Scholarly article on topic 'Physiological and biochemical responses to the exogenous application of proline of tomato plants irrigated with saline water'

Physiological and biochemical responses to the exogenous application of proline of tomato plants irrigated with saline water Academic research paper on "Biological sciences"

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{" Solanum lycopersicum " / Proline / "Foliar spray" / "Salt tolerance" / "Saline water"}

Abstract of research paper on Biological sciences, author of scientific article — B. Kahlaoui, M. Hachicha, E. Misle, F. Fidalgo, J. Teixeira

Abstract In scope of crop salinity tolerance, an experiment was carried out in a field using saline water (6.57dSm−1) and subsurface drip irrigation (SDI) on two tomato cultivars (Solanum lycopersicum, cv. Rio Grande and Heinz-2274) in a salty clay soil. Exogenous application of proline was done by foliar spray at two concentrations: 10 and 20mgL−1, with a control (saline water without proline), during the flowering stage. Significant higher increases in proline and total soluble protein contents, glutamine synthetase (GS, EC6.3.1.2) activities and decreases in proline oxidase (l-proline: O2 Oxidoreductase, EC1.4.3.1) activities were detected in both tomato cultivars when irrigated with saline water (6.57dSm−1) and exogenously applied by the lower concentration of proline. Taking in consideration the obtained results, it was concluded that the foliar spray of low concentration of proline can increase the tolerance of both cultivars of tomato to salinity under field conditions.

Academic research paper on topic "Physiological and biochemical responses to the exogenous application of proline of tomato plants irrigated with saline water"

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Physiological and biochemical responses to the exogenous application of proline of tomato plants irrigated with saline water

Journal of the Saudi Society of Agricultural Sciences

PII: DOI:

Reference:

B. Kahlaoui, M. Hachicha, E. Misle, F. Fidalgo, J. Teixeira

S1658-077X(15)00048-X http://dx.doi.org/10.1016/jossas.2015.12.002 JSSAS 191

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Journal of the Saudi Society of Agricultural Sciences

Received Date: Revised Date: Accepted Date:

19 January 2014 11 December 2015 11 December 2015

Please cite this article as: Kahlaoui, B., Hachicha, M., Misle, E., Fidalgo, F., Teixeira, J., Physiological and biochemical responses to the exogenous application of proline of tomato plants irrigated with saline water, Journal of the Saudi Society of Agricultural Sciences (2015), doi: http://dx.doi.org/10.1016/j.jssas.2015.12.002

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Physiological and biochemical responses to the exogenous application of proline of tomato plants irrigated with saline water

Kahlaoui, Ba Hachicha, Ma., Misle, Eb., Fidalgo, Fc, Teixeira, Jc.

aInstitut National de Recherches en Génie Rural, Eaux et Forêts, 17 rue Hédi Karray, B

Ariana 2080, Université de Carthage, Tunisia.

„ BP ,

bFaculty of Agricultural Sciences and Forestry, Universidad Católica del Maule, Casilla 7AD, Curicó, Chile

cBioFIG - Center for Biodiversity, Functional & Integrative Genomics, Faculdade de

Ciencias, Universidade do Porto, Edificio FC4, Rua do Campo Alegre, s/n 4169 - 007 Porto, Portugal.

Tel., +216. 71. 718 055 Fax, +216. 71. 717 951 Correspondence address, Besma Kahlaoui

E-mail: besma. kahlaoui /

Abstract

In scope of crop salinity tolerance, an experiment was carried out in a field using saline water (6.57dS.m-1) and subsurface drip irrigation (SDI) on two tomato cultivars (Solanum lycopersicum, cv. Rio Grande and Heinz_2274) in a salty clay soil. Exogenous application of proline was done by foliar spray at two concentrations: 10 and 20 mg.L-1, with a control (saline water without proline), during the flowering stage. Significant higher increases of proline and total soluble protein contents, glutamine synthetase (GS, EC6.3.1.2) activities and decreases in proline oxidase (L-proline: O2 Oxidoreductase , EC1.4.3.1) activities were detected in both tomato cultivars when irrigated with saline water (6.57 dS.m-1) and exogenously applied by the lower concentration of proline. Taking in consideration the obtained results, it was concludedthat the foliar spray of low concentration of proline can increase the tolerance of both cultivars of tomato to salinity under field conditions.

Keywords: Solanum lycopersicum; proline; foliar spray; salt tolerance

1. Introduction

Salinity is a major abiotic stress reducing the yield of a wide variety of plants all over the world (Ashraf and Foolad, 2007). Although the level of salt in most irrigation waters is below the threshold for the more sensitive crops, salt accumulation in irrigated soils derived from both irrigation and ground water sources can increase salinity to such levels that can reduce growth and yield of even the more tolerant crops. Salinity poses several problems for plant growth and development, especially for glycophytes, by inducing physiological dysfunction (Shannon et al., 1994). The injurious effects of salinity are associated with germination (Abdul Jaleel et al., 2007), water deficit, ionic imbalance, mineral nutrition, stomata behavior, photosynthetic efficiency, and carbon allocation and utilization (Bohnert et al., 1995).

To counteract the salinity stress, plants developed different adaptive mechanisms (Sairam et al., 2006). An adaptive plant response to salt stress is the synthesis and accumulation of low molecular weight organic compounds in the cytosol and organelles (Ashraf and Harris, 2004; Sairam et al., 2006). These compounds are collectively called compatible osmolytes because they accumulate and act without perturbing intracellular biochemistry, such as enzyme activities in the cell. Some of the compatible osmolytes reported to be affected by salinity stress are: simple sugars (fructose, glucose); disaccharides (trehalose, sucrose); sugar alcohols or polyols (sorbitol, mannitol, galactitol, and cyclic polyols such as myo-inositol, ononitol,

pinnitol); amino acids (proline); quaternary amino acid derivatives (glycine betaine; trigonelline) and sulfonium compounds (Sairam et al., 2006). Of these solutes, the most conspicuous accumulation is of amino acids, in general, and proline, in particular.

Proline accumulation is one of the many plants' adaptations to salinity and water deficit (Kumar et al., 2000; Ramajulu and Sudkakar, 2000). It has also been widely advocated that proline accumulation may be used as a selective parameter for salt stress tolerance (Ramajulu and Sudkakar, 2001). However, proline accumulation cannot be regarded as marker for salt tolerance, as its accumulation represents a general response to stress in many organisms, including higher plants exposed to environmental stresses such as high temperature, drought, and starvation (Misra and Gupta, 2005). High levels of proline enable plants to maintain low water potentials, allowing additional water to be taken up from the environment, thus buffering the immediate effect of water shortages within the organism. Proline also functions as a sink for energy to regulate redox potentials (Blum and Ebercon, 1976), as a hydroxyl radical scavenger (Schobert and Tschesche, 1978), as a solute that protects macromolecules against denaturation (Schobert and Tschesche, 1978), reducing the acidity in the cell (Venkamp et al., 1989), and acts as storage compound and nitrogen source for an after-stress rapid growth (Singh et al., 1973). It has been reported that proline alleviates NaCl-induced stress by enhancing the oxygenase and carboxylase activities of Rubisco (Sivakumar et al., 2000). Recently, it has been reported that proline accumulation protects plants against free radical-induced damage by quenching the oxygen singlet (Teixeira and Fidalgo, 2009). Under this view, the exogenous application of proline promises an alternative/additional way to genetic engineering for improving the yield under environmental stresses (Heuer, 2003; Kahlaoui et al., 2014b).

In plants, proline is synthesized from either glutamate or ornithine. It has been demonstrated that the glutamate pathway is predominant under osmotic stress conditions (Delauney and Verma, 1993). The key enzyme delta 1-pyrroline-5-carboxylate synthetase (P5CS: y-glutamyl kinase, EC 2.7.2.11; glutamate-5-semialdehyde dehydrogenase, EC 1.2.1.41) catalyzes the first two steps of proline biosynthesis from glutamate in plants. The glutamic acid-5-semialdehyde (GSA) produced by these reactions is spontaneously converted into pyrroline-5-carboxylate (PSC), which is then reduced by P5C reductase (P5CR) to proline ( Ramajulu and Sudkakar, 2001; Ashraf and Foolad, 2007; Misra and Saxena, 2009). The control of the accumulation of proline is performed through a catabolism pathway by the proline oxidase (PROX), which catalyzes the conversion of proline to glutamate. The increased or decreased

activities of the enzymes involved in proline metabolism in response to stress has been demonstrated in a wide variety of crop species including green gram (Ramajulu and Sudkakar, 2001), lentil (Misra and Saxena, 2009), tomato (Fujita et al., 2003), peanut (Girija et al., 2002) and sunflower (Manivannan et al., 2007). In addition, the synthesis of proline has been proposed to be a mean of assimilating excess ammonium, acting as an additional way of nitrogen storage (Brugiere et al., 1999). In higher plants, glutamine synthetase (GS, EC 6.3.1.2) is an important enzyme involved in the re-assimilation of NH4+ generated from photorespiration and proteolysis, processes that are increased by s alt stress (Veeranagamallaiah et al., 2007). GS is a key enzyme involved in the assimilation of inorganic nitrogen into organic forms. It catalyzes the ATP-dependent condensation of NH4+ with glutamate to yield glutamine, which then provides nitrogen groups, either directly or via glutamate, for the biosynthesis of all nitrogenous compounds in the plant (Teixeira et al., 2005). GS is widely distributed in plants, occurring in two major forms: one localised in the plastids (GS2), which is the major GS enzyme present in C3 leaves, being virtually absent in non-photosynthesizing tissues; the other isoenzyme is localized in the cytosol (GS1) and is more abundant in non-photosynthetic organs. The GS that accumulates specially in the phloem companion cells has been shown to control proline biosynthesis (Brugiere et al., 2007). GS activity has been well documented in many crops including tomato (Berteli et al., 1995), potato ( Teixeira et al., 2005) and foxtail millet (Veeranagamallaiah et al., 2007).

Considering the promising results reported in different crops, the aim of this research was to investigate the effect of exogenous application of two concentrations of proline on the physiological and biochemical responses of two cultivars of tomato irrigated with subsurface drip irrigation (SDI) and saline water (6.57 dS.m-1). This paper is part of series of experiments performed on two tomato cultivars: a tolerant cultivar Rio Grande (Kahlaoui et and Heinz -2274 cultivar (Kahlaoui et al., 2012).

2. Materials and methods

2.1. Plant material

The experiment was carried out during the summer 2009 (from 02/05 to 27/09). Two tomato cultivars (Solanum lycopersicum) were used: a tolerant cultivar to salinity, Rio Grande (Kahlaoui et al., 2011) and a sensitive one, Heinz_2274 (Kahlaoui et al., 2012).

2.2. Experimental design

The experiment was carried out at the Cherfech Agricultural Experimental Station located 25

km north of Tunis in the Low Valley of Mejerda River. The climate of the region is

Mediterranean with an annual rainfall close to 470 mm and an average yearly

evapotranspiration of 1370 mm (PET Penman). The experiment was set using emitters with

filters and buried at 30 cm depth in subsurface drip irrigation (SDI) system. Transplanting

date was 02/09 in single lines using plants with 5 - 7 leaves of age. Tomato plants were

spaced 1 m between rows and 0.40 m between plants. . This experiment was carried out

according to a randomized design with two factors (cultivar and Pro concentration). Each treatment was replicated three times and each replicate had 10 plants (30 plants per treatment for both cultivars). Treatments were two exogenous applications of Pro (10 and 20 mg.L-1) and a control (without proline application) for each cultivar. Proline spraying was performed 6 times from 30% of flowering state at June. In each treatment, three plants/replications were used in statistical analysis.

2.3. Irrigation water

The irrigation water came from a well with electrical conductivity (ECw) = 6.57 dS. m-1 and SAR=12.8. The water c hemica l characteristics are depicted in Table 1.

2.4. Proline content

The proline (PRO) content was estimated by the method of Bates et al. (1973). Plant material was homogenized in 3% (w/v) aqueous sulfosalicylic acid and the homogenate was centrifuged at 720 x g for 10 min. The supernatant was used for the estimation of proline content. The reaction mixture consisted of 1 ml of acid ninhydrin and 1 ml of glacial acetic acid, which was boiled at 100°C for 1 h. After termination of reaction in ice bath, the reaction mixture was extracted with 2 ml of toluene, and the absorbance read at 520 nm. Proline content was expressed as mg.g-1 DW.

2.5. Protein determination and Glutamine synthetase (GS, EC6.3.1.2) activity assays

Frozen tissue samples were homogenized at 4°C, with 2 vols (w/v) extraction buffer (25 mM Trizma [pH 8.0], 10 mM MgCl2, 1 mM dithiothreitol and 10 % glycerol), and 10 % (w/v) of polyvinylpolypyrrolidone (Teixeira et al., 2005). The homogenates were centrifuged twice at

12 000 x g for 15 min at 6°C. The clear supernatant was recovered to determine Glutamine synthetase activity and soluble protein content. Soluble protein content was determined by the Bradford colorimetric method (1976). GS activities were expressed as |kat.g FW-1.

2.6. PROX [L-proline: O2 Oxidoreductase (EC1.4.3.1)] activity assay

Protein extracts were obtained as for GS. The PROX activity was determined according to a protocol based on Huang and Cavaliery (1979), Rivero et al. (2004) and Nakamura et al. (2007). The assay consisted of 0.1 ml of extract, 0.8 ml of reaction buffer (62.5 mM Tris HCl buffer (pH = 8.5), 6.25 mM MgCl2, 0.625 mM FAD+, 1.25 mM KCN, 1.25 mM of phenazine methosulfate, 75 |M of 2,6-dichlorophenol-indophenol (DCPIP)) and 0.1 ml of 125 mM L-proline. The reaction started with the addition of proline and the decline in absorbance at 600 nm was determined for one min. Proline oxidase was expressed as nkat.mg-1 gFW using s600

nm= 145 cm-1.mM-1.

„ ^ ^

Statistical processing was performed by the software STATISTICA, Version 5 (Statsoft France, 1997). All the recorded parameters were subjected to a factorial analysis of variance with two factors (cultivars and concentration of proline). Means comparison were carried out by the LSD test at the significant level of 0.05.

3. Results

3.1. Proline conte,

The NaCl present in the irrigation water (6.57 dS.m-1) induced the accumulation of free proline in leaves and roots, which was significantly higher in the salt-tolerant cultivar than in the sensitive one (Fig. 1a and b). The highest proline accumulation was obtained for both cultivars when applied at the lowest concentration (10 mg.L-1). The proline content in Rio Grande cultivar was superior than Heinz-2274.

3.2. Total soluble protein content

In leaves and roots the total soluble protein content increased significantly in the salt-tolerant cultivar (Rio Grande) compared to the salt-sensitive one (Heinz-2274) when irrigated with saline water (Fig. 2). The exogenous application of proline significantly affected the total soluble protein content in both cultivars, both in leaves and roots. In leaves, this parameter

increased by 22.7% and 8.7% in Rio Grande and by 11.3% and 24.0% in Heinz_2274 respectively at 10 and 20 mg.L-1 of proline (Fig.2a). In roots, it increased by 24.0% with the lowest concentration of proline and decreased by 25.1% with the highest concentration in Rio Grande. In Heinz-2274, total soluble protein increased by 85.0% and 26.0%, respectively with the 10 and 20 mg.L-1 proline treatments (Fig.2b).

3.3. PROX activity

As shown in Figure 3, PROX activity in leaves and roots increased significantly in both cultivars, more in the sensitive cultivar than in the tolerant one when irrigated with saline water (6.57 dS.m-1). Foliar pulverization of proline had a significant effect on PROX activities in both cultivars of tomato, as PROX activity was significantly reduced at concentrations of 10 mg.L-1 and 20 mg.L-1 of proline in both organs analyzed. The highest decrease was found

with the lowest concentration of proline. 3.4. GS activity

e than in

alyzed. T

GS activity was higher in the Rio Grande than in Heinz-2274 cultivar in the control situation (saline water) both in leaves and roots. In leaves, a significant increase was observed in both tomato cultivars using proline at 10 mg.L-1 (Fig. 4a); it reached 19.2% and 14.8% respectively in Rio Grande and Heinz_2274 cultivars when compared to the control. With the higher proline concentration (20 mg.L-1), GS activity increased by 59.0% in the Heinz-2274 cultivar and did not significantly change in Rio Grande cultivar. In roots, the foliar spray of a low concentration of proline led to an increased GS activity in both tomato cultivars. This increase was about 16.2% and 75.0% in Rio Grande and Heinz-2274, respectively. With the high proline concentration treatment, the GS activity increased by 59.3% in the Heinz-2274 and decreased by 25.7% in the Rio Grande when compared to the control (Fig.4b).

4. Discussion

Biochemical studies have shown that plants under salt stress accumulate a number of metabolites, termed compatible solutes, because they do not interfere with the plant metabolism (Sivakumar et al., 2000). Among these solutes, proline is widely distributed in plants and it accumulates in larger amounts than other amino acids in salt-stressed plants (Ashraf and Foolad, 2007). Proline accumulation is one of the most frequently reported modifications induced by water deficit and salt stress in plants, and it is often considered to be involved in stress tolerance mechanisms (Ashraf and Foolad, 2007; Manivannan et al., 2007).

In the present study, a significant higher increase in proline content was found in leaves and roots of the salt tolerant tomato cultivar (Rio Grande) than in the sensitive one (Heinz-2274) (Fig. 1). The high proline accumulation observed in both organs (roots and leaves) of Rio Grande was probably due to a (a) an increase in GS activity; (b) a decrease in proline degradation; (c) an increase in proline biosynthesis, (d) a decrease in protein synthesis or proline utilization; and (e) an increase in the hydrolysis of proteins (Chen et al., 2001). Similarly to our results, increasing proline accumulation and enhanced tolerance to salinity was also found in alfalfa (Ashraf and Foolad, 2007). As leaves are the major site s of proline biosynthesis, it may explain why the accumulation of proline was more pronounced in leaves than in roots and fruits in our study for both varieties.

The exogenous application of compatible solutes offers a valuable tool for studying mechanisms of salt tolerance. One of these mechanisms depends on the capacity for osmotic adjustment, which allows growth to continue under saline conditions (Heuer, 2003) and, in some plants species, proline plays a major role in osmotic adjustment (Teixeira and Fidalgo, 2009), while in others, such as tomato, proline does not accumulate in leaves to a sufficient level to contribute significantly in the osmotic adjustment (Aziz et al., 1999). Interestingly, in our field experiment, the exogenous application of 10 mg.L-1 of proline lead to a significant increase in the proline accumulation of both organs (leaves and roots) in both tomato cultivars used (Heinz-2274 and Rio Grande) being this increase higher in leaves than in roots for both cultivars, especially in Rio Grande. In various plant species growing under saline conditions, exogenously-supplied proline provided osmoprotection and facilitated growth. According to the report of Kahlaoui et al. (2014a) on tomato crops, the exogenous application of 10 mg L1 of Pro was the most effective in promoting growth in both cultivars of tomato (Rio Grande and Heinz-2274).

Besides the accumulation of proline, which is one the most commonly found metabolic responses of higher plants to salinity (Heuer, 2003), plants are also able to respond and adapt to salt stress through the synthesis of specific proteins which can modify cell metabolism, and the synthesis of stress-induced proteins is part of that stress tolerance mechanism (Veeranagamallaiah et al., 2007). Several salt-induced proteins have been identified in plants species and classified into two distinct groups: (1) salt stress proteins, which accumulate only due to salt stress and (2) stress-associated proteins, which also accumulate in response to heat,

cold, drought, water logging and exposure to high and low amounts of mineral nutrients (Ashraf and Harris, 2004; Veeranagamallaiah et al., 2007), and may play a role in osmotic adjustment (Ashraf and Harris, 2004). In our investigation, the total soluble protein increased significantly more in the salt-tolerant cultivar than in salt-sensitive one when irrigated with saline water (6.57 dS.m-1) (Fig.2). This result is in accordance to the previous work of Ashraf and Harris (2004). Soluble protein content increased in leaves and roots of both tomato cultivars after the foliar pulverization of proline at 10 mg.L-1, being even higher in the leaves of the salt sensitive cultivar (Heinz-2274) when proline was applied at 20 mg.L-1. T he increase in the soluble protein content determined in roots can be the result of the enhanced de novo synthesis of proteins for cell protection (Teixeira et al., 2005), in addition to the beneficial effect of the exogenous proline in stabilizing membranes and the protein synthesis machinery (Khedr et al., 2003) and to a reduced membrane lipid oxidation (Okuma et al., 2004).

Although exogenous application of proline to plants exposed to abiotic stresses generally provides a stress preventing or recovering effect, special attention should be given to the highest proline level (20 mg/L) because this level caused significant reductions in total soluble potein in roots of cultivar Rio Grande compared with control treatment. According to our results, Nanjo et al. (2003) mentioned that the exogenous proline application at high concentrations may be harmful and cause retardation of plant growth. Similarly, Dawood et al. (2014) reported that exogenous application of proline at a concentration of 25 mM partially alleviated the toxicity of diluted seawater on faba bean plants, whereas the 50 mM proline treatment was toxic.

Another important factor controlling proline accumulation levels in plants is its catabolism. The oxidation of proline can be catalyzed by two enzymes (Misra and Gupta, 2005; Misra and Saxena, 2009): proline oxidase, localized in the inner mitochondrial membrane that requires oxygen as the electron acceptor; and proline dehydrogenase (Misra and Gupta, 2005; Misra and Saxena, 2009), localized in the cytosol which functions in the opposite direction of P5CR activity. In this field experiment, proline oxidase (PROX) activity was inhibited to a greater extent in leaves and roots of the salt-tolerant cultivar (Rio Grande) when compared to the saltsensitive one (Heinz-2274) in the control situation (Fig.3), but increased in both organs of the two cultivars when foliar spray of 10 mg.L-1 of proline was performed. Proline oxidase (PROX) activity was inversely correlated with proline content, strongly suggesting that this is the major enzyme controlling proline levels in tomato plants. Reduction in proline oxidase

activity and a simultaneous increase in proline level were also reported in low temperature-stressed sunflower (Manivannan et al., 2007) and tomato (Fujita et al., 2003) under water stress. A relatively higher y-glutamyl kinase activity (proline synthesis, data non shown) and a greater inhibition of the activity of proline oxidase (proline oxidation) present evidence for higher levels of free proline accumulation in both cultivars with the exogenous application of the lowest proline concentration. Our findings evidence that the inhibition of proline oxidase activity in leaves of both cultivars, under foliar spray of proline, not only increase defense as indicated by the literature but also increase proline levels. It is therefore, reinforced that proline can reduce the harmful effects of stress conditions and thus prevents the development of many types of physiological damages.

)oth leav

The present study showed a higher total GS activity in both leaves and roots of the salttolerant cultivar (Rio Grande) than in the salt-sensitive one (Heinz-2274) in the control (Fig. 4). The total GS activity was higher in leaves than in roots in both tomato cultivars. Similar results were obtained by Teixeira et al. (2005) with potato plants. The lower GS activity, detected in roots, can be justified by the fact that both tomato cultivars assimilate most of their inorganic nitrogen in leaves, as suggested by Teixeira et al. (2005) and Teixeira and Fidalgo (2009) on potato plants. A positive correlation between the increased GS activity and increased proline content when proline was exogenously applied at low concentration (10 mg.L-1) was detected. In fact, Venkamp and Lamp (1989) noticed that an increased capacity of organic acid synthesis and GS activity might facilitate the production of glutamine, proline and other organic solutes characteristic of osmotic adjustment under stress conditions. Furthermore, there are reports pointing out that the increased GS activity is associated with increased proline accumulation (Teixeira et al., 2005; Teixeira and Fidalgo, 2009). On the other hand, our findings show a high increase of GS activity in leaves of the salt-sensitive cultivar (Heinz-2274) with the foliar spray of proline at high concentration (20 mg.L-1). This GS activity may contribute to the production of more nitrogenous compounds for storage as an adaptive response to salinity (Teixeira et al., 2005), which is corroborated by the increase in soluble protein content in this organ. In this latter situation, increased GS activity of leaves is a consequence of an increased accumulation of the GS2 isoenzyme, which is localized in photosynthetic cells, where it could be involved in the assimilation of ammonium derived from nitrate reduction and photorespiration. This hypothesis is confirmed by Perez-Garcia et al. (1998), who reported that tomato plants, like other solanaceaous species with very active photosynthetic metabolism, present high levels of GS2 in the leaves.

5. Conclusion

The experiment aimed to investigate the improvement of salt tolerance of tomato in the presence of salinity by the exogenous application of proline. It showed the positive effects of foliar spray of 10 mg.L-1 of proline and subsurface drip irrigation (SDI) on both cultivars of tomato (tolerant to salinity, Rio Grande and a sensitive, Heinz-2274 ), leading to an improvement in biochemical (proline accumulation, soluble proteins, GS and PROX activities) parameters . Based on these results, the use of 10 mg/L of proline sprays can be proposed in commercial formulations to improve the growth and productivity of tomato crops grown under salt stress conditions, but further research confirmation is needed.

Acknowledgments

This study was carried out under Tunisian - Portuguese bilateral collaboration project, the INRGREF/ACSAD project for saline water use in agriculture and PISEAU II project for saline water in Center of Tunisia.

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Figures captions

Fig. 1. Effect of the exogenous application of proline on endogenous proline content in leaves (a), roots (b) and fruits (c) for both tomato cultivars irrigated by SDI and saline water (6.57

807-813.

dS.m-1).

Fig. 2 . Effect of the exogenous application of proline on total soluble protein in leaves (a) and roots (b) for both tomato cultivars irrigated by SDI and saline water (6.57 dS.m-1). Fig. 3. Effect of the exogenous application of proline on proline oxidase (PROX) activity in leaves (a) and roots (b) for both tomato cultivars irrigated by SDI and saline water (6.57 dS.m-1).

Fig. 4. Effect of the exogenous application of proline on glutamine synthetase (GS) activity in leaves (a) and roots (b) for both tomato cultivars irrigated by SDI and saline water (6.57 dS.m-

Table 1. Chemical characteristics of the irrigation water used.

EC Ionic composition (me.L- ) dS/m HCO3 SO2

7.76 6.57 4.15

37.07 14.7

5.11 45.21 12.81

Fig. 1. Effect of the exogenous application of proline on endogenous proline content in leaves (a) and roots (b) for both tomato cultivars irrigated by SDI and saline water (6.57 dS.m-1). Values represent the mean ± SE of three plants per assay and bars with different letters are significantly different at (P < 0.05) according to the LSD test.

Fig. 2 . Effect of the exogenous application of proline on total soluble protein in leaves (a) and roots (b) for both tomato cultivars irrigated by SDI and saline water (6.57 dS.m-1). Values represent the mean ± SE of three plants per assay and bars with different letters are significantly different at (P < 0.05) according to the LSD test.

Fig. 3. Effect of the exogenous application of proline on proline oxidase (PROX) activity in leaves (a) and roots (b) for both tomato cultivars irrigated by SDI and saline water (6.57 dS.m-1). Values represent the mean ± SE of three plants per assay and bars with different letters are significantly different at (P < 0.05) according to the LSD test.

Fig. 4. Effect of the exogenous application of proline on glutamine synthetase (GS) activity in leaves (a) and roots (b) for both tomato cultivars irrigated by SDI and saline water (6.57 dS.m-1). Values represent the mean ± SE of three plants per assay and bars with different letters are significantly different at (P < 0.05) according to the LSD test.