Journal of Applied Botany and Food Quality 92, 258 - 266 (2019), DOI:10.5073/JABFQ.2019.092.036 1Botany Department, Faculty of Science, Suez Canal University, Ismailia, Egypt 2Biology Department, College of Science, Jouf University, Saudi Arabia 3Biology Department, Faculty of Science, Taibah University, Al‑Sharm, Yanbu, Saudi Arabia 4Botany Department, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt 5Botany and Microbiology Department, Faculty of Science, Cairo University, Giza, Egypt 6Department of Agriculture, University of Swabi, Khyber Pakhtunkhwa, Pakistan Calcium availability regulates antioxidant system, physio-biochemical activities and alleviates salinity stress mediated oxidative damage in soybean seedlings Amr A. Elkelish1, Taghreed S. Alnusaire2, Mona H. Soliman3*, Salah Gowayed4, Hoda H. Senousy5, Shah Fahad6 (Submitted: April 17, 2019; Accepted: August 16, 2019) * Corresponding author Summary Salinity is considered as one of the devastating abiotic stress fac‑ tors and global climate change has further worsened the situation. Present experiments were aimed to evaluate the role of calcium (Ca) availability on growth and salinity tolerance mechanisms in soybean. Seedlings were grown with (2 mM Ca) and without Ca supplemen‑ tation and modulation in key physiological and biochemical para‑ meters were studied. Salinity (100 mM NaCl) stress resulted in growth reduction in terms of height and biomass accumulation, which was more pronounced in Ca-deficient plants. Relative to control (Ca defi‑ cient) and NaCl stressed plants, Ca supplemented seedlings exhibited higher relative water content, pigment synthesis and the photosyn‑ thetic efficiency. Ca availability affected the synthesis of proline, glycine betaine and soluble sugars under normal and saline growth conditions. Optimal Ca supplementation up‑regulated the activities of antioxidant enzymes assayed and the contents of non‑enzymatic antioxidants (ascorbate, glutathione, and tocopherol) thereby reflect‑ ing in amelioration of NaCl induced oxidative damage. Moreover, in‑ creased accumulation of phenols due to Ca supplementation and the amelioration of NaCl mediated decline if nitrate reductase activity was observed. More importantly, Ca availability reduced the accu‑ mulation of Na under control and NaCl stressed conditions restrict‑ ing the damging effects on metabolism. Availability of optimal Ca potentially regulates the salinity tolerance mechanisms in soybean by maintaining osmoregulation and antioxidant metabolism. Keywords: Calcium, Antioxidant system, Osmolytes, Lipid peroxi‑ dation, Glycine max L. Introduction Increasing salinity in soils has become one of the serious threats to distribution, survival, and productivity of major crop plants (AshrAf and hArris, 2004). Increased salinity hampers key growth and meta‑ bolic processes including photosynthesis, ion transport their assimi‑ lation and distribution, enzyme activity and osmolyte metabolism (iqbAl et al. 2015). Salinity interferes with carbon and nitrogen me‑ tabolism in plants leading to alterations in the physiological processes responsible for growth and developmental regulation (AhAnger and AgArwAl, 2017; elkelish et al., 2019). Presence of high concen‑ tration of toxic ions like sodium in the soil hampers the uptake of important nutrients including nitrogen, sulfur, calcium, and potas‑ sium (redA et al., 2011). Salinity stress imposes hyper‑osmotic and hyper‑ionic stress in plants leading to membrane disorganization and restriction of functioning in chloroplast and mitochondria. Higher salinity declines the soil osmotic potential thereby prevent the uptake of water into the plant ultimately due to occurance of physiological drought often reflected as reduced growth and development (Ozden et al., 2009). It has been reported that salinity stress increases the generation of toxic reactive oxygen species (ROS) and plants have an antioxidant system comprised of enzymatic and non‑enzymatic components to neutralize ROS and safeguard the cellular function‑ ing (nOctOr and fOyer, 2016). Increased generation of ROS due to stress affects the macromole- cules like proteins, nucleic acids etc. (AhAnger and AgArwAl, 2017) rendering plant cells less efficient. It has been reported that salini- ty generated ROS reduce the rate of photosynthesis, metabolism of nitrogen and carbohydrates often reflected in reduced growth rate and yield (AhAnger and AgArwAl, 2017; sOlimAn et al., 2018). More importantly, salinity influences the expression of proteins regu‑ lating the structural and functional integrity of organelles like chlo‑ roplast and mitochondria (OmidbAkhshfArd et al., 2012). Therefore, for maximally averting the negative effects of salinity stress key mechanisms are initiated to reduce the impact on the growth and productivity of the plant. Salt exclusion through channel proteins, up‑ regulation of ROS scavenging system and accumulation of osmolytes are included among the basic mechanisms leading to enhanced pro‑ tection to structure and function of cellular organelles by regulating ROS and stress signaling (suO et al., 2017). Therefore, strengthening the tolerance mechanisms and understanding their implications on the overall growth performance is requisite for achieving sustainable productivity. Like other beneficial nutrients, interaction between available cal‑ cium (Ca) with the salinity plays an important role in initiating and strengthening of the tolerance mechanism. Salinity reduces uptake of Ca inducing oxidative damage through greater accumulation of ROS. Ca deficiency limits root growth, induces leaf necrosis and curling, affects cellular signaling and can result in blossom end rot, bitter pit and fruit cracking, affect cellular signaling and sensing of other mineral elements like potassium (wAng et al., 2018). Once Na+ is taken up in excess, leakage of K+ and Ca2+ follows resulting in im‑ balance of the cellular ion homeostasis ultimately causing oxidative stress, growth retardation and programmed cell death (cAbOt et al., 2014). The role of optimal Ca availability in growth regulation has not been worked so far. We hypothesized that availability of opti‑ mal Ca can ameliorate the deleterious effects of salinity stress by strengthening the key tolerance mechanisms. Therefore, experiments were conducted using one of the important legume crop, soybean, to investigate the role of Ca availability in salinity stress mitigation by evaluating its involvement in the (a) regulation of the antioxidant system and osmolytes, (b) secondary metabolite accumulation and the (c) mineral nutrition. Calcium availability alleviates salinity stress‑mediated oxidative damage in soybean seedlings 259 Materials and methods Experimental design and treatment A greenhouse experiment was conducted at the Department of Botany of the Faculty of Science, Suez Canal University, Ismailia, Egypt, during November and December 2018. Soybean (Glycine max L.) seeds were sterilized with 3% sodium hypochlorite and sown in earthen pots filled with an equal quantity of reconstituted soil con‑ taining peat, compost, and sand in the ratio of 3:1:1. Concentration of available N, P, K and Ca in soil substrate was 53.21, 20.03, 56.28 and 17.33 mg kg‑1 soil respectively. Pots were wetted by applying 200 mL of full‑strength nutrient solution. Soon after the germination num‑ ber of seedlings per pot was maintained five. Pots were grouped into two categories where one group was irrigated with nutrient solution (100 mL per pot) with Ca (in the form of 2 mM Ca (NO3)2) and an‑ other group without Ca. 100 mM NaCl was supplemented in the form of modified Hoagland solution to induce salinity stress. The compo‑ sition of nutrient solution used was 3 mM KNO3, 2 mM Ca(NO3)2, 2 mM MgSO4, 1 mM NH4H3PO4, 50 μM KCl, 25 μM H3BO4, 2 μM MnCl2, 20 μM ZnSO4, 0.5 μM CuSO4, 0.5 μM (NH4)6Mo7O24, and 20 μM Na2Fe‑EDTA (Per et al., 2016). Therefore, we have following set of treatments: (a) Control (Hoagland solution without Ca). (b) Ca (full strength Hoagland solution). (c) Salt stressed (Hoagland solution without Ca + 100 NaCl). (d) Salt stress + Ca (full strength Hoagland solution + 100 NaCl). Plants were irrigated on every alternate day. Thirty days old plants were harvested and analyzed for photosynthetic attributes, osmo‑ regulatory components, oxidative stress markers, and antioxidants. During the entire experimental time, period pots were kept in a greenhouse and arranged in a complete randomized block design with five replicates for each treatment. Measurement of photosynthetic pigments, stomatal conduc- tance, and photosynthetic efficiency For extraction of chlorophyll and carotenoids, fresh leaf tissue was homogenized in pestle and mortar using 80% acetone (ArnOn, 1949). The homogenate was centrifuged, and an optical density of supernatant was taken at 480, 645 and 663 nm against 80% acetone. For measurement of photosynthetic efficiency and stomatal conduc‑ tance, fully expanded leaf was analyzed using the infrared gas ana‑ lyzer (CID‑340, Photosynthesis System, Bio‑Science, USA). Determination of leaf water content, soluble sugars, proline, and glycine betaine content Relative water content (RWC) of leaves was determined by punching an equal number of leaf discs from each treatment with a sharp cork borer, and their fresh weight (FW) was taken. Thereafter the same leaf discs were floated in petri dishes containing distilled water for 1 hr for measuring the turgid weight (TW). The same discs were oven dried at 80 oC for 24 hrs for the dry weight (DW) measurement (smArt and binghAm, 1974). RWC was calculated by the following formula: A method described by bAtes et al. (1973) was used to estimate the proline content. Dry (500 mg) leaf sample was homogenized in 3% sulphosalicylic acid and subjected to centrifugation for 20 minutes at 3000 g. The supernatant (2 mL) was mixed with 2 mL glacial acetic acid and 2 mL ninhydrin reagent at 100 oC in a water bath for 1 hour. After that samples were cooled on an ice bath and proline was sepa‑ rated using toluene. Absorbance was read spectrophotometrically at 520 nm. Calculation was done from standard curve of proline. For measurement of soluble sugar, content oven dried samples were macerated in 80% ethanol and after centrifugation supernatant was reacted with anthrone reagent following method of SHIELDS and burnett (1960). Calculation was done from standard curve of glu‑ cose. For the determination of glycine betaine (GB) method of grieve and grAttAn (1983) was followed. Dried plant samples were extracted in distilled water and filtered. The filtrate was diluted by H2SO4 (2 N) and cold KI‑I2 reagent was added to an appropriate volume of the aliquot. Samples were centrifuged at 10,000 g for 15 minutes. The supernatant was aspirated, and per‑iodide crystals were dissolved in 1,2‑dichloroethane and after two hours absorbance was taken at 365 nm. The calculation was done using a standard of glycine betaine. Lipid peroxidation and hydrogen peroxide estimation For the measurement of lipid peroxidation, the formation of malon‑ aldehyde (MDA) content was estimated. Fresh leaves were homo‑ genized in 1% trichloroacetic acid (TCA) and the homogenate was centrifuged at 10,000 g for 5 min. Thereafter, 1.0 mL supernatant was mixed with 4 mL 0.5% thiobarbituric acid and mixture was boiled for thirty minutes at 95 oC. Tubes were cooled on an ice bath and centrifuged again for 5 min at 5000 g for clarification. The opti‑ cal density of supernatant was measured at 532 and 600 nm (heAth and PAcker, 1968). Hydrogen peroxide was estimated by macerating 0.5 gm fresh leaf tissue in 5 mL of 0.1% TCA and the homogenate was centrifuged for 15 minutes at 12,000 g. Supernatant (0.5 mL) was mixed with 0.5 mL potassium phosphate buffer (pH 7.0) and potassium iodide (1 mL). Tubes were thoroughly shaken by vortexing and the absorbance was taken at 390 nm (velikOvA et al., 2000). Determination of nitrate reductase Fresh 500 mg tissue was extracted in ice‑cold mortar using 100 mM potassium phosphate buffer (pH 7.5) containing 5 mM cysteine, 1 mM EDTA and 0.5% PVP. The homogenate was centrifuged at 15,000 g for 20 min and the supernatant was used for the deter‑ mination of NR activity. The reaction mixture consisted 100 mM potassium phosphate buffer (pH 7.5), 7 mM KNO3, 10 mM MgCl2, 0.14 mM NADH and the enzyme extract. The reaction was initi‑ ated by addition of NADH and after incubation for 30 minutes at 27 oC, the reaction was terminated by addition of 100 μL zinc ace- tate (0.5M) followed by centrifugation for 10 minutes at 3000 g and subsequent addition of 1% sulfanilamide and 0.01% naphthalene‑ di‑amine‑dihydrochloride (NED). After 20 minutes the absorbance was read at 540 nm (debOubA et al., 2006). Assay of antioxidant enzymes Antioxidant enzymes were extracted by homogenizing 5.0 gm of fresh leaf tissue in pre‑chilled pestle and mortar in phosphate buffer (50 mM, pH 7.0) supplemented with 1% polyvinyl pyrrolidone and 1 mM EDTA. The homogenate centrifuged at 15,000 g for 20 min at 4 oC and supernatant was used as an enzyme source. Protein content in the supernatant was determined by lOwry et al. (1951) method. The assay of superoxide dismutase (SOD, EC 1.15.1.1) was carried by measuring the ability of enzyme extract to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT) at 560 nm (beyer and fridOvich, 1987). The reaction mixture contained 50 mM sodi‑ um phosphate buffer (pH 7.5), 0.1 μM EDTA, L-methionine (13 mM), 75 μM of each NBT and riboflavin, and 100 μL enzyme extract in a final volume of 1.5 mL. Samples were shaken and illuminated for 15 min and the reaction was terminated by switching off the light. The absorbance of the enzyme containing illuminated samples was recorded against their respective non‑illuminated blank at 560 nm. FW ‑ DW RWC - × 100 TW ‑ DW 260 A.A. Elkelish, T.S. Alnusaire, M.H. Soliman, S. Gowayed, H.H. Senousy, S. Fahd The amount of enzyme causing 50% inhibition in NBT photoreduc‑ tion was considered as one unit of SOD and activity was expressed as EU mg‑1 protein. For measuring the activity of ascorbate peroxidase (APX, EC 1.11.1.11) decrease in absorbance was monitored at 290 nm for 3 min in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), ascorbic acid (0.5 mM), hydrogen peroxide (0.1 mM) and 100 μL of enzyme extract in final volume of 1 mL (nAkAnO and AsAdA, 1981). The extinction coefficient of 2.8 mM‑1 cm‑1 was used for calculation. For the assay of glutathione reductase (GR; EC 1.6.4.2) method of fOyer and hAlliwell (1976) was adopted. Briefly, assay mix‑ ture (1.0 mL) contained sodium phosphate buffer (50 mM, pH 7.8), 0.12 mM nicotinamide adenine dinucleotide phosphate (NADPH), 0.5 mM oxidized glutathione (GSSG) and 100 μL enzyme extract. Change in absorbance at 340 nm was followed for 2 min. Activity was expressed as l mol NADPH oxidized min‑1 (units mg‑1 protein) and extinction coefficient for of 6.2 mM‑1 cm‑1 was used for calcula‑ tion. Determination of ascorbate, tocopherol and reduced glutathione For the determination of ascorbate (AsA) content 400 mg fresh plant material of each treatment was extracted in 5 mL of 6% TCA fol‑ lowed by centrifugation at 5000 g for 10 min. To 1 mL supernatant was added 2% dinitrophenylhydrazine (prepared in 9 M H2SO4) and 10% thiourea. The mixture was boiled in a water bath for 15 min and cooled followed by the addition of 5 mL of cooled 80% H2SO4. Thereafter the optical density was measured at 530 nm (mukherjee and chOudhuri, 1983) and calculation was done from a standard curve of ascorbate. The method described by (bAker et al., 1980) was followed for the estimation of tocopherol. 100 mg fresh leaf material was extracted with 5 mL of ethanol and petroleum ether (1.6:2) and an extract was centrifuged at 12,000 g for 10 minutes. 1.0 mL supernatant was mixed with 0.2 mL of 2, 2‑dipyridyl (2%, prepared in ethanol) and left for 5 min in dark for color (red) development. Thereafter 4 mL of distilled water was added and the absorbance was measured at 520 nm. Reduced glutathione (GSH) was estimated by extracting fresh 100 mg leaf tissue in phosphate buffer (pH 8.0) and an extract was centri‑ fuged for 15 minutes at 3000 g. Thereafter 500 μL of supernatant was reacted with 5, 5‑dithiobis‑2‑nitrobenzoic acid and allowed to stand for 10 minutes. Optical density was measured at 412 nm (ellmAn, 1959) and the concentration of GSH was determined from a standard graph of GSH. Estimation of total phenols Dry (500 mg) plant samples were extracted in 80% ethanol and an extract was centrifuged at 10,000 g for 10 minutes. The supernatant was mixed with Folin and Ciocalteau’s phenol reagent in alkaline medium and optical density of the mixture was recorded at 750 nm (slinkArd and singletOn, 1977). The computation was done from the standard curve of catechin. Estimation of ions For estimation of Na and Ca, one gm oven dried samples were acid digested using H2SO4 and HClO4. Digested samples were diluted to 100 mL using distilled water and were read on flame photometer. Statistical analysis Data are mean (±SE) of three replicates and Duncan’s Multiple Range Test was performed using One Way ANOVA for determining the least significant difference (LSD) at p <0.05. Results Ca supplementation protects plant growth and biomass produc- tion under salinity stress Results showing the impact of Ca availability on the height and biomass accumulation under salinity and normal conditions are pre‑ sented in Fig. 1. Relative to control, Ca improved height, fresh and dry weight by 19.30, 19.19 and 19.58% respectively. Salinity (100 mM NaCl) reduced the height (37.67%), fresh (25.79%), and dry (38.81) biomass accumulation over the control plants. Application of Ca sig‑ nificantly reduced the negative effects of NaCl and ameliorated the decline by 17.90, 12.78 and 9.23% over the NaCl stressed counter‑ parts (Fig. 1). Fig. 1: Effect of salinity (100 mM NaCl) on (A) shoot length, (B) fresh and (C) dry weight of soybean (Glycine max L.) with and without cal‑ cium supplementation. Data is mean (±SE) of three replicates and bars with different letters denote significant difference at P ≤ 0.05 Ca supplementation improves photosynthetic efficiency and the pigment synthesis Supplementation of Ca resulted in significant enhancement in the pig- ment synthesis (chlorophylls and carotenoids) and also ameliorated the NaCl induced decline. Relative to control, total chlorophylls, ca‑ rotenoids, photosynthetic rate, and stomatal conductance increased by 19.97, 17.25, 20.15 and 14.44% in Ca treated seedlings. However, NaCl stress reduced chlorophylls (45.18%), carotenoids (30.72%), photosynthetic rate (41.88%) and stomatal conductance (35.98%) over the control plants. The reduction was mitigated by supplemen‑ tation of Ca with an amelioration of 29.64% in total chlorophylls, Calcium availability alleviates salinity stress‑mediated oxidative damage in soybean seedlings 261 14.10% in carotenoids, 22.33% photosynthetic rate and 15.54% in stomatal conductance over the NaCl stressed plants (Fig. 2A‑D). Ca availability improves RWC, and proline, glycine betaine and sugar accumulation under salinity stress In normally grown seedlings, Ca increased the accumulation of pro‑ line (Pro), glycine betaine (GB) and soluble sugar content significant‑ ly over the Ca deficient control plants. An increment of 22.63% in Pro,14.28% in GB and 26.53% sugar content was observed over the control plants. Percent increase in Pro, GB, and sugars due to NaCl stress was 31.01, 28.96, and 37.43% respectively over the control. The maximal increase in Pro (38.39%), GB (39.49%) and sugar (42.14%) content was observed in NaCl + Ca treated plants over the control counterparts (Fig. 3A‑C). A reduction of 27.85% due to salinity was observed in RWC over the control seedlings, however, Ca was effec‑ tive in improving and also mitigating the negative effects of NaCl on RWC. An increase of 7.16% in RWC was observed due to Ca over control and caused an amelioration of 14.57% in seedlings treated with NaCl + Ca over the NaCl stressed plants (Fig. 3D). Fig. 3: Effect of salinity stress (100 mM NaCl) on content of (A) proline, (B) glycine betaine, (C) soluble sugars and (D) relative water content (RWC) in soybean (Glycine max L.) with and without calcium supplementation. Data is mean (±SE) of three replicates and bars with different letters denote significant difference at P ≤ 0.05 Fig. 2: Effect of salinity stress (100 mM NaCl) on (A) total chlorophylls, (B) carotenoids, (C) photosynthetic rate and (D) stomatal conductance in soybean (Glycine max L.) with and without calcium supplementation. Data is mean (±SE) of three replicates and bars with different letters denote significant difference at P ≤ 0.05 262 A.A. Elkelish, T.S. Alnusaire, M.H. Soliman, S. Gowayed, H.H. Senousy, S. Fahd Antioxidant system was up-regulated due to Ca supplementation Supplementation of Ca resulted in a significant increase in the ac‑ tivities of SOD, APX, and GR, and the contents of AsA, GSH, and tocopherol (Fig. 5 and 6). Relative to control, an increase of 21.37% in SOD, 19.73% in APX and 9.46% in GR were observed due to supplementation of Ca. NaCl stress increased the activities of SOD (36.54%), APX (40.00%) and GR (33.40%) over the control, however application of Ca further enhanced the activities. Relative to con‑ trol, an increase of 44.98, 45.37 and 39.26% in SOD, APX and GR were observed due to an application of Ca to NaCl stressed plants (Fig. 5A‑C). Contents of AsA, GSH, and tocopherol increased by 8.89, 9.91 and 21.97% in Ca treated plants over the Ca deficient con‑ trols (Fig. 6A‑C). The maximal increase in GSH (30.06%) and toco‑ pherol (35.36%) was observed in seedlings treated with NaCl + Ca over the control. Salinity reduced AsA (15.57%) while as increased GSH (26.55%) and tocopherol (14.48%) over the control. Reduction in AsA was ameliorated by application of Ca and an amelioration of 4.67% in AsA content was observed in NaCl + Ca treated seedlings over the NaCl stressed plants (Fig. 6A). Ca reduces oxidative damage by reducing the formation of hy- drogen peroxide and lipid peroxidation It was observed that Ca supplementation reduced the generation of H2O2 and lipid peroxidation significantly over the control as well as NaCl stressed plants (Fig. 4 A and B). Accumulation of H2O2 in‑ creased by 37.98% in NaCl stressed plants causing 25.15% increase in lipid peroxidation over the control plants. Supplementation of op‑ timal Ca reduced the generation of H2O2 by 23.29% over the control leading to a declined lipid peroxidation by 13.38% in them. In com‑ parison to NaCl stressed plants amelioration of 17.56% in H2O2 and 11.59% in lipid peroxidation was observed in seedlings treated with NaCl + Ca (Fig. 4 A and B). Ca improves the synthesis of total phenols and enhances nitrate reductase activity Ca supplemented seedlings exhibited an apparent increase in the ac‑ cumulation of total phenols over the Ca deficient normal and salinity stressed plants. Relative to control, Ca supplemented plants showed an increase of 22.39% in total phenol contents which was reduced by 18.33 % due to NaCl stress (Fig. 7). Relative to control, treatment of 100 mM NaCl reduced the activity of nitrate reductase (NR) by 43.25%. However, an amelioration of 11.96% was observed in NaCl + Ca over the NaCl stressed plants. Under normal growth conditions, NR activity increased (11.89%) significantly due to Ca availability (Fig. 8). Ca availability reduces Na uptake and reduces the Na/K ratio Supplementation of Ca reduced the uptake of Na to upper parts (leaf) significantly (Tab. 1). Relative to Ca deficient control, Ca decreased by 35.13% in leaf and 31.75% in root due to NaCl stress, however, accumulated Na in leaf (68.54%) and root (65.98%) more than the control. Relative to control, Ca supplemented seedlings exhibited an increase in the leaf Ca (24.38%), while a decrease in Na content of leaf (28.53%) and root (13.15%). However, in NaCl stressed plants Ca application reduced the Na accumulation by 42.26% and 32.65% in leaf and root tissues (Tab. 1). Discussion Increased soil salinity is considered a critical threat to optimal crop productivity. Such problems have arisen mainly due to the introduc‑ tion of agricultural malpractices like the use of salt‑rich and polluted Fig. 5: Effect of salinity stress (100 mM NaCl) on activity of (A) superoxide dismutase (B) ascorbate peroxidase and (C) glutathione reductase in soybean (Glycine max L.) with and without calcium supplementa‑ tion. Data is mean (±SE) of three replicates and bars with different letters denote significant difference at P ≤ 0.05 Fig. 4: Effect of salinity stress (100 mM NaCl) on (A) hydrogen peroxide and (B) lipid peroxidation (MDA) in soybean (Glycine max L.) with and without calcium supplementation. Data is mean (±SE) of three replicates and bars with different letters denote significant difference at P ≤ 0.05 Calcium availability alleviates salinity stress‑mediated oxidative damage in soybean seedlings 263 water for irrigations, which has resulted in continuous accumulation of salts to toxic levels making the growth and development of grow‑ ing crops difficult. For ensuring better crop productivity under such growth conditions, efficient and robust management practices need to be introduced. In this context, we investigated the potential of soil Ca availability in mitigating the adverse effects of salinity. The indigenous concentration of Ca in nutrient solution proved ef‑ fective in ameliorating the NaCl induced growth decline. NaCl re‑ duces the cellular growth and division by reducing the activity of cyclin‑dependent kinase and CYCB1;2 promoters resulting in the production of smaller meristems (west, 2004). Salinity stress‑medi‑ ated decline in meristem is due to the reduced cellular water content (mAhmOOdzAdeh et al., 2007). In the present study, Ca application effectively regulated the growth of soybean by improving biomass production and growth under salinity stress. Ca is important for cell cycle progression under stress and is an important secondary mes‑ senger. The role of Ca2+ mediated signaling in cell cycle progres‑ sion has been established (mAchAcA, 2010) availability of optimal Ca concentration in the present study may have prevented cell cycle arrest besides regulating the cell proliferation (kAhl and meAns, 2003). As a signaling molecule Ca ion acts as a convergence point of many signaling pathways thereby helping plant cells to repro‑ gram their cellular set up in response to stress signal (tutejA and mAhAjAn, 2007). Ca helps in stress signal perception by membrane receptors, secondary messenger generation, protein phosphorylation and dephosphorylation targeting transcription factors resulting in regulation of gene expression ultimately imparting tolerance to stress (tutejA and mAhAjAn, 2007). Presence of optimal Ca in solution resulted in significant improve‑ ment in the synthesis of chlorophylls and carotenoids resulting in greater photosynthetic efficiency and stomatal conductance. Earlier Xu et al. (2013) have also demonstrated that the exogenous appli‑ cation of CaCl2 ameliorated the negative effects of drought stress on growth by improving the chlorophyll pigment synthesis and the chlorophyll fluorescence and photosynthetic rate. Ca deficiency in‑ tensified the reduction in photosynthetic pigment and performance Fig. 8: Effect of salinity stress (100 mM NaCl) on content the activity of nitrate reductase in soybean (Glycine max L.) with and without cal‑ cium supplementation. Data is mean (±SE) of three replicates and bars with different letters denote significant difference at P ≤ 0.05 Fig. 6: Effect of salinity stress (100 mM NaCl) on content of (A) ascorbic acid, (B) reduced glutathione and (C) tocopherol in soybean (Glycine max L.) with and without calcium supplementation. Data is mean (±SE) of three replicates and bars with different letters denote significant difference at P ≤ 0.05 Fig. 7: Effect of salinity stress (100 mM NaCl) on content of total phenols in soybean (Glycine max L.) with and without calcium supplementa‑ tion. Data is mean (±SE) of three replicates and bars with different letters denote significant difference at P ≤ 0.05 264 A.A. Elkelish, T.S. Alnusaire, M.H. Soliman, S. Gowayed, H.H. Senousy, S. Fahd under salinity conditions. Reduced photosynthetic efficiency due to salinity stress results from the increased chlorophyll degradation re‑ flected in increased chlorophyllase activity and reduced synthesis of chlorophyll intermediates. Also, reduced photosynthetic efficiency due to salinity stress results from ROS induced photoinhibition pri‑ marily through damage to the structure of the pigment‑protein com‑ plex (AhAnger et al., 2017). Optimal supplementation of Ca has ade‑ quately protected the pigment proteins and also have a visible impact on the allocation of key mineral ions like Mg and N for chlorophyll and Rubisco synthesis. Further research is needed to unravel the actual mechanism. Ca2+ regulates photosynthetic electron transport and light‑dependent me‑ tabolism through interplay with thylakoid acidification and redox homeostasis. Reduced NR activity due to salinity has been reported by others as well in crops like Triticum aestivum (AhAnger and AgArwAl, 2017) and Vigna radiata (shAhi and srivAstAvA, 2018), however, Ca mediated regulation of NR activity under salinity has not been worked. Ca considerably ameliorated the salinity mediated decline in NR activity depicting its beneficial role in the protection of enzyme activity. Ca‑mediated growth and photosynthetic improve‑ ment can be attributed to reduced Na uptake in them. Na is toxic to plants at higher concentrations and optimal Ca supplementation mediated decline in its accumulation directly shows its influence on the transport proteins controlling the uptake and the partitioning of Na within the cells mainly Na+/Ca2+ exchanger (drevAl et al., 2005). Salinity affects the transport and homeostasis of Ca in plants by inhibiting Ca2+‑ATPase (geisler et al., 2000). Also, Ca availabil‑ ity may have changed the expression of other membrane proteins like tonoplast H+‑ATPase and Na+/H+ antiport reducing the accumulation of excess levels of Na+. Increased presence of Ca in cells is recog‑ nized by Ca sensors or Ca2+ binding proteins and leads to activation of protein kinases (tutejA and mAhAjAn, 2007). Increased NaCl concentration in soil solution reduces the tissue growth by affecting the water content significantly and in confirma‑ tion to our results are the findings of (POlAsh et al., 2018). Higher accumulation of sugars, Pro and GB in Ca supplemented seedlings resulted in enhanced RWC and also ameliorating the ill effects of sa‑ linity on cellular functioning. Calcium regulates the growth of plants by affecting mineral transport and accumulation of metabolites leading to the maintenance of water content (mOzAfAri, 2013). Ca‑ mediated enhancement in the accumulation of Pro, sugars, and GB resulted in higher RWC preventing the decline in photosynthesis and the hyper‑osmotic effects of salinity on the protein structure (munns and tester, 2008). benhAssAini et al. (2012) have also demon‑ strated salinity mediated enhancement in Pro in Pistacia atlantica. Pro and GB have a role in protecting the protein turnover machinery and improving the expression of stress proteins for greater tolerance. Greater accumulation of compatible osmolytes like Pro, GB, sug‑ ars improves plant performance under stressful conditions through increased RWC. Increased GB accumulation in Ca supplemented plants may have prevented salt mediated photo‑inhibition by protect‑ ing the thylakoid membrane structure and the ATP synthesis lead‑ ing to greater photosynthetic efficiency in them (wAng et al., 2018). sivAkumAr et al (2000) has demonstrated increased carboxylase activity of Rubisco in plants accumulating higher contents of osmo‑ lytes. Pro and GB prevent the salinity mediated ROS initiated lipid peroxidation, deformation, and degradation of a nucleus, chromatin condensation and PCD. Ca supplementation enhanced the synthesis of Pro, sugars, and GB resulting in enhanced RWC, photosynthetic efficiency, and amelioration of oxidative damage. The osmolytes have been believed to strengthen the ROS scavenging systems and stress signaling mechanisms (AhAnger et al., 2018). Salinity stress increased the generation of ROS like H2O2 causing peroxidation of membranes, however, Ca supplementation reduced the generation of H2O2 under normal conditions and also declined the NaCl induced ROS levels. Recently, AhAnger et al. (2018) have also demonstrated reduced membrane stability due to salinity medi‑ ated accumulation of H2O2. Stresses affect the polyunsaturated fatty acids significantly leading to greater membrane instability. The re‑ duction in H2O2 and mediated by Ca have been previously reported to impart a positive impact on the photosynthetic efficiency of plants (sAkhOnwAsee and PhingkAsAn, 2017). Optimal supplementation of Ca may have reduced the activity of lipoxygenase leading to pre‑ vention of any damage to membrane fatty acids besides its appar‑ ent effect on the efficiency of the antioxidant system. In Vicia faba, siddiqui et al. (2018) have also demonstrated up‑regulation of the antioxidant system due to Ca supplementation under cadmium stress. In the present study, it was observed that Ca up‑regulated antioxi‑ dant defense system by up-regulating SOD, APX and GR concomi‑ tant with an increase in the content of GSH, tocopherols, and AsA. Strengthening the antioxidant system prevents cellular oxidative damage by quick elimination of ROS. Ca-induced enhancement in an antioxidant system precludes any damage to chloroplast and mi‑ tochondrial electron transport chain by maintaining optimal H2O2 levels so that their beneficial functions continue (mittler, 2017). In‑ creased SOD activity in Ca supplemented seedlings prevent hydroxyl (OH‑) radical formation resulting in enhanced cellular functioning. Earlier AhmAd et al. (2015) have demonstrated up‑regulation of the antioxidant system in mustard due to calcium supplementation. In‑ creased APX and GR activity in Ca supplemented plants and main‑ tenance of higher concentrations of non‑enzymatic components lead to protection of cells from the oxidative burst through the support of cellular redox state. Antioxidants including low molecular weight antioxidants like AsA, GSH, and tocopherol are rich redox buffers, which interact with numerous cellular components besides other vi‑ tal roles in defense and as enzyme cofactors influencing the growth and development of plants by modulating processes from mitosis and cell elongation to senescence and death (POtters et al., 2004). Ca supplementation strengthens the evidence that redox homeosta‑ sis involves precise ROS - antioxidant interaction as an interface for signals derived from the metabolism and the environment (fOyer and nOctOr, 2005). The antioxidant system was further strength‑ ened by the accumulation of phenols in Ca supplemented seedlings. Therefore, it is inferred from the present study that Ca availability potentially affects the growth and tolerance mechanisms in soybean and could be exploited for enhancing the yield productivity. Tab. 1: Effect of salinity stress (100 mM NaCl) on the uptake of sodium and calcium (mg g‑1 DW) in soybean (Glycine max L.) with and without calcium supplementation. Data is mean (±SE) of three replicates and data followed by different letters denote significant difference at P ≤ 0.05 Na Ca Leaf Root Leaf Root Control 3.866 ± 0.132c 4.493 ± 0.469c 5.908 ± 0.646b 3.420 ± 0.287b NaCl 12.29 ± 1.119a 13.209 ± 0.339a 3.832 ± 0.139d 2.334 ± 0.141d Ca 2.763 ± 195d 3.902 ± 0.105cd 7.813 ± 0.331a 4.990 ± 0.200a NaCl + Ca 7.096 ± 0.193b 8.895 ± 0.573b 4.986 ± 0.126c 3.196 ± 0.176bc Calcium availability alleviates salinity stress‑mediated oxidative damage in soybean seedlings 265 Conclusion It can be said that application of optimal Ca prevents the structural and functional integrity of soybean to counteract the salinity stress mediated growth restrictions. Optimal Ca availability benefited soy‑ bean under salinity stress by restricting the uptake of sodium and in‑ creasing the synthesis of photosynthetic pigments and photosynthetic functioning. Ca supplementation strengthened the indigenous tole‑ rance mechanisms like the synthesis of compatible solutes and up‑ regulation of antioxidant system for preventing damage to cellular macromolecules. NaCl-mediated ROS-generated oxidative damage effects were averted by Ca availability by improving the redox com‑ ponents. By up‑regulating the antioxidant system and the synthesis of osmolytes Ca availability can be exploited for improving the crop productivity. Further studies for unraveling the molecular mecha‑ nisms and the probable crosstalk of Ca with other key molecules in salinity stress mitigation can be handy. References AhAnger, m.A., AgArwAl, r.m., 2017a: Salinity stress induced alterations in antioxidant metabolism and nitrogen assimilation in wheat (Triticum aestivum L) as influenced by potassium supplementation. Plant Phys. Biochem. 115, 449‑460. 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DOI: 10.1371/journal.pone.0068214 ORCID Amr Elkelish https://orcid.org/0000‑0002‑1683‑8436 Mona Soliman https://orcid.org/0000‑0002‑9102‑4790 Address of the corresponding author: Mona H. Soliman: Biology Department, Faculty of Science, Taibah Univer‑ sity, Al‑Sharm, Yanbu El‑Bahr, 46429, Kingdom of Saudi Arabia E‑mail: monahsh1@gmail.com © The Author(s) 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative‑ commons.org/licenses/by/4.0/deed.en). http://dx.doi.org/10.1007/s11099-016-0205-y http://dx.doi.org/10.22271/tpr.2018.v5.i2.029 http://dx.doi.org/10.1104/pp.103.033548 https://doi.org/10.1016/j.plantsci.2011.02.006 http://dx.doi.org/10.1007/s13580-017-0194-1 http://dx.doi.org/10.1080/01904167.2018.1454470 https://doi.org/10.1021/ac60163a053 http://dx.doi.org/10.1016/j.plaphy.2018.05.027 http://dx.doi.org/10.1006/bbrc.2000.4005 https://doi.org/10.1104/pp.53.2.258 http://dx.doi.org/10.1186/s40529-018-0222-1 http://dx.doi.org/10.3390/ijms18051011 http://dx.doi.org/10.4161/psb.2.2.4176 http://dx.doi.org/10.1016/S0168-9452(99)00197-1 http://dx.doi.org/10.3390/ijms19113456 http://dx.doi.org/10.1104/pp.104.040022 http://dx.doi.org/10.1371/journal.pone.0068214 https://orcid.org/0000-0002-1683-8436 https://orcid.org/0000-0002-9102-4790