Botanical Studies (2012) 53: 45-54.
BIOCHEMISTRY
Modulation of antioxidant defense system and NADPH oxidase in Pluchea indica leaves by water deficit stress
Min-Lang CHANG1,3, Li-Jen LIAO2 *, Jia-Hong LIN2, Zin-Huang LIU3, Yuan-Ting HSU4,5,6, and Tse-Min LEE4'5'6'*
1Department of Livestock Management, Branch of Heng-Chun, Institute of Livestock Research, Council of Agriculture Executive Yuan, Heng-Chun 946, Taiiwan
2Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung 80201, Taiwan
3Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan
4Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
5Doctor Degree Program in Marine Biotechnology, National Sun Yst-sen University/Academia Sinica, Kaohsiung 80424, Taiwan
6Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
(Received January 5, 2011; Accepted June 16, 2011)
ABSTRACT. We investigated changes in antioxidant content and antioxidant enzyme activity in polyethylene glycol (PEG)-induced water deficit stressed Pluchea indica leaves. We also used diphenylene iodonium (DPI), a plasma membrane NADPH oxidase suicide inhibitor, to examine the role of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the induction of antioxidant defense systems. PEG decreased water content and the reduction ability of 2,3,5-triphenyltetrazolium chloride (TTC), but increased malondi-aldehyde (MDA), total peroxide, O2- and H2O2 contents, indicating water deficit-dependent oxidative stress. Total ascorbate (AsA), AsA contents, and AsA/oxidized AsA ratios were increased by moderate water deficit (-0.6 MPa) conditions, while total glutathione (GSH), GSH contents and GSH/GSSG ratios increased as water potential decreased. Superoxide dismutase (SOD) activity was not affected after 24 h of PEG treatment but decreased after 48 h. Catalase (CAT) activity increased as water potential decreased while peroxidase (POX) activity increased only at -1.2 MPa. Ascorbate peroxidase (APX) activity increased under moderate water defi­cit and glutathione reductase (GR) activity increased as water potential decreased. DPI depressed the induction of antioxidant accumulation and antioxidant enzyme activity by water deficit, indicating that reactive oxygen species (ROS) signals are involved in activating defense systems in P. indica in response to oxidative stress. We conclude that moderate water deficit stress induces both NADPH oxidase-mediated non-enzymatic and en­zymatic oxidative defense mechanisms in ROS scavenging in P. indica leaves.
Keywords: Antioxidant defense system; NADPH oxidase; Pluchea indica; Reactive oxygen species (ROS); Water deficit.
Abbreviations: APX, Ascorbate peroxidase; AsA, Ascorbate; CAT, Catalase; DPI, Diphenylene iodonium; DTT, Dithiothreitol; GR, Glutathione reductase; GSH, reduced Glutathione; GSSG, oxidized Glutathione; H2O2, Hydrogen peroxide; HO-, Hydroxyl radical; NADPH, Reduced nicotinamide adenine dinucleotide phosphate; MDA, Malondialdehyde; O21, Singlet oxygen; O2-， Peroxide; O2-, Superoxide radical; PEG, Poly­ethylene glycol; POX, Peroxidase; ROS, Reactive oxygen species; SOD, Superoxide dimutase; TCA, Trichlo­roacetic acid; TTC, 2,3,5-Triphenyltetrazolium chloride.
INTRODUCTION
homeostasis of reactive oxygen species (ROS) production from the excitation of O2 to form singlet oxygen (O21) and the transfer of 1, 2 or 3 electrons O2 to form superoxide radical (O2-), hydrogen peroxide (H2O2) and hydroxyl rad­ical (HO-) (Mittler, 2002; Jubany-Mari et al., 2009; 2010). ROS cause the oxidative destruction of the cell compo­nents through oxidative damage of membrane lipids, nu­cleic acid and protein, especially SH-containing enzymes (Imlay and Linn, 1998; Beligni and Lamatina, 1999). To counteract the toxicity of ROS, plants have developed antioxidant defense mechanisms in scavenging cellular
Water deficit stress is a major limiting factor affecting plant growth, development and production (Wilkinson and Davies, 2010). Water deficit stress can induce oxidative stress in plants resulting from the disruption of cellular
*Corresponding authors: E-mail: lijen@nknucc.nknu.edu.tw, Tel: 886-7-7172930 ext. 7313, Fax: 886-7-6051353 (Li-Jen LIAO); E-mail: tmlee@mail.nsysu.edu.tw, Tel: 886-7­5252000 ext. 5110, Fax: 886-7-5251509 (Tse-Min LEE).
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ROS to cope with oxidative stress via the non-enzymatic and enzymatic systems (Bartoli et al., 1999; Mittle, 2002; Mittler et al., 2004). The antioxidants, including water-soluble ascorbate (AsA), reduced glutathione (GSH), water-insoluble a-tocopherol and carotenoids, are a non-enzymatic system for ROS scavenging in cells (Asada, 1992; Noctor and Foyer, 1998; Smirnoff and Wheeler, 2000; Lei et al., 2006). In the enzymatic ROS-scavenging pathways, superoxide dimutase (SOD; EC 1.15.1.1) con­verts 〇2.— to H2O2 and ascorbate peroxidase (APX; EC 1.11.1.11)/glutathione reductase (GR; EC 1.6.4.2) are responsible for operating the ascorbate-glutathione cycle (AGC) in the removal of H2O2 (Asada, 1992). Catalase (CAT; EC 1.11.1.6) (Willekens et al., 1997), and peroxi-dase (POX; EC 1.11.1.7) (Asada, 1992) are also involved in the reduction of H2O2 to H2O.
used DPI, a plasma membrane-bound NADPH oxidase inhibitor to elucidate the possible involvement of NADPH oxidase in the induction of antioxidatant defense systems (Auh and Murphy, 1995; Allan and Fluhr, 1997).
MATERIALS AND METHODS
Plant materials and treatments
Pluchea indica leaves (4.2±0.2 cm length and 1.7±0.1 cm width) were sampled from WuSanTao Mud Volcano Nature Preserve in Yenchao County of Kaohsiung in southern Taiwan. Detached leaves were transferred to the laboratory within 2 h after sampling, washed with distilled water twice, then incubated at 25°C in a plastic box con­taining distilled water for 12 h in the dark to allow any wounds to recover. Detached leaves were prevented from full water submersion during this process. Three healthy leaves without browning were transferred to a Petri dish (10 cm diameter) containing 10 mL distilled water with varying PEG 6000 (Merck, Darmstadt, Germany) concen­trations for water deficit treatment at 0, -0.6, -1.2 and -1.8 MPa. Water potential of -0.6 MPa is moderate water defi­cit, which the leaves can recover fully after release of -0.6 MPa challenge. To elucidate the involvement of NADPH oxidase on water deficit- induced antioxidatant defense system, leaves were pre-treated for 12 h in distilled wa­ter containing 50 μM Diphenylene iodonium (DPI), then transferred to -0.6 MPa solution for another 12 h. In this study, a Petri dish was a replicate and there were three rep­licates for each treatment.
The ability to detoxify H2O2 is pivotal in the antioxi-dant defense mechanisms against ROS in plants (Foyer and Harbinson, 1994). In addition to functioning as a toxic cellular metabolite, H2O2 has been recognized as a signal­ing molecule that mediates stimuli responses in plants (Neill et al., 2002; Miller et al., 2008). When exposed to stresses, the balance of H2O2 production and removal can be disrupted, and the subsequent increase in cellular H2O2 can initiate signaling responses (Overmyer et al., 2003; Laloi et al., 2004; Miller et al., 2008). The oxida-tive burst can also be produced via the increased activity of plasma membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, which transfer electrons from intracellular NADPH to O2 to form O2-, and then dismutated to H2O2, and the influx of H2O2 as a signaling molecule (Hao et al., 2006; Pourrut et al., 2008). Plasma membrane-bound NADPH oxidase is involved in regulating the antioxidant defense mechanisms in plants in response to water deficit stress (Zhao et al., 2001; Jiang and Zhang, 2002a,b; Agrwal et al., 2003).
Relative water contents determination and TTC reduction ability
Relative water contents were determined according to Lee et al. (1996). Fresh weight (f. wt.) of leaves was determined immediately after treatments and then after lyophilization at -60°C to determine dry weight (d. wt.). Lyophilized leaves were stored at -70°C for antioxidant content and antioxidant enzyme activity analyses. Relative water content was calculated as the following equation: (g f. wt.-g d. wt.)/g d. wt.
Pluchea indica (L.) Less (Asteraceae: Inuleae) is a tra­ditional medicinal plant with reported anti-inflammatory, anti-ulcer, anti-amoebic and antioxidant activities (Senet al., 2002; Biswas et al., 2007; Gomes et al., 2007; Pra-manik et al., 2007). It is widespread along the coastline of southwestern Taiwan, commonly occurring in littoral areas such as mangroves, brackish swamps, and tidal flats and is also abundant in the salty, muddy, volcanic areas in Kaoh-siung's Yenchao County in southern Taiwan (Peng et al., 1998). These areas are characterized by high levels of salt and water in the soil, and dry (October to May) and wet (June-September) seasonal climates. manik et al., 2007). It is widespread along the coastline of southwestern Taiwan, commonly occurring in littoral areas such as mangroves, brackish swamps, and tidal flats and is also abundant in the salty, muddy, volcanic areas in Kaoh-siung's Yenchao County in southern Taiwan (Peng et al., 1998). These areas are characterized by high levels of salt and water in the soil, and dry (October to May) and wet (June-September) seasonal climates.
To determine the cellular activities, leaves of approxi­mately 0.05 g f. wt. were incubated in 1.5 mL of 0.8% (w/v) 2,3,5-Triphenyltetrazolium chloride (TTC) solution containing 50 mM potassium phosphate buffer (pH 7.4) and 0.01% (v/v) Tween 20 for 6 h under darkness (Chang et al., 1999). After three washes with 5 mL of distilled wa­ter, intracellular insoluble formazan was extracted twice with 5 mL of 95% ethanol at 80°C for 20 min and the absorbance of ethanol extract was determined at A530. The A530 values of water deficit or DPI-treated leaves were cal-culated as the percentage of control.
Our preliminary examination showed that this shrub fre­quently suffered water deficit stress during the dry season in southern Taiwan. In this work, we investigated the in­duction of the non-enzymatic and enzymatic antioxidatant defense systems in detached P. indica leaves in response to water deficit stress by determining their water-soluble an-tioxidant contents (AsA and GSH) and antioxidant enzyme activities (SOD, CAT, POX, APX and GR) when exposed to varying polyethylene glycol (PEG) concentrations. We
Determination of lipid peroxidation, total perox­ide, O2-, H2O2 and water-soluble antioxidants
0.1 g d. wt. lyophilized leaves were homogenized in 1 mL of 5% (w/v) trichloroacetic acid (TCA). After centrifu-
CHANG et al. ― Pluchea antioxidant defense and NADPH oxidase by water deficit
47
gation at 12,000 xg for 15 min at 4°C, the supernatant was collected as TCA extract for lipid peroxidation and total peroxide determination and the H2O2 and water-soluble antioxidant contents. To determine O2-production rate, lyophilized leaves of 0.05 g d. wt. were homogenized in 1 mL of 50 mM Tris-HCl (pH 7.5), then centrifuged at 12,000 xg for 15 min at 4oC to obtain the supernatant as the O2- determination extract; O2- production rate was as-sayed within 0.5 h after extraction.
15 min at 4oC, the supernatant was used as for an enzyme activity assay. Enzyme activities were determined within 2 h after extraction. The soluble protein contents were deter­mined by the Coomassie blue dye binding method (Brad­ford, 1976), with bovine serum albumin as standard curve.
CAT activity was measured at A420 for H2O2decomposi­tion rate using the extinction coefficient of 40 mM-1 cm-1 according to Kato and Shimizu (1987). Guaiacol POX activity was determined by the formation rate of tetragua-iacol detected at A470 with the extinction coefficient 26.6 mM-1 cm-1 according to Kato and Shimizu (1987). SOD activity was determined by the inhibition of photochemical inhibition of nitro blue tetrazolium according to Giannopo-litis and Ries (1977). APX activity was determined at A290 for oxidized AsA according to the extinction coefficient of 2.8 mM-1 cm-1 according to Nakano and Asada (1981). GR activity was monitored A， for NADPH oxidization as GSSG reduction according to the method of Schaedle and Bassham (1977).
The lipid peroxidation level, that is, malondialdehyde (MDA) content, was determined from the thiobarbituric acid reacting substance (TBARS) contents resulting from the thiobarbituric acid (TBA) reaction as described by Health and Packer (1968). The MDA contents were calcu­lated based on A532-A600 with the extinction coefficient of 155 mM-1 cm-1.
Total peroxide contents were determined by the ab-sorbance of A480 and extinction coefficient of 13.93 μM-1 cm-1 according to Sagisaka (1976). O2 - production rate was determined according to the method described by Able et al. (1998). H2O2 contents were determined based on the decomposition of H2O2 by peroxidase as described
Statistical analysis
SAS (SAS v 8.1, NC, USA) was used in this study. The results present in this study were the mean of 3 replicates with a Petri dish as a replicate. The effects of PEG and treatment time on water contents, MDA contents, total peroxide contents, O2 - production rate, H2O2 contents, water-soluble antioxidant contents and antioxidant en­zyme activity were analyzed by 2-way variance analysis (ANOVA). DPI treatment effects were analyzed by 1-way ANOVA. The significant difference among means was analyzed by Duncan's new multiple range test followed by significant ANOVA at P<0.05.
The total ascorbate (AsA) and reduced AsA content measurements were modified from the method of Hodges et al. (1996). Total AsA contents were determined in 1-mL mixture containing 200 μL of TCA extract, 50 mM potas­sium phosphate buffer (pH 7.4), 3 mM ethylenedinitrilo-tetraacetic acid (EDTA) and 1 mM Dithiothreitol (DTT). The mixture was incubated at 25°C for 10 min, 100 μL of N-ethylmaleimide was added and then 400 μL of 0.61 M TCA, 400 μL of 0.8 M orthophosphoric acid and 400 μL of α,α'-bipyridyl were added. Finally, 200 μL of FeCl3 was added and the reaction mixture was incubated in a 55°C water bath for 10 min, and the absorbance was detected at A525. Reduced AsA contents were determined by adding distilled water instead of DTT and N-ethylmaleimide, then followed the method as described above. Total AsA and reduced AsA contents were estimated from the standard curve of 0-40 nmole L-AsA determined by the above methods for total ascorbate and reduced AsA analysis, respectively. Oxidized AsA contents were calculated by subtracting reduced AsA from total AsA.
RESULTS
Relative water contents, TTC reduction ability, O2- production rate, H2O2 contents, MDA and total peroxide contents
To have a mechanistic causal understanding of PEG-in­duced water deficit injury in P. indica leaves, we estimated statuses of TTC reduction ability, some of the principal ROS, the O2" and H2O2, and the level of lipid peroxida-tion, that is, malondialdehyde (MDA) content, which is an indicator of oxidative damage. PEG (2-way ANOVA, P<0.05) and treatment time (P<0.05) affected water poten­tial and TTC reduction ability, and the interaction of PEG and treatment time on water potential and TTC reduction ability was significant (P<0.05). On exposure to water deficit, the relative water contents decreased as water potential decreased from 0 to -1.8 MPa and appeared a further decline after 48 h treatment as compared to the 24 h treatment (Figure 1A). Similarly, TTC reduction ability also decreased as water potential decreased and the decline in TTC reduction ability was more significant at 48 h than the 24 h treatment (Figure 1B). In contrast, MDA con­tents (Figure 1C), total peroxide contents (Figure 1D), O2 -production rate (Figure 1E) and H2O2 contents (Figure 1F) increased as water potential decreased (P<0.05) and treat-
Total glutathione contents were determined according to Griffiths (1980). Glutathione contents were estimated from the standard curve of 0-20 nmole GSH. After re­moving reduced glutathione (GSH) by 2-vinylpyridine derivatization, oxidized glutathione (GSSG) contents were determined and GSH contents were calculated by subtract­ing GSSG contents from total glutathione contents.
Determination of antioxidant enzyme activity
Lyophilized leaves of 0.01 g d. wt. were homogenized in liquid nitrogen after which 1 mL of 50 mM sodium phosphate buffer (pH 7.0) containing 0.2% Triton X-100, 0.5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF) were added. After centrifugation at 12,000 xg for
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Antioxidant enzyme activities
An analysis of active oxygen-processing enzymes activities showed that SOD was affected by PEG (P<0.05) and treatment time (P<0.05). SOD activities were not affected by PEG after 24 h of treatment, but decreased after 48 h of treatment (Figure 3). CAT activities were affected by PEG (P<0.0001) but not by treatment time (P=0.2562). POX, APX and GR activities were affected by both PEG (2-way ANOVA, P<0.05) and treatment time (P<0.05). The interaction of PEG and treatment time on antioxidant enzyme activities was significant for POX, APX and GR (P<0.05) but not for CAT (P>0.05).
Figure 1. Changes in relative water contents (A), TTC reduc­tion ability (B), MDA contents (C), total peroxide contents (D), O2 - production rate (E) and H2O2 contents (F) in Pluchea indica leaves in response to varying PEG concentrations, i.e. 0, -0.6, -1.2 and -1.8 MPa. Data are present as means士SD (n=3) and different letters indicate significant difference among treatments.
ment time advanced (P<0.05). The interaction of PEG and treatment time on O2 - production rate and total peroxide, MDA and H2O2 contents was significant (P<0.05). These indicate that PEG-induced water deficit can trigger oxida-tive damage in P. indica leaves.
Ascorbate and glutathione contents
We observed changes in antioxidant Ascorbate and glutathione contents during oxidative damage in P. indica leaves. PEG (P<0.05) but not treatment time (P>0.05) affected total AsA and reduced AsA contents while both PEG (P<0.05) and treatment time (P<0.05) affected oxidized AsA contents and reduced AsA/oxidized AsA ratios, the interaction of PEG and treatment time on oxidized AsA contents and reduced AsA/oxidized AsA ratios was significant (P>0.05). After exposure to water deficit, total AsA (Figure 2A) and reduced AsA (Figure 2B) contents and reduced AsA/oxidized AsA ratios (Figure 2D) increased in response to -0.6 MPa, but total AsA, reduced AsA and oxidized AsA contents and reduced AsA/oxidized AsA ratios decreased in response to -1.8 MPa.
Figure 2. Changes in ascorbate: total AsA (A); reduced AsA (B); oxidized AsA (C); reduced AsA/oxidized AsA ratio (D), and glutathione contents: total glutathione (E); GSH (F); GSSG (G); GSH/GSSG ratio (H) in Pluchea indica leaves in response to 0, -0.6, -1.2 and -1.8 MPa. Data are present as means±SD (n=3) and different letters indicate significant difference among treatments.
Both PEG (P<0.05) and treatment time (P<0.05) affected total glutathione, GSH and GSSG contents, and the interaction of PEG and treatment time on total glutathione, GSH and GSSG contents and GSH/GSSG ratios was significant (P<0.05). After 24 h of water deficit treatment, total glutathione (Figure 2E) and GSH (Figure 2F) contents and GSH/GSSG ratios (Figure 2H) increased as water potential decreased from 0 to -1.8 MPa, but GSSG contents only showed a small increase at -1.2 MPa. After 48 h of treatment, total glutathione (Figure 2E) and GSH (Figure 2F) contents also increased, but their increments were relatively smaller than at 24 h treatment. The GSSG contents showed a marked increase after 48 h of water deficit treatment (Figure 2G), resulting in a drop of GSH/GSSG ratios (Figure 2H).
Figure 3. SOD activity in Pluchea indica leaves in response to 0, -0.6, -1.2 and -1.8 MPa. Data are present as means±SD (n=3) and different letters indicate significant difference among treat­ments.
CHANG et al. ― Pluchea antioxidant defense and NADPH oxidase by water deficit
49
CAT activities increased as water potential decreased from 0 to -1.8 MPa (Figure 4A) while POX activities in­creased only at -1.2 MPa, and this increment was more significant after 48 h treatment (Figure 4B). APX activi­ties increased significantly after exposure to -0.6 and -1.2
MPa for 24 h and the magnitude of APX activity incre­ment was reduced after 48 h treatment as compared to the 24 h treatment (Figure 4C). GR activities significantly increased after 24 h of exposure to water deficit, but only increased in response to -1.2 MPa (Figure 4D) after 48 h.
Effects of DPI on water stress-induced antioxi-dant defense system
To elucidate the role of NADPH oxidase in inducing the antioxidatant defense system, we pretreated leaves with 50 μM DPI for 12 h, then transferred to PEG (-0.6 MPa) for another 12 h. When transferred to -0.6 MPa, the magnitude of MDA, total peroxide and H2O2 accumulation in DPI-pretreated leaves was higher than in untreated leaves (ANOVA, P<0.05) (Figure 5). In contrast, the decrease in TTC reduction ability after exposure to -0.6 MPa was more significant in DPI-pretreated leaves than in those without (P<0.05) (Figure 5).
After exposure to water deficit stress, total AsA and oxidized AsA contents in DPI-pretreated leaves showed a more significant decrease than in those not treated (P<0.05), while reduced AsA contents were not affected by DPI pretreatment (P>0.05) (Figure 6). DPI pretreatment induced a greater decrease in total glutathione, GSH and GSSG contents under -0.6 MPa conditions compared to those without DPI (P<0.05) (Figure 6). The GSH/GSSG ratio increased after exposure to -0.6 MPa (P<0.05), but the ratio was same in DPI-pretreated and DPI-free treatments (Figure 6).
Figure 4. CAT (A), POX (B), APX (C) and GR (D) activities in Pluchea indica leaves in response to 0, -0.6, -1.2 and -1.8 MPa. Data are present as means±SD (n=3) and different letters indi­cate significant difference among treatments.
The increase in CAT, APX and GR activities under -0.6 MPa conditions was depressed by DPI pretreatment, but SOD activity was not affected (Figure 7). DPI pretreat-ment increased POX activity (Figure 7).
Figure 6. The effects of DPI (50 μM) pretreatment (12 h) on ascorbate: total AsA (A); reduced AsA (B); oxidized AsA (C); reduced AsA/oxidized AsA ratio (D), and glutathione contents: total glutathione (E); GSH (F); GSSG (G); GSH/GSSG ratio (H) in Pluchea indica leaves after transferred to -0.6 MPa for 12 h. Data are present as means±SD (n=3) and different letters indicate significant difference among treatments.
Figure 5. The effects of DPI (50 fiM) pretreatment (12 h) on relative water contents (A), TTC reduction ability (B), MDA contents (C), total peroxide contents (D), O2 - production rate (E) and H2O2 contents (F) in Pluchea indica leaves after transferred to -0.6 MPa for 12 h. Data are present as means±SD (n=3) and different letters indicate significant difference among treatments.
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stress under moderate water deficit (-0.6 MPa) conditions.
The maintenance of antioxidant pools and enhanced scavenging capacity are essential for plant defense against oxidative stress (Noctor and Foyer, 1998). The present re­sults from an increase in the contents of total and reduced AsA and a higher regeneration rate of reduced AsA as indicated by a rise in reduced AsA/oxidized AsA ratio in P. indica leaves in response to -0.6 and -1.2 MPa (Figure 2) seem to suggest that increased ascorbate pools and the ability in maintaining a high reduced per oxidized ratio of ascorbate are crucial for the proper scavenging of ROS generated under moderate water deficit conditions. It is obvious that sufficient levels of reduced AsA in combina­tion with increased APX activities (Figure 4C) allow P. indica leaves to maintain the balance of their cellular ROS components under water deficit conditions, resulting in low H2O2 accumulation under -0.6 and -1.2 MPa. Because the ascorbate pools and the regeneration ability of oxidized AsA to reduced AsA is decreased under severe water defi­cit (-1.8 MPa) conditions, it is likely that significant oxida-tive damage occurring under these severe conditions is due in part to low capacity in maintaining normal ascorbate biosynthesis and its regeneration to reduced AsA (Figure 2A-D).
Glutathione also accumulated under water deficit conditions but showed a different pattern than did ascorbate. Total glutathione and GSH contents increased as water potential decreased, with the increase more significant after 48 h treatment. Following increasing water deficit stress, increased ability to regenerate glutathione as indicated by accumulated GSH contents and high GSH/GSSG ratio (Figure 2E-H) as well as high GR activities (Figure 4D) demonstrate there was increased capacity to reduce H2O2 by regenerating GSH under moderate water deficit conditions, that is, GSH could be converted to GSSG for the scavenging of ROS and the GSSG can be recycled to GSH. Because GSSG could be regenerated to GSH via GR, a decrease in GR activities after 48 h treatment indicates a reduction in GSH recovery the longer under water deficit stress. This explains the accumulation of GSSG and declined GSH/GSSG ratio after 48 h treatment. Our study results clearly suggest a tightly controlled antioxidant balance in P. indica leaves that helps maintain an optimal redox status under different water deficit conditions. It is obvious that increasing ascorbate and glutathione pools together with higher regeneration rates contribute to the ROS scavenging in P. indica leaves in response to the moderate water deficit conditions while the notable reduction of antioxidant regeneration might be responsible for the occurrence of significant oxidative stress caused by severe water deficit stress.
Figure 7. The effects of DPI (50 μM) pretreatment (12 h) on SOD (A), CAT (B), POX (C), APX (D) and GR (E) activities in Pluchea indica leaves after transferred to -0.6 MPa for 12 h. Data are present as means±SD (n=3) and different letters indicate significant difference among treatments.
DISCUSSION
Pluchea indica, an erect shrub growing in littoral areas of western Taiwan, frequently suffers water deficit stress during the dry season from October to May. The present study was aimed at better understanding the relationship between water deficit stress and oxidative stress in P. in­dica leaves. It has been known that water deficit stress can enhance ROS production resulting in oxidative stress in several plant systems (Sgherri et al., 1993; Smirnoff, 1993; Moran et al., 1994; Sgherri and Navari-Izzo, 1995; Jiang and Zhang, 2002a). We conducted this study due to a lack of information on herbaceous plants responses to water deficit, especially the antioxidant defense responses they use to adapt to the stressful conditions in harsh environ­ments.
The results of the present study provide several lines of evidence showing that PEG-induced water deficit stress can cause oxidative stress in P. indica leaves by increasing lipid peroxidation (MDA), and elevating total contents of peroxide and ROS (O2 - and H2O2) (Figure 1). The magni­tude of oxidative stress is not only increased by decreasing water potential but also enhanced by longer water stress treatment. This study has shown that P. indica leaves have developed defense mechanisms to cope with oxidative
As crucial as is antioxidant ROS scavenging in plant defense against active oxygen species In addition to, the enzyme-mediated conversion of superoxide radical O2˙- to H2O2 and the subsequent detoxification of H2O2 are crucial for (Foyer and Harbinson, 1994; Mittler, 2002). In this study, the maintenance of SOD activity in P. indica leaves.
CHANG et al. ― Pluchea antioxidant defense and NADPH oxidase by water deficit
51
in response to 24 h of water deficit fallowed by a drop at 48 h (Figure 3) suggests that O2 - could be dismutated to H2O2 during the early water stress periods but this O2 - dis-mutation ability was depressed by longer water stress con­ditions. There was a significant O2 - accumulation after 48 h of water stress treatment and thus increased oxidative damage caused by O2 - attack can be expected in response to prolonged water deficit treatment.
system to overcome the subsequent ROS production.
We conclude that the defense mechanisms against oxidative stress occurring in P. indica leaves exposure to water deficit condition are antioxidant availability and an-tioxidatant enzymes activation, and that ROS signals me­diated by plasma membrane-bound NADPH oxidase are involved in the activation of oxidative defense systems. Nitric oxide (NO) is also important to oxidative stress signaling and is likely involved in the regulation of anti-oxidative enzymes in defending against oxidative stress (Arasimowicz and Floryszak-Wieczorek, 2007; Shao et al., 2008a) and interacting with ROS to transmitting the signal (Mittler, 2002; del Rio et al., 2006; Shao et al., 2008b). Al­though we did not apply chemicals such as sodium nitrop-russide (SNP) to release NO (Arasimowicz and Floryszak-Wieczorek, 2007), we speculate that NO may also be involved in controlling the antioxidant defense system in Pluchea indica leaves. We hope to prove this in the near future.
The present results indicate that H2O2 generation fol­lowing the O2 - dismutation in water stress-treated P. indica leaves would be alleviated by APX/GR (Figure 4C-D), and in turn, H2O2 did not accumulate in the condi­tions of both -0.6 and -1.2 MPa. It is known that APX and GR are responsible for the removal of H2O2 as the ascorbate-glutathione cycle. CAT may also be involved in H2O2 removal because its activity increased as water po­tentials decreased (Figure 4A). CAT is likely responsible for excess H2O2 removal under severe water stress condi­tions, while AGC operates under moderate water stress conditions. It is obvious that the H2O2 detoxifying enzyme expression in P. indica leaves is modulated differently depending on water deficit conditions; AGC is activated by moderate water deficit conditions and CAT responds to extreme water stress.
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The present study suggests that antioxidant defense system induction is mediated by H2O2 in P. indica leaves. H2O2 has been known as the signal for plant antioxidatant defense system induction in response to biotic and abiotic stresses (Neill et al., 2002). It is now believed that O2˙- can be generated by mammalian-like plasma membrane neutrophil NADPH oxidase and O2˙- is automatically dismutated to H2O2, which influxes into cellular space as a signal to induce the antioxidant defense system in plants (Auh and Murphy, 1995; Vera-Estrella and Blumwald, 1997). The inhibition of water stress-induced ascorbate and glutathione accumulation and antioxidatant enzyme activity increase by DPI (Figures 6 and 7), the inhibitor of plasma membrane NADPH oxidase, provide evidence that the generation of ROS mediated by NADPH oxidase is involved in the induction of antioxidant defense mechanism in P. indica leaves. This provides an explanation for enhanced H2O2 production in DPI-pretreated P. indica leaves after their transfer to water deficit conditions (Figure 5F). The antioxidative defense systems blockage by DPI has been observed in several plant systems (Doke and Ohashi, 1988; Doke and Miura, 1995; Levine et al., 1994). The regulation of H2O2 production via NADPH oxidase has been suggested in cell suspension cultures of Taxus chinensis treated with a fungal elicitor from Aspergillus niger (Qin et al., 2004). NADPH oxidase is known to mediate the ROS production and signals the up-regulation of abscisic acid (ABA)-induced antioxidant defense systems in maize leaves exposed to water deficit stress (Jiang and Zhang, 2002b). It is most likely that water deficit stress activates the plasma membrane NADPH oxidase in P. indica leaves, resulting in the O2˙- production that automatically dismutates to H2O2 that triggers the antioxidant defense
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鯽魚膽切離葉於缺水環境下抗氧化防禦系統之調節與NADPH
氧化酶活性之關係
張敏郎1,3 廖麗貞2 林佳弘2 劉景煌3 許媛婷4,5,6 李澤民4,5,6
1行政院農業委員會畜產試驗所恆春分所畜產經營系
2國立高雄師範大學生物科技系
3國立中山大學生物科學系
4國立中山大學海洋生物研究所
5國立中山大學與中央研究院合辦海洋生物科技博士學位學程
6國立中山大學亞太海洋頂尖中心
本研究探討鯽魚膽(Pluchea indica (L.) Less.)切離葉片於PEG誘導產生的缺水處理中，其抗氧化 物質及抗氧化酶活性之變化情形，並利用原生質膜NADPH氧化酶抑制劑DPI (diphenylene iodonium)處 理，了解還原態NADPH氧化酶(NADPH oxidase)所扮演之角色。經PEG缺水處理的鯽魚膽葉片其含 水量與TTC活性降低，但malondialdehyde (MDA) 、 total peroxide 、 O2 -與H2O2量增加，顯示鯽魚膽葉 片在缺水逆境確實可誘導氧化逆境的產生。在中度缺水的情況下，total ascorbate (AsA) ， AsA contents及 AsA/oxidized AsA ratios皆增加，且隨著水勢增加，total glutathione (GSH) 、 GSH contents與GSH/GSSG ratios亦增加。而superoxide dismutase (SOD)活性在PEG處理24小時沒有明顯改變，但在48小時後則 增加。catalase (CAT)與glutathione reductase (GR)活性隨著水勢降低而增加，而peroxidase (POX)活性 則只在水勢-1.2 MPa增加，ascorbate peroxidase (APX)活性只在中度缺水時增加。這些藉由缺水逆境所 誘導產生之抗氧化物質的累積與抗氧化酶活性之變化情形可被DPI抑制，顯示鯽魚膽在氧化逆境中可啟 動其防禦系統中之活性氧族（reactive oxygen species ， ROS)訊息分子。因而，鯽魚膽葉片對中度缺水逆 境之反應係藉由NADPH oxidase所媒介之非酵素形式或酵素形式之氧化防禦機制來清除ROS而達成。
關鍵詞：NADPH氧化酶；鯽魚膽；活性氧族；缺水逆境；抗氧化防禦系統。