Protoplasma DOI 10.1007/s00709-014-0691-3
ORIGINAL ARTICLE
Trehalose pretreatment induces salt tolerance in rice (Oryza sativa L.) seedlings: oxidative damage and co-induction of antioxidant defense and glyoxalase systems Mohammad Golam Mostofa & Mohammad Anwar Hossain & Masayuki Fujita
Received: 2 April 2014 / Accepted: 15 August 2014 # Springer-Verlag Wien 2014
Abstract Salinity in the form of abiotic stress adversely effects plant growth, development, and productivity. Various osmoprotectants are involved in regulating plant responses to salinity; however, the precise role of trehalose (Tre) in this process remains to be further elucidated. The present study investigated the regulatory role of Tre in alleviating saltinduced oxidative stress in hydroponically grown rice seedlings. Salt stress (150 and 250 mM NaCl) for 72 h resulted in toxicity symptoms such as stunted growth, severe yellowing, and leaf rolling, particularly at 250 mM NaCl. Histochemical observation of reactive oxygen species (ROS; O2 − and H2O2) indicated evident oxidative stress in salt-stressed seedlings. In these seedlings, the levels of lipoxygenase (LOX) activity, malondialdehyde (MDA), H2O2, and proline (Pro) increased significantly whereas total chlorophyll (Chl) and relative water content (RWC) decreased. Salt stress caused an imbalance in non-enzymatic antioxidants, i.e., ascorbic acid (AsA) content, Handling Editor: Néstor Carrillo Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0691-3) contains supplementary material, which is available to authorized users. M. G. Mostofa : M. Fujita (*) Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan e-mail: [email protected] M. G. Mostofa e-mail: [email protected] M. G. Mostofa Department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh M. A. Hossain Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
AsA/DHA ratio, and GSH/GSSG ratio decreased but glutathione (GSH) content increased significantly. In contrast, Tre pretreatment (10 mM, 48 h) significantly addressed salt-induced toxicity symptoms and dramatically depressed LOX activity, ROS, MDA, and Pro accumulation whereas AsA, GSH, RWC, Chl contents, and redox status improved considerably. Salt stress stimulated the activities of SOD, GPX, APX, MDHAR, DHAR, and GR but decreased the activities of CAT and GST. However, Tre-pretreated salt-stressed seedlings counteracted SOD and MDHAR activities, elevated CAT and GST activities, further enhanced APX and DHAR activities, and maintained GPX and GR activities similar to the seedlings stressed with salt alone. In addition, Tre pretreatment enhanced the activities of methylglyoxal detoxifying enzymes (Gly I and Gly II) more efficiently in salt-stressed seedlings. Our results suggest a role for Tre in protecting against salt-induced oxidative damage attributed to reduced ROS accumulation, elevation of nonenzymatic antioxidants, and co-activation of the antioxidative and glyoxalase systems. Keywords Salt toxicity . Osmoprotectants . Oxidative stress . Trehalose . Antioxidant defense . Glyoxalase system . Oryza sativa L. Abbreviations AsA Ascorbic acid CAT Catalase Chl Chlorophyll DHA Dehydroascorbate Gly Glyoxalase GR Glutathione reductase GSH Reduced glutathione GSSG Oxidized glutathione H2O2 Hydrogen peroxide LOX Lipoxygenase MDA Malondialdehyde
M.G. Mostofa et al.
MG O2.− Pro ROS SOD Tre
Methylglyoxal Superoxide Proline Reactive oxygen species Superoxide dismutase Trehalose
Introduction Plants are intermittently exposed to a plethora of abiotic stresses, which are the most harmful factors constraining plant growth and productivity (Gao et al. 2008). Among abiotic stresses, salinity is considered the most devastating, limiting crop yield especially in arid, semi-arid, and coastal regions. High soil salinity detrimentally affects physiological, biochemical, and molecular events associated with plant growth and development (Parida and Das 2005). Salt stress generally involves breaking ion homeostasis, resulting in ion toxicity, osmotic stress, and nutrient deficiency (Munns and Tester 2008). Salinity, like most other stresses, also induces production of reactive oxygen species (ROS) such as superoxide (O2 − ), hydrogen peroxide (H2O2), hydroxyl radical (OH.), and singlet oxygen (1O2). ROS are highly reactive and toxic when accumulated beyond sublethal levels and can greatly compromise normal metabolism through oxidative damage to photosynthetic pigments, lipids, proteins, carbohydrates, and DNA (Mittler 2002). Membrane lipid peroxidation and loss of membrane integrity due to these ROS is thought to be the prominent effects of salt toxicity in higher plants (Yasar et al. 2008). In addition to ROS, methylglyoxal (MG), a highly mutagenic and cytotoxic compound, has been found to accumulate in plant cells under various stresses including salinity (Yadav et al. 2005a; Hossain et al. 2009). Excess MG can cause cell death by interacting with major biomolecules such as proteins, lipids, carbohydrates, and nucleic acids (Yadav et al. 2005b). Moreover, an increase in MG level further intensifies ROS production by inactivating the antioxidant defense system (Hossain et al. 2012) or by interfering with the photosynthetic electron transport chain (Saito et al. 2011). ROS are inevitable byproducts of essential aerobic metabolisms and must be maintained under sublethal levels for normal plant metabolism. Hence, plants have developed an intrinsic antioxidant defense system involving enzymatic and nonenzymatic antioxidants to protect themselves against oxidative damage (Mittler 2002). Non-enzymatic antioxidants, either hydrophilic such as ascorbic acid (AsA) and glutathione (GSH) or lipophilic such as α-tocopherol and carotenoids, can quench all kinds of ROS. Several enzymes are involved in the detoxification of ROS through a series of complex reactions. Superoxide dismutase (SOD) and the ascorbate–glutathione cycle, comprising ascorbate peroxidase (APX), monodehydroascorbate
reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) constitute the major detoxification system of ROS where O2 − is converted to H2O via H2O2 (Gill and Tuteja 2010). Catalase (CAT) can also reduce H2O2 to water without requiring any reducing equivalents. Glutathionemetabolizing enzymes such as gluathione S-transferase (GST) and glutathione peroxidases (GPX) are associated with the scavenging of lipid peroxides, xenobiotics, and reactive aldehydes produced during abiotic stresses (Roxas et al. 1997). A concerted and balanced operation of these enzymes is crucial for plant survival under stress conditions. It is well documented that antioxidant systems are altered under salt stress, and enhanced antioxidant capacity directly correlates to salt tolerance (Mishra et al. 2013). Plant cells also possess a ubiquitous enzymatic pathway that catalyzes the GSH-dependent detoxification of MG. This pathway consists of two enzymes: glyoxalase I (Gly I) and glyoxalase II (Gly II). Gly I catalyzes the conversion of hemithioacetals formed from a spontaneous reaction between GSH and MG, to S-D-lactoylglutathione (SLG). SLG is then converted to D-lactate by Gly II, which recycles GSH in the system (Yadav et al. 2005b). Genetic manipulation of glyoxalase enzymes improved salinity tolerance in tobacco, rice, and tomato plants (Singla-Pareek et al. 2003, 2008; Álvarez Viveros et al. 2013). Transgenic tobacco plants overexpressing both Gly I and Gly II genes also increased the activities of GSH metabolizing enzymes (GST, GPX, DHAR, and GR), indicating a close interaction between the antioxidant defense and glyoxalase systems (Yadav et al. 2005c). A wealth of studies revealed that efficient induction of the antioxidative and glyoxalase systems offer increased resistance to abiotic stresses (El-Shabrawi et al. 2010; Upadhyaya et al. 2011; Mostofa and Fujita 2013). Salt tolerance is a complex trait that is controlled by multiple genes and involves various biochemical and physiological mechanisms (Zhang and Shi 2013). Osmotic adjustment is an effective mechanism for enduring salt stress-induced hyperosmotic stress (Chen and Jiang 2010). Plants adopt this technique by accumulating various organic compounds, collectively known as osmoprotectants. One such compound is trehalose (Tre), a non-reducing disaccharide of glucose, which plays an important role as a stress protectant in some plants (Garcia et al. 1997; Ali and Ashraf 2011; Duman et al. 2010). In addition to being an energy source, the unique physicochemical properties of Tre efficiently stabilize dehydrated enzymes, proteins, and lipid membranes, as well as protect biological structures from damage during desiccation (Fernandez et al. 2010). Tre has the added advantage of being a signaling and antioxidant molecule. Tre also acts as an elicitor of genes involved in detoxification and stress response (Bae et al. 2005). However, with the exception of resurrection plants, Tre production in most plants is not sufficient to ameliorate stress-induced adverse effects. On the other hand,
Trehalose pretreatment induces salt tolerance in rice
external Tre application increases the internal level of this osmolyte and has been suggested as an alternative approach to induce salinity tolerance (Garcia et al. 1997; Chen and Murata 2002). Exogenous Tre alleviates the adverse effects of various abiotic stresses including salinity in rice, drought in maize, and heat and water deficit in wheat (Nounjan et al. 2012; Ali and Ashraf 2011; Luo et al. 2010; Ma et al. 2013). Most studies about the role of osmoprotectants in modifying antioxidant systems have focused on proline and glycine betaine, but very little is known about Tre in higher plants under stress conditions. Moreover, there have not been any studies about the effect of Tre on the responses of MG detoxification systems in plants under abiotic stress. Therefore, it is worth investigating the role of Tre in the antioxidative defense and glyoxalase systems during abiotic stress tolerance. Rice (Oryza sativa L.) is one of the important crops grown across the world and is also considered a staple food, especially in Bangladesh, China, and India. Salinity is the second most widespread problem next to drought for rice production in these regions. In Bangladesh, scarcity of water, low quality of irrigation water, hot dry climate, and tidal flooding aggravate the existing problems. Rice is a salt-sensitive crop, particularly at the seedling and reproductive stages (Lutts et al. 1995). Therefore, enhancing salt tolerance is of particular interest for sustainable rice production. Tre has been suggested as a global protectant against abiotic stress (ReinaBueno et al. 2012) and might be more beneficial for rice plants over proline during osmotic stress (Garcia et al. 1997). Furthermore, Tre is readily taken up and transported throughout plants when applied exogenously (Luo et al. 2010). Taking this information into account, we conducted a hydroponic experiment on rice seedlings to investigate the effects of exogenous Tre on salt-induced modulation of ROS, lipid peroxidation, proline, relative water content, total chlorophyll, ascorbate, and glutathione content, and the activities of the enzymes involved in the antioxidant defense and glyoxalase systems. We report that enhancing internal Tre content induces salt tolerance by reducing the ROS level and stimulates the antioxidant defense and glyoxalase systems in saltstressed rice seedlings. Furthermore, we believe this is the first reported demonstration that simultaneously inducing the antioxidant defense and glyoxalase systems by external Tre, at least in part, plays an important role in conferring salt tolerance in rice seedlings.
Materials and methods Plant materials, growth condition, and treatments Rice (Oryza sativa L. cv. BR 11) seeds were surface-sterilized with 1 % (v/v) sodium hypochlorite solution for 20 min, washed with distilled water, and imbibed for 24 h. The seeds
were sown on plastic nets floating on distilled water in 250-mL plastic beakers and kept in the dark at 28±2 °C for germination. After 48 h, uniformly germinated seeds were transferred to a growth chamber and grown in a commercial hydroponics solution (Hyponex, Japan) diluted according to the manufacturer’s instructions. The nutrient solution consisted of 8 % N, 6.43 % P, 20.94 % K, 11.8 % Ca, 3.08 % Mg, 0.07 % B, 0.24 % Fe, 0.03 % Mn, 0.0014 % Mo, 0.008 % Zn, and 0.003 % Cu. The seedlings were grown u n d e r c o n t r o l l e d c o n d i t i o n s ( p h o t o n d e n s i t y, 100 μmol m−2 s−1; temperature, 26±2 °C; RH, 65–70 %). Each plastic beaker contained about 50 rice seedlings. The nutrient solution (pH 5.5) was renewed every 4 days. At 12 days, seedlings were pretreated with 10 mM trehalose (Tre) in the hyponex solution for 48 h. Trehalose pretreated and non-pretreated rice seedlings were then subjected to 150 and 250 mM NaCl in hyponex solution to impose moderate and severe salt stress, respectively. These salt concentrations were selected based on a preliminary experiment. A range of NaCl levels (50–300 mM) was applied, and visible toxicity symptoms such as chlorosis (yellowing) and rolling of leaves were considered to choose the intensity of salt stress. Based on literatures (Garcia et al. 1997; Nounjan et al. 2012) and our preliminary experiments with a range of Tre concentrations (5, 10, 15, and 20 mM), we observed that 10 mM Tre was optimally effective in the alleviation of salt-induced toxic symptoms. Therefore, our experiment consisted of six treatments as follows: control, 10 mM trehalose (Tre), 150 mM NaCl (S1), 10 mM trehalose + 150 mM NaCl (Tre+S1), 250 mM NaCl (S2), 10 mM trehalose+250 mM NaCl (Tre+ S2). After 72 h of growth under the above conditions, the second leaf of rice seedlings was harvested to determine various biochemical parameters. Each treatment was replicated three times under the same experimental conditions. Determination of lipid peroxidation, hydrogen peroxide, and proline content Lipid peroxidation of the second leaves was measured by estimating malondialdehyde (MDA) according to the method of Heath and Packer (1968). Fresh leaf samples (0.5 g) were homogenized with 5 % (w/v) trichloroacetic acid (TCA) and centrifuged at 11,500×g for 15 min. The supernatant was mixed with 20 % TCA containing 0.5 % of TBA and heated at 95 °C for 30 min. MDA content was calculated by the difference in absorbance at 532 and 600 nm using an extinction co-efficient of 155 mM−1 cm−1. Hydrogen peroxide (H2O2) was extracted by homogenizing 0.5 g of fresh leaf samples with 50 mM K-phosphate buffer pH (6.5), and the content was determined after reaction with 0.1 % TiCl4 in 20 % H2SO4 following the method of Hossain et al. (2010). Proline (Pro) content was determined according to the method of Bates et al. (1973). Fresh leaf samples (0.5 g) were
M.G. Mostofa et al.
homogenized with 5 mL of 3 % aqueous sulfosalicylic acid, and the homogenate was centrifuged at 11,500×g for 15 min. Supernatant (2 mL) was mixed with 2 mL of glacial acetic acid and 2 mL of acid ninhydrin solution. The resultant mixture was boiled at 100 °C for 1 h and then transferred to ice to stop the reaction. The developed red color was extracted with 4 mL toluene and absorption of the chromophore was measured at 520 nm. Pro concentration was calculated using calibration curve developed with Pro standards. Determination of total chlorophyll and relative water content To estimate total chlorophyll (Chl) content, leaves (0.5 g) were extracted in 80 % chilled acetone, and Chl was estimated according to the method of Arnon (1949). To estimate relative water content (RWC), 20 leaf segments (4–5 cm) were weighed separately to determine fresh weight (FW). Leaf segments were then placed between two layers of filter paper and immersed in deionized water. When the leaf segments became fully turgid, they were gently dried with tissue paper, and turgid weight (TW) was measured. Dry weight (DW) was measured after oven drying at 80 °C for 48 h. RWC was calculated using the following formula—RWC (%) = 100×(FW−DW)/(TW−DW). Histochemical detections of superoxide and H2O2 Superoxide (O2− ) and H2O2 were detected in rice leaves according to the method of Mostofa and Fujita (2013). In brief, the second leaves were stained in 0.1 % nitroblue tetrazolium (NBT) solution or 1 % 3,3′-diaminobenzidine (DAB) solution to detect O2− and H2O2, respectively. After 24 h of incubation, leaves were decolorized by immersing them in boiling ethanol to detect the blue insoluble formazan (forO2− ) or deep brown polymerization product (for H2O2). After cooling, photographs were taken by placing the leaves between two glass plates. Estimation of non-enzymatic antioxidants Fresh leaves (0.5 g) were homogenized in 3 mL of ice-cold 5 % meta-phosphoric acid containing 1 mM EDTA and centrifuged at 11,500×g for 15 min. Reduced and total AsA content were determined following the method of Dutilleul et al. (2003) with minor modifications. To estimate total AsA, the oxidized fraction was reduced by 0.1 M dithiothreitol. Reduced and total AsA content were assayed at 265 nm in 100 mM K-phosphate buffer (pH 7.0) with 1.0 U of ascorbate oxidase (AO). Oxidized ascorbate (DHA)=total AsA−reduced AsA. Based on enzymatic recycling, reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione (GSH+GSSG) were determined according to the method of Griffiths (1980). GSSG was determined after removing
GSH by 2-vinylpyridine derivatization. GSH was measured after subtracting the value of GSSG from total GSH.
Extraction and assay of enzymes To extract enzymes, fresh leaf samples (0.5 g) were homogenized separately with a reaction mixture containing 50 mM Kphosphate buffer (pH 7.0), 100 mM KCl, 1 mM AsA, 5 mM β-mercaptoethanol, and 10 % (w/v) glycerol in pre-chilled mortars and pestles. The homogenate was centrifuged at 11,500×g for 15 min, and the resultant supernatants were collected for analysis of enzyme activities and protein content. All procedures were performed at 0–4 °C. Lipoxygenase (LOX, EC 1.13.11.12) activity was estimated according to the method of Doderer et al. (1992) by monitoring the increase in absorbance at 234 nm using linoleic acid as a substrate. Superoxide dismutase (SOD, EC 1.15.1.1) activity was estimated according to the method of ElShabrawi et al. (2010), which is based on a xanthine–xanthine oxidase system. The reaction mixture contained K-phosphate buffer (50 mM), NBT (2.24 mM), catalase (0.1 units), xanthine oxidase (0.1 units), xanthine (2.36 mM), and enzyme extract. Catalase was added to avoid possible H2O2-mediated inactivation of Cu/Zn-SOD. SOD activity was expressed as units (i.e., amount of enzyme required to inhibit NBT reduction by 50 %) per minute per milligram protein. Catalase (CAT, EC 1.11.1.6) activity was measured according to the method of Hossain et al. (2010). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined by monitoring the decrease in absorbance at 290 nm as AsA was oxidized, according to the method of Nakano and Asada (1981). Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activity was measured by using 1 U of AO, and the oxidation rate of NADPH was determined at 340 nm (Hossain et al. 1984). Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was measured by monitoring the formation of AsA from DHA at 265 nm using GSH (Nakano and Asada 1981). Glutathione reductase (GR, EC 1.6.4.2) activity was measured by monitoring the decrease in the absorbance of NADPH at 340 nm for GSSG-dependent oxidation of NADPH, as described by Foyer and Halliwell (1976). Glutathione S-transferase (GST, EC 2.5.1.18) activity was determined by the method of Hossain et al. (2010). Glutathione peroxidase (GPX, EC: 1.11.1.9) activity was measured as described by Elia et al. (2003) using H2O2 as a substrate. Glyoxalase I (Gly I, EC 4.4.1.5) assay was carried out according to the method of Hossain et al. (2009). The assay mixture contained 100 mM K-phosphate buffer (pH 7.0), 15 mM magnesium sulfate, 1.7 mM GSH, and 3.5 mM MG in a final volume of 0.7 mL. The increase in absorbance was recorded at 240 nm for 1 min, and the activity was calculated using the extinction co-efficient of 3.37 mM−1 cm−1.
Trehalose pretreatment induces salt tolerance in rice
Glyoxalase II (Gly II, EC 3.1.2.6) activity was determined according to the method of Hossain et al. (2010). The reaction mixture contained 100 mM Tris–HCl buffer (pH 7.2), 0.2 mM DTNB, and 1 mM S-D-lactoylglutathione (SLG) in a final volume of 1 mL. The reaction was started by adding SLG, and the activity was calculated using the extinction coefficient of 13.6 mM−1 cm−1. Protein content was determined following the method of Bradford (1976) using bovine serum albumin as a standard. Determination of trehalose content Trehalose content in the second leaves was determined following the method described by Li et al. (2014) with some modifications. The leaves (1.0 g) were homogenized in 5 mL of 80 % (v/v) hot ethanol and centrifuged at 11,500×g for 20 min. The supernatants were dried at 80 °C followed by resuspension in 5 mL distilled water. The solution (100 μL) was mixed with 150 μL 0.2 N H2SO4 and boiled at 100 °C for 10 min to hydrolyze any sucrose or glucose-1-phosphate, etc., and then chilled on ice. NaOH (0.6 N, 150 μL) was added to the above mixture and boiled for 10 min to destroy reducing sugars, and then chilled again. To the above mixture, 2.0 mL of anthrone reagent (0.2 g anthrone per 100 ml of 95 % H2SO4) was added and boiled for 10 min to develop a color, and then chilled again. The absorbance was recorded at 630 nm, and trehalose concentration was calculated as micromoles per gram FW using a standard curve developed with commercial Tre.
Results Lipid peroxidation The level of lipid peroxidation in the leaves was determined as the content of MDA (Table 1). MDA content increased significantly in the S1 and S2 stress groups compared with the control group. The induction of lipid peroxidation was more pronounced in the S2 stress group compared with the S1 stress group. On the other hand, 10 mM Tre pretreatment significantly reduced MDA content in the leaves of the Tre+S1 and Tre+S2 groups compared with the S1 and S2 stress groups, respectively. There was no significant difference in MDA content between the control and the Tre groups.
Hydrogen peroxide (H2O2) level Upon salt imposition, the H2O2 level sharply increased in the leaves (Table 1). The H2O2 level increased by 49 % and 90 % in the S1 and S2 stress groups, respectively, compared with the control group. Tre pretreatment did not increase the level of H2O2 in the Tre group compared with control. In contrast, the H2O2 level decreased significantly in the Tre+S1 and Tre+ S2 groups compared with the S1 and S2 stress groups, respectively.
Proline (Pro) content Statistical analysis The data were subjected to one-way analysis of variance (ANOVA), and different letters indicate significant differences between treatments at p < 0.05, according to Duncan’s multiple-range test (DMRT) using IRRISTAT version 3 (International Rice Research Institute, Biometrics Unit, Manila, Philippines). Data represented in the table and figures are means±standard deviations (SD) of three replicates for each treatment.
Pro content increased significantly in the S1 and S2 stress groups; however, it was remarkably higher (20-fold) in the S2 stress group than that of the control group (Table 1). Interestingly, Tre pretreatment also increased Pro content in the Tre group. On the other hand, Tre pretreatment significantly reduced Pro content in the leaves of the Tre+S1 and Tre+S2 groups compared with the S1 and S2 stress groups, respectively; however, the levels were significantly higher than that of the control group.
Table 1 Effect of exogenous trehalose on MDA, H2O2, Pro content, and LOX activity in rice seedlings with and without NaCl stress Treatment
MDA (nmol g−1 FW)
H2O2 (nmol g−1 FW)
Pro (μmol g−1 FW)
LOX activity (nmol min−1 mg−1 protein)
Control Tre
22.26±1.01ab 20.06±1.10a
25.31±1.56a 24.87±2.16a
0.156±0.003a 0.306±0.010b
12.82±1.73ab 11.46±0.71a
S1 Tre+S1 S2 Tre+S2
28.64±1.55c 22.00±2.17ab 43.81±1.85d 29.73±2.90c
37.78±1.49c 23.39±2.71a 48.06±4.29d 31.58±2.03b
0.679±0.062d 0.524±0.065c 3.137±0.173f 1.746±0.177e
17.39±0.91d 12.83±1.67ab 28.12±2.52e 16.07±1.45c
Data are represented as means±standard deviations from three independent experiments. Different letters within the same column indicate significant differences between treatments at p
ORIGINAL ARTICLE
Trehalose pretreatment induces salt tolerance in rice (Oryza sativa L.) seedlings: oxidative damage and co-induction of antioxidant defense and glyoxalase systems Mohammad Golam Mostofa & Mohammad Anwar Hossain & Masayuki Fujita
Received: 2 April 2014 / Accepted: 15 August 2014 # Springer-Verlag Wien 2014
Abstract Salinity in the form of abiotic stress adversely effects plant growth, development, and productivity. Various osmoprotectants are involved in regulating plant responses to salinity; however, the precise role of trehalose (Tre) in this process remains to be further elucidated. The present study investigated the regulatory role of Tre in alleviating saltinduced oxidative stress in hydroponically grown rice seedlings. Salt stress (150 and 250 mM NaCl) for 72 h resulted in toxicity symptoms such as stunted growth, severe yellowing, and leaf rolling, particularly at 250 mM NaCl. Histochemical observation of reactive oxygen species (ROS; O2 − and H2O2) indicated evident oxidative stress in salt-stressed seedlings. In these seedlings, the levels of lipoxygenase (LOX) activity, malondialdehyde (MDA), H2O2, and proline (Pro) increased significantly whereas total chlorophyll (Chl) and relative water content (RWC) decreased. Salt stress caused an imbalance in non-enzymatic antioxidants, i.e., ascorbic acid (AsA) content, Handling Editor: Néstor Carrillo Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0691-3) contains supplementary material, which is available to authorized users. M. G. Mostofa : M. Fujita (*) Laboratory of Plant Stress Responses, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan e-mail: [email protected] M. G. Mostofa e-mail: [email protected] M. G. Mostofa Department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh M. A. Hossain Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
AsA/DHA ratio, and GSH/GSSG ratio decreased but glutathione (GSH) content increased significantly. In contrast, Tre pretreatment (10 mM, 48 h) significantly addressed salt-induced toxicity symptoms and dramatically depressed LOX activity, ROS, MDA, and Pro accumulation whereas AsA, GSH, RWC, Chl contents, and redox status improved considerably. Salt stress stimulated the activities of SOD, GPX, APX, MDHAR, DHAR, and GR but decreased the activities of CAT and GST. However, Tre-pretreated salt-stressed seedlings counteracted SOD and MDHAR activities, elevated CAT and GST activities, further enhanced APX and DHAR activities, and maintained GPX and GR activities similar to the seedlings stressed with salt alone. In addition, Tre pretreatment enhanced the activities of methylglyoxal detoxifying enzymes (Gly I and Gly II) more efficiently in salt-stressed seedlings. Our results suggest a role for Tre in protecting against salt-induced oxidative damage attributed to reduced ROS accumulation, elevation of nonenzymatic antioxidants, and co-activation of the antioxidative and glyoxalase systems. Keywords Salt toxicity . Osmoprotectants . Oxidative stress . Trehalose . Antioxidant defense . Glyoxalase system . Oryza sativa L. Abbreviations AsA Ascorbic acid CAT Catalase Chl Chlorophyll DHA Dehydroascorbate Gly Glyoxalase GR Glutathione reductase GSH Reduced glutathione GSSG Oxidized glutathione H2O2 Hydrogen peroxide LOX Lipoxygenase MDA Malondialdehyde
M.G. Mostofa et al.
MG O2.− Pro ROS SOD Tre
Methylglyoxal Superoxide Proline Reactive oxygen species Superoxide dismutase Trehalose
Introduction Plants are intermittently exposed to a plethora of abiotic stresses, which are the most harmful factors constraining plant growth and productivity (Gao et al. 2008). Among abiotic stresses, salinity is considered the most devastating, limiting crop yield especially in arid, semi-arid, and coastal regions. High soil salinity detrimentally affects physiological, biochemical, and molecular events associated with plant growth and development (Parida and Das 2005). Salt stress generally involves breaking ion homeostasis, resulting in ion toxicity, osmotic stress, and nutrient deficiency (Munns and Tester 2008). Salinity, like most other stresses, also induces production of reactive oxygen species (ROS) such as superoxide (O2 − ), hydrogen peroxide (H2O2), hydroxyl radical (OH.), and singlet oxygen (1O2). ROS are highly reactive and toxic when accumulated beyond sublethal levels and can greatly compromise normal metabolism through oxidative damage to photosynthetic pigments, lipids, proteins, carbohydrates, and DNA (Mittler 2002). Membrane lipid peroxidation and loss of membrane integrity due to these ROS is thought to be the prominent effects of salt toxicity in higher plants (Yasar et al. 2008). In addition to ROS, methylglyoxal (MG), a highly mutagenic and cytotoxic compound, has been found to accumulate in plant cells under various stresses including salinity (Yadav et al. 2005a; Hossain et al. 2009). Excess MG can cause cell death by interacting with major biomolecules such as proteins, lipids, carbohydrates, and nucleic acids (Yadav et al. 2005b). Moreover, an increase in MG level further intensifies ROS production by inactivating the antioxidant defense system (Hossain et al. 2012) or by interfering with the photosynthetic electron transport chain (Saito et al. 2011). ROS are inevitable byproducts of essential aerobic metabolisms and must be maintained under sublethal levels for normal plant metabolism. Hence, plants have developed an intrinsic antioxidant defense system involving enzymatic and nonenzymatic antioxidants to protect themselves against oxidative damage (Mittler 2002). Non-enzymatic antioxidants, either hydrophilic such as ascorbic acid (AsA) and glutathione (GSH) or lipophilic such as α-tocopherol and carotenoids, can quench all kinds of ROS. Several enzymes are involved in the detoxification of ROS through a series of complex reactions. Superoxide dismutase (SOD) and the ascorbate–glutathione cycle, comprising ascorbate peroxidase (APX), monodehydroascorbate
reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) constitute the major detoxification system of ROS where O2 − is converted to H2O via H2O2 (Gill and Tuteja 2010). Catalase (CAT) can also reduce H2O2 to water without requiring any reducing equivalents. Glutathionemetabolizing enzymes such as gluathione S-transferase (GST) and glutathione peroxidases (GPX) are associated with the scavenging of lipid peroxides, xenobiotics, and reactive aldehydes produced during abiotic stresses (Roxas et al. 1997). A concerted and balanced operation of these enzymes is crucial for plant survival under stress conditions. It is well documented that antioxidant systems are altered under salt stress, and enhanced antioxidant capacity directly correlates to salt tolerance (Mishra et al. 2013). Plant cells also possess a ubiquitous enzymatic pathway that catalyzes the GSH-dependent detoxification of MG. This pathway consists of two enzymes: glyoxalase I (Gly I) and glyoxalase II (Gly II). Gly I catalyzes the conversion of hemithioacetals formed from a spontaneous reaction between GSH and MG, to S-D-lactoylglutathione (SLG). SLG is then converted to D-lactate by Gly II, which recycles GSH in the system (Yadav et al. 2005b). Genetic manipulation of glyoxalase enzymes improved salinity tolerance in tobacco, rice, and tomato plants (Singla-Pareek et al. 2003, 2008; Álvarez Viveros et al. 2013). Transgenic tobacco plants overexpressing both Gly I and Gly II genes also increased the activities of GSH metabolizing enzymes (GST, GPX, DHAR, and GR), indicating a close interaction between the antioxidant defense and glyoxalase systems (Yadav et al. 2005c). A wealth of studies revealed that efficient induction of the antioxidative and glyoxalase systems offer increased resistance to abiotic stresses (El-Shabrawi et al. 2010; Upadhyaya et al. 2011; Mostofa and Fujita 2013). Salt tolerance is a complex trait that is controlled by multiple genes and involves various biochemical and physiological mechanisms (Zhang and Shi 2013). Osmotic adjustment is an effective mechanism for enduring salt stress-induced hyperosmotic stress (Chen and Jiang 2010). Plants adopt this technique by accumulating various organic compounds, collectively known as osmoprotectants. One such compound is trehalose (Tre), a non-reducing disaccharide of glucose, which plays an important role as a stress protectant in some plants (Garcia et al. 1997; Ali and Ashraf 2011; Duman et al. 2010). In addition to being an energy source, the unique physicochemical properties of Tre efficiently stabilize dehydrated enzymes, proteins, and lipid membranes, as well as protect biological structures from damage during desiccation (Fernandez et al. 2010). Tre has the added advantage of being a signaling and antioxidant molecule. Tre also acts as an elicitor of genes involved in detoxification and stress response (Bae et al. 2005). However, with the exception of resurrection plants, Tre production in most plants is not sufficient to ameliorate stress-induced adverse effects. On the other hand,
Trehalose pretreatment induces salt tolerance in rice
external Tre application increases the internal level of this osmolyte and has been suggested as an alternative approach to induce salinity tolerance (Garcia et al. 1997; Chen and Murata 2002). Exogenous Tre alleviates the adverse effects of various abiotic stresses including salinity in rice, drought in maize, and heat and water deficit in wheat (Nounjan et al. 2012; Ali and Ashraf 2011; Luo et al. 2010; Ma et al. 2013). Most studies about the role of osmoprotectants in modifying antioxidant systems have focused on proline and glycine betaine, but very little is known about Tre in higher plants under stress conditions. Moreover, there have not been any studies about the effect of Tre on the responses of MG detoxification systems in plants under abiotic stress. Therefore, it is worth investigating the role of Tre in the antioxidative defense and glyoxalase systems during abiotic stress tolerance. Rice (Oryza sativa L.) is one of the important crops grown across the world and is also considered a staple food, especially in Bangladesh, China, and India. Salinity is the second most widespread problem next to drought for rice production in these regions. In Bangladesh, scarcity of water, low quality of irrigation water, hot dry climate, and tidal flooding aggravate the existing problems. Rice is a salt-sensitive crop, particularly at the seedling and reproductive stages (Lutts et al. 1995). Therefore, enhancing salt tolerance is of particular interest for sustainable rice production. Tre has been suggested as a global protectant against abiotic stress (ReinaBueno et al. 2012) and might be more beneficial for rice plants over proline during osmotic stress (Garcia et al. 1997). Furthermore, Tre is readily taken up and transported throughout plants when applied exogenously (Luo et al. 2010). Taking this information into account, we conducted a hydroponic experiment on rice seedlings to investigate the effects of exogenous Tre on salt-induced modulation of ROS, lipid peroxidation, proline, relative water content, total chlorophyll, ascorbate, and glutathione content, and the activities of the enzymes involved in the antioxidant defense and glyoxalase systems. We report that enhancing internal Tre content induces salt tolerance by reducing the ROS level and stimulates the antioxidant defense and glyoxalase systems in saltstressed rice seedlings. Furthermore, we believe this is the first reported demonstration that simultaneously inducing the antioxidant defense and glyoxalase systems by external Tre, at least in part, plays an important role in conferring salt tolerance in rice seedlings.
Materials and methods Plant materials, growth condition, and treatments Rice (Oryza sativa L. cv. BR 11) seeds were surface-sterilized with 1 % (v/v) sodium hypochlorite solution for 20 min, washed with distilled water, and imbibed for 24 h. The seeds
were sown on plastic nets floating on distilled water in 250-mL plastic beakers and kept in the dark at 28±2 °C for germination. After 48 h, uniformly germinated seeds were transferred to a growth chamber and grown in a commercial hydroponics solution (Hyponex, Japan) diluted according to the manufacturer’s instructions. The nutrient solution consisted of 8 % N, 6.43 % P, 20.94 % K, 11.8 % Ca, 3.08 % Mg, 0.07 % B, 0.24 % Fe, 0.03 % Mn, 0.0014 % Mo, 0.008 % Zn, and 0.003 % Cu. The seedlings were grown u n d e r c o n t r o l l e d c o n d i t i o n s ( p h o t o n d e n s i t y, 100 μmol m−2 s−1; temperature, 26±2 °C; RH, 65–70 %). Each plastic beaker contained about 50 rice seedlings. The nutrient solution (pH 5.5) was renewed every 4 days. At 12 days, seedlings were pretreated with 10 mM trehalose (Tre) in the hyponex solution for 48 h. Trehalose pretreated and non-pretreated rice seedlings were then subjected to 150 and 250 mM NaCl in hyponex solution to impose moderate and severe salt stress, respectively. These salt concentrations were selected based on a preliminary experiment. A range of NaCl levels (50–300 mM) was applied, and visible toxicity symptoms such as chlorosis (yellowing) and rolling of leaves were considered to choose the intensity of salt stress. Based on literatures (Garcia et al. 1997; Nounjan et al. 2012) and our preliminary experiments with a range of Tre concentrations (5, 10, 15, and 20 mM), we observed that 10 mM Tre was optimally effective in the alleviation of salt-induced toxic symptoms. Therefore, our experiment consisted of six treatments as follows: control, 10 mM trehalose (Tre), 150 mM NaCl (S1), 10 mM trehalose + 150 mM NaCl (Tre+S1), 250 mM NaCl (S2), 10 mM trehalose+250 mM NaCl (Tre+ S2). After 72 h of growth under the above conditions, the second leaf of rice seedlings was harvested to determine various biochemical parameters. Each treatment was replicated three times under the same experimental conditions. Determination of lipid peroxidation, hydrogen peroxide, and proline content Lipid peroxidation of the second leaves was measured by estimating malondialdehyde (MDA) according to the method of Heath and Packer (1968). Fresh leaf samples (0.5 g) were homogenized with 5 % (w/v) trichloroacetic acid (TCA) and centrifuged at 11,500×g for 15 min. The supernatant was mixed with 20 % TCA containing 0.5 % of TBA and heated at 95 °C for 30 min. MDA content was calculated by the difference in absorbance at 532 and 600 nm using an extinction co-efficient of 155 mM−1 cm−1. Hydrogen peroxide (H2O2) was extracted by homogenizing 0.5 g of fresh leaf samples with 50 mM K-phosphate buffer pH (6.5), and the content was determined after reaction with 0.1 % TiCl4 in 20 % H2SO4 following the method of Hossain et al. (2010). Proline (Pro) content was determined according to the method of Bates et al. (1973). Fresh leaf samples (0.5 g) were
M.G. Mostofa et al.
homogenized with 5 mL of 3 % aqueous sulfosalicylic acid, and the homogenate was centrifuged at 11,500×g for 15 min. Supernatant (2 mL) was mixed with 2 mL of glacial acetic acid and 2 mL of acid ninhydrin solution. The resultant mixture was boiled at 100 °C for 1 h and then transferred to ice to stop the reaction. The developed red color was extracted with 4 mL toluene and absorption of the chromophore was measured at 520 nm. Pro concentration was calculated using calibration curve developed with Pro standards. Determination of total chlorophyll and relative water content To estimate total chlorophyll (Chl) content, leaves (0.5 g) were extracted in 80 % chilled acetone, and Chl was estimated according to the method of Arnon (1949). To estimate relative water content (RWC), 20 leaf segments (4–5 cm) were weighed separately to determine fresh weight (FW). Leaf segments were then placed between two layers of filter paper and immersed in deionized water. When the leaf segments became fully turgid, they were gently dried with tissue paper, and turgid weight (TW) was measured. Dry weight (DW) was measured after oven drying at 80 °C for 48 h. RWC was calculated using the following formula—RWC (%) = 100×(FW−DW)/(TW−DW). Histochemical detections of superoxide and H2O2 Superoxide (O2− ) and H2O2 were detected in rice leaves according to the method of Mostofa and Fujita (2013). In brief, the second leaves were stained in 0.1 % nitroblue tetrazolium (NBT) solution or 1 % 3,3′-diaminobenzidine (DAB) solution to detect O2− and H2O2, respectively. After 24 h of incubation, leaves were decolorized by immersing them in boiling ethanol to detect the blue insoluble formazan (forO2− ) or deep brown polymerization product (for H2O2). After cooling, photographs were taken by placing the leaves between two glass plates. Estimation of non-enzymatic antioxidants Fresh leaves (0.5 g) were homogenized in 3 mL of ice-cold 5 % meta-phosphoric acid containing 1 mM EDTA and centrifuged at 11,500×g for 15 min. Reduced and total AsA content were determined following the method of Dutilleul et al. (2003) with minor modifications. To estimate total AsA, the oxidized fraction was reduced by 0.1 M dithiothreitol. Reduced and total AsA content were assayed at 265 nm in 100 mM K-phosphate buffer (pH 7.0) with 1.0 U of ascorbate oxidase (AO). Oxidized ascorbate (DHA)=total AsA−reduced AsA. Based on enzymatic recycling, reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione (GSH+GSSG) were determined according to the method of Griffiths (1980). GSSG was determined after removing
GSH by 2-vinylpyridine derivatization. GSH was measured after subtracting the value of GSSG from total GSH.
Extraction and assay of enzymes To extract enzymes, fresh leaf samples (0.5 g) were homogenized separately with a reaction mixture containing 50 mM Kphosphate buffer (pH 7.0), 100 mM KCl, 1 mM AsA, 5 mM β-mercaptoethanol, and 10 % (w/v) glycerol in pre-chilled mortars and pestles. The homogenate was centrifuged at 11,500×g for 15 min, and the resultant supernatants were collected for analysis of enzyme activities and protein content. All procedures were performed at 0–4 °C. Lipoxygenase (LOX, EC 1.13.11.12) activity was estimated according to the method of Doderer et al. (1992) by monitoring the increase in absorbance at 234 nm using linoleic acid as a substrate. Superoxide dismutase (SOD, EC 1.15.1.1) activity was estimated according to the method of ElShabrawi et al. (2010), which is based on a xanthine–xanthine oxidase system. The reaction mixture contained K-phosphate buffer (50 mM), NBT (2.24 mM), catalase (0.1 units), xanthine oxidase (0.1 units), xanthine (2.36 mM), and enzyme extract. Catalase was added to avoid possible H2O2-mediated inactivation of Cu/Zn-SOD. SOD activity was expressed as units (i.e., amount of enzyme required to inhibit NBT reduction by 50 %) per minute per milligram protein. Catalase (CAT, EC 1.11.1.6) activity was measured according to the method of Hossain et al. (2010). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined by monitoring the decrease in absorbance at 290 nm as AsA was oxidized, according to the method of Nakano and Asada (1981). Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activity was measured by using 1 U of AO, and the oxidation rate of NADPH was determined at 340 nm (Hossain et al. 1984). Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was measured by monitoring the formation of AsA from DHA at 265 nm using GSH (Nakano and Asada 1981). Glutathione reductase (GR, EC 1.6.4.2) activity was measured by monitoring the decrease in the absorbance of NADPH at 340 nm for GSSG-dependent oxidation of NADPH, as described by Foyer and Halliwell (1976). Glutathione S-transferase (GST, EC 2.5.1.18) activity was determined by the method of Hossain et al. (2010). Glutathione peroxidase (GPX, EC: 1.11.1.9) activity was measured as described by Elia et al. (2003) using H2O2 as a substrate. Glyoxalase I (Gly I, EC 4.4.1.5) assay was carried out according to the method of Hossain et al. (2009). The assay mixture contained 100 mM K-phosphate buffer (pH 7.0), 15 mM magnesium sulfate, 1.7 mM GSH, and 3.5 mM MG in a final volume of 0.7 mL. The increase in absorbance was recorded at 240 nm for 1 min, and the activity was calculated using the extinction co-efficient of 3.37 mM−1 cm−1.
Trehalose pretreatment induces salt tolerance in rice
Glyoxalase II (Gly II, EC 3.1.2.6) activity was determined according to the method of Hossain et al. (2010). The reaction mixture contained 100 mM Tris–HCl buffer (pH 7.2), 0.2 mM DTNB, and 1 mM S-D-lactoylglutathione (SLG) in a final volume of 1 mL. The reaction was started by adding SLG, and the activity was calculated using the extinction coefficient of 13.6 mM−1 cm−1. Protein content was determined following the method of Bradford (1976) using bovine serum albumin as a standard. Determination of trehalose content Trehalose content in the second leaves was determined following the method described by Li et al. (2014) with some modifications. The leaves (1.0 g) were homogenized in 5 mL of 80 % (v/v) hot ethanol and centrifuged at 11,500×g for 20 min. The supernatants were dried at 80 °C followed by resuspension in 5 mL distilled water. The solution (100 μL) was mixed with 150 μL 0.2 N H2SO4 and boiled at 100 °C for 10 min to hydrolyze any sucrose or glucose-1-phosphate, etc., and then chilled on ice. NaOH (0.6 N, 150 μL) was added to the above mixture and boiled for 10 min to destroy reducing sugars, and then chilled again. To the above mixture, 2.0 mL of anthrone reagent (0.2 g anthrone per 100 ml of 95 % H2SO4) was added and boiled for 10 min to develop a color, and then chilled again. The absorbance was recorded at 630 nm, and trehalose concentration was calculated as micromoles per gram FW using a standard curve developed with commercial Tre.
Results Lipid peroxidation The level of lipid peroxidation in the leaves was determined as the content of MDA (Table 1). MDA content increased significantly in the S1 and S2 stress groups compared with the control group. The induction of lipid peroxidation was more pronounced in the S2 stress group compared with the S1 stress group. On the other hand, 10 mM Tre pretreatment significantly reduced MDA content in the leaves of the Tre+S1 and Tre+S2 groups compared with the S1 and S2 stress groups, respectively. There was no significant difference in MDA content between the control and the Tre groups.
Hydrogen peroxide (H2O2) level Upon salt imposition, the H2O2 level sharply increased in the leaves (Table 1). The H2O2 level increased by 49 % and 90 % in the S1 and S2 stress groups, respectively, compared with the control group. Tre pretreatment did not increase the level of H2O2 in the Tre group compared with control. In contrast, the H2O2 level decreased significantly in the Tre+S1 and Tre+ S2 groups compared with the S1 and S2 stress groups, respectively.
Proline (Pro) content Statistical analysis The data were subjected to one-way analysis of variance (ANOVA), and different letters indicate significant differences between treatments at p < 0.05, according to Duncan’s multiple-range test (DMRT) using IRRISTAT version 3 (International Rice Research Institute, Biometrics Unit, Manila, Philippines). Data represented in the table and figures are means±standard deviations (SD) of three replicates for each treatment.
Pro content increased significantly in the S1 and S2 stress groups; however, it was remarkably higher (20-fold) in the S2 stress group than that of the control group (Table 1). Interestingly, Tre pretreatment also increased Pro content in the Tre group. On the other hand, Tre pretreatment significantly reduced Pro content in the leaves of the Tre+S1 and Tre+S2 groups compared with the S1 and S2 stress groups, respectively; however, the levels were significantly higher than that of the control group.
Table 1 Effect of exogenous trehalose on MDA, H2O2, Pro content, and LOX activity in rice seedlings with and without NaCl stress Treatment
MDA (nmol g−1 FW)
H2O2 (nmol g−1 FW)
Pro (μmol g−1 FW)
LOX activity (nmol min−1 mg−1 protein)
Control Tre
22.26±1.01ab 20.06±1.10a
25.31±1.56a 24.87±2.16a
0.156±0.003a 0.306±0.010b
12.82±1.73ab 11.46±0.71a
S1 Tre+S1 S2 Tre+S2
28.64±1.55c 22.00±2.17ab 43.81±1.85d 29.73±2.90c
37.78±1.49c 23.39±2.71a 48.06±4.29d 31.58±2.03b
0.679±0.062d 0.524±0.065c 3.137±0.173f 1.746±0.177e
17.39±0.91d 12.83±1.67ab 28.12±2.52e 16.07±1.45c
Data are represented as means±standard deviations from three independent experiments. Different letters within the same column indicate significant differences between treatments at p
