Cellular systems for the study of the biosynthesis of polyamines and ethylene,as well as of virus multiplication

Leaves of Chinese cabbage from healthy plants or from those infected with turnip yellow mosaic virus yield protoplasts which convert methionine to protein, S-adenosylmethionine, decarboxylated S-adenosylmethionine, spermidine, spermine and 1-aminocyclopropane-1-carboxylate. The enzyme spermidine synthase is entirely cytosolic and has been purified extensively. An inhibitor of this enzyme, dicyclohexylamine, blocks spermidine synthesis in intact protoplasts, and in so doing stimulates spermine synthesis. Aminoethoxyvinylglycine blocks the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylate, the precursor to ethylene, in protoplasts. This inhibitor markedly stimulates the synthesis of both spermidine and spermine. Essentially all the protoplasts obtained from new leaves of plants infected 7 days earlier are infected. On incubation, such protoplasts convert exogenous methionine to viral protein and viral spermidine whose specific radioactivity is twice that of total cell spermidine. Exogeneous spermidine is also converted to cell putrescine and viral spermidine and spermine. Normal and virus-infected cells are being studied for their content of phenolic acid amides of the polyamines.     

Propylamine transferases in chinese cabbage leaves

We have found spermidine synthase and spermine synthase activities in extracts of leaves of Chinese cabbage (Brassica pekinensis var. Pak Choy) and have developed an assay of the former in crude extracts. The method is based on the transfer of the propylamine moiety of decarboxylated S-adenosylmethionine to labeled putrescine, followed by ion-exchange separation of the labeled amine substrate and product, which are then converted to the 5-dimethylamino-1-napthalene sulfonyl (dansyl) derivatives and further purified and identified by thin layer chromatography. The specific radioactivity of putrescine present in the reaction mixture is determined, as is the radioactivity present in dansyl spermidine. The enzyme is also present in extracts of spinach leaves.

Spermidine synthase has been purified about 160-fold from Chinese cabbage leaves. After partial purification, a rapid coupled enzymic assay has been used to study various properties of the enzyme. The plant enzyme shows maximum activity at pH 8.8 in glycine-NaOH buffer and has a molecular weight of 81,000. The K_m values for decarboxylated S-adenosylmethionine and putrescine are 6.7 and 32 micromolar, respectively. The enzyme activity is inhibited strongly by dicyclohexylamine, cyclohexylamine, and S-adenosyl-3-thio-1, 8-diaminoctane. Of these, dicyclohexylamine is the most potent inhibitor with an I₅₀ at 0.24 micromolar.

     

S-adenosylmethionine decarboxylase and spermidine synthase from chinese cabbage

The enzyme, S-adenosylmethionine (SAM) decarboxylase (EC 4.1.1.50), has been demonstrated in leaves of Chinese cabbage, (Brassica pekinensis var Pak Choy). All of the enzyme can be found in extracts of the protoplasts obtained from the leaves of growing healthy or virus-infected cabbage. The protein has been purified approximately 1500-fold in several steps involving ammonium sulfate precipitation, affinity chromatography, and Sephacryl S-300 filtration. The reaction catalyzed by the purified enzyme has been shown to lead to the equimolar production of CO₂ and of decarboxylated S-adenosylmethionine (dSAM). The K_m for SAM is 38 micromolar. The reaction is not stimulated by Mg⁺⁺ or putrescine, and is inhibited by dSAM competitively with SAM. It is also inhibited strongly by methylglyoxal bis(guanylhydrazone). The enzyme, spermidine synthase (EC 2.5.1.16), present in leaf or protoplast extracts in many fold excess over SAM decarboxylase, has been purified approximately 1900-fold in steps involving ammonium sulfate precipitation, affinity chromatography, and gel filtration on Sephacryl S-300. Standardization of the Sephacryl column by proteins of known molecular weight yielded values of 35,000 and 81,000 for the decarboxylase and synthase, respectively.     

Suppression of ethylene levels promotes morphogenesis in pepper (Capsicum annuum L.)

Ethylene and polyamines (PAs) are two phytohormones that play important roles during in vitro morphogenesis of several plant species. The interaction between ethylene and PAs has been of interest because both have S-adenosylmethionine as a precursor. To study the influence of ethylene and PAs on in vitro morphogenesis of an ornamental pepper, we added an ethylene scavenger, PAs, a PA inhibitor, and compounds that affect ethylene biosynthesis and activity to the regeneration medium. Regeneration frequencies increased in response to treatment with ethylene inhibitors (aminoethoxyvinylglycine and silver thiosulfate) and an ethylene scavenger (mercury perchlorate). Treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid reduced the regeneration frequency, increased callus formation, and increased ethylene levels; similar results were obtained in response to treatment with the PA inhibitor methylglyoxal-bis(guanylhydrazone). By contrast, treatment with PAs (particularly spermidine and spermine) decreased ethylene levels, increased the regeneration frequency, and increased shoot bud formation. These results suggest a coordinated regulation of ethylene and polyamines because the suppression of ethylene levels using ethylene inhibitors, polyamines, or mercury perchlorate increased the in vitro regeneration frequency and morphogenic responses of Capsicum annuum L.     

Putrescine N-methyltransferase – The start for alkaloids

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.     

Ethanol perturbs polyamine metabolism in the phytopathogenic fungus Pyrenophora avenae

Biomass production by the plant pathogenic fungus Pyrenophora avenae was reduced following growth in 1, 3 and 6% ethanol. Although cadaverine concentration was not affected by growth in ethanol, putrescine and spermine concentrations were increased following growth in 3% ethanol and concentrations of spermidine and spermine were substantially increased following exposure to 6% ethanol. These changes were accompanied by significant increases in the activities of the polyamine biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase and in the flux of label from ornithine into the polyamines. Formation of the cadaverine derivatives aminopropylcadaverine and N,N-bis(3-aminopropyl)cadaverine was greatly increased in P. avenae exposed to 6% ethanol, probably via the action of lysine decarboxylase, S-adenosylmethionine decarboxylase and the aminopropyltransferases. There was also a doubling of polyamine oxidase activity following fungal growth in 6% ethanol.     

Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts

Ethylene production in apple fruit and protoplasts and in leaf tissue was inhibited by spermidine or spermine. These polyamines, as well as putrescine, inhibited auxin-induced ethylene production and the conversion of methionine and 1-aminocyclopropane-1-carboxylic acid to ethylene. Polyamines were more effective as inhibitors of ethylene synthesis at the early, rather than at the late, stages of fruit ripening. Ca²⁺ in the incubation medium reduced the inhibitory effect caused by the amines. A possible mode of action by which polyamines inhibit ethylene production is discussed.     

Studies of inhibition of rat spermidine synthase and spermine synthase

1. S-Adenosyl-l-methionine, S-adenosyl-l-homocysteine, 5′-methylthioadenosine and a number of analogues having changes in the base, sugar or amino acid portions of the molecule were tested as potential inhibitors of spermidine synthase and spermine synthase from rat ventral prostate. 2. S-Adenosyl-l-methionine was inhibitory to these reactions, as were other nucleosides containing a sulphonium centre. The most active of these were S-adenosyl-l-ethionine, S-adenosyl-4-methylthiobutyric acid, S-adenosyl-d-methionine and S-tubercidinylmethionine, which were all comparable in activity with S-adenosylmethionine itself, producing 70–98% inhibition at 1mm concentrations. Spermine synthase was somewhat more sensitive than spermidine synthase. 3. 5′-Methylthioadenosine, 5′-ethylthioadenosine and 5′-methylthiotubercidin were all powerful inhibitors of both enzymes, giving 50% inhibition of spermine synthase at 10–15μm and 50% inhibition of spermidine synthase at 30–45μm. 4. S-Adenosyl-l-homocysteine was a weak inhibitor of spermine synthase and practically inactive against spermidine synthase. Analogues of S-adenosylhomocysteine lacking either the carboxy or the amino group of the amino acid portion were somewhat more active, as were derivatives in which the ribose ring had been opened by oxidation. The sulphoxide and sulphone derivatives of decarboxylated S-adenosyl-l-homocysteine and the sulphone of S-adenosyl-l-homocysteine were quite potent inhibitors and were particularly active against spermidine synthase (giving 50% inhibition at 380, 50 and 20μm respectively). 5. These results are discussed in terms of the possible regulation of polyamine synthesis by endogenous nucleosides and the possible value of some of the inhibitory substances in experimental manipulations of polyamine concentrations. It is suggested that 5′-methylthiotubercidin and the sulphone of S-adenosylhomocysteine or of S-adenosyl-3-thiopropylamine may be particularly valuable in this respect.     

Biosynthesis of polyamines in Euglena gracilis

In Euglena gracilis Z the biosynthesis of spermidine and spermine closely resembles the pathways occurring in mammalian tissues and in most microorganisms. l-Ornithine and not l-arginine, as is the case in most plants, is the main precursor of putrescine, and S-adenosylmethionine donates the propylamino moiety for the biosynthesis of spermidine and spermine. Cell-free extracts of Euglena synthesized sym-norspermidine and sym-norspermine from 1,3-diaminopropane and labelled S-adenosylmenthionine. The synthases for the biosynthesis of these two polyamines have a pH optimum of 7.6, like that of spermidine and spermine synthases. Ion exchange chromatography showed two peaks corresponding to the retention times of 2,4-diaminobutyric acid and 1,3-diaminopropane, lower homologues of ornithine and putrescine, respectively. Experiments with dl-2,4-diaminobutyric acid-[4-¹⁴C] did not result in significant incorporation of the label into 1,3-diaminopropane.     

Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase)

The Saccharomyces cerevisiae SPE3 gene, coding for spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3Δ, which has no spermidine synthase activity and has an absolute requirement for spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of spermidine (10⁻⁸ M) or a higher concentration of spermine (10⁻⁶ M).     

Effects of inhibitors of spermidine and spermine synthesis on polyamine concentrations and growth of transformed mouse fibroblasts

1. A number of compounds known to inhibit polyamine biosynthesis at various steps in the biosynthetic pathway were tested for their ability to inhibit growth and decrease polyamine concentrations in virally transformed mouse fibroblasts (SV-3T3 cells). 2. Virtually complete inhibition of growth was produced by the inhibitors of ornithine decarboxylase α-methylornithine and α-difluoromethylornithine and by the inhibitors of S-adenosylmethionine decarboxylase 1,1′-[(methylethanediylidene)dinitrilo]diguanidine and 1,1′-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine). The former inhibitors decreased putrescine and spermidine contents in the cells to very low values, whereas the latter substantially increased putrescine but decreased spermidine concentrations. The inhibitory effects of all of these inhibitors on cell growth could be prevented by the addition of spermidine, suggesting that spermidine depletion is the underlying cause of their inhibition of growth. 3. α-Difluoromethylornithine, which is an irreversible inhibitor of ornithine decarboxylase, was a more potent inhibitor of growth and polyamine production (depleting spermidine almost completely and spermine significantly) than α-methylornithine, which is a competitive inhibitor. This was not the case with the inhibitors of S-adenosylmethionine decarboxylase where 1,1′-[(methylethanediylidene)dinitrilo]diguanidine, a reversible inhibitor, was more active than 1,1′-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine), an irreversible inhibitor. It is suggested that this effect may be due to the lesser uptake and/or greater chemical reactivity of the latter compound. 4. Various nucleoside derivatives of S-adenosylhomocysteine that inhibited spermidine synthase in vitro did not have significant inhibitory action against polyamine accumulation in the cell. These compounds, which included S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphoxide and S-adenosyl-4-thio-butyric acid sulphone did not inhibit cell growth or polyamine content until cytotoxic concentrations were added. 5. 5′-Methylthioadenosine, 5′-isobutylthioadenosine and 5′-methylthiotubercidin, which inhibit aminopropyltransferase activity in vitro, all inhibited cell growth and decreased spermidine content. Although these compounds were most active against spermine synthase in vitro, they acted in the cell primarily to decrease spermidine content. Cell growth could not be restored to normal values by addition of spermidine, suggesting that these nucleosides have another inhibitory action towards cellular proliferation. 6. 5′-Methylthioadenosine and 5′-isobutylthioadenosine are degraded by a phosphorylase present in SV3T3 cells, yielding 5-methylthioribose-1-phosphate and 5-isobutylthioribose-1-phosphate respectively, and adenine. This degradation appears to decrease the inhibitory action towards cell growth, suggesting that the nucleosides themselves are exerting the inhibitory action. 5′-Methylthiotubercidin, which is not a substrate for the phosphorylase and is a competitive inhibitor of it, was the most active of these nucleosides in inhibiting cell growth and spermidine content. 5′-Methylthiotubercidin and α-difluoromethylornithine had additive effects on retarding cell growth, but not on cellular spermine accumulation, also suggesting that the primary growth-inhibiting action of the nucleoside was not on polyamine production. 7. These results support the concept that 5′-methylthioadenosine phosphorylase plays an important role in permitting cell growth to continue by preventing the build-up of inhibitory intracellular concentrations of 5′-methylthioadenosine.     

The synthesis of polyamines from methionine in intact and disrupted leaf protoplasts of virus-infected chinese cabbage

In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-¹⁴C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spormine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [³⁵S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescien, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.     

Effect of polyamines on ethylene production

The polyamines putrescine, cadaverine, spermidine and spermine reduced the amount of ethylene produced by senescing petals of Tradescantia but they did not prevent anthocyanin leakage from these same petals. These polyamines also inhibited auxin-mediated ethylene production by etiolated soybean hypocotyls to less than 7 % of the control. The basic amino acids lysine and histidine reduced the amount of auxin-induced ethylene produced by soybean hypocotyls by ca 50 %. In the hypocotyls, methionine was unable to overcome the inhibition caused by the polyamines. The polyamines spermidine and spermine inhibited ethylene production induced by the application of 1-aminocyclopropane-1-carboxylic acid and they also reduced the endogenous content of this amino acid in the treated tissues.     

Genomic organization of plant aminopropyl transferases

Aminopropyl transferases like spermidine synthase (SPDS; EC 2.5.1.16), spermine synthase and thermospermine synthase (SPMS, tSPMS; EC 2.5.1.22) belong to a class of widely distributed enzymes that use decarboxylated S-adenosylmethionine as an aminopropyl donor and putrescine or spermidine as an amino acceptor to form in that order spermidine, spermine or thermospermine. We describe the analysis of plant genomic sequences encoding SPDS, SPMS, tSPMS and PMT (putrescine N-methyltransferase; EC 2.1.1.53). Genome organization (including exon size, gain and loss, as well as intron number, size, loss, retention, placement and phase, and the presence of transposons) of plant aminopropyl transferase genes were compared between the genomic sequences of SPDS, SPMS and tSPMS from Zea mays, Oryza sativa, Malus x domestica, Populus trichocarpa, Arabidopsis thaliana and Physcomitrella patens. In addition, the genomic organization of plant PMT genes, proposed to be derived from SPDS during the evolution of alkaloid metabolism, is illustrated. Herein, a particular conservation and arrangement of exon and intron sequences between plant SPDS, SPMS and PMT genes that clearly differs with that of ACL5 genes, is shown. The possible acquisition of the plant SPMS exon II and, in particular exon XI in the monocot SPMS genes, is a remarkable feature that allows their differentiation from SPDS genes. In accordance with our in silico analysis, functional complementation experiments of the maize ZmSPMS1 enzyme (previously considered to be SPDS) in yeast demonstrated its spermine synthase activity. Another significant aspect is the conservation of intron sequences among SPDS and PMT paralogs. In addition the existence of microsynteny among some SPDS paralogs, especially in P. trichocarpa and A. thaliana, supports duplication events of plant SPDS genes. Based in our analysis, we hypothesize that SPMS genes appeared with the divergence of vascular plants by a processes of gene duplication and the acquisition of unique exons of as-yet unknown origin.