Ching Huei Kao

Cadmium toxicity is reduced by nitric oxide in rice leaves

We evaluate the protective effect of nitric oxide (NO) against Cadmium (Cd) toxicity in rice leaves. Cd toxicity of rice leaves was determined by the decrease of chlorophyll and protein contents. CdCl2 treatment resulted in (1) increase in Cd content, (2) induction of Cd toxicity, (3) increase in H2O2 and malondialdehyde (MDA) contents, (4) decrease in reduced form glutathione (GSH) and ascorbic acid (ASC) contents, and (5) increase in the specific activities of antioxidant enzymes (superoxide dismutase, glutathione reductase, ascorbate peroxidase, catalase, and peroxidase). NO donors [N-tert-butyl-α-phenylnitrone, 3-morpholinosydonimine, sodium nitroprusside (SNP), and ASC + NaNO2] were effective in reducing CdCl2-induced toxicity and CdCl2-increased MDA content. SNP prevented CdCl2-induced increase in the contents of H2O2 and MDA, decrease in the contents of GSH and ASC, and increase in the specific activities of antioxidant enzymes. SNP also prevented CdCl2-induced accumulation of NH4+, decrease in the activity of glutamine synthetase (GS), and increase in the specific activity of phenylalanine ammonia-lyase (PAL). The protective effect of SNP on CdCl2-induced toxicity, CdCl2-increased H2O2, NH4+, and MDA contents, CdCl2-decreased GSH and ASC, CdCl2-increased specific activities of antioxidant enzymes and PAL, and CdCl2-decreased activity of GS were reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, a NO scavenger, suggesting that protective effect by SNP is attributable to NO released. Reduction of CdCl2-induced toxicity by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H2O2.

Effect of NaCl stress on H2O2 metabolism in rice leaves

The effect of NaCl stress on H2O2 metabolismin detached rice leaves was studied. NaCl (200 mM)treatment did not cause the accumulation ofH2O2 and resulted in no increase in lipidperoxidation and membrane leakage of leaf tissues. The activities of peroxidase, ascorbate peroxidase,superoxide dismutase, and glutathione reductase wereobserved to be greater in NaCl-stressed rice leavesthan in control leaves. However, glycolate oxidasewas lower in NaCl-treated rice leaves than in thecontrol leaves. There was no difference in catalaseactivity between NaCl and control treatments. Theseresults suggest that some antioxidant enzymes can beactivated in response to oxidative stress induced byNaCl.

Enhanced peroxidase activity in rice leaves in response to excess iron, copper and zinc.

The effect of excess Fe(2+) on the peroxidase (POD) activity in detached rice leaves was investigated. FeSO(4) was effective in stimulating POD activity in detached rice leaves under both light and dark conditions. FeSO(4) but not K(2)SO(4) induced POD activity, indicating that POD activity is induced by Fe(2+). FeSO(4)-induced POD activity is not specific for the rice cultivar used in this study. CuSO(4) and ZnSO(4) were also observed to induce POD activity in detached rice leaves. Cycloheximide blocked the enhanced activity of POD by Fe(2+), Cu(2+) or Zn(2+), indicating de novo biosynthesis of the enzyme. Paraquat treatment resulted in a decrease in POD activity. H(2)O(2) had no effect on POD activity in detached rice leaves. It seems that Fe(2+)-, Cu(2+)- or Zn(2+)-induced POD may not be mediated by free radicals. Using isoelectric focusing to separate POD, it was found that excess Fe(2+), Cu(2+) or Zn(2+) induced both quantitative and qualitative metal-specific changes in POD isozyme pattern in detached rice leaves. A new POD isozyme with a pI of 4.81 can be induced by Fe(2+), Cu(2+) and Zn(2+) in detached rice leaves.

Cell wall peroxidase activity, hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings

The changes in cell-wall peroxidase (POD) activity and H2O2 level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively reduced root growth and increased ionically bound cell-wall POD activity. NaCl had no effect on covalently bound cell-wall POD activities. The reduction of root growth by NaCl is closely correlated with the increase in H2O2 level. Exogenous H2O2 was found to inhibit root growth of rice seedlings. Since ammonium and proline accumulation are associated with root growth inhibition caused by NaCl, we determined the effects of NH4Cl or proline on root growth, cell-wall POD activity and H2O2level in roots. External application of NH4Cl or proline markedly inhibited root growth, increased cell-wall POD activity and increased H2O2 level in roots of rice seedlings in the absence of NaCl. An increase in cell-wall POD activity and H2O2 level preceded inhibition of root growth caused by NaCl, NH4Cl or proline. NaCl or proline treatment also increased NADH-POD and diamine oxidase (DAO) activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the H2O2 generation in the cell wall of NaCl- or proline-treated roots. NH4Cl treatment increased NADH-POD activity but had no effect on DAO activity, suggesting that NADH-POD but not DAO is responsible for H2O2 generation in cell wall of NH4Cl-treated roots.

Abscisic acid induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings.

The changes in the activity of peroxidase (POD) extracted from the cell wall and the level of H(2)O(2) of rice seedling roots treated with abscisic acid (ABA) and their correlation with root growth were investigated. Increasing concentrations of ABA from 3 to 18 µM progressively reduce root growth and increase POD activities (using guaiacol or ferulic acid as a substrate) extracted from the cell wall of rice roots. The reduction of root growth by ABA is also correlated with an increase in H(2)O(2) level. Both diamine oxidase (DAO) and NADH peroxidase (NADH-POD) are known to be responsible for the generation of H(2)O(2). ABA treatment increased NADH-POD and DAO activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the generation of H(2)O(2) in the cell wall of ABA-treated roots. An increase in the level of H(2)O(2) and the activity of POD extracted from the cell wall of rice roots preceded root growth reduction caused by ABA. An increase in DAO and NADH-POD activities coincided with an increase in H(2)O(2) in roots caused by ABA. Since DAO catalyzes the oxidation of putrescine, the results that ABA increases the activity of DAO in roots is consistent with those that ABA decreases the level of putrescine. In conclusion, cell wall stiffening catalyzed by POD is possibly involved in the regulation of root growth reduction caused by ABA.

The Effect of Polyethylene Glycol on Proline Accumulation in Rice Leaves

The regulation of proline accumulation in polyethylene glycol (PEG, −1.5 MPa) treated rice leaves was investigated. PEG treatment resulted in a decrease in relative water content, indicating that PEG treatment caused water stress in rice leaves. Proline accumulation caused by PEG was related to protein hydrolysis, an increase in ornithine-δ-amino- transferase activity, an increase in the content of ammonia, and an increase in the contents of the precursors of proline biosynthesis, glutamic acid, ornithine, and arginine. Results also show that abscisic acid accumulation is not required for proline accumulation in PEG-treated rice leaves.

Regulation of proline accumulation in detached rice leaves exposed to excess copper.

Accumulation of proline in response to excess Cu was studied in detached leaves of rice (Oryza sativa). CuSO(4) was effective in inducing proline accumulation in detached rice leaves under both light and dark conditions. CuSO(4) and CuCl(2) were equally effective in inducing proline accumulation, indicating that proline accumulation is induced by Cu. Sulfate salts of Mg, Mn, and Fe were ineffective in inducing proline accumulation in detached rice leaves. Excess Cu had no effect on relative water content of detached rice leaves, suggesting that Cu-induced proline accumulation is unlikely due to water deficit. Proline accumulation induced by excess Cu was related to proteolysis and an increase in Delta(1)-pyrroline-5-carboxylate reductase or ornithine-delta-aminotransferase activity and could not be explained by proline utilization or stress-induced modifications in proline dehydrogenase or Delta(1)-pyrroline-5-carboxylate dehydrogenase. The content of glutamic acid decreased by excess Cu. The increase in arginine but not ornithine was found to be associated with the increase in proline content in Cu-stressed detached rice leaves. CuSO(4) treatment resulted in an increase in abscisic acid content in detached rice leaves. The possibility that proline accumulation induced by excess Cu is mediated through abscisic acid is discussed.

Copper Toxicity in Rice Seedlings: Changes in Antioxidative Enzyme Activities, H2O2 Level, and Cell Wall Peroxidase Activity in Roots

The changes in lipid peroxidation, antioxidative enzyme activity, H2O2 level, and cell wall peroxidase activity in Cu-stressed roots of rice seedlings and their relation with root growth inhibition were investigated. CuSO4 was effective in inhibiting root growth but not shoot growth. Treatment with CuSO4 resulted in an increase in lipid peroxidation and modulated antioxidative enzyme activities in rice roots. CuSO4 increased the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and peroxidase, but had no effect on catalase. CuSO4 also increased H2O2 level and cell wall peroxidase in roots of rice seedlings. Exogenous application of H2O2 resulted in an inhibition of root growth. It appears that growth inhibition of root caused by Cu is associated with H2O2 dependent peroxidase-catalyzed formation of cross-linking among cell wall polymers.

Paraquat toxicity is reduced by nitric oxide in rice leaves

Summary The role of nitric oxide (NO) in plants has been the object of intensive research. In the present work, we evaluated the protective effect of NO against paraquat (PQ) toxicity of rice (Oryza sativa) leaves. PQ toxicity in rice leaves was determined by the decrease of protein content. PQ toxicity of rice leaves was reduced by free radical scavengers such as thiourea, sodium benzoate, ascorbic acid and sodium azide. NO-releasing compounds [N-tert-butyl-α-phenylnitrone (PBN), sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and ascorbic acid + NaNO2] were effective in reducing PQ toxicity in rice leaves. SIN-1 and ascorbic acid + NaNO2 prevented both the PQ-induced increase in content of malondialdehyde and the PQ-induced decrease in activities of active oxygen species-detoxifying enzymes. The protective effect of SIN-1 or ascorbic acid + NaNO2 on PQ toxicity, PQ-induced lipid peroxidation and PQ-decreased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of NO-releasing compounds is attributed to NO released. Reduction of PQ toxicity by NO in rice leaves is most likely mediated through increase in antioxidative enzyme activities and decrease in lipid peroxidation.

Aluminum effects on lipid peroxidation and antioxidative enzyme activities in rice leaves

The effects of aluminum on lipid peroxidation and activities of antioxidative enzymes were investigated in detached rice leaves treated with 0 to 5 mM AlCl3 at pH 4.0 in the light. AlCl3 enhanced the content of malondialdehyde but not the content of H2O2. Superoxide dismutase activity was reduced by AlCl3, while catalase and glutathione reductase activities were increased. Peroxidase and ascorbate peroxidase activities were increased only after prolonged treatment, when toxicity occurred. The results give evidence that Al treatment caused oxidative stress and in turn, it caused lipid peroxidation.