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Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2
Scientia Horticulturae 119 (2009) 163–168
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Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2 M. Ozden a,*, U. Demirel b, A. Kahraman b a b
Harran University, Faculty of Agriculture, Department of Horticulture, 63040 Sanlıurfa, Turkey Harran University, Faculty of Agriculture, Department of Agronomy, 63040 Sanlıurfa, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 June 2008 Received in revised form 22 July 2008 Accepted 22 July 2008
Although proline is one of the major computable organic solutes that accumulate in many plant species in abiotic stresses, a hot debate continues about whether proline accumulation is a reaction to abiotic stress, or a plant’s response is associated with stress tolerance. The effects of proline on antioxidative system in ¨ ku¨zgo¨zu¨’ exposed to oxidative stress by H2O2 was investigated. grape leaves of Vitis vinifera L. cv., ‘O Endogenous proline, hydrogen peroxide (H2O2), malondialdehyde (MDA) concentrations, percentage of electrolyte leakage (EL), and some of the antioxidant enzyme activities; such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) were measured spectrophotometrically. Inhibitory effect of H2O2 on antioxidant enzyme activities, MDA, and EL was found. In the presence of proline, SOD and CAT activities decreased, while POD and APX activities increased. Proline pre-treatment resulted in a decrease in cellular H2O2 content, MDA, and EL, while cellular concentration of proline increased. Based on the finding, it was suggested that proline and H2O2 could play an important role in oxidative stress injury of grapevine leaves grown in vitro culture. Also, proline might have a direct positive effect on antioxidant enzyme system, membrane phase change, MDA, and EL. Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved.
Keywords: Oxidative stress Proline Antioxidant enzymes Lipid peroxidation Electrolyte leakage
1. Introduction Drought, salinity, extreme temperatures, and nutrient imbalances are among the major environmental stresses to crop productivity worldwide (Ashraf and Fooland, 2007). Under optimal growth conditions, reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide (O2 ) and hydroxyl radical (OH ) are continuously produced at low levels mainly in chloroplasts, peroxisomes, and mitochondria of plant cells. The balance between production and removal of ROS are tightly controlled by the antioxidant systems (Apel and Hirt, 2004). However, under severe environmental stress conditions, the reductive enzymatic pathway in plant tissues may be overwhelmed, resulting in oxidative damage to the cell components including chlorophyll, membrane lipids, proteins and DNA, leading finally cell death (Bowler et al., 1992; Dat et al., 2000; Molassiotis et al., 2006). The antioxidant systems consist of three general classes: (a) lipid soluble, membrane associated antioxidants, (b)
* Corresponding author. Tel.: +90 4142470386; fax: +90 4142474480. E-mail address: [email protected] (M. Ozden).
water-soluble reductants, and (c) enzymatic antioxidants such as superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and guaiacol peroxidase (POD; EC 1.11.1.7). SOD, the first enzyme in the detoxifying process, converts O2 radicals to H2O2 and O2. CAT, APX, and non-specific POD scavenge the accumulated H2O2 to nontoxic levels or form water and oxygen (Asada, 1992; Scandolios, 1993; Zhu et al., 2004). H2O2 being the most stable form of the reactive oxygen species can move into cell membrane, and initiate oxidative damage in leaf cells resulting in disruption of metabolic function and loss of cellular integrity. Also, H2O2 changes the redox status of the surrounding cells and gives an antioxidative response by acting as a signal of oxidative stress (Lin and Kao, 1998; Sairam and Srivastava, 2000). One of the common responses of many plant species exposed to different abiotic stresses is the accumulation of compatible organic solutes such as proline, glycine betaine, choline, and O-sulfate (Rhodes and Hanson, 1993; Serraj and Sinclair, 2002). Proline is an amino acid that is a highly soluble, non-toxic, and has a low molecular weight (Ashraf and Fooland, 2007). Proline accumulation has been shown in different abiotic stressed plants. It has been suggested that proline protects plants by functioning as a cellular osmotic regulator between cytoplasm
0304-4238/$ – see front matter . Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.07.031
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and vacuole, and by detoxifying of ROS, thus protecting membrane integrity and stabilizing antioxidant enzymes (Sharp et al., 1990; Bandurska, 1993; Bohnert and Jensen, 1996). Although much effort has been devoted to genetically engineered plants for over production of various osmoprotectants including proline and glycinebetaine, there has been little success in achieving the desired protective levels of these osmolytes in plants. Alternatively, the tolerance level of some plants grown under stress conditions has been increased by exogenous application of various organic solutes. Although this approach might be of great importance for increasing crop production under stress conditions, there are no sufficient data relevant to exogenous application of proline under oxidative stress conditions in the literature. Moreover, proline accumulation has been shown in many plant species grown under stress conditions, the actual role of proline accumulation as well as its physiological importance still remains unclear and a hot debate continues to find the answer whether proline accumulation is a reaction to abiotic stress, or a plant’s response is associated with stress tolerance. Therefore, this study was undertaken to investigate the impacts of exogenous proline on: 1. changes in the activities of antioxidative enzymes including SOD, CAT, POD, and APX; 2. endogenous proline, H2O2, MDA concentrations, and percentage ¨ ku¨zgo¨zu¨’ of cellular EL in grapevine leaves of Vitis vinifera L. cv. ‘O under oxidative stress by H2O2. ¨ ku¨zgo¨zu¨ cultivar is one of the commonly grown grape O cultivars and it is especially utilized for making wine in SouthEastern Anatolia region of Turkey. 2. Materials and methods 2.1. Plant material and stress treatments ¨ ku¨zgo¨zu¨’ used for the Grapevine leaves of Vitis vinifera L. cv., ‘O study were taken from shoots grown in vitro culture. Hardwood ¨ ku¨zgo¨zu¨’) with three nodes, were cuttings (Vitis vinifera L. cv., ‘O obtained at the dormant bud stage from the vineyard of Gaziantep Pistachio Research Institute and were cultured in a growth chamber at 24 1 8C, 60% humidity and 16 h photoperiod. When these plants were more than 2 months old, nodal explants containing a single auxiliary bud were made from the main shoots. The nodal explants (0.5–1 cm), were disinfested in 10% Clorox (0.525% NaOCl) for 15 min and then rinsed three times with sterile distilled water for 5 min each. Thereafter, explants were transferred to MagentaTM GA7 culture vessels (67 mm 67 mm 97 mm h), each containing five explants and 80 ml of shoot induction medium. Shoot induction medium comprised of Murashige and Skoog (MS) (Murashige and Skoog, 1962) supplemented with 1 mg/l of 6-benzyl aminopurine (BAP), 40.53 mg/l adenine sulphate, 218.4 mg/l monobasic sodium phosphate, 3% sucrose, and 0.6% agar. The pH was adjusted to 5.8 prior to autoclaving (121 8C, 1 kg cm 2 for 20 min). The cultures were maintained at 24 1 8C and 16 h photoperiod (150 mmol m 2 s 1) from fluorescent tubes. After 60 days of culture, the 2nd to the 6th fully expanded uniform leaves of the shoots were excised from leaf petiole and used for treatments. Grapevine leaf samples were taken and divided into four groups with three replications, then submerged in a beaker containing 250 ml of the solutions with or without 10 mM of H2O2 and 20 mM of proline (Pro) for different incubation periods. Incubation was carried out in a growth camber at 24 1 8C, and on an orbital rotating plate (60 rpm) in light (150 mmol m 2 s 1).
Oxidative stress treatments were as follows: T1 = incubation in sterile dH2O for 24 h (control); T2 = incubation in H2O2 solution for 6 h (H2O2(6h)); T3 = incubation in H2O2 solution with proline for 6 h (Pro + H2O2(6h)); T4 = pre-incubation in proline solution for 18 h followed by incubation in H2O2 solution for 6 h (Pro(18h) + H2O2(6h)). Subsequently, treated leaves were washed with double dH2O, frozen in liquid N2, and stored at 84 8C until use for the analyses. 2.2. Determination of endogenous proline, H2O2, MDA concentrations, and electrolyte leakage (EL) Proline content was determined according to the modified method of Bates et al. (1973). Proline was extracted from 0.3 g of leaf samples by grinding in 10 ml of 3% sulphosalicylic acid and the mixture was then centrifuged at 10,000 g for 10 min. Two millilitres of the supernatant was added into test tubes to which 2 ml of freshly prepared acid–ninhydrin solution. Tubes were incubated in a water bath at 90 8C for 30 min. The reaction was terminated in ice bath. The reaction mixture was extracted with 5 ml of toluene and vortexed for 15 s. The tubes were allowed to stand for at least for 20 min in darkness at room temperature to allow the separation of toluene and aqueous phase. The toluene phase was then carefully collected into test tubes and absorbance was measured at 520 nm in a spectrophotometer. The concentration of proline was calculated from a standard curve using the following equation: (mg proline in extract/115.5)/g sample = mmol/g fwt. Hydrogen peroxide (H2O2) concentration was determined according to the method by Loreto and Velikova (2001). Leaf samples of 0.3 g were homogenised in 3 ml of 1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000 g and 4 8C for10 min. Subsequently, 0.75 ml of the supernatant was added to 0.75 ml of 10 mM K-phosphate buffer (pH 7.0) and 1.5 ml of 1 M KI. H2O2 concentration of the supernatant was evaluated by comparing its absorbance at 390 nm to a standard calibration curve. The concentration of H2O2 was calculated from a standard curve plotted in the range from 100 to 1000 mmol/ml. H2O2 concentration was expressed as mmol/g fwt. Lipid peroxidation and electrolyte leakage of the leaf samples were measured to assess membrane damage. Lipid peroxidation was measured as the amount of malondialdehyde (MDA) determined by the TBA reaction as described by Heath and Packer (1968). Leaf samples of 0.3 g were homogenised in 4 ml of 1% (w/v) TCA, then centrifuged at 10,000 g for 10 min. To 1.5 ml of the supernatant aliquot, 1.5 ml of 20% (w/v) TCA containing 0.5% (w/v) TBA, were added. The mixture was heated at 95 8C for 30 min and then quickly cooled in an ice bath. The mixtures were centrifuged at 10,000 g for 5 min and their absorbances were measured at 532 nm. The value for non-specific absorption at 600 nm was subtracted from the 532 nm reading. The MDA content was calculated using its extinction coefficient of 155 mM 1 cm 1 and expressed as mmol/g fwt. Electrolyte leakage (EL) was measured by using a conductivity meter. Samples were cut into equal sized pieces (0.3 g per treatment) and placed in 25 mm 150 mm culture vessels containing 15 ml of distilled water, and shaken at 100 rpm on an orbital shaker for 24 h at room temperature. The initial conductance of the bathing solution was measured using a conductivity meter. The tubes were then autoclaved at 115 8C for 10 min and final readings were taken following autoclaving and
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additional 24 h incubation at room temperature. EL (%) was calculated as initial measurements/final measurements 100. 2.3. Enzyme assays Tissue extraction of the samples were prepared for the analyses by homogenizing 1 g of leaf material in 4 ml of ice cold 50 mM Kphosphate buffer (pH 7.0) containing 2 mM Na–EDTA and 1% (w/v) polyvinyl–polypirrolidone (PVP). The homogenate was centrifuged at 10,000 g and 4 8C for 10 min. Afterwards, tissue extract was stored at 84 8C, or immediately used for subsequent analyses of SOD, CAT, POD, APX, and the soluble protein content determined by the Coomassie blue dye binding method (Bradford, 1976) with bovine serum albumin as a standard curve. SOD activity was determined by measuring the ability of the enzyme to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) in the presence of riboflavin in light (Giannopolitis and Ries, 1977). The reaction mixture (3 ml) contained 50 mM K-phosphate buffer (pH 7.8), 13 mM methionine, 75 mM NBT, 4 mM riboflavin, 0.1 mM EDTA, and 0.25 ml enzyme extract. One unit of enzyme activity was determined as the amount of the enzyme to reach an inhibition of 50% NBT reduction rate by monitoring absorbance at 560 nm with spectrophotometer. The test tubes were shaken then placed in a light box consisting of six 15 W fluorescent lamps for 10 min. Reaction was stopped by switching off the light and placing the test tubes into dark. Activities of CAT and POD were measured by the method of Chance and Maehly (1955). For CAT, the decomposition of H2O2 was followed by decline in absorbance at 240 nm. Three millilitres of reaction mixture contained 50 mM phosphate buffer (pH 7.0), 10 mM H2O2 and 50 ml of enzyme extract. The reaction was initiated by adding enzyme extract (Osswald et al., 1992). CAT activity was determined by following the consumption of H2O2 (extinction coefficient, 39.4 mM 1 cm 1) at 240 nm over a 2 min interval. For POD, the oxidation of guaiacol was measured by the increase in absorbance at 470 nm. The assay mixture contained 0.05 ml of guaiacol (20 mM), 2.9 ml of K-phosphate buffer (10 mM, pH 7.0) and 50 ml of enzyme extract. The reaction was initiated with adding 20 ml of H2O2 (40 mM) (Osswald et al., 1992). POD activity was determined by measuring the oxidation of guaiacol in the presence of H2O2 (extinction coefficient, 26.6 mM 1 cm 1) at 470 nm over a 2 min interval. APX activity (EC 1.11.1.11) was determined by the following the decrease of ascorbate and measuring the change in absorbance at 290 nm for a 2 min interval. The reaction mixture contained 50 mM K-phosphate buffer (pH 7.0), 1 mM EDTA–Na2, 0.5 mM ascorbic acid, 0.1 mM H2O2 and 50 ml of crude enzyme extract (Nakano and Asada, 1981). The activity of ascorbate peroxidase was calculated using the extinction coefficient (2.8 mM 1 cm 1). 2.4. Statistical analysis Data presented are mean values S.E.M. for three replicates. The significance of differences was determined LSD using GLM procedure and p 0.05 as significant in SAS (SAS Institute, 1995).
Fig. 1. (A–D) The endogenous proline (A), H2O2 (B), MDA (C), concentrations, and ¨ ku¨zgo¨zu¨’ leaves treated with dH2O, percentage of EL (D) of Vitis vinifera L. cv. ‘O H2O2, proline + H2O2, or pre-treated with proline prior to exposure to oxidative stress by H2O2. Data represented are means of three separate experiments S.E.M. The letters on the top of the bars, which are the same, indicate that there is no statistically significant difference at p < 0.05 level based on LSD test.
3. Results 3.1. Endogenous proline, H2O2, MDA concentrations, and percentage of electrolyte leakage (EL) The effects of oxidative stress and exogenous proline application on endogenous proline, H2O2, MDA concentrations, and percentage of EL in grapevine leaves are shown in Fig. 1A–D. While the concentration of proline was 0.0073 mmol/g fwt under the
condition of no stress, it increased to 0.021 mmol/g fwt when a 6 h of oxidative stress by H2O2 was applied. However, endogenous proline concentration of samples increased to 0.242 mmol/g fwt, after a 6 h of treatment with H2O2 + proline. Pre-incubation of samples in proline for 18 h, followed by subjecting to H2O2 treatment for 6 h resulted in a drastic increase (0.45 mmol/g fwt) of endogenous proline content (Fig. 1A). The endogenous H2O2 content of sample was 20.12 mmol/g fwt under no stress condition.
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On the contrary, it sharply increased to 139.84 mmol/g fwt after a 6 h H2O2 treatment. The addition of proline into the oxidative stress solution caused a decrease in endogenous H2O2 concentration to level of 60.16 mmol/g fwt after a 6 h treatment. Almost similar endogenous proline concentration was observed in leaves (21.57 mmol/g fwt) pre-treated with proline when compared to the control (Fig. 1B). Lipid peroxidation levels of samples, measured as the concentration of MDA, are given in Fig. 1C. The endogenous MDA content of the sample was 10.73 mmol/g fwt when no stress was applied. The results showed that MDA level increased with excessive oxidative stress when compared to control. The highest MDA content (21.89 mmol/g fwt) was obtained from grapevine leaves exposed to oxidative stress without proline. However, significant decreases in MDA content of samples treated with proline + H2O2 were observed. Before exposure to oxidation stress, pre-incubation of leaves with proline reduced MDA content. On the other hand, lipid peroxidation was negligible in excised grape leaves with pre-treated proline compared to control (Fig. 1C). Membrane permeability, estimated as electrolyte leakage (EL), was measured by using a conductivity meter. EL increased significantly under oxidative stress conditions compared to control. While the highest EL was observed under oxidative stress conditions without proline application, the lowest EL was obtained from the control treated with water for 24 h. EL of the samples treated with proline + H2O2 for 6 h was found to be 17.86% injury which is a lower injury rate than that of H2O2 stressed leaves alone (31.12%). The data showed clearly that exogenous proline application under the assay condition significantly lowered electrolyte leakage indicating percentage of cell injury (Fig. 1D). 3.2. Antioxidant enzymes Responses of SOD and CAT enzymes to the different oxidative stress treatments were the opposite of responses of POD and APX under the assay conditions (Fig. 2A–D). Treatment of leaf samples with H2O2 for 6 h drastically increased SOD activity which was 235% of the control value, whereas treatment with proline + H2O2 for 6 h significantly decreased its activity compared to H2O2 treatment alone. Pre-treatment with proline before H2O2 stress continued to decrease SOD activity compared to treatment with both proline + H2O2 and H2O2 treatment alone. Pre-treatment of proline was found to have no significant effect on SOD enzyme activity compared with H2O2 + proline treatment (Fig. 2A). Responses of CAT enzyme to different oxidative stress treatments were similar to SOD activity. The only difference in response of CAT and SOD activity to oxidative stress treatments was that not being a significant difference between CAT activity in control and proline + H2O2 treated leaves (Fig. 2B). Responses of POD and APX to the oxidative stress treatments applied in the assay conditions were completely contrary to the responses of SOD and CAT enzymes (Fig. 2C–D). Leaf samples stressed with H2O2 for 6 h caused to drastic decrease in POD activity which was 33% of control, whereas leaves treated with proline + H2O2 for 6 h significantly enhanced its activity compared to H2O2 treatment alone. Pre-treatment of proline continued to increase in POD activity compared to proline + H2O2 and H2O2 treatments alone. Pre-treatment of proline were found to have no significant effect on POD enzyme activity compared to H2O2 + proline treatments for 6 h (Fig. 2C). The pattern of APX activity against to oxidative stress treatments was similar to POD activity. However, proline + H2O2 treatment was found to have no significant effect on APX compared to H2O2 treatment alone. Pre-treatment of proline caused an increase in APX activity as higher as control (Fig. 2D).
Fig. 2. (A–D) The activity of antioxidant enzymes; SOD (A), CAT (B), POD (C), APX (D) ¨ ku¨zgo¨zu¨’ leaves treated with dH2O, H2O2, proline + H2O2, or in Vitis vinifera L. cv. ‘O pre-treated with proline prior to exposure to oxidative stress by H2O2. Data represented are means of three separate experiments S.E.M. The letters on the top of the bars, which are the same, indicate that there is no statistically significant difference at p < 0.05 level based on LSD test.
4. Discussion Cellular content of MDA, H2O2, and level of EL reflect cellular damage resulting from oxidative stress (Dhindsa et al., 1981). Results of the study showed that treatment of samples with H2O2 caused to an increase in endogenous proline accumulation, MDA, and H2O2 concentrations, and percentage of EL as reported earlier
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in different plant species (Lin and Kao, 1998; Ehsanpour and Fatahian, 2003; Woodward and Bennett, 2005; Sairam et al., 2005; Upadhyaya et al., 2007). The slight increase in proline concentration in H2O2-treated leaf samples may be attributed to the osmoprotectant function of proline as indicated in earlier studies by Matysik et al. (2002), and Upadhyaya et al. (2007). In our experiment, proline content of the leaves increased in relation to the incubation term of proline. The longer incubation of samples with proline, the higher proline content was observed in the treated leaf samples. The concentrations of H2O2, MDA, and percentage of cellular membrane damage were significantly reduced in the leaves of higher proline content. This result showed that when proline was exogenously supplied prior to H2O2 treatment, diffusion of H2O2 into the cells was significantly reduced. These data suggest that proline may exert some action on the cell surface and that one function of accumulated proline is to reduce the diffusion of H2O2 into the grape leaf cells under the assay conditions. Similar phenomenon regarding exogenous proline application was observed in chlorella sp. (chlorophyceae) cells exposed to toxic copper concentrations (Wu, 1998). Proline loaded cells significantly reduced Cu uptake into the cells. Generally, there is a strong positive relationship between stress tolerance and proline accumulation in higher plants (Ashraf and Fooland, 2007). However, more studies should be conducted to determine whether the relationship between stress tolerance and proline accumulation is genotype-specific or it can be changed by changing working conditions. Under normal growth conditions, the antioxidant system is usually sufficient to prevent oxidative damage. On the contrary, enzymatic or non-enzymatic defense system of plants has been activated under stress conditions (Hideg, 1997). SOD, CAT, POD, and APX are the antioxidant enzymes which are increased in their activities under stress conditions, and several cases correlate well with tolerance. Data suggest that the relationship between exogenous H2O2 or proline application and antioxidant enzyme activities is not simple, because responses of SOD and CAT enzymes to H2O2 treatments were just contrary to POD and APX activities. In this study, both SOD and CAT activities of H2O2-treated leaves increased in compared to the control. However, exogenous application of proline caused a decrease in SOD and CAT activities compared to H2O2-treated leaves. In addition, SOD and CAT activities in proline-treated leaves were not significant difference from those in the controls. Unlike SOD and CAT, POD and APX were found to be more sensitive in their response to oxidative stress. The decrease in APX and POD activities might be due to the generation of high level of oxidative stress and possible inactivation of APX and POD activities. Moreover, inhibition of POD and APX activities could be overwhelmed for detoxification of H2O2. Thus, it can be assumed that a decrease in H2O2 content of the cells resulted in the activation of POD and APX. These results indicate that pre-treatment of grape leaves with proline, did not eliminate the inhibitory effect of H2O2 on POD and APX activities but also increased their activities compared to H2O2 treatment. The results of our study indicated that external application of proline counteracted the inhibitory effect of H2O2 on all parameters measured, help the grapevine leaves to avoid oxidative stress by acting as stress protecting compound. Therefore, the results make it possible to recommend ¨ ku¨zgo¨zu¨ grapevine cultivar with proline exogenous treatment of O under oxidative stress condition. 5. Conclusion Based on the results, it may be concluded that H2O2 played direct role in oxidative stress injury to grapevine leaves, also the mechanism by proline reduced oxidative stress in grape leaves was
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2 M. Ozden a,*, U. Demirel b, A. Kahraman b a b
Harran University, Faculty of Agriculture, Department of Horticulture, 63040 Sanlıurfa, Turkey Harran University, Faculty of Agriculture, Department of Agronomy, 63040 Sanlıurfa, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 June 2008 Received in revised form 22 July 2008 Accepted 22 July 2008
Although proline is one of the major computable organic solutes that accumulate in many plant species in abiotic stresses, a hot debate continues about whether proline accumulation is a reaction to abiotic stress, or a plant’s response is associated with stress tolerance. The effects of proline on antioxidative system in ¨ ku¨zgo¨zu¨’ exposed to oxidative stress by H2O2 was investigated. grape leaves of Vitis vinifera L. cv., ‘O Endogenous proline, hydrogen peroxide (H2O2), malondialdehyde (MDA) concentrations, percentage of electrolyte leakage (EL), and some of the antioxidant enzyme activities; such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) were measured spectrophotometrically. Inhibitory effect of H2O2 on antioxidant enzyme activities, MDA, and EL was found. In the presence of proline, SOD and CAT activities decreased, while POD and APX activities increased. Proline pre-treatment resulted in a decrease in cellular H2O2 content, MDA, and EL, while cellular concentration of proline increased. Based on the finding, it was suggested that proline and H2O2 could play an important role in oxidative stress injury of grapevine leaves grown in vitro culture. Also, proline might have a direct positive effect on antioxidant enzyme system, membrane phase change, MDA, and EL. Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved.
Keywords: Oxidative stress Proline Antioxidant enzymes Lipid peroxidation Electrolyte leakage
1. Introduction Drought, salinity, extreme temperatures, and nutrient imbalances are among the major environmental stresses to crop productivity worldwide (Ashraf and Fooland, 2007). Under optimal growth conditions, reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide (O2 ) and hydroxyl radical (OH ) are continuously produced at low levels mainly in chloroplasts, peroxisomes, and mitochondria of plant cells. The balance between production and removal of ROS are tightly controlled by the antioxidant systems (Apel and Hirt, 2004). However, under severe environmental stress conditions, the reductive enzymatic pathway in plant tissues may be overwhelmed, resulting in oxidative damage to the cell components including chlorophyll, membrane lipids, proteins and DNA, leading finally cell death (Bowler et al., 1992; Dat et al., 2000; Molassiotis et al., 2006). The antioxidant systems consist of three general classes: (a) lipid soluble, membrane associated antioxidants, (b)
* Corresponding author. Tel.: +90 4142470386; fax: +90 4142474480. E-mail address: [email protected] (M. Ozden).
water-soluble reductants, and (c) enzymatic antioxidants such as superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and guaiacol peroxidase (POD; EC 1.11.1.7). SOD, the first enzyme in the detoxifying process, converts O2 radicals to H2O2 and O2. CAT, APX, and non-specific POD scavenge the accumulated H2O2 to nontoxic levels or form water and oxygen (Asada, 1992; Scandolios, 1993; Zhu et al., 2004). H2O2 being the most stable form of the reactive oxygen species can move into cell membrane, and initiate oxidative damage in leaf cells resulting in disruption of metabolic function and loss of cellular integrity. Also, H2O2 changes the redox status of the surrounding cells and gives an antioxidative response by acting as a signal of oxidative stress (Lin and Kao, 1998; Sairam and Srivastava, 2000). One of the common responses of many plant species exposed to different abiotic stresses is the accumulation of compatible organic solutes such as proline, glycine betaine, choline, and O-sulfate (Rhodes and Hanson, 1993; Serraj and Sinclair, 2002). Proline is an amino acid that is a highly soluble, non-toxic, and has a low molecular weight (Ashraf and Fooland, 2007). Proline accumulation has been shown in different abiotic stressed plants. It has been suggested that proline protects plants by functioning as a cellular osmotic regulator between cytoplasm
0304-4238/$ – see front matter . Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.07.031
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and vacuole, and by detoxifying of ROS, thus protecting membrane integrity and stabilizing antioxidant enzymes (Sharp et al., 1990; Bandurska, 1993; Bohnert and Jensen, 1996). Although much effort has been devoted to genetically engineered plants for over production of various osmoprotectants including proline and glycinebetaine, there has been little success in achieving the desired protective levels of these osmolytes in plants. Alternatively, the tolerance level of some plants grown under stress conditions has been increased by exogenous application of various organic solutes. Although this approach might be of great importance for increasing crop production under stress conditions, there are no sufficient data relevant to exogenous application of proline under oxidative stress conditions in the literature. Moreover, proline accumulation has been shown in many plant species grown under stress conditions, the actual role of proline accumulation as well as its physiological importance still remains unclear and a hot debate continues to find the answer whether proline accumulation is a reaction to abiotic stress, or a plant’s response is associated with stress tolerance. Therefore, this study was undertaken to investigate the impacts of exogenous proline on: 1. changes in the activities of antioxidative enzymes including SOD, CAT, POD, and APX; 2. endogenous proline, H2O2, MDA concentrations, and percentage ¨ ku¨zgo¨zu¨’ of cellular EL in grapevine leaves of Vitis vinifera L. cv. ‘O under oxidative stress by H2O2. ¨ ku¨zgo¨zu¨ cultivar is one of the commonly grown grape O cultivars and it is especially utilized for making wine in SouthEastern Anatolia region of Turkey. 2. Materials and methods 2.1. Plant material and stress treatments ¨ ku¨zgo¨zu¨’ used for the Grapevine leaves of Vitis vinifera L. cv., ‘O study were taken from shoots grown in vitro culture. Hardwood ¨ ku¨zgo¨zu¨’) with three nodes, were cuttings (Vitis vinifera L. cv., ‘O obtained at the dormant bud stage from the vineyard of Gaziantep Pistachio Research Institute and were cultured in a growth chamber at 24 1 8C, 60% humidity and 16 h photoperiod. When these plants were more than 2 months old, nodal explants containing a single auxiliary bud were made from the main shoots. The nodal explants (0.5–1 cm), were disinfested in 10% Clorox (0.525% NaOCl) for 15 min and then rinsed three times with sterile distilled water for 5 min each. Thereafter, explants were transferred to MagentaTM GA7 culture vessels (67 mm 67 mm 97 mm h), each containing five explants and 80 ml of shoot induction medium. Shoot induction medium comprised of Murashige and Skoog (MS) (Murashige and Skoog, 1962) supplemented with 1 mg/l of 6-benzyl aminopurine (BAP), 40.53 mg/l adenine sulphate, 218.4 mg/l monobasic sodium phosphate, 3% sucrose, and 0.6% agar. The pH was adjusted to 5.8 prior to autoclaving (121 8C, 1 kg cm 2 for 20 min). The cultures were maintained at 24 1 8C and 16 h photoperiod (150 mmol m 2 s 1) from fluorescent tubes. After 60 days of culture, the 2nd to the 6th fully expanded uniform leaves of the shoots were excised from leaf petiole and used for treatments. Grapevine leaf samples were taken and divided into four groups with three replications, then submerged in a beaker containing 250 ml of the solutions with or without 10 mM of H2O2 and 20 mM of proline (Pro) for different incubation periods. Incubation was carried out in a growth camber at 24 1 8C, and on an orbital rotating plate (60 rpm) in light (150 mmol m 2 s 1).
Oxidative stress treatments were as follows: T1 = incubation in sterile dH2O for 24 h (control); T2 = incubation in H2O2 solution for 6 h (H2O2(6h)); T3 = incubation in H2O2 solution with proline for 6 h (Pro + H2O2(6h)); T4 = pre-incubation in proline solution for 18 h followed by incubation in H2O2 solution for 6 h (Pro(18h) + H2O2(6h)). Subsequently, treated leaves were washed with double dH2O, frozen in liquid N2, and stored at 84 8C until use for the analyses. 2.2. Determination of endogenous proline, H2O2, MDA concentrations, and electrolyte leakage (EL) Proline content was determined according to the modified method of Bates et al. (1973). Proline was extracted from 0.3 g of leaf samples by grinding in 10 ml of 3% sulphosalicylic acid and the mixture was then centrifuged at 10,000 g for 10 min. Two millilitres of the supernatant was added into test tubes to which 2 ml of freshly prepared acid–ninhydrin solution. Tubes were incubated in a water bath at 90 8C for 30 min. The reaction was terminated in ice bath. The reaction mixture was extracted with 5 ml of toluene and vortexed for 15 s. The tubes were allowed to stand for at least for 20 min in darkness at room temperature to allow the separation of toluene and aqueous phase. The toluene phase was then carefully collected into test tubes and absorbance was measured at 520 nm in a spectrophotometer. The concentration of proline was calculated from a standard curve using the following equation: (mg proline in extract/115.5)/g sample = mmol/g fwt. Hydrogen peroxide (H2O2) concentration was determined according to the method by Loreto and Velikova (2001). Leaf samples of 0.3 g were homogenised in 3 ml of 1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000 g and 4 8C for10 min. Subsequently, 0.75 ml of the supernatant was added to 0.75 ml of 10 mM K-phosphate buffer (pH 7.0) and 1.5 ml of 1 M KI. H2O2 concentration of the supernatant was evaluated by comparing its absorbance at 390 nm to a standard calibration curve. The concentration of H2O2 was calculated from a standard curve plotted in the range from 100 to 1000 mmol/ml. H2O2 concentration was expressed as mmol/g fwt. Lipid peroxidation and electrolyte leakage of the leaf samples were measured to assess membrane damage. Lipid peroxidation was measured as the amount of malondialdehyde (MDA) determined by the TBA reaction as described by Heath and Packer (1968). Leaf samples of 0.3 g were homogenised in 4 ml of 1% (w/v) TCA, then centrifuged at 10,000 g for 10 min. To 1.5 ml of the supernatant aliquot, 1.5 ml of 20% (w/v) TCA containing 0.5% (w/v) TBA, were added. The mixture was heated at 95 8C for 30 min and then quickly cooled in an ice bath. The mixtures were centrifuged at 10,000 g for 5 min and their absorbances were measured at 532 nm. The value for non-specific absorption at 600 nm was subtracted from the 532 nm reading. The MDA content was calculated using its extinction coefficient of 155 mM 1 cm 1 and expressed as mmol/g fwt. Electrolyte leakage (EL) was measured by using a conductivity meter. Samples were cut into equal sized pieces (0.3 g per treatment) and placed in 25 mm 150 mm culture vessels containing 15 ml of distilled water, and shaken at 100 rpm on an orbital shaker for 24 h at room temperature. The initial conductance of the bathing solution was measured using a conductivity meter. The tubes were then autoclaved at 115 8C for 10 min and final readings were taken following autoclaving and
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additional 24 h incubation at room temperature. EL (%) was calculated as initial measurements/final measurements 100. 2.3. Enzyme assays Tissue extraction of the samples were prepared for the analyses by homogenizing 1 g of leaf material in 4 ml of ice cold 50 mM Kphosphate buffer (pH 7.0) containing 2 mM Na–EDTA and 1% (w/v) polyvinyl–polypirrolidone (PVP). The homogenate was centrifuged at 10,000 g and 4 8C for 10 min. Afterwards, tissue extract was stored at 84 8C, or immediately used for subsequent analyses of SOD, CAT, POD, APX, and the soluble protein content determined by the Coomassie blue dye binding method (Bradford, 1976) with bovine serum albumin as a standard curve. SOD activity was determined by measuring the ability of the enzyme to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) in the presence of riboflavin in light (Giannopolitis and Ries, 1977). The reaction mixture (3 ml) contained 50 mM K-phosphate buffer (pH 7.8), 13 mM methionine, 75 mM NBT, 4 mM riboflavin, 0.1 mM EDTA, and 0.25 ml enzyme extract. One unit of enzyme activity was determined as the amount of the enzyme to reach an inhibition of 50% NBT reduction rate by monitoring absorbance at 560 nm with spectrophotometer. The test tubes were shaken then placed in a light box consisting of six 15 W fluorescent lamps for 10 min. Reaction was stopped by switching off the light and placing the test tubes into dark. Activities of CAT and POD were measured by the method of Chance and Maehly (1955). For CAT, the decomposition of H2O2 was followed by decline in absorbance at 240 nm. Three millilitres of reaction mixture contained 50 mM phosphate buffer (pH 7.0), 10 mM H2O2 and 50 ml of enzyme extract. The reaction was initiated by adding enzyme extract (Osswald et al., 1992). CAT activity was determined by following the consumption of H2O2 (extinction coefficient, 39.4 mM 1 cm 1) at 240 nm over a 2 min interval. For POD, the oxidation of guaiacol was measured by the increase in absorbance at 470 nm. The assay mixture contained 0.05 ml of guaiacol (20 mM), 2.9 ml of K-phosphate buffer (10 mM, pH 7.0) and 50 ml of enzyme extract. The reaction was initiated with adding 20 ml of H2O2 (40 mM) (Osswald et al., 1992). POD activity was determined by measuring the oxidation of guaiacol in the presence of H2O2 (extinction coefficient, 26.6 mM 1 cm 1) at 470 nm over a 2 min interval. APX activity (EC 1.11.1.11) was determined by the following the decrease of ascorbate and measuring the change in absorbance at 290 nm for a 2 min interval. The reaction mixture contained 50 mM K-phosphate buffer (pH 7.0), 1 mM EDTA–Na2, 0.5 mM ascorbic acid, 0.1 mM H2O2 and 50 ml of crude enzyme extract (Nakano and Asada, 1981). The activity of ascorbate peroxidase was calculated using the extinction coefficient (2.8 mM 1 cm 1). 2.4. Statistical analysis Data presented are mean values S.E.M. for three replicates. The significance of differences was determined LSD using GLM procedure and p 0.05 as significant in SAS (SAS Institute, 1995).
Fig. 1. (A–D) The endogenous proline (A), H2O2 (B), MDA (C), concentrations, and ¨ ku¨zgo¨zu¨’ leaves treated with dH2O, percentage of EL (D) of Vitis vinifera L. cv. ‘O H2O2, proline + H2O2, or pre-treated with proline prior to exposure to oxidative stress by H2O2. Data represented are means of three separate experiments S.E.M. The letters on the top of the bars, which are the same, indicate that there is no statistically significant difference at p < 0.05 level based on LSD test.
3. Results 3.1. Endogenous proline, H2O2, MDA concentrations, and percentage of electrolyte leakage (EL) The effects of oxidative stress and exogenous proline application on endogenous proline, H2O2, MDA concentrations, and percentage of EL in grapevine leaves are shown in Fig. 1A–D. While the concentration of proline was 0.0073 mmol/g fwt under the
condition of no stress, it increased to 0.021 mmol/g fwt when a 6 h of oxidative stress by H2O2 was applied. However, endogenous proline concentration of samples increased to 0.242 mmol/g fwt, after a 6 h of treatment with H2O2 + proline. Pre-incubation of samples in proline for 18 h, followed by subjecting to H2O2 treatment for 6 h resulted in a drastic increase (0.45 mmol/g fwt) of endogenous proline content (Fig. 1A). The endogenous H2O2 content of sample was 20.12 mmol/g fwt under no stress condition.
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On the contrary, it sharply increased to 139.84 mmol/g fwt after a 6 h H2O2 treatment. The addition of proline into the oxidative stress solution caused a decrease in endogenous H2O2 concentration to level of 60.16 mmol/g fwt after a 6 h treatment. Almost similar endogenous proline concentration was observed in leaves (21.57 mmol/g fwt) pre-treated with proline when compared to the control (Fig. 1B). Lipid peroxidation levels of samples, measured as the concentration of MDA, are given in Fig. 1C. The endogenous MDA content of the sample was 10.73 mmol/g fwt when no stress was applied. The results showed that MDA level increased with excessive oxidative stress when compared to control. The highest MDA content (21.89 mmol/g fwt) was obtained from grapevine leaves exposed to oxidative stress without proline. However, significant decreases in MDA content of samples treated with proline + H2O2 were observed. Before exposure to oxidation stress, pre-incubation of leaves with proline reduced MDA content. On the other hand, lipid peroxidation was negligible in excised grape leaves with pre-treated proline compared to control (Fig. 1C). Membrane permeability, estimated as electrolyte leakage (EL), was measured by using a conductivity meter. EL increased significantly under oxidative stress conditions compared to control. While the highest EL was observed under oxidative stress conditions without proline application, the lowest EL was obtained from the control treated with water for 24 h. EL of the samples treated with proline + H2O2 for 6 h was found to be 17.86% injury which is a lower injury rate than that of H2O2 stressed leaves alone (31.12%). The data showed clearly that exogenous proline application under the assay condition significantly lowered electrolyte leakage indicating percentage of cell injury (Fig. 1D). 3.2. Antioxidant enzymes Responses of SOD and CAT enzymes to the different oxidative stress treatments were the opposite of responses of POD and APX under the assay conditions (Fig. 2A–D). Treatment of leaf samples with H2O2 for 6 h drastically increased SOD activity which was 235% of the control value, whereas treatment with proline + H2O2 for 6 h significantly decreased its activity compared to H2O2 treatment alone. Pre-treatment with proline before H2O2 stress continued to decrease SOD activity compared to treatment with both proline + H2O2 and H2O2 treatment alone. Pre-treatment of proline was found to have no significant effect on SOD enzyme activity compared with H2O2 + proline treatment (Fig. 2A). Responses of CAT enzyme to different oxidative stress treatments were similar to SOD activity. The only difference in response of CAT and SOD activity to oxidative stress treatments was that not being a significant difference between CAT activity in control and proline + H2O2 treated leaves (Fig. 2B). Responses of POD and APX to the oxidative stress treatments applied in the assay conditions were completely contrary to the responses of SOD and CAT enzymes (Fig. 2C–D). Leaf samples stressed with H2O2 for 6 h caused to drastic decrease in POD activity which was 33% of control, whereas leaves treated with proline + H2O2 for 6 h significantly enhanced its activity compared to H2O2 treatment alone. Pre-treatment of proline continued to increase in POD activity compared to proline + H2O2 and H2O2 treatments alone. Pre-treatment of proline were found to have no significant effect on POD enzyme activity compared to H2O2 + proline treatments for 6 h (Fig. 2C). The pattern of APX activity against to oxidative stress treatments was similar to POD activity. However, proline + H2O2 treatment was found to have no significant effect on APX compared to H2O2 treatment alone. Pre-treatment of proline caused an increase in APX activity as higher as control (Fig. 2D).
Fig. 2. (A–D) The activity of antioxidant enzymes; SOD (A), CAT (B), POD (C), APX (D) ¨ ku¨zgo¨zu¨’ leaves treated with dH2O, H2O2, proline + H2O2, or in Vitis vinifera L. cv. ‘O pre-treated with proline prior to exposure to oxidative stress by H2O2. Data represented are means of three separate experiments S.E.M. The letters on the top of the bars, which are the same, indicate that there is no statistically significant difference at p < 0.05 level based on LSD test.
4. Discussion Cellular content of MDA, H2O2, and level of EL reflect cellular damage resulting from oxidative stress (Dhindsa et al., 1981). Results of the study showed that treatment of samples with H2O2 caused to an increase in endogenous proline accumulation, MDA, and H2O2 concentrations, and percentage of EL as reported earlier
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in different plant species (Lin and Kao, 1998; Ehsanpour and Fatahian, 2003; Woodward and Bennett, 2005; Sairam et al., 2005; Upadhyaya et al., 2007). The slight increase in proline concentration in H2O2-treated leaf samples may be attributed to the osmoprotectant function of proline as indicated in earlier studies by Matysik et al. (2002), and Upadhyaya et al. (2007). In our experiment, proline content of the leaves increased in relation to the incubation term of proline. The longer incubation of samples with proline, the higher proline content was observed in the treated leaf samples. The concentrations of H2O2, MDA, and percentage of cellular membrane damage were significantly reduced in the leaves of higher proline content. This result showed that when proline was exogenously supplied prior to H2O2 treatment, diffusion of H2O2 into the cells was significantly reduced. These data suggest that proline may exert some action on the cell surface and that one function of accumulated proline is to reduce the diffusion of H2O2 into the grape leaf cells under the assay conditions. Similar phenomenon regarding exogenous proline application was observed in chlorella sp. (chlorophyceae) cells exposed to toxic copper concentrations (Wu, 1998). Proline loaded cells significantly reduced Cu uptake into the cells. Generally, there is a strong positive relationship between stress tolerance and proline accumulation in higher plants (Ashraf and Fooland, 2007). However, more studies should be conducted to determine whether the relationship between stress tolerance and proline accumulation is genotype-specific or it can be changed by changing working conditions. Under normal growth conditions, the antioxidant system is usually sufficient to prevent oxidative damage. On the contrary, enzymatic or non-enzymatic defense system of plants has been activated under stress conditions (Hideg, 1997). SOD, CAT, POD, and APX are the antioxidant enzymes which are increased in their activities under stress conditions, and several cases correlate well with tolerance. Data suggest that the relationship between exogenous H2O2 or proline application and antioxidant enzyme activities is not simple, because responses of SOD and CAT enzymes to H2O2 treatments were just contrary to POD and APX activities. In this study, both SOD and CAT activities of H2O2-treated leaves increased in compared to the control. However, exogenous application of proline caused a decrease in SOD and CAT activities compared to H2O2-treated leaves. In addition, SOD and CAT activities in proline-treated leaves were not significant difference from those in the controls. Unlike SOD and CAT, POD and APX were found to be more sensitive in their response to oxidative stress. The decrease in APX and POD activities might be due to the generation of high level of oxidative stress and possible inactivation of APX and POD activities. Moreover, inhibition of POD and APX activities could be overwhelmed for detoxification of H2O2. Thus, it can be assumed that a decrease in H2O2 content of the cells resulted in the activation of POD and APX. These results indicate that pre-treatment of grape leaves with proline, did not eliminate the inhibitory effect of H2O2 on POD and APX activities but also increased their activities compared to H2O2 treatment. The results of our study indicated that external application of proline counteracted the inhibitory effect of H2O2 on all parameters measured, help the grapevine leaves to avoid oxidative stress by acting as stress protecting compound. Therefore, the results make it possible to recommend ¨ ku¨zgo¨zu¨ grapevine cultivar with proline exogenous treatment of O under oxidative stress condition. 5. Conclusion Based on the results, it may be concluded that H2O2 played direct role in oxidative stress injury to grapevine leaves, also the mechanism by proline reduced oxidative stress in grape leaves was
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involved in a direct effect on the antioxidant enzyme system, membrane phase change, lipid peroxidation, and electrolyte leakage. To better understand the actual role of proline in plant cells exposed to stress conditions, the following questions should be answered by further studies; how does proline act on the cell wall or plasmalemma? What is the effective proline concentration range for grapevine cultivars grown under stress conditions? What is the proper plant developmental stage for external proline application? Acknowledgement This study was supported by a research grant from Scientific and Technological Research Council of Turkey (TUBITAK Project no: 107 T 272) References Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Ann. Rev. Plant Biol. 55, 373–399. Asada, K., 1992. Ascorbate peroxidase—a hydrogen peroxide scavenging enzyme in plants. Physiol. Plant 85, 235–241. Ashraf, M., Fooland, M.R., 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 59, 206–216. Bandurska, H., 1993. In vitro and in vivo effect of proline on nitrate reductase activity under osmotic stress in barley. Acta Physiol. Plant 15, 83–88. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil. 39, 205–207. Bohnert, H.J., Jensen, R.G., 1996. Strategies for engineering water-stress tolerance in plants. Trends Biotechnol. 14, 89–97. Bowler, C.M., Van Montagu, M., Inze, D., 1992. Superoxide dismutase and stress tolerance. Ann. Rev. Plant Physiol. Plant Mol. Biol. 43, 83–116. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Chance, B., Maehly, S.K., 1955. Assay of catalase and peroxidase. Methods Enzymol. 2, 764–775. Dat, J., Vandenabeele, S., Vranova, E., Van Montagu, M., Inze, D., Van Breusegem, F., 2000. Dual action of the active oxygen species during plant stress responses. Cell Mol. Life Sci. 57, 779–795. Dhindsa, R.S., Plumb-Dhindsa, P., Thorpe, T.A., 1981. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93–101. Ehsanpour, A.A., Fatahian, N., 2003. Effects of salt and proline on Medicago sativa callus. Plant Cell Tissue Organ Cult. 73, 53–56. Giannopolitis, C.N., Ries, S.K., 1977. Superoxide dismutase. I. Occurrence in higher plants. Plant Physiol. 59, 309–314. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplast. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189– 198. Hideg, E., 1997. Free radical production in photosynthesis under stress conditions. In: Pessarakli, M. (Ed.), Handbook of Photosynthesis. Marcel Decker, New York, pp. 911–930. Lin, J.N., Kao, C.H., 1998. Effect of oxidative stress caused by hydrogen peroxide on senescence of rice leaves. Bot. Bull. Acad. Sin. 39, 161–165. Loreto, F., Velikova, V., 2001. Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol. 127, 1781–1787. Matysik, J., Bhalu, A.B., Mohanty, P., 2002. Molecular mechanism of quenching of reactive oxygen species by proline under water stress in plants. Curr. Sci. 82, 525–532. Molassiotis, A., Sotiropoulos, T., Tanou, G., Diamantidis, G., Therios, I., 2006. Boroninduced oxidative damage, antioxidant and nucleolytic responses in shoot tips culture of the apple rootstock EM9 (Malus domestica Borkh). Environ. Exp. Bot. 56, 54–62. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Plant Physiol. 15, 473–497. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Osswald, W.F., Kraus, R., Hippeli, S., Benz, B., Volpert, R., Elstner, E.F., 1992. Comparison of the enzymatic activities of dehydroascorbic acid reductase, glutathione reductase, catalase, peroxidase and superoxide dismutase of healthy and damaged spruce needles (Picea abies L.). J. Plant Physiol. 139, 742–748. Rhodes, D., Hanson, A.D., 1993. Quaternary ammonium and tertiary sulfonium compounds in higher-plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 44, 357–384. Sairam, R.K., Srivastava, G.C., 2000. Induction of oxidative stress and antioxidant activity by hydrogen peroxide treatment in tolerant and susceptible wheat genotypes. Biol. Plant 43, 381–386.
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