Int. J. Biosci. 2014

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print) 2222-5234 (Online) http://www.innspub.net Vol. 5, No. 7, p. 116-125, 2014

RESEARCH PAPER OPEN ACCESS

Evaluation of two grape cultivars (Vitis vinifera L.) against salinity stress and surveying the effect of methyl and epibrassinolide on alleviation the salinity stress

S. Neda Seif1, Enayat Tafazzoli2 *, Ali-Reza Talaii3, Abdolhossein Aboutalebi4, Vahid Abdosi1

1Department of Horticultural Science, Science and Research Branch, Islamic Azad University, Tehran, Iran 2Department of Horticulture Science, Science and Research Branch, Islamic Azad University, Fars branch, Fars, Iran 3Department of Department of Horticultural Science, Tehran University, Tehran, Iran 4Department of horticultural science, Jahrom branch, Islamic Azad University, Jahrom, Iran

Key words: Vitis vinifera L., cultivar, salinity, Epibrassinolide, Methyle Jasmonate.

http://dx.doi.org/10.12692/ijb/5.7.116-125 Article published on October 06, 2014

Abstract

Salinity is a phenomenon challenging the plantation and growth of grape in arid and semiarid regions. During the present research, tolerance of two grape cultivars(Flame Seedless and Perlette) was evaluated against various sodium chloride salinity levels (0, 25, 50, 75 and 100 mM) and the effect of Epibrassinolide(EBR)(0, 3 and 6 µM) and Methyle Jasmonate(MeJA)(0, 3 and 6 mM) surveyed at these conditions which was conducted based on factorial experiment in the form of Complete Randomized Desighn(CRD) with four Replications. Based on the obtained results, the cultivar, salinity levels and hormonal treatments were significantly effective on surveyed traits. The results from analysis of variance revealed that increased salinity levels led to significant increase in values of lipid peroxidation, electrolyte leackage(EL), proline content and significant decrease in values of photosynthesis and transpiration rate, relative water content(RWC) and the content of chlorophyll a and b in two varieties. Furthermore EBR and MeJA treatments(specially 6mM MeJA) significantly increased proline content, content of chlorophyll a and b, photosynthesis and transpiration rate and decreased EL and lipid peroxidation in two varieties(except at high salinity stress) and mitigated the adverse effects of NaCl in two cultivars. Withoute salinity application Perlette cultivar produced the best values for physiological and morphological indices. In general, Perlette cultivar proved more tolerance against salinity than Flame Seedless cultivar did. * Corresponding Author: Enayat Tafazzoli  [email protected]

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Introduction stresses(Rao et al., 2002). These studies have Iran, the second largest country in the middle East, focused on relieving or overcoming stresses related to has an area of 165 million ha. Approximately, 90% of temperature or water stress for example, the country is classified as arid and semi-arid region, epibrassinolide treatment enhanced tolerance of most of which is faced with low rainfall, high tomato and brome grass to high temperature(Singh evapotranspiration, salinization, shortage of fresh and Shono, 2005). Brassinolide kept rice seedlings in water, erosion, excessive heat and desertification. normal physiological function under high Fresh water resources are declining in the central temperature on rice effectively(Cao and Zhao, 2008). plateau of the country as a result of overusing were shown to have positive effects underground water and sever drought in recent on eggplant, cucumber and maize under chilling years(Cheraghi, 2004). stress(Mandava, 1988; He et al., 1991). In rice, brassinosteroids increased seedling resistance to Land salinization is a major limiting factor for chilling injury(Wang and Zeng, 1993) and increased conventional crop production in the country. the height, root length, root biomass and total Continnuos cropping together with an excessive use biomass of rice under low temperature of chemifertilizers an ill-managed irrigation has conditions(Kim and Sa, 1988; Hirai et al. 1991). Wang turned hundreds of cultivated fertile fields into saline and Zeng(1993) also reported that treatment with 24- ones. These limitations have greate impacts on the epibrassinolide reduced the MDA content and welfare of the farmers whose income is soley increased the proline content of rice under chilling dependent to agriculture. In recent years, increased stress. The increase in chilling resistance was attention has been paid to the use of saline soils and attributed to brassinolide-induced effects on waters for crop production(Banakar and Ranjbar, membrane stability and osmoregulation(Wang and 2010). Zeng, 1993). Schilling et al. (1991) reported that homobrassinolide icreased tap root weight, sucrose Grapevines are considered as moderately sensitive to content and yield of sugar beets grown under drought salinity and the damage is primilarly caused by stress. Similarly, Sairam(1994) found that chloride ions(Walker, 1995). However, grapevine homobrassinolide significantly increased the relative response to salinity depends on several factors, such water content, chlorophyll a content, photosynthetic as rootstock-scion combination, irrigation system, rate, leaf area and biomass production of wheat under soil type and climate. Changing some of these factors moisture stress. Brassinolide was also applied as with the same irrigation water could produce entirely plant regulator on maize to increase tolerance to different results(Fisarakis et al. 2001). drought stress(Li and Van Staden, 1998a, b). Several studies have investigated the effect of Exogenous application of some phytohormones could brassinosteroids on salt-stressed plants. Uner saline also affect directly the plant response to salinity. conditions, 24-epibrassinolide prevented nucleus and Brassinosteroids are natural substances that are chloroplast degradation in wheat by producing a essential for plant growth and development. It is well protective effect on leaf cell ultrastructure(Kulaeva et documented that brassinosteroids can induce abroade al., 1991). The treatment of rice seeds with 24- spectrum of responses among plants, including stem epibrassinolide or 28-homobrassinolide promoted elongation, pollen tube growth, leaf epinasty and yield germination under salt stress. Lengths, fresh and dry increase(Clouse and Sasse, 1998; Kamuro and weights and soluble protein content of the resulting Takatsuto, 1999; Khripach et al., 2000; Mandava, seedlings were enhanced(Anuradna and Rao, 2001). 1998; Sasse, 1999). One of the most interesting Sasse et al. (1995) reported the ability of 24- influences of brassinosteroids is their ability to epibrassinolide to activate the germination of confer resistance to plants against abiotic Eucalyptus camaldulensis seeds under saline stress.

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Application of brassinosteroids increased the salinity private company at jahrom city. After rooting, tolerance in rice, tomato and chickpea(Anuradna and nourishing the scions was done weekly by Basofoliar Rao, 2001; Prakash et al., 1999; Ali et al. 2007). solution(1%). After four months from rooting, treatments were begun by spraying foliar Methyl- are ubiquitously-occuring lipid-derived jasmonate(Sigma Aldrich, St. Louis, USH) (0, 3 and 6 compounds with signal function in plant responces to mM) and Epibrassinolide(0, 3 and 6 µM) with Tween abiotic and biotic stresses, as well as in plant growth 20(0.1%) and NaCl added to the irrigation water(0, and development(Wasternack, 2007). Applied 25, 50, 75 and 100 mM). Hormonal treatments exogenously, they can induce physiological changes applied twice within 14 days. This experiment was identical to characteristic parts of the stress conducted based on factorial experiment(FactorA: responses(Tsonev et al., 1998). Thus, jasmonates variety, FactorB: salinity levels and FactorC: have been identified as stress modulators suppressing hormones) in the form of Randomized Complete or enhancing the stress responses of plants. Desighn(RCD) with four replication. Jasmonate levels were increased with high salinity in Iris hexagona(Wang et al., 2001) and rice, resulting Determination of physiological indicators in the induction of genes involved in stress-related Determination of leaf water status jasmonates biosynthesis(Tani et al., 2008). Fedina Three compound leaves were collected from each and Benderliev(2000) found that exogenously- seedling. Leaf fresh weight was measured applied methyljasmonate supplied simultaneously immediately and then the leaves wer submerged in with NaCl helped algae to counteract salt stress; distilled water at room temperature. After 24 h, the therefore, the toxic ion effects due to salinity could leaves were removed from the water, blotted dry with cause membrane damage and hence trigger the filter paper and weighted to determine saturated release of the lipid pre-cursor for jasmonate fresh weight. The leaves were then dried at 80°C for synthesis. Moreover, Parra-Lobato et al. (2009) 24 h and weighted again. Leaf relative water concluded that exogenous methyljasmonate may be content(LRWC) was calculated as follows: envolved in the oxidative stress processes by LRWC=(fresh weight - dry weight)/(saturated fresh regulating antioxidant enzyme activities. These weight – dry weight) results suggests a role of methyljasmonate in the plant response to saline stress. Determination of leaf electrolyte leakage Leaves were washed with deionized water to remove How ever, not much work has been done on how surface-adhered electrolytes. These were placed in brassinosteroids and jasmonates could help tree closed vials containing 10-ml deionized water and species to overcome abiotic stresses specially salinity incubated at 25°C on a rotary shaker for 24 h. stress. The aim of this study was to test the hypothesis Subsequently, the electrical conductivity of the that exogenous, foliar application of phytohormones solution (s1) was determined. Samples were then could diminish or counteract the metabolic imbalance autoclaved at 120°C for 20 min and the final electrical produced by a high NaCl concentration in the conductivity (s2) was obtained after equilibration at irrigation water. 25°C. The electrolyte leakage (EL) was defined as follows:

Materials and methods EL(%) = (s1/s2) × 100 Plant material, growth conditions and treatments Scions of grapevine(Vitis vinifera L.) cvs. Flame Determination of malondialdehyde(MDA) seedless and Perlette were rooted and grown in Malondialdehyde were determined using the methods plastic pots containing sand and perlite(1:1) under of Zou(2000). In brief, a 0.5 g sample of fresh leaf natural day length in a polyhouse of Parsnarang tissue was ground in a mortar with 10 ml 10%

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Int. J. Biosci. 2014 trichloroacetic acid and a small quantity of quartz. spectrophotometer for chlorophyll a at 663 nm and The homogenate was centrifuged at 4,000 rpm for 10 for chlorophyll b at 645 nm in mg/g of fresh weight of min, then a 2-ml aliquot was removed and mixed with the sample. 2 ml 0.6% thiobarbituric acid(TBA) solution. The solution was incubated at 100°C for 15 min, allowed chlorophyll a = (19.3×A663-0.86×A645) V/100W to cool and then again at 4,000 rpm. Absorbance chlorophyll b= (19.3×A645 – 3.6×A663) V/100W values of the supernatant were recorded at 532, 600 and 450 nm and the MDA and soluble sugar content Statistical analysis were calculated as follows: The analysis of variance was performed using

MDA content (µmol/g FW) = [(6.45 (A532 – A600) – MSTAT-C software. Duncan,s Multiple Range- Test

0.56A450)/1,000] (µmol/ml) × volume of extract was used to determine differences among treatment solution (ml)/fresh weight (g). means at a significance level of p≤0.05.

Determination of proline content Results Quantification of free proline in grape leaves was The increasing salinity caused a significant increase in done according to Bates et al. (1973), using 0.1 gr of proline content of grape leaf (Table 1). While the dried leaf tissues. The plant material was highest value(3.592 mgr/grFW) was measured in homogenized with 3% sulpho-. The 75mM treatment, but there wasn’t any significant homogenate was then filtered and added with glacial difference between proline content in 100mM sodium acetic acidand acid-ninhydrin. After stirring, the chloride(2.109 mgr/grFW) and control sample was incubated at 100°C for 1 h. after 1 hour, treatment(2.248 mgr/grFW) (Table 3). Hormonal toluene was added and absorbance at 520nm was treatments cause an increase in proline content measured by using spectrofluorometer. compared with control treatment(except in 0 and 100 mM sodium chloride). While the highest value of Determination of photosynthesis and transpiration proline was seen in 6mM MeJA(3.329 mgr/grFW) rate and the lowest value(2.340 mgr/grFW) was seen in At the end of the experiment, portable Photosynthesis control treatment (Table 4). Measurement System (ADC BioscientificLCiAnalyser Serial No. 31655, UK) were used to calculate the net The content of chlorophylle a and b was reduced photosynthetic rate and transpiration rate per unit significantly with NaCl treatments although leaf area of the youngest fully expanded leaf of each maximum reduction was induced by 100mM NaCl plant and last but not least, the measurement was (Table 3). EBR and MeJA treatments was effective on conducted between 9AM and 2PM local time under a increasing chlorophylle a compared with control fixed light intensity. treatment while the best results for hormonal treatments was seen at 6mM MeJA (Table 4). Determination of chlorophyll a and b Chlorophyll a and b was measured using Arnon Transpiration and photosynthesis declined method, in this method, as little as a half gram of wet significantly in the face of increasing salinity levels vegetative matter was chopped and thoroughly while the lowest value of transpiration(2.120mmolm- mashed in liquid nitrogen, in a porcelain mortar. As 2s-1) and photosynthesis(2.387 µmolm-2s-1) was much as 20ml of 80% aceton was added to the seen at 100mM sodium chloride level (Table 3). EBR sample, and then the mixture was put into centrifuge and MeJA application has significant effect on device with 6000 rpm speed for 10 minutes. modification of salinity effect on transpiration and Supernatant was transferred into a glass ballon. Some photosynthesis decreasing while the lowest level of of the samples in the ballon were read in transpiration(4.892 mmolm-2s-1) and

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Int. J. Biosci. 2014 photosynthesis(6.190 µmolm-2s-1) was measured at transpiration value in perlette cultivar(9.005mmolm- control treatment (Table 4). The increasing salinity 2s-1) was significantly more than transpiration value levels caused a significant decrease in transpiration of flame cultivar(5.183mmolm-2s-1) (Table 2). and photosynthesis rate of two cultivars but the

Table 1. Analysis of variance on different characteristic of grape affected by salinity, cultivars and hormones.

Sources Degree Of Proline Chlorophyll a Chlorophyll b Transpiration rate Photosynthesis rate RWC (%) MDA EL Of variation freedom (mg/gr FW) (mg/gr FW) (mg/gr FW) (mmolm-2s-1) (µmolm-2s-1) (µmol/gFW) (%) Cultivars 1 0.223 ns 101.731 ** 38.571** 730.11 ** 52.45 ** 1268 ** 0.000 ** 530.4 * Salinity 4 16.205** 98.436 ** 7.501** 613.23 ** 902.0 ** 7510 ** 0.007 ** 93640** Salinity 4 0.454ns 2.397 * 0.343 ns 38.37 ** 13.69 ** 23.24 ns 0.000 ns 11.67 ns × cultivars

Hormones 4 6.459 ** 42.25 ** 3.618 ** 71.98** 250.86 ** 939.6 ** 0.000 ** 2810 ** Cultivars 4 0.108 ns 5.91** 0.009 ns 16.93** 15.25 ** 14.65 ns 0.000 ns 3.261 ns × hormones Salinity 16 1.179 ** 2.33 ** 0.084 ns 15.04 ** 26.91 ** 80.35 ns 0.000 * 1266 ns × hormones Cultivars 16 0.087 ns 0.323 ** 0.012 ns 8.00 ** 9.02 ** 9.329 ns 0.000 ns 32.85 ns × Salinity × hormones Error 150 0.316 ns 0.721 ns 0.333 ns 2.00 ** 2.78 ** 144.7 ns 0.000 ns 14840 ns

C.V (%) 20.11% 23.47% 16.69% 19.94% 19.38% 17.94% 16.06% --- 18.73% *, **, ns: significant at 0.05 , 0.01 probability level and no significant respectively.

Increasing salinity level had a significantly decreasing maximum level(73.86%) of RWC was seen at 6mM effect on percentage of relative water content of grape MeJA treatment which hasn,t any significant different leaf while the lowest value(48.25%) was found at with 3mM concentration(69.71%) wherease the 100mM NaCl level wherease the highest lowest value(62.12%) was at control treatment (Table value(82.15%) was produced at without salinity 4). application treatment(control) (Table 3). The

Table 2. Main effect of Cultivars on different characteristics of grape. parameter Proline Chlorophyll a Chlorophyll b Transpiration Rate Raet of Photosynthesis RWC MDA EL Cultivars (mg/gr FW) (mg/gr FW) (mg/gr FW) (mmolm-2s-1) (µmolm-2s-1) (%) (µmol/gFW) (%) flame seedless 2.760 A 2.905 B 3.019 B 5.183 B 8.095 B 64.57 B 0.02108 A 54.73 A perlette 2.827 A 4.332 A 3.897 A 9.005 A 9.120 A 69.61 A 0.01941 B 51.47 B Values within the each column and followed by the same letter are not different at P˂0.005 by an ANOVA protected Duncan´s Multiple Range- Test.

Our results showed that MDA content and EL value(0.022µmol/gFW) was observed at control percentage was increased in NaCl treatments while treatment that hadn,t any significant difference with the maximum value of MDA(0.038µmol/gFW)was MDA value at 3µM EBR(0.021µmol/gFW). The best observed for the 100mm NaCl treatment and the results for hormonal treatments on MDA content and minimum value(0.004µmol/gFW)was observed for EL percentage was seen at 6µM MeJA (Table 4). More the control treatment (Table 3). In the case of main ever, results showed that MDA value and EL effect of hormonal treatments, the maximum percentage of flame cultivar(0. 021µmol/gFW and

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54.73% respectively) was significantly higher than traits such as proline content, photosynthesis and MDA value and EL percentage of perlette transpiration rate, chlorophylle a and b content and cultivar(0.019µmol/gFW and 51.47% respectively). RWC percentage were more in perlette cultivar than flame seedless cultivar. In addition, perlette cultivar Perlette cultivar exhibited more efficiency with had the lowest percentage of EL and MDA content respect to qualitative factors and highest value for (Table 2).

Table 3. Main effect of salinity on different characteristics of grape. parameter Proline Chlorophyll a Chlorophyll b Transpiration Rate Raet of Photosynthesis RWC (%) MDA (µmol/gFW) EL (%) salinity (mg/gr FW) (mg/gr FW) (mg/gr FW) (mmolm-2s-1) (µmolm-2s-1) 0NaCl 2.248 D 5.290 A 3.941 A 12.30 A 14.21 A 82.15 A 0.0047 E 27.13 E 25mM 2.761 C 4.661 B 3.717 AB 9.135 B 11.92 B 77.20 A 0.011 D 33.95 D 50mM 3.258 B 4.007 C 3.560 B 7.137 C 8.919 C 68.77 B 0.020 C 49.75 C 75mM 3.592 A 2.774 D 3.24 C 4.782 D 5.601 D 59.07 C 0.027 B 69.83 B 100mM 2.109 D 1.360 E 2.832 D 2.12 E 2.387 E 48.25 D 0.0382 A 84.86 A Values within the each column and followed by the same letter are not different at P˂0.005 by an ANOVA protected Duncan´s Multiple Range- Test.

Discussion for proline synthesis. Proline plays a key part in Salt stress adversely affected plant development and maintaining the osmotic pressure and cytoplasmic the results of the corrent study confirmed the enzymes and protects cell membrane from any negative effects of NaCl treatments on all damage through absorbing free radicals. Our results physiological traits. Furthermore MeJA and EBR were similar to earlier reports that proline content effectively alleviated the adverse affects of NaCl. significantly increased in common bean(Khadri et al. 2006) and corn(Yoon et al. 2005) under salt stress. Increasing salinity stress had a significantly MeJA and EBR caused an increase in free proline increasing effect on proline content of the leaves, content. The present results are in tune with work while this was more evident in perlette cultivar than where 24-epibrassinolide increased the resistance flame seedless cultivar. Accumulation of solutes against chilling stress in rice and the tolerance was especially proline, glycin e-betain and sugars is a associated with increased proline levels, as observed common observation under stress conditions(Ashraf by Wang and Zeng(1993) and Rong and Feng(2011) et al., 1994). Proline is an important osmolyte which about water stress in Xanthoceras sorbifolia synthesizing in many micro organisms and plants seedlings. exposed to salinity and drought stress, thus it as a osmoses protector in plant. Proline accumulating in Increasing salinity level had a decreasing effect on plants exposed salinity stress is duo to low activity of chlorophyll content of the leaf, while this was more oxidant enzymes(Sudhakar, 2001). Increasing proline evident in the leaves of flame seedless variety than in is important for osmosis compatibility but also to perlette variety. Many environmental factors control preserving carbohydrates sink in chloroplasts. It is chlorophyll synthesis in plant. Existing there factors known that salinity stress reduces chlorophyll as limiting factors cause to disordering synthesizing content, because the glutamate which is the primary chlorophyll and appearing chlorosis in plant. Nacl constituents of chlorophyll and proline is consumed stress decreased total chlorophyll content of the plant in favor of proline production. Furthermore, salinity by increasing the activity of the chlorophyll degrading stress induce glutamate ligase enzyme to transform enzyme: chlorophyllase(Rao and Rao, 1981), inducing glutamate into proline. Another reason for the destruction of the chloroplast structure and the chlorophyll reduction is the increased use of nitrogen instability of pigment protein complexes(Dubey,

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1997). The decrease in chlorophyll content under this reduction(Sabater and Rodriguez, 1978). saline cinditions is reported by Iqbal et al. (Iqbal et al. Different researcher also believe that decreased 2006) and Ashraf et al. (Ashraf and Foolad, 2005) chlorophyll content may be duo to inhibitory effect of and in several plants such as pea(Ahmad and Jhon, ions accumulated in chloroplast, chlorophyll 2005), wheat(Ashraf and foolad, 2005), degradation by oxidative stress caused by salt, rice(Anuradha and Rao, 2003) and tomato(Al- activation of chlorophyllase enzyme by salinity ions Aghabary et al., 2004). Chlorophyll reduction can and its negative effect on protophyzine. Furthermore attributed to changing Nitrogen metabolism direction increasing salinity level leads to decreased chlorophyll to forming compounds such as proline which used to biosynthesis through increased salt. MeJA and EBR regulating osmoses(Dela-Roza and Maiti, 1995). application enhanced chlorophyll content by Forming protolityc enzymes such as chlorophyllase neutralizing the inhibitory effect of salt stress on which responsible to decompose chlorophyll and pigmentation which could be one of the reasons for damaging photosynthetic structure, is other cause at an increase in the photosynthetic rate.

Table 4. Main effect hormones on different characteristics of grape. Parameter Proline Chlorophyll a Chlorophyll b Transpiration Rate Raet of Photosynthesis RWC (%) MDA (µmol/gFW) EL (%) hormones (mg/gr FW) (mg/gr FW) (mg/gr FW) (mmolm-2s-1) (µmolm-2s-1) treatment 2.340 D 2.496 D 3.167 C 4.892 C 6.190 E 62.12 C 0.0223 A 57.76 A

EBR3 µM 2.516 CD 2.757 D 3.256 C 8.033 A 7.071 D 63.04 C 0.0216 AB 56.31 A 53.37 EBR6 µM 2.721 C 3.572 C 3.397 BC 7.245 B 8.152 C 66.72 BC 0.0204 BC AB MeJA 3 50.64 3.061 B 4.357 B 3.536 B 8.293 A 8.947 B 69.71 AB 0.0195 C mM BC MeJA 6 3.329 A 4.912 A 3.934 A 7.008 B 12.68 A 73.86 A 0.0175 D 47.43 C mM Values within the each column and followed by the same letter are not different at P˂0.005 by an ANOVA protected Duncan´s Multiple Range- Test.

Increasing salinity level causes a rise in leaf In this experiment, increasing salinity level has led to temperature and consequently the stomatas are decreased effect on RWC of the leaves. and closed duo to water limitation stress caused by epibrassinolide and Methyl jasmonate treatments salinity, at the same time duo to synthesis of abscisic significantly increased the relative water content of acid in the root and its translocation to the stomatas. grape leaves in contrast with control treatments In addition, shrinking of the mezophyllic cells under saline stress. Similarly application of contribute to synthesis of and its homobrassinolide increased the relative water translocation to stomatal cells. A drastic decline of content, chlorophyll a content, photosynthetic rate, photosynthesis and transpiration was caused by salt leaf area and biomass production of wheat under stress in cowpea, kidney bean(Murillo-Amador et al., moisture stress(Sairam, 1994). Furthermore in the 2007), and bush bean(Montero et al, 1997) were in other study, brassinolide treatment significantly tune with our results. The current results also increased the leaf relative water content of suggested that MeJA application alleviates salt- Xanthoceras sorbifolia seedlings under water stress. induced changes on photosynthetic and transpiration Osmotic regulation is an indication of response to rates. Similarly, it has been shown that pre-treatment osmotic stress and when there is a water limitation with methyl jasmonate for 3 days before salt caused by salinity stress, osmotic potential is declined treatment diminished the inhibitory effect of NaCl on and this is turn causes the reduction of RWC of the the rate of CO2 fixation(Fedina and Tsonev, 1997; leaves. Osmotic regulation depend upon the cultivar Velitcukova and Fedina, 1998). as well as on decreased rate of water potential and this is safe to say that one of the mechanisms of

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Temperature, drought or salinity stress can result in Refrences oxidative damage to plant cell membranes, MDA is Ahmad P, Jhon R. 2005. Effect of salt stress on one of the end products of lipid peroxidation(Zlatev et growth and biochemical parameters of Pisum al., 2006). The treatment with EBR and MeJA sativum L.. Agronomy and Soil Science 51, 665-672. resulted in decrease in MDA and electrolyte leakage. This suggests that EBR and MeJA may reduce AIn-Lhout F, Zunzunegui M, Diaz M.C, Triado damage of plant cell membranes due to lipid R, Clavijo A, Garcia A, Novo F. 2001. Comparison peroxidation. Several researchers have found that of proline accumulation in two Mediterranean shrubs increased proline levels can protect plants from subjected to natural and experimental water deficit. damage duo to mild or severe water stress. More Plant Soil 203, 175-183. importantly, proline seems to have a protective effect on plants under sever water stress(Ain-Lhout et al., Ashraf MY, Azmi AR, Khan AH, Ala SA. 1994. 2001). Saradhi et al. (1995) reported that proline Effect of water stress on total phenol, peroxidase protects protein structure and membranes from activity and chlorophyll contents in wheat(Triticum damage and reduce enzyme denaturation; this could aestivum L.) Acta Physiology. Plantarum 16, 185-191. minimize damage caused by dehydration. A decrease in protein content in tomato plants grown under Ashraf M, Karim F, Rasul E. 2002. Interactive water stress was reported by Rahman et al. (2004). effects of gibberellic acid (GA3) and salt stress on They postulated that water stress reduces the growth, ion accumulation and photosynthetic synthesis of protein, because of a possible capacity of two spring wheat (Triticum aestivum L.) suppression of the energy supply owing to reductions cultivars differing in salt tolerance. Plant Growth in photosynthesis and the overall adverse effects of Regulators 36, 49-59. the stress on the biochemical processes. Under saline http://dx.doi.org/10.1023/A:1014780630479 stress, EBR and MeJA treatment increased the content of proline to protect plants from damage duo Ashraf M, Foolad MR. 2005. Pre-sowing seed to water stress. treatment-a shotgun approach to improve germination, plant growth and crop yield under saline Conclusion and non-saline conditions. Advances in agronomy 88, The current study clearly demonstrated that MeJA 223-271. and EBR application before exposure of plants to http://dx.doi.org/10.1016/50065-2113(05)88006-x salinity stress may impart changes in the levels of endogenous plant hormones and proline content, Ashraf M, Harris PGC. 2004. Potential which in turn may help the plant to minimize the biochemical indicator of salinity tolerance in plants. adverse effects of salt stress, thus providing an Plant Science 166, 3-16. important do for understanding the defense mechanism of plants against salt stress. Results Bates LS, Waldren RP, Teare ID. 1973. Rapid revealed that perlette cultivar was more tolerant determination of free proline for water stress study. against salinity than flame seedless variety, because Plant Soil 39, 205-207. mechanisms including RWC and proline concentration and lower lipid peroxidation makes it a Del-Ros IM, Maiti RK. 1995. Biochemical tolerant variety for overcoming salinity stress, mechanism in glossy sorghum lines for resistance to wherease flame seedless could not potentially employ salinity stress. Journal of Plant Physiology 146, 515-

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