Varied tolerance to NaCl salinity is related to biochemical changes in two contrasting lettuce genotypes

Abstract

Salt stress perturbs a multitude of physiological processes such as photosynthesis and growth. To understand the biochemical changes associated with physiological and cellular adaptations to salinity, two lettuce varieties (Verte and Romaine) were grown in a hydroponics culture system supplemented with 0, 100 or 200 mM NaCl. Verte displayed better growth under 100 mM NaCl compared to Romaine, but both genotypes registered relatively similar reductions in growth under 200 mM NaCl treatment. Both varieties showed differences in net photosynthetic activity in the absence of salt and 8 days after salt treatment. These differences diminished subsequently under prolonged salt stress (14 days). Verte showed enhanced leaf proline and restricted total cations especially Na+, lesser malondialdehyde (MDA) formation and lignification in the roots under 100 mM NaCl salinity. Membrane damage estimated by electrolyte leakage increased with elevated salt concentrations in roots of both varieties, but Verte had significantly lower electrolyte leakage relative to Romaine under 100 mM NaCl. Moreover, Verte also accumulated greater levels of carotenoids under increasing NaCl concentrations compared to Romaine. Taken together, these findings suggest that the greater tolerance of Verte to 100 mM NaCl is related to the more restricted accumulation of total cations and toxic Na+ in the roots and enhanced levels of antioxidative metabolites in root and leaf tissue.

Introduction

The inhibitory effects of salinity on plant growth include ion toxicity, osmotic stress and mineral deficiencies resulting in morphological, physiological and biochemical perturbations (Hasegawa et al. 2000). Net photosynthesis decreases in response to salinity in different plants including lettuce (Dubey 2005; M’rah et al. 2006; Mahmoudi et al. 2010). A close link between growth and photosynthetic rate was also shown in two wheat (Triticum aestivum L.) genotypes differing in salt tolerance (El-Hendawy et al. 2005). Salinity stress results in the reduction of photosynthesis through stomatal and nonstomatal factors, although the latter are not yet fully understood (Dionisio-Sese and Tobita 2005). There is compelling evidence that salt affects the activity of photosynthetic enzymes and the biosynthesis of chlorophylls and carotenoids (Stepien and Klobus 2006). Carotenoids may have bearing on salt-induced anti-oxidative responses in lettuce in view of their biogenesis in chloroplasts and sequestration as chlorophyll–carotenoid–protein complexes (Gross 1991).

Proline (Pro) accumulation has been correlated with plant tolerance to drought and salinity stresses (Delauney and Verma 1993). In addition to stimulated formation of Pro, salinity is generally known to alter lipid metabolism in plants (Kuiper 1985). Lipid peroxidation correlates to damage provoked by a variety of environmental stresses (Inze’ and van Montagu 1995; Hernandez et al. 2003). Polyunsaturated fatty acids (PUFA) are the main membrane lipid components susceptible to peroxidation and degradation under salt stress (Elkahoui et al. 2005).

Levels of polyphenols, including the two main classes; caffeic acid derivatives and flavonols vary in different cultivars of lettuce (Ke and Saltveit 1988; Hermann 1976). The concentrations of flavonoids and phenolic acids in lettuce are affected by variable environmental conditions (Liu et al. 2007). Despite this body of knowledge, little is known about how biochemical and physiological processes co-ordinately respond to the most common NaCl salinity in lettuce.

In a previous study, we reported significant differences in tissue antioxidants and ion contents between Verte and Romaine lettuce seedlings grown under 0, 100 mM NaCl and 77 mM Na2SO4 (Mahmoudi et al. 2010). Lettuce seedlings can tolerate relatively high concentrations of NaCl for a short time period without developing obvious symptoms (Kim et al. 2008). However, how lettuce plants adapt to prolonged treatment of incremental NaCl supplement is poorly understood. In this article, we report our evaluation of the impact of increasing NaCl salinities on the growth, water content, photosynthetic parameters, ion relation, melanoidine, Pro and polyphenols, as well as on membrane damage (as estimated by electrolyte leakage and MDA) in Verte and Romaine. These two varieties were selected as representatives of different lettuce genotypes with varying salt tolerance, based on initial germination test under increased NaCl concentrations (0–150 mM).

Materials and methods

Selection of lettuce genotypes

In screening for salt tolerance, we tested the effect of NaCl salinity on the germination ratios of four lettuce (Lactuca sativa) varieties (Var Augusta, Vista, Verte de Cobham and Longifolia). The Longifolia (Romaine) variety was the most tolerant to NaCl, while Verte was the most sensitive (Nasri et al. 2010). These two contrasting varieties were selected for the present study to determine the effect of NaCl on growth, and physiological and biochemical parameters.

Plant growth and treatment conditions

Seeds were germinated in Petri dishes at room temperature in the dark. Seven-day old seedlings were irrigated with distilled water during the first week. Uniform seedlings were subsequently cultured individually in a hydroponic system containing a complete Hoagland’s medium (Hoagland and Arnon 1950) diluted eightfold in a greenhouse (16 h light/8 h dark at 20/17°C). Individual plants were grown in control (without salt), 100 mM NaCl, or 200 mM NaCl for 15 days prior to harvest. Fresh weight (FW) and dry weight (DW) of roots and rosette leaves were separately recorded for six plants randomly selected from each variety and treatment combination and used to calculate percent tissue water [(FW–DW)/FW] × 100. The remaining plants were frozen at −80°C for biochemical analysis.

Chlorophyll content and gas exchange

Chlorophyll concentration was determined on 80% acetone (v/v) leaf extracts at 663 and 645 nm (Arnon 1949). Kinetics of net photosynthetic rates and stomatal conductance were measured with a portable LiCor 6200 photosynthesis system (LI-COR, Inc. Lincoln, Nebraska, USA) after 8 and 11 days, and at the end of the salinity treatment on fully expanded healthy leaves randomly selected from five plants for each genotype and treatment combination (mean canopy photon flux density of 450 μmol m−2s−1, ranging from 350 to 550).

Tissue ion content and ion selectivity

Major cations and chloride in dried leaf and roots materials were extracted with 0.5% HNO3 and were assayed with flame photometry as previously described (M’rah et al. 2006). The ability of the plants to maintain tissue K+ or Ca2+ concentration in saline conditions is indicated as the K+ or Ca2+ selectivity. It is defined as the ratio of K+/(K+ + Na+) or Ca2+/(Ca2+ + Na+) in the tissue divided by the ratio of K+/(K+ + Na+) or Ca2+/(Ca2+ + Na+) in the external medium (Ashraf and McNeilly 1990).

Proline accumulation

The proline (Pro) content was determined using a modified method (Bates et al. 1973). A powder of tissue (10 mg DM) from rosette leaves was weighed into 1.5-ml centrifuge tubes. The powder was suspended in 1.5 ml of 3% (w/v) sulphosalicylic acid to precipitate protein. The samples were mixed, centrifuged at 14,000×g for 10 min, and the supernatant transferred to a fresh 1.5-ml tube. An aliquot of 1 ml of supernatant was reacted with 1 ml of glacial acetic acid and 1 ml of ninhydrin reagent (1.25 g ninhydrin in 30 ml of glacial acetic and 20 ml of phosphoric acid 6 M) for 1 h at 100°C before the reaction was stopped by cooling the tubes in ice. The products were extracted with 2 ml of toluene by vortex mixing, the upper (toluene) phase decanted into a glass cuvette and absorbance read at 520 nm. Pro concentrations were calculated from the absorbance of a set of Pro standards (0–20 mg ml−1) assayed in an identical manner.

Membrane permeability (electrolyte leakage)

Electrolyte leakage (EL) was determined as described by Dionisio-Sese and Tobita (1998). Leaf discs of fresh seedlings were cut into 2–3 mm pieces and placed in test tubes containing 10 ml of distilled water. The tubes were incubated in a water bath at 32°C for 2 h and the initial electrical conductivity of the medium (EC1) was measured using a digital conductimeter-type Metrohm. The samples were autoclaved at 121°C for 20 min to release all electrolytes, cooled down to 25°C and the final electrical conductivity (EC2) measured. The electrolyte leakage (EL) was calculated from EL = (EC1/EC2) × 100.

Measurement of malondialdehyde

Lipid peroxidation was measured as the amount of malondialdehyde (MDA) determined by the thiobarbituric acid (TBA) reaction (Heath and Packer 1968). Frozen samples (200 mg) were homogenized with a mortar and pestle in 2 ml of a mixture containing 20% 2-thiobarbituric acid and 0.5% trichloroacetic acid (TCA). An assay mixture containing 2 ml of the supernatant and 2 ml of 0.5% (w/v) TBA in 20% (w/v) TCA was heated at 95°C for 30 min and then rapidly cooled in an ice bath. After centrifugation (4,000g for 30 min at 4°C), the supernatant absorbance was read at 532 nm, and the values corresponding to nonspecific absorption (600 nm) were subtracted. Lipid peroxidation products were measured as the content of TBA-reactive substances. The MDA content (μmol g−1 FW) was calculated according to the molar extinction coefficient of 155/(mM cm−1).

Hydrogen peroxide assay

The hydrogen peroxide (H2O2) content was determined as described (Kotchoni et al. 2006). Fresh leaves (0.1 g) were homogenized in an ice bath with 1 ml of 25 mM H2SO4. The homogenate was centrifuged at 14,000 rpm for 10 min at 4°C. Using PeroXoquant Quantitative Peroxide Assay Kit (Thermo Fisher Scientific, Ottawa, ON, Canada), 20 μl of the supernatant was added to 200 μl of working reagent (WR). The absorbance of the supernatant was measured at 560 nm in a spectrophotometer. The content of H2O2 was calculated by comparison with a standard calibration curve, previously plotted by using different concentrations of H2O2.

Lignin content

Lignin was extracted from frozen ground roots (0.1 g) and quantified using the thioglycolic acid (TGA) method as described elsewhere (Huang et al. 2009).

Melanoidins content

Fresh tissue (0.1 g per plant) was homogenized in 1 ml of distilled water, and the solution was placed in a rotator for 24 h at 4°C and then centrifuged at 1,300g for 15 min. The supernatant was transferred to a new tube, and the extract was scanned with a UV spectrophotometer according to a spectrum of 200–700 nm. The peak was detected at 360 nm as described in Shin et al. (2009).

Carotenoid and polyphenol analysis by HPLC

Extraction and quantification of carotenoids from leaf tissue (0.1 g) by HPLC was carried as previously described (Mahmoudi et al. 2010). Total phenolics were extracted from fresh leaves (0.5 g) with 80% methanol (v/v) and quantified by HPLC as described in Annalisa et al. (2002).

Statistics

Results were expressed as the means of indicated replicates across each parameter. Analysis of variance (ANOVA) was independently performed using collected data. Mean values were compared by Fisher’s least significant difference (LSD) test at P ≤ 0.05.

Results

Effect of NaCl salinity on seedling growth, water content and photosynthesis

Leaf biomass of Verte and Romaine was significantly decreased following salt treatment for 2 weeks (Fig. 1; Table 1). At 100 mM NaCl, the dry biomass of leaves decreased to 60 and 40% of the corresponding controls in Verte and Romaine, respectively. At 200 mM NaCl, growth (DW) decreased to 25 and 24% of control plants in Verte and Romaine, respectively. Root biomass was also significantly decreased in response to NaCl treatment. Root DW was 66 and 40% of corresponding controls in Verte and Romaine when grown at 100 mM NaCl. A similar pattern was found for the two varieties grown under 200 mM NaCl (33% of control in Verte and 20% of control in Romaine).

Fig. 1
figure1

Phenotypes of Verte and Romaine lettuce grown under 0, 100 and 200 mM NaCl for 15 days

Table 1 Growth (per plant), water content, total chlorophyll (mg−1 chl g−1 FW tissue), and carotenoids contents (μg g−1 FW tissue) of Verte and Romaine lettuce grown in the presence of 0, 100 and 200 mM NaCl for 15 days (mean ± SE)

Leaf and root water content was significantly reduced under 200 mM NaCl treatment (Table 1), suggesting that tissue dehydration occurred following exposure to severely high NaCl. Verte leaves had 30% greater leaf water content compared to Romaine at this salt concentration. NaCl salinity appeared to have caused greater osmotic impact on leaves compared to roots (Table 1). No significant differences were found for growth or tissue water content between Verte and Romaine in the absence of NaCl.

Under control conditions, the chlorophyll content of Verte was (1.27 mg chl g−1 FW) and that of Romaine was (0.75 mg chl g−1 FW) (Table 1). We found that Verte contained 1.7-, 1.9- and 1.7-fold greater amounts of total chlorophyll than Romaine under control, and 100 and 200 mM NaCl, respectively. The presence of NaCl salt altered the carotenoid content in both lettuce varieties (Table 1). In fact, total carotenoids increased in Verte by 1.5- and 1.4-fold under 100 and 200 mM NaCl, respectively, while in Romaine, total carotenoids increased by 1.2-fold under 100 mM and decreased under 200 mM NaCl treatments.

We determined the effect of NaCl treatment on photosynthetic assimilation rate at three time points, following 8, 11 and 14 days of NaCl treatment. Our results (Fig. 2) showed that at 100 mM NaCl, assimilation rates (A) were not affected for both varieties by salt treatment with the exception of 8 days where a varietal difference was detected under 0 and 100 mM NaCl. In contrast, an increase in assimilation rate (A) was observed in Romaine after 11 days of salt treatment. The presence of 200 mM NaCl significantly decreased the net photosynthesis in both Verte and Romaine. Significant difference in stomatal conductance was found between Verte and Romaine grown in the presence of 100 and 200 mM NaCl salinities (Fig. 2).

Fig. 2
figure2

Photosynthetic rate (a, μmolm−2 S−1) and stomatal conductance (Gs, mol m−2 S−1) of Verte (empty bar) and Romaine (filled bar) lettuce grown under 0, 100 and 200 mM NaCl for 8, 11 and 14 days (n = 5)

Effect of NaCl on the accumulation of Na+, K+, Ca2+ and Cl

The effect of salt on the accumulation of cations and chloride in leaves and roots is presented in Table 2. In the absence of salt, Verte and Romaine leaves accumulated similar amounts of Na+, K+ and Ca2+ (Table 2). In Verte leaves, sodium concentrations reached 1.7 and 4.4 mmol g−1 DW, while Na+ was 1.7 and 4.27 mmol g−1 DW in Romaine leaves, when plants were grown under 100 and 200 mM NaCl treatments, respectively. When grown under control conditions, roots had similar individual or total ion concentrations. Accumulation of Na+ in roots increased by 16- and 27-fold in Verte under 100 and 200 mM NaCl treatments, respectively, while in Romaine it was only 6- and 15-fold under the same conditions. These results strongly suggest a more restricted retention of cations, particularly the toxic Na+ in the Verte root system in comparison to those of Romaine. These results showed that Na+ accumulation was higher in Verte roots suggesting that Verte may have a greater ability to accumulate and transport Na+ than Romaine.

Table 2 Effect of NaCl salinity on tissue ion content of Verte and Romaine seedlings (14 days) grown in the presence of 0, 100 and 200 mM NaCl for 15 days (mean ± SE, n = 6)

Salinity caused an increase of Cl accumulation in Verte roots by 42- and 142-fold, and in Romaine roots by 49- and 159-fold under 100 and 200 mM NaCl treatments, respectively (Table 2). The presence of NaCl decreased the K+ and Ca2+ levels in leaves of both varieties, especially under 200 mM NaCl. Under control conditions, both tissues of Romaine had slightly more K+ and Ca2+ than those of Verte. The accumulation of K+ in leaves was 2.3- and 4-fold less in Verte under 100 and 200 mM, respectively, but in Romaine it was reduced by 3.7- and 4.3-fold, respectively, under similar conditions. In roots, the decrease of K+ was more pronounced under 200 mM NaCl. On the other hand, the reduction in K+ was independent of the NaCl concentration in Romaine. Similarly, accumulation of Ca2+ was reduced in leaves by 3.4- and 5.2-fold in Verte, compared to 2.7- and 8-fold reduction in Romaine, for both NaCl concentrations.

To aid in the interpretation of differential salinity tolerance in these varieties, cation selectivity was calculated from the tissue ion content (Table 3). Noticeably, a small but significant increase in leaf K/Na selectivity was found in Verte, whereas a significantly greater root Ca/Na selectivity was detected in Romaine under 100 mM NaCl, demonstrating differential selectivities of favorable nutrient ions over toxic Na+ between Verte and Romaine.

Table 3 Effect of NaCl salinity on tissue ion selectivity of Verte and Romaine seedlings (14 days) grown under 0, 100 and 200 mM NaCl for 15 days (mean ± SE, n = 6)

Effect of NaCl on proline content

We measured Pro levels in both varieties under control and saline conditions in order to assess the potential role of Pro in defining tolerance to increasing NaCl concentrations. Without salt, both varieties contained equivalent amount of Pro in leaves (Fig. 3). Under salt stress, leaf Pro content increased significantly at 100 mM NaCl in Verte, while it remained virtually unchanged in Romaine (2.3- and 1.7-fold in Verte in the presence of 100 and 200 mM NaCl, respectively; 1.0 and 1.3-fold in Romaine under the same conditions). These results clearly suggest that reduced sensitivity to 100 mM NaCl in Verte relative to Romaine was at least partially related to transient leaf Pro accumulation.

Fig. 3
figure3

Effect of NaCl on leaf Pro content (μmol g−1 DW). Verte (empty bar) and Romaine (filled bar) lettuce were grown in the presence of 0, 100 and 200 mM NaCl for 15 days (n = 6)

Effect of NaCl salinity on electrolyte leakage and membrane integrity

The extent of membrane damage in leaf and root tissues was estimated by electrolyte leakage (EL). The EL index of leaves and roots of both varieties progressively increased with increasing NaCl concentration (Fig. 4a, b). Relative to the control, salt treatment increased EL by 4- and 6-fold in Verte roots and by 6.7- and 6.8-fold in Romaine roots under 100 and 200 mM NaCl, respectively. In leaves, this index was much higher than in roots in the absence of salt. Leaf EL increased by 1.6 and 3.0 in Verte under 100 and 200 mM NaCl, respectively, while in Romaine leaf EL was increased by threefold compared to the control under both NaCl concentrations.

Fig. 4
figure4

Effect of NaCl on electrolyte leakage (%, n = 5) in leaves (a) and roots (b) and MDA content (μmol g−1FW, n = 12) in leaves (c) and roots (d) of Verte (empty bar) and Romaine (filled bar) lettuce grown under 0, 100 and 200 mM NaCl for 15 days

In the absence of salt, MDA levels were much higher in leaves (about 6 μmol g−1 FW) than in roots (about 1 μmol g−1 FW) in both lettuce varieties (Fig. 4c, d). In Verte, leaf MDA level increased by 4.3-fold in the presence of 200 mM NaCl and by 1.5- and 4.3-fold in Romaine under 100 and 200 mM NaCl, respectively. Without salt, Verte accumulated similar levels of MDA in roots in the absence of salt relative to Romaine. The root MDA level increased 1.3- and 3.2-fold in Verte and 1.3- and 3.9-fold in Romaine grown under 100 and 200 mM NaCl, respectively.

Effect of NaCl salinity on root hydrogen peroxide content

H2O2 contents in root tissue (Fig. 5a) increased under salt stress in both Verte and Romaine. While both varieties had similar levels of H2O2 when grown in the control medium, the presence of salt induced an increase in H2O2 accumulation in Verte roots by 5.64- and 8.44-fold compared to the untreated roots under 100 and 200 mM NaCl, respectively. In Romaine, this increase was 6.69- and 6.40-fold under similar conditions.

Fig. 5
figure5

Tissue biochemical changes in Verte and Romaine lettuce grown under 0, 100 and 200 mM NaCl for 15 days. a Root H2O2 content (μM g−1 FW) of Verte (empty bar) and Romaine (filled bar) (n = 4). b Root lignin content (OD280 g−1 FW) of Verte (empty bar) and Romaine (filled bar) (n = 3). c Leaf melanoidin content (μmol g−1 FW) of Verte (empty bar) and Romaine (filled bar) (n = 4). d Root melanoidin content (μmol g−1 FW) of Verte (empty bar) and Romaine (filled bar) (n = 4)

Effect of NaCl salinity on root lignification

Root lignin content was examined in the two lettuce varieties to determine its potential involvement in salinity adaptation (Fig. 5b). Although 100 mM NaCl treatment had no effect on total lignin content in the two varieties, 200 mM resulted in a significant increase in lignin content in Verte compared to control plants. However, no significant difference in lignin content could be detected between the two varieties under 0 or 100 mM NaCl (Fig. 5b).

Effect of NaCl on melanoidin accumulation

In this study, we investigated the effect of NaCl on melanoidins accumulation in leaves and roots of both lettuce varieties. Without salt (Fig. 5c), the accumulation of melanoidins was much higher in Romaine leaves (50.37 μmol g−1 FW) than in the Verte counterpart (23.71 μmol g−1 FW). Under 100 and 200 mM NaCl, melanoidin levels increased by 1.5- and 3.2-fold in Verte and by 1- and 2-fold, in Romaine, respectively, compared to untreated plants. In roots (Fig. 5d), melanoidin accumulation was similar in both varieties with and without 100 mM NaCl. In response to 200 mM NaCl, Verte accumulated significantly higher melanoidins (29.77 μmol g−1 FW) than Romaine (17.61 μmol g−1 FW).

Effect of NaCl salinity on polyphenol content

In this study, we determined the effect of NaCl on polyphenol content of Verte and Romaine. No significant changes in polyphenols were noticed in Verte and Romaine grown under either of the NaCl treatments (Fig. 6).

Fig. 6
figure6

Polyphenol content (μg g−1 FW) of Verte (empty bar) and Romaine (filled bar) lettuce grown under 0, 100 and 200 mM NaCl for 15 days (n = 3)

Discussion

Plant growth in saline soils is affected by the reduced availability of water due to high osmotic pressure (Bilgin et al. 2008). Under our experimental conditions, Verte lettuce, which was more sensitive during germination and early seedling establishment compared to Romaine (Nasri et al. 2010), was moderately tolerant to 100 mM NaCl as manifested by the restricted translocation of cations from roots to leaves (Table 2), resulting in less growth reductions compared to Romaine variety (Table 1). Similar discrepancy in salt tolerance between germination and later developmental stages were documented in alfalfa (Johnson et al. 1992). Different adaptation mechanisms may be involved in the seed and seedlings warranting further investigations. Furthermore, a reduction in growth and water content in both leaves and roots was observed in Verte and Romaine grown under 200 mM NaCl, where further accumulation of cations occurred in the roots (Table 2). Roots are less vulnerable than leaves to Na+ accumulation, because shoots have a greater propensity than roots to accumulate Na+ and Cl (Tester and Davenport 2003). This is evident from the significantly greater decrease of water content in leaves compared to roots in both varieties under salt stress. However, Romaine had a much lower leaf water content compared to Verte. These data clearly show that both ionic and osmotic impacts are critical contributing factors to the declined growth at 200 mM NaCl, particularly in Romaine.

Salinity provoked strong increases in both Na+ and Cl contents in NaCl-tolerant cell lines (Elkahoui et al. 2005). This is similar to what was observed in our study. Toxic levels of Na+ and Cl in the root environment interfere with the uptake and transport of important nutrients, including K+ and Ca2+ and greatly reduce their availability to the roots. Sodium reduces the influx of Ca2+ by binding it to the plasma membrane, inhibiting influx and increasing efflux of Ca2+, thereby depleting the internal Ca2+ concentration (Cramer et al. 1989). Calcium plays an important role in plant tissues regulating the function of Na+ and K+ (Cachorro et al. 1994; Grattan and Grieve 1993). In saline soils with abundant Ca2+, Ca2+ deficiency arises due to the competitive effects of K+/Na+ and Ca2+/Na+ selectivity that is associated with plant salt tolerance (Ashraf and Naqvi 1991). The Ca2+/Na+ selectivity increased significantly in leaves and roots of both varieties (Table 3). Romaine leaves and roots contained relatively high Ca2+/Na+ selectivity under 100 mM NaCl and Romaine roots also had significantly greater Ca2+/Na+ selectivity under 200 mM NaCl. The greater Ca2+/Na+ selectivity in Romaine (Table 3) was caused by tighter restriction of Na+ absorption in roots (Table 2), greater root–shoot selectivity and translocation of Ca2+ over Na+, serving as an important mechanism of adapting to NaCl stress.

More than 50 enzymes are activated by K+, and Na+ cannot substitute in this role (Bhandal and Malik 1988). Thus, high levels of Na+ or reduced K+/Na+ selectivity can disrupt various enzymatic processes in the cytoplasm. Moreover, protein synthesis requires high concentrations of K+, owing to K+ requirement for tRNA binding to ribosomes (Blaha et al. 2000) and for other aspects of ribosome function (Wyn Jones et al. 1979). The disruption of protein synthesis by elevated concentrations of Na+ appears to be an important cause of damage by Na+. Salinity causes excessive Na+ accumulation in plants and influences the uptake of essential nutrients such as K+ and Ca2+ because of the effect of ion selectivity (He and Cramer 1993; Marschner 1995). Consistent with the literature, our study revealed that increasing NaCl concentration caused a significant decline in K+ and Ca2+ contents in roots and leaves of both varieties. Consequently, crops such as lettuce growing in saline conditions may suffer from Na+ toxicity and K+ and/or Ca2+ deficiency.

Photosynthetic activity is affected by abiotic stresses (Dubey 2005). Declined photosynthetic activities were accompanied by reduced growth in both genotypes under increasing NaCl salinity (Table 1; Fig. 2). There was a simultaneous reduction in photosynthesis and stomatal conductance at 200 mM NaCl, suggesting that the decrease in photosynthesis was attributable to NaCl effects on stomatal closure (Fig. 2). Similar findings were reported in Thellungiella halophila seedlings treated with NaCl (M’rah et al. 2006). In contrast, net photosynthesis was less affected by 100 mM NaCl compared to stomatal conductance, reflecting increased water use efficiency particularly in Verte when exposed to moderate NaCl stress.

Biochemical changes during salt stress were noted in both varieties and may indicate their significance in defining their differential salt tolerance particularly at 100 mM NaCl. Membrane lipid peroxidation results in elevated levels of malondialdehyde (MDA), a generic biomarker for membrane damage (Elkahoui et al. 2005). For instance, MDA content was nearly sixfold lower in roots than in leaves (Fig. 3c, d), suggesting a tissue-dependent difference in the membrane lipid peroxidation process as a result of salinity-induced membrane damage. Leaf Pro transiently peaked in Verte under 100 mM NaCl, which was accompanied by an accumulation of cations. Pro accumulation is frequently associated with plants and diverse microbial species grown in stressful conditions, and regarded as a compatible osmo-protectant (Delauney and Verma 1993). Pro may play a critical role in Verte relative tolerance to moderate NaCl treatment. Melanoidins are commonly present in foods that have undergone some form of non-enzymatic browning, but also accumulate in rice mutant seeds deficient in aldehyde dehydrogenase 7. This enzyme is involved in the removal of excessive aldehydes, including MDA (Shin et al. 2009). Significant increases were detected in leaves of both varieties and in roots of Verte under 200 mM NaCl, which may partly contribute to alleviating NaCl-induced oxidative stress. Although soluble phenolics are an important component of the antioxidant capacity of lettuce (Nicolle et al. 2004), salt stress had no obvious effect on the accumulation of these metabolites in Verte and Romaine (Fig. 6).

Our current study aimed to identify physiological and biochemical changes of two lettuce varieties, Verte (moderately tolerant to NaCl) and Romaine (sensitive to NaCl), and in response to increasing salinity concentrations ranged from 0 to 200 mM NaCl. Collectively, our results suggest that Verte is more tolerant to 100 mM NaCl based on its greater biomass (DW) accumulation and more effective protection systems in leaves (carotenoids and Pro) and roots (melanoidins). Previously, we investigated the greater impact of iso-osmotic Na2SO4 (77 mM) relative to NaCl (100 mM) on lettuce (Mahmoudi et al. 2010). These studies have shown the genetic, ionic and osmotic influence on changes in physiological parameters (growth, water and ion content, photochemical capacity), membrane oxidative damage (MDA, electrolyte leakage), leaf carotenoids, phenolics, melanoidins, Pro and root lignin content. This study further revealed that in the root of both Verte and Romaine, NaCl stimulated the production of H2O2, a toxic reactive oxygen species with deleterious effects in plants (Sairam et al. 1998).

Conclusion

Our results demonstrate that the two varieties, Verte and Romaine, responded differentially to increasing NaCl concentrations. Verte (more sensitive in the germination stage) was more tolerant to 100 mM NaCl than Romaine by preferentially restricting cations in the roots, limiting the accumulation of MDA, and enhancing the accumulation of carotenoids, phenolic antioxidant and Pro. With 200 mM NaCl, the protection machinery was impaired in both varieties, resulting in comparable growth reductions.

Abbreviations

EL:

Electrolyte leakage

MDA:

Malondialdehyde

HPLC:

High-performance liquid chromatography

Pro:

Proline

References

  1. Annalisa R, Patrizia P, Carlotta G, Graziano S, Antonio C, Daniela H (2002) Polyphenols in greenhouse and open-air-grown lettuce. Food Chem 79:337–342

    Article  Google Scholar 

  2. Arnon DI (1949) Copper enzymes in chloroplasts phenol oxidase in Beta vulgaris. Plant Physiol 24:1–15

    PubMed  Article  CAS  Google Scholar 

  3. Ashraf M, McNeilly T (1990) Responses of four Brassica species to sodium chloride. Environ Exp Bot 30:475–487

    Article  CAS  Google Scholar 

  4. Ashraf M, Naqvi M (1991) Growth and ion uptake of four Brassica species as affected by Na/Ca ratio in saline sand culture. Z Pflanzenemiihr Bodenkd 155:101–108

    Article  Google Scholar 

  5. Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207

    Article  CAS  Google Scholar 

  6. Bhandal IS, Malik CP (1988) Potassium estimation, uptake and its role in the physiology and metabolism of flowering plants. Int Rev Cytol 10:205–224

    Article  Google Scholar 

  7. Bilgin O, Baser I, Korkut KZ, Balkan A, Saglam N (2008) The impacts on seedling root growth of water and salinity stress in maize (zea mays indentata sturt.). Bulgarian J Agricul Sci 14:313–320

    Google Scholar 

  8. Blaha G, Stelzl U, Spahn CMT, Agrawal RK, Frank J, Nierhaus KH (2000) Preparation of functional ribosomal complexes and effect of buffer conditions on tRNA positions observed by cryoelectron microscopy. Methods Enzymol 317:292–309

    PubMed  Article  CAS  Google Scholar 

  9. Cachorro P, Ortiz A, Cerda A (1994) Implications of calcium nutrition on the response of Phaseolus vulgaris L. to salinity. Plant Soil 159:205–212

    Article  CAS  Google Scholar 

  10. Cramer GR, Epstein E, Laûchli A (1989) Na–Ca interactions in barley seedlings: relationship to ion transport and growth. Plant Cell Environ 12:551–558

    Article  CAS  Google Scholar 

  11. Delauney AJ, Verma DPS (1993) Proline biosynthesis and osmoregulation in plants. Plant J 4:215–223

    Article  CAS  Google Scholar 

  12. Dionisio-Sese ML, Tobita S (1998) Antioxidant responses of rice seedlings to salinity stress. Plant Sci 135:1–9

    Article  CAS  Google Scholar 

  13. Dionisio-Sese ML, Tobita S (2005) Effects of salinity on sodium content and photosynthetic responses of rice seedlings differing in salt tolerance. J Plant Physiol 157:54–58

    Google Scholar 

  14. Dubey RS (2005) Photosynthesis in plants under stressful conditions In: Photosynthesis Handbooks. CRC Press, New York, pp 717–718

    Google Scholar 

  15. El-Hendawy SE, Hu Y, Schmidhalter U (2005) Growth, ion content, gas exchange, and water relations of wheat genotypes differing in salt tolerances. Aust J Agric Res 56:123–134

    Article  CAS  Google Scholar 

  16. Elkahoui S, Hernández JA, Abdelly C, Ghrir R, Limam F (2005) Effects of salt on lipid peroxidation and antioxidant enzyme activities of Catharanthus roseus suspension cells. Plant Sci 168:607–613

    Article  CAS  Google Scholar 

  17. Grattan SR, Grieve CM (1993) Mineral nutrient acquisition and response by plants grown in saline environments. In: Handbook of plant and crop stress. Marcel Dekker, New York, pp 203–226

  18. Gross J (1991) Pigments in vegetables. Chlorophylls and carotenoids. Avi: Van Nostrand Reinhold Company Inc, New York

    Google Scholar 

  19. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Mol Biol 51:463–499

    Article  CAS  Google Scholar 

  20. He T, Cramer GR (1993) Salt tolerance of rapid-cycling Brassica species in relation to potassium/sodium ratio and selectivity at the whole plant and callus levels. J Plant Nutr 16:1263–1277

    Article  CAS  Google Scholar 

  21. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198

    PubMed  Article  CAS  Google Scholar 

  22. Hermann K (1976) Flavonols and flavones in food plants: a review. J Food Technol 11:433–448

    Article  Google Scholar 

  23. Hernandez JA, Aguilar AB, Portillo B, López-Gómez E, Mataix Beneyto J, García-Legaz MF (2003) The effect of calcium on the antioxidant enzymes from salt-treated loquat and anger plants. Funct Plant Biol 30:1127–1137

    Article  CAS  Google Scholar 

  24. Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Calif Agric Exp Station Circ (Berkley) 347:1–32

    Google Scholar 

  25. Huang J, Bhinu VS, Li X, Dallal Bashi Z, Zhou R, Hannoufa A (2009) Pleiotropic changes in Arabidopsis f5h and sct mutants revealed by large-scale gene expression and metabolite analysis. Planta 230:1057–1069

    PubMed  Article  CAS  Google Scholar 

  26. Inze′ D, van Montagu M (1995) Oxidative stress in plants. Curr Opin Biotechnol 6:153–158

    Article  Google Scholar 

  27. Johnson DW, Smith SE, Dobrenz AK (1992) Genetic and phenotypic relationships in response to NaCl at different developmental stages in alfalfa. Theor Appl Genet 83:833–838

    Article  Google Scholar 

  28. Ke D, Saltveit ME (1988) Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce. Plant Physiol 88:1136–1140

    PubMed  Article  CAS  Google Scholar 

  29. Kim HJ, Fonseca JM, Choi JH, Kubota C, Kwon DY (2008) Salt in irrigation water affects the nutritional and visual properties of Romaine lettuce (Lactuca sativa L.). J Agric Food Chem 56:3772–3776

    PubMed  Article  CAS  Google Scholar 

  30. Kotchoni SO, Kuhns C, Ditzer A, Kirch HH, Bartels D (2006) Overexpression of different aldehyde dehydrogenase genes in Arabidopsis thaliana confers tolerance to abiotic stress and protects plants against lipid peroxidation and oxidative stress. Plant Cell Environ 29:1033–1048

    PubMed  Article  CAS  Google Scholar 

  31. Kuiper PJC (1985) Environmental changes and lipid metabolism of higher plants. Physiol Plant 64:118–122

    Article  CAS  Google Scholar 

  32. Liu X, Ardo S, Bunning M, Parry J, Zhou K, Stushnoff C (2007) Total phenolic content and DPPH radical scavenging activity of lettuce (Lactuca sativa L.) grown in Colorado. Food Sci Technol 40:552–557

    CAS  Google Scholar 

  33. M’rah S, Ouerghi Z, Berthomieu C, Havaux M, Jungas C, Hajji M, Grignon C, Lachaâl M (2006) Effects of NaCl on the growth, ion accumulation and photosynthetic parameters of Thellungiella halophila. J Plant Physiol 163:1022–1031

    PubMed  Article  Google Scholar 

  34. Mahmoudi H, Huang J, Gruber MY, Kaddour R, Lachaâl M, Ouerghi Z, Hannoufa A (2010) The impact of genotype and salinity on physiological function, secondary metabolite accumulation, and antioxidative responses in lettuce. J Agric Food Chem 58:5122–5130

    PubMed  Article  CAS  Google Scholar 

  35. Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic Press, London

    Google Scholar 

  36. Nasri N, Kaddour R, Rabhi M, Plassard C, Lachaal M (2010) Effect of salinity on germination, phytase activity and phytate content in lettuce seedling. Act Physiol Plant doi 10.1007/s11738-010-0625-4

  37. Nicolle C, Carnat A, Fraisse D, Lamaison JL, Rock E, Michel H, Amouroux P, Remesy C (2004) Characterisation and variation of antioxidant micronutrients in lettuce (Lactuca sativa folium). J Sci Food Agric 84:2061–2069

    Article  CAS  Google Scholar 

  38. Sairam RK, Deshmukh PS, Saxena DC (1998) Role of antioxidant system in wheat genotypes tolerance to water stress. Biol Plant 41:387–394

    Article  CAS  Google Scholar 

  39. Shin JH, Kim SR, An G (2009) Rice aldehyde dehydrogenase7 is needed for seed maturation and viability. Plant Physiol 149:905–915

    PubMed  Article  CAS  Google Scholar 

  40. Stepien P, Klobus G (2006) Water relations and photosynthesis in Cucumis sativus L. leaves under salt stress. Biol Plant 50:610–616

    Article  CAS  Google Scholar 

  41. Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Ann Bot 91:503–527

    PubMed  Article  CAS  Google Scholar 

  42. Wyn Jones RG, Brady CJ, Spears J (1979) Ionic and osmotic relations in plant cells. In: Recent Advances in the Biochemistry of Cereals. Academic Press, London, pp 63–103

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Correspondence to Hela Mahmoudi.

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The authors Hela Mahmoudi and Rym Kaddour contributed equally to this work.

Communicated by R. Aroca.

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Mahmoudi, H., Kaddour, R., Huang, J. et al. Varied tolerance to NaCl salinity is related to biochemical changes in two contrasting lettuce genotypes. Acta Physiol Plant 33, 1613–1622 (2011). https://doi.org/10.1007/s11738-010-0696-2

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Keywords

  • Lactuca sativa L.
  • NaCl salinity
  • MDA
  • Proline
  • Polyphenol
  • Photosynthesis