Effect of photoperiod on polyamine metabolism in apical buds of g2 peas in relation to the induction of apical senescence

Polyamine content and arginine decarboxylase activity of apical buds were measured to determine whether polyamines are required to prevent apical senescence in pea. Polyamines were assayed as dansyl derivatives which were separated by reverse phase high performance liquid chromatography and detected by fluorescence spectrophotometry. High polyamine concentrations were found in the vigorous apices of plants grown under a short day photoperiod during which senescence is delayed. As the apex senesced in long days, the amounts of polyamines per organ declined in parallel with decreases in the size of the apical bud. However, a decrease in polyamine concentration, due mainly to a change in spermidine, occurred at the time of marked reduction in bud size and growth rate, but not before the onset of the early symptoms of senescence. No correlation was found with arginine decarboxylase. The results suggest polyamines may be required to support bud growth, but the photoperiodic mechanism which governs apical senescence of G2 peas does not exert control through polyamine metabolism.     

Spermidine acetyltransferase is required to prevent spermidine toxicity at low temperatures in Escherichia coli

Polyamines are required for optimal growth in most cells; however, polyamine accumulation leads to inhibition of cellular growth. To reduce intracellular polyamine levels, spermidine is monoacetylated in both prokaryotes and eukaryotes. In Escherichia coli, the speG gene encodes the spermidine acetyltransferase, which transfers the acetyl group to either the N-1 or N-8 position. In addition to polyamine accumulation, stress conditions, such as cold shock, cause an increase in the level of spermidine acetylation, suggesting an adaptive role for reduced polyamine levels under stressful growth conditions. The effect of spermidine accumulation on the growth of E. coli at low temperature was examined using a speG mutant. At 37 degrees C, growth of the speG mutant was normal in the presence of 0. 5 or 1 mM spermidine. However, following a shift to 7 degrees C, the addition of 0.5 or 1 mM spermidine resulted in inhibition of cellular growth or cell lysis, respectively. Furthermore, at 7 degrees C, spermidine accumulation resulted in a decrease in total protein synthesis accompanied by an increase in the synthesis of the major cold shock proteins CspA, CspB, and CspG. However, the addition of 50 mM Mg(2+) restored growth and protein synthesis in the presence of 0.5 mM spermidine. The results indicate that the level of spermidine acetylation increases at low temperature to prevent spermidine toxicity. The data suggest that the excess spermidine replaces the ribosome-bound Mg(2+), resulting in ribosome inactivation at low temperatures.     

The effect of polyamines on ethylene synthesis during normal and pollination-induced senescence of Petunia hybrida L. flowers

Senescence of Petunia hybrida L. flowers is accompanied by a climacteric pattern in ethylene production and a rapid decline in the levels of putrescine and spermidine during the preclimacteric phase. The decrease in spermidine is caused by the decline in the availability of putrescine which is initially synthesized from L-arginine via agmatine and N-carbamoylputrescine. Inhibition of putrescine and polyamine synthesis resulted in a rapid drop in the levels of putrescine and spermidine without resulting in a concomitant increase in ethylene production. These results indicate that polyamine synthesis is not involved in the control of ethylene synthesis through its effect on the availability of S-adenosylmethionine, and is confirmed by the results obtained with pollinated flowers. Treatment with polyamines may stimulate or suppress ethylene production in the corolla, depending on the concentrations applied. In unpollinated flowers the onset of the climacteric rise in ethylene production was accelerated after treatment with polyamines. However, in pollinated flowers this process was delayed as a result of treatment with low concentrations of polyamines. The effects of exogenous polyamines on ethylene production in both pollinated and unpollinated flowers indicate that ethylene synthesis in these flowers is not regulated by a feedback control mechanism. Although polyamines do not play a key role in the control of ethylene production during the early stages of senescence through their effect on the availability of S-adenosylmethionine, it appears that they play an important role in some of the other processes involved in senescence.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MGBG methylglyoxal bis-(guanylhydrazone) - SAM S-adenosylmethionine     

The putrescine analogue 1-aminooxy-3-aminopropane perturbs polyamine metabolism in the phytopathogenic fungus<Emphasis Type="Italic"> Sclerotinia sclerotiorum</Emphasis>

The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40–50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.Abbreviations AdoMetDC S-Adenosyl-methionine decarboxylase - DFMO -Difluoromethylornithine - MGBG Methylglyoxal bis-[guanyl hydrazone] - ODC Ornithine decarboxylase     

Ethylene evolution during ripening of detached tomato fruit: Its relation with polyamine metabolism

Ethylene and polyamine metabolism, both sharing a common precursor, S-adenosylmethionine (SAM), were investigated during detached tomato (Lycopersicon esculentum Mill. nothovar F1 Lorena) fruit ripening. Putrescine (PUT) was found to be the major polyamine in the fruits, always over 100 nmols/g FW, while spermidine (SPD) was between 7% and 3% of the level of PUT. Spermine (SPM) was not detected at any stage of ripening. The level of PUT and SPD, did not change significantly during ripening in spite of the almost continuous synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene precursor, and only at the last stage of ripening was a drastic decrease in SPD content observed. The results obtained show that the onset of ACC synthesis and its accumulation within the tissue is not a consequence of a decrease in SPD synthesis.     

Activation of casein synthesis by prostaglandins plus spermidine in mammary gland explants of mice.

Prostaglandins B₂, E₂ or F_2α in combination with 0.5 mM spermidine stimulated casein synthesis in mouse mammary gland explants in a prolactin-like manner. Also, methyl GAG, an inhibitor of polyamine synthesis, abolished the stimulation of casein synthesis by prolactin but did not abolish the effect of spermidine plus a prostaglandin.     

The effects of nor-spermidine and dicyclohexylamine on in vitro potato development and free polyamines content

The in vitro micropropagation of potato (Solanum tuberosum L., var. Spunta) on media containing nor-spermidine (nor-SPD), a natural polaymine (PA) or dicyclohexlamine (DCHA), a spermidine synthesis inhibitor was studied to test their effects on plantlet growth and on the level of free cellular polyamines. The triamine nor-SPD, inhibited spermidine synthesis and substantially reduced root growth. DCHA strongly inhibited potato growth but surprisingly the free spermidine level seemed unaffected. These data suggest that dicyclohexylamine acts on the growth and on the development of plants by mechanisms unrelated to polyamine metabolism. Conversely, nor-spermidine was effective in reducing cellular spermidine content and seems to be a spermidine biosynthesis inhibitor in plants.     

Growth and polyamine metabolism inPyrenophora avenae exposed to cyclohexylamine and norspermidine

Summary The effectiveness of inhibitors of polyamine biosynthesis in controlling plant pathogenic fungi is well established. The spermidine synthase inhibitor cyclohexylamine (CHA) and the spermidine analogue norspermidine were evaluated againstin vitro growth of the oat stripe pathogenPyrenophora avenae. Mycelial growth was reduced by 55% upon exposure to 2.0mM CHA while the same concentration of norspermidine reduced growth by 63%. Neither inhibitor had any effect on ODC or AdoMetDC activities, nor the flux of label from ornithine through to the polyamines. Levels of free polyamines in fungal tissue exposed to 0.01 mM norspermidine were unaltered, although 1.0mM CHA did produce a 75% increase in fungal putrescine content. These data suggest that CHA and norspermidine do not reduce fungal growth as a result of a perturbation in polyamine biosynthesis.Abbreviations ODC ornithine decarboxylase - ADC arginine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO adifluoromethylornithine - CHA cyclohexylamine     

Action of methylglyoxal bis (guanyl hydrazone) and related antiprotozoals on Acanthamoeba culbertsoni

Methylglyoxal bis(guanyl hydrazone) (MGBG) and the related diamidine compounds berenil and pentamidine inhibited multiplication of A. culbertsoni. The growth inhibition by MGBG (2.5 mM) in the peptone medium was accompanied by the disappearance of spermidine and a marked reduction in the level of diaminopropane. MGBG and berenil completely inhibited growth in a chemically defined medium at 1 mM and 1-2 microM concentration, respectively. However, there was no decrease in the polyamine levels in the early stages of growth inhibition by these agents. Uptake of putrescine, spermidine and spermine by A. culbertsoni has been demonstrated but addition of exogenous polyamines did not reverse the growth inhibitory action of MGBG and berenil. Inhibition of S-adenosylmethionine decarboxylase and decrease in polyamine synthesis do not seem to be the primary targets for the antiamoebic action of MGBG and berenil.     

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

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.     

Photoperiod-induced changes in gibberellin metabolism in relation to apical growth and senescence in genetic lines of peas (Pisum sativum L.)

In an early-flowering line of pea (G2) apical senescence occurs only in long days (LD), while growth in short days (SD) is indeterminate. In SD, G2 plants are known to produce a graft-transmissible substance which delays apical senescence in related lines that are photoperiod-insensitive with regard to apical senescence. Gibberellic acid (GA₃) applied to the apical bud of G2 plants in LD delayed apical senescence indefinitely, while N⁶-benzyladenine and -naphthaleneacetic acid were ineffective. Of the gibberellins native to pea, GA₉ had no effect whereas GA₂₀ had a moderate senescence-delaying effect. [³H]GA₉ metabolism in intact leaves of G2 plants was inhibited by LD and was restored by placing the plants back in SD. Leaves of photoperiod-insensitive lines (I-types) metabolized GA₉ readily regardless of photoperiod, but the metabolites differed qualitatively from those in G2 leaves. A polar GA₉ metabolite, GA_E, was found only in G2 plants in SD. The level of GA-like substances in methanol extracts from G2 plants dropped about 10-fold after the plants were moved from SD to LD; it was restored by transferring the plants back to SD. A polar zone of these GA-like materials co-chromatographed with GA_E. It is suggested that a polar gibberellin is synthesized by G2 plants in SD; this gibberellin promotes shoot growth and meristematic activity in the shoot apex, preventing senescence.Abbreviations GA gibberellin - GA₃ gibberellic acid - SD short days - LD long days     

Changes in 1-aminocyclopropane-1-carboxylic-acid content of cut carnation flowers in relation to their senescence

The rise in ethylene production accompanying the respiration climacteric and senescence of cut carnation flowers (Dianthus caryophyllus L. cv. White Sim) was associated with a 30-fold increase in the concentration of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petals (initial content 0.3 nmol/g fresh weight). Pretreatment of the flowers with silver thiosulfate (STS) retarded flower senescence and prevented the increase in ACC concentration in the petals. An increase in ACC in the remaining flower parts, which appeared to precede the increase in the petals, was only partially prevented by the STS pretreatment. Addition of aminoxyacetic acid (2 mM) to the solution in which the flowers were kept completely inhibited accumulation of ACC in all flower parts.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA -aminoxyacetic acid - STS silver thiosulfate complex     

Polyamine metabolism in enterocytes isolated from newborn pigs.

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.     

Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

Polyamine Levels During Growth, Sporulation, and Spore Germination of Bacillus megaterium

Spermidine was the major (>95%) polyamine of Bacillus megaterium in all stages of growth, although it could be replaced completely by spermine. Log-phase cells had 40 to 50% as much spermidine, based on ribonucleic acid (RNA) content, as did either stationary-phase cells or dormant spores; similar results were obtained in three other bacilli including an asporogenous mutant. Polyamine levels were essentially the same in B. megaterium grown in rich or poor media, or in media of high or low ionic strength. Polyamine levels were elevated three- to sixfold by exogenous spermidine without a major effect on growth, sporulation, or subsequent spore germination. During germination, the absolute amount of spermidine remained constant for almost 2 h until net RNA synthesis had lowered the polyamine/RNA ratio to a value close to that in log-phase cells. At this time, the spermidine level began to rise, and thereafter spermidine and RNA increased in parallel. This parallel relationship between the spermidine and RNA levels was abolished by actinomycin D, but not by chloramphenicol.     

Epidermal keratinocytes actively maintain their intracellular polyamine levels

Increased cellular polyamine levels are thought to be essential for epidermal keratinocyte proliferation. However, a number of studies report that the induction of keratinocyte proliferation and of ornithine decarboxylase, the rate-limiting enzyme of putrescine, spermidine and spermine biosynthesis, is not concordantly expressed. The relationship between epidermal keratinocyte polyamine synthesis and proliferation was studied in neonatal mouse keratinocyte cultures using specific inhibitors of ODC activity to decrease the intracellular polyamine levels. The ODC inhibitors alpha-methyl ornithine (alpha-Me-Orn), alpha-hydrazino ornithine (alpha-HO) and difluoro-alpha-methylornithine (alpha-DFMO) did not significantly inhibit epidermal keratinocyte proliferation at 5 X 10(-3) to 10(-4) M concentrations. At these doses, only alpha-DFMO was seen to decrease (by 70%) the cellular levels of putrescine, but not of spermidine or spermine. Epidermal keratinocyte growth in the higher dose of 20 mM alpha-DFMO, however, did not decrease the cellular levels of putrescine. Polyamine analyses of the spent medium showed that growth in 10 mM alpha-DFMO decreased the normal epidermal cell transport of putrescine and spermidine into the medium. At 20 mM alpha-DFMO concentration, the keratinocytes actually transported, intracellularly, the putrescine and spermidine that are naturally found in the foetal bovine component of the growth medium. We conclude from these studies that epidermal keratinocyte polyamine levels are determined by both the rate of synthesis, and of the transport of these amines into the extracellular medium. Since epidermal keratinocytes actively maintain specific polyamine levels, it appears that these molecules are essential for epidermal keratinocyte function.     

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.     

Polyamines in grapevine microcuttings cultivated in vitro. Effects of amines and inhibitors of polyamine biosynthesis on polyamine levels and microcutting growth and development

The main free amines identified during growth and development of grapevine microcuttings of rootstock 41 B, (Vitis vinifera cv. Chasselas × Vitis berlandieri) cultivated in vitro were agmatine, putrescine, spermidine, spermine, diaminopropane and tyramine (an aromatic amine). Amine composition differed according to tissue, with diaminopropane the major polyamine in the apical parts, internodes and leaves. Putrescine predominated in the roots. There was also a decreasing general polyamine and specific tyramine gradient along the stem from the top to the bottom. Conjugated amines were only found in roots. The application of exogenous amines (agmatine, putrescine, spermidine, tyramine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these amines can be growth limiting. Diaminopropane (the product of oxidation of spermidine or spermine by polyamine oxydases) strongly inhibited microcutting growth and development. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), led to inhibition of microcutting development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition indicating that polyamines are involved in regulating the growth and development of grapevine microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis from ornithine decarboxylase (ODC), had no effect on microcutting development and growth. We propose that ADC regulates putrescine biosynthesis during microcutting development.     

Regulation of tRNA methyltransferase activities by spermidine and putrescine. Inhibition of polyamine synthesis and tRNA methylation by alpha-methylornithine or 1,3-diaminopropan-2-ol in Dictyostelium.

Inhibitors of polyamine synthesis (alpha-methylornithine and 1,3-diaminopropan-2-ol) were used to study the relationship between polyamine synthesis and specific methylations of tRNA in Dictyostelium discoideum during vegetative growth. Polyamine concentrations were found to be 10 mM for putrescine, 1.6 mM for spermidine and 7 mM for 1,3-diaminopropane throughout the growth stage. On treatment of growing amoebae with alpha-methylornithine or with 1,3-diaminopropan-2-ol (each at 5 mM), the syntheses of putrescine, spermidine and 1,3-diaminopropane were arrested within 4h. After polyamine synthesis had ceased, the incorporation of methyl groups into tRNA was considerably decreased under conditions that had no effect on the incorporation of uridine into tRNA, or on net syntheses of protein and of DNA. The following nucleosides in tRNA were concerned: 1 methyladenosine, 5-methylcytidine, 7-methylguanosine, 2-methylguanosine, N2N2-dimethylguanosine and 5-methyluridine (ribosylthymine). The corresponding tRNA methyltransferases, determined in Mg2+-free enzyme extracts, proved to be inactive unless polyamines were added. Putrescine and/or spermidine at concentrations of 10 mM or 1-2 mM respectively stimulate the transmethylation reaction in vitro to a maximal rate and to an optimal extent at exactly the same concentrations as found in vegetative cells. In contrast, 1,3-diaminopropane, which is formed from spermidine, does not affect the methylation of tRNA in vitro at physiological concentrations. Putrescine and/or spermidine stabilize the tRNA methyltransferases in crude extracts in the presence but not in the absence of the substrate tRNA. The results support the view that S-adenosylmethionine-dependent transmethylation reactions can be regulated by alterations of polyamine concentrations in vivo.