Oxidative responses and genetic stability of date palm Phoenix dactylifera L. Barhi cv. under salinity stress.

Transcription

1 Oxidative responses and geneti staility of date palm Phoenix datylifera L. Barhi v. under salinity stress. Aqeel A.Suhim 1* Karima F. Aas 2* Khearallah M.A. Al-Jaary 1* 1* Date palm Researh Centre- Basra University 2* Environmental Health Dept.- Applied Medial Sienes College - Kerala University Astrat * of the orresponding author: karima.f.aas@gmail.om This study was aimed to investigate the oxidative responses and geneti staility of date palm Phoenix datylifera L. under different irrigation water salinity, date palm off shoots v. Barhi sujeted to different onentrations of NaCl (100, 200, 300 and 400 µm) for 180 days. The otained results showed that, the date palm responses to salinity stress, this responses was inreased of H 2 O 2 level, Peroxidase ativity and Malondialdehyde MDA onentration with inrease NaCl onentration, while opposite trend with memrane staility index, whih H 2 O 2 was inreased from(0.73 µm/g) in ontrol to (2.20 µm/g) in 400 µm treatment, MDA was inreased from (2.35 nmole/g) in ontrol to (nmole/g) in 400 µm treatment, also peroxidase ativity was reahed to (39.59 U/min/g) in 400 µm treatment, while was (20.73 U/min/g) in ontrol and Memrane staility index redution signifiantly from (81.36%) in ontrol to (64.13%) in 400 µm treatment. In terms of geneti staility of date palm under salinity stress, the ISSR markers analysis showed that, the high onentrations of NaCl (200, 300 and 400 µm) produed more polymorphi fragments omparison to ontrol treatment, while the DNA profile was idential etween ontrol and 100 µmtreatment.dendogram was generated using similarity indies of ISSR markers showed, the lowest geneti similarity was found etween 400 µm NaCl onentration and ontrol and 100 µm treatment, followed with oth 200 and 300 µm treatments, whih the ontrol and 100 µm treatment was grouped in one luster, also treatments with 200 and 300 µm grouped in one luster, while the treatment with 400 µm NaCl separated in luster. Keywords: ISSR, Geneti staility, Oxidative stress, Peroxidase, MDA, memrane peroxidation 1-Introdution. Date palm (phoenix datylifera L. 2N=36) elongs to the family Araaeae, it is elieved that the date palm originated in the anient Mesopotamia area (Wrigley, 1995). Date palm trees are widespread in different regions worldwide, partiularly in the Middle East, North Afria, and less intensity in North and South Ameria, Southern Europe, Pakistan and India (Zaid, 2002).This tree is a typial uran tree in Iraq and ultivated over large areas, usually growing y roadsides in industrial, rural, residential and agriultural areas (Al-Jaary et al, 2016). Soil and water salinity onsidered as a major agriultural prolem in many ountries inluding Iraq (Zowain and Ismail, 2013). Salinity an effet of plants through the adverse osmati effet (Munns and Tester, 2008) and toxi effet led to ellular damage (Munns, 2005), and affets several physiologial and iohemial proesses, whih Adulwahid (2012) reported that, the ativity of antioxidant enzymes superoxide dismutase (SOD), Catalase (CAT), asorate peroxidase (APX) and peroxidase (POD), as well as proline was inreased in date palm Hillawi ultivarleaves when irrigated with 200 mm of NaCl, while solule protein dereased under salinity stress, also in vitro responses of date palm Hillawi v. allus to NaCl treatments were investigated (Aass, 2016), the growth of allus was negatively affeted and dereased of atalase ativity while salinity treatments lead to aumulation of proline and Hydrogen peroxide (H2O2). Despite date palm onsidered as a relatively salt tolerane speies (Ramoliya and Pandey, 2003), ut the moleular asis of salt tolerane is poor understood in this tree (Yaish and Kumar, 2015), and pervious work foused on the geneti differentiation of adaptive varieties using DNA marker analysis, suh as random amplified polymorphi DNA (RAPD)(Sedra et al., 1998; Kurup et al., 2009; Aas, 2016) and mirosatellites (Elshili and Korpelainen, 2008). Inter simple sequene repeat (ISSR) markers suessfully used for estimating of geneti diversity in plants when exposed to environmental stress (Khalil, 2013). However, this tehnique has not een used previously to determine the geneti diversity of date palm under salinity stress. The ojetives of the present study were determined some oxidative responses and geneti stailityy using ISSR markers of date palm Barhi ultivar under different salinity onentrations. 70

2 2-Materials and methods This study was onduted in date palm researh entre, Basra University, tissue ulture derived date palm offshoots Barhi ultivar at two years old were seleted, these offshoots were ultured in pots, eah pot filled with 15 kg of soil, and soil hemial harateristis were: ph=7.22, E=4.12, Cation Exhange Capaity (CEC) = 8.5 mole, Organi Matter (OM) = 0.86 % and Soil texture was Loam (silt=45.65, sand=32.44 and lay=21.91). Date palm offshoots sujeted to four onentrations of NaCl were 100, 200, 300 and 400 mm, as well as ontrol treatment, treatments were applied y irrigation using RO water (Reverse Osmosis) whih ph of water was 7.6 and E 1.3 ds/m, plants were irrigated with treatments for 180 days, during the period from 1/6/2019 to 30/152/2016. The experiment was laid aording to Complete Randomized Blok Design (RCBD), with tripliate for eah treatment and results analyzed y using SPSS program V.21. Mean was statistially ompared y LSD test at p<0.05. Oxidative stress responses of treated date palm The responses of date palm to salinity stress were evaluated at 180 days after treatments: H2O2 ontent H2O2 ontent in date palm leaves was determined aording to Sergiev et al. (1997), riefly. A 500mg sample of fresh leaves was homogenized with 5 ml of trihloroaeti aid (TCA; 0.1%: w/v). The homogenate was entrifuged at 13,000 rpm for 15 min, then, 1 ml of the supernatant was added to 0.5 L of potassium phosphate uffer (10 mm, ph =7.0) and 1 ml of potassium iodide(1 M). The asorane of the supernatant was measured at 390 nm. Hydrogen peroxide was used to estalish a standard urve. Malondialdehyde (MDA) ontent. MDA ontent was estimate aording to Heath and Paker (1968); in date palm leaves, 500 mg of leaf tissue was homogenized in 5 ml TCA (0.1%, w/v). Then the homogenate was entrifuged at 10,000 rpm for 5 min, 4 ml of thioariutri aid (TBA; 0.5% w/v) and 1 ml of supernatant prepared in 20% TCA was oiled, after 30 min the reation was terminated y plaing the mixture on ie, then another entrifugation at 10,000 rpm for 15 min was done. The supernatant asorane was measured at 532 and 600 nm, the MDA ontent was alulated using an extintion oeffiient of 155. Memrane staility index (MSI). Memrane staility index (MSI) was measured in date palm leaves g of fresh palm leaves in eah heavy metal stressed and ontrol treatments were ut into small parts and homogenate using 10 ml of deionized water and inuated at room temperature on a rotary shaker for 24 h. Susequently, the initial eletrial ondutivity of the medium (C1) was measured. The samples were plaed in an oven at 90ºC for 2 h to expel all eletrolytes, and ooled at 25ºC, and then the seond eletrial ondutivity (C2) was measured. Leaf memrane staility index was alulatedusing the following formula (Lutts et al., 1996): Memrane Staility Index (MSI %) = Peroxidase ativity: peroxidase ativity in date palm leaves was estimated aording to the method of Kim and Yoo (1996). every single unit of peroxidase atalyzed the oxidation of guaiaol in 1 min (U/min/g). The asorption staility as a result of tetraguaiaol formation was measured at 470 nm. Geneti staility in treated date palm. DNA of date palm Barhi v. leaves was extrated aording to Doyle and Doyle (1990), y using CTAB (etyltrimethyl ammonium romide) method, and DNA was quantified in 1.5% agarose gels to hek out thedna integrity. Eletrophoresis was onduted in a 1X TBEuffer [100 ml 10X TBE (0.89 M Tris ase, 0.89 M Bori aid,20 mm EDTA ph 8.0) and 900 ml distilled water] at 60 V for30 min and then at 120 V for 1.5 h. The DNA was stainedwith 0.5 mg/ml of ethidium romide. DNA onentrationwas measured y Nano-Drop spetrophotometer (Bio-Rad, USA) at A 260 and 280, DNA templates were diluted to 30 ng/μlwith TE uffer. ISSR primers desription and PCR amplifiation Five different primers manufatured y Bioneer South Korea were used for ISSR analysis (Tale 2). Eah polymerase hain reation (PCR) was arried out in a 25 μl volume, ontain 30 ng DNA template, 1.5 mm MgCl 2, 0.32 mm dntps, 1X Taq DNA polymerase uffer, 10 pmol oligonuleotide primer and 2 unit of Taq 71

3 DNA polymerase. Amplifiation was performed in a thermal yler using the following onditions: denaturation at 95 o Cfor 5 min.; 35 yles of 1 min denaturation at 94 o C, 2 min annealing at 36 o C and 2 min extension at 72 o C, and a final extension at 72 o C for 7 min. ISSR markers data analysis DNA markers of Promega 100 DNA Ladder ( , twelve fragments) were used to detet the pries moleular sizes of eah individual fragments using the PhotoCapt MW software (VilerLoumart) and photographed under UV light. Total ands numer, polymorphi and monomorphi fragments were sored visually. The following primers parameters were measured aording to Alansariet al. (2014). 1- Primer effiieny (%) 2- Polymorphism(%) 3- Disrimination power (%) The hange oserved in ISSR profiles, and sored as appearane of new and (1) or disappearane of ands (0) to reate the inary matrix, and geneti similarity index for all pairs treatments was alulated aording to Nie and Li (1979) formula. Geneti Similarity Index (GSI). Where (A) numer of similar fragments in oth treatments, (B) and (C) the total numer of ands in the first and seond treatments. Tale (1) ISSR primers sequene, length and GC ontent. Primer ID Primer sequene Primer length G+C (%) ontent 815 CTC TCT CTC TCT CTC TG CAC ACA CAC ACA CAC AG TCT CTC TCT CTC TCT CA AGA GAG AGA GAG AGA G (CT) T ACA CAC ACA CAC ACA C (CT) T Results and Disussion Oxidative stress responses in date palm leaves under salinity stress. H 2 O 2 and Peroxidase ativity The results in Fig (1) showed the date palm responses to salinity stress y aumulation more H 2 O 2 in leaves, the level of H 2 O 2 was 0.73 µm/g in ontrol plant without signifiant differene ompared to 100 and 200 µmnacl treatments, while the level of H 2 O 2 inreased signifiantly in high onentrations, whih the high level was found in 400 µm NaCl treatment, whih 2.20 µm/g with signifiant differene to other treatments, followed y 300 µm treatment (1.31 µm/g). Similar trend of results were oserved for peroxidase ativity, whih the salinity treatments indued more ativity of peroxidase, and this inrease was gradual with inreased NaCl onentration, the resulting in Fig (2) showed that, the lowest ativity was found in ontrol treatment whih 72

4 20.73 U/min/g without signifiant differene of 100 µm treatment (22.18 U/min/g), while the highest ativity was found in 400 µm treatment (39.59 U/min/g) with signifiant differene to other treatment, followed y treatments 300 µm (32.12 U/min/g) and 200 µm (26.85 U/min/g), with a note that, no signifiant differene etween 200 and 300 µm treatments. The results showed that, H 2 O 2 prodution inreased up to 3 fold in date palm leaves y inreasing NaCl onentration to 400 µm ompared to those in ontrol treatment. H 2 O 2 is one kind of reative oxygen speies (ROS), with different role in plants (Aass and Morris, 2013),among them, it has een suggested that, H 2 O 2 at as ellular signal (Prasadet al., 1994), and aumulation of H 2 O 2 may e due to inreasing of Superoxide dismutase (SOD) enzyme ativity, in whih this enzyme atalyzes the dismutation of anion free radial to O 2 and H 2 O 2 (Meloni et al., 2003). The inrease of hydrogen peroxide levels was aompanied with an inrease in peroxidase ativity, Lin and Kao (2002) stated that, peroxidase enzyme proteting ell from the destrutive influene of H 2 O 2. The inrease of peroxidase ativity in date palm leaves under salinity stress reported in previous works for other ultivar, suh Hillawi (Adulwahid, 2012) and Bartomouda (Darwesh, 2013). Malondialdehyde (MDA) and Memrane staility index (MSI). Malondialdehyde ontent was measured in date palm leaves as indiator of lipid peroxidation, the results in Fig (3) revealed that, no signifiant effet of 100 μm of NaCl in MDA ontent, however, the inrease of NaCl indued MDA aumulation in date palm leaves, MDA ontent was 2.35 nmole/g in ontrol treatment, while reahed to 3.95 nmole/g in 400 μm as highest value with signifiant differene to other treatments, also results showed, the MDA ontent in 300 μm was 2.45 nmole/g without signifiant differene with 200 μm whih 2.39 nmole/g, ut these treatments superior signifiantly of 100 μm and ontrol treatments. The high lipid peroxidation in salinity stressed date palm plants was aompanied with a signifiant redution in memrane staility, the otained results showed in Fig. (4) was indiated to redution MSI from % in non-stressed plant to % in 400 μm treatment with signifiant differene to other treatments, while treatment 300 μm reorded % without signifiant differene ompared 200 μm treatments whih %. MDA is onsidered as a ytotoxi end produt of lipid peroxidation (Agadjanyan et al., 2006), as well known ioti and aioti stress inluding salinity indued ROS prodution and these moleules attak on phospholipid moleule present in polyunsaturated fatty aids (PUFA), and produt MDA whih is responsile of memrane damage (Sharma et al., 2012), also Howladar (2014) reported a well orrelate etween inrease of H 2 O 2 level and lipid peroxidation. Salt stress has also een related to lipid peroxidation and memrane damage (Azooz, 2009). Memrane damage and lipid peroxidation was reported in date palm leaves under other aioti stress suh as heavy metals (Aass et al., 2016; Zouari et al., 2016). LSD 0.05 = 0.21 a Fig (1) H2O2 onentration in (µm/g) date palm Barhi v. leaves under different salinity treatments. 73

5 LSD 0.05 = 4.17 a d d Control Fig (2) Peroxidase ativity (U/min/g) in date palm Barhi v. leaves under different salinity treatments. LSD 0.05 = 0.35 a Control Fig (3) MDA (nmole/g) onentration in date palm Barhi v. leaves under different salinity treatments. 74

6 LSD 0.05 = 3.14 a a Control Fig (4) MSI (%) in date palm Barhi v. leaves under different salinity treatments. Geneti staility in date palm leaves under salinity stress. ISSR primers amplifiation Using five ISSR primers, one ould not amplify DNA, and four gave lear results (818, 822, 834, 855), and these primers were generated a total of 97 reproduile and lear fragments, with average ands for eah primer, 19 out of them were polymorphi, and size of fragments ranging from 200 to 2100 p. The results of primers parameters presented in tale (2) showed that, the primer 818 produed 19 fragments; it is onsidered the lowest ompared to other primers, with 7 polymorphi fragments, and the size of generated fragments etween p, with effiieny of 19.58% and 36.84% for polymorphism and disrimination power, also the results showed the primer 822 reorded the highest numer of a total fragments ompared to other primers, whih this primer was generated 32 fragments, and the size of fragments ranging from 56 to 2100 p, with 4 polymorphi, and reorded an effiieny 32.98%, with 12.5 and % for polymorphism and disrimination power, respetively. Additionally, the tested primer 834 produed 21 fragments as a total numer of fragments, only one fragment was polymorphi, and the size of fragments ranging etween 540 and 200 p, with 21.64, 4.76 and 5.26 % as effiieny, polymorphism and disrimination power, respetively, while the primer 855 generated 25 fragments, 7 out of them as polymorphi fragments, with % effiieny, 28 % polymorphism and 36.84% disrimination power. Aording to ISSR analysis,the results in Fig. (5 a-d) showed the profile of ontrol and 100 μm NaCl treatments was idential, also the 200 and 300 μm profiles was idential, ut with different fragment profile ompared to ontrol treatment, while the inrease of NaCl onentration to 400 μm showed a differene ompared to other fragments profiles of other treatments, a new appearane and disappearane was distinguishale of fragments profile for this treatment. Geneti Similarity Index The results presented in tale (3) showed similarity indies results of ISSR markers analysis, and the data revealed that, the highest average GSI was reorded etween ontrol and 100 μm treatments, whih was 1, followed y 200 and 300 μm treatments, whih was 0.86, while the lowest GSI was reorded etween 400 μm and ontrol treatments, whih was The UPGAM dendogram Fig (6) indiated that, the treatments grouped in three lusters, the first luster had ontrol and 100 μm treatments, seond luster had 200 and 300 μm treatments, while 400 μm treatment was separated in third luster. Results of this work was revealed the suitaility of ISSR tehnique to detet the genotoxiity of salinity stress, this is indiated y previous works, that onduted on other plants, suh as wheat (Lang et al., 2001), rie 75

7 (Kaushik et al., 2003), arely (Khata and Samah, 2013) and sugarane (Markad et al., 2014). Change in DNA profile under high onentrations of NaCl ompared to non-stressed plant ould e attriuted to point mutation; this mutation (inluding insertion or deletion) led to appearane new amplions or disappearane exitingfragments (Atienzar and Jha, 2006; Liu et al., 2012). Tale (2) ISSR primers parameters Primer ID Range of ands size (p) Total ands Polymorphi ands Primer effiieny (%) Primer polymorphism (%) Disrimination power (%) Total Tale (3) Similarity indies of Nie and Li oeffiient of date palm produed y different NaCl onentrations otained from ISSR markers. Treatment Control 100 μm 200 μm 300 μm 400 μm ontrol μm μm μm μm

8 T0 T1 T 2 T3 T4 T0 T1 T 2 T3 T4 Primer 822 Primer 818 T0 T1 T 2 T3 T4 M T4 T3 T 2 T1 T0 M Primer 855 Primer 834 Fig (5): ISSR markers analysis of date palm produed of different NaCl onentrations: M: DNA marker, T0: ontrol; T1: 100 µm; T2: 200 µm; T3: 300 µm; T4: 400 µm. 77

9 Fig (6) Dendogram generated y UPGAM luster method ased on ISSR markers of date palm produed y different onentrations of NaCl treatments. 4-Conlusion The results of this study demonstrated, date palm responded to high onentrations of NaCl ( µm) y aumulation of H2O2 and inrease peroxidase ativity, this indiates to oxidative stress, also salinity stress aused damage to memranes, and that through inrease of MDA onentration and redution of memrane staility index. Also the results showed the possiility of using ISSR markers to detet DNA damage in date palm under salinity stress. 5- Referenes Aass, M.H. (2016). Responses of date palm Phoenix datylifera L. allus to ioti and aioti stress. Emirates J. of Food and Agri., 28(1): Aass M.H., Naema J.D. and Al-Jaary K.M.A. (2016). Biohemial responses to admium and lead stresses in date palm Phoenix datylifera Lplants. AAB Bioflux. 8(3):

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