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Exogenous nitric oxide on antioxidative system and ATPase activities from tomato seedlings under copper stress
Scientia Horticulturae 123 (2009) 217–223
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Exogenous nitric oxide on antioxidative system and ATPase activities from tomato seedlings under copper stress Yikai Zhang a, Xiaojiao Han b,c, Xiuling Chen a, Hong Jin a, Xiumin Cui a,* a
College of Resources and Environment, Shandong Agricultural University, Tai’an 271018, China International Centre for Bamboo and Rattan, Beijing 100102, China c Chinese Academy of Forestry, Beijing 100091, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 November 2008 Received in revised form 20 July 2009 Accepted 28 August 2009
Nitric oxide (NO) serves as a bioactive molecule involved in antioxidant and anti-stress agent in tolerance responses to abiotic stress. Here, we investigated the effects of exogenous sodium nitroprusside (SNP), a NO donor, on both the ROS metabolism and functions of plasma membrane and tonoplast in tomato plants treated with 50 mM CuCl2. The copper stress markedly decreased shoot height, fresh weight, induced significant accumulation of H2O2, and led to serious lipid peroxidation in tomato plants. The application of 100 mM SNP significantly alleviated the growth inhibition, promoted ROS-scavenging enzymes, reduced H2O2 content in tomato plants, and alleviated the inhibition of H+ATPase and H+-PPase in plasma membrane or tonoplast induced by CuCl2. While application of sodium ferrocyanide (an analog of SNP) and sodium nitrate or nitrite (the decomposition product of NO or its donor SNP) which did not release NO, did not show the effects of SNP; furthermore, the effects of SNP were reversed by addition of hemoglobin (a NO scavenger). All together, these results suggested that exogenous NO could be advantageous against copper (Cu) toxicity, and could confer tolerance to heavy metal stress in tomato plants. ß 2009 Published by Elsevier B.V.
Keywords: Tomato Copper stress Nitric oxide Antioxidant enzymes Plasma membrane Tonoplast
1. Introduction Heavy metals are important environmental pollutants and many of them are toxic even at very low concentrations. Cu is an essential micronutrient for growth and development of plants. Like most micronutrients, Cu is needed in small amounts by the plant (Tanyolac et al., 2007). But Cu has become increasingly hazardous due to its involvement among pollutants of agricultural soils, in fungicides, pesticides, fertilizers, animal dung and so on (Kaplan, 1999). In spite of its physiological importance, an increase in Cu content threatens plant health because Cu renders it more toxic causing destruction of the lipid and protein constituent of membrane (Maksymiec, 1997), even at mild excess. Free Cu ions can readily oxidize the thiol group present in the proteins, causing disruption of their structure and functions. The destruction of membrane from root and leaf was reported to result from the overproduction of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical (OH) and superoxide radicals (O2 ) directly (Mazhoudi et al., 1997; Apel and Hirt, 2004). To counteract the toxicity of Cu, plants have
* Corresponding author. E-mail addresses: [email protected], [email protected] (X. Cui). 0304-4238/$ – see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.scienta.2009.08.015
developed various strategies by exudation of organic acid, retention of Cu in roots and immobilization in the cell wall. Besides, plants response to oxidative stress caused by Cu mainly through modulation of antioxidative enzyme activities. For example, catalase (CAT), peroxidase (POD), glutathione reductase (GR), polyphenol oxidase (PPO) were highly induced in tomato after exposure to excessive Cu (Mazhoudi et al., 1997; Martins and Mourato, 2006). Plasma membrane (PM) H+-transporting adenosine triphosphatase (H+-ATPase) is a ‘master enzyme’ located preferentially at the epidermal and cortical cell layers and plays an important role in the establishment and maintenance of ion homeostasis. Tonoplast (V) H+-ATPase and H+-pyrophosphatase (H+-PPase) also plays a central role in the functional association of tonoplast surface charging with H+ efflux/influx and thus the regulation of tonoplast pH in response to a variety of environmental stimulation. It has been reported that PM H+-ATPase was inhibited by heavy metals stress, such as aluminium (Ahn et al., 2001), copper and cadmium (Fodor et al., 1995; BurzyhskL and Kolano, 2003). Nitric oxide (NO) is a small, highly diffusible gas and a ubiquitous bioactive molecule. Its chemical properties make NO a versatile signal molecule that functions through interaction with cellular targets via either redox or additive chemistry (Lamattina et al., 2003). The effects of NO, in some cases, are the result of its
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interaction with ROS. These interactions sometimes can be cytotoxic or cytoprotective depending on the concentration and situation. Now it has widely been accepted that NO protects plant cell against oxidative stress by scavenging Fenton active Fe and regulating activities of antioxidant enzymes (Arasimowicz and Floryszak-Wieczorek, 2007). Under an ordinary physiological condition, superoxide dismutase (SOD) rapidly converts O2 to H2O2 and an oxygen molecule. However, a large amount of NO may combine with O2 to form peroxynitrite (ONOO ), which has been reported to damage lipids, proteins and nucleic acids. Nevertheless, O2 and H2O2 are more toxic than NO and ONOO ; therefore, NO may protect cells from destruction. In accordance, NO has been proposed to have both antioxidant and prooxidant nature (Boveris et al., 2000). Both Cu and NO are redox reactive entities. NO has been reported to counteract the toxicity of ROS generated by excessive cadmium (Hsu and Kao, 2004; Laspina et al., 2005), excessive copper (Yu et al., 2005; Tewari et al., 2008), stimulate the expression of PM H+-ATPase, V-H+-ATPase, V-H+-PPase induced by salt stress (Shi et al., 2007), and dramatically promote the germination of wheat seeds under Cu stress by improving antioxidant capacity (Hu et al., 2007). Particularly, alleviatory role of NO in Cu toxicity is still scanty and needs more attention. While Cu excess has been reported to induce H2O2 generation, NO blocks it. Therefore, in the present study, the objectives of the present experiment were to investigate whether NO is involved in regulation of ROS metabolism and PM H+-ATPase, V-H+-ATPase and V-H+-PPase activity in tomato plant, to elucidate the physiological mechanism of exogenous NO increasing tomato plant tolerance to Cu stress. 2. Materials and methods 2.1. Plant materials and growth conditions Tomato seeds (Lycopersicon esculentum Mill, cv. Meigui) were surface-sterilized by 55 8C sterile distilled water for 15 min, and then were germinated on moist filter paper in the dark at 26 8C for 3 days. The germinated seeds were transferred to the plastic trays filled with vermiculite and grown in greenhouse. Then the seedlings were grown in half-strength Hoagland’s solution. Two-week-old seedlings were planted on perforated polystyrene plates, floating on an aerated Hoagland nutrient solution, which was renewed every 3 days. The plants were precultivated for 3 weeks. Selected uniform size seedlings and transplanted into 5000 ml black plastic containers with five seedlings per container. The experimental design consisted of a control (no added SNP and CuCl2) and five treatments (50 mM CuCl2, indicated by Cu; 50 mM CuCl2 + 100 mM SNP, Cu + SNP; 50 mM CuCl2 + 100 mM sodium nitrite + 100 mM sodium nitrate, Cu + N; 50 mM CuCl2 + 100 mM sodium ferrocyanide, Cu + F; 50 mM CuCl2 + 100 mM SNP + 0.1% bovine hemoglobin, Cu + SNP + Hb), which were arranged in a randomized block design with three replicates, giving a total of 18 containers. 50 mM CuCl2 with or without 100 mM SNP treatments were selected after previous experiments in our laboratory or according to concentrations found in literature (Ouariti et al., 1997; Tewari et al., 2008). In this experiment, Hemoglobin was used as a specific NO scavenger, and sodium nitrite or sodium nitrate and sodium ferrocyanide as the controls of SNP decomposition. The experiment was carried out under natural conditions with the air temperature of 25–32 8C during the day and 19–26 8C during the night. The pH was adjusted to 5.0 0.2 with HCl and KOH. All plants were harvested after 8 days treatment, and separated into leaves and roots.
2.2. Plant growth measurement Growth parameters (shoot height, fresh root weight and fresh shoot weight) were measured after 8 days treatment. 2.3. H2O2 and MDA contents assay H2O2 concentration was determined according to Patterson et al. (1984) with minor modifications. The assay was based on the absorbance change of the H2O2-titanium complex at 415 nm, which was formed by reaction of tissue-H2O2 with titanium tetrachloride. The level of lipid peroxidation in fresh leaf and root was measured in terms of malondialdehyde (MDA) content by the thiobarbituric acid reaction method (Heath and Packer, 1968). 2.4. Antioxidant enzyme extraction and assay For extraction of antioxidative enzymes, leaves and roots were homogenized with 50 mM Na2HPO4-NaH2PO4 buffer (pH 7.8) containing 0.2 mM EDTA and 2% insoluble polyvinylpyrrolidone in a chilled pestle and mortar. The homogenate was centrifuged at 12,000 g for 20 min and the resulted supernatant was used for determination of enzyme activities. The whole extraction procedure was carried out at 4 8C. All spectrophotometric analysis was conducted on a Shimadzu UV-2450 spectrophotometer. Total SOD activity was assayed by the photochemical method described by Rao and Sresty (2000). The 3 ml reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 mM nitroblue tetrazolium, 2 mM riboflavin, 10 mM EDTA and 0.1 ml enzyme extract. One unit of the enzyme activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of nitro blue tetrazolium (NBT) reduction measured at 560 nm. SOD activity was expressed as U g 1 FW. Guaiacol peroxidase (POD) activity was estimated after Nickel and Cunningham (1969) method. Activity was measured by the increase in absorbance at 470 nm due to guaiacol oxidation. The reaction mixture contained 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 1.0 mM H2O2 and 0.1 ml enzyme extract. The activity was expressed as U mg 1 FW min 1. CAT activity was measured according to the method of Cakmak and Marschner (1992) by determination the disappearance of H2O2 by measuring the decrease in an absorbance at 240 nm of a reaction mixture containing 25 mM phosphate buffer (pH 7.0), 10 mM H2O2 and 0.1 ml enzyme extract. CAT activity was expressed as mmol H2O2 mg 1 FW min 1. Activity of APX (ascorbate peroxidase) was measured according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm. The assay mixture contained 0.25 mM AsA, 1.0 mM H2O2, 0.1 mM EDTA, and 0.1 ml enzyme extract in 25 mM phosphate buffer (pH 7.0). The activity of APX was calculated in terms of mmol ASA mg 1 FW min 1. 2.5. Isolation of plasma membrane and tonoplast vesicles Plasma membrane and tonoplast vesicles fraction enriched were isolated from tomato roots and leaves as described by Kasamo (1986) with minor changes. Excised roots and leaves were homogenized (1/2, w/v) with a mortar and pestle in a cold grinding medium containing: 25 mM HEPES–Tris (pH 7.2), 250 mM mannitol, 5 mM EDTA, 5 mM EGTA, 1 mM DTT and 1.5% (w/v) PVP. The whole isolation procedures were carried out at 4 8C. The homogenate was filtered through four layers of cheesecloth and centrifuged at 560 g for 12 min, then the supernatant was centrifuged at 10,000 g for 15 min, and the supernatant was centrifuged at 60,000 g for 30 min to yield a crude membrane
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fraction. The resulting pellet was resuspended with 1 ml in a gradient buffer containing: 20 mM HEPES–Tris (pH 7.5), 5 mM EDTA, 0.5 mM EGTA. The supernatant was layered on top of a step gradient consisting of 1 ml of 45%, 33% and 15% (w/w) sucrose, respectively, and then centrifuged for 2 h at 70,000 g. The tonoplast-enriched fraction was collected at the 15/33% sucrose interface and the plasma membrane-enriched fraction was collected at the 33/45% sucrose interface. Each fraction was centrifuged for 1 h at 100,000 g. The resulting pellet was resuspended in a medium containing: 20 mM HEPES–Tris (pH 7.5), 3 mM MgCl2, 0.5 mM EGTA, 300 mM sucrose, then quickly frozen in liquid nitrogen and stored at 70 8C until used for the enzyme assays. 2.6. Measurement of H+-ATPase in PMs ATP hydrolysis assays were performed as described by Briskin et al. (1987) with minor changes. 0.5 ml the reaction medium containing: 36 mM Tris–Mes (pH 6.5), 30 mM ATP-Na2, 3 mM MgSO4, 1 mM NaN3, 50 mM KNO3, 1 mM Na2MoO4, 0.02% (v/v) Triton X-100, in the presence or absence of 2.5 mM Na3VO4. The reaction was started by adding 50 ml PM vesicles. After 30 min incubation at 37 8C, the reaction was quenched by the addition of 55% (w/v) TCA. The H+-ATPase activity was determined by measuring the release of Pi (Ohinishi et al., 1975). 2.7. Measurement of H+-ATPase and H+-PPase activities in tonoplast H+-ATPase and H+-PPase activities were calculated as the rate of ATP hydrolysis or PPi, respectively, by measuring the amount of released Pi according to Lin and Morales (1977) with minor changes. Activities of tonoplast H+-ATPase were measured in a 0.5 ml reaction medium containing: 36 mM HEPES–Tris (pH 7.5), 30 mM ATP-Na2, 3 mM MgSO4, 0.5 mM NaN3, 50 mM KCl, 0.1 mM Na2MoO4, 0.1 mM Na3VO4, in the presence or absence of 50 mM KNO3. Activities of tonoplast H+-PPase were measured in a 0.5 ml reaction medium containing: 60 mM Tris–HEPES (pH 8.0), 3 mM MgSO4, 5 mM EDTA, 1 mM (NH4)6Mo7O24, 0.5 mM NaN3, 0.1 mM Na3VO4, 30 mM Napyrophosphate, in the presence or absence of 100 mM KCl. The reaction was started by adding 50 ml tonoplast vesicles. After 30 min incubation at 37 8C, the reaction was quenched by 55% TCA and the color developed as in the PM ATPase assay.
Fig. 1. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on shoot fresh weight (A), root fresh weight (B) and shoot height (C) of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
45.6% and 32.1% of control, respectively. Exogenous NO treatment (Cu + SNP) markedly alleviated the inhibition, by 57.3%, 67.3% and 35.9% compared with Cu stress. The alleviating heavy metal stress of exogenous NO was blocked by hemoglobin (Cu + SNP + Hb). Moreover, 100 mM sodium ferrocyanide (Cu + F) and 100 mM sodium nitrate or nitrite (Cu + N) which did not release NO, had no obvious effect on alleviation of heavy metal stress to tomato plants.
2.8. Copper concentration assay 3.2. Hydrogen peroxide concentration and lipid peroxidation Leaf and root samples were oven dried at 70 8C, until they reached a constant weight. At least four plants were analyzed for each treatment. The samples of dried plant material were ashed at 480 8C in a muffle furnace, subsequently digested twice in 10 ml of 3 M HCl at 90 8C, and analyzed by AA370MC flame atomic absorption spectrophotometry. 2.9. Statistics All data presented were means one standard deviation (S.D.) of three replicates. Statistical analyses were performed by analysis of variance (ANOVA) using SAS software. Differences between treatments were separated by the least significant difference (LSD) test at a 0.05 probability level. 3. Results 3.1. Plant growth As shown in Fig. 1, Cu stress decreased shoot fresh weight, root fresh weight and shoot height of tomato plants by 39.1%,
Compared to control, Cu treatment for 8 days significantly increased H2O2 content in tomato roots and leaves by 79% and 121%, respectively. The application of exogenous NO (Cu + SNP) decreased accumulation of H2O2 by 32% and 47% in roots and leaves under heavy metal stress, and the effect of exogenous NO was blocked by addition of hemoglobin (Cu + SNP + Hb). Addition of sodium ferrocyanide (Cu + F) and sodium nitrate or nitrite (Cu + N) had no significant effect on H2O2 content under heavy metal stress (Fig. 2A and B). When plants were subjected to environmental stress, oxidative damage resulted in the level of membrane lipid peroxidation, which could be estimated by MDA content. Similar to H2O2 concentration change, Cu treatment considerably increased MDA content by 71.2% and 80.6% in leaves and roots and the application of NO (Cu + SNP) reduced MDA content by 33.6% and 36.2% in leaves and roots. The effect of exogenous NO was also removed by addition of hemoglobin (Cu + SNP + Hb). Application of sodium ferrocyanide (Cu + F) and sodium nitrate or nitrite (Cu + N) did not markedly affect MDA content (Fig. 2C and D).
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Fig. 2. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on H2O2 content (A), MDA content (C) in leaves and H2O2 content (B), MDA content (D) in roots of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
3.3. Antioxidant enzymes It can be seen that in leaves and roots of tomato plant SOD activity was decreased by 28% and 47% under Cu stress on the 8th day of treatment. Exogenous NO treatment (Cu + SNP) elevated SOD activity by 34% and 82% in leaves and roots. The addition of hemoglobin (Cu + SNP + Hb) removed the effect of exogenous NO, and application of sodium ferrocyanide (Cu + F) and sodium nitrate or nitrite (Cu + N) did not obviously change the SOD activity in Cutreated plants (Fig. 3A and B). Cu treatment inhibited POD activity of both leaves and roots of tomato plant by 28% and 56% after 8 days treatment, and application of NO (Cu + SNP) greatly decreased the inhibition by 42% and 109%, respectively, in leaves and roots. While the addition of hemoglobin removed the effect of exogenous NO, and addition of sodium ferrocyanide and sodium nitrate or nitrite did not obviously change the POD activity (Fig. 3C and D). Similar to the change of POD activity, Cu stress significantly inhibited APX activity by 51% and 53% in both leaves and roots of tomato plant on the 8th day treatment, and exogenous NO (Cu + SNP) greatly decreased the inhibition, by 80% and 95% in leaves and roots compared with Cu stress. APX activity did not show statistically significant differences among Cu treatment, Cu treatment with addition of sodium ferrocyanide, Cu treatment with sodium nitrate or nitrite and Cu treatment with both SNP and hemoglobin (Fig. 3E and F). Contrary to the change of POD and APX activity, CAT activity in both leaves and roots of tomato plant was markedly enhanced by 103% and 54% on the 8th day of treatment by Cu stress. Exogenous NO (Cu + SNP) significantly decreased CAT activity by 45% and 39% in leaves and roots under Cu stress. Moreover, application of hemoglobin with SNP, sodium ferrocyanide and sodium nitrate or nitrite did not significantly influence CAT activity (Fig. 3G and H). 3.4. H+-ATPase activity in plasma membrane H+-ATPase activity in tomato both leaves and roots plasma membrane was determined on the 8th day of treatment. It can be seen that Cu stress inhibited H+-ATPase activity by 43.3% and 53.5%
in both leaves and roots. Application of NO (Cu + SNP) decreased the inhibition by 54.5% and 86.9% in leaves and roots compared with Cu stress, respectively. Addition of sodium ferrocyanide (Cu + F), sodium nitrate or nitrite (Cu + N) and SNP with hemoglobin (Cu + SNP + Hb) had no significant effects on H+ATPase activity in plasma membrane under Cu treatment (Fig. 4A and B). 3.5. H+-ATPase and H+-PPase activity in tonoplast H+-ATPase and H+-PPase activity in tomato both leaves and roots tonoplast was also measured on the 8th day of treatment. In leaves and roots, their activities showed the different change tendency. In leaves, Cu stress promoted tonoplast H+-ATPase and H+-PPase activities by 165% and 141%, respectively. While application of NO (Cu + SNP) decreased tonoplast H+-ATPase and H+-PPase activities, and made them keep at similar level with the control (Fig. 5A and C). Their activity did not show statistically significant differences among Cu treatment, Cu treatment with sodium ferrocyanide, Cu treatment with sodium nitrate or nitrite and Cu treatment with both SNP and hemoglobin. However, in roots, Cu stress inhibited tonoplast H+-ATPase and H+-PPase activities by 57.7% and 57.1%, respectively. Application of NO (Cu + SNP) greatly promoted tonoplast H+-ATPase and H+-PPase activities by 115% and 114% compared with Cu treatment (Fig. 5B and D). 3.6. Cu concentration As shown in Fig. 6, Cu content in both leaves and roots of tomato plant was significantly increased by Cu treatment compared to control (P < 0.05), while application of NO could not markedly affect Cu concentration in both leaves and roots. This observation suggested that exogenous NO supply did not affect Cu uptake. Addition of sodium ferrocyanide and sodium nitrate or nitrite dramatically decreased Cu concentration compared to Cu treatment. Howerever, in roots application of SNP with addition of hemoglobin had no significant effects on Cu content and in leaves application of hemoglobin, sodium ferrocyanide and sodium
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Fig. 3. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on activities of SOD (A), POD (C), CAT (E), APX (G) in leaves and activities of SOD (B), POD (D), CAT (F), APX (H) in roots of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
nitrate or nitrite did not significantly influence Cu content (Fig. 6A and B). 4. Discussion It has been reported that Cu stress could cause an increased production of H2O2 (Weckx and Clijsters, 1996) and induce membrane lipid peroxidation (Mazhoudi et al., 1997). These revealed that Cu treatment greatly induced serious membrane damage in the plant. This experiment showed that oxidative stress of tomato plant induced by excessive Cu was effectively alleviated by application of SNP, while the application of sodium ferrocyanide, sodium nitrate or nitrite and SNP with hemoglobin did not mitigate the oxidative damage. Because SNP is a NO donor, while sodium ferrocyanide is an analog of SNP, sodium nitrate or nitrite is the decomposition product of NO or its donor SNP and hemoglobin is a NO scavenger, therefore, by comparing the results, we could deduce that SNP alleviating the oxidative damage by excessive Cu was ascribed to its releasing NO. As key elements in the defense mechanisms, varying reactions of antioxidative enzymes have been observed under Cu stress conditions in many plants. For example, it has been reported that ROS-scavenging enzyme activity induced by excessive Cu was increased in the case of Cu-tolerant maize (Tanyolac et al., 2007), barley plant (Demirevska-Kepova et al., 2004), tomato shoot
(Martins and Mourato, 2006), beans shoot (Weckx and Clijsters, 1996) and duckweed (Teisseire and Guy, 2000), but was depressed in rice leaves (Chen and Kao, 1999), or was unaffected in tomato roots and stems (Mazhoudi et al., 1997). In the present experiment, activities of SOD, POD and APX in tomato roots and leaves were significantly inhibited on the 8th day by Cu stress, while CAT activity was increased. This result implied that CAT induction might play an important role in tomato tolerance to Cu excess. Some of the observed results were consistent with the reports of Martins and Mourato (2006), and some were not consistent with them. The difference indicated that the influence of Cu stress on the antioxidant enzymes was very complex and related to the plant treatment time, plant tissues, plant species and genotypes. In many studies, it was reported that the function of NO alleviation of oxidative damage was ascribed to induction of various ROS-scavenging enzyme activity (Hsu and Kao, 2004; Hu et al., 2007). In the present work, application of SNP dramatically decreased the inhibited SOD activity by Cu stress. This suggested that application of NO could promote the conversion from O2 into H2O2 and O2, which was an important step in protecting the cell. If the generated H2O2 could not be scavenged efficiently, it could interact with O2 to form highly reactive hydroxyl radicals (OH) that were thought to be primarily responsible for oxygen toxicity in the cell (Shi et al., 2007). Therefore, the efficient scavenging of H2O2 was very important for normal metabolism of
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Fig. 4. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on activity of H+-ATPase in leaves (A) and roots (B) plasma membrane of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
Fig. 6. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on Cu content (A) in leaves and Cu content (B) in roots of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
plant. In the present experiment, application of NO greatly induced the H2O2-scavenging enzymes POD and APX under Cu stress. Similar results had been observed in the experiment of NO increasing the rice leaves tolerance to Cu excess (Yu et al., 2005) and the experiment of NO stimulating wheat seed germination under heavy metal Cu stress (Hu et al., 2007). In this study, Cu stress significantly inhibited PM H+-ATPase activity in both leaves and roots of tomato plant. This would break the electrochemical gradient across the membrane, decrease the ability to keep cell wall acidfication, and inhibit the growth of
plants (Fig. 1). In the meanwhile, V-H+-ATPase and V-H+-PPase activities in roots were greatly inhibited under Cu stress resulted in cytoplasmic H+ concentration increases, and then influenced cytoplasmic pH steady. However, H+-ATPase and H+-PPase activities of tonoplast in tomato leaves were increased, which implied that V-H+-ATPase and V-H+-PPase activitie induction might play an important role in tomato leaves tolerance to Cu stress. Application of NO considerably increased PM H+-ATPase activity in plants, V-H+-ATPase and V-H+-PPase activities in roots. This may facilitate the expulsion of the excess H+, promote the
Fig. 5. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on activities of H+-ATPase (A), H+-PPase (C) in leaf tonoplast and activities of H+-ATPase (B), H+-PPase (D) in root tonoplast of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
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cytoplasm alkalinization and renew cell normal activity partly (Michelet and Boutry, 1995). The results indicated that less severe membrane lipid damage occurred in NO-treated roots of tomato under Cu stress because the membrane lipid oxidative injury caused by elevated active oxygen species under Cu stress was markedly alleviated by NO-enhanced activities of antioxidative enzymes scavenging active oxygen species. It was well known that the degree of lipid peroxidation was closely related to the accumulation of ROS, and the lipid process was one important factor exerting an effect on ATPase under changing environmental conditions (Veselov et al., 2002). As a result of lipid peroxidation induced by Cu excess, ATPase and PPase proteins were disturbed. Therefore, exogenous NO decreasing lipid peroxidation should be considered as one cause maintaining higher ATPase activity. The results of this study showed that exogenous NO, when applied along with excess Cu, could improve plant growth and significantly attenuate Cu-induced oxidative stress as indicated by a down regulation in lipid peroxidation and H2O2 concentration in tomato plants. By determining the activities of SOD, POD, APX and CAT, we learned that NO exerted its protective effect through the activation of some antioxidative enzymes, not by preventing Cu uptake. Therefore, lower lipid peroxidation could maintain better function of the plasma membrane and tonoplast, elevate the activities of H+-ATPase, H+-PPase, and mitigate copper toxicity in tomato plants. Acknowledgements This work was supported by the Science Technology Project of Shandong Provincial Education Department (No. 32353) and the Young Scientist Innovation Science Foundation of Shandong Agricultural University (No. 23420). References Ahn, S.J., Sivaguru, M., Osawa, H., Chung, G.C., Matsumoto, H., 2001. Aluminum Inhibits the H+-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots. Plant Physiol. 126, 1381–1390. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–379. Arasimowicz, M., Floryszak-Wieczorek, J., 2007. Nitric oxide as a bioactive signalling molecule in plant stress responses. Plant Sci. 172 (5), 876–887. Boveris, A.D., Galatro, A., Puntarulo, S., 2000. Effect of nitric oxide and plant antioxidants on microsomal content of lipid radicals. Biol. Res. 33, 159–165. Briskin, D.P., Leonard, R.T., Hodges, T.K., 1987. Isolation of the plasma membrane: markers and general principles. Methods Enzymol. 148, 542–558. BurzyhskL, M., Kolano, E., 2003. In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots. Physiol. Plant. 25, 39–45. Cakmak, I., Marschner, H., 1992. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol. 98, 1222–1227. Chen, L.M., Kao, C.H., 1999. Effect of excess copper on rice leaves: evidence for involvement of lipid peroxidation. Bot. Bull. Acad. Sin. 40, 283–287. Demirevska-Kepova, K., Simova-Stoilova, L., Stoyanova, Z., Ho¨lzer, R., Feller, U., 2004. Biochemical changes in barley plants after excessive supply of copper and manganese. Environ. Exp. Bot. 52, 253–266.
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Fodor, E., Szabo-Nagy, A., Erdei, L., 1995. The effect of cadmium on fluidity and ATPase activity of plasma membrane from sunflower and wheat roots. J. Plant Physiol. 147, 87–92. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189– 198. Hsu, Y.T., Kao, C.H., 2004. Cadmium toxicity is reduced by nitric oxide in rice leaves. Plant Growth Regul. 42, 227–238. Hu, K.D., Hu, L.Y., Li, Y.H., Zhang, F.Q., Zhang, H., 2007. Protective roles of nitric oxide on germination and antioxidant metabolism in wheat seeds under copper stress. Plant Growth Regul. 53, 173–183. Kaplan, M., 1999. Accumulation of Cu in soils and leaves of tomato plants in greenhouses in Turkey. J. Plant Nutr. 22, 237–244. Kasamo, K., 1986. Comparison of plasma membrane and tonoplast H+-translocating ATPase in Phaseolus mungo L. roots. Plant Cell Physiol. 27, 49–59. Lamattina, L., Garcia-Mata, C., Graziano, M., Pagnussat, G., 2003. Nitric oxide: the versatility of an extensive signal molecule. Annu. Rev. Plant Biol. 54, 109–136. Laspina, N.V., Groppa, M.D., Tomaro, M.L., Benavides, M.P., 2005. Nitric oxide protects sunflower leaves against Cd-induced oxidative stress. Plant Sci. 169, 323–330. Lin, T., Morales, M.F., 1977. Application of a one step procedure for measuring inorganic phosphate in the presence of proteins: the actomyosin ATPase system. Anal. Biochem. 77, 10–17. Maksymiec, W., 1997. Effect of copper on cellular processes in higher plants. Photosynthetica 34, 321–342. Martins, L.L., Mourato, M.P., 2006. Effect of excess copper on tomato plants: growth parameters, enzyme activities, chlorophyll, and mineral content. J. Plant Nutr. 29 (12), 2179–2198. Mazhoudi, S., Chaoui, A., Ghorbal, M.H., Ferjani, E.E., 1997. Response of antioxidant enzymes to excess copper in tomato (Lycopersicon esculentum, Mill.). Plant Sci. 127, 129–137. Michelet, B., Boutry, M., 1995. The plasma membrane H+-ATPase: a highly regulated enzyme with multiple physiological functions. Plant Physiol. 108, 1–6. Nakano, Y., Asada, K., 1981. Hydrogen peroxide scavenged by ascorbate-specific peroxidase in spinach chloroplast. Plant Cell Physiol. 22, 867–880. Nickel, R.S., Cunningham, B.A., 1969. Improved peroxidase assay method using Ieuco 2,3,6-trichlcroindophenol and application to comparative measurements of peroxidase catalysis. Ann. Biochem. 27, 292–299. Ohinishi, T., Gall, R.S., Mayer, M.L., 1975. An improved assay of inorganic phosphate in the presence of extralabile phosphate compounds: application to the ATPase assay in the presence of phosphocreatine. Anal. Biochem. 69, 261–267. Ouariti, O., Boussama, N., Zarrouk, M., Cherif, A., Ghorbal, M.H., 1997. Cadmium- and copper-induced changes in tomato membrane lipids. Phytochemistry 45 (7), 1343–1350. Patterson, B.D., Mackae, E.A., Mackae, I.B., 1984. Estimation of hydrogen peroxide in plant extracts using Titanium (IV). Anal. Chem. 139, 487–492. Rao, K.V.M., Sresty, T.V.S., 2000. Antioxidant parameters in the seedlings of pigeon pea (Cajanus cajan L. Millspaugh) in response to Zn and Ni stresses. Plant Sci. 157, 113–128. Shi, Q.H., Ding, F., Wang, X.F., Wei, M., 2007. Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress. Plant Physiol. Biochem. 45, 542–550. Tanyolac, D., Ekmekc, Y., Unalan, S., 2007. Changes in photochemical and antioxidant enzyme activities in maize (Zea mays L.) leaves exposed to excess copper. Chemosphere 67, 89–98. Teisseire, H., Guy, V., 2000. Copper-induced changes in antioxidant enzymes activities in fronds of duckweed (Lemna minor). Plant Sci. 153, 65–72. Tewari, R.K., Hahn, E.J., Paek, K.Y., 2008. Modulation of copper toxicity-induced oxidative damage by nitric oxide supply in the adventitious roots of Panax ginseng. Plant Cell Rep. 27, 171–181. Veselov, A.P., Kurganova, L.N., Likhacheva, A.V., Sushkova, U.A., 2002. Possible regulation effect of lipid peroxidation on the H+-ATPase activity of the plasmalemma under stress conditions. Russ. J. Plant Physiol. 49, 385–389. Weckx, J.E.J., Clijsters, H.M.M., 1996. Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper. Physiol. Plant. 96, 506–512. Yu, C.C., Hung, K.T., Kao, C.H., 2005. Nitric oxide reduces Cu toxicity and Cu-induced NH4+ accumulation in rice leaves. J. Plant Physiol. 162, 319–1330.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Exogenous nitric oxide on antioxidative system and ATPase activities from tomato seedlings under copper stress Yikai Zhang a, Xiaojiao Han b,c, Xiuling Chen a, Hong Jin a, Xiumin Cui a,* a
College of Resources and Environment, Shandong Agricultural University, Tai’an 271018, China International Centre for Bamboo and Rattan, Beijing 100102, China c Chinese Academy of Forestry, Beijing 100091, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 November 2008 Received in revised form 20 July 2009 Accepted 28 August 2009
Nitric oxide (NO) serves as a bioactive molecule involved in antioxidant and anti-stress agent in tolerance responses to abiotic stress. Here, we investigated the effects of exogenous sodium nitroprusside (SNP), a NO donor, on both the ROS metabolism and functions of plasma membrane and tonoplast in tomato plants treated with 50 mM CuCl2. The copper stress markedly decreased shoot height, fresh weight, induced significant accumulation of H2O2, and led to serious lipid peroxidation in tomato plants. The application of 100 mM SNP significantly alleviated the growth inhibition, promoted ROS-scavenging enzymes, reduced H2O2 content in tomato plants, and alleviated the inhibition of H+ATPase and H+-PPase in plasma membrane or tonoplast induced by CuCl2. While application of sodium ferrocyanide (an analog of SNP) and sodium nitrate or nitrite (the decomposition product of NO or its donor SNP) which did not release NO, did not show the effects of SNP; furthermore, the effects of SNP were reversed by addition of hemoglobin (a NO scavenger). All together, these results suggested that exogenous NO could be advantageous against copper (Cu) toxicity, and could confer tolerance to heavy metal stress in tomato plants. ß 2009 Published by Elsevier B.V.
Keywords: Tomato Copper stress Nitric oxide Antioxidant enzymes Plasma membrane Tonoplast
1. Introduction Heavy metals are important environmental pollutants and many of them are toxic even at very low concentrations. Cu is an essential micronutrient for growth and development of plants. Like most micronutrients, Cu is needed in small amounts by the plant (Tanyolac et al., 2007). But Cu has become increasingly hazardous due to its involvement among pollutants of agricultural soils, in fungicides, pesticides, fertilizers, animal dung and so on (Kaplan, 1999). In spite of its physiological importance, an increase in Cu content threatens plant health because Cu renders it more toxic causing destruction of the lipid and protein constituent of membrane (Maksymiec, 1997), even at mild excess. Free Cu ions can readily oxidize the thiol group present in the proteins, causing disruption of their structure and functions. The destruction of membrane from root and leaf was reported to result from the overproduction of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical (OH) and superoxide radicals (O2 ) directly (Mazhoudi et al., 1997; Apel and Hirt, 2004). To counteract the toxicity of Cu, plants have
* Corresponding author. E-mail addresses: [email protected], [email protected] (X. Cui). 0304-4238/$ – see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.scienta.2009.08.015
developed various strategies by exudation of organic acid, retention of Cu in roots and immobilization in the cell wall. Besides, plants response to oxidative stress caused by Cu mainly through modulation of antioxidative enzyme activities. For example, catalase (CAT), peroxidase (POD), glutathione reductase (GR), polyphenol oxidase (PPO) were highly induced in tomato after exposure to excessive Cu (Mazhoudi et al., 1997; Martins and Mourato, 2006). Plasma membrane (PM) H+-transporting adenosine triphosphatase (H+-ATPase) is a ‘master enzyme’ located preferentially at the epidermal and cortical cell layers and plays an important role in the establishment and maintenance of ion homeostasis. Tonoplast (V) H+-ATPase and H+-pyrophosphatase (H+-PPase) also plays a central role in the functional association of tonoplast surface charging with H+ efflux/influx and thus the regulation of tonoplast pH in response to a variety of environmental stimulation. It has been reported that PM H+-ATPase was inhibited by heavy metals stress, such as aluminium (Ahn et al., 2001), copper and cadmium (Fodor et al., 1995; BurzyhskL and Kolano, 2003). Nitric oxide (NO) is a small, highly diffusible gas and a ubiquitous bioactive molecule. Its chemical properties make NO a versatile signal molecule that functions through interaction with cellular targets via either redox or additive chemistry (Lamattina et al., 2003). The effects of NO, in some cases, are the result of its
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interaction with ROS. These interactions sometimes can be cytotoxic or cytoprotective depending on the concentration and situation. Now it has widely been accepted that NO protects plant cell against oxidative stress by scavenging Fenton active Fe and regulating activities of antioxidant enzymes (Arasimowicz and Floryszak-Wieczorek, 2007). Under an ordinary physiological condition, superoxide dismutase (SOD) rapidly converts O2 to H2O2 and an oxygen molecule. However, a large amount of NO may combine with O2 to form peroxynitrite (ONOO ), which has been reported to damage lipids, proteins and nucleic acids. Nevertheless, O2 and H2O2 are more toxic than NO and ONOO ; therefore, NO may protect cells from destruction. In accordance, NO has been proposed to have both antioxidant and prooxidant nature (Boveris et al., 2000). Both Cu and NO are redox reactive entities. NO has been reported to counteract the toxicity of ROS generated by excessive cadmium (Hsu and Kao, 2004; Laspina et al., 2005), excessive copper (Yu et al., 2005; Tewari et al., 2008), stimulate the expression of PM H+-ATPase, V-H+-ATPase, V-H+-PPase induced by salt stress (Shi et al., 2007), and dramatically promote the germination of wheat seeds under Cu stress by improving antioxidant capacity (Hu et al., 2007). Particularly, alleviatory role of NO in Cu toxicity is still scanty and needs more attention. While Cu excess has been reported to induce H2O2 generation, NO blocks it. Therefore, in the present study, the objectives of the present experiment were to investigate whether NO is involved in regulation of ROS metabolism and PM H+-ATPase, V-H+-ATPase and V-H+-PPase activity in tomato plant, to elucidate the physiological mechanism of exogenous NO increasing tomato plant tolerance to Cu stress. 2. Materials and methods 2.1. Plant materials and growth conditions Tomato seeds (Lycopersicon esculentum Mill, cv. Meigui) were surface-sterilized by 55 8C sterile distilled water for 15 min, and then were germinated on moist filter paper in the dark at 26 8C for 3 days. The germinated seeds were transferred to the plastic trays filled with vermiculite and grown in greenhouse. Then the seedlings were grown in half-strength Hoagland’s solution. Two-week-old seedlings were planted on perforated polystyrene plates, floating on an aerated Hoagland nutrient solution, which was renewed every 3 days. The plants were precultivated for 3 weeks. Selected uniform size seedlings and transplanted into 5000 ml black plastic containers with five seedlings per container. The experimental design consisted of a control (no added SNP and CuCl2) and five treatments (50 mM CuCl2, indicated by Cu; 50 mM CuCl2 + 100 mM SNP, Cu + SNP; 50 mM CuCl2 + 100 mM sodium nitrite + 100 mM sodium nitrate, Cu + N; 50 mM CuCl2 + 100 mM sodium ferrocyanide, Cu + F; 50 mM CuCl2 + 100 mM SNP + 0.1% bovine hemoglobin, Cu + SNP + Hb), which were arranged in a randomized block design with three replicates, giving a total of 18 containers. 50 mM CuCl2 with or without 100 mM SNP treatments were selected after previous experiments in our laboratory or according to concentrations found in literature (Ouariti et al., 1997; Tewari et al., 2008). In this experiment, Hemoglobin was used as a specific NO scavenger, and sodium nitrite or sodium nitrate and sodium ferrocyanide as the controls of SNP decomposition. The experiment was carried out under natural conditions with the air temperature of 25–32 8C during the day and 19–26 8C during the night. The pH was adjusted to 5.0 0.2 with HCl and KOH. All plants were harvested after 8 days treatment, and separated into leaves and roots.
2.2. Plant growth measurement Growth parameters (shoot height, fresh root weight and fresh shoot weight) were measured after 8 days treatment. 2.3. H2O2 and MDA contents assay H2O2 concentration was determined according to Patterson et al. (1984) with minor modifications. The assay was based on the absorbance change of the H2O2-titanium complex at 415 nm, which was formed by reaction of tissue-H2O2 with titanium tetrachloride. The level of lipid peroxidation in fresh leaf and root was measured in terms of malondialdehyde (MDA) content by the thiobarbituric acid reaction method (Heath and Packer, 1968). 2.4. Antioxidant enzyme extraction and assay For extraction of antioxidative enzymes, leaves and roots were homogenized with 50 mM Na2HPO4-NaH2PO4 buffer (pH 7.8) containing 0.2 mM EDTA and 2% insoluble polyvinylpyrrolidone in a chilled pestle and mortar. The homogenate was centrifuged at 12,000 g for 20 min and the resulted supernatant was used for determination of enzyme activities. The whole extraction procedure was carried out at 4 8C. All spectrophotometric analysis was conducted on a Shimadzu UV-2450 spectrophotometer. Total SOD activity was assayed by the photochemical method described by Rao and Sresty (2000). The 3 ml reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 mM nitroblue tetrazolium, 2 mM riboflavin, 10 mM EDTA and 0.1 ml enzyme extract. One unit of the enzyme activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of nitro blue tetrazolium (NBT) reduction measured at 560 nm. SOD activity was expressed as U g 1 FW. Guaiacol peroxidase (POD) activity was estimated after Nickel and Cunningham (1969) method. Activity was measured by the increase in absorbance at 470 nm due to guaiacol oxidation. The reaction mixture contained 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 1.0 mM H2O2 and 0.1 ml enzyme extract. The activity was expressed as U mg 1 FW min 1. CAT activity was measured according to the method of Cakmak and Marschner (1992) by determination the disappearance of H2O2 by measuring the decrease in an absorbance at 240 nm of a reaction mixture containing 25 mM phosphate buffer (pH 7.0), 10 mM H2O2 and 0.1 ml enzyme extract. CAT activity was expressed as mmol H2O2 mg 1 FW min 1. Activity of APX (ascorbate peroxidase) was measured according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm. The assay mixture contained 0.25 mM AsA, 1.0 mM H2O2, 0.1 mM EDTA, and 0.1 ml enzyme extract in 25 mM phosphate buffer (pH 7.0). The activity of APX was calculated in terms of mmol ASA mg 1 FW min 1. 2.5. Isolation of plasma membrane and tonoplast vesicles Plasma membrane and tonoplast vesicles fraction enriched were isolated from tomato roots and leaves as described by Kasamo (1986) with minor changes. Excised roots and leaves were homogenized (1/2, w/v) with a mortar and pestle in a cold grinding medium containing: 25 mM HEPES–Tris (pH 7.2), 250 mM mannitol, 5 mM EDTA, 5 mM EGTA, 1 mM DTT and 1.5% (w/v) PVP. The whole isolation procedures were carried out at 4 8C. The homogenate was filtered through four layers of cheesecloth and centrifuged at 560 g for 12 min, then the supernatant was centrifuged at 10,000 g for 15 min, and the supernatant was centrifuged at 60,000 g for 30 min to yield a crude membrane
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fraction. The resulting pellet was resuspended with 1 ml in a gradient buffer containing: 20 mM HEPES–Tris (pH 7.5), 5 mM EDTA, 0.5 mM EGTA. The supernatant was layered on top of a step gradient consisting of 1 ml of 45%, 33% and 15% (w/w) sucrose, respectively, and then centrifuged for 2 h at 70,000 g. The tonoplast-enriched fraction was collected at the 15/33% sucrose interface and the plasma membrane-enriched fraction was collected at the 33/45% sucrose interface. Each fraction was centrifuged for 1 h at 100,000 g. The resulting pellet was resuspended in a medium containing: 20 mM HEPES–Tris (pH 7.5), 3 mM MgCl2, 0.5 mM EGTA, 300 mM sucrose, then quickly frozen in liquid nitrogen and stored at 70 8C until used for the enzyme assays. 2.6. Measurement of H+-ATPase in PMs ATP hydrolysis assays were performed as described by Briskin et al. (1987) with minor changes. 0.5 ml the reaction medium containing: 36 mM Tris–Mes (pH 6.5), 30 mM ATP-Na2, 3 mM MgSO4, 1 mM NaN3, 50 mM KNO3, 1 mM Na2MoO4, 0.02% (v/v) Triton X-100, in the presence or absence of 2.5 mM Na3VO4. The reaction was started by adding 50 ml PM vesicles. After 30 min incubation at 37 8C, the reaction was quenched by the addition of 55% (w/v) TCA. The H+-ATPase activity was determined by measuring the release of Pi (Ohinishi et al., 1975). 2.7. Measurement of H+-ATPase and H+-PPase activities in tonoplast H+-ATPase and H+-PPase activities were calculated as the rate of ATP hydrolysis or PPi, respectively, by measuring the amount of released Pi according to Lin and Morales (1977) with minor changes. Activities of tonoplast H+-ATPase were measured in a 0.5 ml reaction medium containing: 36 mM HEPES–Tris (pH 7.5), 30 mM ATP-Na2, 3 mM MgSO4, 0.5 mM NaN3, 50 mM KCl, 0.1 mM Na2MoO4, 0.1 mM Na3VO4, in the presence or absence of 50 mM KNO3. Activities of tonoplast H+-PPase were measured in a 0.5 ml reaction medium containing: 60 mM Tris–HEPES (pH 8.0), 3 mM MgSO4, 5 mM EDTA, 1 mM (NH4)6Mo7O24, 0.5 mM NaN3, 0.1 mM Na3VO4, 30 mM Napyrophosphate, in the presence or absence of 100 mM KCl. The reaction was started by adding 50 ml tonoplast vesicles. After 30 min incubation at 37 8C, the reaction was quenched by 55% TCA and the color developed as in the PM ATPase assay.
Fig. 1. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on shoot fresh weight (A), root fresh weight (B) and shoot height (C) of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
45.6% and 32.1% of control, respectively. Exogenous NO treatment (Cu + SNP) markedly alleviated the inhibition, by 57.3%, 67.3% and 35.9% compared with Cu stress. The alleviating heavy metal stress of exogenous NO was blocked by hemoglobin (Cu + SNP + Hb). Moreover, 100 mM sodium ferrocyanide (Cu + F) and 100 mM sodium nitrate or nitrite (Cu + N) which did not release NO, had no obvious effect on alleviation of heavy metal stress to tomato plants.
2.8. Copper concentration assay 3.2. Hydrogen peroxide concentration and lipid peroxidation Leaf and root samples were oven dried at 70 8C, until they reached a constant weight. At least four plants were analyzed for each treatment. The samples of dried plant material were ashed at 480 8C in a muffle furnace, subsequently digested twice in 10 ml of 3 M HCl at 90 8C, and analyzed by AA370MC flame atomic absorption spectrophotometry. 2.9. Statistics All data presented were means one standard deviation (S.D.) of three replicates. Statistical analyses were performed by analysis of variance (ANOVA) using SAS software. Differences between treatments were separated by the least significant difference (LSD) test at a 0.05 probability level. 3. Results 3.1. Plant growth As shown in Fig. 1, Cu stress decreased shoot fresh weight, root fresh weight and shoot height of tomato plants by 39.1%,
Compared to control, Cu treatment for 8 days significantly increased H2O2 content in tomato roots and leaves by 79% and 121%, respectively. The application of exogenous NO (Cu + SNP) decreased accumulation of H2O2 by 32% and 47% in roots and leaves under heavy metal stress, and the effect of exogenous NO was blocked by addition of hemoglobin (Cu + SNP + Hb). Addition of sodium ferrocyanide (Cu + F) and sodium nitrate or nitrite (Cu + N) had no significant effect on H2O2 content under heavy metal stress (Fig. 2A and B). When plants were subjected to environmental stress, oxidative damage resulted in the level of membrane lipid peroxidation, which could be estimated by MDA content. Similar to H2O2 concentration change, Cu treatment considerably increased MDA content by 71.2% and 80.6% in leaves and roots and the application of NO (Cu + SNP) reduced MDA content by 33.6% and 36.2% in leaves and roots. The effect of exogenous NO was also removed by addition of hemoglobin (Cu + SNP + Hb). Application of sodium ferrocyanide (Cu + F) and sodium nitrate or nitrite (Cu + N) did not markedly affect MDA content (Fig. 2C and D).
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Fig. 2. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on H2O2 content (A), MDA content (C) in leaves and H2O2 content (B), MDA content (D) in roots of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
3.3. Antioxidant enzymes It can be seen that in leaves and roots of tomato plant SOD activity was decreased by 28% and 47% under Cu stress on the 8th day of treatment. Exogenous NO treatment (Cu + SNP) elevated SOD activity by 34% and 82% in leaves and roots. The addition of hemoglobin (Cu + SNP + Hb) removed the effect of exogenous NO, and application of sodium ferrocyanide (Cu + F) and sodium nitrate or nitrite (Cu + N) did not obviously change the SOD activity in Cutreated plants (Fig. 3A and B). Cu treatment inhibited POD activity of both leaves and roots of tomato plant by 28% and 56% after 8 days treatment, and application of NO (Cu + SNP) greatly decreased the inhibition by 42% and 109%, respectively, in leaves and roots. While the addition of hemoglobin removed the effect of exogenous NO, and addition of sodium ferrocyanide and sodium nitrate or nitrite did not obviously change the POD activity (Fig. 3C and D). Similar to the change of POD activity, Cu stress significantly inhibited APX activity by 51% and 53% in both leaves and roots of tomato plant on the 8th day treatment, and exogenous NO (Cu + SNP) greatly decreased the inhibition, by 80% and 95% in leaves and roots compared with Cu stress. APX activity did not show statistically significant differences among Cu treatment, Cu treatment with addition of sodium ferrocyanide, Cu treatment with sodium nitrate or nitrite and Cu treatment with both SNP and hemoglobin (Fig. 3E and F). Contrary to the change of POD and APX activity, CAT activity in both leaves and roots of tomato plant was markedly enhanced by 103% and 54% on the 8th day of treatment by Cu stress. Exogenous NO (Cu + SNP) significantly decreased CAT activity by 45% and 39% in leaves and roots under Cu stress. Moreover, application of hemoglobin with SNP, sodium ferrocyanide and sodium nitrate or nitrite did not significantly influence CAT activity (Fig. 3G and H). 3.4. H+-ATPase activity in plasma membrane H+-ATPase activity in tomato both leaves and roots plasma membrane was determined on the 8th day of treatment. It can be seen that Cu stress inhibited H+-ATPase activity by 43.3% and 53.5%
in both leaves and roots. Application of NO (Cu + SNP) decreased the inhibition by 54.5% and 86.9% in leaves and roots compared with Cu stress, respectively. Addition of sodium ferrocyanide (Cu + F), sodium nitrate or nitrite (Cu + N) and SNP with hemoglobin (Cu + SNP + Hb) had no significant effects on H+ATPase activity in plasma membrane under Cu treatment (Fig. 4A and B). 3.5. H+-ATPase and H+-PPase activity in tonoplast H+-ATPase and H+-PPase activity in tomato both leaves and roots tonoplast was also measured on the 8th day of treatment. In leaves and roots, their activities showed the different change tendency. In leaves, Cu stress promoted tonoplast H+-ATPase and H+-PPase activities by 165% and 141%, respectively. While application of NO (Cu + SNP) decreased tonoplast H+-ATPase and H+-PPase activities, and made them keep at similar level with the control (Fig. 5A and C). Their activity did not show statistically significant differences among Cu treatment, Cu treatment with sodium ferrocyanide, Cu treatment with sodium nitrate or nitrite and Cu treatment with both SNP and hemoglobin. However, in roots, Cu stress inhibited tonoplast H+-ATPase and H+-PPase activities by 57.7% and 57.1%, respectively. Application of NO (Cu + SNP) greatly promoted tonoplast H+-ATPase and H+-PPase activities by 115% and 114% compared with Cu treatment (Fig. 5B and D). 3.6. Cu concentration As shown in Fig. 6, Cu content in both leaves and roots of tomato plant was significantly increased by Cu treatment compared to control (P < 0.05), while application of NO could not markedly affect Cu concentration in both leaves and roots. This observation suggested that exogenous NO supply did not affect Cu uptake. Addition of sodium ferrocyanide and sodium nitrate or nitrite dramatically decreased Cu concentration compared to Cu treatment. Howerever, in roots application of SNP with addition of hemoglobin had no significant effects on Cu content and in leaves application of hemoglobin, sodium ferrocyanide and sodium
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Fig. 3. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on activities of SOD (A), POD (C), CAT (E), APX (G) in leaves and activities of SOD (B), POD (D), CAT (F), APX (H) in roots of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
nitrate or nitrite did not significantly influence Cu content (Fig. 6A and B). 4. Discussion It has been reported that Cu stress could cause an increased production of H2O2 (Weckx and Clijsters, 1996) and induce membrane lipid peroxidation (Mazhoudi et al., 1997). These revealed that Cu treatment greatly induced serious membrane damage in the plant. This experiment showed that oxidative stress of tomato plant induced by excessive Cu was effectively alleviated by application of SNP, while the application of sodium ferrocyanide, sodium nitrate or nitrite and SNP with hemoglobin did not mitigate the oxidative damage. Because SNP is a NO donor, while sodium ferrocyanide is an analog of SNP, sodium nitrate or nitrite is the decomposition product of NO or its donor SNP and hemoglobin is a NO scavenger, therefore, by comparing the results, we could deduce that SNP alleviating the oxidative damage by excessive Cu was ascribed to its releasing NO. As key elements in the defense mechanisms, varying reactions of antioxidative enzymes have been observed under Cu stress conditions in many plants. For example, it has been reported that ROS-scavenging enzyme activity induced by excessive Cu was increased in the case of Cu-tolerant maize (Tanyolac et al., 2007), barley plant (Demirevska-Kepova et al., 2004), tomato shoot
(Martins and Mourato, 2006), beans shoot (Weckx and Clijsters, 1996) and duckweed (Teisseire and Guy, 2000), but was depressed in rice leaves (Chen and Kao, 1999), or was unaffected in tomato roots and stems (Mazhoudi et al., 1997). In the present experiment, activities of SOD, POD and APX in tomato roots and leaves were significantly inhibited on the 8th day by Cu stress, while CAT activity was increased. This result implied that CAT induction might play an important role in tomato tolerance to Cu excess. Some of the observed results were consistent with the reports of Martins and Mourato (2006), and some were not consistent with them. The difference indicated that the influence of Cu stress on the antioxidant enzymes was very complex and related to the plant treatment time, plant tissues, plant species and genotypes. In many studies, it was reported that the function of NO alleviation of oxidative damage was ascribed to induction of various ROS-scavenging enzyme activity (Hsu and Kao, 2004; Hu et al., 2007). In the present work, application of SNP dramatically decreased the inhibited SOD activity by Cu stress. This suggested that application of NO could promote the conversion from O2 into H2O2 and O2, which was an important step in protecting the cell. If the generated H2O2 could not be scavenged efficiently, it could interact with O2 to form highly reactive hydroxyl radicals (OH) that were thought to be primarily responsible for oxygen toxicity in the cell (Shi et al., 2007). Therefore, the efficient scavenging of H2O2 was very important for normal metabolism of
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Fig. 4. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on activity of H+-ATPase in leaves (A) and roots (B) plasma membrane of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
Fig. 6. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on Cu content (A) in leaves and Cu content (B) in roots of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
plant. In the present experiment, application of NO greatly induced the H2O2-scavenging enzymes POD and APX under Cu stress. Similar results had been observed in the experiment of NO increasing the rice leaves tolerance to Cu excess (Yu et al., 2005) and the experiment of NO stimulating wheat seed germination under heavy metal Cu stress (Hu et al., 2007). In this study, Cu stress significantly inhibited PM H+-ATPase activity in both leaves and roots of tomato plant. This would break the electrochemical gradient across the membrane, decrease the ability to keep cell wall acidfication, and inhibit the growth of
plants (Fig. 1). In the meanwhile, V-H+-ATPase and V-H+-PPase activities in roots were greatly inhibited under Cu stress resulted in cytoplasmic H+ concentration increases, and then influenced cytoplasmic pH steady. However, H+-ATPase and H+-PPase activities of tonoplast in tomato leaves were increased, which implied that V-H+-ATPase and V-H+-PPase activitie induction might play an important role in tomato leaves tolerance to Cu stress. Application of NO considerably increased PM H+-ATPase activity in plants, V-H+-ATPase and V-H+-PPase activities in roots. This may facilitate the expulsion of the excess H+, promote the
Fig. 5. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on activities of H+-ATPase (A), H+-PPase (C) in leaf tonoplast and activities of H+-ATPase (B), H+-PPase (D) in root tonoplast of 8 days treatment tomato plants grown in nutrient solutions without or with 50 mM CuCl2.
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cytoplasm alkalinization and renew cell normal activity partly (Michelet and Boutry, 1995). The results indicated that less severe membrane lipid damage occurred in NO-treated roots of tomato under Cu stress because the membrane lipid oxidative injury caused by elevated active oxygen species under Cu stress was markedly alleviated by NO-enhanced activities of antioxidative enzymes scavenging active oxygen species. It was well known that the degree of lipid peroxidation was closely related to the accumulation of ROS, and the lipid process was one important factor exerting an effect on ATPase under changing environmental conditions (Veselov et al., 2002). As a result of lipid peroxidation induced by Cu excess, ATPase and PPase proteins were disturbed. Therefore, exogenous NO decreasing lipid peroxidation should be considered as one cause maintaining higher ATPase activity. The results of this study showed that exogenous NO, when applied along with excess Cu, could improve plant growth and significantly attenuate Cu-induced oxidative stress as indicated by a down regulation in lipid peroxidation and H2O2 concentration in tomato plants. By determining the activities of SOD, POD, APX and CAT, we learned that NO exerted its protective effect through the activation of some antioxidative enzymes, not by preventing Cu uptake. Therefore, lower lipid peroxidation could maintain better function of the plasma membrane and tonoplast, elevate the activities of H+-ATPase, H+-PPase, and mitigate copper toxicity in tomato plants. Acknowledgements This work was supported by the Science Technology Project of Shandong Provincial Education Department (No. 32353) and the Young Scientist Innovation Science Foundation of Shandong Agricultural University (No. 23420). References Ahn, S.J., Sivaguru, M., Osawa, H., Chung, G.C., Matsumoto, H., 2001. Aluminum Inhibits the H+-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots. Plant Physiol. 126, 1381–1390. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–379. Arasimowicz, M., Floryszak-Wieczorek, J., 2007. Nitric oxide as a bioactive signalling molecule in plant stress responses. Plant Sci. 172 (5), 876–887. Boveris, A.D., Galatro, A., Puntarulo, S., 2000. Effect of nitric oxide and plant antioxidants on microsomal content of lipid radicals. Biol. Res. 33, 159–165. Briskin, D.P., Leonard, R.T., Hodges, T.K., 1987. Isolation of the plasma membrane: markers and general principles. Methods Enzymol. 148, 542–558. BurzyhskL, M., Kolano, E., 2003. In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots. Physiol. Plant. 25, 39–45. Cakmak, I., Marschner, H., 1992. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol. 98, 1222–1227. Chen, L.M., Kao, C.H., 1999. Effect of excess copper on rice leaves: evidence for involvement of lipid peroxidation. Bot. Bull. Acad. Sin. 40, 283–287. Demirevska-Kepova, K., Simova-Stoilova, L., Stoyanova, Z., Ho¨lzer, R., Feller, U., 2004. Biochemical changes in barley plants after excessive supply of copper and manganese. Environ. Exp. Bot. 52, 253–266.
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