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Characterization of γ-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress
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Physiology
Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress Nisreen A. AL-Quraan a,∗ , Fatima Al-batool Sartawe a , Muien M. Qaryouti b a b
Department of Biotechnology and Genetic Engineering, Faculty of Science and Arts, Jordan University of Science and Technology, Irbid 22110, Jordan National Center for Agricultural Research and Extension (NCARE), P.O. Box 639, Amman 19381, Jordan
a r t i c l e
i n f o
Article history: Received 15 November 2012 Received in revised form 21 February 2013 Accepted 22 February 2013 Available online xxx Keywords: GABA GAD Glutamate decarboxylase ␥-Aminobutyric acid Osmotic stress
a b s t r a c t The molecular response of plants to abiotic stresses has been considered a process mainly involved in the modulation of transcriptional activity of stress-related genes. Nevertheless, recent findings have suggested new layers of regulation and complexity. Upstream molecular mechanisms are involved in the plant response to abiotic stress. Plants gain resistance to abiotic stress by reprogramming metabolism and gene expression. GABA is proposed to be a signaling molecule involved in nitrogen metabolism, regulating the cytosolic pH, and protection against oxidative damage in response to various abiotic stresses. The aim of our study was to examine the role of the GABA shunt pathway-specific response in five wheat (Triticum aestivum L.) cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes and Nodsieh) to salt and osmotic stress in terms of seed germination, seedling growth, oxidative damage (malondialdehyde (MDA) accumulation), and characterization of the glutamate decarboxylse gene (GAD) m-RNA level were determined using RT-PCR techniques. Our data showed a marked increase in GABA, MDA and GAD m-RNA levels under salt and osmotic stress in the five wheat cultivars. Um Qayes cultivar showed the highest germination percentage, GABA accumulation, and MDA level under salt and osmotic stresses. The marked increase in GAD gene expression explains the high accumulation of the GABA level under both stresses. Our results indicated that the GABA shunt is a key signaling and metabolic pathway that allows wheat to adapt to salt and osmotic stress. Based on our data, the Um Qayes wheat cultivar is the cultivar most recommended to be grown in soil with high salt and osmotic contents. © 2013 Elsevier GmbH. All rights reserved.
Introduction Higher plants routinely encounter various abiotic stresses such as drought, high salinity, heavy metals, and extreme temperatures, which impair their growth and development (Cho et al., 2006). Environmental stresses may cause various types of physiological responses and oxidative damage in plants. Hydroxyl radicals and reduced oxygen species cause peroxidation of cell membranes in various plants in response to abiotic stress treatments (Kinnersley and Turano, 2000; Locy et al., 2000; Mazzucotelli et al., 2008; ALQuraan et al., 2012). Paraquat treatments in wild type wheat cause oxidative damage to the photosynthetic apparatus through down regulation of photochemical efficiency of photosystem II (PSII) (Ekmekci and Terzioglu, 2005). The effects of long term soil salinity have been studied in many wheat cultivars such as Kharchia 65 (tolerant) and KRL 19 (moderately tolerant) under control conditions and two levels of salinity (Sairam et al., 2002). They found that relative water content, chlorophyll, carotenoids, membrane
∗ Corresponding author. Tel.: +962 2 7201000x23460; fax: +962 2 7201071. E-mail address: [email protected] (N.A. AL-Quraan).
stability index, biomass and grain yield, increased hydrogen peroxide levels, reactive oxygen substances, proline, glycine-betaine, soluble sugars, superoxide dismutase, catalase and glutathione reductase activity were impaired and deteriorated in both cultivars at all stages of growth and development. Wheat (Triticum aestivum L.) was analyzed for temporal accumulation of mRNA during salt stress, osmotic stress and abscisic acid (ABA) treatment (Mazzucotelli et al., 2008). The outcomes of this analysis suggested that wheat has at least two salt stress signal transduction pathways: an ABA-dependent pathway and an ABA-independent pathway. However, because salt stress is accompanied by osmotic stress effects, it is likely that activation and expression of these genes in wheat are controlled by many signaling factors (Nemoto and Sasakuma, 2002). Three wheat (T. aestivum L.) cultivars, Tosun, Bolal (stress tolerant) and C-akmak (stress sensitive) were analyzed for the presence of trehalose, which can serve as a reserve of carbohydrates and as a protectant in response to different stress conditions (El-Bashiti et al., 2005). El-Bashiti et al. showed that trehalose accumulation acts as an osmoprotectant compound in wheat species under salt and drought stress conditions. A salt stress-related gene, TaMYB32, was screened out during the bulk sequencing of full length of cDNAs in wheat (T. aestivum L.) (Zhang
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Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010
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et al., 2009). Homologous analysis found that TaMYB32 had a similarity with R2R3-MYB proteins from rice (Oryza sativa L.) and maize (Zea mays L.) as high as 72.4% and 73.7%, respectively. TaMYB32 was expressed in roots, stems, leaves, pistils, and anthers in wheat. The expression was induced by salt stress according to semiquantitative and real-time reverse transcript PCR analysis (Zhang et al., 2009). In vitro assays revealed that succinate semialdehyde dehydrogenase enzyme (SSADH) is specific for succinate semialdehyde (SSA) and exclusively uses nicotinamide adenine dinucleotide (NAD+ ) to produce NADH. Importantly, both ATP and NADH negatively regulate the activity of the SSADH enzyme. Both products of the reaction catalyzed by SSADH (i.e. succinate and NADH) are substrates of the mitochondrial respiratory chain, which produces ATP as a final product. Therefore, regulation of SSADH by ATP suggests a tight feedback control of the rate of substrates provided by the GABA shunt to the respiratory chain. Consequently, the feedback regulation might also play a role in controlling the steady state levels of GABA, and therefore possible functions of GABA via pathways other than the tricarboxylic acid (TCA) cycle as signaling pathways (Shelp et al., 1999; Locy et al., 2000; Bouché et al., 2003; Bouche and Fromm, 2004). GABA is mainly metabolized via a short pathway composed of three enzymes called the GABA shunt because it bypasses two steps of the TCA cycle. The pathway is composed of the cytosolic enzyme glutamate decarboxylase (GAD), the mitochondrial enzymes GABA transaminase (GABA-TA), and succinic semialdehyde dehydrogenase (SSADH) (Bouche and Fromm, 2004). GABA is thus involved in general nitrogen metabolism and possibly in the storage and/or transport of nitrogen. The GABA shunt is also a way to assimilate carbons from glutamate and to generate C:N fluxes that enter the TCA cycle (Breitkreuz et al., 1999; Barbosa et al., 2010; Akc¸ay et al., 2012). In plants, GAD is activated by acidic pH and GABA accumulates in response to cytosolic acidification, so it is possible that GAD activity could participate in regulating the cytosolic pH of plants (Snedden et al., 1995; Shelp et al., 1999). It has been hypothesized that the degradation of GABA could limit the accumulation of reactive oxygen intermediates under oxidative stress conditions that inhibit certain enzymes of the TCA cycle (Barbosa et al., 2010; Renault et al., 2010; AL-Quraan et al., 2011; Zhang et al., 2011; AL-Quraan et al., 2012). It has been reported that regulatory proteins play pivotal roles in stress signal transduction and gene expression regulation such as transcription factors and protein kinases (Liu et al., 2000; Xiong et al., 2002). In this study, we examined the specific response of five wheat cultivars (Hurani 75, Sham I, Acsad 65, UmQayes, and Nodsieh) to salt and osmotic stresses induced by NaCl, mannitol, and sorbitol treatments in terms of seed germination, seedling growth, oxidative damage in terms of reactive oxygen substance accumulations, GABA accumulation levels, and the expression of glutamate decarboxylse gene (GAD) under salt and osmotic stress using RT-PCR.
Seed sensitivity to salt and osmolarity assay Three separate sets with fifty surface sterilized seeds of each of the five wheat cultivars were planted on synthetic growth soil supplemented with 0, 25, 50, 75, 100 and 200 mM NaCl; 0, 50, 100, 150, 200 and 300 mM mannitol and 0, 50, 100, 150, 200 and 300 mM sorbitol, separately. Emergences of radicle from seeds were scored 10 days post germination. The effects of NaCl, mannitol and sorbitol on seed germination were calculated as the germination percentage and compared to the non-treated plants. The average of three replicate plates was used for each treatment. Oxidative damage assay Three separate sets of two-week-old seedlings of each of the five wheat cultivars were grown on synthetic growth soil supplemented with 0, 25, 50, 75, 100 and 200 mM NaCl; 0, 50, 100, 150, 200, and 300 mM mannitol and 0, 50, 100, 150, 200 and 300 mM sorbitol for 10 days. The level of malondialdehyde (MDA) as a reference for reactive oxygen species in the shoot tissues of the seedlings was determined using the TBARS assay (Heath and Packer, 1968). Three plates with 50 seeds each were used in each replicate of each treatment. Shoot tissues after salt and osmotic treatment were separated and frozen in liquid nitrogen. Tissue (250 mg) was ground using a mortar and pestle, and 0.25 ml of 0.5% (w/v) thiobarbituric acid in 20% (w/v) trichloroacetic acid and 0.25 ml 175 mM NaCl in 50 mM Tris–HCl at pH 8 was added to the ground tissue. Tubes were heated to 90 ◦ C for 25 min. The supernatant was collected after the samples were centrifuged for 20 min at 15,000 rpm. The absorbance of the supernatant was measured at 532 nm. The level of malondialdehyde (MDA) was determined as nmol/mg FW from a standard curve of MDA. The average of three replicate plates was used for each treatment. GABA-metabolite extraction GABA was extracted according to Zhang and Bown (1997) with the following modifications: harvested shoot tissues of two-weekold seedlings of each of the five wheat cultivars grown on synthetic growth soil supplemented with 0, 25, 50, 75, 100 and 200 mM NaCl; 0, 50, 100, 150, 200, and 300 mM mannitol; and 0, 50, 100, 150, 200 and 300 mM sorbitol for 10 days were ground separately with a mortar and pestle, then collected in 1.5 ml microcentrifuge tubes. In each tube, 400 L methanol was added and the samples were mixed for ten min. Liquid from the samples was removed by regular evaporation over night. 500 L of 70 mM lanthanum chloride was added to each tube. The tubes were mixed for 15 min, and subsequently centrifuged at 15,000 rpm for five min. Supernatants were removed to new tubes and mixed with 160 L of 1 M potassium hydroxide (KOH). The tubes were mixed for10 min and then centrifuged at 15,000 rpm for five min. The supernatant containing metabolites was transferred to a new tube and used for GABA level determination.
Materials and methods GABA-metabolite level determination Plant materials and growth conditions Five wheat cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes, and Nodsieh) used in this study were obtained from Jordan National Center for Agricultural Research and Extension (NCARE). All experiments were conducted in a laboratory growth chamber using synthetic growth soil supplemented with salt (NaCl) and osmotic reagents (mannitol and sorbitol). Seedlings were grown under continuous illumination (40 mol m−2 s−1 ) provided by cool white fluorescent lamps at 25 ◦ C.
GABA was measured according to Zhang and Bown (1997) with the following modifications: the reaction mixture contained 50 L of sample extract, 14 L of 4 mM NADP+ , 19 L of 0.5 M potassium pyrophosphate at pH 8.6, 10 L of (2 u/L) GABASE enzyme (GABASE enzyme was suspended in 0.1 M potassium pyrophosphate at pH 7.2 containing 12.5% Glycerol and 5 mM -marcaptoethanol), and 10 L of ␣-ketoglutarate. The change in absorbance at 340 nm after addition of ␣-ketoglutarate was recorded after 90 min incubation at 25 ◦ C using the NanodropSpectorphotometry (ND-1000). The level of GABA (nmol/mg FW)
Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010
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Germinaon %
100 90
Acsad 65
80
Hurani 75
70
Nodsieh
60
um Qayes
50
Sham I
40 30 20 10 0
Concentraon (mM) Fig. 1. Germination percentage of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) grown in synthetic growth media supplemented with five different concentrations of salt and osmotic reagents (NaCl, mannitol and sorbitol). The emergences of radicle from germinating seeds were scored after 10 days. The effect of NaCl, mannitol and sorbitol on seed germination was calculated as described in ‘Materials and methods’.
was determined using the NADPH standard curve. The average of three replicate plates was used for each treatment. Chlorophyll extraction and determination For salt treatments, two-week-old seedlings of each of the five wheat cultivars grown on synthetic growth soil at 25 ◦ C were irrigated with 25, 50, 75, 100, and 200 mM NaCl solution for two weeks at 25 ◦ C. For osmotic treatments, two separate sets of one-weekold seedlings were irrigated with 50, 100, 150, 200, and 300 mM mannitol solution and 50, 100, 150, 200, and 300 mM sorbitol solution, separately. Plants were harvested on the 14th day of solution irrigation from each treatment separately and used for chlorophyll pigment (Chl a and Chl b) extraction and determination according to Jeffrey and Humphrey (1975) with the following modifications: the chlorophyll extract was prepared from 250 mg fresh leaves by grinding in a tissue homogenizer together with 25 ml of ice cold 90% acetone. The homogenate was centrifuged at 4000 rpm for five min. The supernatant was transferred to a separate tube for chlorophyll pigment determination. The absorbance of the extract was read at 647 nm and 664 nm, respectively. The concentrations of chlorophyll a and b and total chlorophyll were calculated according to Jeffrey and Humphrey’s (1975) equation and extension coefficients. The average of three replicate plates was used for each treatment. RNA extraction Total RNA from fresh samples was extracted by using the RNeasy Plant Mini kit from intron biotechnology according to the manufacturer’s instructions. Total RNA was suspended in RNase-free water. RNA concentrations were determined by their absorbance A260 using a nanodrop spectrophotometer (ND-1000). The integrity of RNA was determined after separation of RNA on a 1.5% (w/v) agarose gel after electrophoresis and staining with ethidium bromide and visualized using a UV transilluminator and detection system. GAD mRNA expression levels by reverse transcriptase-PCR Gene-specific primers for the wheat GAD enzyme gene (forward primer 5 -TGC CGG AGA ACT CGA TCC CCA AG-3 ) (reverse primer 5 -CGG TTC TGG AGC TCG GTG GTG AC-3 ) were used for RT-PCR analysis of steady state mRNA levels in all wheat cultivars that
were used in this study under all treatments separately. A onestep reverse transcriptase-PCR (RT-PCR) reaction was performed using primer pairs specific for wheat GAD gene (Mazzucotelli et al., 2006), SuperScriptTM III One-step RT-PCR system with platinum® Taq DNA polymerase according to the manufacturer’s instructions. RT-PCR amplification products were separated on 1% agarose gels and stained with ethidium bromide. The expression level of the GAD gene in wheat was determined. Data statistical analysis Each data point was expressed as the mean ± standard deviation (SD) of three independent experiments. The values were compared and analyzed by one-way analysis of variance (ANOVA) using least significant difference (LSD) multiple comparison tests on the means. Where differences are reported, they are at the 95% confidence level (P < 0.05). Data analysis was performed using SPSS software version 15.0. Results and discussion Wheat seed sensitivity to salt and osmotic stress Fig. 1 shows a marked decrease (P < 0.05) in the germination percentage among the five wheat cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes, and Nodsieh) under salt and osmotic stress compared to the control (no treatment). Hurani 75 showed an observable decrease in germination under salt stress (NaCl) and was decreased up to 50% under the 25 mM NaCl treatment. The data indicated that Hurani 75 showed greater susceptibility to NaCl than to mannitol or sorbitol. Sham I showed a marked decrease (p = 0.003) in seed germination under 300 mM mannitol and sorbitol and decreased up to 50% under the 200 mM NaCl treatment, which indicated that sham I is more susceptible to mannitol and sorbitol than to NaCl. In Acsad 65, seed germination showed a 79% reduction under 200 mM NaCl, while no effect was observed under lower concentrations of NaCl. However, Acsad 65 seed germination decreased under the 300 mM concentrations of mannitol and sorbitol. Acsad 65 was more susceptible to NaCl than to the mannitol and sorbitol treatments. Um Qayes seed germination was reduced 42–57% under 200 mM mannitol. Um Qayes seed germination was reduced up to 77% compared to the control. Nodsieh showed a dramatic decrease (P = 0.007) in germination percentage up to 90% under 200 mM
Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010
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4 3.5
Acsad 65
3
MDA Level nmol/mg FW
Hurani 75 Nodsieh
2.5
Um Qayes 2
Sham I
1.5 1 0.5 0
Concentraon (mM) Fig. 2. The level of malondialdehyde (MDA) as a reference for reactive oxygen species in10-day old shoot tissues of seedlings of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) exposed to NaCl, mannitol and sorbitol separately were determined using the TBARS assay as described in the ‘Materials and methods’. The level of malondialdehyde (MDA) was determined as nmol/mg FW.
NaCl. The percentage reduction reached 48% under 300 mM mannitol and decreased to 71% under the same sorbitol treatment (Fig. 1). Higher plants routinely encounter various abiotic stresses such as drought, high salinity, heavy metals, and extreme temperatures, which impair the growth and development of cultivated plant, and may cause various types of physiological responses and oxidative damage (Cho et al., 2006). Hydroxyl radicals and reactive oxygen species cause peroxidation of cell membranes in various plants in response to abiotic stress treatments (Mazzucotelli et al., 2008; Renault et al., 2010; Zhang et al., 2011; AL-Quraan et al., 2012). Such data are confirmed by the extensive losses of agricultural production worldwide caused by abiotic stress conditions (Boyer, 1982; Bray et al., 2000). Thomas et al. (1995) reported that NaCl and mannitol can penetrate the plant cell and contribute to the decrease of internal osmotic potential during seed germination, allowing the water uptake to occur. Almansouri et al. (2001) showed that salttreated isolated embryos of durum wheat (Triticum durum Desf.) exhibited a lower germination percentage than whole seeds, which suggests the inhibition of germination may occur through direct effect of stress on germinating embryos. Munns (2002) reported that salinity causes a reduction in the growth rate of plants, as it impairs water uptake by plant.
Oxidative damage in wheat seedling in response to salt and osmotic stress The MDA level in the Hurani 75 cultivar was the lowest among the wheat cultivars that were used in this study, as shown in Fig. 2. This finding indicates low accumulation of ROS (reactive oxygen species) in Hurani 75 shoot tissues under the NaCl, sorbitol and mannitol treatments. This is due to physiological adaptation of Hurani 75 to salt and osmotic stress by other mechanisms that might encounter the accumulation of ROS in plant tissues. MDA accumulation in Nodsieh shoot tissues was moderately low under all stress treatments, which was correlated (r = 0.583) with seed sensitivity of the same cultivar under all treatments (Fig. 1). In Um Qayes, the accumulation of MDA was moderate compared to other cultivars, which might be producing ROS adapt to the stress, which correlated with moderate levels of GABA accumulation (GABA r = 0.565, MDA r = 0.401) (Fig. 3). Overall, Um Qayes cultivar was more tolerant to stress than Sham I, which showed the highest level of MDA accumulation, an indication of oxidative damage and impaired plant growth in response to salt and osmotic stress. In a previous study, MDA accumulation took place in plants under stress conditions due to membrane lipid peroxidation (Heath
500 Acsad 65
GABA Level nmol/mg FW
450
Hurani 75
400
Nodsieh
350
Um Qayes Sham I
300 250 200 150 100 50 0
Concentraon (mM) Fig. 3. The level of GABA (␥-aminobutyric acid) metabolite in10-day old seedlings shoot tissues of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) exposed to NaCl, mannitol and sorbitol separately. GABA was extracted and determined as described in the ‘Materials and methods’. The level of GABA was determined as nmol/mg FW.
Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010
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and Packer, 1968). The growth reduction effects of salt stress have also been reported in different maize cultivars at different growth stages (Hichem et al., 2009). Furthermore, growth-reducing effects of different saline regimes in hydroponic culture systems were also observed by Chen et al. (2007). Accumulation of MDA contents coupled with reduced plant growth under salt stress was also reported by Koca et al. (2007) and Li (2009) in tomato and sesame seedlings. AL-Quraan et al. (2011, 2012) found that Arabidopsis thaliana root and shoot tissues accumulate MDA metabolites under various treatments of NaCl, mannitol, hydrogen peroxide and paraquat. GABA (-aminobutyrate) level under salt and osmotic stress in wheat Our study showed that, in the Acsad 65 wheat cultivar, there was a drastic increase (P = 0.028) of the GABA metabolite under 100 mM NaCl and 200 mM sorbitol, and a minor increase under 200 mM mannitol. This indicates that the Acsad 65 cultivar tolerates salt and osmotic stress by sorbitol under higher concentrations through the accumulation of GABA more than under osmotic agents induced by mannitol (Fig. 3). The Hurani 75 wheat cultivar accumulated nearly the same level of GABA as in Acsad 65 under 100 mM NaCl and 200 mM sorbitol. A small quantity of GABA was detected at higher concentrations of NaCl and sorbitol. This may suggest that GABA accumulation level is correlated with a destructive effect of NaCl and sorbitol (r = 0.429), while mannitol had minor effects on inducing the GABA shunt through lower GABA accumulation levels. The highest level of GABA accumulation occurred in the shoot tissues of the Nodsieh cultivar under 75 mM NaCl and 200 mM sorbitol, which suggests a higher tolerance of the Nodsieh cultivar to NaCl treatments. In contrast, the Um Qayes and Sham I cultivars showed higher levels of GABA accumulation under 100 and 200 mM mannitol, respectively, and a drastic decrease in GABA levels under 300 mM mannitol (Sham I P = 0.016, Um Qayes P = 0.042) (Fig. 3). This could be due to the lethal effects of osmotic stress induced by mannitol on this cultivar. The GABA shunt was first reported in plants more than half a century ago in potato (Solanum tuberosum) tubers (Dent et al., 1947). Previous studies showed that the levels of GABA in plants also tend to be elevated during high stress conditions such as hypoxia, darkness, drought and salt stress (Shelp et al., 1999; Bouche and Fromm, 2004; Akc¸ay et al., 2012). In agreement with our results, previous studies have suggested that GABA was the only non-amino acid out of three intermediates of the GABA shunt that increased in response to osmotic stress in wheat (Rentsch et al., 1996). In agreement with our data, AL-Quraan et al. (2012) showed that increased levels of GABA shunt metabolites in response to cold and heat treatments occurred in some Arabidopsis calmodulin mutants. This is correlated with activation of GAD after exposure to temperature stress in response to the intracellular damage and low cytoplasmic pH. In addition, AL-Quraan et al. (in press) reported that high accumulation of GABA metabolite occurred under salt and osmotic stress. GABA shunt metabolites may have a role in osmoregulation and signaling in response to salinity and non-ionic osmotic stress. Bartyzel et al. (2003) showed that GABA increased significantly in wheat seedlings after exposure to 20% polyethylene glycol 6000 for 28 h as an osmotic stress agent. Akc¸ay et al. (2012) found that the cytoplasmic male sterility (CMSII) mutant and WT of Nicotiana sylvestris plants showed better growth under long term salinity, which is related to GABA accumulation and metabolism. Chlorophyll contents reduction under salt and osmotic treatments The highest percentage of Chl a was observed in the Sham I wheat cultivar under 100 mM mannitol treatment. The percentage of Chl a was correlated with high levels of GABA under the same
5
Table 1 Chlorophyll pigment (Chl a) level (nm/mg FW) of 10-day old seedlings shoot tissues of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) exposed to NaCl, mannitol and sorbitol separately. Chlorophyll pigments were extracted and determined according to Jeffrey and Humphrey (1975). [mM]
Acsad 65
Hurani 75
Nodsieh
Um Qayes
Sham I
Control NaCl 25 NaCl 50 NaCl 75 NaCl 100 NaCl 200 Mannitol 50 Mannitol 100 Mannitol 150 Mannitol 200 Mannitol 300 Sorbitol 50 Sorbitol 100 Sorbitol 150 Sorbitol 200 Sorbitol 300
7.05 6.21 2.57 2.13 1.93 0.91 3.52 3.33 3.08 2.39 2.1 2.43 2.22 2 1.45 1.19
8.44 6.22 6.15 5.81 5.74 2.53 10.3 5.57 5.03 4.73 2.59 9.49 7.43 4.57 2.85 1.6
5.72 3.21 3.12 2.86 2.82 2.72 5.12 3.39 2.15 2.15 1.59 4.59 4.37 3.56 2.69 2.62
12.09 10.02 9.62 8.85 5.71 2.26 14.59 9.56 8.82 7.37 7.15 15.05 8.47 7.87 5.6 5.48
8.49 7.09 4.97 2.76 2.7 2.07 1.94 1.7 1.33 1.16 1.06 2.45 2.39 2.29 1.91 1.36
treatment (Table 1). In the Nodsieh cultivar, a correlation (chl a r = −0.409, chl b r = −0.416) was found between the level of GABA accumulation and chlorophyll contents under 75 mM NaCl, which indicated a lethal effect of high NaCl concentration on the Nodsieh cultivar (Tables 1 and 2). In the Hurani 75 cultivar, a higher level of GABA accumulation and chlorophyll b in shoot tissue was observed under the 100 mM NaCl treatment. The Acsad 65 cultivar showed a link between the GABA accumulation and chl b content under the 200 mM mannitol treatment (r = −0.523) (Table 2). Our data suggested that GABA metabolite accumulation had various impacts on plant metabolism under salt and osmotic stresses. This is an indication that the GABA level may act as a signaling molecule or osmoprotectant in various cultivars under abiotic stress. Akc¸ay et al. (2012) showed that the photosynthetic efficiency of the N. sylvestris mutant and WT plants decreased in response to the 100 mM NaCl treatment. Sairam et al. (2002) found that relative water content, chlorophyll, carotenoids, membrane stability index, biomass and grain yield, proline, glycine-betaine, soluble sugars, superoxide dismutase, catalase and glutathione reductase activity were impaired and deteriorated in Kharchia 65 (tolerant) and KRL 19 (moderately tolerant) wheat cultivars at all stages of growth and development in response to long term soil salinity treatments.
Table 2 Chlorophyll pigment (Chl b) level (nm/mg FW) of 10-day old seedlings shoot tissues of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) exposed to NaCl, mannitol and sorbitol separately. Chlorophyll pigments were extracted and determined according to Jeffrey and Humphrey (1975). [mM]
Acsad 65
Hurani 75
Nodsieh
Um Qayes
Sham I
Control NaCl 25 NaCl 50 NaCl 75 NaCl 100 NaCl 200 Mannitol 50 Mannitol 100 Mannitol 150 Mannitol 200 Mannitol 300 Sorbitol 50 Sorbitol 100 Sorbitol 150 Sorbitol 200 Sorbitol 300
4.36 2.6 1.79 1.38 0.89 0.56 1.87 1.85 1.52 1.32 1.29 1.92 1.37 1.28 1.23 1.2
3.42 2.3 1.95 1.79 1.61 1.6 2.81 1.85 1.67 1.59 1.03 2.46 2.22 1.51 0.87 0.57
2.05 1.57 1.06 1.03 1.01 0.74 1.78 1.48 1.33 0.87 0.85 1.3 1.2 1.15 0.79 0.62
6.12 3.95 3.68 2.06 1.28 0.53 5.91 5.33 4.96 4.72 2.44 4.16 3.8 3.7 2.4 1.29
4.54 2.55 2.26 2.12 2.01 1.54 1.53 1.34 1.27 1.14 1.13 3.18 2.34 2.1 1.89 1.45
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Wheat GAD RNA transcripts under salt and osmotic stress Glutamate decarboxylase (GAD) RNA transcripts of wheat were examined in this study under all treatments (Fig. 4). GAD catalyzes the decarboxylation of glutamate to CO2 and ␥-aminobutyrate (GABA) (Fait et al., 2007). Our results showed an association between GAD RNA transcription and the response to osmotic and salt stress in terms of seed germination and GABA and MDA accumulation. The transcription of GAD in terms of RNA levels in the five wheat cultivars was increased under different concentrations of osmotic and salt treatments (Fig. 4). There was high GAD expression and GABA accumulation in the Hurani 75 cultivar under salt stress. In contrast, GAD expression and GABA accumulation were relatively low under osmotic stress when compared to the control and other cultivars under all treatments (Fig. 4). These results suggested that osmotic reagents (sorbitol and mannitol) may have lethal effects on the Hurani 75 cultivar, especially at higher concentrations. It was shown by Mazzucotelli et al. (2008) that post-transcriptional and post-translational mechanisms regulate molecular responses to abiotic stresses, based on alternative splicing and RNA processing, as well as RNA silencing. There was relatively high GAD expression and GABA accumulation under salt and osmotic treatment in the Um Qayes cultivar. The highest GAD expression was observed under mannitol (Fig. 4C). This suggests that the GABA shunt was activated under salt and osmotic stress in terms of metabolite accumulation and GAD expression. In agreement with our results, Akc¸ay et al. (2012) showed that GAD activity rapidly increased in response to NaCl treatments, which was related to an increase in GAD gene expression in the cytoplasmic male sterility (CMSII) mutant and WT of N. sylvestris plants. GAD expression, GABA accumulation and seed germination were low under sorbitol treatments in Sham I (Fig. 4D) and Acsad 65 (Fig. 4F) cultivars. This suggests that sorbitol had a destructive effect on sham I and Acsad 65 shoot tissues, in contrast to high GAD expression under NaCl and mannitol (Fig. 4). High GAD expression under salt and osmotic stress was recorded in the Nodsieh cultivar (Fig. 4E). However, under higher concentrations of sorbitol, GAD expression was very low. This might be due to the destructive effect of sorbitol on the expression machinery at high concentrations, which was also accompanied by low seed germination in the Nosdieh cultivar under the same sorbitol treatment. In contrast, Bartyzel et al. (2003) showed that GAD activity in wheat seedlings did not change when exposed to 20% polyethylene glycol 6000 for 28 h as an osmotic stress agent compared with the control plants. In conclusion, our study showed that abiotic stress is a major limiting agent in plant growth and germination. There was a marked decrease in the germination percentage under salt and osmotic stress compared to the control (no treatment) among the five wheat cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes, and Nodsieh). Also, there was a correlation between the GABA concentration and germination percentage of the five wheat cultivars (i.e. drastic increase of GABA metabolite and MDA level under salt and osmotic stress). A decrease in chlorophyll a and b levels under salt and osmotic reagent treatment was observed. The transcription of GAD in terms of the RNA level increased under different concentrations of osmotic and salt treatments in the five wheat cultivars.
Fig. 4. Glutamate decarboxylase (GAD) RNA transcripts of 10-day old seedlings shoot tissues of five wheat cultivars. (A) Control, (B) Hurani 75, (C) Um Qayes, (D) Sham I, (E) Nodsieh, and (F) Acsad 65 exposed to NaCl, mannitol and sorbitol separately. (J) Internal control primer (CBP20).
Acknowledgments We thank our colleague Dr. Rami AL-Khatib for helpful suggestions and reading of the manuscript. This work was supported by grant number 97/2011 from the Deanship of Research, Jordan University of Science and Technology, Jordan.
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Physiology
Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress Nisreen A. AL-Quraan a,∗ , Fatima Al-batool Sartawe a , Muien M. Qaryouti b a b
Department of Biotechnology and Genetic Engineering, Faculty of Science and Arts, Jordan University of Science and Technology, Irbid 22110, Jordan National Center for Agricultural Research and Extension (NCARE), P.O. Box 639, Amman 19381, Jordan
a r t i c l e
i n f o
Article history: Received 15 November 2012 Received in revised form 21 February 2013 Accepted 22 February 2013 Available online xxx Keywords: GABA GAD Glutamate decarboxylase ␥-Aminobutyric acid Osmotic stress
a b s t r a c t The molecular response of plants to abiotic stresses has been considered a process mainly involved in the modulation of transcriptional activity of stress-related genes. Nevertheless, recent findings have suggested new layers of regulation and complexity. Upstream molecular mechanisms are involved in the plant response to abiotic stress. Plants gain resistance to abiotic stress by reprogramming metabolism and gene expression. GABA is proposed to be a signaling molecule involved in nitrogen metabolism, regulating the cytosolic pH, and protection against oxidative damage in response to various abiotic stresses. The aim of our study was to examine the role of the GABA shunt pathway-specific response in five wheat (Triticum aestivum L.) cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes and Nodsieh) to salt and osmotic stress in terms of seed germination, seedling growth, oxidative damage (malondialdehyde (MDA) accumulation), and characterization of the glutamate decarboxylse gene (GAD) m-RNA level were determined using RT-PCR techniques. Our data showed a marked increase in GABA, MDA and GAD m-RNA levels under salt and osmotic stress in the five wheat cultivars. Um Qayes cultivar showed the highest germination percentage, GABA accumulation, and MDA level under salt and osmotic stresses. The marked increase in GAD gene expression explains the high accumulation of the GABA level under both stresses. Our results indicated that the GABA shunt is a key signaling and metabolic pathway that allows wheat to adapt to salt and osmotic stress. Based on our data, the Um Qayes wheat cultivar is the cultivar most recommended to be grown in soil with high salt and osmotic contents. © 2013 Elsevier GmbH. All rights reserved.
Introduction Higher plants routinely encounter various abiotic stresses such as drought, high salinity, heavy metals, and extreme temperatures, which impair their growth and development (Cho et al., 2006). Environmental stresses may cause various types of physiological responses and oxidative damage in plants. Hydroxyl radicals and reduced oxygen species cause peroxidation of cell membranes in various plants in response to abiotic stress treatments (Kinnersley and Turano, 2000; Locy et al., 2000; Mazzucotelli et al., 2008; ALQuraan et al., 2012). Paraquat treatments in wild type wheat cause oxidative damage to the photosynthetic apparatus through down regulation of photochemical efficiency of photosystem II (PSII) (Ekmekci and Terzioglu, 2005). The effects of long term soil salinity have been studied in many wheat cultivars such as Kharchia 65 (tolerant) and KRL 19 (moderately tolerant) under control conditions and two levels of salinity (Sairam et al., 2002). They found that relative water content, chlorophyll, carotenoids, membrane
∗ Corresponding author. Tel.: +962 2 7201000x23460; fax: +962 2 7201071. E-mail address: [email protected] (N.A. AL-Quraan).
stability index, biomass and grain yield, increased hydrogen peroxide levels, reactive oxygen substances, proline, glycine-betaine, soluble sugars, superoxide dismutase, catalase and glutathione reductase activity were impaired and deteriorated in both cultivars at all stages of growth and development. Wheat (Triticum aestivum L.) was analyzed for temporal accumulation of mRNA during salt stress, osmotic stress and abscisic acid (ABA) treatment (Mazzucotelli et al., 2008). The outcomes of this analysis suggested that wheat has at least two salt stress signal transduction pathways: an ABA-dependent pathway and an ABA-independent pathway. However, because salt stress is accompanied by osmotic stress effects, it is likely that activation and expression of these genes in wheat are controlled by many signaling factors (Nemoto and Sasakuma, 2002). Three wheat (T. aestivum L.) cultivars, Tosun, Bolal (stress tolerant) and C-akmak (stress sensitive) were analyzed for the presence of trehalose, which can serve as a reserve of carbohydrates and as a protectant in response to different stress conditions (El-Bashiti et al., 2005). El-Bashiti et al. showed that trehalose accumulation acts as an osmoprotectant compound in wheat species under salt and drought stress conditions. A salt stress-related gene, TaMYB32, was screened out during the bulk sequencing of full length of cDNAs in wheat (T. aestivum L.) (Zhang
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et al., 2009). Homologous analysis found that TaMYB32 had a similarity with R2R3-MYB proteins from rice (Oryza sativa L.) and maize (Zea mays L.) as high as 72.4% and 73.7%, respectively. TaMYB32 was expressed in roots, stems, leaves, pistils, and anthers in wheat. The expression was induced by salt stress according to semiquantitative and real-time reverse transcript PCR analysis (Zhang et al., 2009). In vitro assays revealed that succinate semialdehyde dehydrogenase enzyme (SSADH) is specific for succinate semialdehyde (SSA) and exclusively uses nicotinamide adenine dinucleotide (NAD+ ) to produce NADH. Importantly, both ATP and NADH negatively regulate the activity of the SSADH enzyme. Both products of the reaction catalyzed by SSADH (i.e. succinate and NADH) are substrates of the mitochondrial respiratory chain, which produces ATP as a final product. Therefore, regulation of SSADH by ATP suggests a tight feedback control of the rate of substrates provided by the GABA shunt to the respiratory chain. Consequently, the feedback regulation might also play a role in controlling the steady state levels of GABA, and therefore possible functions of GABA via pathways other than the tricarboxylic acid (TCA) cycle as signaling pathways (Shelp et al., 1999; Locy et al., 2000; Bouché et al., 2003; Bouche and Fromm, 2004). GABA is mainly metabolized via a short pathway composed of three enzymes called the GABA shunt because it bypasses two steps of the TCA cycle. The pathway is composed of the cytosolic enzyme glutamate decarboxylase (GAD), the mitochondrial enzymes GABA transaminase (GABA-TA), and succinic semialdehyde dehydrogenase (SSADH) (Bouche and Fromm, 2004). GABA is thus involved in general nitrogen metabolism and possibly in the storage and/or transport of nitrogen. The GABA shunt is also a way to assimilate carbons from glutamate and to generate C:N fluxes that enter the TCA cycle (Breitkreuz et al., 1999; Barbosa et al., 2010; Akc¸ay et al., 2012). In plants, GAD is activated by acidic pH and GABA accumulates in response to cytosolic acidification, so it is possible that GAD activity could participate in regulating the cytosolic pH of plants (Snedden et al., 1995; Shelp et al., 1999). It has been hypothesized that the degradation of GABA could limit the accumulation of reactive oxygen intermediates under oxidative stress conditions that inhibit certain enzymes of the TCA cycle (Barbosa et al., 2010; Renault et al., 2010; AL-Quraan et al., 2011; Zhang et al., 2011; AL-Quraan et al., 2012). It has been reported that regulatory proteins play pivotal roles in stress signal transduction and gene expression regulation such as transcription factors and protein kinases (Liu et al., 2000; Xiong et al., 2002). In this study, we examined the specific response of five wheat cultivars (Hurani 75, Sham I, Acsad 65, UmQayes, and Nodsieh) to salt and osmotic stresses induced by NaCl, mannitol, and sorbitol treatments in terms of seed germination, seedling growth, oxidative damage in terms of reactive oxygen substance accumulations, GABA accumulation levels, and the expression of glutamate decarboxylse gene (GAD) under salt and osmotic stress using RT-PCR.
Seed sensitivity to salt and osmolarity assay Three separate sets with fifty surface sterilized seeds of each of the five wheat cultivars were planted on synthetic growth soil supplemented with 0, 25, 50, 75, 100 and 200 mM NaCl; 0, 50, 100, 150, 200 and 300 mM mannitol and 0, 50, 100, 150, 200 and 300 mM sorbitol, separately. Emergences of radicle from seeds were scored 10 days post germination. The effects of NaCl, mannitol and sorbitol on seed germination were calculated as the germination percentage and compared to the non-treated plants. The average of three replicate plates was used for each treatment. Oxidative damage assay Three separate sets of two-week-old seedlings of each of the five wheat cultivars were grown on synthetic growth soil supplemented with 0, 25, 50, 75, 100 and 200 mM NaCl; 0, 50, 100, 150, 200, and 300 mM mannitol and 0, 50, 100, 150, 200 and 300 mM sorbitol for 10 days. The level of malondialdehyde (MDA) as a reference for reactive oxygen species in the shoot tissues of the seedlings was determined using the TBARS assay (Heath and Packer, 1968). Three plates with 50 seeds each were used in each replicate of each treatment. Shoot tissues after salt and osmotic treatment were separated and frozen in liquid nitrogen. Tissue (250 mg) was ground using a mortar and pestle, and 0.25 ml of 0.5% (w/v) thiobarbituric acid in 20% (w/v) trichloroacetic acid and 0.25 ml 175 mM NaCl in 50 mM Tris–HCl at pH 8 was added to the ground tissue. Tubes were heated to 90 ◦ C for 25 min. The supernatant was collected after the samples were centrifuged for 20 min at 15,000 rpm. The absorbance of the supernatant was measured at 532 nm. The level of malondialdehyde (MDA) was determined as nmol/mg FW from a standard curve of MDA. The average of three replicate plates was used for each treatment. GABA-metabolite extraction GABA was extracted according to Zhang and Bown (1997) with the following modifications: harvested shoot tissues of two-weekold seedlings of each of the five wheat cultivars grown on synthetic growth soil supplemented with 0, 25, 50, 75, 100 and 200 mM NaCl; 0, 50, 100, 150, 200, and 300 mM mannitol; and 0, 50, 100, 150, 200 and 300 mM sorbitol for 10 days were ground separately with a mortar and pestle, then collected in 1.5 ml microcentrifuge tubes. In each tube, 400 L methanol was added and the samples were mixed for ten min. Liquid from the samples was removed by regular evaporation over night. 500 L of 70 mM lanthanum chloride was added to each tube. The tubes were mixed for 15 min, and subsequently centrifuged at 15,000 rpm for five min. Supernatants were removed to new tubes and mixed with 160 L of 1 M potassium hydroxide (KOH). The tubes were mixed for10 min and then centrifuged at 15,000 rpm for five min. The supernatant containing metabolites was transferred to a new tube and used for GABA level determination.
Materials and methods GABA-metabolite level determination Plant materials and growth conditions Five wheat cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes, and Nodsieh) used in this study were obtained from Jordan National Center for Agricultural Research and Extension (NCARE). All experiments were conducted in a laboratory growth chamber using synthetic growth soil supplemented with salt (NaCl) and osmotic reagents (mannitol and sorbitol). Seedlings were grown under continuous illumination (40 mol m−2 s−1 ) provided by cool white fluorescent lamps at 25 ◦ C.
GABA was measured according to Zhang and Bown (1997) with the following modifications: the reaction mixture contained 50 L of sample extract, 14 L of 4 mM NADP+ , 19 L of 0.5 M potassium pyrophosphate at pH 8.6, 10 L of (2 u/L) GABASE enzyme (GABASE enzyme was suspended in 0.1 M potassium pyrophosphate at pH 7.2 containing 12.5% Glycerol and 5 mM -marcaptoethanol), and 10 L of ␣-ketoglutarate. The change in absorbance at 340 nm after addition of ␣-ketoglutarate was recorded after 90 min incubation at 25 ◦ C using the NanodropSpectorphotometry (ND-1000). The level of GABA (nmol/mg FW)
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Concentraon (mM) Fig. 1. Germination percentage of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) grown in synthetic growth media supplemented with five different concentrations of salt and osmotic reagents (NaCl, mannitol and sorbitol). The emergences of radicle from germinating seeds were scored after 10 days. The effect of NaCl, mannitol and sorbitol on seed germination was calculated as described in ‘Materials and methods’.
was determined using the NADPH standard curve. The average of three replicate plates was used for each treatment. Chlorophyll extraction and determination For salt treatments, two-week-old seedlings of each of the five wheat cultivars grown on synthetic growth soil at 25 ◦ C were irrigated with 25, 50, 75, 100, and 200 mM NaCl solution for two weeks at 25 ◦ C. For osmotic treatments, two separate sets of one-weekold seedlings were irrigated with 50, 100, 150, 200, and 300 mM mannitol solution and 50, 100, 150, 200, and 300 mM sorbitol solution, separately. Plants were harvested on the 14th day of solution irrigation from each treatment separately and used for chlorophyll pigment (Chl a and Chl b) extraction and determination according to Jeffrey and Humphrey (1975) with the following modifications: the chlorophyll extract was prepared from 250 mg fresh leaves by grinding in a tissue homogenizer together with 25 ml of ice cold 90% acetone. The homogenate was centrifuged at 4000 rpm for five min. The supernatant was transferred to a separate tube for chlorophyll pigment determination. The absorbance of the extract was read at 647 nm and 664 nm, respectively. The concentrations of chlorophyll a and b and total chlorophyll were calculated according to Jeffrey and Humphrey’s (1975) equation and extension coefficients. The average of three replicate plates was used for each treatment. RNA extraction Total RNA from fresh samples was extracted by using the RNeasy Plant Mini kit from intron biotechnology according to the manufacturer’s instructions. Total RNA was suspended in RNase-free water. RNA concentrations were determined by their absorbance A260 using a nanodrop spectrophotometer (ND-1000). The integrity of RNA was determined after separation of RNA on a 1.5% (w/v) agarose gel after electrophoresis and staining with ethidium bromide and visualized using a UV transilluminator and detection system. GAD mRNA expression levels by reverse transcriptase-PCR Gene-specific primers for the wheat GAD enzyme gene (forward primer 5 -TGC CGG AGA ACT CGA TCC CCA AG-3 ) (reverse primer 5 -CGG TTC TGG AGC TCG GTG GTG AC-3 ) were used for RT-PCR analysis of steady state mRNA levels in all wheat cultivars that
were used in this study under all treatments separately. A onestep reverse transcriptase-PCR (RT-PCR) reaction was performed using primer pairs specific for wheat GAD gene (Mazzucotelli et al., 2006), SuperScriptTM III One-step RT-PCR system with platinum® Taq DNA polymerase according to the manufacturer’s instructions. RT-PCR amplification products were separated on 1% agarose gels and stained with ethidium bromide. The expression level of the GAD gene in wheat was determined. Data statistical analysis Each data point was expressed as the mean ± standard deviation (SD) of three independent experiments. The values were compared and analyzed by one-way analysis of variance (ANOVA) using least significant difference (LSD) multiple comparison tests on the means. Where differences are reported, they are at the 95% confidence level (P < 0.05). Data analysis was performed using SPSS software version 15.0. Results and discussion Wheat seed sensitivity to salt and osmotic stress Fig. 1 shows a marked decrease (P < 0.05) in the germination percentage among the five wheat cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes, and Nodsieh) under salt and osmotic stress compared to the control (no treatment). Hurani 75 showed an observable decrease in germination under salt stress (NaCl) and was decreased up to 50% under the 25 mM NaCl treatment. The data indicated that Hurani 75 showed greater susceptibility to NaCl than to mannitol or sorbitol. Sham I showed a marked decrease (p = 0.003) in seed germination under 300 mM mannitol and sorbitol and decreased up to 50% under the 200 mM NaCl treatment, which indicated that sham I is more susceptible to mannitol and sorbitol than to NaCl. In Acsad 65, seed germination showed a 79% reduction under 200 mM NaCl, while no effect was observed under lower concentrations of NaCl. However, Acsad 65 seed germination decreased under the 300 mM concentrations of mannitol and sorbitol. Acsad 65 was more susceptible to NaCl than to the mannitol and sorbitol treatments. Um Qayes seed germination was reduced 42–57% under 200 mM mannitol. Um Qayes seed germination was reduced up to 77% compared to the control. Nodsieh showed a dramatic decrease (P = 0.007) in germination percentage up to 90% under 200 mM
Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010
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1.5 1 0.5 0
Concentraon (mM) Fig. 2. The level of malondialdehyde (MDA) as a reference for reactive oxygen species in10-day old shoot tissues of seedlings of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) exposed to NaCl, mannitol and sorbitol separately were determined using the TBARS assay as described in the ‘Materials and methods’. The level of malondialdehyde (MDA) was determined as nmol/mg FW.
NaCl. The percentage reduction reached 48% under 300 mM mannitol and decreased to 71% under the same sorbitol treatment (Fig. 1). Higher plants routinely encounter various abiotic stresses such as drought, high salinity, heavy metals, and extreme temperatures, which impair the growth and development of cultivated plant, and may cause various types of physiological responses and oxidative damage (Cho et al., 2006). Hydroxyl radicals and reactive oxygen species cause peroxidation of cell membranes in various plants in response to abiotic stress treatments (Mazzucotelli et al., 2008; Renault et al., 2010; Zhang et al., 2011; AL-Quraan et al., 2012). Such data are confirmed by the extensive losses of agricultural production worldwide caused by abiotic stress conditions (Boyer, 1982; Bray et al., 2000). Thomas et al. (1995) reported that NaCl and mannitol can penetrate the plant cell and contribute to the decrease of internal osmotic potential during seed germination, allowing the water uptake to occur. Almansouri et al. (2001) showed that salttreated isolated embryos of durum wheat (Triticum durum Desf.) exhibited a lower germination percentage than whole seeds, which suggests the inhibition of germination may occur through direct effect of stress on germinating embryos. Munns (2002) reported that salinity causes a reduction in the growth rate of plants, as it impairs water uptake by plant.
Oxidative damage in wheat seedling in response to salt and osmotic stress The MDA level in the Hurani 75 cultivar was the lowest among the wheat cultivars that were used in this study, as shown in Fig. 2. This finding indicates low accumulation of ROS (reactive oxygen species) in Hurani 75 shoot tissues under the NaCl, sorbitol and mannitol treatments. This is due to physiological adaptation of Hurani 75 to salt and osmotic stress by other mechanisms that might encounter the accumulation of ROS in plant tissues. MDA accumulation in Nodsieh shoot tissues was moderately low under all stress treatments, which was correlated (r = 0.583) with seed sensitivity of the same cultivar under all treatments (Fig. 1). In Um Qayes, the accumulation of MDA was moderate compared to other cultivars, which might be producing ROS adapt to the stress, which correlated with moderate levels of GABA accumulation (GABA r = 0.565, MDA r = 0.401) (Fig. 3). Overall, Um Qayes cultivar was more tolerant to stress than Sham I, which showed the highest level of MDA accumulation, an indication of oxidative damage and impaired plant growth in response to salt and osmotic stress. In a previous study, MDA accumulation took place in plants under stress conditions due to membrane lipid peroxidation (Heath
500 Acsad 65
GABA Level nmol/mg FW
450
Hurani 75
400
Nodsieh
350
Um Qayes Sham I
300 250 200 150 100 50 0
Concentraon (mM) Fig. 3. The level of GABA (␥-aminobutyric acid) metabolite in10-day old seedlings shoot tissues of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) exposed to NaCl, mannitol and sorbitol separately. GABA was extracted and determined as described in the ‘Materials and methods’. The level of GABA was determined as nmol/mg FW.
Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010
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and Packer, 1968). The growth reduction effects of salt stress have also been reported in different maize cultivars at different growth stages (Hichem et al., 2009). Furthermore, growth-reducing effects of different saline regimes in hydroponic culture systems were also observed by Chen et al. (2007). Accumulation of MDA contents coupled with reduced plant growth under salt stress was also reported by Koca et al. (2007) and Li (2009) in tomato and sesame seedlings. AL-Quraan et al. (2011, 2012) found that Arabidopsis thaliana root and shoot tissues accumulate MDA metabolites under various treatments of NaCl, mannitol, hydrogen peroxide and paraquat. GABA (-aminobutyrate) level under salt and osmotic stress in wheat Our study showed that, in the Acsad 65 wheat cultivar, there was a drastic increase (P = 0.028) of the GABA metabolite under 100 mM NaCl and 200 mM sorbitol, and a minor increase under 200 mM mannitol. This indicates that the Acsad 65 cultivar tolerates salt and osmotic stress by sorbitol under higher concentrations through the accumulation of GABA more than under osmotic agents induced by mannitol (Fig. 3). The Hurani 75 wheat cultivar accumulated nearly the same level of GABA as in Acsad 65 under 100 mM NaCl and 200 mM sorbitol. A small quantity of GABA was detected at higher concentrations of NaCl and sorbitol. This may suggest that GABA accumulation level is correlated with a destructive effect of NaCl and sorbitol (r = 0.429), while mannitol had minor effects on inducing the GABA shunt through lower GABA accumulation levels. The highest level of GABA accumulation occurred in the shoot tissues of the Nodsieh cultivar under 75 mM NaCl and 200 mM sorbitol, which suggests a higher tolerance of the Nodsieh cultivar to NaCl treatments. In contrast, the Um Qayes and Sham I cultivars showed higher levels of GABA accumulation under 100 and 200 mM mannitol, respectively, and a drastic decrease in GABA levels under 300 mM mannitol (Sham I P = 0.016, Um Qayes P = 0.042) (Fig. 3). This could be due to the lethal effects of osmotic stress induced by mannitol on this cultivar. The GABA shunt was first reported in plants more than half a century ago in potato (Solanum tuberosum) tubers (Dent et al., 1947). Previous studies showed that the levels of GABA in plants also tend to be elevated during high stress conditions such as hypoxia, darkness, drought and salt stress (Shelp et al., 1999; Bouche and Fromm, 2004; Akc¸ay et al., 2012). In agreement with our results, previous studies have suggested that GABA was the only non-amino acid out of three intermediates of the GABA shunt that increased in response to osmotic stress in wheat (Rentsch et al., 1996). In agreement with our data, AL-Quraan et al. (2012) showed that increased levels of GABA shunt metabolites in response to cold and heat treatments occurred in some Arabidopsis calmodulin mutants. This is correlated with activation of GAD after exposure to temperature stress in response to the intracellular damage and low cytoplasmic pH. In addition, AL-Quraan et al. (in press) reported that high accumulation of GABA metabolite occurred under salt and osmotic stress. GABA shunt metabolites may have a role in osmoregulation and signaling in response to salinity and non-ionic osmotic stress. Bartyzel et al. (2003) showed that GABA increased significantly in wheat seedlings after exposure to 20% polyethylene glycol 6000 for 28 h as an osmotic stress agent. Akc¸ay et al. (2012) found that the cytoplasmic male sterility (CMSII) mutant and WT of Nicotiana sylvestris plants showed better growth under long term salinity, which is related to GABA accumulation and metabolism. Chlorophyll contents reduction under salt and osmotic treatments The highest percentage of Chl a was observed in the Sham I wheat cultivar under 100 mM mannitol treatment. The percentage of Chl a was correlated with high levels of GABA under the same
5
Table 1 Chlorophyll pigment (Chl a) level (nm/mg FW) of 10-day old seedlings shoot tissues of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) exposed to NaCl, mannitol and sorbitol separately. Chlorophyll pigments were extracted and determined according to Jeffrey and Humphrey (1975). [mM]
Acsad 65
Hurani 75
Nodsieh
Um Qayes
Sham I
Control NaCl 25 NaCl 50 NaCl 75 NaCl 100 NaCl 200 Mannitol 50 Mannitol 100 Mannitol 150 Mannitol 200 Mannitol 300 Sorbitol 50 Sorbitol 100 Sorbitol 150 Sorbitol 200 Sorbitol 300
7.05 6.21 2.57 2.13 1.93 0.91 3.52 3.33 3.08 2.39 2.1 2.43 2.22 2 1.45 1.19
8.44 6.22 6.15 5.81 5.74 2.53 10.3 5.57 5.03 4.73 2.59 9.49 7.43 4.57 2.85 1.6
5.72 3.21 3.12 2.86 2.82 2.72 5.12 3.39 2.15 2.15 1.59 4.59 4.37 3.56 2.69 2.62
12.09 10.02 9.62 8.85 5.71 2.26 14.59 9.56 8.82 7.37 7.15 15.05 8.47 7.87 5.6 5.48
8.49 7.09 4.97 2.76 2.7 2.07 1.94 1.7 1.33 1.16 1.06 2.45 2.39 2.29 1.91 1.36
treatment (Table 1). In the Nodsieh cultivar, a correlation (chl a r = −0.409, chl b r = −0.416) was found between the level of GABA accumulation and chlorophyll contents under 75 mM NaCl, which indicated a lethal effect of high NaCl concentration on the Nodsieh cultivar (Tables 1 and 2). In the Hurani 75 cultivar, a higher level of GABA accumulation and chlorophyll b in shoot tissue was observed under the 100 mM NaCl treatment. The Acsad 65 cultivar showed a link between the GABA accumulation and chl b content under the 200 mM mannitol treatment (r = −0.523) (Table 2). Our data suggested that GABA metabolite accumulation had various impacts on plant metabolism under salt and osmotic stresses. This is an indication that the GABA level may act as a signaling molecule or osmoprotectant in various cultivars under abiotic stress. Akc¸ay et al. (2012) showed that the photosynthetic efficiency of the N. sylvestris mutant and WT plants decreased in response to the 100 mM NaCl treatment. Sairam et al. (2002) found that relative water content, chlorophyll, carotenoids, membrane stability index, biomass and grain yield, proline, glycine-betaine, soluble sugars, superoxide dismutase, catalase and glutathione reductase activity were impaired and deteriorated in Kharchia 65 (tolerant) and KRL 19 (moderately tolerant) wheat cultivars at all stages of growth and development in response to long term soil salinity treatments.
Table 2 Chlorophyll pigment (Chl b) level (nm/mg FW) of 10-day old seedlings shoot tissues of five wheat cultivars (Acsad 65, Hurani 75, Nodsieh, Um Qayes, and Sham I) exposed to NaCl, mannitol and sorbitol separately. Chlorophyll pigments were extracted and determined according to Jeffrey and Humphrey (1975). [mM]
Acsad 65
Hurani 75
Nodsieh
Um Qayes
Sham I
Control NaCl 25 NaCl 50 NaCl 75 NaCl 100 NaCl 200 Mannitol 50 Mannitol 100 Mannitol 150 Mannitol 200 Mannitol 300 Sorbitol 50 Sorbitol 100 Sorbitol 150 Sorbitol 200 Sorbitol 300
4.36 2.6 1.79 1.38 0.89 0.56 1.87 1.85 1.52 1.32 1.29 1.92 1.37 1.28 1.23 1.2
3.42 2.3 1.95 1.79 1.61 1.6 2.81 1.85 1.67 1.59 1.03 2.46 2.22 1.51 0.87 0.57
2.05 1.57 1.06 1.03 1.01 0.74 1.78 1.48 1.33 0.87 0.85 1.3 1.2 1.15 0.79 0.62
6.12 3.95 3.68 2.06 1.28 0.53 5.91 5.33 4.96 4.72 2.44 4.16 3.8 3.7 2.4 1.29
4.54 2.55 2.26 2.12 2.01 1.54 1.53 1.34 1.27 1.14 1.13 3.18 2.34 2.1 1.89 1.45
Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010
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Wheat GAD RNA transcripts under salt and osmotic stress Glutamate decarboxylase (GAD) RNA transcripts of wheat were examined in this study under all treatments (Fig. 4). GAD catalyzes the decarboxylation of glutamate to CO2 and ␥-aminobutyrate (GABA) (Fait et al., 2007). Our results showed an association between GAD RNA transcription and the response to osmotic and salt stress in terms of seed germination and GABA and MDA accumulation. The transcription of GAD in terms of RNA levels in the five wheat cultivars was increased under different concentrations of osmotic and salt treatments (Fig. 4). There was high GAD expression and GABA accumulation in the Hurani 75 cultivar under salt stress. In contrast, GAD expression and GABA accumulation were relatively low under osmotic stress when compared to the control and other cultivars under all treatments (Fig. 4). These results suggested that osmotic reagents (sorbitol and mannitol) may have lethal effects on the Hurani 75 cultivar, especially at higher concentrations. It was shown by Mazzucotelli et al. (2008) that post-transcriptional and post-translational mechanisms regulate molecular responses to abiotic stresses, based on alternative splicing and RNA processing, as well as RNA silencing. There was relatively high GAD expression and GABA accumulation under salt and osmotic treatment in the Um Qayes cultivar. The highest GAD expression was observed under mannitol (Fig. 4C). This suggests that the GABA shunt was activated under salt and osmotic stress in terms of metabolite accumulation and GAD expression. In agreement with our results, Akc¸ay et al. (2012) showed that GAD activity rapidly increased in response to NaCl treatments, which was related to an increase in GAD gene expression in the cytoplasmic male sterility (CMSII) mutant and WT of N. sylvestris plants. GAD expression, GABA accumulation and seed germination were low under sorbitol treatments in Sham I (Fig. 4D) and Acsad 65 (Fig. 4F) cultivars. This suggests that sorbitol had a destructive effect on sham I and Acsad 65 shoot tissues, in contrast to high GAD expression under NaCl and mannitol (Fig. 4). High GAD expression under salt and osmotic stress was recorded in the Nodsieh cultivar (Fig. 4E). However, under higher concentrations of sorbitol, GAD expression was very low. This might be due to the destructive effect of sorbitol on the expression machinery at high concentrations, which was also accompanied by low seed germination in the Nosdieh cultivar under the same sorbitol treatment. In contrast, Bartyzel et al. (2003) showed that GAD activity in wheat seedlings did not change when exposed to 20% polyethylene glycol 6000 for 28 h as an osmotic stress agent compared with the control plants. In conclusion, our study showed that abiotic stress is a major limiting agent in plant growth and germination. There was a marked decrease in the germination percentage under salt and osmotic stress compared to the control (no treatment) among the five wheat cultivars (Hurani 75, Sham I, Acsad 65, Um Qayes, and Nodsieh). Also, there was a correlation between the GABA concentration and germination percentage of the five wheat cultivars (i.e. drastic increase of GABA metabolite and MDA level under salt and osmotic stress). A decrease in chlorophyll a and b levels under salt and osmotic reagent treatment was observed. The transcription of GAD in terms of the RNA level increased under different concentrations of osmotic and salt treatments in the five wheat cultivars.
Fig. 4. Glutamate decarboxylase (GAD) RNA transcripts of 10-day old seedlings shoot tissues of five wheat cultivars. (A) Control, (B) Hurani 75, (C) Um Qayes, (D) Sham I, (E) Nodsieh, and (F) Acsad 65 exposed to NaCl, mannitol and sorbitol separately. (J) Internal control primer (CBP20).
Acknowledgments We thank our colleague Dr. Rami AL-Khatib for helpful suggestions and reading of the manuscript. This work was supported by grant number 97/2011 from the Deanship of Research, Jordan University of Science and Technology, Jordan.
Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010
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Please cite this article in press as: AL-Quraan NA, et al. Characterization of ␥-aminobutyric acid metabolism and oxidative damage in wheat (Triticum aestivum L.) seedlings under salt and osmotic stress. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.02.010