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Calcium application mitigates salt stress in Date Palm (Phoenix dactylifera L.( offshoots cultivars of Berhi and Sayer

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Calcium application mitigates salt stress in Date Palm (Phoenix dactylifera L.( offshoots cultivars of Berhi and Sayer
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    1 Department of Horticulture and Landscape Design, College of Agriculture, University of Basrah, Basrah, Iraq, abbasjasim5@yahoo.com 2 Department of Horticulture and Landscape Design, College of Agriculture, University of Basrah, Basrah, Iraq, muayedfadhil@yahoo.co.uk 3 Department of Date Palm Varieties, Date Palm Research Centre, University of Basrah, Basrah, Iraq, husseinshareef@live.com Acta agriculturae Slovenica, 107 - 1, marec 2016 str. 103 - 112 DOI: 10.14720/aas.2016.107.1.11 Agrovoc descriptors: osmotic stress; calcium fertilizers; fertilizer application; varieties; defence mechanisms; proline; tropical crops; phoenix dactylifera Agris category code : F04, f40, f62 Calcium application mitigates salt stress in Date Palm (  Phoenix dactylifera  L.) offshoots cultivars of Berhi and Sayer Abbas M. JASIM 1 ; Muayed F. ABBAS 2 ; Hussein J. SHAREEF 3 Received September  20, 2015; accepted November 16, 2015. Delo je prispelo 20. septembra 2015, sprejeto 16. november 2015. ABSTRACT The effectiveness of exogenous application of calcium in ameliorating the adverse effects of salt stress (15.9 dS m -1 ) on date palm offshoots ( Phoenix dactylifera  L. cultivars of Berhi and Sayer) was investigated. Ca-fertilisers Polixal and Rexene were applied either as soil amendments or foliar spray. The results showed that Polixal at 30 ml offshoot -1  significantly increased plant height, leaf area, total chlorophyll content, RWC, proline concentration, peroxidase activity, IAA content, K  +  and K  + /Na +  ratio in leaves of Berhi cultivar, whereas catalase activity, ABA and Cl - content were decreased. Also Berhi cultivar responded to soil amendments more than to foliar spray. However, Ca-fertilisers mitigated salt stress in the two cultivars and Berhi cultivar was more salt stress tolerant than Sayer cultivar by maintaining the high ratio of K  + /Na +  and regulating levels of IAA to ABA, in silty clay loam soil. These results suggest that calcium application can improve the defense system under salt stress conditions. Key words: antioxidant enzymes, Date Palm, salt stress, IAA, ABA, calcium application, proline, RWC IZVLE Č EK DODAJANJE KALCIJA ZMANJŠUJE SLANOSTNI STRES PRI KOKOSOVI PALMI (  Phoenix dactylifera  'Berhi’ IN ‘Sayer’) Pri dveh sortah kokosove palme ( Phoenix dactylifera  ‘Berhi’ in ‘Sayer’) je bil preu č evan blažilni u č inek dodajanja kalcija na negativne u č inke slanostnega stresa (15.9 dS m -1 ). Ca-gnojili Polixal in Rexene sta bili dodajani ali kot talni dodatek ali kot listno pršilo. Rezultati so pokazali, da je dodatek Polixal-a v koli č ini 30 ml na rastlino zna č ilno pove č al višino rastlin, listno površino, vsebnost celokupnega klorofila, relativno vsebnost vode (RWC), vsebnost prolina, aktivnost  peroksidaze, vsebnost IAA in K  +  ter razmerje K  + /Na +  v listih sorte Berhi, aktivnost katalaze, vsebnost ABA in Cl - so se zmanjšali. Sorta Berhi se je bolje odzvala na dodatek gnojil v tla kot na foliarna gnojila. Calcijeva gnojila so pri obeh sortah zmanjšala slanostni stres na bogatih peš č eno-ilovnatih tleh, vendar se je sorta Berhi izkazala nanj odpornejša kot sorta Sayer saj je ohranjala ve č  je K  + /Na +  razmerje, kar je izboljšalo tudi razmerje med IAA in ABA. Rezultati te raziskave nakazujejo, da gnojenje s Ca izboljša obrambni sistem rastlin v razmerah slanostnega stresa. Klju č ne besede: antioksidacijski encimi, dateljeva palma, slanostni stres, IAA, ABA, gnojenje s Ca,  prolin, RWC   1 INTRODUCTION Inhibition of plant growth by high amounts of Na +  and Cl −  is one of the main deleterious effects of salt stress. When present in excess amount, Na +  and Cl -  ions enter into plant cells and can exert toxic effects on cell membranes and metabolic activities in the cytosolic part of the cell (Hasegawa et al . 2000; Zhu, 2001; Türkan and Demiral, 2009). The resultant effect of osmotic  Abbas M. JASIM et al.   Acta agriculturae Slovenica, 107 - 1, marec 2016 104 stress and ionic toxicity may lead to secondary effects in plants such as decreased cell expansion,  production of assimilate and membrane functions, decreased cytosolic metabolism with raised  production of ROS, including singlet oxygen ( 1 O 2 ), superoxide (O 2- ), hydroxyl radical (OH   ) and hydrogen peroxide (H 2 O 2 ) (Lindberg et al.,  2012). Calcium plays a fundamental role in plant growth and development. Many extracellular signals and environmental cues including light, abiotic and  biotic stress factors, elicit change in the cellular calcium levels, termed as calcium signatures (Wu et al ., 2013). Calcium ions ameliorate the effect of salt stress by competing with sodium ions for membrane-binding sites. Salt stress reduces N, P, K, and Ca content in tissues; however, the addition of Ca restored the levels of these nutrients. In general, as external Ca 2+  concentrations increase,  Na +  uptake and concentrations decrease while Ca 2+  uptake and concentrations increase because Ca 2+  interferes with non-selective cation channels and restricts Na +  uptake. In addition, as the salt concentration in the root zone increases, the requirement for Ca 2+  increases. However, the uptake of Ca 2+  from the soil may be reduced as a result of ion interactions, precipitation, and increased ionic strength. These factors reduce the activity of Ca 2+  in solution, which reduces the availability of Ca 2+  (Grattan and Grieve 1999, Reddy, 2001 and Louchli and Grattan, 2007). Also,  Na/Ca interactions can affect growth,  photosynthesis, plant nutrition, water and ion transport in plants. The nature of the response will vary depending on the plant genotype (Cramer, 2002). Zekri and Parsons (1990) found that the addition of 1, 5, or 7.5, but not 13.5, mM CaSO 4  to the saline solution significantly decreased the adverse effect of NaCl on shoot growth of sour orange   seedlings. In salt stressed wheat ( Triticum aestivum ‘Samma’) Ca 2+  improved plant height and proline content compared with treatment of 90 mM of NaCl alone (Al-Whaibi et al.  2011).   El-Khawaga, (2013) found that the anti-salinity agents as calcium alleviated the adverse effects of salt stress on the growth of Sewy, Zaghloul and Hayany date palm cultivars. Keeping in view all these aspects, a study was planned to test the effectiveness of Ca-fertilisers in alleviating the undesirable effects of salt stress by evaluating morphological,  physiological and biochemical attributes change in date palm offshoots cultivars of Berhi and Sayer. 2 MATERIALS AND METHODS 2.1 Field experiment The experiment was carried out at the General Authority of Palm station, in Hartha region – Basrah, Iraq (30 o 36.54’N& 30 o 38.60’N to 47 o 44.42’E to 47 o  45.18’E), 24 km from center of Basrah, in 2014. 30 uniform, girth   10 cm vigorous 4-5 years-old ‘Berhi’ and ‘Sayer’ date  palm offshoots were used in the experiment. The selected offshoots were planted at 5x5 m by 15 offshoot for each cultivar. Drip irrigation system was installed. Soil samples were taken from untreated offshoots; also samples of water were taken weekly. Each treatment was replicated three times, with one offshoot for each replicate. The selected offshoots were subjected to spraying foliar and addition to soil treatments to both cultivars at the first week of March as the following: 2.2 Treatments C: Foliar spray of water as control P1: Soil addition of POLIXAL 20-8* (15 ml offshoot -1 ) P2: Soil addition of POLIXAL 20-8 (30 ml offshoot -1 ) R1: Foliar spray of Rexene ca 10   (1000 ppm offshoot -1 ) R2: Foliar spray of Rexene ca 10 (2000 ppm offshoot -1 ) 2.3 Ca-fertilisers compounds * POLIXAL 20-8 : (liquid) 8 % calcium oxide  polyhydrocarboxyl (organic acids) 20 % organic material 20 % total nitrogen 4.70 % for alleviation soil salinity and calcium deficiency from Company of ABONOSUALENCIA .co., Spain    Rexene ca 10 : (Solid) Chelated Calcium EDTA 9.7 % Functional Chemicals B.V. AKzoNobel, Mexio.  Calcium application mitigates salt stress in Date Palm ( Phoenix dactylifera   L. )  offshoots cultivars of Berhi and Sayer    Acta agriculturae Slovenica, 107 - 1, marec 2016 105 2.4 Average of some Environment factors at field Average of electrical conductivity (EC) for soil in study was 15.9 dS m -1 , pH was 8.10. Also, average of water EC was 4.55 dS m -1  and pH 7.91, average of field temperature was 41.6°C. 2.5 Parameters of study Were taken on October 15; all the physiological measurements were performed as following: 2. 5.1 Parameters of vegetative growth 2. 5. 1.1 Increase in offshoot height (cm) Measured by a measuring tape to third fully expanded leaf. The increase in plant height = plant height when sampling - plant height before treatment 2. 5.1. 2 Leaf area (m 2 ) Leaf area (m 2 ) was determined according to Ahmed and Morsy, (1999) in four pinnae taken from the middle parts of each leaf, following the equation: Leaf area (m 2 ) = (0.37 (length   width) + 10.29    No. of pinnae) / 1000 2.5.2 Biochemical constituents 2.5.2.1 Total chlorophyll content The extraction of total chlorophyll was carried out according to Lichtenthaler and Wellburn, (1983). The fresh tissue of leaves was collected and froze then; the leaves (0.25 g) were homogenized with 80 % acetone. The optical density (O.D.) of the extracted chlorophyll was measured at 645 and 663 nm by using spectrophotometer PD-303. Total chlorophyll content was calculated by the following formulae: Total chlorophyll (mg/g) = 20.2 (OD 645) + 8.02 (OD 663) x (Vol. / Wt.) Vol = the final volume (ml) Wt. = sample weight (g) 2.5.2.2 Relative water content (RWC) Leaf samples were weighed (fresh biomass) immediately after harvesting, soaked in distilled water at 25°C for 24 hr to determine the turgid mass then ,the samples were dried in an oven at 80°C for 48 hr and their dry biomass was determined. RWC was calculated by the following equation: RWC = (fresh mass- dry mass) / (turgid mass- dry mass) x 100. 2.5.2.3 Determination of proline concentration   according to   Irigoyen et al. , (1992). 2.5.3 Antioxidant enzyme activity assays 2.5.3.1 Enzyme extraction after Luhova et al.  (2003) 2.5.3.2 Enzyme activity was determined spectrophotometricaly. 2.5.3.3 Peroxidase activity was measured by using a guaiacol assay Angelini et al.  (1990). 2.5.3.4 Catalase activity was measured by hydrogen peroxide assay based on formation of its stable complex with ammonium molibdate (Goth, 1991). 0.2 ml of plant extract was incubated in 1 ml reaction mixture containing 65 mM hydrogen  peroxide in 60 mM potassium phosphate buffer,  pH 7.4 at 25°C for 4 min. The enzymatic reaction was stopped with 1 ml of 32.4 mM ammonium molibdate and the concentration of the yellow complex of molibdate and hydrogen peroxide was measured at 405 nm. Activity was expressed on a fresh mass basis (Units mg protein -1 FW). 2.5.4 Extraction and purification of IAA and ABA Extraction, purification and quantitative determination of free and bound IAA and ABA were done, with minor modifications, according to the methods of Rastegar et al.  (2011). Spectrophotometric techniques were used to determine the amounts of IAA and ABA. One gram fresh weight of each sample was taken and extracted with 60 ml of methanol: chloroform: 2N ammonium hydroxide mixture (12:5:3 v/v/v). Each extract (60 ml) was kept in a bottle in deep freeze for further analysis. Extract was then treated with 25 ml of distilled water and the chloroform phase was discarded. The water-methanol phase was evaporated. The water phase was adjusted to the extract pH value of 2.5 or 7 or 11 with 1N HCl or 1N NaOH respectively and 15 ml ethyl acetate was added at each of three steps. This procedure  provided the isolation of free-form IAA and ABA  Abbas M. JASIM et al.   Acta agriculturae Slovenica, 107 - 1, marec 2016 106 from the extraction solvent. After an incubation  period of 1 hour at 70 °C, the same procedure was used for the isolation of bound-form of IAA and ABA from the extraction solvent. Evaporation of ethyl acetate was performed at 45 °C using a rote-evaporator system (B.chi Instruments). Thin-layer chromatography (TLC) was done using silica gel GF254 (Merck Chemicals, Germany) according to the method of Rastegar et al.,  (2011). TLC separated IAA and ABA were isolated from the glass plaques according to the standard synthetic IAA and ABA Rf values. IAA and ABA were dissolved in 2 ml of methanol for filtration and separation from silica using cotton-glass filled transferring pipettes. Spectrophotometeric assay was done at 280 nm for IAA and 263 nm for ABA and for all standard synthetic IAA and ABA and isolation samples. 2.5.5 Determination of potassium and sodium concentration   was   according to Creser and Parsons, (1979). This solution became transparent and used for determinations of    K and Na   concentrations by emission flame photometer (model 129, Shanghai Lingguang int. trade co., ltd .) 2.5.6 Determination of chloride concentration   Chloride (Cl - ) in plant tissue extracts was determined by potentiometric titration. With use 0.2 g of dried ground leaf tissue and addition of 50 ml 2 % acetic acid with shacking through 30 min and filtered by Whatman No.1, tritrated against 0.01 N silver nitrate using potassium chromate as an indicator to a bricked end point (Kalra, 1998). 2.6 Statistical analysis Randomized completely block design of two date  palm cultivars and five treatments of calcium replicated three times were used to conduct the experiment. Experimental data on all variables were subjected to analysis of variance (ANOVA)  procedures using a statistical package, SPSS version 16.0 (SPSS, Chicago, IL). Revised Least Significant Differences (R.L.S.D.) among treatments was considered at the P   ≤  0.05 levels. 3 RESULTS AND DISCUSSION 3.1 Effect of calcium on plant height, leaf area, total chlorophyll and RWC under salt stress Results presented in Table 1 revealed that calcium treatments significantly ( P   ≤  0.05) increased the height and leaf area of offshoots compared with control. Using polixal at 30 ml   offshoot -1  to Berhi cultivar gave the highest values of height and leaf area (34.3 cm, 1.20 m 2 ), respectively, whereas control with Sayer cultivar recorded the lowest value in this respect (11.3 cm, 0.7 m 2 ), respectively. However, the increase of growth may  be attributed to the expansion of cells and activation of photosynthesis by increased total chlorophyll and RWC, proline concentration and  peroxidase activity. Protective role common to concentration, of Ca +2  might be attributed to its role in the maintenance of the structural integrity of the plasma membrane and thus controlling the uptake of Na +  and Cl. Larkindale and Knight, (2002) suggested that calcium role is in protecting against oxidative damage by the protection of calcium channel blockers and calmodulin inhibitors under heat stress. During our experiments field temperature was up to 40 °C. The resultant transient Ca 2+  increase caused  potential stress signal transduction and led to salt adaptation (Gul and Ajmal, 2006). Effect of calcium on height of the plant is in agreement with that obtained by Al-Whaibi et al.,  (2011) on wheat  plant and effect of calcium on leaf area is in concordance with findings of El-Khawaga, (2013) on date palm. The increased offshoot height of Berhi cultivar may be attributed to its ability to restrict Cl -  movement into the shoot more effectively than the Sayer cultivar. Thus, the concentrations of potentially harmful Cl -  ions would be lower in the photosynthetically active tissues, or different in the genotype of vigorous the growth rate in term of Berhi cultivar, and the largest in Sayer cultivar. Table 1 reveals that the total chlorophyll content and RWC of leaves was increased by polixal at 30 ml offshoot -1 . Analysis of Berhi cultivar gave the highest values of total chlorophyll and RWC (1.8 mg.g -1 , 6.5 g, 83.7 %), respectively, whereas control with Sayer cultivar gave the lowest value in this respect (0.8 mg.g -1 ,  Calcium application mitigates salt stress in Date Palm ( Phoenix dactylifera   L. )  offshoots cultivars of Berhi and Sayer    Acta agriculturae Slovenica, 107 - 1, marec 2016 107 65.3 %). Ca 2+  retarded the loss of chlorophyll,  protein and intercellular space, suggesting that the ion plays a regulatory role in maintaining and controlling membrane structure and function (Hepler, 2005). From those studies and our results suggesting that the Ca 2+  plays a regulatory role in maintaining of chlorophyll, it acts as an antioxidant system regulator by increase proline in chloroplasts for scavenging of ROS. Effect of Ca 2+  is in agreement with that obtained by Jafari et al.,  (2009) on sorghum plant by calcium. The role of Ca 2+  in increased relative water content might be attributed to its ability to regulate the compatible solutes and osmotic adjustment, subsequently increasing turgor. Jafari et al . (2009) suggested that the protective effect of Ca 2+  in salinized plants is probably due to its role in maintaining membrane integrity, because one of the primary effects of salt stress is a disruption of membrane integrity caused by displacement of Ca 2+  from the cell surface by Na + . Table 1:  Averages of plant height   (cm), leaf area ( m 2 ) , total chlorophyll   (mg g -1 ) and RWC (%) of Berhi and Sayer cultivars response to Ca-fertilisers   under salt stress Value represents mean ± standard error of three replicates. C: Foliar spray of Water as control, P1: Soil addition of POLIXAL 20-8* (15ml offshoot -1 ), P2: Soil addition of POLIXAL 20-8 (30 ml offshoot -1 ), R1: Foliar spray of Rexene ca 10   (1000 ppm offshoot -1 ), R2: Foliar spray of Rexene ca 10 (2000 ppm offshoot -1 ) 3.2 Effect of calcium on proline concentration, POD and CAT activities, IAA and ABA under salt stress Table 2 reveals that calcium treatments resulted in significantly ( P   ≤  0.05) higher proline concentration and peroxidase activity of treated leaves compared to control. The application of  polixal 30 ml offshoot -1  to Berhi cultivar led to increase proline concentration and peroxidase activity (15.4 mg g -1 , 7.2 unit mg -1 FW), respectively, compared with control to Sayer cultivar which had the lowest values in this respect (9.1 mg g -1  ,4.2 unit mg -1 FW), when using polixal at 30 ml   offshoot -1 , Experiment with Berhi cultivar gave the lowest value of catalase activity (0.7 units mg protein -1 FW) compared to control to Sayer cultivar, which gave the highest value of catalase activity (2.3 units mg protein -1 FW). Effect of calcium in the alleviation of salt stress and the increase of proline reflects the ability of salt-tolerant offshoot to prevent damage of ROS by maintaining better enzymatic (POD and CAT) and non-enzymatic (proline) defense systems. Proline  plays a major role in osmoadaptation through an increase in osmotic stress that shifts the dominant osmolyte from glutamate to proline (Tripathi et al ., 1998). From the results in this work, it seems that  proline might confer salt stress tolerance to Cultivars Treatments Plant height (cm) Leaf    area ( m 2 ) Total chlorophyll (mg g -1 ) RWC (%)   Berhi Sayer C P1 P2 R1 R2 C P1 P2 R1 R2 15.0±5.0 d  26.6±1.5  b  34.3±2.0 a  25.0±2.0  bc  31.6±2.5 ab  11.3±1.5 d  21.6±2.5 c  30.3±0.57 ab  25.0±2.0  bc  28.6±2.0  b  0.8± 0.02 ef   0.9± 0.06 cde  1.2± 0.16 a  0.9± 0.04 cde  1.1± 0.23 ab  0.7 ± 0.0 f   0.9± 0.01 cde  1.0 ±   0.02  bc 0.8±0.01 def   0.9± 0.02  bcd 0.9±0.02 fg  1.4±0.05  b  1.8±0.03 a  1.0 ±0.05 de  1.1±0.0  c  0.8±0.07 g  1.0± 0.00 d  1.1   ±0.02 c  0.9 ±0.02 ef   0.9 ±0.02 f   67.3±1.2 d  74.3±0.7 c  83.7±0.8 a  78.9±0.5  b  83.3±0.5 a  65.3±0.9 e  75.0±1.2 c  79.5±0.6  b  74.6±0.9 c  78.1±0.6  b  R.L.S.D.  ( P   ≤  0.05) 4.1 0.16 0.06 1.4
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