Effect of NaCl on Stomatal Resistance and Proline, Chlorophyll, Na, Cl and K Concentrations of Lentil Plants

MATERIALS AND METHODS

An experiment was conducted under greenhouse conditions (humidity 65-75%, air temperature 25-30°C and neutral light intensity 340-450 μmol m^-2 sec^-1) in Ankara-Turkey, between April 1st to May 30th, 2006, with lentil (Lens calinaris L. cv: Sultan) plants. Lentil genotype was very sensitive to the salt stress. The experimental soil taken from Aridisol great soil group was calcareous (212 g kg^-1), clay loam in texture (clay 30.49%, silt 41.88% and sand 27.63%), slightly alkaline (pH 8.04, EC 0.108 dS cm^-1; both in 1:2.5 water extract). The soil sample has 42.9 mg kg^-1 exchangeable Na and 6.37 mg kg^-1 water extractable Cl.

The experimental soil (2000 g) was placed into the pots and soil was salinised at the levels of 25, 50 and 100 mM NaCl, before seeds were sown into the pots. Plants grown without NaCl addition were taken as control. For basal fertilizers, 100 mg N kg^-1 as ammonium nitrate, 60 mg P kg^-1 as triple super phosphate and 75 mg K kg^-1 as potassium sulfate were applied to the pots.

Eight seeds of lentil were planted in each pot, thinned to six per pot after germination. The experimental plants were irrigated with de-ionized water, when moisture level decreased below the 60% of field water capacity. Eight weeks after germination, the vegetative growth (shoots plus leaves) was harvested. After weighting the fresh material, plants were washed and 2.0 g of samples were taken for total chlorophyll and proline determinations. The remaining plant samples were dried at 65°C and digested with HNO₃:HClO₄ acid mixture (4:1) in order to determine dry matter and the concentrations of Na, Cl and K.

Sodium and potassium concentrations were determined by flame photometer (Eppendorf Elex 6361 model) described by Miller (1998). Chloride was analyzed by precipitation as AgCl and titration according to Johnson and Ulrich (1959).

For determination of proline concentration the samples were extracted and quantified in Shimadzu UV-1201 model spectrophotometer at 520 nm, as described by Bates et al. (1973).

Total chlorophyll of plants was extracted in 80% (v/v) aqueous acetone and absorption was measured in a Shimadzu UV-1201 model spectrophotometer at 645 and 663 nm (Arnon, 1949).

Stomatal resistance was determined during the span of time (14 and 15 h) before harvest by use of a steady-state porometer (EA 540-026 AP4 model) attached to the abaxial side of leaves. The readings were taken on six fully-expanded leaves per treatment, situated at the different position of the canopy. Likewise, temperature (27°C), relative humidity (65%) and light intensity (340-400 μmol m^-2 sec^-1) were also recorded.

The experiment was arranged in a completely randomized design with three salt treatments and three replicates per treatment. The data were analyzed statistically by using Minitab package program (Minitab Release 10.51) and treatment means were compared using the MSTAT package program (Version 3.00) for Duncan’s Multiple Range Test.

RESULTS

Increasing NaCl concentration gradually decreased the growth of lentil plant, expressed as dry weight (Table 1). A slight reduction of dry weight was observed at the lowest level of NaCl as 12.1% in comparison to the control. In parallel to the increase NaCl concentration the effect of salinity was significant for lentil, i.e., growth reduction of plant at 100 mM NaCl treatment was 45.2% compare with the control.

NaCl salinity exhibited controversial effects on ion concentrations of lentil plants (Table 2). Salt treatments increased significantly (p<0.001) Na and Cl concentrations of plants, whereas potassium concentration and K/Na ratio of plants were decreased.

Total chlorophyll and proline concentrations and stomatal resistance of lentil plants were also affected significantly (p<0.001) by salinity (Table 3). The addition of NaCl in the soil decreased total chlorophyll concentration in plants. On the contrary increased proline concentration and stomatal resistance were determined.

Table 1:	Effects of NaCl on dry weight of lentil plants
Means followed by the different letter(s) are significantly different (Duncan’s Multiple Range Test, p<0.01)

Table 2:	Effects of NaCl on Na, Cl and K concentrations and K/Na ratio of lentil plants
***Significant and p<0.001, Means followed by the same letter(s) are not significantly different (Duncan’s Multiple Range Test, p<0.01)

Table 3:	Effects of NaCl on total chlorophyll and proline concentrations and stomatal resistance of lentil plants
***Significant and p<0.001, Means followed by the same letter(s) are not significantly different (Duncan’s Multiple Range Test, p<0.01)

DISCUSSION

Saline soils and saline irrigations constitute a serious production problem for vegetable crops as these are known to suppress plant growth (Lauchli, 1984; Cusido et al., 1987; Turan et al., 2007a and b). The present study demonstrates that salinity, particularly when applied at high levels (50 and 100 mM NaCl), adversely affect the growth of lentil plants.

In the experiment, salt stress, induced by increasing NaCl in the soil solution, resulted in an increase in Na and Cl concentrations. There is an overwhelming amount of evidence to indicate that NaCl-induced-salinity increases Na and Cl uptake by plants (Chavan and Karadge, 1986; Alpaslan et al., 1999; Taban et al., 1999). The data have shown that K uptake and transport to the aerial part of the lentil plant leaves were significantly reduced with increased salinity. This implies a competition between Na and K absorption in lentil plants, resulting in a Na/K antagonism (Siegel et al., 1980). The reduction in K uptake caused by Na is likely to be the result of the competitive intracellular influx of both ions (Cerda et al., 1995). It is well established that many K transport systems have significant affinity for Na (Schachtman and Liu, 1999).

Measurement of stomatal resistance provides a sensitive tool for determining the degree of stress in plants. In the present study, the measured stomatal resistance at the end of the experimental period showed that under salinity lentil plants close their stomata which lead to enhanced stomatal resistance. Similar results were reported by Turan et al. (2007b). Stomata closure is known to be an effective mechanism for economical water utilisation under salt stress and limitation of the harmful salt ions uptake (Hasegawa et al., 2000).

One of the most important mechanisms exerted by higher plants under salt-stress conditions is the accumulation of compatible solutes such as proline. The salt treatments induced an increase in proline concentration in lentil plants. Proline accumulation under salinity may contribute to osmotic adjustment, protecting cell structure and function and/or may serve as metabolic or energetic reserve in plants (Lewitt, 1980; Harivandi et al., 1982; Chowdhury et al., 1993; Madan et al., 1994).

The total chlorophyll concentration of lentil leaves was reduced by increasing the level of NaCl applied. Similar results were reported for total leaf chlorophyll concentration of Phaseolus vulgaris (Petolino and Leone, 1980), rice (Yeo and Flowers, 1983) and wheat and bean (Turan et al., 2007a and b). This effect of NaCl was attributed to a salt-induced weakening of protein-pigment-lipid complex (Strogonov et al., 1970) and/or increasing chlorophyllase (EC: 3.1.1.14) activity (Stivsev et al., 1973).

Consequently, soil salinity significantly inhibited the growth and resulted in a decrease of dry matter of lentil. However, NaCl caused significant variations in stomatal resistance, proline and total chlorophyll concentrations and mineral compositions by uptaking excess mineral elements.

ACKNOWLEDGMENTS

The authors thank Dr. Arzu Akpınar Bayızıt and Mrs. Cigdem Vardar for technical assistance at this research.