Introduction

Reaumuria vermiculata L. is a xero-halophytic perennial dwarf shrub distributed in many gypseous and saline areas in southern Tunisia (Gorai and Neffati 2007). It reaches 50–100 cm height and occurs along different bioclimatic stages with precipitations varying from 50 to 400 mm (Le Houérou 1993). Reaumuria vermiculata is highly tolerant to drought and salinity, and widely found in tropical and subtropical regions (Le Houérou 1993, 1995). This species, belonging to the Tamariaceae family, avoids salinity or excretes salts via the leaf surface by glandular structures that are similar to hydathodes (Le Houérou 1993) and possesses an environmental interest that consists in its capacity to desalinize saline soils by responsible salt glands. If plants were harvested at the end of the growing season, a significant reduction in soil salinity might be achieved (Gorai and Neffati 2007). Seeds are donned of silken hairs and flowering happens from February to June (Pottier-Alapetite 1979). In their natural habitats, seeds mature at the end of August and germination starts following rains in autumn and early winter, when temperatures were decreasing (Gorai and Neffati 2007). These authors indicated that germination was inhibited by either an increase or decrease in temperature from the most suitable temperature found (15°C) and increasing NaCl-salinity progressivily inhibited seed germination, which was less than 5% at 300 mM.

Environmental abiotic stress conditions, and especially drought and salinity, are currently the major factors responsible for the worldwide deterioration of plant cover and the erosion of soils (Boyer 1982; Owens 2001). The ability of plants to survive and maintain growth under saline conditions is known as salt tolerance. The deleterious effects of salinity on plant growth are associated with low osmotic potential of soil solution, nutritional imbalance, specific ion effect, hormonal imbalance and induction of oxidative stress, or a combination of these factors (Greenway and Munns 1980; Marschner 1995; Parida and Das 2005). Responses of species to salt stress depend on several interacting variables, including the magnitude (salt concentration and time of exposure) of the stress, plant genotype, plant developmental stage and cultural environment (Sultana et al. 1999; Jaleel et al. 2007). Salinity reduces the ability of plants to take up water, causing a reduction in growth along with a suite of metabolic changes (Munns 2002). A metabolic response to salt stress is the synthesis of compatible osmolytes (Ashraf and Foolad 2007). These mediate osmotic adjustment and therefore protect sub-cellular structures and reduce oxidative damage caused by free radicals, produced in response to high salinity (Zhu 2001; Mansour and Salama 2004).

The aim of the present study was to investigate the effects of salinity on growth, water relations and accumulation of organic and inorganic solutes in early seedlings stage of R. vermiculata.

Materials and methods

Plant material and culture conditions

Seeds of R. vermiculata were obtained from plants that were collected from a location near El Fjé, Medenine (33°30′N, 10°39′E; southeast Tunisia) in July 2007. This area is arid to semi-arid with a typical Mediterranean climate, characterized by irregular rainfall events and a harsh dry summer period (Gorai and Neffati 2007).

Seeds were cleaned and stored for 6 months in the seed bank of the Laboratoire d’Ecologie Pastorale at the Institut des Régions Arides (Médenine, Tunisia) in which relative humidity was set at 30% and temperature was maintained at 20°C. When experiments were carried out, seeds were surface sterilized with Na-hypochlorite. The pots were filled with sterilized mixture of sand and soil (1:2), three seeds per pot were planted and the soil irrigated with distilled water until germination. The maximum germination was observed after 15 days. At this stage, the seedlings were thinned to one per pot and irrigated with Hewitt nutrient solution (Hewitt 1966), containing macronutrients: 1.5 mM MgSO4, 1.6 mM KH2PO4, 0.4 mM K2HPO4, 3 mM KNO3, 2 mM NH4NO3 and 3.5 mM Ca(NO3)2. The medium also contained iron as complex EDTA–K–Fe (45 μM) (Jacobson 1951) and micronutrients as a mixture of salts: 8 μM MnCl2; 0.7 μM CuSO4 5H2O; 0.76 μM ZnSO4, 7H2O; 0.3 μM Mo7O24(NH4)6 4H2O and 46 μM H3BO3 (Arnon and Hoagland 1940). Plants were grown in a green-house as follows: 25 ± 1°C temperature, 50% day/75% night relative humidity and 16 h light/8 h dark regime.

Experimental design and NaCl treatments

Individual plants of homogeneous stage of development and size were selected. The experiment was arranged in a completely random design with six NaCl-salinity levels × six replicates. Plants were partitioned into six lots and irrigated with control nutrient solution lacking salt or the same nutrient solution supplemented with 100, 200, 300, 400 or 600 mM NaCl. The choice of salt concentrations was driven on previous studies, where the growth of R. vermiculata seedlings was completely arrested and all of them subsequently died at 800 mM NaCl (Gorai and Neffati 2007; Gorai unpublished data). To avoid osmotic shock, NaCl concentrations were increased stepwise in aliquots of 50 mM day−1. An amount of 200 ml NaCl solution was supplied every 2 days, which equaled the amount that was flushed from the drained pots.

Growth measurements

Two harvests were made, at the beginning of treatment (4-month-old plants) and 30 days later. At the harvests, plants were divided into shoots and roots and their respective fresh mass (FM) was measured immediately. Dry mass (DM) of shoots was determined using a LCD-1 lyophilizer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) and that of roots was obtained after oven drying (60°C, 48 h). Lyophilized samples were used to determine chlorophyll contents, proline and soluble sugars on a dry-weight basis.

The relative growth rate (RGR) was determined as RGR (g g−1d−1) = (ln(W 2) − ln(W 1))/(t 2 − t 1), where W is the dry matter at the beginning (W 1) and the end (W 2) of the 30-day treatment period, and (t 2 − t 1) is the duration of this period (Hunt 1990).

The sensitivity index (SI), i.e., the difference between dry matter production of salt-treated plants irrigated and the control, expressed in percent of the latter, was calculated as SINaCl (%) = [(DMNaCl − DMcontrol)/DMcontrol] × 100. This parameter was more negative when the plant was more sensitive to NaCl (Saadallah et al. 2001).

Chlorophyll contents

Chlorophyll a and b were determined following the method described by Inskeep and Bloom (1985). The extract was analyzed for absorbance at 647 and 664.5 nm on a Jenway 6400 spectrophotometer (Jenway, London, UK). Chlorophyll content (mg g−1 DM) was calculated using the following equations: Chl a = 12.70A 664.5 − 2.79A 647; Chl b = 20.70A 647 − 4.62A 664.5; Total Chl = 17.90A 647 + 8.08A 664.5, where A, absorbance in a 1 cm cuvette.

Water relations

The water content (WC) of shoots and roots was determined as WC (ml H2O g−1 DM) = (FM − DM)/DM. Shoot water potential was measured using a pressure chamber (PMS Instruments Co., Corvallis, OR, USA) after 30 days of salt treatment, according to Scholander et al. (1965).

Ion contents

Ions were extracted from dried, milled plant material with nitric acid (HNO3, 0.5%). Concentrations of Na+ and K+ were determined using an atomic absorption spectrophotometer (Schimazu AA 6800, Schimazu Crop, Kyoto, Japan), while Cl− concentration was determined on the same extract with a chloride meter (Jenway PC LM3, London, UK).

K+/Na+ selectivity ratios

The selectivity ratios of K+ over Na+ (S K/Na) for accumulation, uptake and transport were estimated as: [K/Na]whole plant/[K/Na]medium, [K/Na]root/[Ka/Na]medium and [K/Na]shoot/[K/Na]root, respectively (Gorai et al. 2010a). K and Na represent, respectively, the quantity found in the whole plant (accumulation), roots (uptake) and shoots (transport).

Proline and soluble sugar contents

Soluble sugars were quantified following the phenolsulfuric acid method described by Robyt and White (1987). 100 mg dry weight of shoots was extracted in 80% (v/v) methanol heated to 70°C in a water bath. The extract was then centrifuged at 5,000×g for 10 min. The supernatant was used for the estimation of soluble sugars concentrations. The reaction mixture consisted of 1 ml 5% phenol and 5 ml 98% sulphuric acid. Once the extract had cooled, its absorbance was determined at 490 nm using d-glucose as standard.

Free proline was quantified spectrophotometrically by the ninhydrin method according to Bates et al. (1973). The plant material was homogenized in 3% aqueous sulfosalicylic acid and the homogenate was centrifuged at 14,000g. The supernatant was used for the estimation of the proline concentration. The reaction mixture consisted of 2 ml of acid ninhydrin and 2 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 4 ml of toluene, and absorbance was read at 520 nm using l-proline as standard.

Tissue osmolarity due to K+ plus Na+ and associated anions

A conservative estimate of tissue osmolarity (M) due to K+ plus Na+, and associated anions, supposed univalent and soluble, were calculated as: 2 × ([Na+] + [K+])/[H2O], where brackets design ion (mmol g−1 DM) or water (ml g−1 DM) contents (Glenn and Brown 1998).

Vacuolar compartmentation

To appreciate the Na+ compartmentalization degree in foliar tissues, we have correlated shoot water content with its Na+ content according to Oertli’s hypothesis (Flowers et al. 1991). When Na+ is compartmentalized inside the cells, it was used for the osmotic adjustment and its vacuolar accumulation induced a supplement tissue hydration; however, if Na+ is remained in the cell walls, it involved tissue dehydration.

Statistical analysis

Data were analysed using SPSS statistical package (SPSS 2002). Data were tested for normal distribution using the Shapiro–Wilk test, and heterogeneities of variance within treatments were tested using Levene’s test. When necessary, log transformations were used to normalise distributions. A comparison of means was carried out using Duncan test at P < 0.05.

Results

Growth attributes

During NaCl-salinity exposure, salts were secreted from the salt glands in leaves and deposited as crystals on the upper surface of leaves of treated plants, whereas there was no deposition in controls. NaCl-salinity significantly reduced both shoot and root dry matter of R. vermiculata (P < 0.01). Plant growth was considerably reduced at the highest salt concentrations (600 mM NaCl) either in shoots and roots, ca. 39 and 52% of the control value, respectively (Fig. 1a). Shoot fresh:dry weight ratio was significantly decreased with increase in salt level, but the highest salt level caused a sharp decline in this attribute. However, root fresh:dry weight ratio seemed to be unaffected by salt (Fig. 1b). The RGR followed a similar trend as that observed for dry matter production, and decreased with increasing salinity in both shoots and roots (P < 0.01). Root growth was more sensitive to NaCl than shoot growth (Fig. 1c). To appreciate the sensitivity of R. vermiculata to salinity, the SI was determined for shoots and roots (Fig. 1d). The roots presented with the more negative SI were considered as the highest sensitive tissue.

Fig. 1
figure 1

Changes in tissue a dry mass (g plant−1), b fresh:dry weight ratio, c relative growth rate (g g−1 day−1), and d sensitivity index (%) of Reaumuria vermiculata when 4-month old plants were subjected for 30 days to salinity at varying NaCl-salinity concentrations (0–600 mM) in nutrient solution. Data represent mean ± 95% confidence limits, n = 5. Different letters indicate significant differences between treatments at P < 0.05 according to the Duncan test

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The chlorophyll content of R. vermiculata subjected at varying NaCl-enriched nutrient solutions is shown in Fig. 2. Salt stress significantly decreased chl a, chl b and Total chl contents (P < 0.001) with increase in salt level (Fig. 2a–c), whereas chl a/b ratio was significantly increased (P < 0.001) at higher NaCl concentrations (Fig. 2d).

Fig. 2
figure 2

Mean chlorophyll content (mg g−1 DM shoot tissue) of Reaumuria vermiculata when 4-month old plants were subjected for 30 days to salinity at varying NaCl concentrations (0–600 mM) in nutrient solution. Data represent mean ± 95% confidence limits, n = 5. Different letters indicate significant differences between treatments at P < 0.05 according to the Duncan test

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Water relations

As shown in Fig. 3a, NaCl-salinity significantly affected shoot water content of R. vermiculata (P < 0.001). At 600 mM NaCl, shoot water content was drastically decreased (ca. 32% of the control value). In roots, water content was not affected by salt exposure and did not exceed 4 ml g−1 DM. There was a positive correlation between shoot WC and shoot DM, with R 2 = 0.987 (Fig. 3b). A one-way ANOVA indicated that treatments significantly affected water potential of salinized plant shoots relative to the control ones. At the highest NaCl concentration, shoot water potential dropped to −3.4 MPa (P < 0.001). There was a strong relationship between shoot water potential and the NaCl-enriched nutrient solutions, with R 2 = 0.911 (Fig. 3c).

Fig. 3
figure 3

Changes in a tissue water content (ml H2O g−1 DM), and regression plots for b shoot dry matter (g plant−1) and its water content (ml H2O g−1 DM) and c shoot water potential (–MPa) at varying NaCl-salinity concentrations (0–600 mM) in nutrient solution of Reaumuria vermiculata when 4-month old plants were subjected for 30 days to salt stress. Lines describing the dependencies were obtained using a linear regression. Data represent mean ± 95% confidence limits, n = 5. Different letters indicate significant differences between treatments at P < 0.05 according to the Duncan test

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Ions and organic solutes accumulation

Reaumuria vermiculata seedlings in the presence of NaCl-enriched nutrient solution accumulated higher amounts of Na+ in the shoots (P < 0.001) as compared to that in roots (P < 0.001). Sodium concentration in the shoots ranged from 1.08 to 2.68 mol l−1 at 100 mM and 600 mM NaCl, respectively, whereas that in the roots varied from 0.18 to 0.36 mol l−1, respectively (Fig. 4a). Chloride concentration in leaves displayed a similar pattern as that observed for Na+, but at lower levels than sodium (P < 0.001). However, there were no significant differences in Cl− concentration in roots with increasing NaCl supply (Fig. 4b). Increasing NaCl-salinity had no adverse effect on potassium concentration in shoots and high amounts were reached at the highest NaCl concentration (P < 0.05), whereas that in roots significantly decreased as compared to the controls (P < 0.01) (Fig. 4c).

Fig. 4
figure 4

Changes in Na+ (a), Cl− (b) and K+ (c) concentrations (mmol l−1 tissue water) in shoots and roots of Reaumuria vermiculata when 4-month old plants were subjected for 30 days to salinity at varying NaCl-salinity concentrations (0–600 mM) in nutrient solution. Data represent mean ± 95% confidence limits, n = 5. Different letters indicate significant differences between treatments at P < 0.05 according to the Duncan test

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Increasing NaCl concentration of nutrient solutions had a significant effect on proline concentration in shoots (P < 0.001), whereas that of soluble sugars seemed to be unaffected (P > 0.05) (Fig. 5a, b). At 600 mM NaCl, proline concentration represented ca. ninefold of the control value.

Fig. 5
figure 5

Mean concentration (mmol l−1 tissue water) of proline (a) and soluble sugars (b) in shoots of Reaumuria vermiculata grown on nutrient solution supplied with different NaCl-salinity concentrations (0–600 mM). Data represent mean ± 95% confidence limits, n = 5. Different letters indicate significant differences between treatments at P < 0.05 according to the Duncan test

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K+/Na+ selectivity ratios

To assess the ability of R. vermiculata for selective uptake of K+ with increasing osmolarity of solutions, the S K/Na was calculated and given in Fig. 6. A one-way ANOVA of the S K/Na ratios revealed that NaCl-salinity significantly affected accumulation of K+ in the whole plant (P < 0.001), uptake by roots (P < 0.001) and transport by shoots (P < 0.001). At all salinity levels, S K/Na for transport in shoots was lower than those of uptake and accumulation. At the highest NaCl concentrations, S K/Na for uptake, accumulation and transport represented ca. 4.5-, 3.0- and 1.5-fold of 100 mM NaCl-enriched nutrient solution, respectively.

Fig. 6
figure 6

Changes in the K/Na selectivity ratios (S K/Na) for accumulation, uptake and transport in Reaumuria vermiculata when 4-month old plants were subjected for 30 days to salinity at varying NaCl concentrations (0 to 600 mM) in nutrient solution. Data represent mean ± 95% confidence limits, n = 5. Different letters indicate significant differences between treatments at P < 0.05 according to the Duncan test

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Vacuolar compartmentation

Figure 7 shows the relationship between shoot Na+ and water content in R. vermiculata. Shoot hydration was correlated with the accumulation of Na+. Shoot water content fell as sodium concentration rose although not as much as in roots. Because the large accumulation of Na+ ions resulted in tissue dehydration, it is likely that this cation was remained in the cell walls.

Fig. 7
figure 7

Relationship between shoot water content (ml H2O g−1 DM) and its sodium content (μmol Na+ g−1 DM) of Reaumuria vermiculata when 4-month old plants were subjected for 30 days to salinity at varying NaCl concentrations (0–600 mM) in nutrient solution. A line describing the dependency was obtained using a polynomial regression, R 2 = 0.52. Values are from six treatments with five replicates (n = 30)

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Tissue osmolarity due to K+ plus Na+ and associated anions

The osmolarity in shoots remained significantly higher than in the medium for all NaCl concentrations (P < 0.001); however, in roots only for NaCl concentrations lower than 300 mM NaCl. The concentration of soluble ions in shoots ranged from 0.77 to 5.80 M at 0 and 600 mM NaCl, respectively, whereas that in roots varied from 0.37 to 0.82 M.

Discussion

In the field, R. vermiculata naturally inhabits inland salines throughout the Tunisian desert and its wadies, where it is very common. A number of morphological characteristics associated with dehydration have been observed as adaptations under arid environment, including small leaf size and extremely thick cuticle (Gorai personnal observation). This study has demonstrated that growth of R. vermiculata seedlings was reduced by increasing NaCl concentrations in nutrient solution. Nevertheless, this species was able to survive at about sea-level concentrations of 600 mM NaCl. This corroborates previous studies on other halophytes, showing optimal growth in mediums lacking salts (Barhoumi et al. 2007; Naidoo et al. 2008). The loss of chlorophyll is often considered as a marker of a cellular component of salt stress (Singh and Dubey 1995). Thus, our data support the hypothesis that leaf cells of R. vermiculata were stressed when the plants were grown in a salty medium.

Many halophytes have salt glands that secreted excess stress-inducing ions that invade the plant and maintain internal ion concentration at lower level (Hogarth 1999; Tester and Davenport 2003; Naidoo et al. 2008). In R. vermiculata salts were secreted by salt glands present in leaves and deposited as crystals on the upper surface of leaves of NaCl-treated plants (Gorai personnal observation). As initially hypothesized by Oertli (1968) and later confirmed (Flowers et al. 1991), in the absence of efficient compartmentalization of Na+ by leaf cells, their concentration in the leaf apoplast may reach excessive values, leading to leaf cell dehydration (Munns and Passioura 1984) and membrane disruption (Speer and Kaiser 1991). This was also true in the present study, as shown by the negative relationship between Na+ tissue concentration and the shoot hydration (Fig. 7). It is possible that a proportion of Na+ ions which accumulated within the shoots of R. vermiculata remained in the cell walls, bringing about an efflux of water from the protoplast to the apoplast and the subsequent loss of this water by transpiration. It was found by Ramadan (1998) that salt glands of R. hirtella secrete a variety of ions, but NaCl is the most abundant salt excreted. Sodium and chloride, which were the predominating ions in the soil solution of the root environment, constituting ca. 89% of salts secreted. This author estimated that more than 67% of the absorbed NaCl was secreted during the day, and the rest is accumulated by the plant leaves.

Water relations in plants are affected by salinity (Hasegawa et al. 2000). The inaptitude of plants to hydrate their tissues appropriately on saline soils causes a physiological drought. Several research reports that plants grown under salt stress manifest acclimation to succeed in their establishment, by lowering both water and osmotic potentials in the leaves (Sultana et al. 1999; Koyro 2006). Osmotic adjustment by net accumulation of solutes in cells in response to a fall in the water potential of their environment can in part offset this deterioration of growth conditions. In R. vermiculata, shoot water potential reaching the lowest values (down to −3.4 MPa) at the highest salt level (600 mM NaCl). A conservative estimate of the concentration of osmotically active (soluble) ions in the tissues was obtained assuming that these ions comprised K+ and Na+ plus the anions associated with them (assumed to be univalent) (Glenn and Brown 1998). The data of Fig. 8 show that the concentration of soluble ions in shoots reached high values with 5.8 M at the highest NaCl concentration (600 mM). In Thellungiella halophila, a halophytic species, M’rah et al. (2006) showed that the concentration of these ions reached only 1.3 M when treated with NaCl concentration of 200 mM. Proline is a compatible solute that accumulates in response to osmotic stress, and the accumulation of this osmolyte represents an important adaptive response to salt and drought stress (Ashraf and Harris 2004; Parida and Das 2005). In our study, R. vermiculata subjected to salt stress accumulated ninefold more proline than controls. By studying the metabolic acclimation of R. soongorica during water loss, Liu et al. (2007) showed that elevated levels of proline may assist in osmotic adjustment during desiccation.

Fig. 8
figure 8

Regression plots for tissue osmolarity of Reaumuria vermiculata when 4-month old plants were subjected for 30 days to salinity at varying NaCl concentrations (0–600 mM) in nutrient solution. Lines describing the dependencies were obtained using a logarithmic regression. Data represent mean ± 95% confidence limits, n = 5. Different letters indicate significant differences between treatments at P < 0.05 according to the Duncan test

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Many studies on halophytes and some tolerant glycophytes plants showed that a high S K/Na for transport is a salt tolerance criterion (Gorham et al. 1990; Shachtman et al. 1991; Wolf et al. 1991; Yeo 1998; Debez et al. 2004; Gorai et al. 2010b). Reaumuria Vermiculata appears to be able to maintain a high S K/Na for accumulation in the whole plant and the uptake by roots; however, the transport in shoots reached lower values when salinity increases in the medium. The ability of this species to maintain a substantial growth rate under saline conditions is directly related to an efficient S K/Na. Zhu (2001) reported that the capacity of plants to counteract salinity stress strongly depends on the status of their potassium nutrition. Maintenance of a high K+ cytosolic concentration with increasing salinity apparently occurs via highly selective pathways at the root–soil interface and is an important determinant of salt tolerance (Maathuis and Amtmann 1999; Rodriguez-Navarro 2000). Ion selectivity also occurs in salt glands. For example, in R. hirtella grown under natural saline conditions, Ramadan (1998) found that the secretion mechanism has a high specifcity for Na+ and Cl− and a low specifcity for secretion of K+, Ca2+, Mg2+ and SO4 2−, and PO4 3− and NO3 − seems to be unavailable for secretion, possibly because of their rapid incorporation and utilization in the metabolism. Sporobolus spicatus was found to secrete 93% NaCl by weight of salts secreted by plants from four different sites while K+, Ca2+, Mg2+ and SO4 2− constituted only 5% of salts (Ramadan 2001).

In conclusion, this study shows that R. vermiculata grown in the presence of NaCl-salinity appears to be able to survive up to 600 mM and could accumulate large amounts of ions in its shoots without damage. This accumulation leads to (1) the tissue dehydration, and (2) the chlorophyll loss. The more negative values of shoot water potential were achieved by the accumulation of osmotically active solutes. Further investigations are necessary to understand the strategies for adaptation of Reaumuria grown under saline environments.