Scholarly article on topic 'Alleviating the adverse effects of NaCl stress in maize seedlings by pretreating seeds with salicylic acid and 24-epibrassinolide'

Alleviating the adverse effects of NaCl stress in maize seedlings by pretreating seeds with salicylic acid and 24-epibrassinolide Academic research paper on "Biological sciences"

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{"Salicylic acid" / 24-Epibrassinolide / Salinity / Maize / "Antioxidant system" / Anatomy}

Abstract of research paper on Biological sciences, author of scientific article — Ramadan A. Agami

Abstract The effects of bio-regulators salicylic acid (SA) and 24-epibrassinolide (EBL) as seed soaking treatment on the growth traits, content of photosynthetic pigments, proline, relative water content (RWC), electrolyte leakage percent (EC%), antioxidative enzymes and leaf anatomy of Zea mays L. seedlings grown under 60 or 120mM NaCl saline stress were studied. A greenhouse experiment was performed in a completely randomized design with nine treatments [control (treated with tap water); 60mM NaCl; 120mM NaCl; 10−4 M SA; 60mM NaCl+10−4 M SA; 120mM NaCl+10−4 M SA; 10μM EBL; 60mM NaCl+10μMEBL or 120mM NaCl+10μM EBL] each with four replicates. The results indicated that NaCl stress significantly reduced plant growth traits, leaf photosynthetic pigment, soluble sugars, RWC%, and activities of catalase (CAT), peroxidase (POX) as well as leaf anatomy. However, the application of SA or EBL mitigated the toxic effects of NaCl stress on maize seedlings and considerably improved growth traits, photosynthetic pigments, proline, RWC%, CAT and POX enzyme activities as well as leaf anatomy. This study highlights the potential ameliorative effects of SA or EBL in mitigating the phytotoxicity of NaCl stress in seeds and growing seedlings.

Academic research paper on topic "Alleviating the adverse effects of NaCl stress in maize seedlings by pretreating seeds with salicylic acid and 24-epibrassinolide"

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Alleviating the adverse effects of NaCl stress in maize seedlings by pretreating seeds with salicylic acid and 24-epibrassinolide

Ramadan A. Agami *

Agricultural Botany Department, Faculty of Agriculture, Fayoum University, 63514-Fayoum, Egypt

ARTICLE INFO ABSTRACT

The effects of bio-regulators salicylic acid (SA) and 24-epibrassinolide (EBL) as seed soaking treatment on the growth traits, content of photosynthetic pigments, proline, relative water content (RWC), electrolyte leakage percent (EC%), antioxidative enzymes and leaf anatomy of Zea mays L. seedlings grown under 60 or 120 mM NaCl saline stress were studied. A greenhouse experiment was performed in a completely randomized design with nine treatments [control (treated with tap water); 60 mM NaCl; 120 mM NaCl; 10-4 M SA; 60 mM NaCl + 10-4 M SA; 120 mM NaCl + 10-4 M SA; 10 |M EBL; 60 mM NaCl + 10 |MEBL or 120 mM NaCl + 10 |M EBL] each with four replicates. The results indicated that NaCl stress significantly reduced plant growth traits, leaf photosynthetic pigment, soluble sugars, RWC%, and activities of catalase (CAT), peroxidase (POX) as well as leaf anatomy. However, the application of SA or EBL mitigated the toxic effects of NaCl stress on maize seedlings and considerably improved growth traits, photosynthetic pigments, proline, RWC%, CAT and POX enzyme activities as well as leaf anatomy. This study highlights the potential ameliorative effects of SA or EBL in mitigating the phytotoxicity of NaCl stress in seeds and growing seedlings.

© 2013 SAAB. Published by Elsevier B.V. All rights reserved.

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Article history:

Received 26 May 2013

Received in revised form 16 July 2013

Accepted 22 July 2013

Available online xxxx

Edited by J. van Staden

Keywords:

Salicylic acid

24-Epibrassinolide

Salinity

Antioxidant system Anatomy

1. Introduction

Salinity stress is one of the most serious abiotic stress factors limiting crop productivity. Salinity disrupts plant morpho-physiological processes due to osmotic disturbance and ionic stress (Vinocur and Altman, 2005). The negative impact of salinity on plant growth and metabolism has been attributed, principally, to enhanced Na+ ion uptake, which causes an excess of Na+ ions in plant tissues (Abbas et al., 1991). Salt stress can restrict photosynthesis by decreasing green pigments (Sudhir and Murthy, 2004). Salt stress can reduce activity of various enzymes involved in nitrogen metabolism thus reducing plant nitrogen status (Munns et al., 2006; Soussi et al., 1998). Salt stress causes increase in reactive oxygen species (ROS) which thereby accelerate toxic reactions like lipid peroxidation, protein degradation and DNA mutation (McCord, 2000). To alleviate these oxidative effects, plants generate different kinds of antioxidants like superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) (Noreen and Ashraf, 2009).

Corn (Zea mays L.) is cultivated in many countries and it is a major source of food for both human and animals worldwide. Improvement in corn relies on better understanding of the corn itself, including its genome, physiology and behavior in growth and development (Xu et al., 2004). Because of the restricted resources of fresh water from River Nile, the use of saline water becomes important, so it is necessary to improve the salinity tolerance of maize plants to increase

* Tel.: +20 1152429790, +20 1003230804; fax: +20 84 6334964. E-mail address: ramadanagami@yahoo.com.

0254-6299/$ - see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/mi 016/j.sajb.2013.07.019

its yield under stress conditions. Therefore, it is important to understand different ways that plants can tolerate salinity. In this respect, various compounds exploited to alleviate the plant stress, the salicylic acid and 24-epibrassinolide are recognized as novel groups of phytohor-mones to regulate the plant growth and their productivity.

Salicylic acid (SA) naturally occurs in plants in very low amounts and participates in the regulation of physiological processes such as stomatal closure, nutrient uptake, protein synthesis, inhibition of ethylene biosynthesis and transpiration (Khan et al., 2003; Raskin, 1992; Shakirova et al., 2003). Also, SA plays an important role in a biotic stress tolerance and considerable interests have been focused on SA due to its ability to induce a protective effect on plants under stress (Gunes et al., 2007). Many studies support that SA induced increases in the resistance of maize (Hussein et al., 2007; Tuna et al., 2007) and wheat (Arfan et al., 2007; Bhupinder and Usha, 2003; Sakhabutdinova et al., 2003; Shakirova and Bezrukova, 1997) to salinity and osmotic stress, respectively.

Brassinosteroids (BRs) are a novel phytohormonal group of steroids and are distributed throughout the plant kingdom (Krishna, 2003; Montoya et al., 2005). Brassinosteroids play a prominent role in various physiological processes, like cell division and expansion, xylem differentiation, stem elongation and root growth (Cao et al., 2005; Kartal et al., 2009; Khripach et al., 2000). Moreover, BRs are also reported to have an ameliorative effect on plants subjected to environmental stress such as cold stress (Hu et al., 2008; Liu et al., 2009), heat stress (Ogweno et al., 2008) and oxidative damage (Cao et al., 2005). Under salt stress, the effects of BRs were mainly focused on plant growth and antioxidant systems (Ali et al., 2008a, 2008b; Anuradha and Rao, 2001; Arora et al.,

2008; Rady, 2011). The potential application of BRs in agriculture is based on their ability to increase crop yield, also due to stress amelioration. It is clear that SA and EBL mediated physiological and anatomical changes in plant, which play a vital role during environmental stresses. Owing to obvious evidence of the adverse effects of NaCl stress on plant growth, it was hypothesized that the novel bio-regulators SA and EBL used in this study as a seed soaking can overcome the injurious effects of NaCl stress on maize plants. Therefore, the main objective of this study was to examine whether or not SA or EBL could alleviate the injurious effects of NaCl stress and regulate maize plant growth by adjusting the osmoprotectants (proline and soluble sugars) and antioxidant enzymes activities involved in stress tolerance.

2. Materials and methods

2.1. Plant material, treatments, and growth conditions

Two pot experiments were conducted at the Faculty of Agriculture, Fayoum, (Southeast Fayoum; 29° 17'N; 30° 53'E) Egypt during two successive seasons. Seeds of maize (Z. mays L., Giza 10) were obtained from Agricultural Research Center, Egypt. Seeds of uniform size were washed with distilled water after surface sterilizing with 10% sodium hypochlorite solution. Seeds were presoaked for 12 h, either in 10-4 M salicylic acid, 10 |jM 24-epibrassinolide or distilled water as a control. The selection of the concentrations was based on preliminary study (data not shown). The concentrations of salicylic acid (10-4 M) and 24-epibrassinolide (10 |jM) used generated the best response. Therefore, they were selected for the experiment. Five seeds were sown in each plastic pot (20 cm diameter, 25 cm deep) on the 15th June 2011 and 15th June 2012. Each pot was filled with a sandy soil that had been washed with commercial HCl, and then washed with deionized water to remove all remnants of the acid. After germination the seedlings were thinned to one plant per pot. When maize seedlings were at the three-true leaf stage the seedlings were supplemented with NaCl (60 or 120 mM) along with the nutrient solution. The concentrations of NaCl above 120 mM proved lethal. Therefore, the concentration below the lethal concentrations (i.e. 60 or 120 mM) was used in the experiment. All pots were arranged in complete randomized design in greenhouse. There were six pots per replicate and four replicates per treatment. Thus, the nine treatments per experiment involved 216 pots. Seedlings were irrigated with half-strength (0.5 x) Hoagland's solution (1.5 L pot-1) every 3 days throughout the duration of the experiments. Average day and night temperatures were 32° ± 5 °C and 21° ± 3 °C, respectively. The relative humidity ranged from 63.6 to 68.7%, and natural light day-length ranged from 11 to 12 h. Samples were collected at 50 days from planting to assess chlorophylls and carot-enoids of leaves, proline of leaves, total soluble sugars of leaves, electrolyte leakage percent, relative water content percent and antioxidant enzymes of plant leaves. The other parameters were determined at the end of experiment (after 60 days from planting). The plants were removed from the pots along with the sand and were dipped in a bucket filled with water. The plants were moved smoothly to remove the adhering sand particles and the length of shoots were measured by using a meter scale. Number of leaves was counted. The leaf area was recorded by using a digital leaf meter (Ll-3000 Portable Area meter Produced by LI-COR Lincoln, NE, USA). The plants were weighed to record the plant fresh mass. The plants were then placed in an oven run at 80 °C for 24 h. These dried plants were weighed to record the plant dry mass.

2.2. Photosynthetic pigments, soluble sugars and proline contents determination

Leaf chlorophyll and carotenoids (mg g-1 fresh matter) were determined using a colorimetric Arnon's (1949) method.

Leaf soluble sugars (mg g-1 fresh matter) were assessed by the method of A.O.A.C. (1990).

Proline content (mg g-1 fresh matter) in maize leaves was measured by rapid colorimetric method as suggested by Bates et al. (1973). Proline was extracted from 0.5 g of fresh leaf samples by grinding in 10 ml of3% sulphosalicylic acid and the mixture was then centri-fuged at 10,000 xg for 10 min. Supernatant (2 ml) was added into test tubes to which 1 ml of freshly prepared acid-ninhydrin solution was added. Tubes were incubated in a water bath at 90 °C for 30 min. The reaction was terminated in ice-bath. The reaction mixture was extracted with 5 ml of toluene and vortexed for 15 s. The tubes were allowed to stand for at least 20 min in darkness at room temperature to allow the separation of toluene and aqueous phase. The toluene phase was then carefully collected into test tubes and toluene fraction was read at 520 nm. The proline concentration in the sample was determined from a standard curve using analytical grade proline and calculated on fresh weight basis.

2.3. Electrolyte leakage determination

The total inorganic ions leaked out in the leaves were estimated by the method of Sullivan and Ross (1979). Twenty leaf discs were taken in a boiling tube containing 10 ml of deionized water and electrical conductivity (ECa) was measured. The content was heated at 45 °C and 55 °C for 30 min each in a water bath and electrical conductivity (ECb) was measured. Later the content was again boiled at 100 °C for 10 min and electrical conductivity (ECc) recorded. The electrolyte leakage was calculated by using the formula:

Electrolyte leakage (%) = ECb-ECa/ECc x 100 k.

2.4. Determination of relative water content

The relative water content (RWC) was determined in fresh leaf discs of 2 cm2 diameter, excluding midrib. Discs were weighed quickly and immediately floated on deionized distilled water (DDW) in Petri dishes to saturate them with water for the next 24 h, in dark. The adhering water of the discs was blotted and turgor mass was noted. Dry mass of the discs was recorded after dehydrating them at 70 °C for 48 h. RWC was calculated by placing the values in the following formula (Hayat et al., 2007):

RWC = Fresh mass-dry mass x100.

Turgor mass- dry mass

2.5. Antioxidant enzyme activity determinations

Leaves of 7-week-old plants were excised, weighed (1.0 g fresh weight) and ground with a pestle in an ice-cold mortar with 10 ml 50 mM phosphate buffer (pH 7.0). The homogenates were centrifuged at 20,000 xg for 30 min at 4 °C. The supernatant filtered through two layers of cheese-cloth were used for the assays of enzymatic activities.

The catalase (CAT) activity in leaves of 6-week-old plants was determined by the method suggested by Luck (1975). CAT activity was assayed by estimating the residual H2O2 by oxidation with KMnO4 titrimetrically. The reaction mixture consisted of 3 ml of phosphate buffer (0.1 M, pH 7.0), 30 of H2O2 (5 mM) and 1 ml of enzyme extract. It was then incubated in a test tube at 25 °C for 1 min, reaction stopped by adding 10 ml of 0.35 M H2SO4 and the residual H2O2 estimated by titrating the reaction mixture against 0.01 M KMnO4. The end-point for the titration was a faint purple color which persisted for at least 15 s. A blank was prepared by adding enzyme extract to an acidified solution of reaction mixture at zero time. The enzyme activity was expressed as moles of H2O2 10 min-1 g-1 of fresh weight of leaves.

Table 1

Effects of salicylic acid (SA) and 24-epibrassinolide (EBL) on growth traits (shoot length, number of leaves plant-1, leaves area plant-1, plant fresh mass and plant dry mass) of maize plants under NaCl stress.

Treatments Shoot length (cm) Number of leaves plant 1 Leaf area plant 1 (cm2) Plant fresh mass (g) Plant dry mass (g)

0 (control) 68.6 ± 0.57cd 6.6 ± 0.57a 138.8 ± 1.9c 27.5 ± 0.34c 3.5 ± 0.10a

60 mM NaCl 60.3 ± 0.57e 5.6 ± 0.57bc 74.0 ± 1.7g 12.1 ± 0.47g 1.4 ± 0.05d

120 mM NaCl 56.6 ± 1.5f 5.3 ± 0.57c 61.3 ± 3.2h 9.2 ± 0.55h 0.97 ± 0.02e

10-4MSA 84.0 ± 1.0a 6.6 ± 0.57a 172.0 ± 4.2a 31.5 ± 0.30a 3.4 ± 0.05a

60 mM NaCl + 10-4 M SA 69.0 ± 1.7c 6.6 ± 0.57a 105.3 ± 3.3d 18.5 ± 0.10d 2.2 ± 0.32b

120 mM NaCl + 10-4MSA 68.3 ± 0.57cd 6.0 ± 0.57abc 85.9 ± 2.8e 13.8 ± 0.26e 1.7 ± 0.05c

10 MM EBL 76.3 ± 1.1b 6.6 ± 0.57a 150.0 ± 3.9b 29.0 ± 0.80b 3.5 ± 0.15a

60 mM NaCl + 10 mMEBL 67.0 ± 1.0d 6.3 ± 0.57ab 81.5 ± 1.7ef 13.0 ± 0.51ef 1.5 ± 0.15cd

120 mM NaCl + 10 mMEBL 60.3 ± 0.57e 6.0 ± 0.001 abc 80.3 ± 1.3f 12.7 ± 0.34fg 1.4 ± 0.05d

LSD < 0.05 1.80 0.87 4.94 0.78 0.24

Mean values (n = 5) ± SD in each column followed by different lower-case letters are significantly different (P < 0.05) by Duncan's multiple range test.

The peroxidase (POX) activity in leaves was estimated using the method of Thomas et al. (1981). POX was assayed using guaiacol as the substrate. The enzyme extract was prepared in a similar way to the one used for the extraction of CAT. The reaction mixture consisted of 3 ml of phosphate buffer (0.1 M, pH 7.0), 30 of H2O2 (20 mM), 50 M of enzyme extract and 50 Ml of guaiacol (20 mM). The reaction mixture was incubated in a cuvette for 10 min at room temperature. The optical density was measured at 436 nm. The enzyme activity was expressed as number of absorbance units g-1 fresh weight of leaves.

2.6. Anatomical study

For anatomical study, samples were taken at the age of 60 days from the middle of fourth leaf from apex. Samples were killed and fixed in F.A.A. solution (50 ml 95% ethyl alcohol + 10 ml formalin + 5 ml glacial acetic acid + 35 ml distilled water) for 48 h. Thereafter, samples were washed in 50% ethyl alcohol, dehydrated and cleared in tertiary butyl alcohol series, embedded in paraffin wax of 54-5°6C m.p. Cross sections, 20 |jm thick, were cut by a rotary microtome, adhered by Haupt's adhesive and stained with the crystal violet-erythrosin combination (Sass, 1961), cleared in carbol xylene and mounted in Canada balsam. The sections observed and documented using an upright light microscope (AxioPlan, Zeiss, Jena, Germany). Measurements were done, using a micrometer eyepiece and average of 5 readings were calculated.

2.7. Statistical and data analysis

Treatments were arranged in a completely randomized design with 9 treatments. Analysis of variance was performed using the SPSS software package. Analysis of variance (ANOVA) was performed on the data to determine the least significant difference (LSD) among treatment at P < 0.05 and Duncan's multiple range tests were applied for comparing the means (Duncan, 1955).

3. Results

3.1. Seedling growth traits

The presence of 60 or 120 mM NaCl significantly reduced the growth traits (length of shoot, no. of leaves plant-1, leaf area plant-1 and plant fresh and dry mass) in maize plants (Table 1). Compared to the control, the above mentioned parameters were significantly decreased by 17.4%, 19.9%, 55.8%, 66.5% and 72.2%, respectively under level, 120 mM NaCl. On the other hand, application of SA or EBL, as seed soaking in absence of NaCl stimulated the growth traits that were significantly higher than the control (P < 0.05). Salicylic acid or EBL also improved the growth of the plants grown under NaCl and the values were generally significantly higher than those of the plants grown under stress alone. However, the response generated by SA was more effective than that generated by ELB.

3.2. Photosynthetic pigments, soluble sugars and proline

The stress generated by 60 or 120 mM NaCl resulted in a significant decrease in the levels of chlorophyll a,b and carotenoids (Table 2). The high NaCl level was more toxic compared to the low one. However, these attributes were improved by SA or EBL, both in the presence and absence of NaCl. Soaking maize seed in SA or EBL significantly overcame the toxicity generated by NaCl-stress and the response generated under stress-free condition was significantly higher than that generated under the stress. SA treatments were more effective than the EBL one.

A difference in total soluble sugar content between treatments was observed (Table 2). Increase in salinity levels from 60 to 120 mM significantly reduced total soluble sugar contents of maize leaves. The percentages of decrease were 22.4% and 25.9%, respectively compared to control. The data herein obtained revealed that soaking maize seed in SA or EBL stimulated the accumulation of total soluble sugars as compared with the corresponding salinity level. The highest total soluble sugar content was recorded by SA treatment under stress-free condition as compared to the other treatments. However, SA induced a more

Effects of salicylic acid (SA) and 24-epibrassinolide (EBL) on leaf photosynthetic pigments [chlorophyll fractions; chloro. a, chloro. b and carotenoids contents (mg g-1 F.W.), total soluble sugars contents (mg g-1 F.W.) and proline contents (mg g-1 F.W.) of maize plants under NaCl stress].

Treatments Chloro.a (mg g 1 F.W.) Chloro.b (mg g-1 F.W.) Carotenoids (mg g 1 F.W.) Total soluble sugars (mg g-1 F.W.) Proline (mg g 1 F.W.)

0 (control) 0.91 ± 0.01b 0.23 ± 0.01c 0.29 ± 0.005c 37.0 ± 0.49c 0.26 ± 0.005g

60 mM NaCl 0.43 ± 0.01e 0.16 ± 0.01d 0.21 ± 0.005f 28.7 ± 0.52g 2.2 ± 0.06b

120 mM NaCl 0.21 ± 0.01f 0.09 ± 0.005e 0.14 ± 0.005g 27.4 ± 0.65h 2.6 ± 0.20a

10-4 M SA 1.5 ± 0.05a 0.25 ± 0.01b 0.28 ± 0.005c 42.9 ± 0.62a 0.35 ± 0.03f

60 mM NaCl + 10-4 M SA 0.88 ± 0.01b 0.23 ± 0.005c 0.29 ± 0.005c 35.2 ± 0.40d 0.89 ± 0.01d

120 mM NaCl + 10-4MSA 0.73 ± 0.01c 0.23 ± 0.005c 0.26 ± 0.005e 34.6 ± 0.45d 1.2 ± 0.08c

10 mMEBL 0.88 ± 0.01b 0.43 ± 0.01a 0.48 ± 0.005a 40.1 ± 0.20b 0.35 ± 0.03f

60 mM NaCl + 10 mMEBL 0.77 ± 0.005c 0.25 ± 0.005b 0.32 ± 0.005b 31.7 ± 0.36e 0.70 ± 0.02e

120 mM NaCl + 10 mMEBL 0.67 ± 0.005d 0.22 ± 0.005c 0.27 ± 0.005d 30.3 ± 0.36f 0.79 ± 0.02df

LSD < 0.05 0.036 0.012 0.009 0.811 0.132

pronounced effect than EBL. The level of proline in maize leaves significantly enhanced in response to NaCl stress compared to the control (Table 2). The quantity of proline was found to be higher in plants subjected to high NaCl level than plants subjected to the low NaCl one. The maximum quantity of proline (2.6 mg-1 g) was found in the plants which were subjected to 120 mM NaCl in comparison with other treatments.

33. Relative water content (RWC), electrolyte leakage (%) and enzyme activities

The RWC was significantly decreased gradually with the increase of NaCl level (Table 3). The percentages of decrease were 12.4% and 16.8%, for 60 and 120 mM NaCl, respectively as compared to the control. Nevertheless, application of SA or EBL as seed soaking in absence or presence of NaCl improved the RWC and the values were significantly higher than those of the plants grown under stress alone. A maximum RWC (81.7%) was recorded in the plants, exposed simultaneously to both SA and NaCl when compared with other treatments. A different pattern of response was observed when electrolyte leakage was studied in SA or EBL treated seed plants, in the presence or absence of NaCl (Table 3). Under stress-free medium, SA slightly affected the electrolyte leakage, but this parameter significantly increased under stress condition as compared to the control. The presence of NaCl caused a significant increase in electrolyte leakage, compared to the control. A maximum electrolyte leakage was recorded in the plants, exposed to 120 mM NaCl. The exposure of maize seeds to SA or EBL caused a significant decline in the electrolyte leakage, compared to the stressed plants that were not treated with SA or EBL. However, treatment of the stressed plants with SA or EBL partially neutralized the toxicity and caused a significant improvement in the membrane stability.

The changes in the activities of the antioxidative enzymes, catalase and peroxidase in response to NaCl stress either alone or in combination with each of SA or EBL are recorded (Table 3). Results indicated that, cat-alase and peroxidase were significantly decreased under stress conditions compared to control. Priming of maize seed by soaking in SA or EBL improves stress resistance by the increase in CAT and POX activities as compared with the corresponding salinity level. The higher activities were recorded in response to SA in CAT and POX in absence of NaCl stress. At the same time, SA has more pronounced effect than EBL treatment on catalase or peroxidase activity.

3.4. Anatomy of leaves

Table 4 and Fig. 1 show that, increasing NaCl level caused gradual decrease in midvein, blade, mesophyll, adaxial and abaxial epidermis thickness as well as average diameter of vessels, while cuticle thickness was increased compared to the control. However, soaking maize seeds in either SA or EBL caused considerable improvement in the above mentioned characteristics and countered the adverse effect of NaCl stress as compared with the corresponding salinity level. The maximum cuticle

thickness and average diameter of the vessels was observed in EBL treated plants in absence NaCl stress as compared to other treatments.

4. Discussion

Exposure of maize plants to NaCl stress induces various physiological and biochemical mechanisms related with plant growth and development. Reduction in growth of maize plants grown under salt stress may result from its effect on dry matter allocation, ion relations, water status, biochemical reactions and/or a combination of many physiological factors (Sohan et al., 1999). The reduction in leaf area of maize plants under salt stress can be considered as a voidance mechanisms, which minimizes water loss when the stomata closed. It is known that reduction in leaf area in salt-stressed plants can be explained by a decrease in leaf turgor, changes in cell wall properties and a decreased in photosynthetic rate (Rodriguez et al., 2005). The susceptibility of maize plants to high concentration of NaCl is demonstrated by growth reduction and loss of fresh and dry mass as shown in our results. These effects are probably due to an excessive increase and translocation of Na+ and Cl- ions to the leaf tissue, which cause alterations in the osmotic potential (Passos et al., 2005). Salinity decreased the growth and leaf water potential of Brassica napus (Naeem et al., 2010). In this study application of SA or EBL, as seed soaking in absence or presence of NaCl stress stimulated the growth traits compared to the control. These positive results were obtained as a result of SA or EBL overcoming the harmful effects of NaCl stress by the induction of assimilating area, photosynthetic pigments and protein biosynthesis which consequently delayed leaf senescence which is induced by salt stress. SA-application reduced damaging action of salinity on plant growth and accelerates growth processes after removal stress factors (Gunes et al., 2007; Shakirova et al., 2003). Also, Anuradha and Rao (2001) reported that, seed application with BR reduced the adverse effect of salt stress on growth of rice by restored pigment levels, increased nitrate reductase, nucleic acids and proteins. In the present study, the growth inhibition was found to be associated with NaCl stress induced decrease in the contents of photosynthetic pigments and the relative water content. Salt stress affects photosynthetic components such as enzymes, chlorophyll and carotenoids, and this depends on the severity and stress duration (Lakshmi et al., 1996; Misra et al., 1997). In addition, reduction in the content of chlorophyll a and b in leaves of salt stressed maize plants could be attributed to increased activity of ion accumulation (Parida et al., 2004). However, decrease in carotenoids lead to degradation of B-carotene and formation of zeaxanthins, which are apparently involved in protection against photoinhibition (Sharma and Hall, 1991). The treatment of maize seed with SA or EBL significantly increased photosynthetic pigments and overcame the toxicity generated by NaCl-stress. High concentrations of these pigments might be explained by the fact that SA and EBL had a protective effect on the leafs ultrastructure and preventing nucleus and chloroplast degradation (Kulaeva et al., 1991) which delays leaf senescence/abscission. Many researchers reported that EBL and SA caused increases in photosynthetic

Table 3

Effects of salicylic acid (SA) and 24-epibrassinolide (EBL) on relative water content (RWC), electrolyte leakage (EL%), the activities of catalase (|mol H2O210 min-1 g-1 F.W.) and peroxidase (units 10 min-1 g-1 F.W.) of maize plants under NaCl stress.

Treatments Relative water content Electrolyte leakage Catalase activity Peroxidase activity

0 (control) 80.7 ± 0.85b 14.4 ± 0.56f 35.5 ± 0.49c 1.2 ± 0.70c

60 mM NaCl 70.7 ± 0.45f 19.9 ± 0.80c 25.8 ± 0.81g 0.76 ± 0.01def

120 mM NaCl 67.1 ± 0.55g 24.4 ± 50a 22.1 ± 0.30h 0.67 ± 0.02f

10-4 M SA 81.7 ± 0.61a 14.9 ± 0.32ef 47.1 ± 0.35a 2.6 ± 0.13a

60 mM NaCl + 10-4 M SA 74.8 ± 0.35c 17.0 ± 0.15d 31.2 ± 0.40d 0.86 ± 0.02d

120 mM NaCl + 10-4MSA 71.8 ± 0.15e 22.3 ± 0.45b 28.8 ± 0.35e 0.75 ± 0.02ef

10 |M EBL 81.1 ± 0.55ab 15.6 ± 0.32e 45.8 ± 0.25b 2.4 ± 0.10b

60 |M NaCl + 10 |M EBL 73.9 ± 0.37d 17.7 ± 0.41d 28.7 ± 0.43e 0.82 ± 0.02de

120 |M NaCl+ 10 |M EBL 70.8 ± 0.45f 19.9 ± 0.61c 27.2 ± 0.64f 0.72 ± 0.01def

LSD < 0.05 0.83 0.84 0.82 0.10

Table 4

Effects of salicylic acid (SA) and 24-epibrassinolide (EBL) on anatomical structure of maize plants leaf.

Treatments Characters

Midvein thick. Blade thick Mesophyll Adaxial epidermis Abaxial epidermis Cuticle thick Average diameter

(|im) (|im) thick (|m) thick (|m) thick (|m) (|im) of vessels (|m)

0 (control) 720 ± 5ab 240 ± 3d 150 ± 3c 60 ± 2c 30 ± 3a 7.5 ± 1.0c 43.8 ± 3c

60 mM NaCl 320 ± 8h 200 ± 4f 120 ± 2e 50 ± 2d 30 ± 2a 12.5 ± 2ab 31.7 ± 1.1e

120 mMNaCl 410 ± 6g 190 ± 2g 130 ± 4d 40 ± 3e 20 ± 1b 12.5 ± 1ab 41.3 ± 2.6c

10-4MSA 700 ± 8c 310 ± 6a 240 ± 5a 50 ± 3d 20 ± 2b 12.5 ± 1ab 52.5 ± 1.7a

60 mM NaCl + 10-4 M SA 730 ± 9a 250 ± 7c 150 ± 4c 70 ±4b 30 ± 4a 12.5 ± 1ab 48.8 ± 1.9b

120 mMNaCl + 10-4MSA 620 ± 5e 220 ± 5e 120 ± 2e 70 ± 2b 30 ± 2a 10 ± 1bc 40.6 ± 0.9c

10 |M EBL 710 ± 8bc 200 ± 6f 100 ± 3f 70 ± 3b 30 ± 2a 15 ± 3a 54.4 ± 0.8a

60 mMNaCl + 10 |M EBL 590 ± 10f 300 ± 8b 170 ± 6b 100 ± 5a 30 ± 3a 10 ± 1bc 36.9 ± 2d

120 mMNaCl + 10 |MEBL 650 ± 15d 170 ± 3 h 100 ± 2f 60 ± 4c 10 ± 1c 12.5 ± 1.5ab 43.1 ± 3c

LSD < 0.05 14.9 9.0 6.3 5.6 4.1 2.6 3.5

Mean values (n = 5) ± SD in each column followed by different lower-case letters are significantly different (P < 0.05) by Duncan's multiple range test.

rate and carboxylating enzymes (Anuradha and Rao, 2001; Khodary, 2004; Rady, 2011). Exposure of plants to NaCl stress significantly reduced total soluble sugar contents of maize leaves. The change in sugar contents under salt stress has already been reported for a number of plant species (Hassanein et al, 2009; Khattab, 2007). This reduction concluded that salt stress may inhibit the photosynthetic activity and/

or increased partial utilization of carbohydrates into other metabolic pathways (Hassanein et al., 2009). In the present investigations application of SA or EBL stimulated the accumulation of total soluble sugars as compared with the corresponding salinity level. In salt stressed maize plants, SA might be assumed to inhibit polysaccharide hydrolyzing enzyme system and/or accelerate the incorporation of soluble sugars

Fig. 1. Transections of maize leaf blade as affected by application of salicylic acid (SA) and 24-epibrassinolide (EBL) under NaCl stress. A) untreated plant, B) 120 mMNaCl, C) 10 4 MSA D) 120 mM NaCl + 10-4 M SA, E) 10 |jM EBL, F) 120 mM NaCl + 10 |jM EBL b, blade; pc, parenchyma cells and v, vessel.

into polysaccharides (Khodary, 2004). Wu et al. (2008) stated that increased BR levels favor sucrose accumulation in the leaf and starch in the seed and regulate the effects of sugar metabolism in leaves and seeds. During the present work, proline content significantly increased in maize seedlings exposed to increased salt stress. There is a strong correlation between increased cellular proline levels and capacity to survive the effects of high environmental salinity. Proline contents of maize leaves showed increased trend after SA or EBL application under NaCl stress. Seed soaking in SA or EBL alleviated the negative effects of NaCl on leaf photosynthetic pigments and increased the tolerance of maize seedlings to salinity by raising the contents of carotenoids as anti-oxidant and the contents of proline and total soluble sugars as osmoprotectants. During the present work, RWC significantly decreased in maize seedlings exposed to increased salt stress. Application of SA or EBL in absence or presence of NaCl improved the RWC. Agarwal et al. (2005) reported that RWC capacity increased in wheat with SA treatment. BRs are known to improve water relations such as increase relative water content and water uptake (Ali et al., 2005) leading to an increase in relative water content. The presence of NaCl caused a significant increase in electrolyte leakage, compared to the control. This result already observed by several authors on various crops (Ghoulam et al., 2002; Kaya et al., 2001; Lutts et al., 1996) would be associated to chain reactions initialized by free radicals (Mazliak, 1983). Soaking maize seed to SA or EBL caused a significant decline in the electrolyte leakage, compared to the stressed plants that were not treated with SA or EBL. However, treatment of the stressed plants with SA or EBL partially neutralized the toxicity and caused a significant improvement in the membrane stability. These results are concordant with Yildirim et al. (2008) for cucumber, who determined that SA facilitated the maintenance membrane functions. This facilitation could be attributed to the induction of antioxidant responses and elevated Ca+2 uptakes that protects the plant from the oxidative damage by SA (El-Tayeb, 2005; Senaratna et al., 2000). Maintaining integrity of the cellular membranes under salt stress is considered an integral part of the salinity tolerance mechanism (Stevens et al., 2006). Also, BRs may help membrane integrity by enhancing the level of the antioxidant system that protects the plant from the oxidative damage (Ali et al., 2008b; Arora et al., 2008). In the present investigation, activities of antioxidant enzymes (peroxidase and catalase) in maize plants were decreased under salt stress while increased after SA or EBL application. The reduction in catalase and peroxidase activities indicated that these enzymes were unable to completely neutralize H2O2 that resulted from the oxi-dative salt stress (Shalata and Neumann, 2001). Salinity accumulates the ROS especially H2O2 in plant cells. The metabolism of H2O2 is dependent on various functionally interrelated antioxidant enzymes such as catalase and peroxidases. These enzymes are involved in the elimination of H2O2 from stressed cells (Kim et al., 2005). The elevation in the activities of antioxidant enzymes by BRs is a gene regulated phenomenon. Cao et al. (2005) demonstrated that, on the basis of molecular, physiological and genetic approaches the elevation in antioxidant enzymes was the consequence of enhanced expression of DET2 gene, which enhanced the resistance to oxidative stress in Arabidopsis. Previous reports also showed that the application of BRs modified antioxidant enzyme activities; under water stress (Li and van Staden, 1998), salinity (Ali et al., 2007) and cadmium stress (Hayat et al., 2007). Chen et al. (1997) found that application of homobrassinolide increased superoxidse dismutase and peroxidase activities in rice. In our results the increase in NaCl salinity levels greatly reduced midvein, blade, mesophyll, adaxial and, abaxial epidermis thickness and average diameter of vessels. The decrease in mesophyll thickness of the NaCl stressed leaves may arise from a reduction in mesophyll cells. The decrease in lamina thickness can also be related to smaller volumes of mesophyll cells in salinity stressed leaves. Similarly, Todd et al. (1974) observed that leaf thickness had decreased as a result of water stress. Merkulov et al. (1997) revealed that leaf blade of sugar beet genotypes subjected to drought stress showed smaller epidermal cells, thicker cuticle and a

greater number of layers of smaller mesophyll cells. However, soaking maize seeds in either SA or EBL caused considerable improvement in the above mentioned attributes and countered the adverse effect of NaCl stress as compared with the corresponding salinity level. BRs play prominent roles in various physiological processes, like cell division and expansion, xylem differentiation, stem elongation (Cao et al., 2005; Kartal et al., 2009; Khripach et al., 2000).

5. Conclusions

Seed soaking in SA or EBL was beneficial for the early vegetative growth of NaCl-stressed maize plants. Application of SA or EBL significantly improved all parameters of growth, and elevated levels of the antioxidant system (catalase, peroxidase and carotenoids) and the osmoprotectant soluble sugars, thus improving the tolerance of maize plants to NaCl stress, occurred through the protection of photosynthetic machinery.

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