Senescence of Rice Leaves XXX Levels of Endogenous Polyamines and Dark-Induced Senescence of Rice Leaves

The role of endogenous polyamines in the control of dark-inducedsenescence of detached rice leaves was investigated by quantitatinglevels of various polyamines by HPLC. Putrescine, spermidineand spermine were all present throughout senescence. Neithercadaverine nor 1,3-diaminopropane was detected. During dark-inducedsenescence, there was a marked decrease in levels of putrescineand an increase in those of spermidine and spermine. The rateof production of ethylene increased markedly upon excision ofleaves. -Difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO) caused a reduction in levels of putrescine, yet had noeffect on levels of spermidine and spermine. Neither DFMA norDFMO had any effect on senescence or on the production of ethylene.Treatment with dicyclohexylamine (DCH) and methylglyoxal bis-(guanylhydrazone)(MGBG) reduced levels of spermine and increased those of putrescinein detached leaves. After treatment with DCH or MGBG, both senescenceand the production of ethylene were significantly promoted.The current results suggest that endogenous polyamines may notplay a significant role in the control of dark-induced senescenceof rice leaves. This conclusion is supported by the furtherobservations that (a) benzyladenine, which is known to retardsenescence, decreased levels of putrescine but had no effecton those of spermidine and spermine; and (b) ABA, which promotedsenescence, increased levels of putrescine and had no effecton those of spermidine and spermine. (Received March 30, 1991; Accepted June 27, 1991)     

Effect of paclobutrazol on water stress-induced ethylene biosynthesis and polyamine accumulation in apple seedling leaves

Ethylene biosynthesis and polyamine content were determined in [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol] (paclobutrazol) pre-treated and non-treated water-stressed apple seedling leaves. Paclobutrazol reduced water loss, and decreased endogenous putrescine spermidine content. Gibberellic acid (GA) counteracted the inhibitory effect of paclobutrazol on polyamine content. Paclobutrazol also prevented accumulation of water stress-induced 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), ethylene production and polyamines in apple leaves. α-Difluoromethylarginine (DFMA), but not α-difluoromethylornithine (DFMO), inhibited the rise of putrescine and spermidine in stressed leaves. S-Adenosylmethionine (SAM) was maintained at a steady state level even when ethylene and the polyamines were actively synthesized in stressed apple seedling leaves. The conversion of ACC to ethylene did not appear to be affected by paclobutrazol treatment.     

Stimulation of ethylene production in detached rice leaves by vanadate

The effect of vanadate on ethylene biosynthesis in detached rice leaves was investigated. Vanadate at pH 5.0–7.0 effectively enhanced ethylene production within 3 h of its application. It promoted the conversion of ACC to ethylene. Treatment with vanadate did not decrease ACC level until late stage of incubation, i.e. at 12 h after incubation. Molybdate, an inhibitor of phosphatase had no or much less stimulatory effect on ethylene production than did vanadate at comparable concentrations. Azide, an inhibitor of F₁-ATPase, inhibited ethylene production in detached rice leaves. FC and vanadate were observed to be synergisticly increased ethylene production in detached rice leaves. In conclusion, plasma membrane H⁺-ATPase does not seem to be involved in ethylene biosynthesis in detached rice leaves.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - FC Fusicoccin     

Osmotic stress and water stress have opposite effects on putrescine and proline production in excised rice leaves

The effects of water stress and osmotic stress (sorbitol treatment) on the production of putrescine and proline in excised rice leaves were compared. Osmotic stress and water stress were found to affect differentially the levels of putrescine and proline in excised rice leaves. Putrescine accumulation is induced by osmotic stress, whereas proline accumulation is induced by water stress. The effects of ABA on the levels of proline and putrescine are similar to those of water stress, whereas the effects of jasmonic acid methyl ester (JA-Me) are similar to those of osmotic stress. Water stress results in an increase of endogenous ABA is excised rice leaves. However, neither osmotic stress nor JA-Me has effect on endogenous ABA levels in excised rice leaves. Of particular interest is the finding that proline levels increase when putrescine levels induced by osmotic stress or JA-Me are reduced by D-arginine and -methylornithine. L-arginine and L-ornithine applied exogenously also cause an increase in proline levels. It seems that L-arginine and L-ornithine are preferentially utilized as precursors for putrescine accumulation in excised rice leaves treated with osmotic stress and JA-Me, and for proline accumulation in excised rice leaves exposed to water stress and ABA.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - HPLC high performance chromatography - JA-Me jasmonic acid methyl ester - PVP poly-vinylpyrrolidone     

Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4-[C]methionine into spermidine in aged orange peel discs

The flux of radioactivity from 3,4-[(14)C]methionine into S-adenosyl-l-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylene by 100 millimolar phosphate and 2 millimolar Co(2+) was studied in aged peel discs of orange (Citrus sinensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightly reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-[(14)C]methionine into spermidine.     

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.

     

Effects of exogenous ethylene on ethylene production in citrus leaf tissue

Exogenous ethylene stimulated ethylene production in intact citrus (Citrus sinensis L. Osbeck cv. “Washington Navel”) leaves and leaf discs following a 24-hour exposure. Studies with leaf discs showed that ethylene production decreased when ethylene was removed by aeration. The extent of stimulation was dependent upon the concentration of exogenous ethylene (1-10 microliters per liter). Silver ion blocked the autocatalytic effect of ethylene at concentrations of 0.5 millimolar and lower, but increased ethylene production at higher concentrations. The stimulating effect of ethylene resulted from the enhancement of both 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. Whereas autocatalysis was evident following 24 hours incubation, autoinhibition of wound- and mannitol-induced ethylene production was observed during the first 24-hour incubation. Ethylene treatment during this period resulted in a marked decrease in ACC levels and ethylene production rates. Furthermore, in leaf discs treated for 24 hours with ethylene, ethylene production rates increased greatly during the first 2 hours after removal of exogenous ethylene by aeration. This increase was eliminated if the discs were transferred to propylene instead of air, indicating that the autocatalytic effect of ethylene is counteracted by its autoinhibitory effect. It is suggested that autocatalysis involves increased synthesis of ACC synthase and the enzyme responsible for the conversion of ACC to ethylene, whereas autoinhibition involves suppression of the activity of these two enzymes.     

Enhancement of extractable ethylene at light/dark transition in primary leaves of paclobutrazol-treated Phaseolus vulgaris seedlings

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol], a triazole growth retardant, increased the 1-aminocyclopropane-1-carboxylic acid (ACC) level and resulted in reduced ethylene production, estimated as ethylene release in a closed system or by vacuum-extraction, in the primary leaves of Phaseolus vulgaris L. cv. Juliska seedlings exposed to light. At the light/dark transition, a definite enhancement of the endogenous ethylene level was observed by vacuum-extraction of primary leaves of treated plants and the ethylene deficiency of retardant-treated leaves ceased. The concentration of ACC after the light/dark transition followed the pattern for ethylene, and the increase in ACC content was paralleled by a decrease in malonyl-ACC.
It is concluded that the internal level of ethylene is not necessarily lower in the primary leaves of paclobutrazol-treated bean plants, but under special environmental conditions in vivo it may reach that of the control.     

ACC Synthesis as the Activated Step Responsible for the Rise of Ethylene Production Accompanying Citrus Exocortis Viroid Infection in Tomato Plants

Ethylene production was stimulated during the period when systemic symptoms appeared in tomato plants infected with citrus exocortis viroid (CEV). Neither methionine nor S-adenosylmethionine increased ethylene production in leaf discs. In contrast, 1-aminocyclopropane-l-carboxylic acid (ACC) stimulated ethylene production notably. Whether viroid infection acted upon ACC production, its conversion to ethylene, or both, was studied by determining the time course of the concentration of ACC and its in vivo production and conversion rates. During early symptoms, ACC synthesis increased and then remained steady during the development of symptoms, but no difference in the capacity of conversion of ACC to ethylene between healthy and CEV-infected tissues was observed. This indicates that ethylene production in tomato leaves showing systemic symptoms to CEV is activated at the level of ACC production.     

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     

Ethylene and ACC levels in developing grains are related to the poor appearance and milling quality of rice

The possible relationship between the levels of ethylene and 1-aminocylopropane-1-carboxylic acid (ACC) in the grains and the quality of rice (Oryza sativa L.) were investigated by using 12 rice cultivars. The results showed that both the ethylene evolution rate and ACC content in grains during the grain filling period correlated negatively with head rice production and positively with chalky kernels, chalky size, and chalkiness. The levels of ethylene and ACC were not significantly correlated with alkali spreading value and amylose content. Application of ethephon, an ethylene-releasing agent, or ACC to panicles at the early grain filling stage significantly reduced the rates of brown rice, milled rice, and head rice, and significantly increased the percentage of chalky kernels, chalky size, and chalkiness. Application of aminoethoxyvinylglycine, an inhibitor of ACC synthase, had the opposite effect. Chalkiness appears to be a senescence related phenomenon which is stimulated by ethylene. The results suggest that ethylene and ACC in grains play an important role in regulating rice quality, and that grain appearance and milling quality would be improved though the reduction of ethylene and ACC in grains during grain filling.     

Effects of Low O(2) Root Stress on Ethylene Biosynthesis in Tomato Plants (Lycopersicon esculentum Mill cv Heinz 1350)

Low O₂ conditions were obtained by flowing N₂ through the solution in which the tomato plants (Lycopersicon esculentum Mill cv Heinz 1350) were growing. Time course experiments revealed that low O₂ treatments stimulated 1-aminocyclopropane-1-carboxylate (ACC) synthase production in the roots and leaves. After the initiation of low O₂ conditions, ACC synthase activity and ACC content in the roots increased and reached a peak after 12 and 20 hours, respectively. The conversion of ACC to ethylene in the roots was inhibited by low levels of O₂, and ACC was apparently transported to the leaves where it was converted to ethylene. ACC synthase activity in the leaves was also stimulated by low O₂ treatment to the roots, reaching a peak after 24 hours. ACC synthase levels were enhanced by cobalt chloride and aminooxyacetic acid (AOA), although they inhibited ethylene production. Cobalt chloride enhanced ACC synthase only in combination with low O₂ conditions in the roots. Under aeration, AOA stimulated ACC synthase activity in both the roots and leaves. However, in combination with low O₂ conditions, AOA caused a stimulation in ACC synthase activity in the leaves and no effect in the roots.     

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     

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     

Changes m Ethylene Production in Relation to l-Aminocyclopropane-l-Carboxylic Acid and Its Malonyl Conjugate in Waterlogged Wheat Plants

When wheat seedlings were subjected to waterlogging, 1-aminocyelopropane-l-carboxylic acid (ACC), an ethylene precursor, accumulated in large quantity in roots. In shoots, ACC and ethylene production also increased, but declined with the prolonged periods of waterlogging. However, ACC content in roots maintained in high level during the whole period of waterlogging. Drainage caused a drastic drop in both ACC content and ethylene production in waterlogged plants to control level. 1-(malonylamino) cyclopropane-l-carboxylic acid (MACC) level in roots subjected to waterlogging showed little changes. However, MACC content in shoots kept increasing during the 9-days period of waterlogging. At later period of waterlogging (longer than 5 days) when ACC and ethylene production bad dropped, the. level of MACC continued to increase. Draining stopped this increasing, but did not reduced its level. When exogenous ACC was introduced into the leaves via transpiration stream, the ability of leaves of waterlogged plant to convert ACC to MACC was much higher than control. The data presented showed that at the later stage of waterlogging, the conversien of a great quantity of ACC to MACC in waterlogged wheat plants is the cause of the reduction of ethylene production and ACC content. It was suggested that the formation of MACC is another way of regulation in ethylene biosynthesis. Among leaves of different ages, the enhancement of ethylene, ACC and MACC content was more pronounced in older leaves than in younger laves during the waterlogging period. The physiological significance of adaptation to waterlogging stress was discussed.     

Induction by ozone of ethylene production and an ACC oxidase cDNA in rice (Oryza sativa L.) leaves

Exposure to ozone at 1 µl l^–1 for 6 h induced ethylene production in rice (Oryza sativa L. cv. Hitomebore) leaves. The stimulation of ethylene production was detectable 2 h after the start of the exposure to ozone, and lasted for 6 h after the exposure. A 429-bp cDNA fragment encoding ACC oxidase was obtained by RT-PCR from ozone-treated rice leaves. Its nucleotide sequence and deduced amino-acid sequence had 97.2% and 94.4% identity, respectively, to those of OS1A1COX, which was previously obtained from deepwater rice. The abundance of the cDNA increased in accordance with the induction of ethylene production by the exposure to ozone.     

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     

Contrasting effects of ethylene perception and biosynthesis inhibitors on germination and seedling growth of barley (Hordeum vulgare L.)

The effects of the plant growth regulator ethylene, and of ethylene inhibitors, on barley (Hordeum vulgare L.) germination and seedling growth were investigated. Exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) at 100 microM enhanced ethylene production by barley seedlings and stimulated shoot growth, whereas both germination and seedling growth were inhibited by antagonists of ethylene perception (75 microM silver ions, 100 microM 2,5-norbornadiene (NBD)). In contrast, germination was unaffected by, and root and shoot growth of seedlings was strongly stimulated by inhibitors of ethylene biosynthesis (10 microM cobalt chloride, 10 microM aminoethoxyvinylglycine (AVG)). Since the ethylene and polyamine biosynthetic pathways are linked through S:-adenosylmethionine, this prompted further explorations into the role of polyamines in germination and seedling growth. Exogenous polyamines (putrescine, spermidine and spermine) at 1 microM concentration stimulated barley seedling growth in a similar fashion to the ethylene biosynthetic inhibitors. Both polyamines and ethylene biosynthetic inhibitors reversed the inhibitory effects of ethylene perception inhibitors on germination and seedling growth. Blocking endogenous ethylene production with aminoethoxyvinylglycine enhanced the free putrescine and spermidine content of germinating barley grains. Thus endogenous polyamines may play a complementary, growth-promotive, role to ethylene in the normal course of barley germination. Further, experiments that have been carried out using inhibitors of ethylene biosynthesis may have to be re-evaluated to take the possible effect of polyamines into account.     

Ethylene production and embyogenesis from anther cultures of barley (Hordeum vulgare)

Summary Ethylene production was measured in cultured barley (Hordeum vulgare L.) anthers. The pattern of ethylene production and the content of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were different among cultivars. Ethylene production appeared to be related to embryogenesis (callus and embryo production). In cultivars in which anthers had low amounts of ACC and produced ethylene slowly, the addition of ethylene promotors (Ethrel or ACC) increased embryogenesis. However, in the cultivar Klages, in which anthers had high amounts of ACC and produced ethylene rapidly, the addition of an ethylene production inhibitor (putrescine) increased embryogenesis. Thus, an optimum level of ethylene production appears to be important for embryogenesis. The differences in anther response and callus production among cultivars may be due to both the capacity to produce ethylene and the sensitivity to high ethylene levels.     

Comparative Study of the Metabolism of 1-Aminocyclopropane-1-carboxylic Acid and Senescence of Water-Stressed and ABA-Treated Excised Rice Leaves

Changes in the metabolism of 1-aminocyclopropane-l-carboxylicacid (ACC) during senescence in the light in turgid, water-stressed,and ABA-treated, excised rice leaves were examined. The decreasesin levels of Chl and protein were more rapid in the water-stressedand in the ABA-treated leaves than in the turgid leaves. Inturgid leaves, levels of proline remained very low, but theyincreased considerably as a result of water stress or treatmentwith ABA. The production of ethylene was strongly inhibitedby water stress and by ABA through the inhibition of the synthesisof ACC and/or the conversion of ACC to ethylene. In turgid leaves,the level of 1-(malonylamino)cyclopropane-l-carboxylic acid(MACC) increased with time during incubation in the light. Waterstress resulted in a pattern of accumulation of MACC similarto that in the turgid control. However, ABA blocked the malonylationof ACC. (Received July 27, 1989; Accepted March 12, 1990)