Nitric oxide reduces Cu toxicity and Cu-induced
NH
4
+
accumulation in rice leaves
Chia Chi Yu, Kuo Tung Hung, Ching Huei Kao
Department of Agronomy, National Taiwan University, Taipei, Taiwan, China Received 7 January 2005; accepted 10 February 2005
Summary
Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important antioxidant. Here we investigated the protective effect of NO against the toxicity and NH4
+
accumulation in rice leaves caused by excess CuSO4 (10 mmol L1). It was found that free radical scavengers (sodium benzoate,
thiourea, and reduced glutathione) reduced the toxicity and NH4+accumulation in rice
leaves caused by excess CuSO4. NO donor sodium nitroprusside (SNP) was also
effective in reducing CuSO4-induced toxicity and NH4+accumulation in rice leaves. The
protective effect of SNP on the toxicity and NH4+accumulation can be reversed by
2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide, a NO scavenger, suggesting that the protective effect of SNP is attributable to NO released. Results obtained in the present study suggest that reduction of CuSO4-induced toxicity and
NH4+accumulation by SNP is most likely mediated through its ability to scavenge active
oxygen species.
&2005 Elsevier GmbH. All rights reserved.
Introduction
Copper (Cu) is an essential element in plants involved in various biochemical processes, but excess Cu (above 0.01 mmol L1 in an incubation medium; Wainwright and Woolhouse, 1977) is
harmful to most plants (Fernandes and Henriques, 1991). Due to its widespread industrial and agri-cultural use, Cu pollution is a major environmental problem. Excess Cu can generate active oxygen species (AOS), such as H2O2and O2
d
(Girotti, 1985;
Fernandes and Henriques, 1991;Chen et al., 2000)
www.elsevier.de/jplph KEYWORDS Active oxygen species; Excess copper; Lipid peroxidation; Nitric oxide; Oryza sativa
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.02.003
Abbreviations: AOS, Active oxygen species; APX, Ascorbate peroxidase; CAT, Catalase; c-PTIO, 2-(4-carboxy-2-phenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide; FW, Fresh weight; GR, Glutathione reductase; GS, Glutamine synthetase; GSH, Reduced glutathione; MDA, Malondialdehyde; NO, Nitric oxide; PAL, Phenylalanine ammonia-lyase; PBN, N-Tert-butyl-a-phenylnitrone; POX, Peroxidase; SB, Sodium benzoate; SIN-1, 3-morpholinosydonimine; SNP, Sodium nitroprusside; SOD, Superoxide dismutase; TU, Thiourea
Corresponding author. Fax: 886 2 23620879. E-mail address: [email protected] (C. Huei Kao).
and is able to stimulate the production of dHO in a Fenton-type reaction (Sandmann and Bo¨ger, 1980). Cu-increased lipid peroxidation has been demon-strated in Avena sativa (Luna et al., 1994), Brassica napus (Baryla et al., 2000), Helianthus annuus (Gallego et al., 1996), Holcus lanatus ( Hartley-Whitaker et al., 2001), Lycopersicon esculentum (Mazhoudi et al., 1997), Nicotiana plumbaginifolia (Savoure´ et al., 1999), Oryza sativa (Lindon and Henriques, 1993; Chen and Kao, 1999), Phaseolus vulgaris (Weckx and Clijsters, 1996;Cuypers et al., 2000), and Silene cucubalus (De Vos et al., 1993). Thus, excess Cu leads to oxidative stress in plant cells.
NH4+ is a central intermediate of nitrogen
metabolism (Miflin and Lea, 1976). Glutamine synthetase (GS) is the key enzyme in NH4
+
assimila-tion and catalyzes the ATP-dependent condensaassimila-tion of NH4
+
with glutamate to produce glutamine (Miflin and Lea, 1976). Previously, we demonstrated that NH4+accumulation in rice leaves subjected to excess
Cu is attributable to a decrease in GS activity (Chen and Kao, 1998). GS in plants has been reported to be particularly prone to degradation under oxida-tive stress conditions (Ortega et al., 1999;Palatnik et al., 1999;Chien et al., 2002;Ishida et al., 2002). In oat chloroplasts, it has been shown that specific activity of a thylakoid-bound endopeptidase in-creased under photooxidative environmental con-ditions or treatment with dOH or H2O2 generating
systems (Casano and Trippi, 1992, Casano et al., 1990,1994). Phenylalanine ammonia-lyase (PAL) catalyzes the elimination of NH4
+
from phenylala-nine and produces trans-cinnamate (Hahlbrock and Grisebach, 1979). NH4+ released in the
PAL-cata-lyzed reaction is known to be trapped in the glutamine molecule by the action of GS (Razel et al., 1996).Sakurai et al. (2001) provided evidence showing that GS is partly coupled to the reaction of PAL in developing leaves. It has been shown that CdCl2 treatment results in an increase in NH4+
accumulation, an increase in the specific activity of PAL and a decrease in the activity of GS in rice leaves (Hsu and Kao, 2004). Kumar and Knowles (2003) demonstrated that the specific activity of PAL induced by wounding is related to the ability to produce superoxide radicals. Previously, we have shown that NH4
+
accumulation in rice leaves subjected to excess Cu is attributable to a decrease in GS activity and suggested, but did not prove, that NH4+ accumulation is part of an overall
expression of oxidative damage caused by an excess of Cu (Chen and Kao, 1998).
Nitric oxide (NO) is a free radical involved in numerous and diverse physiological processes in mammals (Torreilles, 2001) and plants (Lamattina
et al., 2003). Evidence has been obtained for the involvement of NO in plant growth and develop-ment, as well as in defense responses (Durner and Klessig, 1999;Wojtaszek, 2000;Beligni and Lamat-tina, 2001; Lamattina et al., 2003). NO seems to exert a protective function also during abiotic stress. It is reported that NO increases drought tolerance of some species (Garcia-Mata and La-mattina, 2001; Cheng et al., 2002; Neill et al., 2002), salt stress in rice (Uchida et al., 2002), Lupinus lutenus and reed (Zhao et al., 2004), heat stress in rice (Uchida et al., 2002), and Cd stress in rice (Hsu and Kao, 2004) and Lupinus lutenus (Kopyra and Gwo´z´dz´, 2003).
Several recent publications demonstrate that NO counteracts the toxicity of AOS generated by diquat or paraquat in potato and rice (Beligni and Lamattina, 1999; Hung et al., 2002) and oxidative stress induced by abscisic acid (Hung and Kao, 2003), methyl jasmonate (Hung and Kao, 2004), and excess Cd (Hsu and Kao, 2004). Orozco-Ca´rdenas and Ryan (2002) demonstrated that NO blocks H2O2production induced by jasmonic acid in
tomato leaves. In the present investigation, we examined the effect of NO on Cu toxicity and NH4+
accumulation caused by excess copper in rice leaves.
Material and methods
Plant material and chemicals
Rice (Oryza sativa L., cv. Taichung Native 1) seeds were sterilized with 2.5% sodium hypochlor-ite for 15 min and washed extensively with distilled water. Seeds were then germinated in Petri dishes with wetted filter paper at 37 1C under dark conditions. After 48 h, uniformly germinated seeds were selected and cultivated in a 500 mL beaker containing half-strength Kimura B solution as described previously (Chu and Lee, 1989). The hydroponically cultivated seedlings were grown for 12 days in a Phytotron with natural sunlight at 30 1C day/25 1C night and 90% relative humidity. The apical 3 cm of the third leaf were used in all experiments. A group of ten segments were floated in a Petri dish containing 10 mL of test solution and application was carried out at 27 1C in the light (40 mmol m2s1).
Test solutions included CuSO4, NO donors, and
the NO scavenger 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). N-Tert-butyl-a-phenylnitrone (PBN), 3-morpholinosy-donimine (SIN-1), sodium nitroprusside (SNP) and a
solution containing ascorbic acid (Asc) and NaNO2
were used as NO donors. All chemicals were purchased from Sigma Co (St. Louis, MO, USA).
Determination of protein, H
2O
2, lipid
peroxidation, NH
4+, and Cu
The toxicity of detached rice leaves exposed to CuSO4 (10 mmol L1) was followed by monitoring
decrease of protein content. For protein extrac-tion, leaf segments were homogenized in a 50 mmol L1 sodium phosphate buffer (pH 6.8).
The extracts were centrifuged at 17,600gn for
20 min and supernatants used for protein measure-ments afterBradford (1976)and for the determina-tion of the activities of antioxidant enzymes. The H2O2 content was measured colorimetrically as
described by Jana and Choudhuri (1981). H2O2
was extracted by homogenizing leaf tissue with phosphate buffer (50 mmol L1, pH 6.5) containing 1 mmol L1 hydroxylamine. The homogenate was centrifuged at 6000gn for 25 min and the
super-natant mixed with 0.1% titanium chloride in 20% (v/ v) H2SO4. The mixture was then centrifuged at
6000gn for 25 min and absorbance measured at
410 nm. The H2O2content was calculated using the
extinction coefficient 0.28 mmol1cm1. Malondial-dehyde (MDA), routinely used as an indicator of lipid peroxidation, was extracted with 5% (w/v) trichloroacetic acid and determined according to
Heath and Packer (1968). NH4+ was determined as
described previously (Chien and Kao, 2000). For the determination of Cu, leaves were dried at 65 1C for 48 h and ashed at 550 1C for 48 h. The ash was incubated with 31% HNO3 and 17.5% H2O2at 70 1C
for 12 h and dissolved in 1 mol L1HNO3. Cu content
was then quantified using an atomic absorption spectrophotometer (Model AA-6800, Shimadzu, Kyoto, Japan).
Enzyme assays
The assays of antioxidant enzymes have been described previously (Hurng and Kao, 1994). Per-oxidase (POX) activity was measured using a modification of the procedure of MacAdam et al. (1992). Activity was calculated using the extinction coefficient [26.6 (mmol L1)1cm1at 470 nm] for tetraguaiacol. Catalase (CAT) activity was assayed by measuring the initial rate of disappearance of H2O2 (Kato and Shimizu, 1987). The activity was
calculated using the extinction coefficient [40 (mmol L1)1cm1 at 240 nm] for H2O2 (Kato and
Shimizu, 1987). Superoxide dismutase (SOD) and ascorbate peroxidase (APX) were determined
ac-cording to Paoletti et al. (1986) and Nakano and Asada (1981), respectively. Glutathione reductase (GR) was determined by the method ofFoster and Hess (1980). One unit of activity for CAT, POX, SOD, APX, and GR was defined as the amount of enzyme which degraded 1 mmol H2O2 per min, caused the
formation of 1 mmol tetraguaiacol per min, inhib-ited 50% the rate of NADH oxidation observed in control, degraded 1 mmol of ascorbic acid per min, and decreased 1A340per min, respectively.
For extraction of glutamine synthetase (GS), leaf samples were homogenized with 10 mmol L1 Tris–HCl buffer (pH 7.6, containing 1 mmol L1 MgCl2, 1 mmol L1 EDTA and 1 mmol L1
2-mercap-toethanol) using a chilled pestle and mortar. The homogenate was centrifuged at 15,000gnfor 30 min
and supernatants used for determination of GS activity after Oaks et al. (1980). One unit of GS activity is defined as 1 mmol L-glutamate
g-mono-hydroxamate formed per min. Phenylalanine am-monia-lyase (PAL) was extracted and determined according toHyodo and Fujinami (1989). One unit of PAL activity was defined as the amount of enzyme, which caused the formation of 1 nmol trans-cinnamic acid per h. For protease extraction, leaf samples were homogenized in a pre-chilled pestle and mortar with 10 mmol L1Tris–HCl buffer (pH 7.4) containing 10 mmol L12-mercaptoethanol at 4 1C. The homogenate was centrifuged at 15,000gn for 30 min and supernatants used for
protease assay after Sheoran and Garg (1978). One unit of protease activity was defined as the amount of enzyme which increased A280 by
0.01 per h.
Statistical analysis
Statistical differences between measurements (n ¼ 4) of different treatments were analyzed following Duncan’s multiple range test or Student’s t-test.
Results
In the present study, the toxicity in detached rice leaves caused by excess CuSO4 was assessed by
monitoring the decrease in protein content. In our previous work, we observed that CuSO4and CuCl2
were equally effective in causing the loss of chlorophyll and protein in detached rice leaves in the light (Chen and Kao, 1999). InFig. 1 the time courses of protein and MDA contents in detached rice leaves treated with either water or 10 mmol L1 CuSO4 in the light are shown. The
Figure 1. Changes in the contents of protein (A), MDA (B), H2O2(C), and Cu (D) in rice leaves treated with CuSO4.
Detached rice leaves were treated with either water or 10 mmol L1CuSO4in the light. Values are means7SE (n ¼ 4).
Asterisks denote values that are significant at Po0:05.
Figure 2. Effect of SB, TU, and GSH pre-treatments on the contents of protein (A), MDA (B), and H2O2(C, D) in rice
leaves treated with CuSO4. Detached rice leaves were pre-treated with water, 1 mmol L 1
SB, 5 mmol L1 TU or 5 mmol L1 GSH for 6 h and then treated with water or 10 mmol L1 CuSO4 for 18 h (A, B, D) or 2 h (C). Values are
means7SE (n ¼ 4). Values with the same letter are not significantly different at Po0:05, according to Duncan’s multiple range test.
promotion of protein loss by CuSO4was evident 4 h
after treatment (Fig. 1A). The MDA content in CuSO4-treated leaves was observed to be greater
than that in water-treated controls throughout the entire duration of incubation (Fig. 1B), indicating that Cu toxicity in detached rice leaves was linked to lipid peroxidation. Lipid peroxidation is caused by AOS (Thompson et al., 1987). The H2O2content
remained unchanged for 4 h, and subsequently increased in control leaves (Fig. 1C) but rapidly increased at 2 h and then declined after CuSO4
-treatment (Fig. 1C). This decline is most likely due to a Cu-catalyzed Fenton reaction (Sandmann and Bo¨ger, 1980). It appears that short-term treatment (2 h) with CuSO4 increased the H2O2 content
whereas long-term treatment (24 h) caused a decrease (Fig. 1C). To determine if the observed toxicity, lipid peroxidation and change in H2O2was
related to an increase of Cu in leaves, the internal Cu concentration was measured. The Cu content of control leaves remained unchanged during 24 h of
incubation in the light (Fig. 1D). However, Cu content in CuSO4-treated detached rice leaves
increased with increasing duration of incubation (Fig. 1D). The increase in Cu content in CuSO4
-treated leaves was evident 2 h after treatment (Fig. 1D).
When rice leaves were pre-treated with free radical scavengers such as SB, TU, and GSH, it was found that they inhibited the reduction in protein content (Fig. 2A) and the increase in MDA content (Fig. 2B) caused by CuSO4. They were also effective
in preventing early increase (Fig. 2C) and late decrease (Fig. 2D) in H2O2 content caused by
CuSO4. These results support the involvement of
AOS as the chemical species inducing toxicity of rice leaves by excess Cu.
Plant cells are equipped with several AOS detoxifying enzymes to protect them against oxidative damage. These include SOD, APX, GR, CAT, and POX (Foyer et al., 1997). The striking increase in lipid peroxidation seen in rice leaves
Figure 3. Changes in the specific activities of SOD (A), APX (B), GR (C), CAT (D), and POX (E) in rice leaves treated with CuSO4. Detached rice leaves were treated with either water or 10 mmol L
1
CuSO4in the light. Values are means7SE
treated with excess Cu may be a reflection of the changes of the specific activities of antioxidant enzymes. As shown inFigs. 3A and E, CuSO4-treated
rice leaves had higher specific activities of SOD and POX than the controls at 2 and 8 h, respectively, after treatment. Low GR specific activity in CuSO4
-treated rice leaves was observed at 2 h after incubation (Fig. 3C). However, higher APX specific activity in CuSO4-treated rice leaves was observed
only at 24 h after treatment (Fig. 3B). Figure 3D
shows that CuSO4-treated rice leaves had higher
CAT specific activity at 12 h and lower CAT specific activity at 24 h than the controls. Pre-treatment of free radical scavengers (SB, TU, and GSH) inhibited the increase in SOD and POX specific activities (Figs. 4A and C) and the decrease in GR specific activity (Fig. 4B) caused by excess Cu. These results further suggest that excess Cu causes strong oxidative stress in rice leaves.
There are reports that NO counteracts oxidative stress in plants (Beligni and Lamattina, 1999;
Beligni et al., 2002; Cheng et al., 2002; Hung et al., 2002; Hung and Kao, 2003,2004; Hsu and Kao, 2004). When rice leaves were pre-treated with NO donor SNP for 6 h, it was observed that the reduction in protein content (Fig. 5A) as well as the increase in MDA content (Fig. 5B) were inhibited. Similar results were obtained by using other NO donors such as PBN, SIN-1, and a mixture of Asc and NaNO2 (data not shown). SNP
pre-treatment was also observed to be effective in preventing early increase (Fig. 5C) and late decrease (Fig. 5D) in H2O2. Furthermore, SNP
pre-treatment inhibited the increase in SOD and POX specific activities (Figs. 6A and C) and the decrease in GR specific activity (Fig. 6B) caused by excess Cu. In the present investigation, NO donors rather than NO per se were used. In order to determine whether the protective effect caused by SNP pre-treatment was the result of a release of NO, 100 mmol L1 c-PTIO, a NO-specific scavenger, was
applied together with 100 mmol L1 SNP. It was found that the protective effect of SNP on CuSO4
-induced toxicity, increase in MDA content, changes in H2O2 content and antioxidant enzyme specific
activities could be reversed by c-PTIO (Figs. 5 and 6). Clearly, the effect of NO donor SNP is attributable to free NO.
Figure 7Ashows that the increase in NH4+content
by CuSO4 was evident 8 h after treatment. NH4+
accumulation caused by CuSO4was observed to be
preceded by a decrease in the activity of GS and an increase in the specific activity of PAL and protease (Fig. 7B–D). When rice leaves were pretreated with free radical scavengers (SB, TU, and GSH), it was found that they inhibited the increase in the content of NH4
+
(Fig. 8A), the decrease in the activity of GS (Fig. 8B), and the increase in the specific activities of PAL and protease (Figs. 8C and D), suggesting that oxidative stress is involved in NH4+ accumulation in CuSO4-treated rice leaves. If
this assumption is correct, then SNP pre-treatment can be expected to counteract the CuSO4-induced
increase in NH4+content, decrease in the activity of
GS, and increase in the specific activities of PAL and Figure 4. Effect of SB, TU, and GSH pre-treatments on
the specific activities of SOD (A), GR (B), and POX (C) in rice leaves treated with CuSO4. Detached rice leaves
were pre-treated with water, 1 mmol L1SB, 5 mmol L1 TU or 5 mmol L1GSH and then treated with either water
or 10 mmol L1 CuSO4 for 18 h. Values are means7SE
(n ¼ 4). Values with the same letter are not significantly different at Po0:05, according to Duncan’s multiple range test.
protease, and c-PTIO can be expected to reverse the SNP effect. As shown inFig. 8, this is indeed the case.
Discussion
Copper-mediated AOS formation has been de-monstrated in isolated chloroplasts of Spinacia oleracea (Sandmann and Bo¨ger, 1980), in intact roots of Silene cucubalus (De Vos et al., 1993) and Oryza sativa (Chen et al., 2000), in detached leaves of Avena sativa (Luna et al., 1994), and in intact leaves of Phaseolus vulgaris (Weckx and Clijsters, 1996). It has also been shown that excess Cu induced lipid peroxidation (De Vos et al., 1993;
Luna et al., 1994;Gallego et al., 1996;Weckx and Clijsters, 1996; Mazhoudi et al., 1997; Chen and Kao, 1999; Savoure´ et al., 1999; Cuypers et al., 2000; Hartley-Whitaker et al., 2001). These find-ings suggest that excess Cu causes oxidative stress in plants. Our results presented here not only show that CuSO4 rapidly increases the content of H2O2
(Fig. 1C), but also demonstrate that it causes an increase in the specific activities of SOD and POX and a decrease in the specific activity of GR (Figs. 3A, C, and E). Furthermore, protein loss and lipid peroxidation were observed in CuSO4-treated rice
leaves (Figs. 1A and B). All these results suggest that excess Cu causes oxidative stress and that Cu-induced toxicity is mediated through oxidative stress. This suggestion is further supported by observations that free radical scavengers (SB, TU, and GSH) inhibit CuSO4-induced toxicity (Fig. 2A),
lipid peroxidation (Fig. 2B), early increase in H2O2
content (Fig. 2C), increase in SOD and POX specific activities (Figs. 4A and C) and decrease in GR specific activities (Fig. 4B).
There is only limited information about the mechanism of Cu-induced H2O2 production. SOD is
known to catalyze the dismutation of superoxide into H2O2and O2. CuSO4treatment rapidly induced
a significant increase in SOD specific activity in rice leaves (Fig. 3A). It appears that Cu-induced early accumulation of H2O2in rice leaves may be, at least
in part, due to Cu-enhanced SOD specific activity. In several plant systems, the accumulation of H2O2
Figure 5. Effect of SNP pre-treatment on the contents of protein (A), MDA (B), and H2O2(C, D) in rice leaves treated
with CuSO4. Detached rice leaves were pre-treated with water, 100 mmol L1SNP or 100 mmol L1SNP+100 mmol L1
c-PTIO for 6 h and then treated with water or 10 mmol L1 CuSO4for 18 h (A, B, D) or 2 h (C). Values are means7SE
(n ¼ 4). Values with the same letter are not significantly different at Po0:05, according to Duncan’s multiple range test.
appears to be mediated by the activation of a plasma-membrane NADPH oxidase complex ( Oroz-co-Ca´rdenas and Ryan, 1999; Pei et al., 2000;
Zhang et al., 2001;Jiang and Zhang, 2002). In the present study, we have not investigated whether early rapid H2O2 production is augmented by the
stimulation of plasma-membrane NADPH oxidase. Further research is necessary to clarify this point.
In the present study, we found that NO reduced early increase in H2O2 content and lipid
peroxida-tion in rice leaves caused by excess Cu (Figs. 5B and C). These results are in agreement with our previous work, in which we demonstrated that NO counteracted paraquat-increased lipid peroxida-tion in rice leaves (Hung et al., 2002). Because lipid peroxidation is a consequence of AOS produc-tion (Thompson et al., 1987), NO may act as an AOS scavenger in CuSO4-treated rice leaves.
Ferrer and Barcelo (1999)demonstrated that the NO donor SNP (5 mmol L1) and NO (55 mmol L1s1) itself were able to inhibit POX activity in the xylem of Zinnia elegans. However, our results show that SNP treatment alone did not affect the specific activity of POX in rice leaves (data not shown). Thus, the reduction of CuSO4-induced increase in
the specific activity of POX by SNP is unlikely due to a direct NO-mediated inhibition of POX.
Of particular interest in the present study is the finding that CuSO4treatment resulted in a decrease
in the activity of GS and an increase in the specific activities of PAL and protease (Figs. 7B–D), which preceded the occurrence of NH4+accumulation (Fig.
8A). It appears that CuSO4-induced NH4+
accumula-tion is mediated through the increase in the specific activities of PAL and protease and the decrease in the activity of GS in rice leaves. NH4+released from
the PAL reaction is known to be trapped in glutamine by the activity of GS (Sakurai et al., 2001). It has been shown that GS in plants is particularly prone to proteolysis under oxidative stress conditions (Ortega et al., 1999; Palatnik et al., 1999; Chien et al., 2002; Ishida et al., 2002), the specific activity of protease increases under photooxidative environmental conditions and treatment with dOH generating system or H2O2
(Casano and Trippi, 1992;Casano et al.,1990,1994), and the specific activity of PAL induced by wound-ing is related to the ability to produce superoxide radicals (Kumar and Knowles, 2003). We have previously shown that paraquat, an AOS generating chemical, increased lipid peroxidation, decreased GS activity, and increased NH4+in rice leaves in the
light (Chien et al., 2002). Here, we demonstrated that free radical scavengers (SB, TU, and GSH) and NO donor SNP were able to counteract the CuSO4
-induced decrease in the activity of GS (Fig. 8B), increase in the specific activities of PAL and protease, and increase in NH4+ content (Fig. 8). It
appears that changes in the content of NH4+, the
activity of GS, and the specific activities of PAL and protease in rice leaves caused by excess Cu are the consequence of oxidative damage.
In the present study, we clearly show that exogenous application of NO donor SNP counteracts Figure 6. Effect of SNP and SNP+c-PTIO pre-treatments
on the specific activities of SOD (A), GR (B) and POX (C) in rice leaves treated with CuSO4. Detached rice leaves
were pre-treated with water, 100 mmol L1 SNP or 100 mmol L1 SNP+ 100 mmol L1 c-PTIO for 6 h and then
treated with water or 10 mmol L1CuSO4for 18 h. Values
are means7SE (n ¼ 4). Values with the same letter are not significantly different at Po0:05, according to Duncan’s multiple range test.
Figure 8. Effect of SB, TU,GSH, SNP, and SNP+c-PTIO pre-treatments on the content of NH4+(A), the activity of GS (B),
and the specific activities of PAL (C) and protease (D) in rice leaves treated with CuSO4. Detached rice leaves were
pre-treated with water, 1 mmol L1SB, 5 mmol L1TU, 5 mmol L1GSH, 100 mmol L1SNP or 100 mmol L1SNP+100 mmol L1
c-PTIO for 6 h and then treated with water or 10 mmol L1CuSO4for 18 h. Values are means7SE (n ¼ 4). Values with the
same letter are not significantly different at Po0:05, according to Duncan’s multiple range test.
Figure 7. Changes in the content of NH4+(A), the activity of GS (B), and the specific activities of PAL (C) and protease
(D) in rice leaves treated with CuSO4. Detached rice leaves were treated with either water or 10 mmol L1CuSO4in the
Cu-induced toxicity and NH4+ accumulation in rice
leaves. It is not known if endogenous NO plays a role in regulating Cu-induced toxicity and NH4 +
accumulation in rice leaves. To determine a possible involvement of endogenous NO in melior-ating the effects caused by CuSO4, c-PTIO was used
to deplete endogenous NO. This removal of NO did not increase toxicity symptoms and NH4+
accumula-tion (data not shown), indicating that endogenous NO plays no role in meliorating Cu-induced effects in leaves. Possibly, the amount of endogenous NO in rice leaves is too low to exert a protective role.
Acknowledgements
This work was supported by the National Science Council of the Republic of China (NSC90-2313-B002-267).
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