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.     

Salicylic acid inhibits the biosynthesis of ethylene in detached rice leaves

The effects of salicylic acid (SA) on ethylene biosynthesis in detached rice leaves were investigated. SA at pH 3.5 effectively inhibited ethylene production within 2 h of its application. It inhibited the conversion of ACC to ethylene, but did not affect the levels of ACC and conjugated ACC. Thus, the inhibitory effect of SA resulted from the inhibition of both synthesis of ACC and the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - SA salicylic acid     

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.     

Light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in leaves is mediated through carbon dioxide

The mechanism of light-inhibited ethylene production in excised rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L.) leaves was examined. In segments of rice leaves light substantially inhibited the endogenous ethylene production, but when CO₂ was added into the incubation flask, the rate of endogenous ethylene production in the light increased markedly, to a level which was even higher than that produced in the dark. Carbon dioxide, however, had no appreciable effect of leaf segments incubated in the dark. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, was not significantly affected by lightdark or CO₂ treatment, indicating that dark treatment or CO₂exerted its effect by promoting the conversion of ACC to ethylene. This conclusion was supported by the observations that the rate of conversion of exogenously applied ACC to ethylene was similarly inhibited by light, and this inhibition was relieved in the presence of CO₂. Similar results were obtained with tobacco leaf discs. The concentrations of CO₂ giving half-maximal activity was about 0.06%, which was only slightly above the ambient level of 0.03%. The modulation of ACC conversion to ethylene by CO₂ or light in detached leaves of both rice and tobacco was rapid and fully reversible, indicating that CO₂ regulates the activity, but not the synthesis, of the enzyme converting ACC to ethylene. Our results indicate that light inhibition of ethylene production in detached leaves is mediated through the internal level of CO₂, which directly modulates the activity of the enzyme converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid Recipient of a Republic of China National Science Council Fellowship     

Ethylene synthesis and growth of tomato hypocotyls: Induction by auxin and fusicoccin and inhibition by vanadate

The effects of fusicoccin (FC) on growth and ethylene synthesis of tomato (Lycopersicon esculentum Mill.) hypocotyls were compared to those of indole-3-acetic acid (IAA). Fusicoccin promoted both growth and ethylene production maximally at <2M. Growth was stimulated to a slightly greater extent by FC as compared to IAA, while ethylene synthesis rates in response to FC were about 50% less than those induced by IAA. Cycloheximide (0.5 M) inhibited auxin-induced growth by 80% but had no effect on FC-induced growth; ethylene production was inhibited to the same extent (58%) when induced by either IAA or FC. Both IAA and FC caused tissue contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-ACC to increase, indicating that like IAA, FC induces ethylene synthesis by stimulating the formation of ACC. Orthovanadate, a potent inhibitor of proton-translocating plasma membrane ATPases, reduced both IAA- and FC-induced growth and ethylene synthesis at concentrations less than 1 mM, with ethylene synthesis being approximately 10 times more sensitive to inhibition than growth. Vanadate did not affect tissue ACC levels, slightly reduced total ACC production, and inhibited conversion of ACC to ethylene. However, significant inhibition of in vivo ethylene-forming enzyme activity required high concentrations of vanadate (1 mM) and was less effective than inhibition by cobaltous ion. The site of action of vanadate in inhibiting ethylene synthesis remains unclear, but the ion did not prevent the elevation of tissue ACC levels in response to IAA or FC. It is unlikely, therefore, that stimulation of plasma membrane H⁺-ATPase activity is required for the induction of ACC synthase by IAA and FC.     

Role of ethylene in the senescence of detached rice leaves

The role of ethylene in the senescence of detached rice leaves in relation to their changes in 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production was studied. In freshly excised rice leaf segments, ACC level and ethylene production rates were very low. Following incubation, the rates of ethylene production increased and reached a maximum in 12 h, and subsequently declined. The rise of ethylene production was associated with a 20- to 30-fold increase in ACC level.

Ethylene seems to be involved in the regulation of the senescence of detached rice leaves. This conclusion was based on the observations that (a) maximum ethylene production preceded chlorophyll degradation, (b) ACC application promoted chlorophyll degradation, (c) inhibitors of ethylene production and ethylene action retarded chlorophyll degradation, and (d) various treatments such as light, cycloheximide, α,α-dipyridyl, Ni²⁺, and cold temperature, which retarded chlorophyll degradation, also inhibited ethylene production.

Abscisic acid promoted senescence but significantly decreased ethylene production, whereas benzyladenine retarded senescence but promoted ethylene production. This is interpreted to indicate that abscisic acid treatment increased the tissue sensitivity to ethylene, whereas benzyladenine treatment decreased it.

     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Ethylene synthesis and growth of tomato hypocotyls: Induction by auxin and fusicoccin and inhibition by vanadate

The effects of fusicoccin (FC) on growth and ethylene synthesis of tomato (Lycopersicon esculentum Mill.) hypocotyls were compared to those of indole-3-acetic acid (IAA). Fusicoccin promoted both growth and ethylene production maximally at <2μM. Growth was stimulated to a slightly greater extent by FC as compared to IAA, while ethylene synthesis rates in response to FC were about 50% less than those induced by IAA. Cycloheximide (0.5 μM) inhibited auxin-induced growth by 80% but had no effect on FC-induced growth; ethylene production was inhibited to the same extent (58%) when induced by either IAA or FC. Both IAA and FC caused tissue contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-ACC to increase, indicating that like IAA, FC induces ethylene synthesis by stimulating the formation of ACC. Orthovanadate, a potent inhibitor of proton-translocating plasma membrane ATPases, reduced both IAA- and FC-induced growth and ethylene synthesis at concentrations less than 1 mM, with ethylene synthesis being approximately 10 times more sensitive to inhibition than growth. Vanadate did not affect tissue ACC levels, slightly reduced total ACC production, and inhibited conversion of ACC to ethylene. However, significant inhibition of in vivo ethylene-forming enzyme activity required high concentrations of vanadate (1 mM) and was less effective than inhibition by cobaltous ion. The site of action of vanadate in inhibiting ethylene synthesis remains unclear, but the ion did not prevent the elevation of tissue ACC levels in response to IAA or FC. It is unlikely, therefore, that stimulation of plasma membrane H⁺-ATPase activity is required for the induction of ACC synthase by IAA and FC.     

Effects of Exogenous 1,3-Diaminopropane and Spermidine on Senescence of Oat Leaves : II. Inhibition of Ethylene Biosynthesis and Possible Mode of Action

The effects of the polyamines spermidine and 1,3-diaminopropane on ethylene biosynthesis and chlorophyll (Chl) loss were studied in peeled leaves of oat (Avena sativa L., var. Victory) incubated in the dark. Peeling off the epidermal cells induces an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity, resulting in an enhanced ACC and ethylene formation. Both polyamines inhibit ethylene biosynthesis from methionine by inhibiting ACC synthase activity and, more effectively, the conversion of ACC to ethylene. They also inhibit Chl loss occurring between 24 and 48 h of dark incubation; but, as shown by inhibitor experiments, inhibition of Chl loss does not result from inhibition of ethylene formation. Ethylene production and Chl loss, both associated with senescence, require membrane integrity; thus, treatments which promote deterioration of membranes inhibit both processes. Ca²⁺ in the incubation medium competitively reduces the polyamine-mediated inhibition of ACC conversion and Chl loss. The data suggest that polyamines initially attach to membranes, thereby inducing changes which, in turn, lead to inhibition of ethylene biosynthesis and retardation of senescence.     

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.     

The effects of 2,2′-bipyridine and other metal chelators on the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in rice

Effects of metal chelators, 2,2-bipyridine, 8-hydroxyquinoline and 1,10-phenenthroline, on the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in detached leaves of light-grown rice (Oryza sativa) seedlings and detached shoots of etiolated rice seedlings were investigated. Metal chelators strongly inhibited the in vivo ACC oxidase activity in detached leaves and detached etiolated shoots. This inhibition could be partially recovered by Fe²⁺. Our results support the notion that Fe²⁺ is an essential cofactor for the conversion of ACC to ethylene in vivo.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - BP 2,2-bypyridine - HQ 8-hydroxylquinoline - MJ methyl jasmonate - PA 1,10-phenanthroline - Put putrescine     

Abscisic acid and Ethrel abolish the inhibition of adventitious root formation of paclobutrazol-treated bean primary leaf cuttings

Paclobutrazol (PB), a triazole growth retardant and an inhibitor of gibberellin biosynthesis, reduced at 17 μM concentration the adventitious root formation of bean primary leaf cuttings. Treatments with 5 μM ABA or 4 μM Ethrel, an ethylenereleasing compound, restored the rooting of PB-treated cuttings. Ethylene production and the content of the precursor 1-aminocyclopropane-l-carboxylic acid (ACC) were enhanced in root-forming tissues of PB-treated petioles 48 h after ABA application. The effect of ABA could be abolished by 10 μM CoCl₂, an inhibitor of ACC oxidase. Thus, ABA might stimulate rooting through its effect on ethylene release. 2 mM silver thiosulphate, an inhibitor of ethylene action, decreased the rooting of PB-treated cuttings similarly to Co²⁺, but failed to negate the ABA effect. These data indicate that the effect of PB on rhizogenesis is not associated directly with the inhibition of the biosynthesis of gibberellins Acknowledgements: We are grateful to Gabriella Biró. This work was supported by the Hungarian National Science Research Foundation (OTKA), Project No. 462.     

Characterization of ethylene production in developing strawberry fruit

Ethylene production, ACC content, and ACC oxidase activity were determined in strawberry fruit harvested at different stages of development and in fruit harvested green and developed in vitro in solutions containing sucrose. In fruit harvested at progressive stages of development from green through full ripe, ethylene production and ACC oxidase activity decreased whereas ACC content increased between the white and pink stages. Fruit detached at the green stage and developed to full ripe by immersion of the cut pedicel in sucrose solutions exhibited an increase in ACC content, decreased ethylene production, and no change in ACC oxidase activity. Detached green fruit provided with sucrose containing 0.5 mM silver (STS) had elevated ethylene production and more ACC oxidase activity than did fruit incubated without the silver salt. Green fruit provided with sucrose containing 1 mM ACC showed markedly increased ACC content, ACC oxidase activity, and ethylene production. These increases were noted following 4 days incubation in ACC, and were more pronounced after 11 days, at which time fruit of all treatments had attained a full-ripe stage of development. Calyx tissue exhibited more ACC oxidase activity, less ACC content, and similar ethylene production compared with receptacle tissue. ACC synthase could not be detected in fruit harvested at different developmental stages or in fruit detached and developed in vitro.abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - HQS 8-hydroxyquinoline hemisulfate - SAM S-adenosyl methionine - STS silver thiosulfate     

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.     

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.     

Exogenous Polyamines Stimulate Ethylene Synthesis by Soybean Leaf Tissues

Exogenous supply of spermine (Spm) markedly stimulated ethyleneevolution from intact soybean leaves of leaf discs, stronglyincreased the level of free 1-aminocyclopropane-1-carboxylicacid (ACC), and slightly stimulated ethylene forming-enzyme(EFE) activity Spm treatment also resulted in leaf epinastyand accelerated leaf senescence Ethylene stimulation was depressed,but not abolished, by light, and was suppressed by inhibitorsof ACC synthase and EFE activity Spermidine had a less pronouncedstimulatory effect on ethylene production whereas the diaminesputrescine and diaminopropane were without effect These resultscontrast with other reports indicating that di- and polyaminesinhibit ethylene biosynthesis in plants, and extend our previousresults on detached tobacco leaves exogenously treated withpolyamines Glycine max, ethylene, polyamines     

Ethylene production in rice bronzing leaves induced by ferrous iron

Bronzing, a nutritional disorder of rice plants which is widely distributed in tropical lowlands, was induced by dipping the cut end of rice leaves into FeSO₄ solution (pH 3.5). Ethylene production; the activities of peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase; and the effects of Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, and 1-aminocyclopropane-1-carboxylate, were investigated in the course of bronzing development. It was found that ethylene production could be stimulated up to about 20 times that of the control by Fe²⁺, and a peak could be reached at about 24 h after incubation. The Fe²⁺-treated leaves also had 10-fold higher peroxidase activity than the control, whereas in vitro enzyme activity was inhibited by Fe²⁺. Cycloheximide retarded in vivo stimulation of peroxidase, indicating that in vivo stimulation resulted from inducing de novo synthesis of the enzyme. No changes in the activities of phenylalanine ammonia-lyase and polyphenol oxidase were observed. The results, obtained from the incubation of leaves with Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, or 1-aminocyclopropane-1-carboxylate, showed that ethylene production was the effect of Fe²⁺ stress and that it was not involved in the process of bronzing development, which is probably an acclimation process to enable plants to cope with stress. The accelerated peroxidase activity may be associated with bronzing development.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - EFE ethylene forming enzyme - PAL phenylalanine ammonia-lyase - POD peroxidase - PPO polyphenol oxidase - SE standard error     

The effect of plant-hormone pretreatments on ethylene production and synthesis of 1-aminocyclopropane-1-carboxylic acid in water-stressed wheat leaves

Excised wheat (Triticum aestivum L.) leaves, when subjected to drought stress, increased ethylene production as a result of an increased synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and an increased activity of the ethyleneforming enzyme (EFE), which catalyzes the conversion of ACC to ethylene. The rise in EFE activity was maximal within 2 h after the stress period, while rehydration to relieve water stress reduced EFE activity within 3 h to levels similar to those in nonstressed tissue. Pretreatment of the leaves with benzyladenine or indole-3-acetic acid prior to water stress caused further increase in ethylene production and in endogenous ACC level. Conversely, pretreatment of wheat leaves with abscisic acid reduced ethylene production to levels produced by nonstressed leaves; this reduction in ethylene production was accompanied by a decrease in ACC content. However, none of these hormone pretreatments significantly affected the EFE level in stressed or nonstressed leaves. These data indicate that the plant hormones participate in regulation of water-stress ethylene production primarily by modulating the level of ACC.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - BA N⁶-benzyladenine - EFE ethylene-forming enzyme - IAA indole-3-acetic acid     

Implication of Ethylene in the Release of Secondary Dormancy in Amaranthus caudatus L. Seeds by Gibberellins or Cytokinin

Ethephon (Eth), gibberellin A₃, A_{4 + 7} (GA₃, GA_{4 + 7}), and 6-benzyladenine (BA) removed secondary dormancy of Amaranthus caudatus seeds. The GAs and BA potentiated the effect of ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene biosynthesis precursor, in terms of the rate or final percent of germination. Aminoethoxyvinylglycine (AVG), an ACC synthase activity inhibitor, was observed to simultaneously inhibit the release from dormancy effected by GA₃ or BA as well as the ethylene production stimulated by these regulators. Breaking of secondary dormancy by GA₃, GA_{4 + 7} or BA was prevented by 2,5-norbornadiene (NBD), an inhibitor of ethylene binding. Ethylene completely or markedly reversed the inhibitory effect of NBD. We thus conclude that the removal of secondary dormancy in Amaranthus caudatus seeds by gibberellin or benzyladenine involves ethylene biosynthesis and action.