Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.     

Polyamines influence morphogenesis and caffeine biosynthesis in in vitro cultures of <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

The influence of polyamines, polyamine inhibitors and ethylene inhibitors were tested in Coffea canephora for in vitro morphogenetic response and caffeine biosynthesis. Coffea canephora produced non-embryogenic and embryogenic calli. Somatic embryos were produced only from the embryogenic callus. Endogenous polyamine pools were estimated in these tissues. Somatic embryos were subjected to secondary embryogenesis under the influence of putrescine, silver nitrate and specific inhibitors of polyamine biosynthesis. Estimation of endogenous total polyamines revealed that embryogenic callus contained 11-fold more spermine and 3.3-fold higher spermidine when compared to non-embryogenic callus. Incorporation of polyamines resulted in 58% explant response for embryogenesis when compared to control with 42% response. Incorporation of silver nitrate resulted in 65% response for embryogenesis. Incorporation of polyamine biosynthetic pathway inhibitors DFMO and DFMA resulted in 83% reduction in embryogenic response with concomitant increase in caffeine levels by two-fold as compared to control. These results have clearly demonstrated that polyamines play a crucial role in embryogenesis and caffeine biosynthesis.     

Inhibition of polyamine biosynthesis by alpha-difluoromethyl ornithine potentiates the cytotoxic effects of arabinosyl cytosine in HeLa cells

The object of this study was to examine the effect of inhibition of polyamine biosynthesis on the cell cycle traverse of HeLa cells using α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ornithine decarboxylase. The results of this study indicate that DFMO inhibits HeLa cell growth by causing a decrease in the intracellular levels of putrescine and spermidine without any significant effect on concentration of spermine. The inhibition is readily reversible by exogenous supply of putrescine to the medium. The DFMO treatment also results in an accumulation of cells in S phase. Further, the use of an S phase-specific drug like Ara-C following DFMO treatment results in a synergistic killing of the tumor cells as revealed by the inhibition of cell growth. These observations suggest that exploitation of regulation of the cell cycle by the depletion of polyamines with the use of inhibitors like DFMO might help in designing better therapeutic regimes in combination with other cytotoxic drugs.     

Polyamine depletion and growth inhibition ofCryptococcus neoformans by α-difluoromethylornithine and cyclohexylamine

The ability of two known inhibitors of polyamine synthesis,-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and cyclohexylamine, an inhibitor of spermidine synthase, to inhibit thein vitro growth and polyamine synthesis of clinical isolates ofCryptococcus neoformans was examined. Treatment ofC. neoformans with either DFMO or cyclohexylamine resulted in depletion of cellular polyamines and inhibition of growth.Cryptococcus neoformans was shown to lack detectable spermine and to require high concentrations of spermidine, but not putrescine, for growth. The growth inhibition by DFMO and cyclohexylamine was reversed by exogenous polyamines. These findings document the ability of cyclohexylamine and DFMO to inhibit polyamine synthesis and growth in clinically important isolates ofC. neoformans.     

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     

Identification of a novel acetylated form of branched-chain polyamine from a hyperthermophilic archaeon Thermococcus kodakarensis

Long/branched-chain polyamines are unique polycations found in thermophiles. The hyperthermophilic archaeon Thermococcus kodakarensis contains spermidine and a branched-chain polyamine, N⁴-bis(aminopropyl)spermidine, as major polyamines. The metabolic pathways associated with branched-chain polyamines remain unknown. Here, we used gas chromatography and liquid chromatography-tandem mass spectrometry analyses to identify a new acetylated polyamine, N⁴-bis(aminopropyl)-N¹-acetylspermidine, from T. kodakarensis; this polyamine was not found in other micro-organisms. The amounts of branched-chain polyamine and its acetylated form increased with temperature, indicating that branched-chain polyamines are important for growth at higher temperatures. The amount of quaternary acetylated polyamine produced was associated with the amount of N⁴-bis(aminopropyl)spermidine in the cell. The ratio of acetylated to non-acetylated forms was higher in the stationary phase than in the logarithmic growth phase under high-temperature stress condition.     

Growth inhibition of pathogenic yeast isolates by α-difluoromethylornithine: An inhibition of ornithine decarboxylase

A large body of evidence exists suggesting that polyamines can play essential roles in cellular growth and differentiation. We examined the ability of -difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, the major rate-limiting enzyme in polyamine biosynthesis, to inhibit the growth of Candida albicans, C. tropicalis, and C. parapsilosis. Substantial growth-inhibition was observed for all three species at DFMO concentrations ranging from 1 to 100 mM. C. tropicalis was significantly more susceptible to DFMO than C. albicans or C. parapsilosis. Depletion of cellular polyamine pools was seen in all 3 species following exposure to DFMO and polyamine depletion enhanced the susceptibility of the organisms to DFMO. The action of DFMO was specifically antagonized by exogenous polyamines. These data suggest that polyamines are important in the growth of Candida spp. and that inhibitors of polyamine biosynthesis may be useful as antifungal agents.     

Fungicidal Activity of Two Spermidine Analogues

The potential use of polyamine analogues as inhibitors of polyamine biosynthesis to control plant pathogenic fungi is well established. However, all of this information relates to the use of putrescine analogues and no data exist for spermidine analogues. In the present work, two spermidine analogues. N¹- and N⁸-acetylspermidine were evaluated against powdery mildew on barley. Post-inoculation treatments reduced infection by 69.7% and 51.5%. respectively. Since the barley powdery mildew fungus cannot be grown axenically. mode of action studies were undertaken using the oat leaf-stripe pathogen Pyrenophora avenae. Neither of the analogues had any effect on polyamine biosynthesis in P. avenae grown in vitro. Although the mechanism(s) by which inhibitors affect in vivo fungal growth and in vitro growth may differ, it is unlikely that the antifungal properties of the analogues are the result of a perturbation in polyamine biosynthesis.     

Selective regulation of polyamine metabolism with methylated polyamine analogues

Polyamine metabolism is intimately linked to the physiological state of the cell. Low polyamines levels promote growth cessation, while increased concentrations are often associated with rapid proliferation or cancer. Delicately balanced biosynthesis, catabolism, uptake and excretion are very important for maintaining the intracellular polyamine homeostasis, and deregulated polyamine metabolism is associated with imbalanced metabolic red/ox state. Although many cellular targets of polyamines have been described, the precise molecular mechanisms in these interactions are largely unknown. Polyamines are readily interconvertible which complicate studies on the functions of the individual polyamines. Thus, non-metabolizable polyamine analogues, like carbon-methylated analogues, are needed to circumvent that problem. This review focuses on methylated putrescine, spermidine and spermine analogues in which at least one hydrogen atom attached to polyamine carbon backbone has been replaced by a methyl group. These analogues allow the regulation of both metabolic and catabolic fates of the parent molecule. Substituting the natural polyamines with methylated analogue(s) offers means to study either the functions of an individual polyamine or the effects of altered polyamine metabolism on cell physiology. In general, gem-dimethylated analogues are considered to be non-metabolizable by polyamine catabolizing enzymes spermidine/spermine-N ¹-acetyltransferase and acetylpolyamine oxidase and they support short-term cellular proliferation in many experimental models. Monomethylation renders the analogues chiral, offering some advantage over gem-dimethylated analogues in the specific regulation of polyamine metabolism. Thus, methylated polyamine analogues are practical tools to meet existing biological challenges in solving the physiological functions of polyamines.     

Polyamine uptake in carrot cell cultures

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca²⁺. The effects of Ca²⁺ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca²⁺ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca²⁺ concentration range between 10 micromolar and 1 millimolar. La³⁺ nullified the stimulatory effect of 10 micromolar Ca²⁺ on putrescine uptake and that of 1 millimolar Ca²⁺ on spermidine uptake. La³⁺ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule.     

Depletion of 9L rat brain tumor cell polyamine content by treatment with d,l-α-difluoromethylornithine inhibits proliferation and the G1 to S transition

d,l-α-Difluoromethylornithine (DFMO), an irreversible inactivator of ornithine decarboxylase, inhibited 9L monolayer culture rat brain tumor cell proliferation at concentrations as low as 1 mM DFMO to about 25% of control growth when cells were seeded at an initial density of 5 × 10⁵/flask. DFMO reduced intracellular putrescine content to <5% of control by 8 h and spermidine content to <5 % of control by 48 h post-treatment. Cytostasis caused by 10 or 25 mM DFMO could both be reversed and blocked by addition of exogenous putrescine. Cells pretreated for 48 h with DFMO and then replated in its absence could not enter exponential growth until polyamine production resumed. Addition of exogenous putrescine at the time of replating allowed pretreated cells to resume exponential growth at the same time as controls. Flow cytometry revealed that the fraction of cells in G1 increased until polyamine accumulation resumed, implying the presence of a G1-S block. Within 6 h of replating, there was a decrease in the fraction of control cells in G1. These observations support the hypothesis that entry of 9L cells into S phase depends on an adequate intracellular pool of polyamines.     

A role for polyamines in glucose-stimulated insulin-gene expression.

The aim of the present study was to evaluate the possible role for polyamines in the glucose regulation of the metabolism of insulin mRNA of pancreatic islet cells. For this purpose islets were prepared from adult mice and cultured for 2 days in culture medium RPMI 1640 containing 3.3 mM- or 16.7 mM-glucose with or without the addition of the inhibitors of polyamine biosynthesis difluoromethylornithine (DFMO) and ethylglyoxal bis(guanylhydrazone) (EGBG). Culture at the high glucose concentration increased the islet contents of both insulin mRNA and polyamines. The synthesis of total RNA, total islet polyamines and polyamines associated with islet nuclei was also increased. When the combination of DFMO and EGBG was added in the presence of 16.7 mM-glucose, low contents of insulin mRNA, spermine and spermidine were observed. Total islet polyamine synthesis was also depressed by DFMO + EGBG, unlike islet biosynthesis of polyamines associated with nuclei, which was not equally decreased by the polyamine-synthesis inhibitors. Total RNA synthesis and turnover was not affected by DFMO + EGBG. Finally, actinomycin D attenuated the glucose-induced enhancement of insulin mRNA, and cycloheximide counteracted the insulin-mRNA attenuation induced by inhibition of polyamine synthesis. It is concluded that the glucose-induced increase in insulin mRNA is paralleled by increased contents and rates of polyamine biosynthesis and that an attenuation of the increase in polyamines prevents the increase in insulin mRNA. In addition, the results are compatible with the view that polyamines exert their effects on insulin mRNA mainly by increasing the stability of this messenger.     

Cytological events induced by the inhibition of polyamine biosynthesis in thin cell layers of tobacco

Summary The proliferative growth of thin cell layers ofNicotiana tabacum cultured on a rhizogenic medium was markedly disturbed when polyamine biosynthesis was inhibited. Treatments with polyamine inhibitors led to cell expansion, accompanied by thinning of the cell wall and inhibition of cell division, and frequent cases of nucleolar extrusion, mainly in the parenchymal layer in contact with the medium. Nucleolar extrusion was not correlated with cell expansion. The highest incidence of nucleolar extrusion occurred when the pathways of putrescine biosynthesis were inhibited and when spermidine synthesis, via S-adenosylmethionine decarboxylase, was blocked. The duration of the growth phase with nuclear amitotic divisions was prolonged in the presence of the inhibitors and root meristem formation delayed. When polyamines were added with the inhibitors, all reactions proceeded as in the controls.Abbreviations CHA cyclohexylamine - DFMA DL--difluoromethyl-arginine - DFMO DL--difluoromethylornithine - LS longitudinal section - MGBG methylglyoxal-bis(guanylhydrazone) - PA polyamine - Pu putrescine - RLS radial longitudinal section - S.E. standard error - Spd spermidine     

Isolation of a polyamine transport deficient cell line from the human non-small cell lung carcinoma line NCI H157

In an effort to study the mechanism underlying the observed phenotype-specific response of human lung cancer cell lines to a polyamine analogue, N¹,N¹²-bis(ethyl)spermine(BESpm), we have isolated a BESpm resistant cell line from the BESpm-sensitive large cell lung carcinoma line NCIH157. The mutant line exhibits identical growth rates in the presence or absence of the analogue. However, the overall growth of mutant cells reaches stationary phase earlier than that of the parental cells. In contrast to the parental cells, where a superinduction of spermidine/spermine N¹-acetyltransferase (SSAT) is associated with BESpm toxicity, treatment of this resistant line with BESpm did not induce SSAT mRNA or enzyme activity. BESpm treatment was not effective in depleting the intracellular polyamine pools and very low intracellular BESpm levels were detected. This BESpm resistance is not mediated by multidrug resistance (MDR) protein, since these cells maintain their sensitivity to the antineoplastic agent adriamycin. Treatment of these cells with methylglyoxal bis(guanylhydrazone) (MGBG), an AdoMetDC inhibitor which enters cell using polyamine transport system, shows no inhibition of cell growth. Our data suggest that these mutant cells are deficient in polyamine transport. Consistent with this hypothesis, exogenous polyamines did not prevent difluoromethylomithine (DFMO) induced growth inhibition in the mutant cells. © 1996 Wiley-Liss, Inc.     

Oligogalacturonides enhance cytokinin-induced vegetative shoot formation in tobacco explants, inhibit polyamine biosynthetic gene expression, and promote long-term remobilisation of cell calcium

Long-sized oligogalacturonides (OGs) are cell wall fragments that induce defence and developmental responses. The Ca²⁺-dependent “egg-box” conformation is required for their activity, and polyamines may prevent them from adopting this conformation. Although OGs are known to inhibit auxin-induced growth processes, their effect on cytokinin-induced ones requires investigation. In the present work OGs were shown to promote cytokinin (benzyladenine, BA)-induced vegetative shoot formation from tobacco leaf explants, independent of the presence of CaCl₂ in the medium and of auxin (indoleacetic acid, IAA) supply. The effect of polyamines, putrescine (PU) and spermidine (SD) supplied with/without their biosynthetic inhibitors (DFMO, CHA) was also investigated, and showed that spermidine enhanced adventitious vegetative shoot formation, but only on medium containing Ca²⁺ and IAA. Treatments with inhibitors blocked this promotive effect. OGs did not alter free polyamine concentrations, but caused a moderate increase of conjugated ones, and exhibited an early inhibitory effect on polyamine biosynthetic gene expression. OGs, but not SD, caused long-term changes in calcium-associated epifluorescent signals in the cell walls, and, later, inside the cells of specific tissues. Electron microscopy analysis (ESI system) demonstrated that calcium accumulated in the cell walls and vacuoles of OG-cultured explants. The relationship between OGs, cytokinin, calcium, and polyamines in adventitious vegetative shoot formation is discussed.     

Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N⁴-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N⁴-bis(aminopropyl)spermidine, an isomer of N⁴-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N⁴-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N⁴-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap–time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N⁴-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N⁴-bis(aminopropyl)spermidine from spermidine via N⁴-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N⁴-aminopropylspermidine and N⁴-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments.     

Polyamine catabolism in carcinogenesis: potential targets for chemotherapy and chemoprevention

Polyamines, including spermine, spermidine, and the precursor diamine, putrescine, are naturally occurring polycationic alkylamines that are required for eukaryotic cell growth, differentiation, and survival. This absolute requirement for polyamines and the need to maintain intracellular levels within specific ranges require a highly regulated metabolic pathway primed for rapid changes in response to cellular growth signals, environmental changes, and stress. Although the polyamine metabolic pathway is strictly regulated in normal cells, dysregulation of polyamine metabolism is a frequent event in cancer. Recent studies suggest that the polyamine catabolic pathway may be involved in the etiology of some epithelial cancers. The catabolism of spermine to spermidine utilizes either the one-step enzymatic reaction of spermine oxidase (SMO) or the two-step process of spermidine/spermine N ¹-acetyltransferase (SSAT) coupled with the peroxisomal enzyme N ¹-acetylpolyamine oxidase. Both catabolic pathways produce hydrogen peroxide and a reactive aldehyde that are capable of damaging DNA and other critical cellular components. The catabolic pathway also depletes the intracellular concentrations of spermidine and spermine, which are free radical scavengers. Consequently, the polyamine catabolic pathway in general and specifically SMO and SSAT provide exciting new targets for chemoprevention and/or chemotherapy.     

Effects of inhibitors of ornithine and S-adenosylmethionine decarboxylases on L6 myoblast proliferation

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.     

Polyamines and cytokinins in celery embryogenic cell cultures

The effect of inhibitors of polyamine biosynthesis on the development of embryogenic cell cultures of celery (Apium graveolus L.) was studied. Several developmental stages of somatic embryos were compared for differences in the content and biosynthesis of free polyamines and for cytokinin content. Cyclohexylamine and particularly methylglyoxal bis(guanylhydrazone), inhibited both cell division and the organization of polar embryos from globular embryos. Difluoromethylornithine slightly promoted embryo development, especially cell division.The free putrescine content of globular embryos was 6-fold that of fully differentiated plantlets, and that of spermidine 2-fold. Only a slight increase in the spermine content was found with embryo development. These differences were confirmed by data from polyamine biosynthesis. Incorporation of ¹⁴C-arginine into polyamines was slightly higher than that of ¹⁴C-ornithine. Over 96% of this incorporation was detected in the putrescine fraction. Incorporation of ¹⁴C into putrescine in globular embryos was 3 to 4-fold that in fully-differentiated plantlets. Incorporation into spermidine and spermine was, however, higher in plantlets than in globular embryos.Cytokinin analysis revealed considerable differences in the biological activity between the developmental stages of embryogenesis. This could be due to endogenous cytokinins and/or BA taken up from the maintenance medium. Cytokinin levels decreased with increased embryo development. Most of the detected cytokinin-like activity co-chromatographed with BA and its metabolites. Some as yet unidentified peaks of activity were recorded in the globular embryos.The results are considered with respect to the possible participation of polyamines and cytokinins in the development of embryogenic cell cultures of celery. It is suggested that the onset of embryogenesis is characterized by a high content of putrescine and cytokinins, while a decrease in putrescine synthesis and cytokinin content, and an increase in spermidine and spermine content, accompany further embryo development and plantlet formation.Abbreviation ADC arginine decarboxylase - ODC ornithine decarboxylase - 2,4-D dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DFMO difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone) - CHA cyclohexylamine - BA benzyladenine - BAR benzyladenine riboside     

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.