Use of iso-osmotic solution to understand salt stress responses in lentil (Lens culinaris Medik.)

South African Journal of Botany 113 (2017) 346–354

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Use of iso-osmotic solution to understand salt stress responses in lentil (Lens culinaris Medik.) M.S. Hossain a, M.U. Alam a, A. Rahman b, Mirza Hasanuzzaman b, K. Nahar c, J. Al Mahmud a, M. Fujita a,⁎ a b c

Laboratory of Plant Stress Responses, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka 1207, Bangladesh Department of Agricultural Botany, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka 1207, Bangladesh

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Article history: Received 21 April 2017 Received in revised form 28 July 2017 Accepted 5 September 2017 Available online xxxx Edited by A Andreucci Keywords: Abiotic stress Drought Ion homeostasis Ionic stress Oxidative stress Salinity

a b s t r a c t Lentil, an important source of protein for human consumption, is considered as a salt susceptible crop species. Thus the present study was carried out to evaluate the most important lentil physiological characters that induce salt tolerance. Salt stress results in osmotic stress immediately followed by ionic toxicity. We used iso-osmotic solutions with different kinds of osmotica i.e. salt (NaCl and KCl), and polyethylene glycol (PEG) to identify the specific response of osmotic stress and ionic toxicity. All of these altered seedling height, chlorophyll, malondialdehyde (MDA), hydrogen peroxide (H2O2), proline, reduced ascorbate (AsA) and total glutathione content, and ion uptake. Moreover, these osmotica also altered the activities of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR), except monodehydroascorbate reductase (MDHAR). Among different osmotica, only NaCl exhibited leaf chlorosis, reduction in K+ and disruption in ion homeostasis, and increased MDA, H2O2 and proline content than isoosmotic KCl and PEG, indicating susceptibility of lentil seedling to salt stress. Addition of Ca along with NaCl showed no chlorosis and improved K+ content. These results demonstrate that prevention of Na-induced K depletion in root might enhance salt tolerance in lentil. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Soil salinity affects 20% of the total global irrigated land and 33% of irrigated agricultural land which has led to severe decrease in crop production in those salt affected areas (Negrao and Tester, 2016). Moreover, it is predicted that a half of cultivable lands would be afflicted with salinity by 2050, since every year 10% of arable land are being affected by salinity for natural and anthropogenic reasons (Jamil et al., 2011). Total crop production rate may fall presumably with the increase of saline area, posing threat to food security since the requirement of food is assumed to increase by 70 to 110% for ever growing population by 2050 (Munns et al., 2012; Hasanuzzaman et al., 2016). Salt stress affects plant growth, immediately creating osmotic stress and later, ionic toxicity (Munns and Tester, 2008). Salinity-induced osmotic stress

Abbreviations: AO, Ascorbate oxidase; APX, Ascorbate peroxidase; AsA, Ascorbic acid; CAT, Catalase; CDNB, 1-Chloro-2,4-dinitrobenzene; DAB, Diaminobenzidine; DHA, Dehydroascorbic acid; DHAR, Dehydroascorbate reductase; DTNB, 5,5-Dithio-bis-(2nitrobenzoic) acid; Gly, Glyoxalase; GR, Glutathione reductase; GSH, Reduced glutathione; GSSG, Oxidized glutathione; MDA, Malondialdehyde; MDHAR, Monodehydroascorbate reductase; MG, Methylglyoxal; PEG, Polyethylene glycol; Pro, Proline; ROS, Reactive oxygen species; SLG, S-D-lactoyl-glutathione; SOD, Superoxide dismutase; TBA, Thiobarbituric acid; TCA, Trichloroacetic acid. ⁎ Corresponding author. E-mail address: [email protected] (M. Fujita).

http://dx.doi.org/10.1016/j.sajb.2017.09.007 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.

causes a reduction in leaf growth by inhibiting cell expansion and cell division. This is followed by chlorosis, necrosis and senescence of mature leaf as a result of ionic toxicity (Carillo et al., 2011); additionally, the excess amount of Na+ disrupts ion homeostasis. Furthermore, it interferes with protein synthesis and enzyme activity because Na+ can compete with K+ for major substrate binding sites in the enzymes (Carillo et al., 2011; Shabala and Pottosin, 2014). To cope with osmotic stress, plants decline water loss by reducing stomatal conductance, leading plants to suffer from CO2 unavailability required for the functioning of the Calvin Cycle. As a result, light absorption exceeds the demand required for photosynthesis and photorespiration, which subsequently boost reactive oxygen species (ROS) production. Accumulation of Na+ and Cl− at toxic levels also contributes to ROS production by interfering with repairing damage of photosystem II due to excess light (Allakhverdiev et al., 2002; Miller et al., 2010; Bose et al., 2014). Overproduced ROS causes oxidative damage to DNA, proteins and lipids, and in severe case, cell death occurs (Hasanuzzaman et al., 2013; Mishra et al., 2013). However, the antioxidant defense system of plants controls the excess ROS generation and prevents cell from damaging its vital constituents. The non-enzymatic antioxidants such as ascorbic acid, (AsA), glutathione (GSH), phenolic compounds, alkaloids, and αtocopherols and enzymes such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) function together

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to maintain ROS below the level that causes cellular injury (Gill and Tuteja, 2010; Hasanuzzaman et al., 2012). Upregulation of the antioxidant system may provide tolerance to plants under salt stress condition by maintaining cellular redox homeostasis (Gill and Tuteja, 2010; Hasanuzzaman et al., 2011; Nahar et al., 2016). Since the osmotic component of salt stress mimics drought stress, to thoroughly understand the mechanism of salt stress, it is necessary to differentiate between damages caused by the osmotic component and ionic component of salt stress. A rapid response due to osmotic stress is growth reduction, often occurs within minutes, and within a few days, ion specific damage appears on leaf showing necrosis, chlorosis and senescence because the excess amount of Na+ accumulation in cytosol occurs at higher rate than the rate of compartmentalization of cell to reduce ion toxicity in the cell (Munns, 2002). To dissect salinity-induced damages, different types of osmotica such as PEG, mannitol, sorbitol, KCl and NaCl are used. Iso-osmotic PEG-6000 and NaCl treatment during vegetative and reproductive stages could not affect the growth and yield of rice, except that K+ content reduced under salt stress (Castillo et al., 2007). However, PEG-6000 caused greater growth inhibition of leaf and accumulation of ABA compared with NaCl (Chazen et al., 1995). Seed germination was considerably reduced in maize due to PEG-6000, but completely failed due to NaCl of similar osmotic potential (Mohammadkhani and Heidari, 2008). NaCl, but not iso-osmotic PEG resulted in the instability of photosystem-II (PSII) and the reduction of PSII energy conversion efficiency (Muranaka et al., 2002). In contrast, NaCl caused fewer damages on sorghum growth, Ca2 + metabolism and photosynthetic gas exchange than KCl (Wang et al., 1999). Interestingly, plant responses to these osmotica greatly differ with genotypes. For example, a lentil genotype, Pantelleria (PAN) has showed tolerance against NaCl whereas it showed susceptible response against PEG (Muscolo et al., 2015). Lentil (Lens culinaris) is a beneficial crop in many ways: a cheap source of protein for human consumption, having the capability of nitrogen fixation and rich in metabolites which are of pharmacological importance (Sidari et al., 2008; Afzal et al., 2014). Among the glycophytes, lentil is very sensitive to salt like other legumes (Ashraf and Waheed, 1990). Therefore, lentil growth and development are negatively affected by salinity stress. Salinity can reduce lentil yield up to 50% by negatively affecting yield attributes (Ayoub, 1977; Golezani and Yengabad, 2012). Development of salt tolerant genotype through breeding is an urgent task for breeders. Thus, identification of physiological attributes that confer salt tolerance may reduce time and increase the success rate of breeding program. Addressing this problem, in this experiment we differentiated the damages caused by osmotic and ionic component of salt stress. Thus we could find out the most appropriate physiological attributes only for salt tolerance in lentil.

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these osmotica, six-day-old seedlings were subjected to these isoosmotic solutions. The control plants were grown in the Hyponex solution only. Nutrient solutions were changed every two days. Seedlings were harvested 4 days after treatment. To understand the response of these osmotica, picture was taken 6 days after treatment. To observe the protective role of Ca, 2 mM CaCl2 was added along with 100 mM NaCl. The experiment was conducted using a completely randomized design with three replications. All chemicals used in this study were purchased from Wako, Japan. 2.2. Observation of seedling growth To determine seedling growth, shoot length, root length, and fresh weight (FW) were measured. Fresh weight and length were expressed as mg seedling−1 and cm respectively. 2.3. Determination of water content To determine water content (WC) of shoot, fresh weight (FW) and dry weight (DW) were measured. For DW, seedlings were oven dried at 80 °C until the weight became constant. Finally, WC was calculated using the following formula: WC (%) = {(FW − DW)∕DW} × 100. 2.4. Determination of ion content Sodium (Na+), potassium (K+), calcium (Ca2 +), and magnesium (Mg2 +) ion contents were determined according to Rahman et al. (2016a). Plant samples were oven dried at 80 °C for a period until weight becomes constant. 100 mg of dried root and shoot were ground and digested separately with an acid mixture, nitric acid and perchloric acid (5:1) at 70 °C for 48 h. Then mineral contents were measured by using an atomic absorption spectrophotometer (Hitachi Z-5000; Hitachi, Japan). 2.5. Determination of chlorophyll content For determining chlorophyll (chl) content, a leaf sample (0.5 g) was homogenized with 10 ml of 80% acetone using a mortar and pestle. After centrifugation at 9000 × g for 10 min, absorbance of supernatant was measured with a UV-spectrophotometer at 663 and 645 nm for chl a and chl b contents, respectively as described by Arnon (1949). 2.6. Determination of proline content Proline (Pro) content was quantified according to a widely used method by Bates et al. (1973). 2.7. Determination of osmotic potential

2. Materials and methods 2.1. Plant materials and treatments Lentil (L. culinaris Medik cv. BARI Lentil-7) seeds were collected from Bangladesh Agricultural Research Institute (BARI), Bangladesh. Lentil seeds were placed on 6-layered moistened filter paper in Petri dishes. Then the Petri dishes were transferred to a germination chamber for 72 h. Germinated seedlings were then transferred and grown in a growth chamber under an irradiance of 350 μmol (photon) m−2 s−1, a temperature of 25 ± 2 °C, and a relative humidity of 65–70%. One Petri dish contained 35 seedlings. Hyponex (Tokyo, Japan) solution was diluted 5000-fold and applied as the nutrient solution. To create iso-osmotic stress according to Sosa et al. (2005), 100 mM NaCl, 100 mM KCl and 12% (w/v) PEG 6000 were prepared with the nutrient solution. To observe the effect of severe osmotic stress, 20% (w/v) was used. To determine the response of lentil seedling upon exposure to

To determine osmotic potential, leaves were homogenized using ice cold mortar and pestle followed by centrifugation at 12,000 × g for 10 min. Plant extract was then used to determine osmolarity (c) using a K-7400 semi-micro osmometer according to Rahman et al. (2016b). Osmotic potential was calculated by converting osmolarity to osmotic potential using the following formula.   −1 ψП ðMPaÞ ¼ −c mOsmol kg  2:58  10−3 2.8. Determination of electrolyte leakage Electrolyte leakage was measured according to Dionisio-Sese and Tobita (1998). Shoot sample (0.2 g) was cut into smaller pieces and then placed in a test tube containing 15 ml distilled deionized water. Covering with caps, test tubes were heated at 40 °C for 10 min. After

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cooling, initial electric conductivity (EC1) was measured using a Eutech CON 700 conductivity meter, Singapore. Again, test tubes were heated at 121 °C for 20 min using an autoclave. After cooling at room temperature, the final electric conductivity (EC2) was recorded. Electrolyte leakage was calculated using the following formula:

Electrolyte leakage ð%Þ ¼ EC1 ∕EC2  100

2.9. Determination of lipid peroxidation and hydrogen peroxide levels Following the method of Heath and Packer (1968), malondialdehyde (MDA, a product of lipid peroxidation) content was quantified by observing the difference in absorbance at 532 nm and 600 nm and calculated using an extinction coefficient of 155 mM−1 cm−1 and expressed as nmol of MDA g−1 DW. Hydrogen peroxide content was determined according to Yu et al. (2003) by observing the absorbance at 410 nm and calculated using an extinction coefficient of 0.28 mM−1 cm−1. 2.10. Determination of reduced ascorbate and total glutathione content To determine AsA and total GSH, a 0.5 g shoot was homogenized in 3 ml ice-cold 5% meta-phosphoric acid containing 1 mM EDTA. After centrifugation at 11,500 × g for 15 min at 4 °C, the supernatant was used in further process. After neutralizing the supernatant with 0.5 M potassium phosphate (K-P) buffer (pH 7.0), reduced AsA was assayed using a spectrophotometer at 265 nm in 100 mM K-P buffer (pH 7.0) with 1.0 U of ascorbate oxidase (AO) (Nahar et al., 2016). To calculate ascorbate, a specific standard curve of AsA was used. To measure, oxidized glutathione or glutathione disulfide (GSSG) and total glutathione, we followed the method of Griffiths (1980) based on enzymatic recycling. Both reduced AsA and total GSH contents were calculated from standard curve. 2.11. Determination of protein Protein concentration was measured according to Bradford (1976) using bovine serum albumin (BSA) as a protein standard. 2.12. Enzyme extraction and assay For enzyme assay, shoots (0.5 g) were homogenized in 50 mM K-P buffer (pH 7.0) containing 100 mM KCl, 1 mM ascorbate, 5 mM βmercaptoethanol, and 10% (w/v) glycerol using a pre-chilled mortar and pestle. The homogenates were centrifuged at 11,500 × g for 15 min and the supernatant was used to determine protein content and enzyme activity. During extraction, all procedures were performed at 0–4 °C. To determine ascorbate peroxidase (APX, EC: 1.11.1.11) activity, enzyme extract was added to reaction buffer containing 50 mM K-P buffer (pH 7.0), 0.5 mM AsA, 0.1 mM H2O2, 0.1 mM EDTA. The decreased absorbance at 290 nm for 1 min was observed and APX activity was calculated using an extinction coefficient of 2.8 mM−1 cm−1 (Nakano and Asada, 1981). According to the method of Hossain et al. (1984), monodehydroascorbate reductase (MDHAR, EC: 1.6.5.4) activity was assayed and calculated using an extinction coefficient of 6.2 mM−1 cm−1. Dehydroascorbate reductase (DHAR, EC: 1.8.5.1) activity was determined according to the method of Nakano and Asada (1981). To determine glutathione reductase (GR, EC: 1.6.4.2) activity, enzyme extract was added to reaction buffer containing 0.1 M K-P buffer (pH 7.8), 1 mM EDTA, 1 mM GSSG, and 0.2 mM NADPH. The decreased absorbance at 340 nm for 1 min was observed and GR activity was

calculated using an extinction coefficient of 6.2 mM−1 cm− 1 (Foyer and Halliwell, 1976). Catalase (CAT, EC: 1.11.1.6) activity was assayed as described by Hasanuzzaman et al. (2011) and calculated using an extinction coefficient of 39.4 mM−1 cm−1. 2.13. Statistical analysis The data were subjected to analysis of variance (ANOVA) and the mean differences were compared by Fisher's least significant difference (LSD) using XLSTAT v.2016 software (Addinsoft, 2016). Differences at P ≤ 0.05 were considered significant. 3. Results 3.1. Phenotypic appearance and plant growth Four days after iso-osmotic treatment, plant responded differently with different osmotica. Among different iso-osmotic solutions, only NaCl induced chlorosis and shoot tip wilting (Fig. 1a). Surprisingly, there was no such chlorosis in the case of iso-osmotic KCl and PEG, even in the case of 20% PEG which creates severe osmotic stress than that of iso-osmotic 12% PEG (Fig. 1a). Determination of total chlorophyll content revealed that NaCl reduced chlorophyll content by 50%. Similarly, chlorophyll reduction was observed in the case of iso-osmotic KCl and PEG but this reduction was not significant compared to control (Table 1). Imposition of NaCl, KCl and PEG reduced plant height significantly compared to control (Table 1). Furthermore, severe growth reduction was observed in 20% PEG treated seedling where shoot growth reduced by 41% from control. Similarly, NaCl inhibited growth by 33% whereas iso-osmotic KCl and PEG inhibited growth by 22 and 21%, respectively (Table 1). However, root length was not affected by any of these treatments (Table 1). Seedling fresh weight (FW) was greatly affected by these osmotica. Seedling FW dropped by 44% compared with control due to iso-osmotic NaCl, though not significant, this reduction was higher in NaCl treated seedlings than that of iso-osmotic KCl and PEG (Table 1). Therefore, these results indicated that the effect of NaCl on growth reduction was more critical than KCl and 12% PEG even though they have similar osmotic potential. 3.2. Effect on water content, osmotic potential and proline Shoot water content declined significantly under osmotic stress created by NaCl, KCl and PEG. Among different osmotica, NaCl resulted in the highest decline of water content which was 11% compared with control (Table 1). In spite of being an iso-osmotic solution, KCl and 12% PEG declined water content by 3 and 4% respectively which were significantly different from iso-osmotic NaCl (Table 1). Shoot osmotic potential increased with addition of different osmotica. Iso-osmotic NaCl, KCl and PEG caused 285, 124, and 22% increase, respectively, in osmotic potential compared to control (Table 1). Proline content increased markedly in a dose dependent manner with 12 and 20% PEG. However, NaCl being iso-osmotic with 12% PEG enhanced proline content by 162 and 27% over control and 20% PEG, respectively (Table 1). 3.3. Effect on electrolyte leakage As shown in Table 1, NaCl and KCl treated seedlings showed 68 and 41% electrolyte leakage, respectively whereas control seedling showed 11% electrolyte leakage. However, there was no remarkable change in electrolyte leakage between control and PEG treated seedlings.

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Fig. 1. Phenotypic appearance of lentil seedlings under different treatments (a) four days after stress treatment; (b) six days after stress treatments. Here, NaCl, KCl and D12 indicate isoosmotic solution of 100 mM NaCl, 100 mM KCl, and 12% PEG, respectively and D20 indicates 20% PEG.

3.4. Effect on oxidative damage Malondialdehyde (an indicator of lipid peroxidation) and H2O2 contents increased due to NaCl, KCl and PEG treatment, indicating oxidative stress in lentil seedlings (Fig. 2a, b). Iso-osmotic NaCl increased MDA content by 218% and H2O2 content by 15% over control (Fig. 2a). Among different osmotica, NaCl produced more MDA whereas higher H2O2 was produced by 20% PEG and NaCl (Fig. 2b). 3.5. Effect on antioxidants Reduced ascorbate (AsA) and total glutathione (GSH) contents significantly altered due to stress treatments. NaCl declined the AsA content by 87% over control (Fig. 3a). However, total GSH content increased by 260% from control under 100 mM NaCl stress (Fig. 3b). There were no marked changes between iso-osmotic KCl and PEG in terms of AsA content. Importantly, being iso-osmotic with KCl and PEG, NaCl-induced alterations in AsA and total GSH contents were notably different from the changes due to other osmotica (Fig. 3a, b). 3.6. Effect on different enzymes Ascorbate peroxidase enzyme activity was considerably inhibited by iso-osmotic KCl, not by iso-osmotic NaCl or PEG. There was a 23% reduction in APX activity due to KCl, however, no significant change was observed among treatment groups compared to control (Fig. 4a). Though MDHAR activity was enhanced by stress treatments, the change in MDHAR was not significant between iso-osmotic treatment and control

seedlings (Fig. 4b). Lentil seedling treated with NaCl, KCl and PEG had higher DHAR activity than control. Dehydroascorbate reductase enzyme activity was increased by 286% as a result of NaCl treatment in relation to control. In addition, KCl and 20% PEG treated seedling showed 75 and 64% higher DHAR activity than control (Fig. 4c). Among different treatments, only NaCl treated seedling had the highest GR activity which was 162% than control. On the other hand, KCl and PEG treatment modified GR activity which was not significant compared to control (Fig. 4d). There was a significant fall of CAT activity due to NaCl. Along with NaCl, KCl also inhibited the CAT activity compared to control. In contrast, both iso-osmotic PEG and 20% PEG induced CAT activity were not significant from control (Fig. 4e).

3.7. Effect on ion balance Treatment with 100 mM NaCl boosted Na+ content both in shoots and roots of lentil seedlings as shown in Table 2. Similarly, K+ increased sharply both in shoots and roots when seedlings were treated with 100 mM KCl. A severe decrease (by 94%) in the amount of K+ in root was observed in NaCl treated seedling, though application of NaCl increased the K+ content in shoot (Table 2). Na+/K+ ratio increased only in the case of NaCl treated seedlings. Ca content in shoot declined by 27 and 24% when lentil seedlings were treated with iso-osmotic NaCl and KCl while iso-osmotic PEG augmented Ca content in shoot (Table 2). Severe reduction (by 80 and 91%) in Mg2+ content was observed in root of iso-osmotic KCl and NaCl treated seedlings compared with control seedlings (Table 2).

Table 1 Effect of iso-osmotic solutions on growth parameter, shoot water content (RWC), proline content, chlorophyll content of lentil seedlings. Means (±SD) were calculated from three replicates for each treatment. Values with different letters are significantly different at P ≤ 0.05 applying Fisher's LSD test. Here, NaCl, KCl and D12 indicate iso-osmotic solution of 100 mM NaCl, 100 mM KCl, and 12% PEG, respectively and D20 indicates 20% PEG. Treatments

Shoot length (cm)

Root length (cm)

Fresh weight (mg seedling−1)

Shoot water content (%)

Osmotic potential (MPa)

Proline (μmol g−1 DW)

Total chlorophyll (mg g−1 DW)

Electrolyte leakage (%)

Control NaCl KCl D12 D20

11 ± 0.5a 7 ± 0.5c 8 ± 0.2b 8 ± 0.4b 6 ± 0.5d

6 ± 0.2a 6 ± 0.8a 6 ± 0.5a 7 ± 0.3a 6 ± 0.9a

136 ± 6a 75 ± 11b 94 ± 3b 92 ± 10b 76 ± 18b

84 ± 1a 75 ± 2c 81 ± 2b 80 ± 1b 81 ± 0.5b

−0.71 ± 0.06d −2.4 ± 0.2a −1.4 ± 0.05b −0.85 ± 0.04d −1.2 ± 0.08c

3.3 ± 0.9d 8.7 ± 0.8a 5.3 ± 0.2c 5.4 ± 0.9c 6.8 ± 0.6b

94 ± 16a 47 ± 2b 68 ± 4ab 77 ± 18a 90 ± 21a

11 ± 3c 68 ± 5a 41 ± 2b 14 ± 0.4c 14 ± 2c

M.S. Hossain et al. / South African Journal of Botany 113 (2017) 346–354

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Fig. 2. Effect of iso-osmotic solutions on (a) lipid peroxidation (MDA content); (b) H2O2 content. Means (±SD) were calculated from three replicates for each treatment. Values with different letters are significantly different at P ≤ 0.05 applying Fisher's LSD test. Here, NaCl, KCl and D12 indicate iso-osmotic solution of 100 mM NaCl, 100 mM KCl, and 12% PEG, respectively and D20 indicates 20% PEG.

3.8. Effect of Ca on chlorophyll, MDA, proline and ion homeostasis To further explore the salt specific effect underlying the greater damage of chl in lentil plants, we observed changes in chl, MDA, Pro, Na+, K+, Ca2+, and Mg2+ after supplementation of CaCl2. Addition of Ca surprisingly prevented chlorophyll damage as shown in Fig. 5 while salinity treated plant without Ca showed a two-fold chl reduction compared with control. Furthermore, salt stressed plant supplemented with Ca accumulated 1.2-fold lower proline (Fig. 5b) and had a 2.5-fold lower MDA content (Fig. 5a) compared with salt stressed plant without Ca, indicating lower oxidative and osmotic stress in Ca treated plant. Application of Ca along with salt reduced Na content in shoot but not in root (Table 3), increased K+ and Mg2+ content by 359 and 55% in root (Table 3), increased Ca content by 100% in shoot but not in root (Table 3) of lentil compared with salt treated lentil plant alone. NaCl treatment increased the ratio whereas Ca application reduced the Na/K ratio in the seedling under salt stress.

4. Discussion In this study, we showed that in spite of being an iso-osmotic solution, NaCl was responsible for more damage in lentil seedling growth and development than that of other iso-osmotic solutions prepared with KCl and PEG, sometimes even prepared with 20% PEG (lower osmotic potential than iso-osmotic 12% PEG), indicating seedling sensitivity to ionic component of salinity rather than osmotic component. By affecting the ROS detoxification system and ion homeostasis, these osmotica resulted in damage at the whole plant level starting from the cell. However, lentil seedling responses varied with the type of osmotica

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i.e. NaCl, KCl and PEG even though iso-osmotic, furthermore, also varied even with different doses of the same osmoticum for example 12% and 20% PEG. The probable reasons of the variation in plant response towards KCl, NaCl and PEG have been discussed below. Iso-osmotic solutions were used in this experiment to create osmotic stress by lowering the osmotic potential of the growing medium. Osmotic stress reduces the nutrient uptake, water content and CO2 assimilation, increases ABA accumulation, suppresses GA synthesis as a result reduction in shoot growth, and limits leaf expansion and plant biomass production (Munns, 2002; Munns and Tester, 2008). In our study, seedlings treated with different osmotica exhibited reduction in shoot length, fresh weight and water content but not in root length (Table 1). Therefore, our results suggested that osmotic stress created by the osmotica limited the growth of lentil seedlings and the reduction in growth was proportional to osmotic stress confirmed when 20% PEG caused greater growth reduction than 12% PEG. One important finding of our result is that only iso-osmotic NaCl but not other osmotica reduced chl content in lentil seedlings after a 4-day stress treatment. Moreover, with the increase in stress duration from 4 to 6 days, a severe chl degradation was also observed phenotypically in iso-osmotic NaCl followed by KCl and 12% PEG treated seedlings (Fig. 1b). Therefore, we suggest that ionic osmotica such as NaCl, KCl has role in chl degradation. NaCl-induced chl reduction has also been found by El-Monem and Sharaf (2008) and Tepe and Aydemir (2015) in lentil. The possible reasons for chl degradation are that NaCl upregulates the chl degrading enzyme chlorophyllase and reduces δaminolevulinic acid (ALA) content, a precursor of chl biosynthesis (Santos, 2004; Akhzari and Aghbash, 2013), while drought stress prevents chlorophyll formation by reducing ALA and inhibiting ALA dehydratase (ALAD) activity (Jain et al., 2013). On the contrary, iso-

1.5 b

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KCl

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Fig. 3. Effect of iso-osmotic solutions on (a) reduced ascorbate (AsA); (b) total GSH content. Means (±SD) were calculated from three replicates for each treatment. Values with different letters are significantly different at P ≤ 0.05 applying Fisher's LSD test. Here, NaCl, KCl and D12 indicate iso-osmotic solution of 100 mM NaCl, 100 mM KCl, and 12% PEG, respectively and D20 indicates 20% PEG.

M.S. Hossain et al. / South African Journal of Botany 113 (2017) 346–354

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Fig. 4. Effect of iso-osmotic solutions on different enzyme activities; (a) APX activity; (b) MDHAR activity; (c) DHAR activity; (d) GR activity; (e) CAT activity. Means (±SD) were calculated from three replicates for each treatment. Values with different letters are significantly different at P ≤ 0.05 applying Fisher's LSD test. Here, NaCl, KCl and D12 indicate iso-osmotic solution of 100 mM NaCl, 100 mM KCl, and 12% PEG, respectively and D20 indicates 20% PEG.

osmotic PEG-induced severe chl reduction was observed in sugarcane and maize by Chazen et al. (1995) and Patade et al. (2011), suggesting greater inhibitory role of PEG over iso-osmotic NaCl on leaf growth due to higher viscosity of PEG. It is worth to note that chl content should be determined based on dry weight basis specially for the plants with small leaflets like lentil when, for example, number of leaflets in 1 g of stressed plant is higher than control plants due to drastic reduction in water content of stress plant, consequently stressed plants may show higher chl content. Accumulation of proline is a common metabolic adjustment to maintain water relation in plant tissue under salinity and drought (Alam et al., 2014; Rahman et al., 2016b). Wyn Jones and Storey

(1978) reported that higher accumulation of Pro occurred in a barley cultivar, Arimar, under iso-osmotic NaCl than PEG caused by either extracellular Na+ and Cl− induced dehydration effect or by ion toxicity. This finding is in line with our result where iso-osmotic NaCl accumulated more Pro than other osmotica (Table 1). NaCl-induced Pro accumulation observed in lentil by El-Monem and Sharaf (2008) and Golezani and Yengabad (2012). However, no significant change in Pro was observed between iso-osmotic PEG (20%) and NaCl (150 mM) by Patade et al. (2011). In addition Ahmad et al. (2007) found higher amount of Pro accumulation in iso-osmotic PEG treated rice callus than NaCl. Reduction in osmotic potential of cell is necessary for maintaining water balance under salinity and drought stresses. To reduce osmotic

Table 2 Effect of iso-osmotic solutions on Na, K, Ca and Mg content, and Na+/K+ ratio in shoot and root of lentil seedlings. Means (±SD) were calculated from three replicates for each treatment. Values with different letters are significantly different at P ≤ 0.05 applying Fisher's LSD test. Here, NaCl, KCl and D12 indicate iso-osmotic solution of 100 mM NaCl, 100 mM KCl, and 12% PEG, respectively and D20 indicates 20% PEG. Treatments

Control NaCl KCl D12 D20

Na content (μmol g−1 DW)

Na+/K+ ratio

K content (μmol g−1 DW)

Ca content (μmol g−1 DW)

Mg content (μmol g−1 DW)

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

4 ± 0.2b 2608 ± 155a 7 ± 0.8b 4 ± 2b 4 ± 1b

58 ± 2b 1711 ± 187a 23 ± 3b 52 ± 7b 42 ± 0.4b

299 ± 4c 339 ± 33b 591 ± 9a 343 ± 16b 315 ± 25bc

443 ± 12b 26 ± 11c 539 ± 38a 451 ± 15b 432 ± 15b

0.01 ± 0.001b 8 ± 0.67a 0.01 ± 0.002b 0.01 ± 0.005b 0.01 ± 0.005b

0.13 ± 0.001b 71 ± 23a 0.04 ± 0.01b 0.12 ± 0.01b 0.10 ± 0.003b

100 ± 6b 72 ± 3c 75 ± 2c 135 ± 10a 139 ± 4a

125 ± 12b 179 ± 13a 80 ± 2d 115 ± 6bc 105 ± 2c

147 ± 19b 126 ± 11c 120 ± 7c 161 ± 3ab 176 ± 9a

107 ± 6a 9 ± 4d 22 ± 11c 116 ± 2a 77 ± 4bc

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b 100

400 a

Chl content (mg g−1 DW)

MDA content (nmol g−1 DW)

a

300 200 b

b

100 0 NaCl

b 50

c

25

NaCl+Ca

16

Proline content (µmol g −1 DW)

75

0 Control

c

a

Control

NaCl

NaCl+Ca

d a

12

b 8 c

4

0 Control

NaCl

NaCl+Ca

NaCl

NaCl+CaCl2

Fig. 5. Effect of exogenous Ca under salt stress on (a) MDA content; (b) chlorophyll content; (c) proline content and (d) phenotypic appearance 4 days after stress treatment. Means (±SD) were calculated from three replicates for each treatment. Values with different letters are significantly different at P ≤ 0.05 applying Fisher's LSD test. Here, NaCl and NaCl + Ca indicate 100 mM NaCl and 100 mM NaCl with 2 mM CaCl2, respectively.

potential, plant accumulates osmolytes, sugars, polylols and sometimes inorganic molecules because inorganic molecules can adjust osmotic potential more efficiently than organic solutes (Slama et al., 2008; Sucre and Suárez, 2011). Therefore, in our experiment, we observed a greater reduction in osmotic potential in NaCl and KCl treated seedlings than in PEG treated seedlings. Reduction in osmotic potential under isoosmotic NaCl and PEG was also found in rice by Ozfidan-Konakci et al. (2015). Electrolyte leakage in plant indicates the magnitude of tissue injury resulting from stress. It is mediated by K+ efflux and the counter ions − 3− 2− (Cl−, HPO2− 4 , NO3 , C6H5O7 , C4H4O5 ) that are required for balancing positively charged K+ ions. Along with oxidation of cell membrane, activation of K-permeable channels is the main reason behind this phenomenon (Demidchik et al., 2014). In our study, higher electrolyte leakage was observed in NaCl treated plant (Table 1) because NaCl caused higher oxidative damage in plants. Our result is similar with Bandeoğlu et al. (2004) where they found 200 mM NaCl treatment increased electrolyte leakage in lentil seedlings. Both salinity and drought cause overproduction of ROS (Mittler, 2002). This ROS causes oxidative damage by protein oxidation, lipid oxidation, and DNA damage. To understand the response of different osmotica, we further checked the level of oxidative damage in the cell. We found severe oxidative damage indicated by higher level of MDA produced due to iso-osmotic NaCl and KCl but no oxidative stress occurred in the case of iso-osmotic PEG (Fig. 2a). However, Patade et al. (2011) found no oxidative stress in sugarcane under iso-osmotic PEG

and NaCl. In another study, higher MDA content was found in shoots of Haloxylon aphyllum due to PEG rather than iso-osmotic NaCl (Rakhmankulova et al., 2014). One possible reason of this contrasting finding with our result is that the plant used in their experiment was a halophyte which requires NaCl for growth and development while lentil is a glycophyte that does not require salt for growth. Under normal growth, production of ROS is unavoidable and sometimes ROS plays a role in different physiological processes by signaling. However, excessive ROS is harmful (Mittler, 2017). To check this ROS, plants possess an antioxidant defense system which consists of antioxidant metabolites and antioxidant enzymes, which can detoxify overproduced ROS. In this study, the alterations in AsA and total GSH, and CAT, APX, DHAR, MDHAR and GR activities indicated the imbalance in ROS homeostasis. Like our result (Fig. 3a, b), PEG and NaCl-induced ascorbate reduction and glutathione increase were observed by Nahar et al. (2015) in mungbean and Rahman et al. (2016b) in rice. The reduction of AsA is due to scavenging of H2O2 which overproduced under stress condition; as a result AsA converted to DHA whereas GSH, plays a role in AsA regeneration, scavenging ROS, content is proportional to the level of oxidative stress because GSH tried to maintain redox balance in the cell. In our experiment, iso-osmotic NaCl-induced ROS imbalance is more severe than iso-osmotic KCl and PEG, which indicates the additional role of iso-osmotic NaCl in redox imbalance in cell. Catalase enzyme converts excess H2O2 into water. Thus higher activity of this enzyme helps to reduce excess level of H2O2. In this study, CAT activity increased due to PEG treatment but decreased due to KCl and

Table 3 Effect of exogenous Ca on Na, K, Ca and Mg content, and Na+/K+ ratio in shoot and root of lentil seedlings. Means (±SD) were calculated from three replicates for each treatment. Values with different letters are significantly different at P ≤ 0.05 applying Fisher's LSD test. Here, NaCl and NaCl + Ca indicate 100 mM NaCl and 100 mM NaCl with 2 mM CaCl2, respectively. Treatments

Control NaCl NaCl + Ca

Na content (μmol g−1 DW)

K content (μmol g−1 DW)

Na+/K+ ratio

Ca content (μmol g−1 DW)

Mg content (μmol g−1 DW)

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

4 ± 0.2c 2716 ± 65a 2405 ± 84b

49 ± 6c 2025 ± 266b 2499 ± 85a

299 ± 5c 336 ± 4b 391 ± 3a

442 ± 12a 32 ± 5c 147 ± 18b

0.01 ± 0.001c 8 ± 0.21a 6 ± 0.20b

0.11 ± 0.1c 63 ± 5a 17 ± 4b

100 ± 6b 69 ± 3c 140 ± 9a

125 ± 12b 172 ± 16a 129 ± 2b

147 ± 20a 119 ± 2b 138 ± 2ab

107 ± 6a 18 ± 6b 28 ± 2b

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NaCl, indicating excessive K+ and Na+ might inhibit CAT activity (Fig. 4e). NaCl-induced reduction in CAT activity was observed in lentil by Bandeoğlu et al. (2004). The opposite result was found by Patade et al. (2011) where iso-osmotic PEG decreased CAT activity while NaCl increased CAT activity. However, both NaCl and PEG can downregulate the CAT activity. Ascorbate peroxidase also converts H2O2 into water and DHA using AsA. APX activity declined due to KCl treatment whereas APX activity increased (not significantly) due to iso-osmotic PEG and NaCl (Fig. 4a). This result indicates an ion specific role of osmotica on APX enzyme. Our result is in line with Patade et al. (2011) where they found increased activity of APX due to iso-osmotic PEG and NaCl. Glutathione reductase and DHAR are involved in GSH and AsA generation. Higher GR and DHAR activity was observed in NaCl treated seedling indicating the need of AsA and GSH to reduce oxidative damages (Fig. 4c, d). Rahman et al. (2016b) reported that 200 mM NaCl treatment upregulated the DHAR and GR activity in rice seedling which corroborated with our result. Both drought and salinity stress disrupt the ion homeostasis in plants, as a result plants do not get proper nutrition for growth and development. That is why we checked the role of different osmotica on ion homeostasis in lentil seedlings. As shown in Table 2, NaCl treatment increased Na+ content in both root and shoot, decreased K+ and Mg2+ content in root, and reduced Ca2+ content in shoot of lentil seedlings. These results indicate Na+ toxicity in seedlings. Higher amount of Na+ in growth medium depolarizes the plasma membrane, displaces Ca2+ from plasma membranes, activates guard cell outward rectifying potassium channels (GORK) to uptake Na+, induces K+ efflux and disrupts Na+/K+ ratio (Lahaye and Epstein, 1969; Blumwald et al., 2000; Demidchik and Tester, 2002). Moreover, overproduced ROS induces K+ leakage by activating permeable non-selective cationic channels (NSCC) (Anschutz et al., 2014). Therefore, in our experiment severe K+ reduction was observed in iso-osmotic NaCl mainly due to NaCl and overproduced ROS. Potassium reduction in lentil root was also reported by Mamo et al. (1996) and El-Monem and Sharaf (2008). Anschutz et al. (2014) suggested that reduction of K+ in cytosol activates endonucleases and proteases which in turn cause cell death. At the same time, NaCl-induced Ca2+ decline leads to loosening of cell wall and disruption of Ca2+ ion homeostasis in plants, affecting Ca2+ dependent signaling pathways (Rengel, 1992). Based on results, it is hypothesized that the reason of more damages in iso-osmotic NaCl treated lentil seedling than isoosmotic PEG was mainly due to disruption of ion homeostasis which was not severe in PEG treated lentil seedlings. To prove the hypothesis that severe reduction in K+ was the major reason for NaCl-induced damages in lentil seedlings, we applied 2 mM external CaCl2 as it has a role in ion homeostasis and antioxidant defense system (Rahman et al., 2016a). Application of CaCl2 improved K+, Ca2 + and Mg2 + content while salt stress reduced their content (Table 3). Furthermore, no chlorosis was observed (Fig. 5b, d) and NaCl-induced osmotic and oxidative stress was alleviated due to addition of Ca2 + (Fig. 5a, c). Our results are in agreement with Rahman et al. (2016a) who reported that exogenous CaCl2 (2 mM) enhanced salt stress tolerance in rice by ameliorating antioxidant defense and ion homeostasis. To maintain ion homeostasis under NaCl stress, exogenous Ca reduces the transport of Na+ from root to shoot by blocking fast vacuole channel, increases K+ content by blocking NSCC and GORK channel, thus additional Ca2 + in growth medium under salt stress condition delays the damages caused by NaCl (Shabala et al., 2006; Nedjimi and Daoud, 2009; Wu and Wang, 2012; Shabala, 2013). So it is clear from our result that prevention of K leakage from root might enhance salt tolerance. 5. Conclusion Iso-osmotic solutions such as NaCl, KCl and PEG lead to inhibition of lentil growth and development by affecting osmotic adjustment,

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antioxidant defense and nutrient uptake. The results suggest that isoosmotic NaCl results in dramatic reduction in chl content and K+ content, severe oxidative damage, severe osmotic stress and disruption in ion homeostasis while iso-osmotic PEG or KCl-induced damages were not so critical. Therefore, to develop saline tolerant lentil variety, prevention of NaCl-induced K+ leakage should be focused. Author contributions MSH conceived, designed, and performed the experiment and prepared the manuscript. MUA, AR, JAM and KN actively participated in executing the experiment. MH designed the experiment, analyzed the data and edited the manuscript. MF conceived, designed, and monitored the experiment. All authors read and approved the final manuscript. Acknowledgment This research was funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We thank Mr. Peter Lutes, Mr. M. H. M. Borhannuddin Bhuyan, Ms. Taufika Islam Anee and Ms. Tasnim Farha Bhuiyan, Faculty of Agriculture, Kagawa University, Japan for a critical review and editing the English of the manuscript. We also thank Dr. Md. Motiar Rohman, Bangladesh Agricultural Research Institute, Gazipur, Bangladesh for providing lentil seeds. References Afzal, F., Khan, T., Khan, A., Khan, S., Raza, H., Ihsan, Ahanger, M.A., Kazi, A.G., 2014. Nutrient deficiencies under stress in legumes. In: Azooz, M.M., Ahmad, P. (Eds.), Legumes Under Environmental Stress: Yield, Improvement and Adaptations. Wiley, pp. 53–65. Ahmad, M.S.A., Javed, F., Ashraf, M., 2007. Iso-osmotic effect of NaCl and PEG on growth, cations and free proline accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes. Plant Growth Regulation 53, 53. Alam, M.M., Nahar, K., Hasanuzzaman, M., Fujita, M., 2014. Exogenous jasmonic acid modulates the physiology, antioxidant defense and glyoxalase systems in imparting drought stress tolerance in different Brassica species. Plant Biotechnology Reports 8, 279–293. Allakhverdiev, S.I., Nishiyama, Y., Miyairi, S., Yamamoto, H., Inagaki, N., Kanesaki, Y., Murata, N., 2002. Salt stress inhibits the repair of photo damaged photosystem II by suppressing the transcription and translation of psbA genes in Synechocystis. Plant Physiology 130, 1443–1453. Anschutz, U., Becker, D., Shabala, S., 2014. Going beyond nutrition: regulation of potassium homeostasis as a common denominator of plant adaptive responses to environment. Journal of Plant Physiology 171, 670–687. Arnon, D.T., 1949. Copper enzymes in isolated chloroplasts polyphenol oxidase in Beta vulgaris. Plant Physiology 24, 1–15. Ashraf, M., Waheed, A., 1990. Screening of local/exotic accessions of lentil (Lens culinaris Medic.) for salt tolerance at two growth stages. Plant and Soil 128, 167–176. Ayoub, A.T., 1977. Salt tolerance of lentil (Lens esculenta). Journal of Horticultural Science 52, 163–168. Bandeoğlu, E., Eyidoğan, F., Yücel, M., Öktem, H.A., 2004. Antioxidant responses of shoots and roots of lentil to NaCl-salinity stress. Plant Growth Regulation 42, 69–77. Bates, L.S., Waldren, R.P., Teari, D., 1973. Rapid determination of free proline for water stress studies. Plant and Soil 39, 205–207. Blumwald, E., Aharon, G.S., Apse, M.P., 2000. Sodium transport in plant cells. Biochimica et Biophysica Acta (BBA) - Biomembranes 1465, 140–151. Bose, J., Rodrigo-moreno, A., Shabala, S., 2014. ROS homeostasis in halophytes in the context of salinity stress tolerance. Journal of Experimental Botany 65, 1241–1257. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Carillo, P., Annunziata, M.G., Pontecorvo, G., Fuggi, A., Woodrow, P., 2011. Salinity stress and salt tolerance. In: Shanker, A. (Ed.), Abiotic Stress in Plants — Mechanisms and Adaptations. InTech, pp. 21–38. Castillo, E.G., Tuong, T.P., Ismail, A.M., Inubushi, K., 2007. Response to salinity in rice: comparative effects of osmotic and ionic stresses. Plant Production Science 10, 159–170. Chazen, O., Hartung, W., Neumann, P.M., 1995. The different effects of PEG 6000 and NaCl on leaf development are associated with differential inhibition of root water transport. Plant, Cell & Environment 18, 727–735. Demidchik, V., Tester, M., 2002. Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots. Plant Physiology 128, 379–387. Demidchik, V., Straltsova, D., Medvedev, S.S., Pozhvanov, G.A., Sokolik, A., Yurin, V., 2014. Stress-induced electrolyte leakage: the role of K+-permeable channels and involvement in programmed cell death and metabolic adjustment. Journal of Experimental Botany 65, 1259–1270. Dionisio-Sese, M.L., Tobita, S., 1998. Antioxidant responses of rice seedlings to salinity stress. Plant Science 135, 1–9.

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