doi: 10.14720/aas.2017.109.2.12 Original research article / izvirni znanstveni članek Salinity induced changes in water relations, oxidative damage and morpho-physiological adaptations of pistachio genotypes in soilless culture Zahra MIRFATTAHI1, Soheil KARIMI1*, Mahmoud Reza ROOZBAN1 Received November 19, 2016; accepted August 03, 2017. Delo je prispelo 19. novembra 2016, sprejeto 03. avgusta 2017. ABSTRACT Selecting salt tolerant rootstocks is a sustainable approach for developing fruit trees in salinity prone areas. 60-day-old seedlings of Pistacia vera 'Akbari' and 'Ghazvini', and P. vera 'Ghazvini' x P. atlantica (GxA) were subjected to 0, 50, 100 and 150 mM NaCl in half strength Hoagland's nutrient solution. After 45 days, the growth, water relations, and oxidative damage parameters were investigated. Salt stress reduced plant biomass, height, crown diameter and leaf number, but increased specific leaf area (SLA) of the seedlings. Under salt stress, the growth of 'Akbari' seedlings was higher than the other genotypes. Accumulation of malondialdehyde (MDA) and proline was observed in the leaves of salt affected seedlings. 'Ghazvini' seedlings had the highest MDA concentration and the lowest cell membrane stability in their leaves. Degredation of photosynthetic pigments under salt stress was lower in the leaves of 'Akbari' seedlings than that in other genotypes. Increase in leaf succulence was observed in 'Akbari' and GxA seedlings in response to salt stress. Relative water content and concentration of anthocyanins in the leaves of pistachio genotypes remained unchanged under salt stress. The results revealed that monitoring leaf abscission, SLA, leaf succulence, MDA concentration, and photosynthetic pigments provide suitable contrast for screening salt tolerance in pistachio. Furthuremore, 'Akbari' was found to be the most salt tolerant genotype. Key words: interspecific hybrid; leaf pigments; morphophysiological adaptation; salt stress; oxidative stress; Pistacia vera IZVLEČEK S SLANOSTJO VZPODBUJENE SPREMEMBE V VODNEM REŽIMU, OKSIDATIVNE POŠKODBE IN MORFOLOŠKO-FIZIOLOŠKE PRILAGODITVE GENOTIPOV PISTACIJE V BREZTALNEM GOJENJU Izbor na slanost tolerantnih podlag je primeren pristop pri razvoju sadnih dreves na območjih podvrženih zasoljevanju. 60-dni stare sejanke pistacije (Pistacia vera 'Akbari' in 'Ghazvini', ter P. vera 'Ghazvini' x P. atlantica (GxA)) so bile izpostavljene 0, 50, 100 in 150 mM NaCl v polovični Hoaglandovi hranilni raztopini. Parametri rasti, vodnih razmer in oksidativnih poškodb so bili preučeni po 45 dneh. Solni stres je zmanjšal biomaso rastlin, višino, premer krošnje in število listov, a povečal specifično listno površino (SLA) sejank. Pod solnim stresom je bila rast sejank 'Akbari' večja kot drugih genotipov. Akumulacija malondialdehida (MDA) in prolina je bila opažena v listih od soli prizadetih sejank. Sejanke 'Ghazvini' so imele največjo koncentracijo MDA in najmanjšo stabilnost celičnih membran listov. Razgradnja fotosinteznih pigmentov v listih sejank 'Akbari'je bila v solnem stresu manjša kot pri drugih genotipih. Povečanje sukulence listov kot odziv na solni stres je bilo opaženo pri sejankah 'Akbari' and GxA. Relativna vsebnost vode in vsebnost antocianinov v listih sta pri vseh genotipov pistacije ostali nespremenjeni v solnem stresu. Izsledki so odkrili, da daje spremljanje odpadanja listov, SLA, listne sukulence, koncentracije MDA in fotosinteznih pigmentov primernen nabor znakov za odkrivanje tolerance na sol pri pistaciji. 'Akbari' je bil prepoznan kot na sol najbolj tolranten genotip. Ključne besede: medvrstni križanci; listni pigmenti; morfološko-fiziološke adaptacije; solni stres; oksidativni stres; Pistacia vera 1 INTRODUCTION Salinity is one of the most serious constraints to agricultural crop production in arid and semi-arid areas. More than 1.5 Mha of irrigated lands are taken out of production each year due to high salt accumulation in the soil (Munns and Tester, 2008). Salinity reduces the growth and development of plant by affecting water availability to plant, absorption of mineral nutrients, and ion homeostasis (Parida and Dos, 2005). Furthermore, 1 Department of Horticultural Science, College of Aburaihan, University of Tehran, Tehran, Iran; Corresponding author: skarimi@ut.ac.ir Acta agriculturae Slovenica, 109 - 2, september 2017 str. 357 - 362 Zahra MIRFATTAHI et al. burst of regeneration of reactive oxygen species (ROS) under this situation damages proteins, lipids and the genetic material (Gill and Tuteja, 2010). Morphological, physiological, and biochemical adaptations to salt stress, such as stomatal closure, osmotic adjustment, ion exclusion and compartmentation, and increase in the antioxidative activity have been widely discussed in the literature (Hishida et al., 2014). Pistachio (Pistacia vera L.) is originally native to arid and semi-arid regions of the middle east and Persia. Although the plant is relatively salt tolerant (Ferguson et al., 2002), intensified salt built up in the soil of these regions due to using poor quality water for irrigation in association with frequent drought periods has reduced its production over recent decades (Karimi and Rahemi, 2012). Selecting salt tolerant rootstocks is an effective approach for sustainable development of pistachio in such salt prone areas. In this regard, understanding the effects of salinity on pistachio is of a crucial importance for establishing a successful rootstock breeding program. Although there are many reports on evaluating salt tolerance of pistachio genotypes and related species, some controversies in results of these researches can be found. The issue is mainly due to physico-chemical differences in growing media, and using convenient criteria for evaluating salt tolerance of pistachio which are mainly developed for herbaceous annual crop species. However, performing such experiments in soilless culture may reduce experimental errors by increasing uniformity of growing medium. Absence of complex interactions between soil, the stressor, and plant in soilless condition enhances repeatability of results and provide a clear understanding of the plant responses to salinity. Therefore, in this study the effects of salt stress were evaluated on the growth, morpho-physiological adaptations, and biochemical characteristics of pistachio seedlings in soilless culture in order to screen salt tolerance. Moreover, effectiveness of different criteria for screening salt tolerance in pistachio seedlings was investigated. 2 MATERIAL AND METHODS 2.1 Plant material and experimental conditions This study was conducted at the Department of Horticultural Science of University of Tehran in 201415. Seeds of pistachio 'Ghazvini' and 'Akbari' were obtained from Pistachio Research Station of Damghan, Iran. Seeds of interspecific hybrid P. vera 'Ghazvini' x P. atlantica Desf. (GxA) were obtained by controlled hybridization (Morovati, 2013). The seeds were soaked for 24 hrs and then treated with 0.2 % captain fungicide for 4 hrs. The seeds were transferred to clean containers with moistened filter paper and allowed to germinate for 5 days at room temperature. Three germinated seeds were sown in 4.5 liter plastic pots containing 3.0 kg of coco-peat and perlite mixture (2:1 volume ratio) in a greenhouse with average day/night temperatures of 30/24 °C and air humidity of 10/22 %. The pots were irrigated with tap water for about 30 days. At four leaved stage, the seedlings started to receive half-strength Hoagland's nutrient solution (Hogland and Arnon, 1950) for a 60-day period and then subjected to four NaCl concentrations (0, 50, 100 and 150 mM) in the nutrient solution for 45 days. The seedlings were irrigated with the nutrient solutions every 48 hrs. To avoid salt build-up in the pots, the plants were irrigated enough to ensure drainage of 30 % of the solutions. 2.2 Growth parameters After 45 days of exposure to the salt stress plant height, leaf number and crown diameter of the seedlings were measured. Then, the plants were harvested and their fresh mass and dry mass were determined. Specific leaf area (SLA) was measured by determining dry mass of fifteen leaf discs (0.90 cm diameter) from the 4-5th fully expanded leaves from shoot top according to Eq. 1: Eq. 1 2.3 Water relations Leaf water potential (TLeaf) was measured at midday (11:00-12:00) using a portable pressure chamber device (Soil Moisture Equipment Corp., USA). TLeaf was measured immediately after excising the 4-5th fully expanded leaves from the top of the stem. Leaf relative water content (RWC) was measured by punching fifteen discs (0.90 cm diameter) from the developed leaves. The discs were weighted (FM), floated on distillated water for 24 hrs (at 4 C in dark) to obtain turgid mass (TM), and finally their dry mass was recorded 72 hrs after placing at 70 °C (DM). RWC was calculated according to Eq. 2: RWC = Eq. 2 Leaf water content was expressed as the percentage equivalent of the ratio of the mass of water (FM - DM) to the leaf dry matter (Md) (Eq. 3). fm-DM Leaf water content = ——— X 1UU Eq. 3 Md Area basis leaf water content (succulence index) was expressed as the ratio of the mass of water to the area of 292 Acta agriculturae Slovenica, 109 - 2, september 2017 Salinity induced changes in water relations, ... adaptations of pistachio genotypes in soilless culture the leaf sample (LAS). In this order, fifteen foliar discs (0.90 cm diameter) from the developed leaves of shoot top were used (Eq. 4). FMDM Succulence = , . _— Eq. 4 LAS 2.4 Leaf proline concentration Leaf proline concentration was measured according to the method described by Bates et al. (1973). Leaf tissue (0.1 g) was extracted in 10 ml of 3 % sulphosalicylic acid and then, 2 ml of ninehydrin reagent and 2 ml acetic acid were added to 2 ml of the extract. Then, the samples were heated in boiling water for 60 min. Four milliliters of toluene was added to each sample and vortexed for 15-20 seconds. The absorbance of toluene phase was measured at 532 nm using a spectrophotometer (Perkin Elmer, Lambada 25, USA). The concentration of proline was determined according to an external standard curve. 2.5 Oxidative damage parameters Cell membrane stability index (CMS) was determined by measurement of electrolyte leakage from leaf samples. Fifteen leaf discs (0.90 cm diameter) were excised from fully expanded young leaves. The leaf discs were washed three times in deionized water and incubated in 15 ml deionized water (40°C) for 30 min. The initial conductance (CO of the incubation solution was measured using an electrical conductance meter. Leaf tissue in the incubation solution was killed by placing the samples in boiling water for 10 min. The conductance of the solution (Cmax) was determined at room temperature. CMS was calculated by using the following formula (Eq. 5): CMS=l-(C7CinflJxl00 Eq.5 Lipid peroxidation was assessed by measurement of malondialdehyde (MDA) concentration in fully expanded young leaves, according to the method described by Heath and Parker, (1986). Leaf tissue (200 mg) was homogenized in 10 ml of 0.1 % trichloroacetic acid (TCA) and centrifuged at 1000 g for 5 min. Then 4 ml TCA acid 20 % and thiobarbituric acid 0.15 % were added to 1 ml of the supernatant. The absorbance was measured at 532 nm by spectrophotometry (PerkinElmer, Lambda 25, USA). 2.6 Leaf pigments In addition to evaluation of leaf greenness by using a SPAD 502 chlorophyll meter (Minolta Co., Japan), concentrations of chlorophylls and carotenoids were measured in the 4-5th developed leaves according to the method described by Lichtenthaler (1987). Fresh tissue (15x0.64 cm2 leaf discs) was extracted in 80 % acetone and after centrifuging at 4800 rpm for 20 min, the absorption of supernatant was read at 470, 647 and 664 nm using a spectrophotometer (PerkinElmer, Lambda 25, USA). The concentrations of chlorophyll a (Chl a) and b (Chl b), and carotenoids were calculated according to the following formulas (Eq. 6-8): Chl a = 12.25A664 - 2.79A647 Eq. 6 Chl b = 21.51A647 - 5.10A664 Eq. 7 Carotenoids = (1000A470 - 1.8Ca - 85.02Cj)/198 Eq. 8 For measuring anthocyanins, 500 mg fresh leaf tissue was extracted in 10 ml of methanol acidified with 1 % HCl at 4 oC for 24 hrs. The absorbance of the extract was determined at 550 nm using a spectrophotometer (PerkinElmer, Lambda 25, USA). Anthocyanin concentration was calculated using an extinction coefficient of 33000 mol-1 cm-1 (Wagner, 1979). 2.7 Statistical analyses The experiment was conducted as factorial (3 genotypes x 4 NaCl concentrations) based on a completely randomized design with three replications. Three pots were considered in each replication and mean of these pots were considered as a replication for each treatment. In sum, 108 pots were used in the experiment. The data were subjected to ANOVA and the means were compared using Duncan's multiple range test (DMRT) at P < 0.05. The statistical analyses were performed using the SPSS software (v. 21.0). 3 RESULTS The effects of salt stress on growth of pistachio seedlings are represented in Table 1. Shoot height, crown diameter, leaf number, and dry mass of the seedlings were significantly reduced under 100 and 150 mM NaCl treatments. Shoot height and leaf number of 'Akbari' seedlings were significantly higher than the other genotypes. Trunk diameter of 'Akbari' seedlings was significantly higher than the other genotypes and the lowest value was found in GxA. Fresh mass of the seedlings significantly decreased by increasing NaCl concentration in the nutrient solution. Among the genotypes, seedlings of 'Akbari' had the highest fresh and dry mass at the end of the experiment; no significant differences were observed between fresh and dry mass of 'Ghazvini' and GxA seedlings. SLA significantly increased under 100 and 150 mM NaCl treatments. The highest SLA was found in GxA and 'Akbari' seedlings had the lowest SLA. Acta agriculturae Slovenica, 109 - 2, september 2017 Zahra MIRFATTAHI et al. Table 1: The effects of salt stress and plant genotype on growth of pistachio seedlings Plant height Crown diameter Leaf number Fresh mass Dry mass SLA (cm) (mm) (g) (g) (cm2 g-1) Genotype 40.3at 0.010b Akbari 5.79a 22.9a 81.0a 36.1a Ghazvini 35.1b 4.78b 19.2b 56.6b 24.7b 0.011ab GxA 32.9b 4.44c 18.5b 47.8b 20.7b 0.012a NaCl (mM) 0.010b 0 39.9a 5.43a 24.6a 77.2a 35.0a 50 35.8ab 5.12ab 21.7ab 65.5b 28.4ab 0.010b 100 33.8b 4.87bc 18.8bc 53.1c 23.6b 0.012a 150 34.2b 4.63c 16.2c 48.5c 21.5c 0.012a ANOVA Genotype ** ** * ** ** * NaCl Level * ** ** ** ** ** NaClxGenotype ns ns ns ns ns ns Difference among the treatments was analyzed by 3 genotypes x 4 salt stress ANOVA; ns, *, ** indicate nonsignificant, and significant differences at 0.05 and 0.01, respectively. The data are means of 3 replicates; Mean separation was performed according to DMRT (P < 0.05) and similar letters indicate no significant difference between mean values. Relative water content (RWC) and water potential (^Leaf) of the leaves were not affected by the NaCl treatments. However, TLeaf was significantly higher in the leaves of 'Akbari' seedlings (-0.68 MPa) and the lowest value (-0.892 MPa) was found in 'Ghazvini' leaves (Table 2). Leaf water content and succulence were significantly affected by the interactive effects of salt stress and plant genotype (P < 0.01). Water content significantly increased in the leaves of 'Akbari' and GxA under 100 and 150 mM NaCl stress up to 21 and 41 percent, respectively. However, no significant changes were observed in leaf water content of 'Ghazvini' under salt stress (Figure 1). Unlike the other genotypes, area basis leaf water content was significantly reduced in the leaves of 'Ghazvini' under 150 mM NaCl by 16.2 percent (Figure 1). Figure 1: The interactive effects of salt stress and genotype on water content and succulence of the leaves of pistachio seedlings. Mean separation according to DMRT at P < 0.05 (n = 3) Significant increase in leaf proline concentration was observed under severe salt stress. Among the genotypes, 'Ghazvini' and 'Akbari' had more leaf proline content than GxA (Table 2). Cell membrane stability index (CMS) remained unchanged under salt stress, too (Table 2). However, CMS in the leaves of 'Ghazvini' was significantly lower than the other genotypes. Significant accumulation of malondialdehyde (MDA) concentration was found in the leaves of the seedlings under salt stress and the highest increase in MDA concentration (45.9 %) was observed under 150 mM NaCl treatment. GxA had the highest leaf MDA concentration (15.5 mmol g-1), and the lowest concentration (10.56 mmol g-1) was found in 'Akbari' (Table 2). 292 Acta agriculturae Slovenica, 109 - 2, september 2017 Salinity induced changes in water relations, ... adaptations of pistachio genotypes in soilless culture Table 2: The effects of salt stress and plant genotype on relative water content (RWC), water potential (¥Leaf), proline, cell membrane stability index (CMS), and concentration of malondialdehyde (MDA) in the leaves of pistachio seedlings RWC ^Leaf Proline CMS MDA (%) (MPa) (pmol g -1) (%) (mmol g-1) Genotype -0.680at 267.8ab 10.65b Akbari 83.3 90.7a Ghazvini 84.0 -0.892b 296.7a 83.5b 12.26ab GxA 85.3 -0.744ab 218.1b 92.1a 15.50a NaCl (mM) 231.2b 10.72b 0 84.2 -0.851 88.8 50 84.0 -0.683 258.2b 90.7 11.94ab 100 85.3 -0.755 271.0ab 88.2 13.25ab 150 83.6 -0.851 283.3a 87.0 15.65a ANOVA Genotype ns ** * ** * NaCl Level ns ns * ns * NaClxGenotype ns ns ns ns ns Difference among the treatments was analyzed by 3 genotypes x 4 salt stress ANOVA; ns, *, ** indicate nonsignificant, and significant differences at 0.05 and 0.01, respectively. The data are means of 3 replicates; Mean separation was performed according to DMRT (P < 0.05) and similar letters indicate no significant difference between mean values. Table 3 represents the effects of salt stress and plant genotype on concentration of leaf pigments. Chlorophyll a:b ratio (Chl a:b) significantly increased in the leaves of pistachio seedlings in response to salt stress. Chl a:b in the leaves of 'Akbari' was significantly lower than the other genotypes. Leaf greenness, which was measured by SPAD, significantly reduced under salt stress. However, no significant difference was observed in leaf color of the genotypes. Salt stress significantly reduced total chlorophyll concentration in the leaves. 'Akbari' had the highest leaf chlorophyll concentration than the other genotypes. Concentration of anthocyanins and carotenoids in the leaves remained unchanged under salt stress, however, concentration of carotenoids in the leaves of 'Akbari' was higher than 'Ghazvini' and GxA. Table 3: The effects of salt stress and plant genotype on concentration of pigments in the leaves of pistachio seedlings SPAD Chl a:b Total Chls Carotenoids Anthocyanins (mg cm-2) (mg cm-2) (mmol g-1) Genotype 0.395bt Akbari 55.0 29.2a 2.23a 280.9 Ghazvini 55.9 0.420a 25.4b 2.03b 282.1 GxA 56.1 0.423a 23.6b 1.90b 282.3 NaCl (mM) 0.396b 0 57.8a 27.9a 2.13 289.5 50 55.4b 0.422a 23.8b 1.90 279.3 100 54.6b 0.421a 26.8ab 2.08 283.3 150 54.9b 0.416a 25.2b 2.06 275.3 ANOVA Genotype ns ** ** ** ns NaCl Level * ** * ns ns NaClxGenotype ns ns ns ns ns Difference among the treatments was analyzed by 3 genotypes x 4 salt stress ANOVA; ns, *, ** indicate nonsignificant, and significant differences at 0.05 and 0.01, respectively. The data are means of 3 replicates; mean separation was performed according to DMRT (P < 0.05) and similar letters indicate no significant difference between mean values. Acta agriculturae Slovenica, 109 - 2, september 2017 Zahra MIRFATTAHI et al. Chlorophylls:carotenoids ratio (Chl:Crt) was 'Akbari' and 'Ghazvini' seedlings. A significant significantly affected by the interactive effect of salt decrease in Chl:Crt ratio was found in the leaves of stress and plant genotype (P < 0.01). Chl:Crt ratio G*A under salt stress (Figure 2). remained unchanged in the leaves of salt stressed □ 0 Q50 H100 ■ 150 mM NaCl 14 - Akbari Ghazvini G>