Additive effects of Na⁺ and Cl^– ions on barley growth under salinity stress

Experiment 1. The separate effects of Na⁺ and Cl^– ions at different concentrations on growth of barley in solution culture

A solution culture experiment was conducted to assess the effect of different concentrations of Na⁺ and Cl^– ions on the growth of Barque73 and Tadmor. Ten concentrations (0, 10, 20, 40, 60, 80, 100, 120, 140, and 160 mM) of Na⁺-dominant and Cl^–-dominant Hoagland's solutions were prepared by dissolving a mixture of Na⁺ salts (Na₂SO₄, Na₂HPO₄, and NaNO₃) or a mixture of Cl^– salts (CaCl₂, MgCl₂, and KCl) in Milli-Q water (conductivity ≥18.2 MΩ cm⁻¹). All treatment solutions had a background of modified Hoagland's solution for nutrient supply, the composition of which (in mM) was: NH₄NO₃ (0.2), KNO₃ (5), Ca (NO₃)₂ (2), MgSO₄ (2), KH₂PO₄ (0.1), NaFe(III)-hydroxyethyl ethylenediamine triacetic acid (HEDTA) (0.05), H₃BO₃ (0.01), MnCl₂ (0.005), ZnSO₄ (0.005), CuSO₄ (0.0005), and Na₂MoO₃ (0.0001). Solutions were changed every 10 d and the pH of the solution was monitored several times each week and maintained within a range of 6.0–6.5. The experiment was conducted in an air-conditioned growth chamber with day/night temperatures of approximately 25/18 °C. The intensity of photosynthetically active radiation was measured using a Li-Cor quantum sensor (model LI-1000, Li-Cor, Lincoln, NE, USA) and varied from 550 to 600 mmol m⁻² s⁻¹. At 10–12 d after germination, when the third leaf was beginning to appear, the Na⁺ and Cl^– treatments were introduced in increments of 20 mM over 8 d. Supplementary Ca²⁺ (5 mM) as CaCl₂ was added to the Na⁺ treatment to prevent Ca⁺² deficiencies in plants (Genc et al., 2010).

The growth system of the plants was modified from Casey et al. (2003) and consisted of a polypropylene mesh supported at the bottom of a flanged tube (4 cm diameter×8 cm depth) which, in turn, formed the bottom of the tube. This assembly was inserted into a lid which rested on top of a PVC pot (10.4 cm diameter×32 cm depth) filled with nutrient solution. Each lid had a 0.5 cm drilled hole for a capillary air tube (see Supplementary Fig. S1 at JXB online). Uniformly sized seeds of each genotype were surface-sterilized in 70% ethanol for 1 min, followed by soaking in 3% sodium hypochlorite for 5 min, then rinsed three times with deionized water. Four seeds were placed on the mesh and, after the seedlings were established, they were thinned to one seedling. The tubes were then filled with cylindrical black polycarbonate pellets (approximately 2–4 mm long and 1–2 mm in diameter) to support the plant stem and prevent light penetration to the nutrient solution. At this stage the level of the solution was allowed to drop to about 1.5 cm below the mesh. The experimental design was a factorial, completely randomized design comprising 19 salt treatments (control and 9 rates Na⁺/Cl^–)×two barley genotypes with three replicates.

Experiment 2. The separate effects of Na⁺ and Cl^– ions at high concentration on barley growth using solution culture

A solution culture experiment was conducted to assess the relative importance of Na⁺ and Cl^– ions to salt toxicity. The varieties, Barque73, Clipper, Sahara, and Tadmor were used because of their different sensitivities to salt stress and abilities to exclude Na⁺ and Cl^– ions. A factorial experiment consisted of six different treatments: control, 120 mM NaCl, 120 mM NaCl+0.07 mM 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS), 120 mM Na⁺-gluconate, 120 mM Cl^–-dominant, and 120 mM Na⁺-dominant Hoagland's solutions. The effect of Na⁺ independently of Cl^– was examined in three ways: by using DIDS, which is a non-permeating amino acid that inhibits Cl^– transport (Lin, 1981; Lin and Kao, 2001); by using Na⁺-gluconate because gluconate is an anion that is unable to permeate the cell membrane; and by using the Na⁺-Hoagland solution. The solutions were designed to have similar EC and osmotic potentials (Table 1). The growth conditions were identical to those of Experiment 1. The experimental design was a factorial completely randomized design comprising six treatments×four barley genotypes with three replicates.

Experiment 3. The separate effects of Na⁺ and Cl^– ions at high concentration on barley growth using soil culture

An experiment was conducted using field soil to assess the effect of high concentrations of Na⁺ and Cl^– ions on the growth of Barque73, Clipper, Sahara, and Tadmor. The A horizon (topsoil) soil of a sandy loam red Chromosol (Isbell, 1996) collected from Roseworthy (34.51 °S, 138.68 °E, South Australia) was used. Following collection, the soil was air-dried and ground to pass through a 5 mm sieve. A soil-water characteristic curve was determined using the pressure plate method (Klute, 1986) and the soil moisture content at field capacity (–10 kPa, equivalent to 37% w/w) was estimated.

The experiment was designed to compare growth in saline soils at similar soil electrical conductivities (EC) and soil osmotic potential (Ψ_O) but different ionic compositions. To achieve this, it compared the effects of 120 mmol kg⁻¹ Na⁺ (applied as a range of sulphate, nitrate, and phosphate salts), 120 mmol kg⁻¹ Cl^– (applied as a range of calcium, magnesium, and potassium salts), and 120 mmol kg⁻¹ NaCl at a similar EC in a soil-based system (Table 2). The Cl^–, Na⁺, and NaCl-dominant saline soils were prepared by dissolving a mixture of Na⁺ salts (20 mM Na₂SO₄, 20 mM Na₂HPO₄, and 40 mM NaNO₃), a mixture of Cl^– salts (20 mM CaCl₂, 20 mM MgCl₂, and 40 mM KCl), and NaCl salt (120 mM NaCl), respectively, in milliQ H₂O and spraying each solution on a 2 cm layer of soil to reach field capacity moisture content. Each soil was covered with plastic to control evaporation and left for 5 d at 25 °C to reach equilibrium, then mixed thoroughly and air-dried (Tavakkoli et al., 2010a). Basal fertilizer was thoroughly mixed with the soil at the following concentrations (in mg pot⁻¹): NH₄NO₃ (380), KH₂PO₄ (229), CaCl₂ (131), MgCl₂ (332), CuCl₂ (10.7), ZnCl₂ (11), Na₂MoO₄ (6.84), and H₃BO₃ (15). All soils with basal fertilizer were wetted to field capacity with deionized water and allowed to equilibrate for 4 d at 25 °C. Samples of the four synthesized soils were moistened to field capacity (water potential at –10 kPa) and centrifuged at 4000 g for 30 min to extract the soil solution which was passed through 0.25 μm filter paper. Electrical conductivity, Ψ_O and ion concentrations of the solutions were measured (Table 2).

The plants were grown in pots, 10.4 cm in diameter and 32 cm deep in which there were two layers of soil; 2200 g of air-dried soil (subsoil) which contained the salt treatment with 800 g of untreated soil above (topsoil). Each layer was packed to a bulk density of 1.35 Mg m⁻³. The subsoil and topsoil were separated by a 3 cm layer of plastic beads (120 g) to prevent salt rising to the topsoil through capillarity action. The top 3 cm of the pot was also covered by plastic beads to minimize the water evaporation from the soil surface. Uniformly sized seeds of each genotype were surface-sterilized in 70% ethanol for 1 min, followed by soaking in 3% sodium hypochlorite for 5 min, then rinsed three times with deionized water. Four barley seeds were sown in each pot and thinned to one per pot after 5 d. The experiment was conducted under the same growth conditions as described in Experiment 1. The pots were weighed and watered to field capacity regularly and daily water use calculated. The experimental design was a factorial, completely randomized design comprising four treatments×four barley genotypes with three replicates, giving a total of 48 pots.

Experiment 1

Plants were harvested after 49 d, the fresh and dry weights of the shoots were recorded, and the whole shoot moisture content was calculated. The shoots were digested in 40 ml of 1% HNO₃ at 95 °C for 6 h in a 54-well HotBlock (Environmental Express, Mt Pleasant, South Carolina, USA). The concentration of Na⁺ and K⁺ in the digested samples was determined using a flame photometer (Model 420, Sherwood, Cambridge, UK). Chloride concentrations of the digested extracts were determined using a chloride analyser (Model 926, Sherwood Scientific, Cambridge, UK). Recovery of Na⁺, Cl^–, and K⁺ from plant standards (Australasian Soil and Plant Analysis Council) were 94, 98, and 93%, respectively.

Experiments 2 and 3

Leaf chlorophyll content was measured using a hand-held SPAD 502 meter (Minolta, Osaka, Japan) in Experiment 3. Average SPAD chlorophyll readings were calculated from four measurements from the leaf tip to the leaf base of the fourth and the fifth leaf. An infrared, open gas exchange system LI-6400 (Li-Cor, Inc., Linclon, NE, USA) coupled with an integrated fluorescence chamber head (Li-6400-40 leaf chamber fluorometer; Li-Cor Inc., Nebraska, USA) was used to measure instantaneous gas exchange and chlorophyll fluorescence. All measurements were made on the fifth leaf, 4–5 h into a 9 h photoperiod on the day after the plants were rewatered to field capacity. The settings were chosen to match growth chamber conditions. Leaf temperature was maintained at 25 °C, light intensity was set at 800 μmol photons m⁻² s⁻¹ with a red/blue light source, and the CO₂ concentration was set at 400 μmol mol⁻¹. Leaf to air VPD was maintained at 1.1 kPa. The photosynthetic rate (A), stomatal conductance (g_s), transpiration (T), and substomatal CO₂ concentration (C_i) were recorded. Fluorescence parameters were estimated from the measurements of chlorophyll fluorescence on light-adapted leaves (Genty et al., 1989). The operating quantum efficiency of PSII photochemistry, ΦPSII, was calculated as (F′m − F′)/F′m. The efficiency of open PSII reaction centres (F′v/F′m) was calculated as (F′m − F′o)/F′m and the photochemical quenching (qP) factor was determined as (F′m − F′)/(F′m−F′o). The non-photochemical quenching (NPQ) was expressed as Fm/F′m−1 where F′m is the maximal fluorescence during a saturating flash of light >7 mmol m^-2 s^-1, and F′o is the minimal fluorescence of a momentarily darkened leaf.

To measure the osmotic potential (Ψ_s) of leaf sap, a disc of Whatman GF/B glass micro-fibre paper was placed in the barrel of a 2 ml plastic syringe so that it covered the outlet hole. A fresh leaf was then put in the barrel, the plunger was re-inserted, and the tip of the syringe was sealed with Blu-Tack^®. The syringe was frozen in liquid nitrogen and stored until measurement. The frozen leaf was thawed to ambient temperature, the plunger and Blu-Tack were removed and the barrel of the syringe was placed in a 15 ml centrifuge tube, with its tip resting inside a 1.5 ml Eppendorf tube. After centrifugation at 2500 g for 10 min at 4 °C, the osmolality of a 10 μl sample collected in the Eppendorf tube was measured by a calibrated vapour pressure osmometer (Model 5520; Wescor, Inc., Utah, USA).

Water use was determined as the difference between the water added to the pots to replace that lost and that retained in the plant (due to growth). Transpiration efficiency (TE=A/T) was determined by measurements of A and T on single leaves and water use efficiency (WUE) was determined by gravimetric determinations of growth and water loss on whole plants. Plants were harvested 49 d after sowing, 1 d after watering to field capacity. Fresh weight was measured before plants were dried at 70 °C for 72 h and dry weights were recorded. Shoot moisture content was estimated from the difference between fresh weight and dry weight. The nutrient concentration of the whole shoot was measured after digestion in nitric acid/hydrogen peroxide in a closed vessel (50 ml polypropylene centrifuge tube) using a hot block digestion system. Elemental analyses of plant samples were performed using inductively coupled plasma-optical emission spectrometry (Zarcinas et al., 1987). Chloride concentrations of the digested extracts were determined using a chloride analyser (Model 926, Sherwood Scientific, Cambridge, UK).

Whole shoot ion concentration and leaf osmotic potential

For plants grown in the control treatment, tissue Na⁺ concentration was 4–10 mmol kg⁻¹ DW and no difference was apparent among the four genotypes. The Na⁺ concentration in all varieties increased with increasing concentration of Na⁺ in treatment solutions (Table 3). Sahara had the highest Na⁺ concentration of all the genotypes (∼1040 mmol kg⁻¹ DW). The Na⁺ concentration in shoots of Clipper was about 5% less than Sahara, while Barque73 and Tadmor had the lowest Na⁺ concentration (∼720 mmol kg⁻¹ DW) (Table 3) which is consistent with previous hydroponic screening of these varieties (E Tavakkoli, unpublished data). Potassium concentrations were not significantly affected by NaCl and Na⁺ treatments and did not differ significantly among the four varieties but were about 10% higher in Cl^–-dominant Hoagland's solutions due to using KCl to prepare this salt solution. The K⁺:Na⁺ ratio never fell below 1.1, which is the ratio considered critical for maintaining metabolic activity (Munns, 1985). Chloride concentrations in the shoot tissue of all genotypes were 10–15 mmol kg^-1 DW in the control and 2 mmol kg⁻¹ DW in the Na⁺-dominant Hoagland's solutions and the Na⁺-gluconate treatments. The concentration increased significantly when grown under Cl^–-dominant Hoagland's solutions and NaCl treatments, but the responses of genotypes differed significantly (Genotype×Treatment interaction, P <0.001). Consistent with our previous results, Barque73 showed greater Cl^– exclusion (relative to Na⁺) whereas the shoot Cl^– concentration of the other three genotypes increased to levels similar to their shoot Na⁺ concentration. Treatments with NaCl in the presence of DIDS resulted in about 75–83% reduction in shoot Cl^– concentrations, but did not affect Na⁺ concentrations (Table 3).

Plant biomass and water relations

The four genotypes did not differ significantly in shoot dry weight when grown in non-saline solution for 49 d. Increased levels of salts in the solution reduced shoot dry weights of all the varieties significantly but the responses of varieties differed (Fig. 2). In Barque73 the shoot dry weight was reduced by 24% in Na⁺ (NaCl+DIDS, Na⁺-gluconate, and Na⁺-Hoagland) and 10% in Cl^– treatments (Cl^–-Hoagland), respectively, however, the reduction due to NaCl was about 34%. The dry weight of Clipper was reduced by 18%, 23%, and 47% in Na⁺, Cl^–, and NaCl treatments, respectively. Sahara had a similar salt tolerance to Barque73 in the NaCl treatment but the growth reduction in Na⁺ treatments was 10%, whereas it was reduced by 23% in the Cl^– treatment. Tadmor had the lowest shoot dry weight under NaCl salinity compared with the other three (43%). It showed similar shoot dry weight under high Na⁺ concentration as Barque73, however, the growth reduction under Cl^– treatments was 40% indicating a significant effect of Cl^– to shoot dry weight depression. Adding DIDS to NaCl increased the shoot dry weight of plants by 10%, 45%, 37%, and 62% in Barque73, Clipper, Sahara, and Tadmor, respectively, compared with NaCl in the absence of DIDS (Fig. 2). The pattern of growth of four genotypes in Na⁺-gluconate was very similar to that obtained in Na⁺-dominant Hoagland's solution. Leaf osmotic potential also decreased 2-fold in the saline treatments compared to control (Table 3). The reduction in Ψ_s was similar in Na⁺, Cl^–, and NaCl treatments. Sahara had significantly higher Ψ_s compared with the other three genotypes.

Soil properties

The Na⁺-treated and NaCl-treated soils had comparable levels of Na⁺ in the soil solution and the Cl^–-treated and NaCl-treated had similar concentrations of Cl^– (Table 2). The use of different salt solutions in the four treatments resulted in some variation in the concentrations of Ca²⁺, Mg²⁺, K⁺, P, and S. However, in all cases, the variation in plant nutrient concentrations was much lower than those in the soil solution (Tables 2, 4) and all were within the physiologically normal range for barley (Reuter and Robinson, 1997). While the effects of this variation in the other cations and anions on the growth of the plants in the different salt treatment can not be discounted completely, the much greater range in the final concentrations of Na⁺ and Cl^– in the plants means that the responses to salt that were observed in barley were largely due to the effects of Na⁺, Cl^–, and NaCl.

Whole shoot ion concentration

For plants in the control treatments, the mean tissue Na⁺ concentration was 88 mmol kg⁻¹ DW and there was no significant difference among the four genotypes (Table 4). As soil Na⁺ concentration increased, so too did tissue Na⁺ concentration in all genotypes. Large genotypic variation was observed in shoot Na⁺ concentration: Clipper had the lowest Na⁺ concentration (450 mmol kg⁻¹) whereas Sahara had the highest (750 mmol kg⁻¹), and Tadmor and Barque73 were intermediate with shoot Na⁺ concentration of about 685 mmol kg⁻¹ DW. The concentration of shoot Cl^– increased in Cl^– and NaCl soils, and the Cl^– concentrations were consistently greater than the tissue Na⁺ concentrations in control and NaCl treatments with the exception of Barque73 grown under NaCl. The responses of the four genotypes differed significantly (Genotype×Treatment interaction, P <0.001) (Table 4). The Cl^– concentrations in Tadmor and Sahara were over 2–3 times greater than Barque73 and Clipper. Potassium concentration of all genotypes decreased significantly when exposed to Na⁺ or NaCl. Sahara consistently maintained a higher concentration of K⁺ and a much higher K⁺:Na⁺ ratio (>1.1), whereas this ratio fell below 1.1 for the other varieties, with Clipper having the lowest ratio (Table 4). This negative effect of Na⁺ on K⁺ was indicated between treatments within each genotype, however, among the genotypes there is a positive relationship between Na⁺ and K⁺. In other words, genotypes that exclude Na⁺ also seem to have lower K⁺, and the competitive relationship operates within a genotype but not between genotypes. Changes in calcium and magnesium tissue concentration followed a very similar pattern as K⁺, and it decreased in all genotypes when grown in Na⁺ and NaCl-dominant soils.

Plant biomass and water use

Increases in Na⁺, Cl^– or NaCl concentration of the soil solution significantly (P <0.05) reduced shoot dry weight in all genotypes but the genotypic responses were different (Table 4). The shoot dry matter of Barque73, Tadmor, and Clipper was reduced by up to 20–25% in the Na⁺-dominant soil, whereas the reduction in Sahara was only 11%. The biomass reduction under Cl^–-dominant soil was 10%, 30%, 22%, and 45% in Barque73, Clipper, Sahara, and Tadmor, respectively. The biomass reductions due to NaCl salinity reflected the additive effects of Na and Cl: they ranged from 30% in Barque73 and Sahara to 45% and 65% in Clipper and Tadmor, respectively.

Daily water use increased consistently throughout the experiment in the control treatments of all four genotypes (Fig. 3). Salinity reduced daily water use with the largest reduction occurring in NaCl treatments. The greater sensitivity of Tadmor to salt is evident as is the greater effect of Cl^– on water use. Similar to shoot dry weights (Table 4), the reduction in total water use due to the presence of NaCl was greater than that due to Na⁺ and Cl^– individually, and was equal to the additive effect of the two. The cumulative water use at the final measurement can be used to quantify the relative effects of Na⁺, Cl^–, and NaCl to total plant water uptake and, consequently, growth. Total water use in Barque73 in the Na⁺ and Cl^– and NaCl treatments was reduced by 21%, 10%, and 30%, respectively, whereas in Tadmor the reductions were 20%, 45%, and 60%, respectively (Fig. 3b, d). The cumulative water use of Clipper was reduced by 22% and 25% by Na⁺ and Cl^– treatments, respectively, and a 45% reduction occurred under NaCl. Sahara was more sensitive to Cl^– than Na⁺ as indicated by a 20% reduction in cumulative water use under Cl^– treatment compared with a 10% reduction under Na⁺ treatment (Fig. 3f). Leaf osmotic potential also decreased in the salt treatments compared with the control. Sahara and Barque73 had inherently higher Ψ_s than Tadmor and Clipper in the control (Table 4). Sahara maintained a significantly higher Ψ_s than the other varieties under salt stress.

Chlorophyll content, gas exchange, and chlorophyll fluorescence parameters

The leaf chlorophyll content of all genotypes was increased in Na⁺-dominant soil (15–19%. whereas it was reduced by between 20% and 36% in Clipper, Sahara, and Tadmor in Cl^–-dominant and NaCl-dominant soils and remained unchanged in Barque73 (Table 5). The gas exchange parameters of all genotypes decreased in all salt treatments. The rate of photosynthesis (A) of plants grown in Na⁺-dominant soil decreased by 20–25% in Barque73, Clipper, and Tadmor, and by 10% in Sahara (Table 5). In Cl^–-dominant soil A was reduced by 40% in Tadmor, 23% in Clipper and Sahara, and 10% in Barque73. The greatest reduction in A occurred in the NaCl treatment and followed a similar pattern to shoot dry weight and cumulative plant water use. Clipper and Tadmor had significantly lower A than Barque73 and Sahara in the Na⁺ and NaCl treatments. Stomatal conductance (g_s) of all genotypes followed a similar pattern of reduction to A. The relative intercellular CO₂ concentration (C_i/C_a) of all genotypes decreased by 2.5–4% when grown in the Na⁺ treatment. This value decreased by only 6% in Barque73 under Cl^– treatment, but there was a larger reduction of C_i/C_a in Tadmor (40%), Sahara (22%), and Clipper (16%). The changes of C_i/C_a under NaCl salinity was similar or just slightly less than Cl^– treatment which indicates the reduction of C_i/C_a under salinity was driven by Cl^– concentration (Table 5).

The barley varieties used in this study showed some significant differences in the instantaneous rates of net transpiration (T) by single leaves which followed a similar pattern to changes in the A. The transpiration efficiencies (TE) generated from these data on individual leaves therefore varied little (Table 5): the difference between the maximum and minimum values of the TE for the different cultivars was about 15% of the maximum (P <0.05). There was a significant increase in TE under Na⁺ treatments in all genotypes and under Cl^– only in Tadmor, however, the TE of Barque73 and Clipper was not affected by NaCl treatment while it increased in Sahara and Tadmor (P <0.05). The varieties also differed significantly in their water-use efficiencies (WUE) when these were estimated for whole plants (Table 4). The WUE was correlated with the growth among the four varieties, however, Na⁺, Cl^–, and NaCl treatments did not significantly affect WUE.

The Cl^– and NaCl salts had a marked effect on the four key fluorescence parameters measured but the effects varied with genotypes (Table 6). The presence of Cl^– had a larger effect than Na⁺ in all varieties. Values for the efficiency of light harvesting of PSII (F′v/F′m) of Clipper and Sahara were reduced by 20–25% in the Cl^– treatment and by 40% in Tadmor; the reduction in Barque73 was smaller, but still significant (P <0.05). A similar pattern was observed for qP and ΦPSII under both treatments. Significant relationships between A and both (F′v/F′m) (r=0.89) and ΦPSII (r=0.92) were found for Clipper, Sahara, and Tadmor, indicating that these parameters may not be independent or are co-regulated (Tables 5, 6). As the proportion of PSII reaction centres that remained open (qP) was reduced in Clipper, Sahara, and Tadmor, the portion of fluorescence quenching associated with thermal energy dissipation (NPQ) increased significantly, but the effect was larger in Tadmor. These values changed similarly for Barque73 under Cl^– and NaCl treatments, but the changes are smaller in magnitude (Table 6).