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Journal of Stress Physiology & Biochemistry, Vol. 16, No. 4, 2020, pp. 35-48 ISSN 1997-0838 Original Text Copyright © 2020 by Ebtesam A.Q.
ORIGINAL ARTICLE
Effect of Exogenous Trehalose on Physiological Responses of Wheat Plants Under Drought Stress
Ebtesam Ahmed Qaid
Department of Biology, Faculty of Science, University of Taiz, Taiz, Yemen
*E-Mail: [email protected]
Received June 9, 2020
Drought stress causes physiological changes in plant morphological growth and development. The protective role of trehalose (Tre) was investigated through exogenously applied to seedlings of wheat cv. Sakha 93 plants. Pots experiment were divided into four groups: control, Tre treatment, drought stress and drought stress with Tre treatment. Samples were collected in two stages after 7 and 14 days from drought stress to determine the fresh weight, dry weight, chlorophyll pigments, relative water content, electrolytic leakage, proline, malondialdehyde, antioxidant enzyme activities, and carbohydrates content. Drought stress reduced many growth and physiological characters. It significantly increased specific activities of guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) and significantly decreased the catalase (CAT) activity. It caused significantly increased in the Tre content while reduced sucrose and starch contents. Combination exogenous applied Tre (40 mM) with drought stress improved some growth parameters and photosynthetic pigments. Application of Tre markedly decreased proline and malondialdehyde contents, GPX; APX activities whereas increased CAT activity. Tre treatment appeared increased in the internal Tre content, on the other hand; exogenous Tre maintained the sucrose and starch contents in the seedlings of wheat plants under drought stress. The results concluded that applied Tre alleviated the adverse effect of drought stress on wheat plants by enhancing the antioxidant defense system and conservation of the membrane stability.
Key words: Alleviate, Antioxidant Enzymes, Combination, Morphological, Physiological, Protective
Abbreviations: APX: Ascorbate peroxidase, CAT: catalase, GPX: guaiacol peroxidase, MDA: Malondialdehyde, ROS: Reactive oxygen species, RWC: Relative Water content
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Wheat (Triticum aestivum L.) is the most important food crop in many developing countries. Wheat grains are a concentrated source of vitamins, minerals, and protein (Rauf et al., 2007). Its productivity has noticeably affected by most of the environmental constraints.
Among abiotic stress, drought causes marked changes in molecular, biochemical, and physiological processes that affecting plant growth and development (Farooq et al., 2010). It has an adverse effect on plant photosynthetic efficiency that due to closing stomata which limits CO2 diffusion into leaf, or inhibits Rubisco activity and breaks down energy balance and break down the distribution during photosynthesis (Rapacz et al., 2010).
In plants, the links between the production of reactive oxygen species (ROS) and photosynthetic metabolism are particularly important. The ROS has been shown to cause membrane integrity loss, oxidative damage biomolecules including protein, lipids, and DNA which leads to the release of malondialdehyde (MDA) and the regulation of antioxidant enzymes (Zhang et al., 2007). The plants can avoid the damage caused by drought stress through enhancement of the antioxidant defense system that scavenges ROS and compatible solutes accumulation (Alam et al., 2014). Enhanced antioxidant enzymes activities such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) improve drought tolerance in many crop plants (Alam et al., 2013).
Many substances are accumulated in plants under biotic and abiotic stresses like soluble sugars and free amino acids. Among different compatible solutes, Tre is a non-reducing disaccharide that is important storage carbohydrate and stress protection metabolites in yeast, bacteria, and certain fungi (Elbein et al., 2003), (Gancedo & Flores, 2004). Only trace amounts of Tre were detected in higher plant tissues (Garg et al., 2002). Tre improves the response of plants to drought, nutrition element deficiency, or salinity stresses (Chang et al., 204). Tre is likely to function through its ability to act as a direct and indirect scavenger of ROS, conferring protection to the machinery of protein synthesis (Zhu,
2002). Tre may have an indirect effect on carbohydrates metabolism by interfering with photosynthetic capacity and the conversion and utilization of other sugars (Ranwala & Miller, 2009).
Exogenously applied Tre is readily accumulated and transported by leaf or roots tissues and displays significant roles as osmoprotectants (Luo, et al., 2010). Application of Tre for improvement of environmental stress tolerance was reported in several plants such as Zea mays under salt stress (Zeid, 2009), (Li et al., 2014)]; Arabidopsis thaliana against drought stress (Stolker, 2010); rice seedlings under salt stress (Nounjam et al., 2012)]; Catharanthus roseus has grown under saline condition (Chang et al., 2014). and Brassica species under drought stress (Alam et al., 2014).
The present study investigated the role of exogenous application of Tre in reducing the adverse effect of drought on growth parameters, different antioxidant enzymes in cv. Sakha 93 plants under drought stress.
MATERIALS AND METHODS
Plant Seeds
Wheat seeds (Triticum aestivum cv. Sakha 93) were purchased from Agriculture Research Center, Giza, Egypt.
Greenhouse Experiment
Seeds of Sakha 93 cultivar were sterilized with ethanol (70%) and washed with distilled water then surface sterilized with 20% sodium hypochloride (v/v) for 20 min, and thoroughly rinsed with sterilized distilled water. Fifteen seeds were sown in each pot (12 cm in diameter) contained 500g of sterilized perlite and vermiculate (1: 1, w/w). The average environmental conditions were: temperature, 25 ± 3 °C; light, 5000 Lux; RH, 70% during entire period of experimentation.
Seedlings were grown up to 10 days irrigated with sterilized tap water (70% field capacity) then thinning was started so that five plants of uniform size were maintained in each pot. Pots were divided into four groups:
Control plants were irrigated with the half strength of Hoagland solution
Treated Tre plants were irrigated with half strength of Hoagland solution supplemented with 40 mM Tre
Drought Plants were subjected to drought by withholding irrigation for 7 and 14 days
Drought +Tre Plants were treated with 40 mM Tre, then subjected to drought by withholding irrigation for 7 and 14 days
Plants were subjected to drought after 20 days from planting. Samples were collected in two stages after 7 and 14 days from drought stress to determine various physiological and biochemical parameters. Each treatment was replicated three times under the same experimental conditions. To determine FW, 10 shoots were separated and weighed to determine FW (mg/ plant). To quantify DW, 10 shoots from each treatment were oven-dried at 60 °C for 3 days and weighed, then expressed as mg/plant. Relative Water content (RWC)
RWC was measured according to Jones and Turner, (1978). Freshly excised leaves were weighed to obtain leaves samples weight (FW). Then, the samples were immediately transferred to the deionized water for 3 h under normal room light and temperature. After 3 h the samples were taken out of the water and well dried of any surface moisture quickly and lightly with filter paper and weighed to obtain fully turgid weight (TW). The samples were oven-dried at 70°C for 48 h and weighed (after being cooled) to determine the dry weight (DW). RWC was calculated by the following formula: RWC (%) = [(FW-DW)/(TW-DW)]x100 Determination of Photosynthetic Pigments
Fresh leaves were extracted with 80% acetone and supernatant were obtained by centrifuging at 5,000g. the absorbance of the supernatants was were taken with UV-visible spectrophotometer at 663, 644, and 470 nm and concentrations of chlorophyll a, b, and carotenoids were calculated according to Metzner et al. (1965). The concentration of each pigment was calculated from the following formula:
Chlorophyll a = 10.3 E663 - 0.918 E644 Chlorophyll b = 19.7 E644 - 3.87 E663 Carotenoids = 4.2 E470 (0.0264 Ch a + 0.426 Ch b)
The results were expressed as цд pigment /ml extract, then calculated as mg pigment/g fresh weight. Determination of Proline Content
Proline (Pro) content in leaf tissues was determined according to the protocol of Bates et al. (1973). Fresh leaf tissue (0.5 g) was homogenized well in 10 ml of 3% sulfosalicylic acid in ice. The homogenate was centrifuged at 11,500xg for 15 min. Two ml of the filtrate was mixed with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid. The mixture was placed at 100°C in the water bath for 1 h, then transferred in to test tube and kept in ice to be cooled, after it was cooled, 4 ml of toluene was added and mixed thoroughly by vortex mixture. After some time chromophore containing toluene was read spectrophotometrically at 520 nm. Purified proline with different known concentrations was used to make a standard curve by comparing with the proline content of plant samples were calculated. Determination of Electrolyte Leakage
Electrolyte leakage (EL) of tissue was measured according to the method described by Gilley and Fletcher, (1997), using conductance meter (Model CD -4301, Lutron). EL was calculated as a ratio of the conductivity before and after boiling. Determination of MDA Level
The level of lipid peroxidation was measured by estimating malondialdehyde (MDA), using thiobarbituric acid (TBA) according to Dhindsa and Matowe, (1981). The MDA level was calculated by using the molar extinction coefficient of 155 mM-1cm-1 and expressed as Mmol of MDA g-1 fresh weight. Determination of Protein
Soluble protein was determined according to Lowry et al. (1951) as follows:
Mixture of alkaline sodium carbonate solution (2% Na2CO3 in 0.1 N NaOH), 0.5 % CuSO4 and 1.0 potassium and sodium tartrate was prepared by the ratio of (98 : 1 : 1) (v/v/v). To 1.0 ml of the plant extract, 0.5 ml of the previous mixture was added and allowed to stand at room temperature for 10 min then; 0.5 ml of diluted
folin reagent 1: 9 (v/v) was added and mixed rapidly. After 30 min the absorbance was read at 750 nm. The standard curve of protein was carried out using egg albumin as standard protein. Enzymes Extraction and Assay
Leaves were ground using pre-chilled mortar and pestle, and ice-cooled 100 mM K-phosphate pH 7.8, 60 mg PVP (Polyvinyl pyrolidene), were added. The homogenate was vortexed and centrifuged at 12000 g for 15 min at 4°C. The supernatant was considered as enzyme crude extract and used to measure the activities of GPX, APX, and CAT.
The specific activity was calculated after estimation of total soluble proteins according to Lowry et al., (1951).
Assay of guaiacol peroxidase (GPX)
The activity of GPX was determined according to the method of Velikova et al.,2000. The reaction mixture of 3 ml contained 50 mM K-phosphate buffer PH 7, 0.2% guaiacol (w/v) and 0.04 ml enzyme extract was prepared. The absorbance at 470 nm was measured 5 min after the addition of 3 mM H2O2. The activity of GPX was calculated as mM of guaiacol reduced using the extinction coefficient of 26.6 mm-1cm-1. The specific activity was calculated as mM guaiacol reduced min-1 mg-1 protein.
Assay of Ascorbic peroxidase (APX)
For APX assay, the method described by Nakano and Asada, (1980) was used.
The reaction mixture of 3 ml contained 0.5 mM ascorbic acid, 0.1 mM EDTA (Ethylene diamine tetra acetic acid), and 0.1 ml enzyme extract. The reaction was initiated by adding 1.5 mM H2O2. The absorbance of the reaction mixture was measured at 290 nm. The specific activity of APX was calculated using the extinction coefficient of 2.8 mM-1 cm-1 and expressed as mM ascorbate oxidized min-1 mg protein. Assay of Catalase activity (CAT)
CAT activity was estimated by 3 ml reaction mixture contained 10 mM k- phosphate buffer pH7, 0.1 ml enzymes extract, and 0.035% H2O2. The activity of CAT was calculated based on the decline in the absorbance at 240 nm as the decomposition of H2O2 and used the
extinction coefficient of 40 mM-1 cm-1, and represented as mM H2O2 reduced min-1 mg-1 protein according to Velikova et al. (2000).
Extraction and Determination of Trehalose Extraction of trehalose:
Trehalose extraction was carried out according to Ferreira et al. (1997), by boiling 100mg frozen tissue in 2 ml ethanol, evaporated at 60 °C, the residue was dissolved in 5 ml of 5 mM H2SO4, centrifuged at 10,000 g for 10 min and filtered. Filtrate was heated in a boiling water bath for one hour to hydrolyze sucrose in the extract because the sucrose retention time is the same as that of trehalose. Then pH was adjusted to 7.0, the solution was evaporated and the residue was dissolved in distilled water.
Determination of trehalose: Trehalose content was determined by the method described by Cizmarik et al., (2004). The determination of trehalose was carried out using HPLC (Hewlett Packard, HP 1090 liquid chromatography), using Hypersil, 100 x 3 mm, 3^m column.
The separation was carried out at 32 °C using a flow rate of 0.8 ml/ min with acetonitrile: H2O (85: 15) as the mobile phase. The elution was detected with a Diode Array Detector (DAD) at 192 nm. Extraction and Determination of Sucrose: Extraction of sucrose
Sucrose was extracted by grinding 0.1 g of tissue, mixing with 10 mg PVP, 80% ethanol. The homogenate has been incubated at 70°C for 20 min. After centrifugation at 4,000 g for 10 min, the supernatant was separated and the pellet was re-extracted twice with 80% ethanol at 70 °C for 30 min. The supernatant was pooled and evaporated. The residue was dissolved in 1 mL of deionized H2O.
Determination of Sucrose The content of sucrose was determined using the previously mentioned HPLC method (Cizmarik et al., 2004).
Extraction and Determination of Starch Extraction of starch:
Starch was extracted according to the method of
Grotelueschen and Smith, (1967). 0.1 g of fine dry powder of shoots was extracted with 5 ml of 80 % ethanol, by boiling the sample in 95 °C water bath for 10 min, centrifuged at 2,500 g for 5 min. For complete extraction, the previously mentioned steps were repeated twice. The supernatants were pooled and used for the determination of total soluble sugars especially fructan content. The remaining residues were extracted by adding 5 ml of 0.005 N H2SO4, heated in the water bath at 95 °C for one hour and centrifuged at 4,000 for 10 min.
Determination of starch:
Released glucose was determined in the supernatant by glucose oxidase peroxidase kit according to the methoddescribed by Bergmeyer and Bernt, (1974). Statistical Analysis
Results are the mean of three measurements for each treatment. All obtained data were subjected to oneway analysis of variance (ANOVA), and the mean differences were compared by Duncan's multiple range test (DMRT) using SPSS software version 10. Different letters indicate significant differences between treatments at p < 0.05.
RESULTS
Plant Growth Parameters
Plant growth parameters such as fresh weight (FW), and dry weight (DW), were determined to estimate the negative effects of drought stress on plant growth and the potential mitigation effects of Tre on the drought-stress wheat plant (cv. Sakha 93).
Data in Table 1 indicate that drought stress and trehalose treatment caused a significant effect on FW, DW, and leaves area. FW of drought-stress wheat plants decreased by 22.16%, 26.60% whereas DW decreased by 20.32% and 25.23% at days 7 and 14, respectively, compared with control. However, wheat plants subjected to drought stress that pretreated with 40 mM Tre showed significantly mitigation in FW which reaching to 13% and 15.11% and DW that reached to 10.74% and 13.84% at days 7 and 14, respectively compared with control.
Wheat plants under drought stress displayed inhibition in leaf area which reaching to 32.24% at 14
days compared with control whereas the inhibition was 19.27% in the case of plants that pretreated with 40 mM Tre.
Tre application (40 mM) did not alter RWC compared with control. RWC significantly decreased in wheat plants that suffered from drought stress which reached to the highest value (23.14%) at 14 day compared with control. Whereas pretreated with Tre caused alleviation from the adverse factor of drought stress on RWC of wheat plants which reached to 12.12% compared with control.
Data in Table 2 cleared the effect of drought stress and exogenous Tre application on chlorophyll a, b and carotenoid contents in wheat plants. Drought stress showed marked reduction in chlorophyll a, b and carotenoid contents of wheat plant cv. Sakha 93 at 7 and 14 days. The highest reduction in Chl a, b and carotenoid contents were observed at 14 days which was 24.43%, 27.74% and 45.97%, respectively. However, pretreatment with Tre (40 mM) significantly improved chlorophyll a, b and carotenoid contents under water stress which was 8.61%, 15.24%, and 25.28%, respectively compared with control.
The pattern of change in EL% in wheat plants was recorded in Table 2. As compared to control plants, the significant increment of EL% was 59.5% at 14 days after water stress. The application of Tre reduced the negative effect of water stress on EL% which was 15.14% at 14 days compared with control. Proline content:
Under drought stress, the proline content was drastically increased in cv. Sakha 93, the level was 2 and 1.8 fold than control at 7 and 14 day, respectively. Feeding with Tre significantly decreased the proline level in wheat plants which reached to 1.2 fold at the two ages, but a level still higher than the control as showing in Figure 1. MDA Content
Data in Figure 2 show that MDA level markedly increased in wheat plants, the highest value of MDA level was recorded at 14 days from drought stress compared with the control. Exogenous Tre treatment caused significant decrement in the MDA content
compared to drought-stressed plants, the percentage of alleviation of drought stress was reached to 50% in the case of MDA compared to control. Antioxidant Enzymes
Data presented in Figure 3 show that the specific activities of guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT) in the shoot of cv. Sakha 93.
The effect of Tre treatment (40 mM) on antioxidant enzyme activities was non-significant at the first time of the experiment compared with control. On the other hand, Tre treatment exhibited increasing in GPX, APX and CAT activities during the second period of the experiment. Drought stress markedly increased specific activities of GPX and APX and significantly decreased the CAT activity. The highest value of GPX and APX activities was 42% and 79%, respectively compared with the control after 14 day from drought stress treatment.
On the other hand, the reduction in specific activity of CAT was 77% at the same time of drought duration compared to the control.
Externally applied Tre (40 mM) significantly increased the specific activities of GPX and APX and markedly decreased in specific activity of CAT. Carbohydrates Contents
Results in Table 3 revealed that Tre, sucrose and starch contents in cv. Sakha 93 leaves. Tre treatment alone led to a significant increase in the content of internal Tre, which reaching to 5-folds; sucrose and starch during the two times of the experiment which reaching to 132%, 8.5% and 17%, respectively at the second time of treatment compared to control. Tre level was increased while sucrose and starch levels were reduced in leaves of cv. Sakha 93 grown under water stress. The highest value of Tre level was 3 fold compared with control after 14 day of drought stress.
Table (1): Effect of drought and exogenous Tre (40 mM) combined with drought on growth parameters; Fresh weight (mg/plant), Dry weight (mg/plant), leaves area (cm2/plant) and RWC in wheat seedlings 7 and 14 days after drought stress.
Duratio n Treatment Parameters
FreshWeight (mg/ plant) Dry Weight (mg/plant) Leaves area (cm2 / plant) RWC %
7 Day Control 643.67 ± 20.11 a 67.00 ± 2.40 a 14.20 ± 0.78 a 90.75 ± 1.11 a
Control + Tre 635.66 ± 22.33 a 65.95 ± 2.21 a 14.63 ± 0.40 a 93.26 ± 2.18 a
Drought 501.00 ± 17.96 c 53.38 ± 2.19 c 11.09 ± 0.32 c 77.07 ± 4.28 c
Drought+ Tre 559.89 ± 16.16 b 59.80 ± 1.48 b 12.72 ± 0.56 b 84.25 ± 0.66 b
14 Day Control 757.37 ±22.96 b 76.80 ± 1.95 a 18.11 ± 0.14 a 89.67 ± 1.67 a
Control + Tre 814.70 ± 23.04 a 79.87 ± 1.99 a 18.87 ± 0.30 a 92.54 ± 5.71 a
Drought 555.90 ± 23.56 d 57.42 ± 2.82 c 12.27 ± 0.37 c 68.92 ± 0.78 c
Drought+ Tre 642.93 ± 25.65 c 66.17 ± 3.15 b 14.62 ± 0.51 b 78.80 ± 0.95 b
Mean values was calculated from three replicates for each treatment. Different letters within each column indicate a significant difference between treatments at P<0.05.
Table (2): Effect of drought and exogenous Tre (40 mM) combined with drought on photosynthetic pigments; Chl.a, Chl. b and carotenoids and EL% in wheat seedlings 7 and 14 days after drought stress.
Duratio n Treatment Photosynthetic pigments EL %
Chl.a Chl.b Carotenoid
7 Day Control 0.963 ± 0.010 b 0.354 ± 0.014 b 0.085 ± 0.004 b 31.06 ± 1.31 c
Control + Tre 1.057 ± 0.040 a 0.406 ± 0.006 a 0.097 ± 0.001 a 31.13 ± 0.61 c
Drought 0.821 ± 0.014 d 0.277 ± 0.011 d 0.066 ± 0.001 d 42.43 ± 2.08 a
Drought+ Tre 0.912 ± 0.012 c 0.312 ± 0.012 c 0.072 ± 0.001 c 34.03 ± 0.66 b
14 Day Control 0.929 ± 0.016 b 0.328 ± 0.005 a 0.087 ± 0.002 b 37.12 ± 2.78 c
Control + Tre 0.998 ± 0.001 a 0.326 ± 0.006 a 0.103 ± 0.002 a 35.97 ± 1.24 c
Drought 0.702 ± 0.013 d 0.237 ± 0.008 c 0.047 ± 0.003 d 59.21 ± 1.02 a
Drought+ Tre 0.849 ± 0.011 c 0.278 ± 0.014 b 0.065 ± 0.000 c 42.74 ± 0.59 b
Mean values was calculated from three replicates for each treatment. Different letters within each column indicate a significant difference between treatments at P<0.05.
Table (3 ) : Effect of drought and exogenous Tre (40 mM) combined with drought on trehalose, sucrose and starch contents in wheat seedlings 7 and 14 days after drought stress.
Duration Treatment Trehalose Sucrose starch
7 Day Control 0.227 ± 0.012 d 9.48 ± 0.23 b 8.64 ± 0.12 b
Control + Tre 0.435 ± 0.010 c 10.72 ± 0.70 a 9.01 ± 0.11 a
Drought 0.647 ± 0.033 b 8.67 ± 0.14 c 7.54 ± 0.08 c
Drought+ Tre 0.907 ± 0.037 a 9.76 ± 0.13 b 8.73 ± 0.03 b
14 Day Control 0.226 ± 0.010 d 14.11 ± 0.31 b 9.18 ± 0.06 b
Control + Tre 0.525 ± 0.009 c 15.31 ± 0.35 a 10.78 ± 0.19 a
Drought 0.942 ± 0.021 b 12.28 ± 0.28 c 7.73 ± 0.05 c
Drought+ Tre 1.140 ± 0.050 a 14.39 ± 0.14 b 9.23 ± 0.12 b
Mean values was calculated from three replicates for each treatment. Different letters within each column indicate a significant difference between treatments at P<0.05.
GControE QConJttre
□ Drought □ brought*! re
8
a
7 Day 14 Day
Figure (1): Effect of drought and exogenous Tre (40 mM) combined with drought on proline content in wheat seedlings 7 and 14 days after drought stress. Values are mean of three replicates ± SD. Bars with different letters are significantly different at P<0.05.
Figure (2): Effect of drought and exogenous Tre (40 mM) combined with drought on MDA content in wheat seedlings 7 and 14 days after drought stress. Values are mean of three replicates ± SD. Bars with different letters are significantly different at P<0.05.
Figure (3): Effect of drought and exogenous Tre (40 mM) combined with drought on guaicol peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT) activities in wheat seedlings 7 and 14 days after drought stress. Values are mean of three replicates ± SD. Bars with different letters are significantly different at P<0.05.
DISCUSSION
Plant Growth Parameters
In our results, drought stress reduced FW, DW, leaf RWC, and leaf area of cv. Sakha 93 plants. However, reduction growth parameters were restored by exogenous Tre supplementation under drought stress as
achieved by ameliorating leaf RWC, fresh and dry weight. Similar findings were documented previously by Tre applied with abiotic stress (Alam et al., 2014; Ali, 201; Ali & Ashraf, 2011; Mostofa et al., 2015; Ahmed et al., 2016; Akram et al., 2016).
Drought stress decreased FW, DW of the shoot,
shoot height, and leaf area, of wheat plants whereas a combination of Tre or maltose with drought stress enhanced the growth parameters (Ibrahim & Abdellatif, 2016).
Under Cu-stress, plant height, fresh and dry weights, RWC significantly decreased in rice seedlings, on the other hand, Tre pretreatment could enhance tolerance of rice seedlings to Cu-stress (Mostofa et al., 2015).
Although drought stress caused reduction chlorophyll pigments in cv. Sakha 93 plants, but the interaction of Tre addition and drought stress alleviated the photosynthetic pigment contents. The same results were observed in many plants under abiotic stress: maize under salinity stress (Zeid, 2009); rice seedlings under Cu-stress (Mostofa et al., 2015); Brassica seedlings under drought stress (Alam et al., 2014); wheat under drought stress (Ahmed et al., 2016); radish under drought stress (Akram et al., 2016). Tre has positive influence on rate photosynthesis through interaction with the sugar-signaling network (Ali & Ashraf, 2011). Drought stress has an indirect effect on stomata closure that due to a decrease in leaf CO2 concentration. A low level of CO2 may cause a reduction in NADP+ available to accept electrons from the photosynthetic system (PSI and PSII). Thereby initiate O2 reduction accompanied by ROS production (Hernandez et al., 2000). Electrolytic leakage (EL%)
The degree of cell membrane injury induced by abiotic stress may be easily estimated through membrane leakage to cell electrolytes. Our study, EL% increased in cv. Sakha 93 plants by drought stress, meanwhile application of Tre reduced the values of EL% in stressed wheat plants. The same results were recorded in previous (Quan et al., 2004; Valentovic et al., 2014; Aldesuquy & Ghanem, 2015; Li et al., 2014). Proline content:
Proline level increased in leaves of cv. Sakha 93 plant under drought stress; the similar results were recorded in several reports (Nahar et al., 2013; Alam et al., 2014; Mostofa et al., 2015; Ibrahim & Abdellatif, 2016; Ahmed et al., 2016). Many researchers reported that many of compatible solutes such as proline (Pro),
Tre and soluble sugar accumulate in higher concentrations in most plants to overcome of adverse effects of stress conditions. Pro plays a vital role in somatic adjustment, stress signal transduction.
Exogenously applied Tre with drought stress reduced Pro level in cv. Sakha 93 leaves. These results are agreement with (Nounjan et al., 2012; Alam et al., 2014; Mostofa et al., 2015;; Ahmed et al., 2016). Tre prevented wheat plants from adverse effects of drought stress through it could provide osmotic protection to cells by adjustment the accumulated Tre to optimal level so that wheat plants did not need to increase the Pro level. On the other hand, these results are disagreement with reports that recoded applied Tre with drought stress increased Pro content in wheat plants (Balla et al., 2011; Ibrahim, Abdellatif, 2016). MDA content:
MDA induced by drought stress was observed in cv. Sakha 93 and other previous studies (Ali & Ashraf, 2011; Alam et al., 2014; Aldesuquy & Ghanem, 2015; Ahmed et al., 2016). MDA content was used as an indicator of lipid peroxidation which led to membrane fluidity and increases membrane permeability (Liu et al., 2011). Exogenously applied Tre reduced MDA level in wheat plants. These results are agreement with (Ma et al., 2013; Alam et al., 2014; Aldesuquy & Ghanem, 2015). Tre was an osmoprotectant that stabilizes macromolecules, protein and lipids membranes and protects biological structures from the damage to water leakage (Garg & Manchand, 2009). In contrast, the application of Tre or maltose with drought stress remarkably increased the MDA level in wheat plants (Ibrahim & Abdellatif, 2016). Antioxidant Enzymes
ROS directly affected on cellular membranes of cells and organelles through react with unsaturated fatty acids that led to peroxidation of essential membrane lipids, consequently leakage of electrolytes and cellular contents and increasing MDA concentration (Candar & Tarhan, 2003). Antioxidants are the first line of defense against free radical damage.
Generally, APX and CAT participated in ascorbate and glutathione cycle. Superoxide dismutase generated
H2O2 that was catalyzed into H2O by CAT and peroxidases.
Our results for GPX and APX activities were similar with other reports such as in Brassica (Alam et al., 2014); in wheat (Aldesuquy & Ghanem, 2015; Ibrahim & Abdellatif, 2016). On the other hand, CAT activity significantly reduced in wheat plants under drought stress. Our results are supported by previous researches, (Sorial et al., 2010; Alam et al., 2014).
The same results was observed in some reports in Lemna gibba under Cd stress (Duman et al., 2011); in Oryza stativa under salt stress (Nounjan et al., 2012); in Brassica juncea under drought stress (Alam et al., 2014) in Triticum aestivum under drought stress [(Aldesuquy & Ghanem, 2015; Ibrahim & Abdellatif, 2016; Ahmed et al., 2016). Tre acts as a signaling molecule under abiotic stress which induced ROS production in plants, consequently, ROS sends signals to activate enzymatic antioxidants which have a vital role in scavenging of ROS due to alleviate oxidative stress (Fernandez et al., 2010).
Carbohydrates Contents
Soluble sugar may be playing a key role in osmotic adjustment of the cellular level of plants under abiotic stress (Gupta & Kaur, 2005).
Exogenously applied Tre before exposing the plants to drought stress had a fluctuant effect on internal Tre, sucrose and starch content. The level of Tre was increased into 4 fold whereas levels of sucrose and starch were similar to control.
This result was supported by previous reports; Tre level increased into 145-fold in seedlings of Arbidopsis thaliana that pretreated by Tre (30 mM) (Bae et al., 2005); Tre accumulation up to 385.25 |g/ g FW in Capparis ovate exposed to drought stress (IIhan et al., 2014). Endogenous Tre increased internal Tre to 2-folds in Oryza sativa seedlings that were pretreated with Tre before exposure to Cu-stress. On the other hand, Tre application maintained the levels of sucrose and starch at the same level as control.
In contrast, some reports disagreed with our results. Tre application increased accumulation of sucrose in some plants under abiotic stress: 3-folds in seedlings of
Arbidopsis thaliana (Bae et al., 2005)]; in Catharanthus roseus under salt stress [10], in internodes of sugarcanes that grown in Tre treatment (Glassop et al., 2007).
The vital function of Tre in plants is the metabolic regulation, control of metabolization of sucrose to several pathways important for metabolism, structural and storage functions of plant cells (Ahmed et al., 2013). The addition of Tre increased accumulation of starch which is due to increase the activity of AD-glucose pyrophosphorylase (AGPase), a major enzyme controlling starch synthesis (Wingler et al., 2000).
CONCLUSION
In conclusion, our results indicate that the drought had a negative influence on membrane characteristics, enzymatic antioxidant and growth parameters of wheat plants. On the other hand, exogenous application of Tre combined with drought appeared to ameliorate the effect of drought by decreasing membrane damage. Tre pretreatment induced growth improvement in wheat plants under drought stress that could be associated with a significant increase in Tre level that regulating carbohydrates.
ACKNOWLEDGEMENT
I would like to express my deepest thanks to my country (Republic of Yemen), special my University (Taiz University) for allowing me to carry out this research and financial support for me during this work.
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