Impaginato 161 Adv. Hort. Sci., 2019 33(2): 161-170 DOI: 10.13128/ahs-23363 The responses of enzymatic and non- enzymatic antioxidant systems of scion on different rootstocks under water stress deficit M. Keshavarzi , A. Shekafandeh (*) Horticultural Science, College of Agriculture, University of Shiraz, Iran. Key words: chlorophyll, Ficus carica, graft, growth. Abstract: Selecting of the specific type of rootstock is an appropriate and short- term method for increasing drought tolerance by improving the antioxidant systems in plants. In this research, the responses of the antioxidant systems of fig scion ‘Sabz’ were investigated on various rootstocks at different irrigation levels. Graft combinations were ‘Sabz’ on ‘Sabz’ (Sa/Sa), ‘Siah’ (Sa/Si) and ‘Torch’ (Sa/T) rootstocks, plus ‘Sabz’, ‘Siah’ and ‘Torch’ cultivars with no graft- ing, for a total of six groups. The plants were irrigated with 4 levels of 25, 50, 75 and 100% of water requirement (WR) for a duration of 12 weeks. The experi- ment was performed in a randomized complete design with 5 replications per treatment. The results showed that the ‘Torch’ rootstock induced the greatest amount of anthocyanin, glutathione and ascorbic acid in ‘Sabz’ (Sa/T) at 25% WR. Superoxide dismutase and catalase activities of ‘Sabz’ grafted on ‘Siah’ were more evidence compared to ‘Sabz’ grafted on ‘Torch’ rootstock at 25% of WR. The cv. Sabz grafted on both ‘Siah’ and ‘Torch’ rootstocks indicated higher chlorophyll content, chlorophyll stability index and shoot growth than cv. Sabz with no grafting. As a result, both rootstocks (T and Si) with the activation of enzymatic and non-enzymatic antioxidant systems caused the scion protects its integrity and be able to tolerate more water stress. 1. Introduction Water scarcity is one of the most important environmental stresses, with its greatest impact on agriculture worldwide, especially in arid and semiarid regions (Knapp et al., 2001; Alizadeh et al., 2011). In recent years, the issue of climate change, combined with global warming, has been a major contributor to the increased water scarcity and plant losses in many parts of the world (Kramer and Boyer, 1995). Since most of a plant’s processes are directly or indirectly affected by water, it is clear that most plants are affected by moderate to long-lasting drought throughout their life cycles (Bhattacharjee and Saha, 2014). The effects of drought stress on plants depend on genotype, rate and severity of the stress, age and stage of plants growth and development (Rostami and (*) Corresponding author: shefakan@shirazu.ac.ir Citation: KESHAVARZI M., SHEKAFANDEH A., 2019 - The responses of enzymatic and non-enzymatic antioxidant systems of scion on different root- stocks under water stress deficit. - Adv. Hort. Sci., 33(2): 161-170 Copyright: © 2019 Keshavarzi M., Shekafandeh A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 6 June 2018 Accepted for publication 14 December 2018 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(2): 161-170 162 Rahemi, 2013). Plant tolerance to water stress depends on their morphological, physiological and biochemical mechanisms that determine the responses of the plant under stress conditions (Penella et al., 2014). The fig (Ficus carica L.) belongs to the Moraceae family, a perennial fruit tree, which generally has a high tolerance to water deficit (Faghih and Sabet Sarvestani, 2001). This tree is managed in two ways, irrigated or rain-fed, and is cultivated in many parts of the world with diverse climatic conditions such as Iran, Turkey and other Mediterranean countries. The total area in the world cultivated with figs is reported to be about 388368 hectares with a production of 918813 tons per year (El-Shazly et al., 2014). Iran, with an annual production of more than 76414 tons of fig, ranks fourth in the world (FAO, 2012). Most fig trees in Iran are cultivated in Estahban, a semi-arid region in the southeast of Fars province (Shirbani et al., 2013). Drought stress stimulates the accumulation of active oxygen species in plants. These reactive oxy- gen species are active forms of the oxygen molecule that are produced by the excitation of an oxygen molecule, or the transfer of one, two or three elec- trons to oxygen molecules, which ultimately lead to the formation of superoxide (O2 -), hydrogen peroxide (H2O2) and hydroxyl radical (OH) (Gill and Tuteja, 2003) These reactive oxygen species may be the first step in the degradation of oxidative processes, such as lipid peroxidation, chlorophyll breakdown, protein oxidation, and nucleic acid damage (Anjum et al., 2011; Gu et al., 2013). Plants have specific defense mechanisms that provide protection on the molecu- lar level. These mechanisms comprise non-enzymatic antioxidants, such as ascorbic acid (ASA), glutathione (GSH) and anthocyanin and enzymatic antioxidants include catalase (CAT) and superoxide dismutase (SOD) (Guerfel et al., 2009; Liu et al., 2012 a). Superoxide dismutase is a major scavenger that counters superoxide damage. Catalase catalyzes the decomposition of the excess of hydrogen peroxide to water and oxygen and protects cells from oxidative damage. These two enzymes play an important role in the degradation of reactive oxygen species (Mittler, 2002). With regard to the climate change models that predict an increase in drought and excessive groundwater depletion in the future, water scarcity appears to be the main limiting factor in agri- culture (Alizadeh, 2005). In this regard, the use of drought tolerant rootstocks can play an important role in managing water absorption, water use effi- ciency, survival potential, growth capacity and the success of grafting in dry conditions. Different rootstocks show different levels of toler- ance in response to drought, and their ability to have high tolerance depend on improving the vegetative growth of the scion, the horizontal and vertical expansion of the roots, the ability to absorb water and minerals and antioxidant system activities for scavenging of active oxygen species (Ballesta et al., 2010; Corso and Bonghi, 2014). How the rootstock genotypes may be effective in response to water deficit stress has been investigat- ed in many studies on fruit trees, such as apples and grapes (Alizadeh et al., 2011; Liu et al., 2012 b; Corso and Bonghi, 2014). Most of these researches focus on the effects of rootstock on vegetative growth, fruit yield and quality, nutrition and hormones, and the scion’s water content in response to drought. However, there are limited investigations on how rootstock affects the antioxidant systems of the scion in response to water deficit. The aim of this study was to investigate the effect of different fig root- stocks on the enzymatic and non-enzymatic antioxi- dant systems of the ‘Sabz’ scion in response to differ- ent levels of irrigation. 2. Materials and Methods In early November, offshoots semi-hard woodcut- tings of different cultivars (‘Sabz’, ‘Siah’ and ‘Torsh’) were disinfected with a commercial bleach (Clorax) solution containing 5% NaOCl and 2% benomyl for 20 min. Then, they were wrapped in a wet cotton cloth and placed at 4°C for 15 days to ensure that their chilling requirement was satisfied. After that, the bottoms of the cuttings treated with IBA solution (1000 mg/L) and cultured in cartonplast boxes con- taining perlite. They were put in a greenhouse with natural sunlight, relative humidity of 64% and an average temperature of 38/16±2°C day and night respectively. After 2 weeks, rooted cuttings were transferred to 1 L plastic pots containing a mixture of soil: peat: sand (1:1:1, v/v/v). After 8 weeks, when the length of the new shoots reached 20 cm, cultivar Sabz as scion were grafted (cleft grafting) onto the three root- stocks (‘Sabz’, ‘Siah’ and ‘Torsh’). The place of graft- ing was firmly enclosed with cellophane. To maintain moisture, grafted plants were covered with plastic bags to ensure grafting success. After 15 days, the plastic bags were removed and the cellophanes were Keshavarzi and Shekafandeh - Scion antioxidants responses on different rootstocks 163 opened. Subsequently, all grafted and non-grafted plants (controls) were transferred to 10 L pots with- out drainage that contained the aforementioned ratio of the soil mixture. The field capacity of the soil used for potting was determined according to the protocol described by Richards (1949). The pots were irrigated daily at field capacity by the help of a bal- ance. After complete deployment, the experiment was carried out by selecting 40 plants of each grafted combination (‘Sabz’/ ‘Sabz’, ‘Sabz’/ ‘Siah’, ‘Sabz’/ ‘Torsh’) and 40 plants from cultivars with no grafting (‘Sabz’, ‘Siah’ and ‘Torsh’) in a randomized complete design with 4 levels of irrigation 100%, 75%, 50% and 25% of water requirement (WR). Three months after the start of treatments, various indices were mea- sured as follows. Anthocyanin Extraction solution containing methanol, water and concentrated chloric acid (HCl) was prepared by a ratio of 80: 20: 1. Leaf samples were kept in the extract solution at 4°C in the dark for 48 h. Then, the extract passed through a Whatman filter paper (No. 1) and was read at 530 and 657 nm. Anthocyanin content was measured using the microplate reader spectrophotometer according to the method described by Alexieva et al. (2001). The amount of anthocyanin was calculated as μg g-1 fresh weight. Glutathione The method used by Moron et al. (1979) was fol- lowed to measure glutathione concentration. An amount of 200 mg of fresh leaves was homogenized with 2 ml of cooled Trichloroacetic Acid (TCA) (5%), and they were centrifuged for 30 min at 15,000 rpm at 4°C. From supernatant extract, 75 μl was trans- ferred to a vial containing 300 μl sodium phosphate buffer (0.2 M, pH 8) and 750 μl DTNB (5, 5-di-tiobis- 2-nitrobenzoic acid) 0.6 mM. The extract was read at 412 nm by a spectrophotometer microplate reader. Glutathione concentration was calculated using stan- dard curve. Ascorbic acid To determine the concentration of ascorbic acid, 1 g of fresh leaf tissue was thoroughly crushed in 5 ml of TCA (10% cold), and then centrifuged at 3500 rpm for 20 min. The supernatant was isolated and diluted to reach 10 ml. One ml of aqueous extract was mixed with 0.2 ml of DTC reagent (2, 4-di-nitrohydrazine, thiourea, copper sulfate) and was incubated at 37°C for 3 h. Then, 1.5 ml of sulfuric acid (65%) was added and mixed thoroughly. The extract was allowed to stand at room temperature for 20 min. The absorbance was read at 520 nm using a spectropho- tometer. The ascorbic acid was calculated in μg g-1 F.W. (Elavarthi and Martin, 2010). Antioxidant enzymes To prepare leaf extract containing enzymatic antioxidant, 200 mg of fresh leaf tissue was ground with liquid nitrogen in a mortar. Then, 1.2 ml of K2PO4 buffer 0.2 M (pH 7.8) containing 0.1 mM EDTA (Ethylenediaminetetraacetic acid, C10H16N2O8) was added to the samples and homogenized. The samples were centrifuged at 15,000 g for 20 min at 4°C. The supernatants were separated and then the same operation was repeated on residual. The resulting extract was used for determining the activity of antioxidant enzymes (Elavarthi and Martin, 2010). Catalase activity Catalase activity was determined by the method of Aebi (1984) as described by Elavarthi and Martin (2010) by measuring the amount of hydrogen perox- ide (H2O2) degradation via reducing the absorbance at 240 nm with a spectrophotometer microplate reader. To this purpose, 3 ml of the reaction mixture was made up of 2 ml of leaf extract, diluted with 50 mM K2SO4 buffer at pH=7 and H2O2 10 mM, which reached 3 ml by distilled water, the reaction started and the absorbance of the samples were recorded for 1 min. Superoxide dismutase Superoxide dismutase activity (SOD) was evaluat- ed using a modified nitro-blue tetrazolium (NBT) method (Elavarthi and Martin, 2010). Accordingly, 2 ml of the reaction mixture including 50 mM phos- phate buffer (pH= 7.8), 2 mM EDTA, 9.9 mM methionine, 55 μM NBT (Nitro blue tetrazolium chlo- ride, C10H330Cl2N10O6 ), 0.025% triton X-100 to was added to 40 μl of diluted sample (×2) and then, added 20 μl of Riboflavin (1 mM). The reaction began by exposing the samples under a fluorescent tube (15 Watts) for 10 min. The blank received the same chemical mixture but without leaf samples through- out the steps. The absorbance of the specimens was read at 560 nm in a microplate reader spectropho- tometer and one unit of enzyme activity was taken as the quantity of enzyme, which reduced the absorbance reading of the sample to 50% in compari- son to control. Finally, SOD was calculated as U g-1 fresh weight. Chlorophyll stability index Chlorophyll stability index (CSI) was measured Adv. Hort. Sci., 2019 33(2): 161-170 164 according to the method used by Murty and Majumber (1962). The fresh leaf samples were emerged in 20 ml of distilled water and placed in a warm water bath of 56±1°C for 30 min, and the chlorophyll contents of the samples were determined. The chlorophyll stability index was obtained from the following formula. CSI (%)= 1-(chlorophyll content without heating/chlorophyll con- tent after heating) × 100. Chlorophyll content Total chlorophyll, chlorophyll a and chlorophyll b content of leaves were measured using the Dimethyl sulfoxide (DMSO) method introduced by Hiscox and Israelstam (1979). Leaf samples (100 mg fresh leaf) were submerged in 7 ml of dimethyl sulfoxide solu- tion and put in the dark for 17 h. After, they were incubated in an oven at 60°C. By adding 3 ml of dimethyl sulfoxide to the samples, the volume was adjusted to 10 ml. Then, 200 μl of the samples extract were transferred to a plate and read by microplate reader spectrophotometry at 633 and 645 nm wavelengths. The following formulas were used in order to determine the total chlorophyll concen- tration as mg g-1 fresh weight (FW). Ch a (mg g-1 FW)= 12.7(A663) - 2.69(A645) x volume made w.t of the sample x 10 Ch b (mg g-1 FW) = 22.9(A645) - 4.68(A663) x volume made w.t of the sample x 10 TCh (mg g-1 FW)= 20.2(A645) + 8.02 (A663) x volume made w.t of the sample x 10 In these formulas, Cha represents chlorophyll a, Chb = chlorophyll b, TCh = total chlorophyll; A = absorbance in the wavelength (nm). Cell membrane injury The cell membrane damage index, measured by electrolyte leakage, is an indicator of estimating the tolerance of cellular protoplasts and the ability of the membrane to maintain integrity under conditions of water scarcity (Bajji et al., 2002). This index was mea- sured using the method followed by Kocheva and Georgiev (2003). A punching machine was used in order to prepare leaf samples measuring 1 cm in diameter. The leaf sample discs of 1 cm in diameter were washed three times with distilled water to remove surface contamination. Then they were transferred to vials containing 20 ml of deionized water and kept at 10°C for 24 h. After measuring their electrical conductivity, they were placed in an autoclave of 120°C for 15 min, after cooling at 25°C, the electrical conductivity was re-measured. Cell membrane damage (CMI) was obtained from the fol- lowing formula. CMI ( %) = [1- ( T1 / T2)] / [1- (C1 / C2)]×100 T and C are the stressed and control samples, respectively, while 1 and 2 are the primary and sec- ondary EC measurements. The experiment had a factorial layout, based on 4 irrigation levels and 6 combinations of grafted and non-grafted plants in a randomized complete design with 5 replications and 2 plants per replicate. Data analysis was performed using SAS software version 9.3 and Duncan’s Multiple Range Test at 5% of proba- bility was used for the means comparison. 3. Results Analysis of variance showed that the interaction between cultivars and different levels of irrigation were significant on all measured traits except antho- cyanin (Table 1). Anthocyanin The results showed that increasing drought stress from 100% to 25% of WR the anthocyanin level increased in all grafted and non-grafted plants (Fig. Table 1 - The variance analysis of some traits of fig cultivars under water stress condition Source of variation df Mean square SOD CAT Ant GSH ASC Cultivar (A) 5 0.0259 ** 10.9 ** 0.008 ** 14.8 ** 68.3 ** Water stress (B) 3 0.4569 ** 5.5 ** 0.014 ** 3.8 ** 42.2 ** A × B 15 0.0293 ** 2.3 ** 0.0001 ns 3.3 ** 15.8 ** Error 96 0.0028 0.044 0.0001 0.55 1.29 C.V. - 3.63 19.33 14.07 22.41 10.64 SOD= Superoxide dismutase; CAT= catalase; GSH= glutathione; ASC= ascorbic acid; Ant= Anthocyanin. **significant at 1% probability, NS; non-significant. Keshavarzi and Shekafandeh - Scion antioxidants responses on different rootstocks 165 1A). A comparison of cultivars with no grafting (‘Sabz’, ‘Siah’, and ‘Torsh’) showed that ‘Torsh’ and ‘Siah’ had anthocyanin levels higher than the amount found in the ‘Sabz’. However, ‘Torsh’ rootstock increased the amount of the anthocyanins in the scion (‘Sabz’) compared to the non-grafted ‘Sabz’ cul- tivar (Fig. 1B). Glutathione The amount of glutathione in the leaves of non- grafted ‘Torsh’ increased with increasing the stress intensity from 100% to 25% of WR, while the ‘Siah’ cultivar reduced the glutathione significantly. The decrease in glutathione concentration in the leaf of the ‘Sabz’ non-grafted and the ‘Sabz’/‘Sabz’ combina- tion was not significant with increasing drought stress (Fig. 2A). Furthermore, both ‘Siah’ and ‘Torsh’ rootstocks significantly increased the glutathione level of the ‘Sabz’ scion at 75% of WR. However, there was no a significant difference in glutathione rate between 75%, 50% and 25% of WR in Sa/To combination. Ascorbic acid The results showed with increasing stress from 100% to 25% of WR, no increase in ascorbic acid con- tent was observed in non-grafted ‘Siah’ and ‘Sabz’ cultivars. The non-grafted ‘Torsh’ showed an increase of 47.63% in the ascorbic acid content in the severe water stress compared with control. The ‘Sabz’ and ‘Siah’ rootstocks reduced the ascorbic acid content of the ‘Sabz’ scion at 50% and 25% of WR, compared to the non-grafted ‘Sabz’. Nonetheless, ‘Torsh’ root- stock also reduced the ascorbic acid content of the ‘Sabz’ scion when 100% of WR was supplied, but this rootstock caused a significant increase in the ascorbic acid content (15.55%) of the ‘Sabz’ scion in the 25% of WR (Fig. 2B). Superoxide dismutase Superoxide dismutase activity increased with increasing drought stress in all non-grafted and graft- ed cultivars (Fig. 3A). Comparison of cultivars with no grafting showed that ‘Siah’ at 50% of WR had the highest enzyme activity compared with ‘Torsh’ and ‘Sabz’ cultivars. At 25% of WR, the SOD activity in the ‘Sabz’ rootstock was 1.78 times higher than the ‘Siah’ rootstock, and in the ‘Siah’ rootstock it was 1.63 times higher than in the ‘Torsh’ rootstock. Each of the three rootstocks reduced the activity of the enzyme compared to the non-grafted ‘Sabz’ cultivar when supplied with 50% and 25% of WR, although the dif- ferences in these values were not statistically signifi- cant (Fig. 3A). Catalase activity The activity of catalase enzyme increased signifi- cantly in all grafted and non-grafted cultivars in Fig. 1 - Main effects of irrigation levels (A) and grafted and non- grafted cultivars (B) on leaf anthocyanin rate. Sa= Sabz; Si= Siah; To= Torsh. Values are means ± SE (n=5). Fig. 2 - Interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50, 25% WR) on: Glutathione (A) and Ascorbic acid (B). Sa= Sabz; Si= Siah; To= Torsh. Values are means ± SE (n=5). 166 Adv. Hort. Sci., 2019 33(2): 161-170 response to intense drought stress (Fig. 3B). Comparison of non-grafted cultivars showed that catalase enzyme activity in ‘Torsh’ at 75%, 50% and 25% of WR was significantly higher than the two other cultivars (‘Sabz’ and ‘Siah’), which did not differ significantly. However, only ‘Siah’ rootstock increased the activity of catalase enzymes (by 31.13%) in the ‘Sabz’ scion when 25% of WR was applied compared to the non-grafted ‘Sabz’ cultivar. Analysis of variance showed that the interaction between cultivars and different levels of irrigation were significant on all measured traits (Table 2). Total leaf chlorophyll The results showed that in non-grafted cultivars, ‘Torsh’ at 100%, 75% and 50% of WR showed higher chlorophyll contents than the other two cultivars. In grafted rootstocks, ‘Siah’ and ‘Torsh’ significantly increased the chlorophyll content in the leaves of the scion ‘Sabz’ in 100% and 75% of WR. ‘Siah’ rootstock also increased the chlorophyll content of the ‘Sabz’ scion by 42.52% in 50% of WR compared with non- grated ‘Sabz’ (Fig. 4A). Chlorophyll stability index The results showed that the chlorophyll stability index decreased as the drought stress intensified Table 2 - The variance analysis of total Chl, CSI, CMI, shoot FW and Shoot DW of fig cultivars under water stress condition Fig. 3 - Interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50, 25% water requirement: WR) on Superoxide dismutase (SOD) acti- vity (A) and Catalase activity (B). Sa= Sabz; Si= Siah; To= Torsh. Values are means ± SE (n=5). Source of variation df Mean Square Total Chl CSI CMI Shoot FW Shoot DW Cultivar (A) 5 685.6 ** 5449.9 ** 469.7 ** 13355.4 ** 1895.5 ** Water stress (B) 3 1963.6 ** 8539.5 ** 11707.8 ** 22405.1 ** 2749.35 ** A × B 15 108.2 ** 134.7 ** 180.4 ** 691.71 ** 83.24 ** Error 96 12.45 41.75 26.47 119.36 19.38 C.V. - 23.83 12.45 5.59 13.03 12.77 Chl= chlorophyll; CSI= chlorophyll stability index; CMI= cell membrane injure; FW= fresh weight; DW= dry weight. **significant at 1% probability Fig. 4 - Interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50 and 25% WR) on leaf chlorophyll content (A) and chlorophyll sta- bility index (B). Sa= Sabz; Si= Siah; To= Torsh. Values are means ± SE (n=5). Keshavarzi and Shekafandeh - Scion antioxidants responses on different rootstocks 167 (Fig. 4B). Among the non-grafted cultivars, ‘Torsh’ showed more stability in the leaf chlorophyll content than the other two cultivars at different levels of WR. ‘Sabz’ scion on the ‘Siah’ and ‘Torsh’ rootstocks showed the higher chlorophyll stability than non- grafted ‘Sabz’ and ‘Sabz’/‘Sabz’ graft combination at all levels of WR (Fig. 4B). Cell membrane injury The results showed that high levels of water stress increased leaf cell membrane damage in all grafted and non-grafted plants (Fig. 5). The differences in the damage to the cell membranes of the ‘Siah’, ‘Torsh’ and ‘Sabz’ were not significant at 100% of WR, but the 75% and 50% of WR induced significantly greater damages to the leaf cell membrane of ‘Siah’ than the other two cultivars. At 25% of WR, the non-grafted ‘Sabz’ cultivar showed lower levels of membrane damage in its leaf cells, compared to ‘Siah’ and ‘Torsh’. The results also showed that the effect of the ‘Sabz’, ‘Torsh’, and ‘Siah’ rootstocks on the ‘Sabz’ scion was not significant at all levels of water require- ment, compared to the non-grafted ‘Sabz’ cultivar (Fig. 5). Shoot fresh and dry weight The results showed that with increasing water stress deficit the shoot growth decreased in all non- grafted and grafted combinations of plants. However, ‘Siah’ and ‘Torsh’ rootstocks are associated with higher scion shoot fresh and dry weight compared to non-grafted (Sa) and self-grafted (Sa/Sa) Sabz cultivar (Fig 6. A and B). 4. Discussion and Conclusions The increase in anthocyanin content in stressed leaves in this study confirms the protective role of anthocyanin against sunlight and active oxygen species in stressed plants (Gholami et al., 2012 a). Stimulation of anthocyanin production has been proven by osmotic pressure and it is believed (in most cases) that tissues that contain more antho- cyanins are more tolerant to drought (Chalker-Scott, 1999). Anthocyanins are able to protect cells against environmental damage through protection of cell membranes, organelles and nucleic acids (Neil et al., 2002). In this study, ‘Torsh’ rootstock increased the leaf anthocyanin in the ‘Sabz’ scion. Accordingly, the type of rootstock can increase the production of antho- cyanins and thus counteract the reactive species of oxygen, increasing the ability of the plant to tolerate conditions of water stress. In the green tissues of plants, ascorbic acid is a major antioxidant soluble in water. Leaf ascorbic acid changes seasonally, but as the leaf ages it remains at a constant level. However, exposure to stress can sig- nificantly change this situation and increase it (Sircelj and Batic, 2007). Ascorbic acid, like carotenoids, plays an important role in protecting the photosyn- thetic system against the harmful effects of reactive Fig. 5 - Interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50 and 25% of WR) on membrane injury index. Sa= Sabz; Si= Siah; To= Torsh. Values are means ± SE (n=5). Fig. 6 - Interaction of fig grafted and non-grafted combinations and different levels of irrigation (100, 75, 50 and 25% WR) on shoot fresh weight (A) and shoot dry weight (B). Sa= Sabz; Si= Siah; To= Torsh. Adv. Hort. Sci., 2019 33(2): 161-170 168 oxygen species (ROS). In this research, the amount of glutathione and ascorbic acid increased in ‘Torsh’ rootstock, and this effect was transmitted to the ‘Sabz’ scion, which may be due to the rapid reaction of this rootstock to severe drought stress and the need for glutathione and ascorbic acid, which can confront ROS more efficiently. Even though the changes in glutathione and ascorbic acid were not significant at different levels of irrigation in the non- grafted ‘Sabz’ cultivar, by grafting the ‘Sabz’ scion on the ‘Siah’ rootstock, the glutathione level in the ‘Sabz’ scion increased significantly in 75% of water requirement and did not show any changes in ascor- bic acid. In this regard, Tausz et al., (2004) also reported that drought stress leads to a decrease in glutathione concentration, and the oxidation and reduction of compounds ultimately leads to the sys- tem’s degradation. An increase in the amount of glu- tathione in conditions of water shortage may be nec- essary to adjust the level of ascorbic acid in the plant (Gholami et al., 2012 b). It seems that increased levels of glutathione in non-grafted ‘Torsh’ cultivar and ‘Sabz’ grafted on ‘Torsh’ rootstock have been effective in regulating ascorbic acid levels at mild and severe stress levels. Sircelj et al. (2005) also reported an increase in the level of glutathione and ascorbic acid at moderate levels, indicating compatibility with oxidative stress in apple trees, and stated that the decrease in glu- tathione levels at severe stress levels indicates severe oxidative stress. Many researchers have shown that drought stress leads to oxidative stress in the plant (Mittler, 2002; Gill and Tuteja, 2010; Gholami et al., 2012 b; Shirbani et al., 2013). Excessive forms of stress can damage the plant by producing reactive oxygen species. The antioxidant defense system and the breakdown of active oxygen species are known to be under dry con- ditions (Zarafshar et al., 2014). Under mild and mod- erate water deficit conditions, a number of compati- ble plant species increase the activity of enzymatic antioxidants such as superoxide dismutase and cata- lase, although severe drought stress may cause dam- age to cells by stronger stimulations or impairments via reactive oxygen species that suppress enzymatic antioxidant activity (Guerfel et al., 2008). Superoxide dismutase and catalase are active enzymes for the elimination and degradation of harmful oxygen species in plants and maintain oxida- tive equilibrium during oxidative stress (Gill and Tuteja, 2010). Superoxide dismutase enzymes have been reported to play an important role in the antioxidant metabolism of plants under environmen- tal stress conditions, such as water deficit, via regu- lating their gene expression or activities (Xu et al., 2010). High SOD activity in ‘Siah’ and ‘Sabz’ non- grafted cultivars were achieved in 50% and 25% of WR respectively, and the activity of catalase in the non-grafted ‘Torsh’ cultivar and in the ‘Sabz’/‘Siah’ graft combination (at 25% of WR) were also compar- atively high. These results depend on the plant’s bet- ter protection against oxidative damage caused by water stress. It seems that the scion of the ‘Sabz’ cul- tivar on the ‘Siah’ rootstock led to greater improve- ments in the antioxidant system. Chlorophyll content has a positive correlation with the rate of photosynthesis. Therefore, the decrease in chlorophyll content under drought stress condition is a common symptom of oxidative stress, which may be due to photo-oxidation of pigments and chloro- phyll degradation (Anjum et al., 2011; Shirbani et al., 2013). The reduction of chlorophyll content was observed in all cultivars under stress in this study. According to Guerfel et al. (2009) the reduction of chlorophyll content can be attributed to its suscepti- bility to environmental stresses, especially drought. Chlorophyll levels tend to decrease or otherwise remain unchanged during the period of drought stress in many species. This, however, depends on the duration and severity of the drought (Anjum et al., 2011). In this study, chlorophyll contents at 75% and 50% of WR decreased less sharply in ‘Sabz’/Siah graft combination. This indicates that the light dissipation and antioxidant systems may prevent the degrada- tion of chlorophyll molecules (Niu et al., 2008). Sircelj et al. (2005) also reported no reduction in the chloro- phyll content of apple leaves ‘Elastar’ under water stress is due to a strong antioxidant system and an efficient light dissipation system. Anjum et al. (2011) attributed the decrease in the content of chlorophyll to the chloroplast membrane damage, swelling, lamella distortion and the occurrence of small droplets of lipids under drought stress conditions. The chlorophyll stability index is an indicator of measuring the membrane’s integrity and the pig- ments stability under stress conditions (Ananthi et al., 2013). Surendar et al. (2013) reported that a decrease in chlorophyll content under stress was due to the destruction of the chloroplast membrane with increasing phosphatase activity, which is located on the membrane. In this study, intense water stress (25% of WR) reduced the chlorophyll stability in all grafted and non-grafted rootstocks compared to Keshavarzi and Shekafandeh - Scion antioxidants responses on different rootstocks 169 100% of WR. This could be due to the degradation of chlorophyll when proteolytic enzymes, such as the chlorophyllase enzymes, are produced. The values of the high chlorophyll stability index in the non-grafted ‘Torsh’ cultivar and the ‘Sabz’/‘Siah’ or ‘Sabz’/’Torsh’ graft combinations indicate a higher chlorophyll con- tent in the leaves, which leads to an increase in the rate of photosynthesis and the production of more dry weight which helps the plant with stand dehydra- tion (Babu et al., 2008; Ananthi et al., 2013; Surendar et al., 2013). Under conditions of water scarcity, the cell mem- brane of the leaves are subject to changes such as increased permeability and reduced selectivity, which can be observed through an increase in elec- trolyte leakage (Zarafshar et al., 2014). In this study, the overall increase for leakage under stressful condi- tions was observed in all grafted and non-grafted rootstocks as compared to fully irrigated plants. The non-grafted ‘Siah’ cultivar experienced a greater damage to the cell membrane of its leaf, com- pared to the non-grafted ‘Sabz’ and ‘Torsh’ cultivars. This indicates that the rootstock’s ability to maintain the integrity of its cell membrane in severe stress con- ditions is considered an important factor in determin- ing tolerance to drought (Bolat et al., 2014). Undesirable performance of the cell’s metabolism during periods of drought stress leads to the stimula- tion of reactive oxygen species or the disruption of systems that prevent or reduce the activity of reactive oxygen species, which would damage the cell mem- brane and increase electrolyte leakage (Guerfel et al., 2008; Karimi et al., 2013). Therefore, the fact that damage to the cell membranes of the non-grafted ‘Sabz’ cultivar occurred less than in the non-grafted ‘Siah’ rootstock may indicate a more tolerance to drought in the ‘Sabz’ cultivar. However, Shahidi-Rad et al. 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