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.     

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.     

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.     

Regulation of ammonium-induced proline accumulation in detached rice leaves

Accumulation of proline in response to NH₄Cl was studied indetached leaves of rice (Oryza sativa cv. Taichung Native1). Increasing concentrations of NH₄Cl from 50 to 200mMprogressively increased proline content and this was correlated with theincrease in ammonium content. Proline accumulation induced by NH₄Clwas related to proteolysis, an increase in ornithine--aminotransferaseactivity, a decrease in proline dehydrogenase activity, and a decrease inproline utilisation and could not be explained by NH₄Cl-inducedmodification in ¹-pyrroline-5-carboxylate reductase activity.The content of glutamic acid was decreased by NH₄Cl, whereas theincrease in arginine and ornithine contents was found to be associated with theincrease in proline content in NH₄Cl-treated detached rice leaves.     

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.     

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.     

Relationship between ammonium accumulation and senescence of detached rice leaves caused by excess copper

The possibility that ammonium (NH ₄ ⁺ ) accumulation is linked to the senescence of detached rice (Oryza sativa) leaves induced by copper (Cu) was investigated. CuSO₄ was effective in promoting senescence of detached rice leaves. Both CuSO₄ and CuCl₂ induced NH ₄ ⁺ accumulation in detached rice leaves, indicating that NH ₄ ⁺ accumulation is induced by copper. Sulfate salts of Mg, Mn, Zn, and Fe were ineffective in inducing NH ₄ ⁺ accumulation in detached rice leaves. The senescence of detached rice leaves induced by Cu was found to be prior to NH ₄ ⁺ accumulation. Free radical scavengers, such as glutathione and thiourea, inhibited senescence caused by Cu and at the same time inhibited Cu-induced NH ₄ ⁺ accumulation. The current results suggest that NH ₄ ⁺ accumulation is not associated with senescence induced by Cu, but is part of the overall expression of oxidative damage caused by an excess of Cu. Evidence was presented to show that copper-induced ammonium accumulation in detached rice leaves 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.     

The Relation between Accumulation of Abscisic Acid and Proline in Detached Rice Leaves

The relation between abscisic acid (ABA) and proline accumulation was investigated in detached rice (Oryza sativa L.) leaves. In darkness, proline content increased about 2-, 2,5- and 6-fold after 24, 48 and 72 h. ABA content reached maximum after 48 h. In the light, proline content remained almost unchanged until 48 h and subsequently increased slightly. ABA content in the light was lower than in darkness, but the maximum was also after 48 h. During 12-h exposure to decreased air humidity, proline content gradually increased, but ABA content increased about 25-fold after 4 h and declined thereafter. Exogenous application of ABA resulted in an increase in proline content in detached rice leaves under both light and darkness.     

Antioxidative responses and proline level in leaves and roots of pea plants subjected to nickel stress

The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST) as well as proline content were studied in leaves and roots of 14 day-old pea plants treated with NiSO₄ (10, 100, 200 μm) for 1, 3, 6 and 9 days. Exposure of pea plants to nickel (Ni) resulted in the decrease in CuZnSOD as well as total SOD activities in both leaves and roots. The activity of APX in leaves of plants treated with 100 and 200 μm Ni increased following the 3rd day after metal application, while in roots at the end of the experiment the activity of this enzyme was significantly reduced. In both organs CAT activity generally did not change in response to Ni treatment. The activity of GST in plants exposed to high concentrations of Ni increased, more markedly in roots. In both leaves and roots after Ni application accumulation of free proline was observed, but in the case of leaves concentration of this amino acid increased earlier and to a greater extent than in roots. The results indicate that stimulation of GST activity and accumulation of proline in the tissues rather than antioxidative enzymes are involved in response of pea plants to Ni stress.     

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.     

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 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.     

Polyamines content and physiological and biochemical responses to ladder concentration of nickel stress in Hydrocharis dubia (Bl.) Backer leaves

Influence of ladder concentration of nickel (Ni) on the leaves of Hydrocharis dubia were studied after 3 days treatment. The accumulation of Ni, the content of polyamines, proline, malondialdehyde (MDA) and soluble protein, as well as the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in the leaves were investigated. The result indicated that the toxicity of Ni manifested in respective aspect of physiological and biochemical characters. Significant increase of Ni concentration in the leaf tissue was observed, which was concentration dependent. Visible symptoms of Ni toxicity: chlorosis and necrosis occurred following the 3rd day. Meantime, treatment with Ni resulted in the increase in the generation rate of O₂^•− in the leaves. SOD and CAT activities decreased significantly in response to Ni treatment, it was possibly the reason of accumulation of O₂^•−. However, a several-fold decrease in POD activities was found. Our results indicated that because of prolonged increases in O₂^•− level, oxidative damage, measured as the level of lipid peroxidation, occured in the leaves of Ni treated fronds. The changes of the content of polyamines (PAs) were also investigated in the leaves of Hydrocharis dubia. Ni treatment significantly increased the putrescine (Put) level and lowered spermidine (Spd) and spermine (Spm) levels, thereby significantly reducing the ratio of free (Spd + Spm)/Put in leaves, which has been considered as the signal under stress. Although the trend that PS-conjugated PAs and PIS-bound PAs changed the same as free PAs, they changed in more less extent.     

Proline protects <Emphasis Type="Italic">Atropa belladonna</Emphasis> plants against nickel salt toxicity

Atropa belladonna L. plants were grown in water culture for 8 weeks before the nutrient medium was supplemented with NiCl₂ to final concentrations of 0 (control treatment), 50, 100, 150, 200, 250, and 300 μM. After 4 days of plant growing in the presence of nickel chloride, the content of water, proline, Ni, Fe, free polyamines, as well as lipid peroxidation rates were measured. The addition of 100–150 μM Ni to the medium significantly reduced the fresh weight increments and water content in comparison with these parameters for untreated plants; 200 μM Ni caused serious, although nonlethal damage to the plants, whereas 250 and 300 μM Ni proved to be lethal. In the aboveground organs, the major part of Ni was accumulated in the apical leaves. When the plants were treated with 200 μM Ni, the Ni content in apical leaves was 220 μg/g dry wt, while Ni content in roots reached 1500 μg/g dry wt. The treatment of plants with proline in the presence of 200 μM Ni inhibited Ni accumulation in tissues. The proline-treated plants exhibited elevated iron content in leaves and especially in roots and were characterized by comparatively low rates of lipid peroxidation and by sustained leaf water status. When 200 μM Ni was applied, the content of free putrescine decreased, while the contents of spermine and spermidine in leaves increased appreciably with respect to the control values. The toxic effect of nickel was accompanied not only by an enhanced accumulation of high- molecular-weight polyamines but also by their oxidative degradation, which was evident from the 14-fold increase in the content of 1,3-diaminopropane. The protective effect of exogenous proline in the presence of high nickel concentrations was manifested in lowered lipid peroxidation rates, alleviation of iron deficiency, and in retarded oxidative degradation of polyamines.     

Direct Regeneration of Shoots from Hairy Root Cultures of Centaurium erythraea Inoculated with Agrobacterium rhizogenes

Effect of free radical scavengers and metal chelators on polyethylene glycol (PEG, osmotic potential −1.5 MPa) induced oxidative damage in detached rice leaves was investigated. PEG treatment resulted in a decrease in relative water content and an increase in proline content, and lipid peroxidation. PEG treatment also decreased chlorophyll and protein contents. Free radical scavengers (ascorbate, sodium benzoate, reduced glutathione, and thiourea) retarded and metal chelators [2,2′-bipyridine (BP), 8-hydroxyquinoline, and 1,10-phenanthroline] prevented PEG-induced oxidative damage. Furthermore, the protective effect of BP was reversed by adding Fe²⁺ and Cu²⁺, but not by Mn²⁺ or Zn²⁺. The protective effect of BP is most likely mediated through chelation of iron. It seems that oxidative damage induced by PEG may require the participation of iron. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Toxicity in leaves of rice exposed to cadmium is due to hydrogen peroxide accumulation

The production of H₂O₂ in detached rice leaves of Taichung Native 1 (TN1) caused by CdCl₂ was investigated. CdCl₂ treatment resulted in H₂O₂ production in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase (NOX), prevented CdCl₂-induced H₂O₂ production, suggesting that NOX is a H₂O₂-genearating enzyme in CdCl₂-treated detached rice leaves. Phosphatidylinositol 3-kinase inhibitors wortmanin (WM) or LY294002 (LY) inhibited CdCl₂-inducted H₂O₂ production in detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY, suggesting that phosphatidylinositol 3-phosphate is required for Cd-induced H₂O₂ production in detached rice leaves. Nitric oxide donor sodium nitroprusside (SNP) was also effective in reducing CdCl₂-inducing accumulation of H₂O₂ in detached rice leaves. Cd toxicity was judged by the decrease in chlorophyll content. The results indicated that DPI, IMD, WM, LY, and SNP were able to reduce Cd-induced toxicity of detached rice leaves. Twelve-day-old TN1 and Tainung 67 (TNG67) rice seedlings were treated with or without CdCl₂. In terms of Cd toxicity (leaf chlorosis), it was observed that rice seedlings of cultivar TN1 are Cd-sensitive and those of cultivar TNG67 are Cd-tolerant. On treatment with CdCl₂, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Prior exposure of TN1 seedlings to 45^oC for 3 h resulted in a reduction of H₂O₂ accumulation, as well as Cd tolerance of TN1 seedlings treated with CdCl₂. The results strongly suggest that Cd toxicity of detached leaves and leaves attached to rice seedlings are due to H₂O₂ accumulation.     

Cadmium toxicity of rice leaves is mediated through lipid peroxidation

Oxidative stress, in relation to toxicity of detached rice leaves,caused by excess cadmium was investigated. Cd content inCdCl₂-treated detached rice leaves increased with increasingdurationof incubation in the light. Cd toxicity was followed by measuring the decreasein chlorophyll and protein. CdCl₂ was effective in inducing toxicityand increasing lipid peroxidation of detached rice leaves under both light anddark conditions. These effects were also observed in rice leaves treated withCdSO₄, indicating that the toxicity was indeed attributed to cadmiumions. Superoxide dismutase (SOD), ascorbate peroxidase (APOD), and glutathionereductase (GR) activities were reduced by excess CdCl₂ in the light.The changes in catalase and peroxidase activities were observed inCdCl₂-treated rice leaves after the occurrence of toxicity in thelight. Free radical scavengers reduced CdCl₂-induced toxicity and atthe same time reduced CdCl₂-induced lipid peroxidation and restoredCdCl₂-decreased activities of SOD, APOD, and GR in the light. Metalchelators (2,2-bipyridine and 1,10-phenanthroline) reducedCdCl₂ toxicity in rice leaves in the light. The reduction ofCdCl₂ toxicity by 2,2-bipyridine (BP) is closely associatedwith a decrease in lipid peroxidation and an increase in activities ofantioxidative enzymes. Furthermore, BP-reduced toxicity of detached riceleaves,induced by CdCl₂, was reversed by adding Fe²⁺ orCu²⁺, but not by Mn²⁺ or Mg²⁺.Reduction of CdCl₂ toxicity by BP is most likely mediated throughchelation of iron. It seems that toxicity induced by CdCl₂ mayrequire the participation of iron.     

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--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.