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Protective effect of nitric oxide on light-induced oxidative damage in leaves of tall fescue
ARTICLE IN PRESS Journal of Plant Physiology 167 (2010) 512–518
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Protective effect of nitric oxide on light-induced oxidative damage in leaves of tall fescue Yuefei Xu a, Xiaoling Sun a, Jingwei Jin b, He Zhou a,n a b
Department of Grassland Science, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China College of Resources and Environment, China Agricultural University, Beijing 100193, China
a r t i c l e in f o
a b s t r a c t
Article history: Received 24 May 2009 Received in revised form 26 October 2009 Accepted 26 October 2009
Nitric oxide (NO) is an important signaling molecule involved in many physiological processes. In this study, the effect of NO on oxidative damage caused by high levels of light was investigated in leaves of two varieties of tall fescue (Arid3 and Houndog5). Leaves of Houndog5 were more susceptible to high-light stress than Arid3 leaves. Pretreatment of these leaves with NO donor sodium nitroprusside (SNP), prior to exposure to high-light stress, resulted in reduced light-induced electrolyte leakage and reduced contents of malondialdehyde, hydrogen peroxide (H2O2) and superoxide radicals (O2 ). The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) increased in both varieties in the presence of SNP under high-light stress, but lipoxygenase (LOX) activity was inhibited. These responses could be reversed by pretreatment with the NO scavenger 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). A pronounced increase in nitric oxide synthase (NOS) activity and NO release was found in light-tolerant Arid3 plants after exposure to high-light stress, while only a small increase was observed in more sensitive Houndog5. Pretreatment with the NOS inhibitor No-nitro-Larginine (LNNA) resulted in increased oxidative damage under high-light stress, with more injuries occurring in Arid3 than Houndog5. These results suggest that high-light stress induced increased NOS activity leading to elevated NO. This NO might act as a signaling molecule triggering enhanced activities of antioxidant enzymes, further protecting against injuries caused by high intensity light. This protective mechanism was found to more efficiently acclimate light-tolerant Arid3 than lightsensitive Houndog5. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Antioxidant enzymes High-light Nitric oxide Oxidative damage Tall fescue
Introduction Light is essential for plant growth and development, but when the amount of absorbed light exceeds the amount required for photosynthesis, the excess light can be harmful because it causes the accumulation of reactive oxygen species (ROS) (Asada, 2006), such as hydrogen peroxide (H2O2), superoxide radical (O 2 ), hydroxyl radical (HO) and singlet oxygen (O12). If these ROS are not removed immediately, they can cause lipid peroxidation and damage to the cell membrane. To avoid ROS-induced cellular
Abbreviations: APX, ascorbate peroxidase; CAT, catalase; GR, glutathione reductase; HL, high-light; H2O2, hydrogen peroxide; LNNA, No-nitro-L-arginine; LOX, lipoxygenase; MDA, malondialdehyde; NO, nitric oxide; NOS, nitric oxide synthase; O2 , superoxide radical; PPFD, photosynthetic photo flux density; PTIO, 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; REL, relative electrolyte leakage; ROS, reactive oxygen species; SNP, sodium nitroprusside; SOD, superoxide dismutase n Corresponding author. Tel./fax: +86 10 62734122. E-mail address: [email protected] (H. Zhou). 0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2009.10.010
injury, plants utilize various antioxidative enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POD: EC 1.11.1.7), ascorbate peroxidase (APX: EC 1.11.1.11), glutathione reductase (GR: EC 1.6.4.2) as well as low molecular weight antioxidants such as ascorbate (ASC), glutathione (GSH), a-tocopherol and flavonoids (Mittler, 2002; Apel and Hirt, 2004). Nitric oxide (NO) is an important inter- and intracellular signaling molecule involved in many plant physiological processes (Lamattina et al., 2003; Lamotte et al., 2005). It is also a reactive nitrogen species, and its concentration-depending effects on different cell types were shown to be either protective or toxic (Beligni and Lamattina, 1999). NO is involved in regulating growth and developmental processes, such as seed germination, de-etiolation, cell senescence and programmed cell death (Beligni and Lamattina, 2000; Neill et al., 2003). Moreover, NO was found to mediate plant responses to abiotic stress, caused by heat, drought, salinity, UV-B radiation or heavy metals (An et al., 2005; Laspina et al., 2005; Song et al., 2006; Shi et al., 2007; Vital et al., 2008; Zhao et al., 2008). In animal cells, most of the NO is
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synthesized by nitric oxide synthase (NOS). Recently, NOS was also detected in plants (Cueto et al., 1996; Barroso et al., 1999; Guo et al., 2003; Crawford, 2006; Corpas et al., 2008; Chaki et al., 2009). Tall fescue (Festuca arundinacea) is a widely used cold-season turf grass. It is often grown under fluctuating photosynthetic photon flux densities due to the impact of vegetation canopies, buildings or different weather patterns. In a preliminary experiment, two varieties of tall fescue, Arid3 and Houndog5, were found to exhibit distinct photo-acclimation behavior. Houndog5 was photobleached under high-light (HL) conditions while Arid3 was not affected, suggesting that Houndog5 is more susceptible to HL stress than Arid3. HL stress leads to an enhanced generation of ROS. Increased activities of ROS-scavenging enzymes were reported under HL stress that mitigated oxidative damage (Burritt and Mackenzie, 2003; Ali et al., 2005a; Jiang et al., 2005). Previous studies also demonstrated that the second messenger NO was able to increase the activity of antioxidant enzymes under different abiotic stress forms, but there is no report on a possible effect of NO on antioxidant enzymes under HL stress. The objective of this study was to elucidate the role of NO (applied exogenous NO or depleted endogenous NO) in alleviating lightinduced oxidative damage in leaves of two varieties of tall fescue (Arid3 and Houndog5).
Materials and methods Plant materials and treatments Seeds of tall fescue [Festuca arundinacea (Schreb.) cvs. Arid3 and Houndog5] were obtained from Beijing Clover Seed & Turf CO. Ltd., China. Seeds were surface sterilized in 1.8% (v/v) sodium hypochlorite, rinsed several times in distilled water, and germinated on moistened filter paper at room temperature for 7 d. Seedlings were selected and placed into 5 L black plastic containers containing 4 L of nutrient solution. Each plastic container contained six plants. Seedlings cultured hydroponically in a continuously aerated nutrient solution containing 4 mM Ca(NO3)2, 4 mM KNO3, 1 mM KH2PO4, 2 mM MgSO4, 46 mM H3BO3, 10 mM MnSO4, 50 mM Fe-EDTA, 1.0 mM ZnSO4, 0.05 mM H2MoO4 and 0.95 mM CuSO4. The nutrient solution pH was adjusted close to 6.5 by adding H2SO4 or KOH. Nutrient solution was renewed once a week. The plants were grown in a plant incubator at a day/night temperature 25/20 1C, a relative humidity of 70%, a day/night regime of 14/10 h and a photosynthetic photo flux density (PPFD) at the height of the plants of 100 mmol m 2 s 1. Light was provided by a fluorescent lamp. Stress treatments were carried out after 21 d of pre-culture. The plants were treated under high light (500 mmol m 2 s 1 PPFD), at the same time, control plants were treated under low light (100 mmol m 2 s 1 PPFD). Sodium nitroprusside (SNP; Sigma, USA) was used as NO donor, and one millimolar (mM) SNP produced the NO concentration with value 0.78 70.06 mM (Wodala et al., 2008). The potassium salt of 2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; Sigma, USA) was used as NO scavenger. No-nitro-L-arginine (LNNA; Sigma, USA) was used as nitric oxide synthase (NOS) inhibitor. SNP at concentrations from 50 to 500 mM, LNNA at 150 mM or PTIO at 200 mM were applied to tall fescue seedlings through the roots incubated (An et al., 2005; Laspina et al., 2005; Sun et al., 2007) in 4 L of nutrient solution (regenerated once a day) with HL treatments. One hundred mM NaCN, NaNO2, NaNO3, ferrocyanide and ferricyanide were used as additional controls. The 21-d-old seedlings were incubated in the solutions for 7 d at a day/night temperature 25/20 1C, a relative humidity of 70%, a day/night
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regime of 14/10 h. After 7 d of treatments, plants were harvested and frozen in liquid nitrogen, and then stored at 80 1C for further analysis. Electrolyte leakage measurement Relative electrolyte leakage (REL) was determined by the modified method according to Song et al. (2006). The fresh leaves (0.5 g) were washed in deionized water and placed in petri dishes with 5 mL of deionized water at 25 1C for 2 h. After the incubation, the conductivity was measured (C1). Then, the samples were boiled for 20 min and conductivity was read again (C2). REL was expressed as a percentage of the total conductivity after boiling (REL%=C1/C2 100). Determination of hydrogen peroxide and superoxide radical Hydrogen peroxide contents were measured according to Veljovic-Jovanovic et al. (2002). Leaves (0.5 g) were ground in liquid nitrogen and the powder was extracted in 2 mL 1 M HClO4 in the presence of 5% PVP. The homogenate was centrifuged at 12,000g for 10 min, and the supernatant was neutralized with 5 M K2CO3 (pH 5.6) in the presence of 0.1 mL of 0.3 M phosphate buffer (pH 5.6).The solution was centrifuged at 12,000g for 1 min, and the sample was incubated for 10 min with 1 U ascorbate oxidase to oxidize ascorbate prior to assay. The reaction mixture contained 0.1 M phosphate buffer (pH 6.5), 3.3 mM DMAB, 0.07 mM MBTH, 0.3 U POX and 200 mL supernatant. Changes in absorbance at 590 nm were monitored at 25 1C. Superoxide radical production rate was determined by the modified method according to Elstner and Heupel (1976). Leaves (1.0 g) were homogenized in 3 mL of 50 mM potassium phosphate buffer (pH 7.8) and centrifuged at 12,000g for 20 min. The incubation mixture contained 1 mL of supernatant, 1 mL of 50 mM potassium phosphate buffer (pH 7.8) and 1 mL of 1 mM hydroxylaminoniumchloride and the mixture was incubated in 25 1C for 20 min. The mixture was subsequently incubated with 2 mL of 17 mM sulphanilic acid and 2 mL of 7 mM a-naphthyl amine at 25 1C for 20 min. The final solution was mixed with an equal volume of ethyl ether, and the absorbance of the pink phase was read at 530 nm. The production rate of superoxide radical was calculated based on a standard curve. Analysis of lipid peroxidation Membrane lipid peroxidation was estimated by the level of malondialdehyde production with a slight modification of the thiobarbituric acid method of Buege and Aust (1978). Leaves (0.5 g) were homogenized with a mortar and pestle in 10% trichloroacetic acid, and then the homogenate was centrifuged at 4000g for 30 min. A 2 mL aliquot of supernatant was mixed with 2 mL of 10% trichloroacetic acid containing 0.5% thiobarbituric acid. The mixture was heated at 100 1C for 30 min. The absorbance of the supernatant was measured at 532 nm with a reading at 600 nm subtracted from it to account for non-specific turbidity. Antioxidant enzyme activity Leaves (1.0 g) were homogenized with a mortar and pestle at 4 1C in 5 mL 50 mM phosphate buffer (pH 7.8) containing 1 mM EDTA and 2% PVP. The homogenate was centrifuged at 12,000g for 20 min at 4 1C and the supernatant was used for the following enzyme activity assays. The protein contents in the supernatant
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were determined according to the method of Bradford (1976) with BSA as standard. Total superoxide dismutase (SOD) activity was measured by NBT method of Beauchamp and Fridovich (1971). One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm. Catalase (CAT) activity was determined by following the consumption of H2O2 at 240 nm (E= 39.4 mM 1 cm 1) by the method of Aebi (1984). Ascorbate peroxidase (APX) activity was determined according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm (E =2.8 mM 1 cm 1). Glutathione reductase (GR) activity was measured according to the method of Foyer and Halliwell (1976), which depends on the rate of decrease in the absorbance of NADPH at 340 nm (E=6.2 mM 1 cm 1). LOX activity assay Lipoxygenase activity (LOX, EC 1.13.11.12) was determined according to Page et al. (2001) with some modification. Leaves (1.0 g) were ground in 1 mL homogenizing buffer containing 50 mM phosphate buffer (pH 6.5), 10% PVP, 0.25% Triton X-100 and 1 mM PMSF. The 1 mL reaction mixture contained 50 mM Tris–HCl buffer (pH 6.5), 0.4 mM linoleic acid and 10 mL extract. The absorbance was measured at 234 nm (E =25 mM 1 cm 1). NO content and NOS activity determination NO content determination was performed according to Murphy and Noack (1994) with some modifications. The fresh leaves (0.5 g) were incubated with 100 units of catalase and 100 units of superoxide dismutase for 5 min to remove endogenous ROS before addition of 5 mL oxyhaemoglobin (5 mM). After 2 min incubation, NO concentrations were estimated by following the conversion of oxyhaemoglobin to methaemoglobin spectrophotometrically at 577 and 591 nm. NOS activity was determined according to the method of Murphy and Noack (1994) with some modifications. Leaves (1.0 g) were homogenized in 2 mL of homogenization buffer (50 mM pH 7.4 Tris–HCl containing 0.5 mM EDTA, 1 mM leupeptin, 1 mM pepstatin, 7 mM gluathione and 0.2 mM PMSF). After centrifuging at 10,000g for 20 min (4 1C), the supernatant was collected and recentrifuged at 100,000g for 45 min. The supernatant was used for NOS determination. Reaction mixture (total volume of 1 mL) in 10 mM Hepes (pH 7.0) contained 200 mL enzyme extract, 1 mM arginine, 1 mM magnesium diacetate, 1 mM CaCl2, 1 mM CaM and 4 mM FAD. This was incubated at 37 1C for 30 min. Reactions were stopped by heat inactivation at 50 1C for 10 min. To ensure that this assay accurately reflected NOS activity, duplicate reactions were performed in presence or absence of nitro-L-arginine. Since this arginine analog inhibits NOS activity, any metHb generated in its presence was produced by non-specific HbO2 oxidation. NOS activity was determined by subtracting the amount of metHb produced in the presence of this inhibitor from the amount generated in its absence. The reaction was started by addition of NADPH to 100 mM and H4B to 10 mM. The change in absorbance was recorded at 401 nm (Chandok et al., 2003). Statistical analysis Each experiment was repeated at least three times. Values were expressed as means7SD. Statistical analyses were performed by analysis of variance (ANOVA). Means were separated using Duncan0 s multiple range test at 5% level of significance.
Results Effect of NO on electrolyte leakage Electrolyte leakage was remarkably enhanced in both plants 7 d after of HL treatments (Fig. 1A). Electrolyte leakage in Arid3 and Houndog5 leaves increased by 36.1% and 81.4%, respectively. Treatments of plant leaves with NO donor sodium nitroprusside (SNP) before HL stress resulted in a significant decrease of electrolyte leakage in leaves of Arid3 and Houndog5. NO scavenger 2-(4-carboxy-2-phenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide (PTIO) was utilized to further clarify the role of SNP. The results indicate that addition of PTIO or SNP +PTIO enhanced electrolyte leakage to similar levels in both plants leaves under HL stress; in other words, PTIO scavenged endogenous NO as well as exogenous NO supplied by SNP. Supplementation with NOS inhibitor LNNA strongly enhanced electrolyte leakage in leaves of Arid3, but had no effect on Houndog5 leaves, indicating that significant activated NOS existed in Arid3 leaves under HL stress, but not in leaves of Houndog5, and that application of LNNA inhibited NOS activity in Arid3 leaves. Apart from NO, SNP may also generate other residual products, such as NaCN, NaNO2, NaNO3, ferrocyanide and ferricyanide. To determine which product functions in reducing electrolyte leakage, 100 mM NaCN, NaNO2, NaNO3, ferrocyanide and ferricyanide were added separately. The results showed that none of the above-mentioned residual products had remarkable effects on electrolyte leakage in both plants leaves under HL stress (Fig. 1B). Since the effects of NO on plants are concentration dependent, different SNP concentrations (50–500 mM) were applied. The results show that SNP concentrations from 50 to 300 mM alleviated electrolyte leakage, with 100 mM being most effective under HL stress. However, 500 mM SNP was found to be toxic, leading to a great increase of electrolyte leakage in both plants leaves under HL stress (Fig. 1C). As a result, in the following experiments, we applied 100 mM SNP to as the NO donor. Effect of NO on lipid peroxidation The content of malondialdehyde (MDA) is an indicator of lipid peroxidation and oxidative damage to membrane. Transfer to HL increased MDA contents by 42.9% and 111.2% in leaves of Arid3 and Houndog5, respectively. Supplementation with NO donor, SNP before HL stress remarkably (at P o0.05) reduced MDA contents in both plants, but when NO was removed or NOS was inhibited (LNNA, PTIO or SNP +PTIO addition) MDA contents rose evidently, indicating that severe lipid peroxidation was caused (Fig. 1D). Effect of NO on H2O2 and O 2 production HL stress caused significant accumulations (at Po0.05) of H2O2 and O 2 in leaves of tall fescue (Fig. 2). Seven days after HL treatments, levels of H2O2 and O 2 increased by 66.7% and 63.6% in leaves of Arid3, 200.3% and 166.3% in Houndog5 leaves, respectively. Supplementation with SNP effectively reduced production, while addition of LNNA, PTIO or H2O2 and O 2 levels, especially in Arid3 SNP+PTIO increased H2O2 and O 2 leaves. Effect of NO on antioxidative enzymes All antioxidant enzymes measured showed increased activities 7 d after HL treatments, especially in Arid3 leaves (Fig. 3). The
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Fig. 1. (A) Effect of NO on electrolyte leakage in leaves of Arid3 and Houndog5 under high-light stress. CK, 100 mmol m 2 s 1 PPFD; HL, 500 mmol m 2 s 1 PPFD; HL+ SNP, HL+ 100 mM SNP; HL+ LNNA, HL +150 mM LNNA; HL +PTIO, HL+ 200 mM PTIO; HL+ SNP+ PTIO, HL +100 mM SNP+ 200 mM PTIO. (B) Effect of residual products of NO decomposition on electrolyte leakage in leaves of Arid3 and Houndog5 under high-light stress. (C) Changes of electrolyte leakage in the presence of different SNP concentrations in leaves of Arid3 and Houndog5 under high-light stress. (D) Effect of NO on MDA contents in leaves of Arid3 and Houndog5 under high-light stress. Mean value7 SD (n= 3). Bars with different letters are significantly different at the 5% level.
Fig. 2. Effect of NO on levels of H2O2 (A) and superoxide radical (B) in leaves of Arid3 and Houndog5 under high-light stress. Mean value 7SD (n =3). Bars with different letters are significantly different at the 5% level.
activities of SOD, CAT, APX and GR increased by 78.0%, 153.3%, 57.3%, 57.6% and 14.3%, 34.4%, 10.1%, 13.9% in leaves of Arid3 and Houndog5, respectively. Pretreatment with SNP remarkably increased SOD, CAT, APX and GR activities (at Po0.05), especially in Houndog5 leaves; While LNNA, PTIO or SNP+ PTIO addition greatly reduced these antioxidant enzymes activities, especially in Arid3 leaves.
both plants, whereas supplementation with NOS inhibitor (LNNA), NO scavenger (PTIO) or both (SNP+PTIO) elevated LOX activities by 56.1%, 59.4% and 65.0% in leaves of Arid3, respectively, but had little effect on those in Houndog5 leaves. It was evident that both NO and NOS were involved in the decrease of LOX activity, apparently little NO or NOS exist in Houndog5 leaves under HL stress (Fig. 4).
Effect of NO on lipoxygenase (LOX) activity
NO production and NOS activity
Transfer to HL also caused increased LOX activity, 30.7% and 90.3% greater than the control in leaves of Arid3 and Houndog5, respectively. Addition of NO donor, SNP reduced LOX activity in
To further elucidate the relationship between NO release and HL stress, NO production and NOS activity were measured. HL stress greatly enhanced NOS activity and NO generation in leaves
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Fig. 3. Effect of NO on the activities of antioxidant enzymes in leaves of Arid3 and Houndog5 under high-light stress: (A) SOD, (B) CAT, (C) APX and (D) GR. Mean value7 SD (n= 3). Bars with different letters are significantly different at the 5% level.
Fig. 4. Effect of NO on LOX activity in leaves of arid3 and Houndog5 under highlight stress. Mean value7 SD (n= 3). Bars with different letters are significantly different at the 5% level
of Arid3, but had no significant effect on Houndog5 leaves (Fig. 5A). Application of NO scavenger PTIO merely reduced NO content, whereas NOS inhibitor LNNA reduced NOS activity as well as NO release, revealing that NOS is essential for NO synthesis, and NO release was repressed when NOS activity was inhibited (Fig. 5B).
Discussion Photoinhibition occurs when plants are exposed to a photosynthetic photo flux density (PPFD) higher than that required for
CO2 fixation, which leads to increased ROS generation (Asada, 2006). Seven days HL treatments gave rise to increased H2O2 and formation, lipid peroxidation and membrane damage, O 2 especially in leaves of Houndog5 (Fig. 1A and D). Similar observations were made by Ali et al. (2005a) in Phalaenopsis. LOX is another important initiator of membrane damage and responsible for lipid peroxidation and the formation superoxide (Saravitz and Siedow, 1996; Ali et al., 2005b). HL stress enhanced LOX activity in both plants, especially in Houndog5 leaves (Fig. 4). As a result, it can be concluded that Houndog5 is more susceptible to HL stress than Arid3, and this result is in agreement with our observation in a preliminary experiment. Tolerance to HL stress is viewed as being associated with an increase in antioxidant enzyme activities (Burritt and Mackenzie, 2003; Jiang et al., 2005). Both varieties of tall fescue exhibited increases in the activities of antioxidant enzymes (SOD, CAT, APX and GR). SOD is a major scavenger of O 2 , catalyzing the dismutation of superoxide radicals to H2O2 and O2. CAT, APX and GR are important H2O2 detoxifying enzymes. Increases in the activities of all antioxidant enzymes suggest that ROS induced these increases in different cellular compartments (Logan et al., 1998). Under HL stress, a greater increase in antioxidant enzymes activities and lower levels of ROS were found in Arid3 leaves than in Houndog5 leaves, indicating that Arid3 alleviated oxidative injuries through raising antioxidant enzymes activities to scavenge newly produced ROS. NO can counteract oxidative damage and displays a protective effect against various stressful conditions (Arasimowicz and Floryszak-Wieczorek, 2007). Addition of exogenous NO with SNP significantly enhanced antioxidant enzymes activities and reduced ROS levels and LOX activities, prevented lipid peroxidation and membrane damage, whereas a reversed pattern was found when the NO scavenger PTIO was applied. This is consistent with the postulated role of NO as a signaling molecule involved in inducing increases in the activities of antioxidant enzymes
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Fig. 5. (A) NO production and (B) NOS activity in leaves of Arid3 and Houndog5 under high-light stress. Mean value 7 SD (n= 3). Bars with different letters are significantly different at the 5% level.
(Laspina et al., 2005; Shi et al., 2007; Zhang et al., 2009) and decrease in LOX activity (Song et al., 2006; Zhao et al., 2008) to inhibit lipid peroxidation and membrane damage caused by environmental stresses. Additionally, NO itself can detoxify ROS, and generating the peroxynitrite such as reacting with O 2 ion (ONOO ). In the physiological pH range, ONOO can be protonated and decomposes to a nitrate anion and a proton, or react with H2O2 to yield a nitrite anion and oxygen (Martinez et al., 2000; Wendehenne et al., 2001). Recent research also revealed that NO can induce APX and CAT antioxidant genes in Arabidopsis suspension cells (Huang et al., 2002). Arid3 suffered severe peroxidative damage when endogenous NO was removed by PTIO, which confirms the role of NO in photoprotection. On the other hand, NO was also found to inhibit photosynthetic electron transport and modulate reaction-center-associated non-photochemical quenching in plants (Wodala et al., 2008). Animal cells synthesize NO primarily through the activity of NOS. However, at present, the mechanism of NO production in plant cells is still a controversial issue (Corpas et al., 2006, Crawford et al., 2006, Zemojtel et al., 2006; Moreau et al., 2008). Previous studies have shown that the NO pool in plants includes enzymatic sources like NOS-like enzymes and nitrate reductase (NR), and non-enzymatic sources (Neill et al., 2003). NR was shown to be involved in NO production in several physiological situations, such as ABA-induced stomatal closure (Bright et al., 2006) and auxin-induced lateral root development (Kolbert et al., 2008). However, most reports support that NO generation is mainly mediated by a putative NOS-like enzyme, which catalyses the formation of NO from L-arginine, rather than NR (Tian et al., 2007; Corpas et al., 2008). Furthermore, several studies have identified NOS-dependent NO generation during plants exposure to stress conditions (Corpas et al., 2006, 2008; Valderrama et al., 2007; Zhao et al., 2007; Chaki et al., 2009). LNNA, an inhibitor of NOS, was reported to reduce NOS activity and NO release in Arabidopsis and Pisum (Qu et al., 2006; Tian et al., 2007; Zhao et al., 2007; Zhang et al., 2009). Application of LNNA severely reduced NOS activity and, thus, inhibited NO synthesis (Fig. 5), which eventually led to oxidative damage. This indicates that the LNNA-sensitive NOS-like enzyme is responsible for the majority of NO production in leaves of tall fescue. NOS activity was also influenced by exogenous NO application. Addition of SNP slightly reduced NOS activity, and this might be due to the negative feedback regulation of NOS by NO, as has been observed in maize and pea under UV-B irradiation (Zhang et al., 2003; An et al., 2005; Qu et al., 2006). The gene(s) encoding NOS in plants is still not known. AtNOS1 of Arabidopsis was initially reported to encode a protein containing NOS activity (Guo et al., 2003). However, this role of AtNOS1
has recently been questioned (Zemojtel et al., 2006), but Crawford et al. (2006) in their reply confirmed that the involvement of AtNOS1 in the synthesis or accumulation of NO might be direct or indirect. AtNOS1 was renamed AtNOA1 (for NITRIC OXIDE ASSOCIATED PROTEIN 1) to avoid confusion with animal NOS genes. Recently, Zhao et al. (2007) confirmed the presence of decreased NOS activity and NO production in the Arabidopsis noa1 mutant. However, Moreau et al. (2008) demonstrated that AtNOA1 was not a NOS, but specifically binds and hydrolyzes GTP, a function that is necessary for the complementation of the knockout mutant. These controversial results indicate that the search for the elusive plant NOS is not over yet. In conclusion, ROS metabolism is important for tall fescue during acclimatization to HL stress. The acquisition of tolerance to HL stress by two varieties of tall fescue is owed to significantly increased antioxidant enzymes activities. As a bioactive antioxidant, NO protects tall fescue leaves against light-induced oxidative damage by reacting with ROS directly or inducing activities of ROS-scavenging enzymes, especially in Arid3 plants, which are better adapted to HL stress.
Acknowledgement This work was supported by Key Projects in the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (No. 2006BAD16B09-2).
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Protective effect of nitric oxide on light-induced oxidative damage in leaves of tall fescue Yuefei Xu a, Xiaoling Sun a, Jingwei Jin b, He Zhou a,n a b
Department of Grassland Science, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China College of Resources and Environment, China Agricultural University, Beijing 100193, China
a r t i c l e in f o
a b s t r a c t
Article history: Received 24 May 2009 Received in revised form 26 October 2009 Accepted 26 October 2009
Nitric oxide (NO) is an important signaling molecule involved in many physiological processes. In this study, the effect of NO on oxidative damage caused by high levels of light was investigated in leaves of two varieties of tall fescue (Arid3 and Houndog5). Leaves of Houndog5 were more susceptible to high-light stress than Arid3 leaves. Pretreatment of these leaves with NO donor sodium nitroprusside (SNP), prior to exposure to high-light stress, resulted in reduced light-induced electrolyte leakage and reduced contents of malondialdehyde, hydrogen peroxide (H2O2) and superoxide radicals (O2 ). The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) increased in both varieties in the presence of SNP under high-light stress, but lipoxygenase (LOX) activity was inhibited. These responses could be reversed by pretreatment with the NO scavenger 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). A pronounced increase in nitric oxide synthase (NOS) activity and NO release was found in light-tolerant Arid3 plants after exposure to high-light stress, while only a small increase was observed in more sensitive Houndog5. Pretreatment with the NOS inhibitor No-nitro-Larginine (LNNA) resulted in increased oxidative damage under high-light stress, with more injuries occurring in Arid3 than Houndog5. These results suggest that high-light stress induced increased NOS activity leading to elevated NO. This NO might act as a signaling molecule triggering enhanced activities of antioxidant enzymes, further protecting against injuries caused by high intensity light. This protective mechanism was found to more efficiently acclimate light-tolerant Arid3 than lightsensitive Houndog5. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Antioxidant enzymes High-light Nitric oxide Oxidative damage Tall fescue
Introduction Light is essential for plant growth and development, but when the amount of absorbed light exceeds the amount required for photosynthesis, the excess light can be harmful because it causes the accumulation of reactive oxygen species (ROS) (Asada, 2006), such as hydrogen peroxide (H2O2), superoxide radical (O 2 ), hydroxyl radical (HO) and singlet oxygen (O12). If these ROS are not removed immediately, they can cause lipid peroxidation and damage to the cell membrane. To avoid ROS-induced cellular
Abbreviations: APX, ascorbate peroxidase; CAT, catalase; GR, glutathione reductase; HL, high-light; H2O2, hydrogen peroxide; LNNA, No-nitro-L-arginine; LOX, lipoxygenase; MDA, malondialdehyde; NO, nitric oxide; NOS, nitric oxide synthase; O2 , superoxide radical; PPFD, photosynthetic photo flux density; PTIO, 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; REL, relative electrolyte leakage; ROS, reactive oxygen species; SNP, sodium nitroprusside; SOD, superoxide dismutase n Corresponding author. Tel./fax: +86 10 62734122. E-mail address: [email protected] (H. Zhou). 0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2009.10.010
injury, plants utilize various antioxidative enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POD: EC 1.11.1.7), ascorbate peroxidase (APX: EC 1.11.1.11), glutathione reductase (GR: EC 1.6.4.2) as well as low molecular weight antioxidants such as ascorbate (ASC), glutathione (GSH), a-tocopherol and flavonoids (Mittler, 2002; Apel and Hirt, 2004). Nitric oxide (NO) is an important inter- and intracellular signaling molecule involved in many plant physiological processes (Lamattina et al., 2003; Lamotte et al., 2005). It is also a reactive nitrogen species, and its concentration-depending effects on different cell types were shown to be either protective or toxic (Beligni and Lamattina, 1999). NO is involved in regulating growth and developmental processes, such as seed germination, de-etiolation, cell senescence and programmed cell death (Beligni and Lamattina, 2000; Neill et al., 2003). Moreover, NO was found to mediate plant responses to abiotic stress, caused by heat, drought, salinity, UV-B radiation or heavy metals (An et al., 2005; Laspina et al., 2005; Song et al., 2006; Shi et al., 2007; Vital et al., 2008; Zhao et al., 2008). In animal cells, most of the NO is
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synthesized by nitric oxide synthase (NOS). Recently, NOS was also detected in plants (Cueto et al., 1996; Barroso et al., 1999; Guo et al., 2003; Crawford, 2006; Corpas et al., 2008; Chaki et al., 2009). Tall fescue (Festuca arundinacea) is a widely used cold-season turf grass. It is often grown under fluctuating photosynthetic photon flux densities due to the impact of vegetation canopies, buildings or different weather patterns. In a preliminary experiment, two varieties of tall fescue, Arid3 and Houndog5, were found to exhibit distinct photo-acclimation behavior. Houndog5 was photobleached under high-light (HL) conditions while Arid3 was not affected, suggesting that Houndog5 is more susceptible to HL stress than Arid3. HL stress leads to an enhanced generation of ROS. Increased activities of ROS-scavenging enzymes were reported under HL stress that mitigated oxidative damage (Burritt and Mackenzie, 2003; Ali et al., 2005a; Jiang et al., 2005). Previous studies also demonstrated that the second messenger NO was able to increase the activity of antioxidant enzymes under different abiotic stress forms, but there is no report on a possible effect of NO on antioxidant enzymes under HL stress. The objective of this study was to elucidate the role of NO (applied exogenous NO or depleted endogenous NO) in alleviating lightinduced oxidative damage in leaves of two varieties of tall fescue (Arid3 and Houndog5).
Materials and methods Plant materials and treatments Seeds of tall fescue [Festuca arundinacea (Schreb.) cvs. Arid3 and Houndog5] were obtained from Beijing Clover Seed & Turf CO. Ltd., China. Seeds were surface sterilized in 1.8% (v/v) sodium hypochlorite, rinsed several times in distilled water, and germinated on moistened filter paper at room temperature for 7 d. Seedlings were selected and placed into 5 L black plastic containers containing 4 L of nutrient solution. Each plastic container contained six plants. Seedlings cultured hydroponically in a continuously aerated nutrient solution containing 4 mM Ca(NO3)2, 4 mM KNO3, 1 mM KH2PO4, 2 mM MgSO4, 46 mM H3BO3, 10 mM MnSO4, 50 mM Fe-EDTA, 1.0 mM ZnSO4, 0.05 mM H2MoO4 and 0.95 mM CuSO4. The nutrient solution pH was adjusted close to 6.5 by adding H2SO4 or KOH. Nutrient solution was renewed once a week. The plants were grown in a plant incubator at a day/night temperature 25/20 1C, a relative humidity of 70%, a day/night regime of 14/10 h and a photosynthetic photo flux density (PPFD) at the height of the plants of 100 mmol m 2 s 1. Light was provided by a fluorescent lamp. Stress treatments were carried out after 21 d of pre-culture. The plants were treated under high light (500 mmol m 2 s 1 PPFD), at the same time, control plants were treated under low light (100 mmol m 2 s 1 PPFD). Sodium nitroprusside (SNP; Sigma, USA) was used as NO donor, and one millimolar (mM) SNP produced the NO concentration with value 0.78 70.06 mM (Wodala et al., 2008). The potassium salt of 2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; Sigma, USA) was used as NO scavenger. No-nitro-L-arginine (LNNA; Sigma, USA) was used as nitric oxide synthase (NOS) inhibitor. SNP at concentrations from 50 to 500 mM, LNNA at 150 mM or PTIO at 200 mM were applied to tall fescue seedlings through the roots incubated (An et al., 2005; Laspina et al., 2005; Sun et al., 2007) in 4 L of nutrient solution (regenerated once a day) with HL treatments. One hundred mM NaCN, NaNO2, NaNO3, ferrocyanide and ferricyanide were used as additional controls. The 21-d-old seedlings were incubated in the solutions for 7 d at a day/night temperature 25/20 1C, a relative humidity of 70%, a day/night
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regime of 14/10 h. After 7 d of treatments, plants were harvested and frozen in liquid nitrogen, and then stored at 80 1C for further analysis. Electrolyte leakage measurement Relative electrolyte leakage (REL) was determined by the modified method according to Song et al. (2006). The fresh leaves (0.5 g) were washed in deionized water and placed in petri dishes with 5 mL of deionized water at 25 1C for 2 h. After the incubation, the conductivity was measured (C1). Then, the samples were boiled for 20 min and conductivity was read again (C2). REL was expressed as a percentage of the total conductivity after boiling (REL%=C1/C2 100). Determination of hydrogen peroxide and superoxide radical Hydrogen peroxide contents were measured according to Veljovic-Jovanovic et al. (2002). Leaves (0.5 g) were ground in liquid nitrogen and the powder was extracted in 2 mL 1 M HClO4 in the presence of 5% PVP. The homogenate was centrifuged at 12,000g for 10 min, and the supernatant was neutralized with 5 M K2CO3 (pH 5.6) in the presence of 0.1 mL of 0.3 M phosphate buffer (pH 5.6).The solution was centrifuged at 12,000g for 1 min, and the sample was incubated for 10 min with 1 U ascorbate oxidase to oxidize ascorbate prior to assay. The reaction mixture contained 0.1 M phosphate buffer (pH 6.5), 3.3 mM DMAB, 0.07 mM MBTH, 0.3 U POX and 200 mL supernatant. Changes in absorbance at 590 nm were monitored at 25 1C. Superoxide radical production rate was determined by the modified method according to Elstner and Heupel (1976). Leaves (1.0 g) were homogenized in 3 mL of 50 mM potassium phosphate buffer (pH 7.8) and centrifuged at 12,000g for 20 min. The incubation mixture contained 1 mL of supernatant, 1 mL of 50 mM potassium phosphate buffer (pH 7.8) and 1 mL of 1 mM hydroxylaminoniumchloride and the mixture was incubated in 25 1C for 20 min. The mixture was subsequently incubated with 2 mL of 17 mM sulphanilic acid and 2 mL of 7 mM a-naphthyl amine at 25 1C for 20 min. The final solution was mixed with an equal volume of ethyl ether, and the absorbance of the pink phase was read at 530 nm. The production rate of superoxide radical was calculated based on a standard curve. Analysis of lipid peroxidation Membrane lipid peroxidation was estimated by the level of malondialdehyde production with a slight modification of the thiobarbituric acid method of Buege and Aust (1978). Leaves (0.5 g) were homogenized with a mortar and pestle in 10% trichloroacetic acid, and then the homogenate was centrifuged at 4000g for 30 min. A 2 mL aliquot of supernatant was mixed with 2 mL of 10% trichloroacetic acid containing 0.5% thiobarbituric acid. The mixture was heated at 100 1C for 30 min. The absorbance of the supernatant was measured at 532 nm with a reading at 600 nm subtracted from it to account for non-specific turbidity. Antioxidant enzyme activity Leaves (1.0 g) were homogenized with a mortar and pestle at 4 1C in 5 mL 50 mM phosphate buffer (pH 7.8) containing 1 mM EDTA and 2% PVP. The homogenate was centrifuged at 12,000g for 20 min at 4 1C and the supernatant was used for the following enzyme activity assays. The protein contents in the supernatant
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were determined according to the method of Bradford (1976) with BSA as standard. Total superoxide dismutase (SOD) activity was measured by NBT method of Beauchamp and Fridovich (1971). One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm. Catalase (CAT) activity was determined by following the consumption of H2O2 at 240 nm (E= 39.4 mM 1 cm 1) by the method of Aebi (1984). Ascorbate peroxidase (APX) activity was determined according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm (E =2.8 mM 1 cm 1). Glutathione reductase (GR) activity was measured according to the method of Foyer and Halliwell (1976), which depends on the rate of decrease in the absorbance of NADPH at 340 nm (E=6.2 mM 1 cm 1). LOX activity assay Lipoxygenase activity (LOX, EC 1.13.11.12) was determined according to Page et al. (2001) with some modification. Leaves (1.0 g) were ground in 1 mL homogenizing buffer containing 50 mM phosphate buffer (pH 6.5), 10% PVP, 0.25% Triton X-100 and 1 mM PMSF. The 1 mL reaction mixture contained 50 mM Tris–HCl buffer (pH 6.5), 0.4 mM linoleic acid and 10 mL extract. The absorbance was measured at 234 nm (E =25 mM 1 cm 1). NO content and NOS activity determination NO content determination was performed according to Murphy and Noack (1994) with some modifications. The fresh leaves (0.5 g) were incubated with 100 units of catalase and 100 units of superoxide dismutase for 5 min to remove endogenous ROS before addition of 5 mL oxyhaemoglobin (5 mM). After 2 min incubation, NO concentrations were estimated by following the conversion of oxyhaemoglobin to methaemoglobin spectrophotometrically at 577 and 591 nm. NOS activity was determined according to the method of Murphy and Noack (1994) with some modifications. Leaves (1.0 g) were homogenized in 2 mL of homogenization buffer (50 mM pH 7.4 Tris–HCl containing 0.5 mM EDTA, 1 mM leupeptin, 1 mM pepstatin, 7 mM gluathione and 0.2 mM PMSF). After centrifuging at 10,000g for 20 min (4 1C), the supernatant was collected and recentrifuged at 100,000g for 45 min. The supernatant was used for NOS determination. Reaction mixture (total volume of 1 mL) in 10 mM Hepes (pH 7.0) contained 200 mL enzyme extract, 1 mM arginine, 1 mM magnesium diacetate, 1 mM CaCl2, 1 mM CaM and 4 mM FAD. This was incubated at 37 1C for 30 min. Reactions were stopped by heat inactivation at 50 1C for 10 min. To ensure that this assay accurately reflected NOS activity, duplicate reactions were performed in presence or absence of nitro-L-arginine. Since this arginine analog inhibits NOS activity, any metHb generated in its presence was produced by non-specific HbO2 oxidation. NOS activity was determined by subtracting the amount of metHb produced in the presence of this inhibitor from the amount generated in its absence. The reaction was started by addition of NADPH to 100 mM and H4B to 10 mM. The change in absorbance was recorded at 401 nm (Chandok et al., 2003). Statistical analysis Each experiment was repeated at least three times. Values were expressed as means7SD. Statistical analyses were performed by analysis of variance (ANOVA). Means were separated using Duncan0 s multiple range test at 5% level of significance.
Results Effect of NO on electrolyte leakage Electrolyte leakage was remarkably enhanced in both plants 7 d after of HL treatments (Fig. 1A). Electrolyte leakage in Arid3 and Houndog5 leaves increased by 36.1% and 81.4%, respectively. Treatments of plant leaves with NO donor sodium nitroprusside (SNP) before HL stress resulted in a significant decrease of electrolyte leakage in leaves of Arid3 and Houndog5. NO scavenger 2-(4-carboxy-2-phenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide (PTIO) was utilized to further clarify the role of SNP. The results indicate that addition of PTIO or SNP +PTIO enhanced electrolyte leakage to similar levels in both plants leaves under HL stress; in other words, PTIO scavenged endogenous NO as well as exogenous NO supplied by SNP. Supplementation with NOS inhibitor LNNA strongly enhanced electrolyte leakage in leaves of Arid3, but had no effect on Houndog5 leaves, indicating that significant activated NOS existed in Arid3 leaves under HL stress, but not in leaves of Houndog5, and that application of LNNA inhibited NOS activity in Arid3 leaves. Apart from NO, SNP may also generate other residual products, such as NaCN, NaNO2, NaNO3, ferrocyanide and ferricyanide. To determine which product functions in reducing electrolyte leakage, 100 mM NaCN, NaNO2, NaNO3, ferrocyanide and ferricyanide were added separately. The results showed that none of the above-mentioned residual products had remarkable effects on electrolyte leakage in both plants leaves under HL stress (Fig. 1B). Since the effects of NO on plants are concentration dependent, different SNP concentrations (50–500 mM) were applied. The results show that SNP concentrations from 50 to 300 mM alleviated electrolyte leakage, with 100 mM being most effective under HL stress. However, 500 mM SNP was found to be toxic, leading to a great increase of electrolyte leakage in both plants leaves under HL stress (Fig. 1C). As a result, in the following experiments, we applied 100 mM SNP to as the NO donor. Effect of NO on lipid peroxidation The content of malondialdehyde (MDA) is an indicator of lipid peroxidation and oxidative damage to membrane. Transfer to HL increased MDA contents by 42.9% and 111.2% in leaves of Arid3 and Houndog5, respectively. Supplementation with NO donor, SNP before HL stress remarkably (at P o0.05) reduced MDA contents in both plants, but when NO was removed or NOS was inhibited (LNNA, PTIO or SNP +PTIO addition) MDA contents rose evidently, indicating that severe lipid peroxidation was caused (Fig. 1D). Effect of NO on H2O2 and O 2 production HL stress caused significant accumulations (at Po0.05) of H2O2 and O 2 in leaves of tall fescue (Fig. 2). Seven days after HL treatments, levels of H2O2 and O 2 increased by 66.7% and 63.6% in leaves of Arid3, 200.3% and 166.3% in Houndog5 leaves, respectively. Supplementation with SNP effectively reduced production, while addition of LNNA, PTIO or H2O2 and O 2 levels, especially in Arid3 SNP+PTIO increased H2O2 and O 2 leaves. Effect of NO on antioxidative enzymes All antioxidant enzymes measured showed increased activities 7 d after HL treatments, especially in Arid3 leaves (Fig. 3). The
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Fig. 1. (A) Effect of NO on electrolyte leakage in leaves of Arid3 and Houndog5 under high-light stress. CK, 100 mmol m 2 s 1 PPFD; HL, 500 mmol m 2 s 1 PPFD; HL+ SNP, HL+ 100 mM SNP; HL+ LNNA, HL +150 mM LNNA; HL +PTIO, HL+ 200 mM PTIO; HL+ SNP+ PTIO, HL +100 mM SNP+ 200 mM PTIO. (B) Effect of residual products of NO decomposition on electrolyte leakage in leaves of Arid3 and Houndog5 under high-light stress. (C) Changes of electrolyte leakage in the presence of different SNP concentrations in leaves of Arid3 and Houndog5 under high-light stress. (D) Effect of NO on MDA contents in leaves of Arid3 and Houndog5 under high-light stress. Mean value7 SD (n= 3). Bars with different letters are significantly different at the 5% level.
Fig. 2. Effect of NO on levels of H2O2 (A) and superoxide radical (B) in leaves of Arid3 and Houndog5 under high-light stress. Mean value 7SD (n =3). Bars with different letters are significantly different at the 5% level.
activities of SOD, CAT, APX and GR increased by 78.0%, 153.3%, 57.3%, 57.6% and 14.3%, 34.4%, 10.1%, 13.9% in leaves of Arid3 and Houndog5, respectively. Pretreatment with SNP remarkably increased SOD, CAT, APX and GR activities (at Po0.05), especially in Houndog5 leaves; While LNNA, PTIO or SNP+ PTIO addition greatly reduced these antioxidant enzymes activities, especially in Arid3 leaves.
both plants, whereas supplementation with NOS inhibitor (LNNA), NO scavenger (PTIO) or both (SNP+PTIO) elevated LOX activities by 56.1%, 59.4% and 65.0% in leaves of Arid3, respectively, but had little effect on those in Houndog5 leaves. It was evident that both NO and NOS were involved in the decrease of LOX activity, apparently little NO or NOS exist in Houndog5 leaves under HL stress (Fig. 4).
Effect of NO on lipoxygenase (LOX) activity
NO production and NOS activity
Transfer to HL also caused increased LOX activity, 30.7% and 90.3% greater than the control in leaves of Arid3 and Houndog5, respectively. Addition of NO donor, SNP reduced LOX activity in
To further elucidate the relationship between NO release and HL stress, NO production and NOS activity were measured. HL stress greatly enhanced NOS activity and NO generation in leaves
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Fig. 3. Effect of NO on the activities of antioxidant enzymes in leaves of Arid3 and Houndog5 under high-light stress: (A) SOD, (B) CAT, (C) APX and (D) GR. Mean value7 SD (n= 3). Bars with different letters are significantly different at the 5% level.
Fig. 4. Effect of NO on LOX activity in leaves of arid3 and Houndog5 under highlight stress. Mean value7 SD (n= 3). Bars with different letters are significantly different at the 5% level
of Arid3, but had no significant effect on Houndog5 leaves (Fig. 5A). Application of NO scavenger PTIO merely reduced NO content, whereas NOS inhibitor LNNA reduced NOS activity as well as NO release, revealing that NOS is essential for NO synthesis, and NO release was repressed when NOS activity was inhibited (Fig. 5B).
Discussion Photoinhibition occurs when plants are exposed to a photosynthetic photo flux density (PPFD) higher than that required for
CO2 fixation, which leads to increased ROS generation (Asada, 2006). Seven days HL treatments gave rise to increased H2O2 and formation, lipid peroxidation and membrane damage, O 2 especially in leaves of Houndog5 (Fig. 1A and D). Similar observations were made by Ali et al. (2005a) in Phalaenopsis. LOX is another important initiator of membrane damage and responsible for lipid peroxidation and the formation superoxide (Saravitz and Siedow, 1996; Ali et al., 2005b). HL stress enhanced LOX activity in both plants, especially in Houndog5 leaves (Fig. 4). As a result, it can be concluded that Houndog5 is more susceptible to HL stress than Arid3, and this result is in agreement with our observation in a preliminary experiment. Tolerance to HL stress is viewed as being associated with an increase in antioxidant enzyme activities (Burritt and Mackenzie, 2003; Jiang et al., 2005). Both varieties of tall fescue exhibited increases in the activities of antioxidant enzymes (SOD, CAT, APX and GR). SOD is a major scavenger of O 2 , catalyzing the dismutation of superoxide radicals to H2O2 and O2. CAT, APX and GR are important H2O2 detoxifying enzymes. Increases in the activities of all antioxidant enzymes suggest that ROS induced these increases in different cellular compartments (Logan et al., 1998). Under HL stress, a greater increase in antioxidant enzymes activities and lower levels of ROS were found in Arid3 leaves than in Houndog5 leaves, indicating that Arid3 alleviated oxidative injuries through raising antioxidant enzymes activities to scavenge newly produced ROS. NO can counteract oxidative damage and displays a protective effect against various stressful conditions (Arasimowicz and Floryszak-Wieczorek, 2007). Addition of exogenous NO with SNP significantly enhanced antioxidant enzymes activities and reduced ROS levels and LOX activities, prevented lipid peroxidation and membrane damage, whereas a reversed pattern was found when the NO scavenger PTIO was applied. This is consistent with the postulated role of NO as a signaling molecule involved in inducing increases in the activities of antioxidant enzymes
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Fig. 5. (A) NO production and (B) NOS activity in leaves of Arid3 and Houndog5 under high-light stress. Mean value 7 SD (n= 3). Bars with different letters are significantly different at the 5% level.
(Laspina et al., 2005; Shi et al., 2007; Zhang et al., 2009) and decrease in LOX activity (Song et al., 2006; Zhao et al., 2008) to inhibit lipid peroxidation and membrane damage caused by environmental stresses. Additionally, NO itself can detoxify ROS, and generating the peroxynitrite such as reacting with O 2 ion (ONOO ). In the physiological pH range, ONOO can be protonated and decomposes to a nitrate anion and a proton, or react with H2O2 to yield a nitrite anion and oxygen (Martinez et al., 2000; Wendehenne et al., 2001). Recent research also revealed that NO can induce APX and CAT antioxidant genes in Arabidopsis suspension cells (Huang et al., 2002). Arid3 suffered severe peroxidative damage when endogenous NO was removed by PTIO, which confirms the role of NO in photoprotection. On the other hand, NO was also found to inhibit photosynthetic electron transport and modulate reaction-center-associated non-photochemical quenching in plants (Wodala et al., 2008). Animal cells synthesize NO primarily through the activity of NOS. However, at present, the mechanism of NO production in plant cells is still a controversial issue (Corpas et al., 2006, Crawford et al., 2006, Zemojtel et al., 2006; Moreau et al., 2008). Previous studies have shown that the NO pool in plants includes enzymatic sources like NOS-like enzymes and nitrate reductase (NR), and non-enzymatic sources (Neill et al., 2003). NR was shown to be involved in NO production in several physiological situations, such as ABA-induced stomatal closure (Bright et al., 2006) and auxin-induced lateral root development (Kolbert et al., 2008). However, most reports support that NO generation is mainly mediated by a putative NOS-like enzyme, which catalyses the formation of NO from L-arginine, rather than NR (Tian et al., 2007; Corpas et al., 2008). Furthermore, several studies have identified NOS-dependent NO generation during plants exposure to stress conditions (Corpas et al., 2006, 2008; Valderrama et al., 2007; Zhao et al., 2007; Chaki et al., 2009). LNNA, an inhibitor of NOS, was reported to reduce NOS activity and NO release in Arabidopsis and Pisum (Qu et al., 2006; Tian et al., 2007; Zhao et al., 2007; Zhang et al., 2009). Application of LNNA severely reduced NOS activity and, thus, inhibited NO synthesis (Fig. 5), which eventually led to oxidative damage. This indicates that the LNNA-sensitive NOS-like enzyme is responsible for the majority of NO production in leaves of tall fescue. NOS activity was also influenced by exogenous NO application. Addition of SNP slightly reduced NOS activity, and this might be due to the negative feedback regulation of NOS by NO, as has been observed in maize and pea under UV-B irradiation (Zhang et al., 2003; An et al., 2005; Qu et al., 2006). The gene(s) encoding NOS in plants is still not known. AtNOS1 of Arabidopsis was initially reported to encode a protein containing NOS activity (Guo et al., 2003). However, this role of AtNOS1
has recently been questioned (Zemojtel et al., 2006), but Crawford et al. (2006) in their reply confirmed that the involvement of AtNOS1 in the synthesis or accumulation of NO might be direct or indirect. AtNOS1 was renamed AtNOA1 (for NITRIC OXIDE ASSOCIATED PROTEIN 1) to avoid confusion with animal NOS genes. Recently, Zhao et al. (2007) confirmed the presence of decreased NOS activity and NO production in the Arabidopsis noa1 mutant. However, Moreau et al. (2008) demonstrated that AtNOA1 was not a NOS, but specifically binds and hydrolyzes GTP, a function that is necessary for the complementation of the knockout mutant. These controversial results indicate that the search for the elusive plant NOS is not over yet. In conclusion, ROS metabolism is important for tall fescue during acclimatization to HL stress. The acquisition of tolerance to HL stress by two varieties of tall fescue is owed to significantly increased antioxidant enzymes activities. As a bioactive antioxidant, NO protects tall fescue leaves against light-induced oxidative damage by reacting with ROS directly or inducing activities of ROS-scavenging enzymes, especially in Arid3 plants, which are better adapted to HL stress.
Acknowledgement This work was supported by Key Projects in the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (No. 2006BAD16B09-2).
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