Impaginato 131 1. Introduction The ability of plants to tolerate salinity stress is facilitated by a series of biochemical pathways which maintain or absorb water, protect plants chloroplast function and sustain an ionic balance. Some of these pathways include the synthesis of active osmotic metabolites (Zhifang and Loescher, 2003). Some pro- teins and enzymes destroy free radicals (Mittova et al., 2003). Carica papaya L. is the only species belonging to the Carica genus. It can be found in tropical region of America. The cultivation of this crop is common in southern Mexico, Central America and South America, as well as most countries in the tropics. Papaya is a fast-growing tree (it produces fruit in the third year after planting), extremely sensi- tive to cold, and planted only in the tropics. Papaya fruit is rich in carotenoids, vitamins B, C, lycopene and mineral fibers. The skin, flesh and seeds of this product contain a number of phenolic compounds. Salinity stress in soil or water, especially in hot, arid regions, could limit plant growth and reduce its yield (Koca et al., 2007). Plants growing in areas with extreme salinity are divided into halophytes and gly- cophytes. Most glycophyte plants do not have the ability to tolerate salinity stress (Sairam and Tyagi, 2004). During salinity stress, all the main processes, including photosynthesis, lipid metabolism and ener- gy, are affected (Sairam and Tyagi, 2004). The first response is to reduce the development rate of leaf area and then complete cease. However, growth process resumes as soon as the problem is fixed (Parida and Das, 2005). The plant either tolerates the stress, or avoids it. The former generally occurs at the cell level while the latter occurs at the plant level. During salinity stress, a plant can undergo dormancy (avoidance) or make certain cellular adjustments in order to resist drought stress (Yokoi et al., 2002). The Adv. Hort. Sci., 2017 31(2): 131-139 DOI: 10.13128/ahs-21090 Effects of salinity stress on certain morphological traits and antioxidant enzymes of two Carica papaya cultivars in hydroponic culture A. Refahi, A.R. Shahsavar (*) Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran. Key words: antioxidant enzymes, Carica papaya, hydroponic, morphological factors, salinity stress. Abstract: Carica papaya L. is the only species belonging to the Carica genus. Salinity stress in soil or water, especially in hot, arid regions, could limit plant growth and reduce its yield. This research studied six-month old seedlings of two culti- vars of papaya (‘Sinta’ and ‘Solo’), in solid, disease-free form for two weeks inside a half-dose of Hoagland solution. Results obtained from the effects of salinity stress indicated that the longest root and shoot were observed in the control treatment in ‘Sinta’. Moreover, there was no significant difference between the two cultivars in terms of root length, shoot length, fresh weight of roots and fresh weight of shoots in different salinity levels. The highest dry weights of roots and shoots were found in the ‘Sinta’ control treatment, while the lowest was observed in ‘Solo’ 6 dS/m treatment. There was no significant difference between the two cultivars in terms of dry weights of roots and shoots. Finally, the interac- tion of salinity levels showed that increasing salinity in both cultivars led to higher peroxidase, catalase, superoxide dis- mutase and ascorbic peroxides activity. By increasing the salinity level, the total protein and proline greatly increased in both cultivars, where the maximum value was found in the ‘Sinta’ 6 dS/m salinity treatment, and this was significantly different from other treatments. A comparison of the different salinity levels showed that there was a significant differ- ence between the 6 dS/m salinity treatment and other treatments. (*) Corresponding author: shahsava@shirazu.ac.ir Received for publication 16 January 2017 Accepted for publication 21 June 2017 Copyright: © 2017 Author(s). This is an open access article 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. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2017 31(2): 131-139 132 aim of this study is to survey the effect of salinity stress on certain morphological traits and also on antioxidant enzymes of two Carica cultivars in hydro- ponic culture condition. 2. Materials and Methods Six-month old seedlings of two cultivars of papaya (‘Sinta’ and ‘Solo’), solid and disease-free, were placed for two weeks in a glass containing 700 ml of a half-dose Hoagland solution, and were then trans- ferred to the hydroponic system. Afterwards, 500 ml of the half-dose Hoagland solution per seedling was added to the hydroponic system. After two weeks, the salinity treatments were initiated at pH=6 as salinity stress continued for 8 weeks. Then, the leaf samples were placed in aluminium foil and frozen at - 20°C for further measurements of protein and antioxidant enzymes. Plant height was measured with a ruler. At the end of the experiment, the fresh weight of plant organs was measured and then rinsed through distilled water and finally kept in the oven at 70°C until the dry weight was stabilized. Then, shoots and roots dry weights were measured. A factorial experiment was conducted based on com- pletely randomized design with 5 replications in the greenhouse of the Horticultural Science Department, University of Shiraz. The factors included salinity treatments: 0 (Control), 2, 4, 6, 8 and 10 dS/m NaCl which were added to half dose Hoagland solution and 2 cultivars of papaya (‘Sinta’ and ‘Solo’). Data analysis was done using SAS (version 9.2; SAS Institute, Cary, NC, USA), mean comparisons were carried out using LSD test at 5% of probability. Extraction for measuring the amount of protein and antioxidant enzymes For extraction, 0.5 g of root or leaf sample was first ground in liquid nitrogen and then 2 ml of extraction buffer was added and homogenized in a porcelain mortar. Then this mixture tube was cen- trifuged at 13,000 rpm for 15 min at 4°C. The upper phase was isolated for the purpose of reading the protein content and enzyme activity. For the prepa- ration of the extraction buffer (50 ml), 0.607 g of tris(hydroxylmethyl)aminomethane and 0.05 g of Polyvinylpyrrolidone (PVP) were dissolved into 45 ml of distilled water (pH 8.0). Total protein The Bradford assay (1976) was used to determine the protein concentration. To measure the protein concentration, 20 ml extract was diluted in 80 µl of extraction buffer; 5 ml of fresh reagent Coomassie was added, stirred for 2 min, and finally, after 5 min, the optical density was read at a wavelength of 595 nm. Besides, the extraction buffer was used as con- trol. The concentration of protein in the sample was obtained according to the absorption, using the standard curve. Bovine serum albumin was used as the standard and total soluble protein concentra- tions were expressed in mg g -1 fresh weight. Proline The method of Bates et al. (1973) was employed to measure the concentration of proline. According to this method, 0.5 g of leaves from each sample was placed in 10 ml of an aqueous solution of sulfosali- cylic acid (3%) and the mixture was completely homogenized in a porcelain mortar. Then, the homogenized mixture was filtered through paper no. 2. In the next stage, 2 ml of solution was mixed with 2 ml of a reagent, creatininedimenhydrinate and 2 ml of acetic acid was added to each tube. Then, the samples were placed in bain-marie bath for 1 h at a temperature of 100°C and were immediately trans- ferred into an ice bath for a few minutes. Afterwards, 4 ml of toluene was added to each tube and the sam- ples were stirred through Vortex for 15 to 20 s until they were completely homogeneous. The super- natant phase was used to determine proline concen- tration based on the proline standard curve in the spectrometer at a wavelength of 520 nm. Proline concentration was calculated using L-proline for the standard curve. Guaiacol peroxidase (POD) activity To measure the quantitative concentrations of this enzyme, the method of Chance and Mahly (1955) was used with minor modifications. Measurements were done according to the oxidation of guaiacol by that enzyme. In this method, 33 mol of extract was dissolved into 1 ml of a peroxidase solu- tion containing 13 Mm guaiacol, 5 mM hydrogen per- oxide (H2O2) and 50 mM potassium phosphate buffer (pH=7), and the absorbance values were read for one minute at 10-s intervals and at a wavelength of 470 nm. To prepare 1000 ml of potassium phosphate buffer, 39 ml potassium phosphate and 50 mM monohydrate were mixed with 61 ml potassium dihy- drogen and 50 mM phosphate. Ascorbic peroxidase (APX) activity To measure the quantitative concentrations of this enzyme, the method of Nakano and Asada Refahi and Shahsavar - Salinity stress on morphological traits and antioxidant enzymes of two Carica papaya cultivars 133 (1981) was used. According to this method, 50 ml of the extract was mixed with 1 ml of ascorbic peroxi- dase containing 50 mM potassium phosphate buffer (pH=7), 0.1 mM EDTA, 0.5 mM ascorbic acid (ASA), and 0.15 mM peroxide hydrogen (H2O2). Then, the absorption at a wavelength of 290 nm was read with- in one minute through a spectrophotometer. An enzymatic unit of ascorbic peroxidase is equivalent to a dissolution of 1 mM ascorbic acid in a minute. Catalase (CAT) activity To measure the quantitative concentrations of this enzyme, the method of Nakano and Asada (1981) was used. According to this method, 50 ml of the extract was mixed with 1 ml of catalase contain- ing 50 mM potassium phosphate buffer (pH=7) and 15 mM hydrogen peroxide (H2O2). Then, the absorp- tion at a wavelength of 240 nm was read within one minute through a spectrophotometer. An enzymatic unit of catalase is equivalent to a dissolution of 1 mM hydrogen peroxide (H2O2) in a minute. Superoxide dismutase (SOD) activity To measure the quantitative concentrations of this enzyme, the method of Beauchamp and Fridovich (1971) was used. The measurement was based on the ability of SOD enzyme to stop the pho- tochemical reduction of NBT by superoxide radicals in the presence of riboflavin in light. According to this method, 50 ml of the extract was mixed with 1 ml of superoxide dismutase containing 50 mM potassium phosphate buffer (pH 7.8), 75 mM of NBT, 13 mM of L-methionine, 0.1 mM of EDAT and 2 mM riboflavin. It should be noted that the solution was stored sepa- rately in a dark container and after the addition of soluble extract and measurement solution, superox- ide dismutase was added to Qt. The mixture reacted when placed in the light chamber for 15 min. The solution was then placed in a spectrophotometer and the absorbance was measured and read at a wave- length of 560 nm. 3. Results The interaction between salinity levels and culti- vars on root length showed that the highest root length was found in the control treatment in Sinta, while the minimum value was found in the 6 dS/m treatment of both ‘Sinta’ and ‘Solo’. Seedlings of both cultivars in the 8 and 10 dS/m treatments exhib- ited no salinity resistance and were dried, therefore, these two treatments were excluded from the results; results also showed no significant differences between the two cultivars, in terms of root length, at various salinity levels (Fig. 1). As shown in figure 2, the highest shoot length was observed in the ‘Sinta’ control treatment, which was significantly different from other treatments. Moreover, there was no significant difference between the two cultivars in terms of salinity levels. The control group of both cultivars generally had a significant difference with other treatments, while there existed no significant difference between con- centrations of 2, 4 and 6 dS/m. Evaluation of salinity effect on roots fresh weight between the two cultivars showed that the highest and lowest values were observed in the control treat- ment and the Sinta 6 dS/m treatment, respectively. There was no significant differences between the two cultivars in terms of root fresh weight. The compari- son of the various salinity levels, showed a significant difference between the control and other salinity treatments, while there was no significant difference Fig. 1 - Interaction of cultivar and salinity levels on roots length. The means followed by the same letters were not signifi- cantly different at p≤0.05. Fig. 2 - Interaction of cultivar and salinity levels on shoots length. The means followed by the same letters were not significantly different at p≤0.05. Adv. Hort. Sci., 2017 31(2): 131-139 134 between treatments 2, 4 and 6 (dS/m) (Fig. 3). In this study maximum root dry weight was observed in the ‘Sinta’ control (Fig. 4) while the mini- mum was observed in the ‘Solo’ 6 dS/m treatment. The comparison between the two cultivars showed a significant difference between them in terms of roots dry weight. The mean salinity concentrations indicat- ed that the control had a significant difference with other concentrations, while there was no significant difference between concentrations of 2 and 4 dS/m. Evaluation of treatments applied in this study on shoots fresh weight indicated that control treatment in both ‘Sinta’ and ‘Solo’ had the highest values, while treatments 4 and 6 dS/m had the lowest val- ues. A comparison of the two cultivars showed no significant differences in term of shoot fresh weight. In the comparison of salinity concentrations, no sta- tistically significant difference was found between the control and 2 dS/m (Fig. 5). According to figure 6, the findings of this study regarding the shoots dry weight showed that the highest dry weight was found in the control ‘Sinta’, where there was no significant difference between this treatment and the 2 and 4 dS/m salinity treat- ments. The lowest shoot dry weight was found in the ‘Solo’ 6 dS/m salinity treatment. A comparison of the two cultivars represented a significant difference in the shoots dry weight. Moreover, the comparison of various salinity levels showed no significant differ- ence between the control and 2 dS/m salinity treat- ments for both cultivars. The interaction effect of cultivar and salinity levels in this study showed that an increase in the salinity levels enhanced peroxidase activity in both cultivars. The highest enzyme activity was observed in ‘Sinta’ 6 dS/m salinity treatment while the lowest was in ‘Solo’ control treatment. A comparison of values between the cultivars showed a significant difference in terms of peroxidase activity. Moreover, the com- parison of various salinity levels showed a significant difference between the 6 dS/m salinity treatment and other treatments for both cultivars (Fig. 7). According to figure 8, the increased salinity levels in both cultivars enhanced catalase activity. The max- imum level of this enzyme was found in the ‘Sinta’ 6 Fig. 3 - Interaction of cultivar and salinity levels on root fresh weight. The means followed by the same letters were not significantly different at p≤0.05. Fig. 4 - Interaction of cultivar and salinity levels on root dry weight. The means followed by the same letters were not significantly different at p≤0.05. Fig. 5 - Interaction of cultivar and salinity levels on shoot fresh weight. The means followed by the same letters were not significantly different at p≤0.05. Fig. 6 - Interaction of cultivar and salinity levels on shoot dry weight. The means followed by the same letters were not significantly different at p≤0.05. Refahi and Shahsavar - Salinity stress on morphological traits and antioxidant enzymes of two Carica papaya cultivars 135 dS/m salinity treatment, which showed a significant difference with other treatments, while the minimum values were observed in the control treatment of both cultivars. A comparison of the two cultivars showed no significant differences. In the showed results, there was a significant difference between the different levels of salinity in the 6 dS/m treat- ment and the control 2 dS/m, while there was no sig- nificant difference when compared to the 4 dS/m treatment. In this study, the interaction effect of salinity stress on superoxide dismutase activity showed an increase at higher salinity levels in both cultivars. The maximum activity of this enzyme was found in the Solo 6 dS/m salinity treatment which was not signifi- cantly different when compared with other treat- ments and the ‘Sinta’ 4 and 6 dS/m salinity treat- ments. The comparisons between the cultivars were significantly different. The comparison of different salinity levels showed no significant difference between the 4 and 6 dS/m treatments (Fig. 9). Results reported in figure 10 showed that as salin- ity levels increased, the ascorbic peroxidase activity also increased. An evaluation of the interaction of salinity levels on the enzyme activity showed that the highest value was found in Sinta 6 dS/m treatment while there was no significant difference with the same treatment in ‘Solo’. The comparison between the cultivars showed no significant difference. Moreover, a comparison of various salinity levels showed a significant difference between 6 dS/m salinity treatment and other treatments for both cul- tivars. From figure 11, the interaction effect of salinity levels on activity of total protein in this study showed that as salinity levels increased there was higher pro- tein content in both cultivars. The maximum value was observed in ‘Sinta’ of 6 dS/m salinity treatment, which was significantly different from other treat- ments, while the lowest was in the control for the two cultivars which were not significantly different. No significant difference was found when the culti- Fig. 7 - Interaction of cultivar and salinity levels on peroxidase activity. The means followed by the same letters were not significantly different at p≤0.05. Fig. 8 - Interaction of cultivar and salinity levels on catalase activity. The means followed by the same letters were not significantly different at p≤0.05. Fig. 9 - Interaction of cultivar and salinity levels on superoxide dismutase activity. The means followed by the same let- ters were not significantly different at p≤0.05. Fig. 10 - Interaction of cultivar and salinity levels on ascorbic per- oxidase activity. The means followed by the same letters were not significantly different at p≤0.05. Adv. Hort. Sci., 2017 31(1): 131-139 136 vars were compared. As for the various salinity levels, the results showed that the 6 dS/m salinity treatment was significantly different from other treatments. Evaluation of interaction effect between salinity levels on proline showed an increase in salinity level in both cultivars, followed by higher proline. In fact, the maximum amount of proline was observed in the Sinta 6 dS/m salinity treatment which was significant- ly different from other treatments. There was no sig- nificant difference when both cultivars were com- pared. Moreover, the comparison of various salinity levels showed a significant difference between the 6 dS/m salinity treatment and other treatments for both cultivars (Fig. 12). 4. Discussion and Conclusions As mentioned in the results, the vegetative indica- tors decreased upon initiation of salinity treatments. Literature indicates that plants, especially glyco- phytes, are highly sensitive during their early vegeta- tive growth, for instance chickpea (Khan et al., 2016), pepper (Penella et al., 2016), grapevine (Ikball et al., 2014), tomato (Manai et al., 2014), rice (Horie et al., 2012), cantaloupe (Botia et al., 2005). Increased salt leads to Na+ sediment into root growth area, thus reducing the ability to select K+ versus Na+ for root cells, which ultimately reduces their growth rate (Zhong and Lauchli, 1994). When exposed to saline conditions, plants show reduced uptake and low tis- sue retention of K+. (Chakraborty et al., 2012; Gharsallah et al., 2016). Accordingly, K+ is considered as a key regulatory elements in plant metabolic process by promoting Na+ exclusion and osmotic adjustment (Chakraborty et al., 2016; Gharsallah et al., 2016). Reduced shoot growth due to salinity usu- ally appears as shoots with low growth and reduced leaf area (Lauchli and Epstein, 1990). Saline condi- tions reduce root growth and reduce water move- ment through the root with a decrease in hydraulic conductivity (Acosta-Motos et al., 2017). Root hydraulic conductance is expressed in terms of root dry weight. Root dry weight values which determine the root length and surface area, may vary greatly, thus affecting the water absorption (Jonathan et al., 2006; Zobel et al., 2007; Acosta-Motos et al., 2017). The results of our study are in accordance with these results. Decreasing in fresh weight or dry weight has been observed in all plant tissues subjected to salt stress especially in the aerial part (Acosta-Motos et al., 2017). The stem growth is also reduced by salinity conditions. One of the important reason for decreas- ing root and shoot growth under saline condition could be the decreasing of nitrogen uptake in response to external NaCl salinity due to antagonism between Na + and NH4 + or between Cl- and No3 - (Parihar et al., 2015; Salachna and Piechocki, 2016). Another reason responsible for the reduction of veg- etative index in the shoots, similar to what was men- tioned for roots, is ion imbalance and increased ratio of Na to Ca (Neves-Piestun and Bernstein, 2005). In the current experiments, the concentration of pro- line, which has an important role in eliminating free radicals and enzymes, increased at higher salinity lev- els. Accumulation of proline under salinity conditions has been indicated to correlate with salt tolerance (Mansour and Ali, 2017). In fact, to eliminate osmotic stress created by high salinity, plants need to synthesize compatible organic solutes such as proline in the cytosol (Gharsallah et al., 2016). Besides its role as an osmolyte, proline con- tributes to scavenging ROS, stabilizing sub cellular structures and functioning as a signal (Szabados and Fig. 11 - Interaction of cultivar and salinity levels on protein con- tent. The means followed by the same letters were not significantly different at p≤0.05. Fig. 12 - Interaction of cultivar and salinity levels on proline. The means followed by the same letters were not significant- ly different at p≤0.05. Refahi and Shahsavar - Salinity stress on morphological traits and antioxidant enzymes of two Carica papaya cultivars 137 Savoure, 2010). Generally, plants under salinity con- ditions need to sustain their water potential (below potential of ground water) because they can contin- ue absorbing water from the soil in order to maintain their turgor (Tester and Davenport, 2003). To main- tain osmotic potential as well as ionic balance, plant cells during stress tend to accumulate any substance compatible with metabolism which does not inter- fere with other biochemical processes and are active in osmotic terms (Zhifang and Loescher, 2003). These substances cover a wide range of compounds such as carbohydrates, proteins and amino acids, including proline. This amino acid accumulates at a higher con- centration than other amino acids in plant cells (Abraham et al., 2003). It is possible that proline as a signaling molecule or regulator can activate the response as an adjustment process (Maggio et al., 2002). Hence, given the foregoing facts, there is a positive relationship between proline and curtailed damage of salt sensitivity, where the current study was consistent with previous works. In relation to salt damage to the overall growth of plant, the con- centration of enzymes eliminating free radicals is very important. In their resistance against the dam- aging effects of reactive oxygen species, plants pos- sess anti-free radical enzymes such as catalase, per- oxidase, superoxide dismutase and other enzymes which eliminate reactive oxygen species and free rad- icals (Mittova et al., 2003). In our study, the concen- tration of eliminating free radicals enzymes such as peroxidase, catalase, superoxide dismutase and ascorbic peroxidase increased at higher salinity lev- els. The increases in theses enzymes activities are an adaptive trait to overcome salt damage by reducing toxic levels of H2O2 and provide protection against oxidative stress (Chawla et al., 2013; Gharsallah et al., 2016). Catalase, ascorbic peroxidase and gluta- tion peroxidase have been reported as antioxidant enzymes in different plant tissues (Chawla et al., 2013). During salinity stress, the balance between pro- duction and consumption of reactive oxygen species (ROS) is disrupted, leading to the formation of con- densation oxide (Spychalla and Desborough, 1990). ROS has the potential to damage cellular structures, perchloric acids, fats and proteins (Valko et al., 2006). In some salt-tolerant plants, increased in catalase activity have been recored after increasing NaCl, such as those described in myrtle, suggesting increased photorespiratory activity (Acosta-Motos et al., 2015, 2017). Catalase is often related to an enhanced toler- ance to salt stress (Gao et al., 2008; Gharsallah et al., 2016). Similarly, ascorbate peroxidase activity under salinity stress increases (Mittova et al., 2004; Gharsallah et al., 2016). These responses to salinity were the results of differentially increased activities of ascorbate peroxidase and catalase over that of superoxide dismutase (Mittova et al., 2004; Gharsallah et al., 2016). The results of our study were in accordance with these results. The results of this study indicated also that as salinity levels increased, there was higher protein content in both cultivars. This result was in agree- ment with that of Abdel-Haleem (2007) who report- ed as increase in protein band which might be involved in mungbean tolerance. Plants growing in saline environments show distinct changes in the pattern of synthesis and accumulation of proteins. Salinity causes either decreased or increase in the level of soluble proteins or completely disappears in some proteins when compared to the control treat- ment (Win and Zaw, 2017). Salinity is an important stress in arid and semi- arid region that reduces crops productivity, including that of the cultivars of Carica papaya L. here investi- gated (‘Sinta’ and ‘Sola’). Shoots and roots growth were decreased under salinity conditions. There was no significant difference between the two cultivars in terms of root length, shoot length, fresh weight of roots and shoots in different salinity levels. Interestingly, on the other hand, in both cultivars increasing salinity led to higher peroxidase, catalase, superoxide dismutase and ascorbic peroxidase activi- ty to provide protection against oxidative stress. Also the increases in those enzymes activities are an adaptative trait to overcome salt damage by reduc- ing toxic levels of H2O2. Furthermore, in this experi- ment the concentration of proline, which has an important role in eliminating free radicals, increased at higher salinity levels in both cultivars; plants need to synthesize compatible organic solutes such as pro- line. Finally, in this study increasing salinity led to higher protein content in both cultivars; proteins play a major role in salt stress acclimation and plant cellular adjustment. Salinity causes either decrease or increase in the level of soluble proteins. 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