration 128.42mMof NaCl. (Figure 1).

 

 

Figure 1 Effect of different NaCl levels on germination rate in tomato cultivars. Values indicated Tolerance index (IT)

  

1.2 Effect of salt treatment on seedling length
In the absence of treatment, the different varieties have average lengths ranging between 9.66 and 11.17 cm. The highest length was recorded in Rio Grande variety (11.47 cm) with a standard deviation of 3.03. PICO variety showed a relatively short length (9.93 cm), the other varieties have lengths ranging from 9.93 to 11.17cm. Reducing the length of the seedlings was relatively large in presence at 51.33 mM compared with control. All varieties were affected at 51.33 mM concentration and records low-lengths with very similar values ranging between 4.46 and 5.33 cm. There continues the superiority of the Rio Grande variety with a length of 7.86cm. (Table 2).

 

 

Table 2 Effect of different NaCl levels on seedling length (cm). Measurements were taken two-weeks old and data indicated as means ± standard deviation

  

The length is strongly affected by a salt concentration equivalent to T2. The reduction is marked for all varieties compared to the control, it was important in Agora (0.13 cm). Rio Grande variety indicate a value of 1.13 cm, the other varieties recorded values ranging between 0.20 and 0.79 cm. A complete inhibition of the emergence of the root for young seedling among all varieties in presence of 256.54 mM of NaCl (Table 3) Barbara et al (2015) indicates that generally exposure to NaCl stress significantly reduced length of grass pea seedling organs (root and shoot).

 

 

Table 3 One-way ANOVA analyses of the effect of salt stress on different morphometric parameters in tomato cultivars. a. Treatments effect on germination rate

  

Concerning a ratio (Figure 2), results showed values varied between 0.42 and 0.68 for the tolerance index T1 / T0. Two groups were distinguished. The first group was represented by two tolerate varieties "RG" and "PIC" with 0.68 and 0.60 values respectively. The rest of varieties present the second group, with a moderate tolerance, values varied between 0.42 and 0.60. Regarding the tolerance index T2 / T0, we observed that MCH variety appears as salt tolerant to this treatment and the other varieties are salt stress-slightly tolerant or completely sensitive. For the treatment T3, the growth was completely inhibited among all genotypes. Seed germination, seedling growth were all reduced even by low to moderate salinity concentrations and completely inhibited at lethal level (256.54 mM).

 

 

Figure 2 Salt stress effects on seedling length for all genotypes. Values indicated Tolerance index (IT)

  

In this experiment, the effect of salt stress on both components was considered. Data for statistical analysis with one-way analysis of variance (ANOVAs) presented in Table 3 indicated a significant difference at P≤0.05 among treatments and varieties for seeds vigor and only effect of treatments for seedling length. Homogeneous group were established with Newman - keuls test. (Data not shown).
 
1.3.1 Effect of NaCl on isozyme esterase and protein pattern
In order to determine the nature of the esterase and protein responses of tomato to salt stress during seedling emergence and seedling growth, we measured the esterase activity and protein expression in seedling of two cultivars under the different Na Cl concentration. We subsequently selected the MRMD variety as salt stress-slightly sensitive and the MCH as salt stress-tolerant cultivar; the choice of these two varieties for further biochemical analysis was based on the tolerance index T2/T0, which appears as a better ratio to discriminate between the tolerant and sensitive genotypes.
 
1.3.2 Effect of NaCl on isozyme esterase pattern
Esterases (ESTs) hydrolyse ester linkage of different metabolites and are presented ubiquitous in all developmental stages of plants in many isoforms (Rasol et al., 1999 ; Swapna 2003) changes in their expression and activity were observed under abiotic stress (de Carvalho et al., 2003; Syros et al., 2005; Thonar et al., 2009).
 
Globally, esterase expression in plantlets aged 1 to 9 days depends on the development stage. In the other hand, this expression was highly delayed under salt stress. For example in MCH, when plants aged 9 days were subjected at 128.42 mM (T2) and 256.54 mM (T3) salt concentration profiles were similar to those in the control of the second and the third days respectively (Figure 3B).

 

 

Figure 3 Effect of salinity levels on esterase (EST) activities and isoform patterns on native-PAGE of tomato cultivars, A: cv. Marmande (MRMD) and B: cv. Merveille des Marchés (MCH) at different salinity levels at different day’s growth stage. Tracks: 1-9 (control), seedlings untreated at different stage old (one to nine days respectively) and tracks 10-12, treated seedling with different NaCl concentrations (51.33 mM, 128.42 mM, and 256.84 mM respectively) with 9 days of growth

  

In Merveille des Marchés (MCH) (salt-tolerant), comparing with control esterase activities increased at all salinity levels (Figure 3B) in contrast in slightly sensible cultivar MRMD, esterase activities were less at 51.33 mM level. The plantlets of this cultivar treated at different level of NaCl showed a changes in esterase profiles which was characterized by induction of new esterase isoform (EST 1-Rf_0.11and EST 3-Rf_0.41) at both 128 mM and 256.84 mM levels. However, some of these isoform disappear such as EST 2-Rf_0.25, which has been, shifts under 256.84 mM concentration. The results also showed that the activity of some previously present isoforms diminish their intensity, as in the case of EST 4-Rf_0.78) (Figure 3B). Swapna (2003) revealed that salt stress caused reduction in esterase activity in embryo stage but it increased in tillering stage. This author concluded that tolerant rice variety under salt stress showed major difference in activity of esterase in comparison with sensible rice variety. In slightly sensible cultivar Marmande (MRMD), esterase pattern profiles showed 6 bands named (EST 1-Rf_0.07 to EST 6-Rf_0.93) (Figure 3A).
 
1.3.3 Effect on NaCl on protein banding pattern
Amini et al., (2007) stippled that when tomato plants were treated with salt, one the up-regulated protein was a salt tolerance with molecular weight of 25 KD. Moreover, it has already been reported that salt and osmotic stresses can increase the expression of stress proteins. Zhang et al., (2013), Studying effect of slat stress on protein profile in different tissues of Broussonetia papyriefera, revealed that changes in protein profiles were observed under salt stress which was characterized by induction of new protein bands as well as diminish of the previously present protein bands. In addition, the increase or decrease of intensity of protein bands in response to NaCl stress were also found. Pawar and Pannaeerselvam (2015) sowed that NaCl stress resulted in the reduction of amount of proteins. In addition, qualitative differences in the expression of proteins were observed with increase in the level of salt stress in a mangrove Xylocarpus granatum Koen plants. In our study, results showed a change in protein profiles in plantlets treated with different salt concentrations using SDS-PAGE.
 
In Marmande (MRMD), four bands named Prt-1 to Prt-4 (Figure 4 A) but in MCH only tree proteins (Prt-1 to Prt-3) were visualized (Figure 4 B). As shown in Figure 3 A, when slightly sensible variety (MRMD) was exposed at all levels of salt stress Prt 1-Rf_ 0.55 was shift comparing with control and Prt 3- Rf_0.68 was shift at 256.84 mM concentration comparing with 51.33 and 128.42 mM concentrations (Figure 4 A). In contrast, the intensity of Prt 1-RF_0.28 was increased at the same concentration (256 mM) in tolerant cultivar MCH (Figure 4 B). This trend was in accordance with Amini et al., (2007). These authors suggested that protein changes in salt tolerant tomato cv. Shiraz are related to increasing salt tolerance. Zhang et al., (2013) considered that changes in proteins profiles induced by NaCl might be that the translation of the mRNAs was inhibited or stimulated by increased concentrations, or it is due to regulation mRNA transcription.

 

 

Figure 4 SDS-PAGE of proteins (Prt) profiles of tomato cultivars, A: cv. Marmande (MRMD) and B: cv. Merveille des Marchés (MCH) at different salinity levels at different day’s growth stage. Tracks: 1-9 (control), seedlings untreated at different stage old (one to nine days respectively) and tracks 10-12, treated seedling with different NaCl concentrations (51.33 mM, 128.42 mM, and 256.84 mM respectively) with 9 days of growth

  

2 Conclusion
Altogether, these results indicated that under salinity conditions the seed vigor and seedling growth were better in tolerant genotypes than in sensible one. High level of salt induces denaturation of proteins isoforms in sensible genotypes. In contrast, tolerant genotypes increase of presently consecutive expression proteins when plants were exposed to salt stress allowing them to store form of nitrogen for the reutilization when stress is over. Another interesting finding in the current work was the distinction in antioxidant esterase activities between sensible and tolerant tomatoes genotypes. A elevates expressed of esterase isoforms is more important in the tolerant genotype than in the sensitive one. Moreover, high levels of salt stress inhibited completely some of them in both genotypes.
 
Taken together, study provides information on morphometric and biochemical variations on tomatoes under salt stress. It may strengthen our understanding of the mechanisms by which salinity affects plant growth and development. To conclude, the appearance of the regulation and translation of the mRNAS under salt stress of cultivated tomatoes plants needs further study.
 
3 Materials and Methods
3.1 Plant materials
To undertake this study, tomato seeds of eight genotypes were used including: "Marmande" (MRMD), "Saint Pierre" (STP), "Rio-Grande" (RG), "Heinz" (HZ), "Pico de Aneto" (PIC), "Merveille des marchés" (MCH), "Elgon" (ELG) and one hybrid "Agora F1" (AG).
 
3.2 Screening levels of NaCl.
Vicent et al., (2013) have indicated that under artificial stress conditions, plants are suddenly exposed to high saline concentration (e.g. 100 or 200mM of NaCl) or in increasing steps (25, 50, and 75 mM). For our investigation, four levels of NaCl were tested, control: 0, T1: 51.33 mM, T2: 128.42 mM, and T3: 256.54 mM in order to select the tolerant and the sensitive genotype.
 
3.3 Measurements of physiological parameters
Seed germination and growth measurements
Two different parameters were taken into account in order to assess the morphometric variability after two weeks of germination under stress: (a) rate of germination (vigor seeds) (b) length of seedling aged for 15 days old. For each parameter, ratio tolerance index IT (Treated / Control) was calculated. Seeds are disinfected by sodium hypochlorite 5% for five minutes and then rinsed three times with distilled water prior to germination.  The seeds were placed in Petri dishes, lined with a paper filter cloth by the different concentrations, at a rate of 30 seeds per variety per treatment. Four repetitions are performed over time, with a staff of 120 seeds per repetition and by variety. 480 seeds per variety were analyzed. Cultures were irrigated with different solutions containing the four treatments and placed in obscurity at a temperature of 24°C for seven days to initiate germination. Cultures were transferred at 24 ± 2 °C in 16 h continuous lights.
 
Depending on the conditions of ISTA rules (1993), the seed is regarded as germinated if the length of the root is equal to or greater than 1 millimeter.
 
Experimental design and Data analysis
The factorial experiment (8×4×2) was designed in a randomized completely block design (RCBD) with four replicates.All data were analyzed statistically using the Statistical Analysis program Statistica 6.1 version (Stat Soft, Inc. France). Data were recorded for the Mean ± SD (standard deviation). Data for seeds vigor were normalized by a transformation using Log10 (x + 1). All data were subjected to one-way ANOVA followed by Newman-keuls post-hoc test. The mean and standard deviation values were calculated and compared by multiple range data. Differences at P ≤ 0.05 were considered significant.
 
3.4 Biochemical approach
Protein pattern changes and isozyme Esterase variations under salt stress were evaluated. The analysis was performed on 1 to 9 days old seedling from the beginning of germination (controlled conditions) compared with treated seedling aged for 9 days at three treatments (T1, T2 and T3).
 
Protein estimation and SDS-PAGE analysis
Extraction of protein for gel electrophoresis was done from five seedlings from control and treated plants were macerated in mortar on ice with pestles in homogenization buffer containing [25 mM Tris-HCl, pH 6.8, 5% ß- mercaptoethanol, 2% (w/v) SDS and 10% (w/v) glycerol]. The extracts were denatured at a temperature above 100°C for three minutes. After 20 minutes of centrifugation at 15 000 rpm, the supernatant is recovered and precipitated overnight at 4°C by addition of 4 volumes of cold water. The extracts were stored in liquid nitrogen for subsequent use. Samples were resolved in 10% SDS-PAGE following the procedure of Laemmli (1970) and stained with coomassie Brilliant Blue R-250 (Sigma). Scanned protein banding pattern gels were analyzed and Rfs were determined by Software Gel ANALYZER (Istvan Lazar Hungary Copyright 2010).
 
Native PAGE and Esterase activity analysis
Seedling esterase extraction of the treatment lot and control were ground in liquid nitrogen in cooled mortar and homogenized in Tris-KCl buffer pH 6.8.
 
The mixture was centrifuged at 15 000 rpm for 20 min at 4°C. The extract was centrifuged two more times to remove lipids and debris. The supernatants were kept on liquid nitrogen until used.
 
The esterase activity was determined in native - polyacrylamide gel electrophoresis performed in vertical slab apparatus. The gel slab consisted of spacer gel made of 4% and a separating gel of 8% prepared in acrylamide Tris-Borate-EDTA buffer pH 6.8. The running buffer was 25 M Tris-Glycine pH 8.6, under 150 V at 4°C for 12 h (Fyad-Lameche, 1999).
 
Acknowledgments
This work was supported by the Algerian Ministry of Environment and durable development (Research project number 270). We are very grateful two anonymous referees and editor or this paper for their helpful and constructive comments.
 
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