Effects of Irradiance and Copper on the Activity of Ascorbate Oxidase in Detached Rice Leaves

The effects of copper on the activity of ascorbic acid oxidasc (AAO) in detached rice leaves under both light and dark conditions and in etiolated rice seedlings were investigated. CuSO₄ increased AAO activity in detached rice leaves in both light and darkness, however, the induction in darkness was higher than in the light. In the absence of CuSO₄, irradiance (40 μmol m^-2 s^-1) resulted in a higher activity of AAO in detached rice leaves than dark treatment. Both CuSO₄ and CuCl₂ increased AAO activity in detached rice leaves, indicating that AAO is activated by Cu. Sulfate salts of Mg, Mn, Zn and Fe were ineffective in activating AAO in detached leaves. CuSO₄ was also observed to increase AAO activity in the roots but not in shoots of etiolated rice seedlings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ammonium ion, ethylene, and NaCl-induced senescence of detached rice leaves

The possibility that NH₄ ⁺ accumulation is linkedto the senescence of detached rice (Oryza sativa) leavesinduced by NaCl was investigated. NaCl was effective in promoting senescenceandin increasing NH₄ ⁺ content of detached rice leaves.NaCl-promoted senescence is mainly due to the effect of both Na⁺ andCl^- ions. NaCl had no or slight effect on relative water content,suggesting that an osmotic effect is unlikely to be a major factor contributingto senescence of these leaves. NaCl-induced NH₄ ⁺accumulation was due to enhanced nitrate reduction and decreased glutaminesynthetase activity. Exogenous NH₄Cl, which caused an accumulationofNH₄ ⁺ in detached rice leaves, also promoted senescence.Itwas found that an increase in NH₄ ⁺ content preceded theoccurrence of senescence caused by NaCl. Results also show that NaCl-promotedsenescence is unlikely to be due to the lack of glutamate, glutamine,aspartate,and asparagine. The current results suggest that NH₄ ⁺accumulation is linked to NaCl-induced rice leaf senescence. Since ethylene isknown to be a potent promoter of leaf senescence, we also investigated the roleof ethylene in the regulation of NH₄ ⁺-promoted senescenceof detached rice leaves. NaCl or NH₄Cl treatment resulted in adecrease of ethylene production. Evidence was presented to show thatNH₄ ⁺ accumulation in detached rice leaves does not changetissue sensitivity to ethylene. Clearly, the possible involvement of ethyleneinNH₄ ⁺-promoted senescence is excluded.     

Ammonium accumulation is associated with senescence of rice leaves

The relationship between ammonium accumulation and senescence of detached rice leaves was investigated. Ammonium accumulation in detached rice leaves coincided closely with dark-induced senescence. Exogenous NH4Cl and methionine sulfoximine, which caused an accumulation of ammonium in detached rice leaves, promoted senescence. Treatments such as light and benzyladenine, which retarded senescence, decreased ammonium level in detached rice leaves. Abscisic acid, which promoted senescence, increased ammonium level in detached rice leaves. The current results suggest that ammonium accumulation may be involved in regulating senescence. Evidence was presented to show that ammonium accumulated in detached rice leaves increases tissue sensitivity to ethylene. The accumulation of ammonium in detached rice leaves during dark-induced senescence is attributed to a decrease in glutamine synthetase activity and an increase in reduction of nitrate.     

Light-Dependent Ammonium Ion Toxicity of Rice Leaves in Response to Phosphinothricin Treatment

Ammonium ion accumulation in detached rice leaves treated with phosphinothricin (PPT), an inhibitior of glutamine synthetase (GS), was investigated in the light and darkness. PPT treatment increased NH₄ ⁺ content and induced toxicity in rice leaves in the light but not in darkness, suggesting the importance of light in PPT-induced NH₄ ⁺ toxicity in detached rice leaves. PPT treatment in the light resulted in a decrease of activities of the cytosolic form of GS and the chloroplastic form of GS. The photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced NH₄ ⁺ accumulation induced by PPT in the light. In darkness, PPT-induced NH₄ ⁺ accumulation and toxicity were observed in the presence of glucose or sucrose.     

Water stress, ammonium, and leaf senescence in detached rice leaves

Ammonium accumulation in relation to water stress-promoted senescence of detached rice leaves was investigated. The effect of water stress on the senescence of detached rice leaves is associated with the accumulation of ammonium. The accumulation of ammonium in detached rice leaves by water stress is attributed to a decrease in glutamine synthetase activity. Ammonium accumulation in detached rice leaves, induced by water stress, was accompanied by an increase in tissue sensitivity to ethylene which, in turn, accelerated leaf senescence.     

The toxicity induced by phosphinothricin and methionine sulfoximine in rice leaves is mediated through ethylene but not abscisic acid

The possible role of ethylene and abscisic acid (ABA) in regulating thetoxicity of detached rice leaves induced by phosphinothricin (PPT) andmethionine sulfoximine (MSO), both known to be glutamine synthetase (GS)inhibitors, was studied. During 12 h of incubation, PPT and MSOinhibited GS activity, accumulated NH₄ ⁺ and inducedtoxicity of detached rice leaves in the light but not in darkness. PPT and MSOtreatments also resulted in an increase of ethylene production and ABA contentin a light dependent way. Addition of fluridone, an inhibitor of ABAbiosynthesis, reduced ABA content in rice leave but did not preventNH₄ ⁺ toxicity of rice leaves induced by PPT and MSO.Cobalt ion, an inhibitor of ethylene biosynthesis, affected PPT- andMSO-inducedtoxicity of detached rice leaves but had no effect on PPT- and MSO-inducedNH₄ ⁺ accumulation. Results suggest that ethylene but notABA may be responsible for PPT- and MSO-induced toxicity of detached riceleaves.     

Effects of Juglone on Growth of Muskmelon Seedlings with Respect to Physiological and Anatomical Parameters

The effect phosphinothricin (PPT), an inhibitor of glutamine synthetase (GS), on proline accumulation in detached rice leaves was investigated. During 12 h incubation, PPT inhibited GS activity and induced accumulation of NH₄ ⁺, and accumulation of proline in the light but not in darkness. Proline accumulation caused by PPT in the light was related to protein hydrolysis, and increase in the contents of precursors of proline, ornithine and arginine. Abscisic acid accumulation was not required for proline accumulation in PPT-treated rice leaves. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hydrogen peroxide is involved in methyl jasmonate-induced senescence of rice leaves

The role of H₂O₂ in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H₂O₂ production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H₂O₂, was observed to be effective in inhibiting MJ‐induced senescence and MJ‐increased malondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ‐induced H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂‐generating enzyme in MJ‐treated detached rice leaves. DPI and IMD also inhibited MJ‐promoted senescence and MJ‐increased MDA content in detached rice leaves. Phosphatidylinositol 3‐kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ‐induced H₂O₂ production and senescence of detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)‐sensitive and those of cultivar Tainung 67 (TNG67) are JA‐insensitive. On treatment with JA, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ‐induced H₂O₂ production in detached rice leaves is abscisic acid (ABA)‐independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ‐ and ABA‐induced H₂O₂ production and senescence of detached rice leaves, suggesting that the action of MJ and ABA is ethylene‐dependent.     

Ammonium Ion, Ethylene, and Abscisic Acid in Polyethylene Glycol-treated Rice Leaves

Polyethylene glycol (PEG)-treatment decreased chlorophyll and protein contents and increased NH₄ ⁺ content due to decreased glutamine synthetase activity in detached rice leaves. PEG-treatment also increased abscisic acid (ABA) content and decreased ethylene production. Addition of fluridone, an inhibitor of ABA biosynthesis, reduced ABA content in rice leaves but did not prevent chlorophyll and protein loss in rice leaves induced by PEG. Silver thiosulfate, an inhibitor of ethylene action, was effective in preventing PEG-promoted chlorophyll and protein loss, but had no effect on PEG-induced NH₄ ⁺ accumulation. The current results suggest that NH₄ ⁺ accumulation in rice leaves induced by PEG increases leaf sensitivity to ethylene, which in turn results in an enhancement of chlorophyll and protein loss. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ammonium in relation to proline accumulation in detached rice leaves

Ammonium accumulation in relation to prolineaccumulation in detached rice leaves under stressconditions was investigated. Ammonium accumulation indark-treated detached rice leaves preceded prolineaccumulation. Ammonium accumulation caused by waterstress coincided closely with proline accumulation indetached rice leaves. Exogenous NH₄Cl andmethionine sulfoximine (MSO), which caused anaccumulation of ammonium in detached rice leaves,increased proline content. It was found that prolinein NH₄Cl- or MSO-treated rice leaves is lessutilized than in water-treated rice leaves (controls). These results are in agreement with the observationthat a decrease in proline utilization contributes tothe accumulation of proline in dark-treated and waterstressed rice leaves. Although ammonium contentincreased in Cd- and Cu-treated rice leaves, theincrease in ammonium content was only observed afterthe increase in proline content.     

Phosphatidylinositol 3-phosphate is required for abscisic acid-induced hydrogen peroxide production in rice leaves

Rice leaves produce H₂O₂ in response to abscisic acid (ABA), which results in induction of senescence and accumulation of NH₄⁺. The upstream steps of the ABA-induced H₂O₂ production pathway in rice leaves remain largely unclear. In animal cells, H₂O₂ production in neutrophils is activated by phosphatidylinositol 3-phosphate (PI3P), a product of phosphatidylinositol 3-knase (PI3K). In the present study, we examined whether PI3P plays a role in H₂O₂ production in rice leaves exposed to ABA. We found that PI3K inhibitors LY 294002 (LY) or wortmannin (WM) inhibited ABA-induced H₂O₂ production, senescence and NH₄⁺ accumulation. Hydrogen peroxide almost completely rescued the inhibitory effect of LY or WM. It appears that PI3P plays a role in ABA-induced H₂O₂ production, senescence, and NH₄⁺ accumulation in rice leaves.     

Inhibition of methyl jasmonate-promoted senescence in rice leaves by a metal chelator, 2,2′-bipyridine

The possible mediatory role of transition metals in methyl jasmonate- (MJ-)induced senescence of rice leaves was investigated. Metal chelators(2,2-bipyridine, 8-hydroxylquinoline and 1,10-phenanthroline) reducedMJ-promoted senescence of rice leaves. The reduction of MJ-promoted senescenceby 2,2-bipyridine(BP) is closely associated with the decrease in lipidperoxidation and increase in activity of superoxide dismutase (SOD). Our resultssuggest that iron or copper plays a major role in MJ-promoted senescence ofdetached rice leaves. BP-reduced senescence of detached rice leaves induced byMJ was reversed by adding Fe²⁺ or Cu²⁺, but notby Mn²⁺ or Mg²⁺. Reduction of MJ-promotedsenescence of detached rice leaves by BP is most likely mediated throughchelation of iron or copper and an increase in SOD activity.     

γ‐Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH₄⁺) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH₄⁺ toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH₄⁺ concentrations above 0.75 mM inhibited the growth of rice and caused NH₄⁺ accumulation in both shoots and roots. Use of excessive NH₄⁺ also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH₄⁺ conditions, exogenous γ‐aminobutyric acid (GABA) treatment limited NH₄⁺ accumulation in rice seedlings, reduced NH₄⁺ toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH₄⁺. Furthermore, we found that the activity of glutamine synthetase/NADH‐glutamate synthase (GS; EC 6.3.1.2/NADH‐GOGAT; EC1.4.1.14) in root increased gradually as the NH₄⁺ concentration increased. However, when the concentration of NH₄⁺ is more than 3 mM, GABA treatment inhibited NH₄⁺‐induced increases in GS/NADH‐GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH₄⁺. These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA‐dependent alleviation of ammonium toxicity in rice seedlings.     

Hydrogen peroxide is required for abscisic acid-induced NH4+ accumulation in rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced NH₄⁺ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH₄⁺ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH₄⁺ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced accumulation of NH₄⁺ in rice leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-α-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H₂O₂ contents in rice leaves, inhibited ABA-induced increase in the content of NH₄⁺. Similarly, the changes of enzymes responsible for NH₄⁺ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H₂O₂ was found to increase NH₄⁺ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H₂O₂ is involved in ABA-induced NH₄⁺ accumulation in rice leaves.     

Endogenous abscisic acid levels are not linked to methyl jasmonate-promoted senescence of detached rice leaves

Both abscisic acid (ABA) and jasmonates are known to promote leaf senescence. Since ABA and jasmonates have both chemical and physiological similarities, we are interested to know whether senescence of detached rice leaves induced by methyl jasmonate (MJ) is mediated through an increase in endogenous ABA levels. In darkness, the endogenous level of ABA in detached rice leaves remained unchanged in the first day of incubation in water and increased about 5 times its initial value in the second day. However, the pattern of senescence, as judged by protein loss, was rapid during the first day. MJ significantly promoted senescence of detached rice leaves. Contrary to our expectation, endogenous ABA levels decreased in MJ-treated detached rice leaves. Similar to the effect of MJ, endogenous ABA levels decreased in detached rice leaves which were induced to senesce by treatment with NH₄Cl. These results suggest that endogenous ABA levels are not linked to MJ-induced senescence of detached rice leaves.     

The effect of NaCl on proline accumulation in rice leaves

The regulation of proline accumulation in detached leaves of rice(Oryza sativa cv. Taichung Native 1) was investigated.Increasing concentrations of NaCl from 50 to 200 mM progressivelyincreased proline content in detached rice leaves. NaCl induced prolineaccumulation was mainly due to the effect of both Na⁺ andCl^– ions. Proline accumulation caused by NaCl was related toprotein proteolysis, an increase in ornithine--aminotransferaseactivity,a decrease in proline dehydrogenase activity, a decrease in prolineutilisation,and an increase in the content of the precursors of proline biosynthesis,ornithine and arginine. Results also show that proline accumulation caused byNaCl was associated with ammonium ion accumulation.     

The participation of hydrogen peroxide in methyl jasmonate-induced NH(4)(+) accumulation in rice leaves

Ammonium is a central intermediate in the nitrogen metabolism of plants. We have previously shown that methyl jasmonate (MJ) not only increases the content of H(2)O(2), but also causes NH(4)(+) accumulation in rice leaves. More recently, H(2)O(2) is thought to constitute a general signal molecule participating in the recognition of and the response to stress factors. In this study, we examined the role of H(2)O(2) as a link between MJ and subsequent NH(4)(+) accumulation in detached rice leaves. MJ treatment resulted in an accumulation of NH(4)(+) in detached rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease appear to be the enzymes responsible for the accumulation of NH(4)(+) in MJ-treated detached rice leaves. Dimethylthiourea (DMTU), a chemical trap for H(2)O(2), was observed to be effective in inhibiting MJ-induced NH(4)(+) accumulation in detached rice leaves. Scavengers of free radicals (sodium benzoate, SB, and glutathione, GSH), nitric oxide donor (N-tert-butyl-alpha-phenylnitrone, PBN), the inhibitors of NADPH oxidase (diphenyleneiodonium chloride, DPI, and imidazole, IMD), and inhibitors of phosphatidylinositol 3-kinase (wortmannin, WM, and LY 294002, LY), which have previously been shown to prevent MJ-induced H(2)O(2) production in detached rice leaves, inhibited MJ-induced NH(4)(+) accumulation. Similarly, changes in enzymes responsible for NH(4)(+) accumulation induced by MJ were observed to be inhibited by DMTU, SB, GSH, PBN DPI, IMD, WM, or LY. Seedlings of rice cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)-sensitive and those of cultivar Tainung 67 (TNG67) are JA-insensitive. On treatment with JA, H(2)O(2) accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ- and abscisic acid-induced accumulation of NH(4)(+) and changes in enzymes responsible for NH(4)(+) accumulation in detached rice leaves, suggesting that the action of MJ and ABA is ethylene dependent.     

Proline accumulation induced by excess nickel in detached rice leaves

The regulation of proline accumulation in detached rice leaves exposed to excess NiSO₄ was investigated. NiSO₄ treatment increased proline and Ni contents but had no effect on relative water content, indicating that proline accumulation in Ni-exposed detached rice leaves was due to Ni uptake per se, rather than to water stress. Proline accumulation caused by NiSO₄ was related to protein hydrolysis, a decrease in proline dehydrogenase activity, and a decrease in proline utilization. It seems that an increase in the content of ammonia and an increase in the activities of Δ¹-pyrroline-5-carboxylate reductase and ornithine-δ-aminotransferase play minor if any role in Ni-induced proline accumulation in detached rice leaves.     

Inducible nitrate reductase of rice plants as a possible indicator for nitrification in water-logged paddy soils

When following the pattern of the disappearance of NH ₄ ⁺ –N from ammonium sulfate applied to the flooded soil-rice plant system (field and greenhouse experiments) during a growing season, it was observed that the lowest NH ₄ ⁺ –N level coincided with the highest value of NR activity in the leaves. Nitrate was detected in both the root and shoot systems of the rice plants and autotrophic nitrifiers (Nitrosomonas and Nitrobacter) were particularly abundant. Since it was also demonstrated in this work that the NR activity of rice plants grown with nitrate fertilization (growth chamber culture experiments) was inducible by its substrate, it can be assumed that NH ₄ ⁺ –N oxidation takes place in the water-logged soil studied. Therefore, the occurrence of the nitrification process following NH ₄ ⁺ –N fertilizer application can be predicted by thein vitro orin situ evaluation of the NR activity of the rice leaf as an indicator.     

Changes in ammonium ion content and glutamine synthetase activity in rice leaves caused by excess cadmium are a consequence of oxidative damage

Ammonium ion accumulation and the decrease in glutamine synthetase (GS)activity induced by CdCl₂ were investigated in relation to lipidperoxidation in detached rice leaves. CdCl₂ was effective inincreasing ammonium ion content, decreasing GS activity and increasing lipidperoxidation. Free radical scavengers (glutathione, thiourea, sodium benzoate)and an iron chelator (2,2-bipyridine) were able to inhibit the decreasein GS activity and ammonium ion accumulation caused by CdCl₂ and atthe same time inhibit CdCl₂-induced lipid peroxidation. Paraquat,which is known to produce oxygen radicals, decreased GS activity, increasedammonium ion content, and increased lipid peroxidation. GS1 appears to be thepredominant isoform present. Excess Cd caused a decrease in GS1 but not in GS2in detached rice leaves. An increase in lipid peroxidation preceded ammoniumionaccumulation and the decrease in GS1 activity. These results suggest that thedecrease in GS activity and the accumulation of ammonium ions in detached riceleaves are a consequence of oxidative damage caused by excess Cd.