Exogenous application of 24-epibrassinolide and spermine ...curresweb.com/mejas/mejas/2019/mejas.2019.9.4.22.pdfSep 04, 2019 · Wheat plants of cv. Giza 168 were subjected to well-watered

Middle East Journal of Applied Sciences EISSN: 2706 -7947 ISSN: 2077- 4613 DOI: 10.36632/mejas/2019.9.4.22

Volume : 09 | Issue :04 |Oct.-Dec.| 2019 Pages: 1063-1080

Corresponding Author: Zeid, F. A., Soils, Plant Biotechnology Research Laboratories (PBRL), Plant Physiology Division, Department of Agricultural Botany, Faculty of Agriculture, Cairo University, Giza, Egypt.

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Exogenous application of 24-epibrassinolide and spermine improves growth and yield of drought stressed wheat (Triticum aestivum L.)

Zeid F. A., El Shihy O. M. and Ibrahim F. A. Plant Biotechnology Research Laboratories (PBRL), Plant Physiology Division, Department of Agricultural Botany, Faculty of Agriculture, Cairo University, Giza, Egypt.

Received: 30 Sept. 2019 / Accepted 25 Nov. 2019 / Publication date: 10 Dec. 2019 ABSTRACT The present study was conducted to investigate the effect of foliar application of 24-epibrassinolide (EBL) and spermine (Spm) on growth, yield, chemical constituents and the activity of antioxidant enzymes in wheat plants subjected to drought stress conditions. Wheat plants of cv. Giza 168 were subjected to well-watered conditions and drought stress conditions (75% and 50% of field capacity) with and without (0.5 μM EBL + 100 μM Spm) foliar application at 45 and 75 days after sowing during two successive seasons 2013/2014 and 2014/2015. Drought stress significantly reduced relative water content (RWC), membrane stability index, concentration of total chlorophylls, total crude protein, growth, and yield of wheat plants, particularly under 50% of field capacity. However, foliar application (0.5 μM EBL + 100 μM Spm) significantly improved the above parameters in drought stressed wheat plants. Moreover, drought stress significantly increased lipid peroxidation, electrolyte leakage, and concentration of total sugars, total free amino acids, glycinebetaine and proline in wheat plants. However, lipid peroxidation and electrolyte leakage were significantly decreased in stressed plants treated with foliar application, meanwhile, concentration of total sugars, total free amino acids, glycinebetaine and proline were significantly increased. The activity of antioxidant enzymes; superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, were slightly increased in response to drought treatments, while they significantly increased in response to foliar application in drought stressed wheat plants. The obtained results in this study confirmed the effective influences of 24-epibrassinolide (EBL) and spermine (Spm) for improving drought tolerance of wheat plant by increasing osmopotectants and enhancing ROS scavenging antioxidant defense machinery which resposible for protecting cell enzymes, organelles and cell membranes. Keywords: Wheat, drought stress, 24-epibrassinolide, spermine, antioxidant enzymes

Introduction

Global warming problem is one of the major sources of drought stress. With the rising of this problem, the probability of drought stress occurrence was increased and become a widespread problem that adversely affects crop productivity worldwide. Drought induces oxidative stress in plants through accumulation of reactive oxygen species (ROS) which leads to damage of the lipid membrane. To cope up with these damages it is necessary to keep the level of reactive oxygen species under control. Therefore plants increase the levels of non-enzymatic and enzymatic antioxidant systems to protect cells from oxidative damages under drought stress (Mittler, 2002). However, under severe stress conditions, the plants fail to maintain the equilibrium status of antioxidants and ROS synthesis (Gill and Tuteja 2010).

Brassinosteroids (BRs) are considered as a new group of plant growth hormones that perform a variety of physiological roles like growth, seed germination, rhizogenesis, senescence, and resistance to plants against various abiotic stresses (Rao et al., 2002). BRs are a new type of polyhydroxysteroidal phytohormones with significant growth-promoting influence (Vardhini, 2012a and b; Bajguz and Piotrowska-Niczyporuk, 2014). Sixty BRs related compounds have been identified (Haubrick and Assmann, 2006). However, brassinolide (BL), 28-homobrassinolide (28-HomoBL) and 24-epibrassinolide (24-EpiBL) are the three bioactive BRs those are widely used in most physiological and experimental studies (Vardhini et al., 2006). Reports are extensive on the role of BRs and related compounds in plant drought tolerance (Li and Van Staden, 1998 a,b; Li et al., 1998, 2008 and 2012; El-

Middle East J. Appl. Sci., 9(4): 1063-1080, 2019 EISSN: 2706 -7947 ISSN: 2077- 4613 DOI: 10.36632/mejas/2019.9.4.22

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Khallal, 2002; Vardhini and Rao, 2003a,b, 2005; Zhang et al.,2008; Behnamnia et al., 2009; Fariduddin et al., 2009; Farooq et al., 2010; Yuan et al., 2010; Anjum et al., 2011; Mahesh et al., 2013).

The application of polyamines (PAs) is an effective approach for enhancing stress tolerance of crops (Chai et al., 2010; Shi et al., 2010; Xu et al., 2011b). PAs are cationic molecules that are essential for plant growth and are involved in seed germination, root growth, flower and fruit development, cell division, elongation, replication, transcription, translation, and membrane and cell wall stabilization (Hussain et al., 2011). The biosynthesis of PAs starts with the decarboxylation of ornithine or arginine to yield the diamine putrescine (Put). Spermidine (Spd) and spermine (Spm) are formed by the sequential addition of aminopropyl groups to Put and Spd, respectively (Kusano et al., 2008). These PAs are present in the free forms, conjugated to small molecules, and covalently bound to macromolecules. One of the mechanisms for stress tolerance is the accumulation of PAs in plant cells. Besides their function in regulating plant growth, PAs are also associated with the response of plants to diverse environmental stresses such as drought, salinity, temperature, and metal toxicity (Duan et al., 2008; Kubis, 2008; Zhang et al., 2009; Xu et al., 2011a, Hussain et al., 2011).

Wheat (Triticum aestivum L.) is the most important growing cereal crop in Egypt. Although wheat production per unit area in Egypt has significantly increased during the last years, wheat production supplies about 50% of its annual domestic demand. Therefore, it is very essential to increase wheat productivity. Extending wheat growing outside the Nile valley is the first effort toward overcoming wheat problems. However, most of the area outside the Nile Valley suffers from the scarcity of fresh water and depends on limited water sources. Therefore, there is a need to find means for enhancing the ability of wheat to tolerate drought stress and consequently increasing its productivity under drought stress conditions. Thus, the present study was conducted to investigate the effect of foliar application of 24-epibrassinolide (EBL) and spermine (Spm) on growth, yield, chemical constituents and the activity of antioxidant enzymes in wheat plants subjected to drought stress conditions. Wheat plants of cv. Giza 168 were subjected to well-watered conditions and drought stress conditions (75% and 50% of field capacity) with and without (0.5 μM EBL + 100 μM Spm) foliar application at 45 and 75 days after sowing during two successive seasons 2013/2014 and 2014/2015.

sand Method sMaterial

The present work was carried out in the wire greenhouse and Plant Biotechnology Research Laboratories, Plant Physiology Division, Department of Agricultural Botany, Faculty of Agriculture, Cairo University. This study was conducted to investigate the effect of foliar application of 24-epibrassinolide (EBL) and spermine (Spm) on growth, yield, chemical constituents and the activity of antioxidant enzymes in wheat plants subjected to drought stress conditions. Preliminary experiments were carried out to determine the effective concentration of (EBL) and (Spm) either individual or in combination of both compounds for foliar application on wheat plants grown under drought stress conditions, hence, the obtained results of growth and yield of wheat plants during two successive seasons 2011/2012 and 2012/2013 proved that the combination of (EBL + Spm) with this concentrations (0.5 μM EBL + 100 μM Spm) was the most effective treatment, therefore, this treatment was used in the main experiments. Thus, wheat plants of cv. Giza 168 were subjected to well-watered conditions and drought stress conditions (75% and 50% of field capacity) with and without (0.5 μM EBL + 100 μM Spm) foliar application at 45 and 75 days after sowing during two successive seasons 2013/2014 and 2014/2015.

Wheat grains of cv. Giza 168 were obtained from the Department of Wheat Research, Agricultural Research Center, Giza, Egypt. Wheat Grains were hand planted on 10th of Nov., in both seasons; 2013/2014 and 2014/2015. The grains were sown in plastic pots 25 cm in diameter, filled with 10 kg of a mixture of (2:1, soil:sand, v/v); the clay loam soil was collected from the Agricultural Experiments Station, Faculty of Agriculture, Cairo University. 20 pots were used for each treatment; each pot was fertilized with 1.4 g calcium super phosphate (15.5% P2O5), 1.1 g potassium sulfate (48% K2O) before sowing, in addition to 2.5 g of ammonium nitrate (33.5% N) which was divided into four dosages (10% before sowing, 20% at 35 days after sowing (DAS), 30% at 55 (DAS) and 40% at 75 (DAS), as recommended by the Ministry of Agriculture and Land Reclaimation. The pots were irrigated with tap water until complete germination (10 days), and then the plants were later thinned to leave only three plants/pot. After planting, irrigation was applied at the appropriate times with tap water to maintain


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soil moisture near maximum water-holding capacity for 30 days. Drought stress treatments were carried out 30 days after sowing, the plants were exposed to three soil water conditions: control treatment (100% field capacity), water-stressed conditions (75% and 50% of field capacity). Foliar application treatments

Wheat plants of each treatment (20 pots) were sprayed at 45 and 75-days old; each time with one liter of 0.00 (double distilled water) or 0.5 μM EBL + 100 μM Spm; 24-epibrassinolide (EBL; C28H48O6, MW = 480.7; Sigma) and spermine (Spm; C10H26N4, MW = 202.3; Sigma). Tween-20 (0.05%) was added as surfactant at the time of treatment.

During the experimental growth period, two samples were taken at 60 and 90 days after sowing from each treatment. At each sampling date, 15 plants (3 plants/pot x 5 pots) from each treatment were taken using a stream of water to insure minimal losses of root system. The plants were then carefully cleaned to remove any adherent dirt and divided into two groups; the first group contains 9 plants used for growth analysis and the second group contains 6 plants used for chemical analyses. The plants of the first group were separated into shoots and roots and the following growth characters were recorded: 1-Plant height 2- Root Length 3-Average number of tillers/plant 4-Average number of leaves/plant

Thereafter, the plant parts were dried in an electric oven at 70 ºC for 48 hr, and the crude dry weight of both shoots and roots were determined. At harvest (145 days after sowing) the following yield components were recorded: 1- Grain yield (g)/ plant. 2- Weight of 1000 grains (g). Chemical analyses

In each sample, shoots of 6 plants were chemically analyzed in order to determine their chemical composition. Determination of relative water content (RWC)

Leaves were harvested and their fresh weight (FW) was determined immediately. They were floated on deionized water overnight at 4 °C and their turgescent weight (TW) was recorded. Finally, leaves were dried at 70 °C and their dry weight (DW) was recorded. Relative water content was calculated using the following formula:

RWC (%) = (FW-DW/TW-DW) x100, (Gouiaa et al., 2012). Determination of lipid peroxidation

Oxidative damage to lipids was expressed as equivalents of malondialdehyde (MDA) contents. Fresh leaves (0.5 g) were homogenized in 80% ethanol and centrifuged at 3000x g for 10 min at 4 ºC. The pellet was extracted twice with 80% ethanol. The supernatant was added to a test tube with an equal volume of the solution comprised of 20% trichloroacetic acid, 0.01% butylated hydroxy toluene and 0.65% thiobarbituric acid. The mixture was heated at 95 ºC for 25 min and cooled to room temperature. Absorbance was recorded at 440, 532 and 600 nm. The MDA content was calculated according to Hodges et al., (1999). Estimation of electrolyte leakage

Electrolyte leakage (EL) was measured according to (Cao et al., 2007) and refined according to (Parvin et al., 2015). The plant leaf segments were weighed (0.1g) and taken in a falcon tube with 25 ml deionized water. The tubes were shaken on a gyratory shaker at room temperature for 2 h. The initial electrical conductivity (EC1) of the solution was measured by using a conductivity detector. These samples were then autoclaved at 120 ºC for 20 min to release all the electrolytes from the tissues completely. Samples were then cooled to 25 ºC and the final electrical conductivity (EC2) of the


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resulting solution was measured. The percentage of electrolyte leakage was calculated according to the formula:

EL (%) = EC1/ EC2 x100. Estimation of Leaf cell membrane stability index

Leaf membrane stability index (MSI) was determined according to the method of Premchandra et al., (1990) as modified by Sairam (1994). Leaf discs (200 mg) were taken in 10 ml of double distilled water in two sets. One set was heated at 40 ºC for 30 min in a water bath and the electrical conductivity bridge (C1) was measured on a conductivity meter. The second set was boiled as 100 ºC on a boiling water bath for 10 min and its conductivity was also measured on the conductivity bridge (C2). (MSI) was calculated using this formula:

MSI = [1- (C1/C2) ] x 100 Determination of total chlorophylls

Fresh leaves were cut into pieces and 100 mg put into a bottle containing 12 ml of 80% acetone and kept on a shaker (150 rpm/min) for 6 hours in the dark at room temprature, afterwords, the bottles kept in refregerator at 4 ºC. After 24 hours absorbance of leaf tissue extract was measured at wave length 663 nm and 645 nm. The total chlorophylls was calculated using the following equation according to Arnon (1949):

Total chlorophylls (g/l) = 0.0202 x A645 + 0.00802 x A663 A = absorbance at 645 or 663 nm Determination of total sugar (T.S.)

0.2 g of ground dry leaves was accurately weighed and extracted with 20 ml of 80 % ethyl alcohol by heating on boiling water bath under reflex condenser. After filtration the alcohol was evaporated from the extract under reduced pressure and the residue was transferred into 50 ml volumetric flask then made up to the volume with distilled water. Total sugars were determined by phosphomolybdic acid method according to (A.O.A.C., 1975). The extract (50 ml) mixed with 5 ml HCl (lN) then heated at 50 ºC under reflex condenser for one hour. The solution was neutralized with sodium bicarbonate (2N) and made up to 100 ml volume with distilled water and to determine the total sugars one ml of the extract was mixed with 1 ml of copper sulphate reagent and 1 ml of alkaline tartrate solution, and then heated in a boiling water bath for 10 min. The reaction mixture was cooled under running tap water then, 2 ml of phosphomolybdic acid reagent was added and the developed blue color was measured using Jasco model V-530 UV/VIS Spectrophotometer at 540 nm (A.O.A.C., 1975). Determination of total free amino acids

For preparation of the ethanol, 0.2 grams of dry leaves were weighted and extracted with 25 ml of 70 % boiling ethanol for about 10 min, then filtered through a centered glass funnel (G3). The residue was re-extracted and filtered twice with 70% boiling ethanol, then was adjusted to 100 ml with 70 % ethanol. The total free amino acids were determined by using ninhydrin reagent, as described by (Moore and Stein, 1954). Determination of total crude protein

The total nitrogen of dry leaves was determined by using the modified micro- Kjeldahel method as described by Peach and Tracy (1956). Crude protein was calculated by multiplying N percent by the factor 5.83 according to Merrill and Watt (1973). Determination of glycinebetaine and proline

Glycinebetaine was determined as reported by Grieve and Grattan (1983). Dried ground leaves (0.1 g) were treated with 5 ml of toluene-water mixture (0.5% toluene). Test tubes were shaken mechanically for 24 h at 25 ºC. The extract was filtered and made up to a volume of 100 ml; then 1 ml of the solution was treated with 1 ml 2 N HCl. An aliquot of the solution from this extract (0.5 ml) was taken and treated with 0.1 ml of potassium triiodide solution. It was then shaken in an ice bath for 90 min and then ice-cooled water (2 ml) was added with 4 ml 1,2-dichloroethane. By stirring, two layers were formed. The lower colored layer was taken and its optical density was read at 365 nm.


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Proline was determined by following the method of Bates et al., (1973). Fresh leaves were

extracted in sulphosalicylic acid, an equal volume of glacial acetic acid and ninhydrin solutions were added to the extract. The sample was heated at 100 ºC, and then 5 ml of toluene were added. The absorbance of the toluene layer was read at 528 nm, on a spectrophotometer. Proline (Sigma) was used for the standard curve. Assay of antioxidant enzymes activity

Wheat fresh leaf tissue (0.5 g) was homogenized in 5 ml of 100 mM phosphate buffer (pH 7.0) containing 1% Polyvinylpyrrolidone (PVP) and 1 mM EDTA and then centrifuged at 15,000 x g for 10 min at 5 ºC. The supernatant was collected and used for determination of antioxidant enzyme activities.

Superoxide dismutase (SOD, EC 1.15.1.1)

Superoxide dismutase activity was assayed by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) as described by Becana et al., (1986). The reaction mixture contained 50 mM Na-phosphate buffer (pH 7.8), 0.1 mM EDTA, 14.3 mM methionine, 82.5 µM NBT, 2.2 µM riboflavin and 50 µl enzyme extract. The reaction was initiated by placing the test tubes under 15 W fluorescent lamps. The reaction was terminated after 10 min by removing the reaction tubes from the light source. Non-illuminated and illuminated reactions without supernatant served as calibration standards. The absorbance was read at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of NBT reduction under assay conditions. Catalase (CAT, EC 1.11.1.6)

Catalase activity was assayed as described by Chance and Maehly (1955). The reaction mixture consisted of phosphate buffer (pH 6.8), 0.1 M H2O2 and 1.0 ml enzyme extract as a substrate. Changes in absorbance of the reaction solution at 240 nm were sequentially read every 20 s for 1 min. The disappearance of H2O2 was detected by titrating the reaction mixture against 0.1 N potassium permanganate solution. The reaction mixture without enzyme was treated as blank. One unit of CAT activity was defined as that amount of enzyme which breaks down 1 µmol of H2O2 min-1 under the described assay conditions. Ascorbate peroxidase (APX, EC 1.11.1.11)

Ascorbate peroxidase activity was assayed according to the method of Ramel et al., (2009) by monitoring the rate of ascorbate oxidation at 290 nm (ɛ, 2.8 mM-1 cm-1). The reaction mixture (3 ml) contained 1.5 ml of 0.1 M potassium phosphate buffer (pH 6.8), 0.5 ml of 6 mM ascorbate, 0.5 ml of 12 mM H2O2 and 0.5 ml of enzyme extract. Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4)

Monodehydroascorbate reductase activity was carried out as described by Hossain et al., (1984) by monitoring the change in absorbance at 340 nm due to NADH oxidation (ɛ, 6.2 mM-1 cm-1) for 4 min. The reaction mixture (1 ml) contained 90 mM K-phosphate buffer (pH 7.0), 0.0125% Triton X-100, 0.2 mM NADH, 2.5 mM ascorbate, 0.25 units ascorbate oxidase and enzyme extract. Dehydroascorbate reductase (DHAR, EC 1.8.5.1)

Dehydroascorbate reductase activity was assayed following the procedure described by Doulis et al., (1997) by measuring the reduction of dehydroascorbate at 265 nm for 4 min (ɛ,14 mM-1cm-1). The reaction mixture consisted of 90 mM K-phosphate buffer (pH 7.0), 1 mM EDTA, 5.0 mM GSH and enzyme extract. The reaction was initiated by the addition of 0.2 mM dehydroascorbate. Glutathione reductase (GR, EC 1.8.1.7)

Glutathione reductase activity was measured according to Foyer and Halliwell (1976), which depends on the rate of the decrease in the absorbance of NADPH at 340 nm (ɛ,6.2 mM-1cm-1). The reaction mixture (1 ml) contained 100 mM sodium phosphate buffer (pH 7.8), 0.5 mM GSSG, 50 µl extract and 0.1 mM NADPH.


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Statistical analysis Data were statistically analyzed using factorial experiments and the mean values were compared

using the least significant difference test (L.S.D.) at 5% and 1% levels (Snedecor and Cochran, 1980). Combined analysis was made for chemical analyses because the results of the two seasons followed a similar trend. Results and Discussion

In this study, improving wheat tolerance to drought stress via exogenous application of EBL + Spm was investigated. Thus, the effects of exogenous (0.5 μM EBL + 100 μM Spm) on growth, yield, chemical constituents and the activity of antioxidant enzymes of wheat plants grown under drought stress treatments (75% and 50% of field capacity) in comparison with the control plants were detected in two samples at 60 and 90 days after sowing during two successive seasons (2013/2014 and 2014/2015). 1. Effects of exogenous (EBL+ Spm) on growth and yield of wheat plants grown under drought stress conditions a. Plant height

Plant height of wheat plants was obviously affected by drought stress and results in Fig.1 clearly revealed that the reduction in plant height was increased by increasing drought level and recorded the maximum reduction at the highest level of drought (50% field capacity) in all samples during both seasons. However, foliar application with (0.5 μM EBL + 100 μM Spm) resulted in increases in plant height and these increases reached the significant level in plant height under (75% field capacity) water treatment and a highly significant increase under (50% field capacity) water treatment in comparison with the control plants in all samples during both seasons

b. Root length Results in Fig.1 showed that, root length of wheat plants was significantly decreased by increasing

the level of drought stress. The maximum reduction was recorded with the (50% field capacity) water treatment in all samples during both seasons. However, foliar application with (EBL + Spm) resulted in increases in root length and these increases reached the significant level under (75% field capacity) water treatment only at 90 days after sowing during both seasons, while under (50% field capacity) water treatment these increases reached the significant level at 60 days after sowing and the highly significant level at 90 days after sowing in comparison with the control plants during both seasons.

c. Number of tillers

The results of the produced number of tillers of wheat plants under drought stress revealed that drought stress caused a significant reduction in number of tillers/plant and this reduction was positively correlated with the severity of drought stress as shown in Fig.1. However, foliar application with (EBL + Spm) resulted in increases in number of tillers/plant and these increases reached the highly significant level under (75% and 50% field capacity) water treatments at 60 and 90 days after sowing during in comparison with the control plants in both seasons. d. Number of leaves

The results in Fig.1 showed that, the number of leaves/plant was slightly decreased due to drought stress and reached the significant level only at 90 days after sowing during both seasons. However, foliar application with (EBL + Spm) resulted in increases in number of leaves/plant and these increases reached the highly significant level under (75% field capacity) only at 90 days after sowing during both seasons, while these increases reached the significant and highly significant level under (50% field capacity) water treatment at 60 and 90 days after sowing during in comparison with the control plants in both seasons, respectively.


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Fig. 1. Determination of changes in plant height (cm), root length (cm), leaves number plant/1, and

tillers number plant/1 of wheat plants at 60 and 90 days after sowing of wheat plants were subjected to different foliar application treatments [control (double distilled water) and 0.5 μM EBL + 100 μM Spm] under different water stress treatments [control (100% of field capacity) and drought stress treatments (75% and 50% of field capacity)]. At harvest, grain yield (g)/plant and weight of 100 grains (g) were determined. Every column in each graph represents the mean (±SE) of three replicates. Asterisks indicate significant differences at the 0.05 (*) and 0.01 (**) levels compared with the non-treated plants.

e. Shoot dry weight The obtained results in Fig.1 confirmed the detrimental impact of drought on growth of root and

shoot and consequently the accumulation of dry matter in these organs of stressed wheat plants. Thus, the reduction in dry weight of shoot was recorded and reached the significant and highly significant


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level under (75% field capacity) at 60 and 90 days after sowing during both seasons, respectively. Moreover, the reduction in shoot dry weight was reached the highly significant level under (50% field capacity) water treatment at 60 and 90 days after sowing during both seasons. As a result of foliar application with (EBL + Spm) highly significant increases were recorded in shoot dry weight under (75% and 50% field capacity) water treatments at 60 and 90 days after sowing in comparison with the control plants during both seasons. f. Root dry weight

The results in Fig.1 also confirmed the reduction in root dry weight which was reached the significant and highly significant level under (75% field capacity) at 60 and 90 days after sowing during both seasons, respectively. While, the reduction in root dry weight was reached the highly significant level under (50% field capacity) water treatment at 60 and 90 days after sowing during both seasons. However, foliar application with (EBL + Spm) resulted in significant and highly significant increases which were recorded in root dry weight under (75% and 50% field capacity) water treatments at 60 and 90 days after sowing in comparison with the control plants during both seasons, respectively.

The effect of drought stress on growth of wheat plants could be attributed to over-production of ROS which can destabilize the cell membrane and can cause direct damage to DNA, pigments, proteins, lipids, and other essential cellular molecules, leading to cell death and loss of biomass (Miller et al., 2010). In contrast to negative effects of drought stress, foliar application with (0.5 μM EBL + 100 μM Spm) improved growth and development of stressed wheat plants and the obtained results evedently indicate to a synergistic influence of this treatment on all growth characters; plant height, root length, number of tillers/plant, number of leaves/plant, shoot dry weight and root dry weight.

In this context, BRs have to their credit a host of roles in general plant growth and development. BRs can activate the cell cycle during seed germination (Zadvornova et al., 2005), control progression of cell cycle (González-Garcia et al., 2011), induce exaggerated growth in hydroponically grown plants (Arteca and Arteca, 2001), and also control proliferation of leaf cells (Nakaya et al., 2002). Furthermore, the application of polyamines (PAs) is an effective approach for enhancing stress tolerance of crops (Chai et al., 2010; Shi et al., 2010; Xu et al., 2011b, Radhakrishnan and Lee, 2013). PAs are cationic molecules that are essential for plant growth and are involved in seed germination, root growth, flower and fruit development, cell division, elongation, replication, transcription, translation, and membrane and cell wall stabilization (Hussain et al., 2011). g. Yield components

Due to the obvious effects of drought stress on growth and development of wheat plants in this study, yield reduction was significantly occurred. Thus, results of yield components in Fig.1 showed that, drought stress resulted in a significant and highly significant reduction in number of grains/plant under (75% and 50% field capacity) water treatments in comparison with the control plants in both seasons, respectively. Moreover, the reduction in grain yield (g)/plant and weight of 1000 grains was recorded and reached the highly significant level under (75% and 50% field capacity) water treatments in comparison with the control plants in both seasons. However, foliar application with (EBL + Spm) resulted in highly significant increases in all yield components. The results of growth characters and yield components clearly proved that, drought stress conditions significantly disrupted the growth of wheat plants and consequently the yield of these plants was significantly reduced in comparison with the control (well-watered condition). However, the EBL + Spm treatment improved the growth and yield of stressed plants in comparison with non-treated stressed plants (control treatment) and these results confirmed the positive effects of EBL and Spm on enhancing drought tolerance in wheat plant. Thus, the EBL + Spm treatment was significantly increased plant height, root length, number of leaves/plant, number of tillers/plant, shoot and root dry weight, in addition to yield components; number of grains/plant, grain yield (g)/plant and 1000 grains weight (g) under drought stress conditions. In addition, these results confirmed that, the EBL + Spm treatment was alleviated the detrimental effects of drought stress on wheat plants and recorded a highly significant increase in plant yield of both drought stressed and non-stressed plants. In this context, the yield components like grain number and grain size were decreased under pre-anthesis drought stress treatment in wheat (Edward and Wright, 2008). Hasheminasab et al., (2012), reported that, growth and grain yield of wheat plants significantly decreased under drought stress conditions.


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Moreover, the obtained results in this study, on the positive impact of 24-epibrassinolide and spermine on drought stressed wheat plants are in accordance with those previously reported by several workers, thus, exogenous EBL + Spm treatment has the ability to regulate plant growth (Duan et al., 2008; Kubis, 2008; Zhang et al., 2009; Chai et al., 2010; Shi et al., 2010; Hussain et al., 2011; Xu et al., 2011a and b) and substantially enhances wheat yield and its stress tolerance by inducing cellular changes that are related to stress tolerance like stimulate nucleic acid and protein synthesis (Dhaubhadel et al., 2002), activate ATPase pump (Khripach et al., 2003), increase antioxidant enzyme activities and osmoprotectants accumulation (Ozdemir et al., 2004), induce other hormone responses (Vert et al., 2005), regulate stress-responsive genes expression (Kagale et al., 2007) and induce photosynthetic efficiency and the translocation of photosynthesis to the sink (Shahbaz et al., 2008). Therefore, it could be concluded that, exogenous EBL+Spm evidently proved an effective influence on improvement of growth and productivity of drought stressed wheat plants. 2. Effects of exogenous (EBL+ Spm) on chemical constituents and the activity of antioxidant enzymes of wheat plants grown under drought stress conditions a. Relative water content, membrane stability index (%), lipid peroxidation and electrolytes leakage

In this study, drought stress caused a significant decrease in relative water content (%) and membrane stability index (%) in leaves of wheat plants at 60 and 90 days after sowing. However, the EBL + Spm treatment was significantly increased relative water content (%) and membrane stability index (%) in treated plants in comparison with non-treated plants under drought stress conditions as shown in Fig. 2. On the contrary, the lipid peroxidation and electrolytes leakage level were significantly increased in leaves of wheat plants at 60 and 90 days after sowing under drought stress conditions as shown in Fig. 2, meanwhile, the EBL + Spm treatment caused a highly significant decrease in the lipid peroxidation and electrolytes leakage level in treated plants in comparison with non-treated plants under drought stress conditions.

Therefore, with these evidences it could be concluded that, the EBL + Spm treatment improved drought tolerance in wheat plants which retained more water content in their cells and maintained the stability of cell membrane. The obtained results here are in accordance with several reports, thus, lipid peroxidation (LP) is considered as the most damaging factor in every living organism under various stresses (Renu and Devarshi, 2007). LP determined as malondialdehyd (MDA) content. Hasheminasab et al., (2012) reported that, under drought stress LP increased significantly in wheat as a result of oxidative stress and consequently cell membrane injury has also been reported by various studies (Zlatev et al., 2006; Turkan et al., 2005; Amjad et al., 2011).

Membrane damage is sometimes taken as the major parameter to estimate the level of lipid destruction under various stresses. The degree of cell membrane injury induced by drought stress may be easily estemated through measurements of electrolyte leakage from the cell (Sarvajeet and Narendra, 2010; Ahmadizadeh et al., 2011). Membrane stability index (MSI) sgnificantly decreased under drought stress in wheat (Hasheminasab et al., 2012), the decrease in membrane stability reflected the extent of lipid peroxidation caused by reactive oxygen species (Sariam and Sirvastava, 2001). Higher MSI and antioxidant activity with lower LP and H2O2 have been reported in drought tolerant genotypes of wheat (Renu and Devarshi, 2007), bean (Zlatev et al., 2006) and Mulberry (Ramachandra et al., 2004). The content of MDA was higher in plants treated with PEG. High concentrations of MDA decrease the membrane fluidity and damage membrane proteins, thus inactivating receptors, enzymes, and ion channels (Gill and Tuteja, 2010). The application of Spm significantly reduced the MDA levels in osmotically stressed and unstressed plants. Similar results were observed in Spm-treated rice plants under osmotic stress conditions (Farooq et al., 2009). High MDA levels indicate that plants are affected by oxidative stress. The reduction in MDA content by Spm treatment indicates decreased membrane lipid peroxidation. To date, there is relatively little information on the effect of Spm on the regulation of antioxidant concentrations during plant adaptation to osmotic stress (Yamaguchi et al., 2007; Farooq et al., 2009; Shi et al., 2010).


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Fig. 2: Determination of changes in relative water content (%), lipid peroxidation, electrolyte leakage

(%) and membrane stability index (%) in leaves of wheat plants at 60 and 90 days after sowing. Wheat plants were subjected to different foliar application treatments [control (double distilled water) and 0.5 μM EBL + 100 μM Spm] under different water stress treatments [control (100% of field capacity) and drought stress treatments (75% and 50% of field capacity)]. Every column in each graph represents the mean (±SE) of three replicates. Asterisks indicate significant differences at the 0.05 (*) and 0.01 (**) levels compared with the non-treated plants.

b. Concentration of total chlorophylls, total crude protein, total sugars, total free amino acids, proline and glycinebetaine

In the present study, results of the concentration of total chlorophylls (mg/g D.W.) and total crude protein (mg/g D.W.) indicated that, drought stress caused a significant decrease in leaves of wheat plants at 60 and 90 days after sowing. However, the EBL + Spm treatment significantly enhanced the concentration of total chlorophylls and total crude protein in treated plants in comparison with non-treated plants under drought stress conditions as shown in Fig 3. The obtained results are in accordance with previuos reports, thus, drought stress, among other changes, has the ability to reduce the tissue concentrations of chlorophylls and carotenoids (Havaux, 1998; Kiani et al., 2008), primarily with the production of ROS in the thylakoids (Niyogi, 1999; Reddy et al., 2004). Moreover, reports dealing with the strategies to improve the pigments contents under water stress are entirely scarce. However, the obtained results here provide evidences to conclude that, the potection of the plant cells from oxidative stress due to the effective role of the EBL and Spm as a synergistic influence was proved.

Drought stress caused a significant increase in total sugars (mg glucose/g D.W.), total free amino acids (mg/g D.W.), free proline (mg/g D.W.) and glycinebetaine (mg/g D.W.) in leaves of wheat plants at 60 and 90 days after sowing as shown in Fig 3. Moreover, the EBL + Spm treatment significantly increased the concentration of total sugars, total free amino acids, free proline and glycinebetaine in treated plants in comparison with non-treated plants under drought stress conditions. These results proved that, the EBL + Spm treatment lead to increase the concentration of these important compounds in leaf cells of drought stressed wheat plants which reflects the positive effects of this treatment in improving drought tolerance in wheat plants. It is well known that growth of plants under drought stress is less than unstressed plants and this of course due to the inhibition of protein synthesis in the cell. Many reports indicated that the biosynthesis of some amino acids was dramatically decreased by increasing the level of drought consequently concentration of protein recorded significant decreases in comparison to control plants. Inhibition of protein biosynthesis means that free amino acids which did not incorporated into polypeptides will accumulate in the cell, and this can explain why a high


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concentration of free amino acids are accumulated in cells of stressed plants. The concentration of these active compounds; total chlorophylls, total crude protein, total sugars, total free amino acids, free proline and glycinebetaine was significantly increased in leaves of treated wheat plants with (EBL + Spm) under drought stress conditions. Moreover, in response to this treatment the maintenance of plasma membrane integrity was confirmed. Non-enzymatic antioxidants comprise of ascorbic acid, phenolics, proline, flavonoids, β-carotenes and reduced glutathione (GSH). Proline is the most common osmoprotectant that accumulates in plants in response to water stress. Accumulation of protective solutes like proline and glycine betaine is a unique plant response to drought stress. Also proline is considered as a potent antioxidant and potential inhibitor of programmed cell death (Bates et al., 1973; Pireivatloum et al., 2010).

Fig. 3. Determination of changes in total Chlorophylls (mg/g D.W.), total sugars (mg glucose/g D.W.),

total free amino acids (mg/g D.W.), total crude protein (mg/g D.W.), free proline (mg/g D.W.) and glycinebetaine (mg/g D.W.) in leaves of wheat plants at 60 and 90 days after sowing. Wheat plants were subjected to different foliar application treatments [control (double distilled water) and 0.5 μM EBL + 100 μM Spm] under different water stress treatments [control (100% of field capacity) and drought stress treatments (75% and 50% of field capacity)]. Every column in each graph represents the mean (±SE) of three replicates. Asterisks indicate significant differences at the 0.05 (*) and 0.01 (**) levels compared with the non-treated plants.

Under drought stress conditions, free proline content significantly increased in plants as reported

in several studies; in wheat (Geravandi et al., 2011), in tomato (Behnamnia et al., 2009), in bean (Turkan


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et al., 2005). Free proline accumulation takes place in plants subjected to drought stress. It acts as osmolyte for osmotic adjustment during drought as well as stabilizes sub-cellular structures such as membrane and proteins, scavenges free radicals and buffers cellular redox potential under stress (Schafleitner, 2007).

Accumulation of proline under water stress protects the cell by balancing the osmotic potential of cytosol with that of vacuole and external environment. Pireivatloum et al., (2010) reported that, proline is an important osmolyte to adjust the plant under drought condition. In addition, proline can be considered as potent non-enzymatic antioxidants that plants need to counteract the inhibitory effects of ROS.

d. Effects of exogenous (EBL+ Spm) on the activity of antioxidant enzymes In this study, drought stress caused an obvious increase in the anti-oxidative enzyme activities;

APX, MDHAR, DHAR, CAT, GR and SOD, as shown in Fig 4. However, the EBL + Spm treatment lead to highly significant increase in the activity of these anti-oxidative enzymes in treated plants in comparison with non-treated plants under drought stress conditions.

These results evedently proved that, exogenous EBL + Spm alleviated the detrimental effects of drought stress on wheat plants and decreased the accumulation of ROS by enhancing their scavenging through elevation of antioxidant enzymes activity. Many studies reported that activity of antioxidant enzymes can be correlated with the drought tolerance capability of wheat plants (Hasheminasab et al., 2012; Abdullah and Ghamdi, 2009).

ROS produced under stress conditions are scavenged by antioxidants, allowing normal growth. GSH is one of the nonenzymatic antioxidants, which, besides its involvement in stress response, plays a role in cell differentiation (Rausch and Wachter, 2005). The central role of antioxidants such as GSH in the acclimation of plants to osmotic stress enables them to be used as stress markers. Benavides et al., (2000) reported that Spm application effectively restored the GSH content in paraquat-treated sunflower leaves. Antioxidant defense systems are rapidly activated in plants to protect themselves against unfavorable environmental stress conditions (Gill and Tuteja, 2010). ROS metabolism is interrelated with antioxidant enzymes such as POD, CAT, and SOD. Field and pot experiments of 0.2 mgl−1 BL application to 1-year-old Robinia pseudoacacia seedlings grown under drought stress increased the activity of SOD, POD and CAT, and the contents of soluble sugars and free proline (Li et al., 2008). Radhakrishnan and Lee (2013) indicated that, low levels of GSH in plants affected by osmotic stress, with the GSH level recovering after treatment with Spm. However, the CAT activity was lower in osmotically stressed plants, whereas treatment with Spm resulted in elevated CAT activity. These results are consistent with those of Farooq et al., (2009a), who found that Spm treatment alleviated the effect of osmotic stress by enhancing CAT activity in rice plants, which might resulted in the elimination of H2O2. Endogenous PAs counteract oxidative stress in plants by acting as free radical scavengers or by binding with antioxidant enzyme molecules to scavenge the ROS and enhance the enzyme activity (Alcazar et al., 2010). Whereas no significant change in the activity of SOD was observed in plants treated individually with PEG and Spm, the combined effects of PEG and Spm accelerated the activity of SOD in soybean plant. Yang et al., (2011) reported an increased level of SOD activity with exogenous Spm treatment of Potamogeton malaianus plants under cadmium stress. Among the detoxifying enzyme systems, SOD plays an essential role in scavenging ROS, and H2O2 produced via SOD action is further scavenged by POD and CAT (Jaleel et al., 2009). Shi et al., (2010) observed that SOD activity in Spm-treated plants was significantly higher under dehydration stress conditions than in the controls. Radhakrishnan and Lee, (2013), reported that, plants enhance their antioxidant capacity to counteract the effects of osmotic stress. The accumulation of MDA, GSH, CAT, SOD, POD, and PPO changed in Spm-treated plants compared to controls.

The phytohormones brassinosteroids and polyamines can regulate the protective responses of plant to various stresses 24-Epibrassinolide and spermine modify antioxidant enzymes and non-enzymatic antioxidants in plants under different stress conditions Talaat and Shawky, 2012 and 2013. Talaat et al., (2015) reported that, dual application of Spm + EBL significantly alleviated drought-induced inhibition in the activities of monodehydroascorbate reductase and dehydroascorbate reductase as well as in the ratios of AsA/DHA and GSH/GSSG. Overall, dual application improved the plant drought tolerance and decreased the accumulation of ROS by enhancing their scavenging through elevation of antioxidant enzymes activity and improving the redox state of ascorbate and glutathione.


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Also, Hanafy Ahmed et al., (2017) reported that, Put and EBL increased antioxidant enzymes CAT, POX and SOD activities.

Finally, the obtained results in this study all together provide strong evidences drive us to conclude that, a synergistic influence on growth and development of drought stressed wheat plants can attributed to exogenous EBL + Spm which accompanied with enhancement in plant metabolism and consequently the productivity.

Fig. 4. Determination of changes in the activities of superoxide dismutase (SOD), catalase (CAT),

ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) in leaves of wheat plants at 60 and 90 days after sowing. Wheat plants were subjected to different foliar application treatments [control (double distilled water) and 0.5 μM EBL + 100 μM Spm] under different water stress treatments [control (100% of field capacity) and drought stress treatments (75% and 50% of field capacity)]. Every column in each graph represents the mean (±SE) of three replicates. Asterisks indicate significant differences at the 0.05 (*) and 0.01 (**) levels compared with the non-treated plants.

Conclusions Drought stress is a major agricultural threat that adversely affects crop production and may lead

to an imbalance between antioxidant defenses and the amount of ROS, resulting in oxidative stress. To survive such stress, plants have invoked various defense mechanisms to respond and adapt to water stress. Emerging evidence indicates that, in many species, the phytohormones brassinosteroids and polyamines can regulate the protective responses of plant to various stresses. They control plant stress


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responses and regulate stress- responsive gene expression. 24-Epibrassinolide and spermine modify antioxidant enzymes and non-enzymatic antioxidants in plants under different stress conditions. Therefore, in this study the obtained results all together can explain and confirm the positive role of exogenous EBL+Spm in enhancing growth and yield of drought stressed wheat plants. Moreover, the obtained results are in accordance with several reports on the role of BRs and polyamines (PAs) in enhancing plant stress tolerance through increasing the concentration of some active substances in the cells of stressed plants such as; chlorophylls, proteins, total sugars, total free amino acids, proline and glycinebetaine, in addition to the increases in the activity of antioxidant enzymes; APX, MDHAR, DHAR, CAT, GR and SOD, which resposible for protecting cell enzymes, organelles and cell membranes through reducing the oxidative stress by enhancing the ROS scavenging antioxidant defense machinery in plant cell.

In conclusion, the obtained results in this study proved the beneficial effects of foliar application treatment with (0.5 μM 24-epibrassinolide + 100 μM spermine) on growth characters, chemical constituents and consequently the productivity of wheat plants under drought stress conditions. These effects may attribute to the protective role of 24-epibrassinolide and spermine in plant cells from the oxidative stress induced by drought. Therefore, the obtained results confirmed the effective influences of 24-epibrassinolide (EBL) and spermine (Spm) for improving drought tolerance of wheat plant by increasing osmopotectants and enhancing ROS scavenging antioxidant defense machinery which resposible for protecting cell enzymes, organelles and cell membranes. Therefore, with these evidences it could be concluded that, the massive impact of the EBL and Spm which could be concluded as a synergistic influence can attributed to the crosstalk between EBL and Spm with all endogenous phytohormones and finally their impact on the gene expression network to potect the cell from oxidative stress.

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Exogenous application of 24-epibrassinolide and spermine ...curresweb.com/mejas/mejas/2019/mejas.2019.9.4.22.pdfSep 04, 2019 · Wheat plants of cv. Giza 168 were subjected to well-watered