African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12268

Full Length Research Paper

Germination, growth and physiological responses of Senegalia senegal (L.) Britton, Vachellia seyal (Delile) P. Hurter and Prosopis juliflora (Swartz) DC to salinity stress in greenhouse conditions

Dioumacor Fall
  • Dioumacor Fall
  • Centre National de Recherches Forestières (CNRF)/Institut Sénégalais de Recherches Agricoles (ISRA), Route des Pères Maristes, BP 2312, Dakar, Sénégal
  • Google Scholar
Niokhor Bakhoum
  • Niokhor Bakhoum
  • Laboratoire Commun de Microbiologie IRD/ISRA/UCAD (LCM), Centre de Recherche de Bel-Air, BP 1386, Dakar, Sénégal.
  • Google Scholar
Fatoumata Fall
  • Fatoumata Fall
  • Laboratoire Commun de Microbiologie IRD/ISRA/UCAD (LCM), Centre de Recherche de Bel-Air, BP 1386, Dakar, Sénégal.
  • Google Scholar
Fatou Diouf
  • Fatou Diouf
  • Laboratoire Commun de Microbiologie IRD/ISRA/UCAD (LCM), Centre de Recherche de Bel-Air, BP 1386, Dakar, Sénégal.
  • Google Scholar
Mamadou O. Ly
  • Mamadou O. Ly
  • Centre National de Recherches Forestières (CNRF)/Institut Sénégalais de Recherches Agricoles (ISRA), Route des Pères Maristes, BP 2312, Dakar, Sénégal
  • Google Scholar
Mayécor Diouf
  • Mayécor Diouf
  • Centre National de Recherches Forestières (CNRF)/Institut Sénégalais de Recherches Agricoles (ISRA), Route des Pères Maristes, BP 2312, Dakar, Sénégal
  • Google Scholar
Djamel Gully
  • Djamel Gully
  • Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM)/Institut de Recherche pour le Développement (IRD) TA A-82/J, Campus International de Baillarguet, 34398 Montpellier, France.
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Valérie Hocher
  • Valérie Hocher
  • Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM)/Institut de Recherche pour le Développement (IRD) TA A-82/J, Campus International de Baillarguet, 34398 Montpellier, France.
  • Google Scholar
Diégane Diouf
  • Diégane Diouf
  • Laboratoire Commun de Microbiologie IRD/ISRA/UCAD (LCM), Centre de Recherche de Bel-Air, BP 1386, Dakar, Sénégal.
  • Google Scholar


  •  Received: 09 June 2016
  •  Accepted: 18 August 2016
  •  Published: 14 September 2016

 ABSTRACT

Salinity is among the most widespread environmental threats to global plant production, especially in arid and semi-arid climates. Thus, the selection of salt tolerant species is necessary for sustainable plant productivity. The purpose of this study was to measure and understand the salt tolerance of three multipurpose trees used in reforestation programs in many Sahelian countries (Senegalia senegal, Syn. Acacia senegal; Vachellia seyal, Syn. A. seyal, and Prosopis juliflora). The effect of salinity was evaluated at seed germination stage on Petri dishes containing water agar (0.9%, w/v) with seven concentrations of NaCl (0, 86, 171, 257, 342, 428, and 514 mM). Our results showed that all the species had a germination rate higher than 85% at 257 mM. However, it decreased at 342 mM with a reduction of 70 and 20%, respectively for S. senegal and V. seyal. For plants growth and physiological responses, seedlings were individually cultivated in plastic bags (25×12 cm) containing non-sterile soil and watered with four salt solutions (0, 86, 171 and 257 mM NaCl). Four months after the plants’ cultivation, the results showed that for all species, the salinity reduced significantly the height, the collar diameter, the shoot and root dry biomass as well as the total chlorophyll, K+ and K+/Na+ ratio. In the meantime, proline content, Cl- and Na+ accumulation in leaves were increased. It was also found that S. senegal and V. seyal tolerated high concentrations of NaCl (257 mM) and developed physiological and molecular mechanisms, such as salt tolerance genes (NHX1), which allow them to be considered as moderated salt tolerant species and seemed to be potential species for the restoration of salt-affected land as P. juliflora.

Key words: Multipurpose leguminous trees, abiotic stress, salt tolerance, Senegal.


 INTRODUCTION

Soil salinization is an emerging environmental problem around the world and represents a major limiting factor for plants production and ecological environment, especially in arid and semi-arid regions (Zahran, 1999). Over 953 million ha of land are salt-affected throughout the world, covering about 8% of the world’s land surface (Singh, 2009). In Senegal, 1 700 000 ha of the 3 800 000 ha of the agricultural lands are salt-affected (FAO-LADA, 2009). Numerous studies have shown that high NaCl concentrations in the growth medium of plants generate primary and secondary effects that negatively affect plant growth and development. Several physiological functions, including photosynthesis, mineral nutrition and carbohydrate metabolism have been shown to be affected by the high salinity (Chen et al., 2008). Primary effects are ionic toxicity and osmotic stress. Ionic toxicity occurs because of the high Na+ and Cl- concentrations in the cytoplasm of cells. The lowering of the water potential that causes turgor reduction and cellular water loss also induces osmotic stress. Secondary effects of NaCl stress include inhibition of K+ uptake, membrane dysfunction and generation of reactive oxygen species (ROS) in the cells (Rout and Shaw, 2001; Ghoulam et al., 2002; Agarwal and Pandey, 2004; Upadhyay and Panda, 2005). Salinity modifies photosynthetic parameters, including osmotic and leaf water potential, transpiration rate, leaf temperature, and relative leaf water content (RWC); salt also affects. The components of photosynthesis such as enzymes, chlorophylls, and carotenoids are also affected by the salt. Changes in these parameters depend on the severity and duration of stress (Misra et al., 1997) and on plant species (Liu et al., 2011).

Senegalia senegal (L.) Britton, Vachellia seyal (Delile) P. Hurter and Prosopis juliflora (Swartz) DC are trees species widely found in arid and semi-arid zone of Senegal. S. senegal is a multipurpose perennial legume, widespread in the semi-arid zone of tropical Africa and the Middle East. S. senegal has huge potential in agroforestry systems, fuelwood production, forage, medicinal products and gum production (Von Maydell, 1986). In this respect, more attention is given to S. senegal which is among the selected forest species for the Great Green Wall, from Senegal to Djibouti (GMV, 2009). V. seyal contributes to soil fertility in agroforestry systems. It produces fodder and gum and is also an important source of rural energy because of its role in the production of both firewood and charcoal. In Senegal, V. seyal is widely distributed in the salt-affected coastal steppes. P. juliflora is used in agroforestry systems. This legume tree is an important component of the system, because it serves as a source of high quality animal feed. It can be sold for fuel and timber, as well as it improves soil physiochemical and biological properties, generating “fertility islands” or “resource islands” beneath its canopy (Reyes-Reyes et al., 2002; Diedhiou et al., 2009; Dossa et al., 2010). It also has been described as salt-tolerant species (Basavaraja, 2007).

Adaptation of plants during germination and early seedling stages in saline environments is crucial for the establishment of species (Debez et al., 2004; Vicente et al., 2004). After seeds germination, seedlings are the most vulnerable in the life cycle of plants (Kolb and Barsch, 2010). Thus, for the successful establishment of plants in saline environments, seeds must remain viable at high salinity in an imposed secondary dormancy and germinate when salinity decreases (Vicente et al., 2004). Furthermore, seedlings will be able to grow in these conditions. One key mechanism of salinity tolerance is the ability to remove Na+ ions from the cytosol and its sequestration into the vacuole to limit cell damage. The transport of Na+ into vacuoles is thought to be mediated by vacuolar Na+/H+ antiporters of the NHX family, which are driven by the electrochemical gradient of protons (Gaxiola et al., 1999; Pardo et al., 2006). Several studies showed that NHX1 overexpression increased salinity tolerance in plants (Apse et al., 1999; Tian et al., 2006; Brini et al., 2007; Chen et al., 2015).

In Senegal, like in most arid and semi-arid regions, reforestation of salty lands has become a priority. S. senegal, V. seyal and P. juliflora are leguminous multipurpose trees selected in many reforestation programs. However, few studies have been done on the adaptation of these species to environmental conditions in relation to climate change such as drought and salinity. The objective of our study was to evaluate the effect of NaCl on S. senegal, V. seyal and P. juliflora germination, growth and some physiological and molecular traits in order to measure and understand their salt tolerance; with the aim to facilitate the on-going initiatives of Senegalese Forest Department to find suitable species for plantation in the salt affected areas in a context of climate change.


 MATERIALS AND METHODS

Effect of NaCl on S. senegal, V. seyal and P. juliflora seeds germination

Seeds of S. senegal (Provenance Dahra-2013), V. seyal (Provenance Ndiaffate-2013) and P. juliflora (Provenance Ndiaffate-2013) were provided by the National Centre for Forestry Research (CNRF) of the Senegalese Institute of Agricultural Research (ISRA). P. juliflora was chosen as a reference of wood species for salt tolerance. Seeds scarification and germination were performed as described by Fall et al. (2008). Seeds (n=20) were germinated on Petri dishes containing water agar (0.9% w/v) with the different concentrations of sodium chloride (NaCl). Seven concentrations of NaCl were tested: 0, 86, 171, 257, 342, 428 and 514 mM (Fall et al., 2009). The experiment was repeated three times (20 seeds × 3 = 60 seeds) for each salt treatment. Seeds were considered as germinated when emerging radical was visible. The number of germinated seeds was counted daily for 10 days. The germination rate was expressed in mean final percent germination, calculated from cumulative germinated seeds on the final day of assessment to that of the total number of seeds in the sample at different salinity levels.

Growth and physiological responses of S. senegal, V. seyal and P. juliflora seedlings under saline conditions

Growth conditions and salt treatment

Seeds were germinated as described on water agar. Seedlings were then individually cultivated in plastic bags (25×12 cm) containing 1.3 kg of non-sterile soil (Table 1) collected from Sadioga (Centre of the groundnut basin of Senegal, 16° 23’ 18 W, 14° 03’ 53 N). The experiment was carried out in greenhouse conditions at the Laboratoire Commun de Microbiologie IRD/ISRA/UCAD (LCM) of Dakar (Senegal) in Bel-Air (14°44’N, 17°30’W) under natural sunlight (35°C day, 27°C night, with 14 h photoperiod). The relative humidity was about 75%. Accordingly to the results on the species germination rate and the level of salinity of our study site (EC = 0.414 to 34.3 mS/cm), four concentrations of NaCl (0, 86, 171, and 257 mM) were tested. A randomized experimental design was used with four treatments and 10 replicates per treatment. Salt stress treatment was performed one month after transplantation. Seedlings were gradually exposed to NaCl in order to minimize any salinity shock. NaCl concentrations were increased by 43 mM per day until reaching the required final concentration. The salinity of the leachate from representative pots was monitored regularly with a salinometer (Digit 100 ATC Salinity pocket refractometer, CETI, Optical Instruments, Belgium) to ascertain actual NaCl concentrations within the rooting medium.

 

 

Plants growth measurement

Three months after salt stress application, plants growth was evaluated by measuring plant height, collar diameter, shoot (leaves + stems) and root dry biomass.

Physiological traits measurements

Relative water content: Relative water content (RWC) estimation was done by incubating stem fragment (5 cm) in 15 ml distilled water for 24 h and calculated according to Yamasaki and Dillenburg (1999) after 96 h at 80°C in a stove.

Leaf water potential: Water potential is defined as the potential energy per unit mass of water with reference to pure water at zero potential (atmospheric pressure and 20°C), (Campbell, 1977). Leaf water potential (LWP) was measured using a Scholander pressure chamber (Scholander et al., 1965).

Total chlorophyll content: The total chlorophyll content was evaluated from 100 mg of fresh leaves according to Arnon (1949) method. The total chlorophyll content was calculated as follow: C = [20.2 (A645) + 8.02 (A663)] × V/M; where, V and M are the extraction volume (L) and weight (mg) of crushed leaves, respectively.

Proline content: Free proline content was determined by spectrophotometry from 100 mg leaf samples according to Monnevaux and Nemmar (1986). The proline concentration on a fresh-matter basis was obtained from a calibration graph prepared with a series of standard proline solutions.

Concentration of Na and K in roots and leaves: Concentrations of Na and K were determined by atomic absorption spectro-photometer after HNO3-H2O2 digestion. Chloride was extracted by contact with boiling deionized water and colorimetric assay was done using the method of mercuric thiocyanate and ferric nitrate.

Salt tolerance index of S.senegal, V. seyal and P. juliflora at germination and growth in greenhouse conditions: Salt tolerance index (STI) was calculated as the ratio of the parameters salt stressed plants versus those of control plants (Cano et al., 1998).

STI (%) = (MV at Cx / MV at C0) × 100

Where, MV is the measured variable, C0 is the control, and Cx is a given concentration of salt.

Identification of a vacuolar antiport Na+/H+ (NHX1) in S. senegal, V. seyal and P. juliflora

Total RNA extraction

The seedlings of S. senegal, V. seyal and P. juliflora were grown on a mix of peat and vermiculite (2/1) during three weeks. Total RNA was extracted from young leaves using the RNeasy total RNA isolation kit (Qiagen). About 100 mg of fresh leaves were excised and ground using liquid nitrogen to fine powder. One milliliter of RTL buffer containing 10 mg of polyvinylpyrrolidone (PVPP) and 10 ml de Mercaptoethanol was added to the powder. The mix was incubated at 56°C for 2 min in a water bath. The lysate (650 ml) was transferred to a QIAshredder column and centrifuged at room temperature (25°C) at 18000 rpm for 2 min. The supernatant was collected in a fresh Eppendorf tube and 0.5 volumes of alcohol (96%) were added and mixed very well. The mixture was transferred on a spin column and centrifuged at 10 000 rpm for 45 s. Total RNA were washed with 700 ml of RW1 buffer by centrifugation at 10 000 rpm for 20 s and with 500 ml of RPE buffer at 10 000 rpm for 20 s. Total RNA was eluted with 30 ml of RNase-free water. To remove contaminating DNA, RNAs were treated with RNase-free DNase. The integrity of total RNA was estimated by a bioanalyzer before reverse transcription. One microgram of RNA was reverse-transcribed using Supercript II reverse transcriptase. The reverse transcription (RT) reactions were performed at 42°C for 50 min and using oligo-dT by following the instruction of the manufacturer.

NHX1 gene amplification

Since any specific primers were described in the literature to amplify NHX1 gene for the three species, degenerated primers were used. Thus, the complete sequences of NHX1 gene in several salt tolerant and non-tolerant species were searched in Genbank. After alignment of the obtained sequences, primers were designed in the highly conserved regions. Thus, one forward primer (NHX1F_5’-TTYAATGCHGGSTTTCARG-3’) and 3 reverse primers (NHX1R1_5’-GABGTDGCATCATTHACAACWCC-3’; NHX1R2_5’-ACCTCDCGATCWGTNGART GC-3’ and NHX1R3_5’-TGRGACRTVACAATMCCAC-3’) were designed and theoretical sizes for three combinations were estimated. Two microliters of cDNAs were used as templates for PCR amplification with degenerated primers. Three couples of primers were tested (NHX1F/NHX1R1, NHX1F/NHX1R2 and NHX1F/NHX1R3). Samples were denatured for 3 min at 94°C and then run for 30 cycles of 30 s each at 94 and 50°C, 45 s at 72°C with a final extension of 7 min at 72°C. The PCR products were separated by 1% agarose gel electrophoresis (Figure 2).

Data statistical analysis

A one-way analysis of variance (ANOVA) analysis was performed for all data sets using XLSTATTM for Windows statistical data analysis package (version 2009, Addinsoft, Paris, France). Student-Newman-Keuls´s post-hoc test was employed to determine if significant (P ≤ 0.05) differences occurred in parameters measured between salinity treatments, and also the STI of species.


 RESULTS

Effect of NaCl on S. senegal, V. seyal and P. juliflora seeds germination

The effect of NaCl on S. senegal, V. seyal and P. juliflora seeds germination, evaluated by the percentage of germinated seeds after 10 days, is as shown in Figure 1.

 

 

 

The results show that for all species, the germination rate decreased with an increase of the NaCl concentration. Nevertheless, the negative effect of NaCl varied according to species. Below 257 mM, the germination rate of species was nearly equal to 100%. However, from this concentration, a difference in salinity tolerance was observed between species. For example, at 428 mM of NaCl, a reduction was observed in the germination rate of 100 (no germination), 92 and 48%, respectively for S. senegal, V. seyal and P. juliflora. At 514 mM NaCl, no germination was observed in V. seyal while a germination rate of 18% was observed for P. juliflora.

Effect of NaCl on S. senegal, V. seyal and P. juliflora seedlings growth

Salinity reduced height, shoot and root dry biomass of S. senegal, V. seyal and P. juliflora seedlings as shown in Table 2. However, the collar diameter seemed to increase with NaCl concentration. The low concentrations of NaCl (86 mM) seemed to increase the growth of P. juliflora. No significant negative effect of salinity was observed on seedlings height of the three species and on P. juliflora shoots dry biomass (SDB). In contrast, a significant negative effect was noted on SDB of S. senegal and V. seyal seedlings. For S. senegal, the average SDB was 0.95 g plant-1 at control and this value gradually decreased throughout the increasing salt concentrations, and reached to 0.28 g plant1 at 257 mM NaCl. In V. seyal, the SDB was 1.16 gplant-1 at 0 mM NaCl against 0.28 g plant-1 at 257 mM NaCl. At 257 mM, when compared to the control, the species had approximately the same percentages reduction in height with 24, 21 and 21%, respectively for P. juliflora, S. senegal and V. seyal. The percentages reduction in shoots dry weights were 76, 71 and 37%, respectively for V. seyal, S. senegal, and P. juliflora (Table 2). RDB at 257 mM NaCl decreased by 71, 57 and 51%, respectively for P. juliflora, S. senegal and V. seyal when compared with the seedlings control.

 

 

Physiological responses of S. senegal, V. seyal and P. juliflora seedlings under saline conditions

Chlorophyll, proline, leaves Cl-, Na+ and K+ contents were not evaluated in S. senegal at 257 mM NaCl because seedlings had no leaves. Table 3 showed total chlorophyll and proline contents, RWC and LWP of S. senegal, V. seyal and P. juliflora seedlings. The chlorophyll content decreased in S. senegal and V. seyal with the increase in NaCl concentration. However, the chlorophyll content seemed to be increased in P. juliflora.

 

 

The reduction rate in chlorophyll content at 171 mM NaCl was 65 and 9%, respectively in S. senegal and V. seyal when compared with controls, while an increase of 11% was observed in P. juliflora at the same concentration of NaCl. In contrast to chlorophyll, free proline content increased with the NaCl concentration. This increase of proline content was more pronounced, respectively in P. juliflora, S. senegal and V. seyal for NaCl concentrations tested, with respectively 208, 95 and 73%. No significant difference (excepted S. Senegal at 257 mM) was observed in RWC of seedlings grown under salinity stress compared to those grown in non-saline soil.

However, RWC seemed to increase in S. senegal and decrease in V. seyal and P. juliflora seedlings when increasing NaCl concentration. LWP was more negative under salinity stress for all species. Significant difference was noted between controlled and stressed plants for all species (Table 3). At 171 mM NaCl, LWP was negatively increased by 75, 26 and 22%, respectively in P. juliflora, S. senegal and V. seyal. Our results indicated that Cl- and Na+ content in leaves and roots increased with salinity (Table 4). The accumulation of Cl- and Na+ was higher in leaves than roots in S. senegal and V. seyal, while it is higher in roots than leaves in P. juliflora. However, the accumulation of K+ was higher in leaves than roots for all species (Table 4). At 171 mM NaCl, leaf Cl- content was increased by 6, 164 and 278%, respectively in P. juliflora, S. senegal and V. seyal. However, leaf Na+ content was increased by 471, 1093 and 3050%, respectively in P. juliflora, V. seyal and S. senegal. The K+ content and the K+/Na+ ratio were reduced in roots and leaves of S. senegal, V. seyal and P. juliflora. The K+ content was significant higher in leaves than roots for all species.

 

 

Salt tolerance index of S. senegal, V. seyal and P. juliflora at germination and growth

Results show that salt tolerance index (STI) decreased with increasing NaCl concentration at germination and growth (Table 5). For germination, no significant difference was noted between STI of species for NaCl concentrations less than or equal to 257 mM NaCl with 89, 85 and 92%, respectively in S. senegal, V. seyal and P. juliflora. Nevertheless, when the NaCl concentration increased, a difference in STI occurred. For S. senegal, STI was 30% at 342 mM and became 0% from 428 mM of NaCl, while V. seyal and P. juliflora had an STI of 80 and 88%, respectively at 342 mM. At 514 mM, the STI of V. seyal became 0%; however, P. juliflora had a STI of 18% (Table 5). For growth (shoot dry biomass), STI of S. senegal was higher than V. seyal for all NaCl concentrations. At 86 mM NaCl, no significant difference was observed between S. senegal and P. juliflora which had STI significantly higher than V. seyal. At 257 mM NaCl, STI was 24, 29 and 63%, respectively for V. seyal, S. senegal and P. juliflora.

 

 

Identification of NHX1 gene in S. senegal, V. seyal and P. juliflora

Results showed that the three primers generated amplicons for all species except for P. juliflora with NHX1F/NHX1R1 couple. NHX1F/NHX1R1, NHX1F/ NHX1R2 and NHX1F/NHX1R couples produced good amplicons with the expected size with an approximately size of 300, 500 and 600 pb, respectively.

Any difference in terms of amplicon size was obtained among species for the same primer.


 DISCUSSION

Our results show that for all species, the percentage of germination decreased as the degree of salinity increased. Reduction in germination by increasing salinity levels has been described by numerous studies (El-Tayeb, 2005; Abari et al., 2011; Tsegay and Gebreslassie, 2014; Sharma and Vimala, 2016). High concentration of NaCl in the salt solution increases its osmotic potential and the germination rate reduction could be attributed to the osmotic effect of NaCl, which limits the seed hydration (Tobe et al., 2000; El-Keblawy and Al-Rawai, 2005). It could also be due to the toxic effect of NaCl on seed embryo or endosperm cell membranes (Bliss et al., 1986; Khajeh-Hosseini et al., 2003). The inhibition of germination can be due to high absorption of Na and Cl ions during seed germination (Taiz and Zeiger, 2002). On the basis of their salt tolerance at germination, the species can be arranged as follows: P. juliflora > V. seyal > S. senegal.

It was found that the seedlings growth (height, shoot and root dry weight) was affected by salinity and that the salt effect depends on salinity level and species. Results for height, shoot and root dry weight in response to increasing salinity level suggested that there was maximum reduction in shoot dry weight for V. seyal and S. Senegal, while the highest reduction of root dry weight was observed in P. juliflora. According to Alam et al. (2004), it is possible that the decrease in plant growth in saline condition was due to several reasons. One possibility is that salinity reduced photosynthesis, which in turn limited the supply of carbohydrates needed for growth (da Silva et al., 2011). A second possibility is that salinity reduced shoot and roots growth by reducing turgor in expanding tissues resulting from lowered water potential in root growth medium. Third, a disturbance in mineral supply, either an excess or deficiency, induced by changes in concentrations of specific ions in the growth medium, might have affected directly the growth (Lazof and Bernstein, 1998; Zhu, 2002). The decrease in shoot biomass and the yellow leaves might be due to disturbance in carbohydrates allocation in shoots. Our results for reduction of seedlings growth of all species with increase in NaCl concentration are in conformity with the finding of several authors (Abari et al., 2011; Saini et al., 2012; Sharma and Vimala, 2016). The low concentration of NaCl (86 mM) seemed to increase P. juliflora seedlings growth. This result corroborated those obtained by Viégas et al. (2004) on P. juliflora seedlings in hydroponic condition. The absence and the falling leaves at 257 mM NaCl, observed respectively in S. senegal and V. seyal, reduced the photosynthetic area which result in the reduction of plant growth and yield. On the basis of tolerance at growth (shoot biomass production), the species can be arranged as follows: P. juliflora > S. senegal > V. seyal. This ranking was confirmed by STI, which were 63, 29 and 24%, respectively in P. juliflora, S. senegal and V. seyal at 257 mM NaCl. The total chlorophyll (a + b) content decreased significantly with increasing NaCl concentration for all species. The decrease in chlorophyll content under salt stress is a commonly reported phenomenon. It is related to its adverse effects on membrane stability (Ashraf and Bhatti, 2000), the weakening of protein-pigment-lipid complex (Ayala-Astorga and Alcaraz-Meléndez, 2010) and the increasing chlorophyllase (EC: 3.1.1.14) activity (Lakhdar et al., 2008). Also, Santos (2004) pointed out that the induced decrease in chlorophyll content in severely NaCl stressed leaves is mainly due to a decrease of 5-aminolinolic acid (ALA) synthesis, and therefore to limitations of chlorophyll synthesis. These observations corroborated with the results obtained by several authors (Hardikar and Pandey, 2008; Turan et al., 2009; Molazem et al., 2010; Heidari, 2012; Taibi et al., 2016). In contrast, the increase in NaCl concentration seemed to increase leaf total chlorophyll of P. juliflora suggesting that it was more tolerant in saline medium than S. senegal and V. seyal whose chlorophyll content decreased.

Free proline content increased with NaCl concentration for all species. Salt tolerant plants are distinguished by their capacity to accumulate high concentrations of compatible osmotica such as glycine betaine, proline, in cytoplasm to balance the osmotic pressure of ions in the vacuoles. Increasing leaf proline content under salinity stress might be caused by induction or activation of proline synthesis from glutamate or decrease in its utilization in protein synthesis or enhancement in protein turnover. The high accumulation of proline indicated that S. senegal (95%) and V. seyal (73%) seemed to have the capacity to tolerate salinity as P. juliflora (208%). Similar results were observed in many plant species such as Acacia auriculiformis (Diouf et al., 2005), date palm (Sané et al., 2005), rice (Shereen et al., 2007), maize (Cha-um and Kirdmanee, 2009), walnut (Akça and Samsunlu, 2012), and potato (Jaarsma et al., 2013).       

Relative water content (RWC) and leaf water potential (LWP) are the basic parameters of plant water status. Water status is the main factor affecting the plants growth and development. Even if no significant difference was observed, the decrease of RWC in V. seyal and P. juliflora seedlings grown under salinity stress, compared to those grown in non-saline soil, indicated that salinity resulted in dehydration at cellular level and dehydration symptoms were greater in higher NaCl concentration treatment because of the increase in cellular water loss. This is a common reaction to salinity similar to those reported for other species such as pepper plants (Navarro et al., 2003), Avicennia germinans (L.) (Suárez and Medina, 2008). Therefore, plants should have osmotic adjustment inside the cell, since turgor maintenance is required for cell expansion and the biochemical, physiological and developmental processes (Flowers, 2004). Even so, the RWC seemed to increase in S. senegal with NaCl concentrations. This exception can be explained by the reduction and/or the absence of leaves, which reduced the transpiration area along the water retention in cells. Water potential in S. senegal, V. seyal and P. juliflora became increasingly more negative with the corresponding increase in media salinity, indicating that these species osmotically adjust in response to increases in salinity. Water potential was more negatively increased by 75, 26 and 22%, respectively in P. juliflora, S. senegal and V. seyal, suggesting that P. juliflora adjust better its osmotic pressure followed by S. senegal and V. seyal.

According to Greenway and Munns (1980), NaCl, the predominant form of salt in most saline soils, enhances Na+ and Cl- contents and consequently affects the uptake of other mineral elements. Our results showed that Cl- and Na+ accumulation increased in roots and leaves when increasing NaCl concentration while K+ accumulation and K+/Na+ ratio decreased, but more Na+ was taken up than Cl, indicating that S. senegal, V. seyal and P. juliflora are an ion accumulators. The strong Na+ accumulation in leaves could be responsible for their loss as observed in S. senegal and V. seyal. Previous studies have shown that salinity increases Na+ and Cl- and decreased K+ and K+/Na+ in plant leaves (Saghir et al., 2002; Hosseini and Thengane, 2007; Taffouo et al., 2010; Silini et al., 2016) found. This implies a competition between Na+ and K+ absorption in plants, resulting in a Na+/K+ antagonism (Mori et al., 2011). The reduction in K+ uptake caused by Na+ is likely to be the result of the competitive intracellular influx of both ions (Tripathi and Müller, 2015). It is well established that many K transport systems have significant affinity for Na+ (Rodriguez-Navarro and Rubio, 2006).

Our preliminary results on molecular studies show the presence of salt tolerant gene NHX1 in S. senegal, V. seyal and P. juliflora. To our knowledge, this is the first study of the tonoplast associated Na+/H+ antiporter in these species. This result indicated that these species have the ability to sequester the Na+ in the vacuole which in turn, will enhance their adaptation to salinity. The overexpression of NHX1 in saline conditions will increase their tolerance to salinity as shown by several studies (Gouiaa et al., 2012; Baltierra et al., 2013; Hasegawa, 2013; Panahi et al., 2013; Hu and Wu, 2014; Chen et al., 2015).


 CONCLUSION

The results show that salt stress decreased germination rate, growth, total chlorophylls content, relative water content, leaf water potential, K+ accumulation and K+/Na+ ratio in S. senegal, V. seyal and P. juliflora. Salt stress increased proline, Cl- and Na+ accumulation in all species. Nevertheless, results showed that species maintained a good germination rate and an overall growth at high level of salinity. In summary, the results show that S. senegal, V. seyal and P. juliflora accumulated Na+ to achieve a negative water potential gradient and also accumulate proline as an osmoprotectant or to achieve osmotic balance in the cytoplasm. According to our results, S. senegal and V. seyal should be considered as species that might be used to phytoremediate degraded saline lands as P. juliflora. However, molecular studies will be continued to evaluate the expression salt tolerant gene (NHX1) in saline conditions and field trials will be conducted to confirm their ability.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.


 ACKNOWLEDGMENTS

This work was funded by the ‘Fonds d’Impulsion de la Recherche Scientifique et Technologique (FIRST)’ of the Ministry of Higher Education and Research of Senegal and the Laboratoire Mixte International «Adaptation des Plantes et Microorganisms associés aux Stress Environnementaux» (LMI-LAPSE). They also thank Mrs. Awa DIALLO and Dado WAGUE, students at the University Assane SECK of Ziguinchor (Senegal) for their technical assistance and Dr Moussa DIENG, PhD (Michigan State University, USA), for the English revision.



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