Manipulation of the polyamine content and senescence of apical buds of G2 peas

The effect of various treatments on the apical senescence and polyamine content of apical buds of G2 peas was analysed. Defruiting prevented senescence and increased bud size and polyamine content. Exogenous applications of GA₂₀ enhanced bud size and spermidine concentration. Applied spermidine had a slight effect on spermidine level but did not delay senescence. ACC strongly induced adecrease in bud size and, at 10 mM, apical senescence. This was accompanied by a steady decline in the level of all polyamines though their concentration remained constant until 10 mM ACC, where a drop was noted. Spermidine in the presence of ACC modulated the effect of ACC on the bud size while returning the internal polyamine content to control levels. AVG, an inhibitor of ACC synthesis produced pronounced increases in putrescine though no apparent effect on apical bud growth. Polyamine synthesis inhibitors were without effect on growth or internal polyamine content. The internal polyamine content appeared to correlate with apical bud size and vigor but did not show any consistent relationship to apical bud senescence.     

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.     

Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture

Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.Abbreviations ADC arginine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DCHAS dicyclohexylammonium sulfate - SAMDC S-adenosylmethionine decarboxylase     

Changes in free polyamines and related enzymes during stipule and pod wall development in Pisum sativum

Level of free polyamines, their key metabolic enzymes, and other features related to ageing were examined during stipule and pod wall development in pea (Pisum sativum). Free polyamine titre (per unit fresh mass) in both the organs, the specific activities of arginine decarboxylase and ornithine decarboxylase in the pod wall, gradually decreased with maturation. In stipule, these enzymes attained peak activity at 15 days after pod emergence and declined thereafter. Ornithine decarboxylase activity was greater in pod wall than in stipule; while, arginine decarboxylase activity was higher in stipule. Activity of degradative enzyme diamine oxidase increased with the onset of senescence in both the organs. Chlorophyll and electrical conductance had a inverse relationship throughout the experimental period, whereas, the chlorophyll content was directly related with polyamine levels in both stipule and pod wall during aging. On the other hand, protein and RNA contents were positively correlated with free polyamines throughout the test period in stipule, but in the pod wall this was true only for the later stages of development.     

Effects of Polyamines and Kinetin on In Vitro Frond Senescence in Lemna aequinoctialis 6746

Both polyamines and kinetin could retard the loss of chlorophyll during dark-induced senescence in excised frond of Lernna aequinoctialis 6746. The effect of polyamines on retarding the chlorophyll loss was stronger than that of kinetin. Kinetin remarkably inhibited the loss of soluble proteins and the increase of protease activity, while no similar effects were observed from polyamines. An inhibitor of polyamine biosynthesis, methylglyoxal bis- (guanyl- hydrazone) (MGBG), slightly increased the loss of chlorophyll and soluble proteins. During senescience, both the increase of putrescine (Put) content and the decrease of spermidine (Spd) content were inhibited by kinetin at the concentration of 0.05 mmol/L, but the spermine (Spm) level was not affected by kinetin. The arginine decarboxylase (ADC) activity was dominant in frond of Lemna aequinoctialis 6746. Kinetin slightly increased ADC activity, while it had no marked effect on ornithine decarboxylase (ODC) and s-adenosylmethionine decarboxylase (SAMDC). The possible relationship between polyamines and cytokinins in retarding senescence was also discussed.     

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.     

Long-distance translocation of polyamines in phloem and xylem of Ricinus communis L. plants

Polyamine content and enzyme activities in the biosynthetic and degradative pathways of polyamine metabolism were investigated in sieve-tube sap, xylem sap and tissues of seedlings and adult plants of Ricinus communis L. Polyamines were present in tissues and translocation fluids of both seedlings and adult plants in relatively high amounts. Only free polyamines were translocated through the plant, as indicated by the finding that only the free form was detected in the phloem and the xylem sap. Removal of the endosperm increased the polyamine content in the sieve-tube exudate of seedlings. The level and pattern of polyamines in tissue of adult leaves changed during leaf age, but not, however, in the sieve-tube sap. Xylem sap was relatively poor in polyamines. Polyamine loading in the phloem was demonstrated by incubating cotyledons with [¹⁴O]putrescine and several unlabelled polyamines. Feeding cotyledons with cadaverine and spermidine led to a decrease in the level of putrescine in sieve-tube sap, indicating a competitive effect. Comparison of polyamine content in the tissue and export rate showed that the export would deplete the leaves of polyamines within 1–3 d, if they were not replenished by biosynthesis. Polyamine biosynthesis in Ricinus proceeds mostly via arginine decarboxylase, which in vitro is 100-fold more active than ornithine decarboxylase. The highest arginine decarboxylase, ornithine decarboxylase and diamine oxidase activities were detected in cotyledons, while in sieve-tube sap only a slight arginine decarboxylase activity was found. Received: 18 March 1997 / Accepted: 20 August 1997     

多胺与激动素对稀脉浮萍离体叶状体衰老的影响

多胺与ＫＴ 都可抑制暗诱导衰老的稀脉浮萍（Ｌｅｍ ｎａ ａｅｑｕｉｎｏｃｔｉａｌｉｓ）离体叶状体的叶绿素损失,且多胺的作用大于ＫＴ。ＫＴ 还显著抑制蛋白质的损失与蛋白酶活性的上升,而多胺对此却无大的影响。０．０５ ｍ ｍ ｏｌ／Ｌ的甲基乙二醛二脒基－腙（ＭＧＢＧ）轻微促进叶绿素和蛋白质的损失。０．０５ ｍ ｍ ｏｌ／Ｌ的ＫＴ 可抑制衰老过程中腐胺（Ｐｕｔ）的上升和亚精胺（Ｓｐｄ）的下降,而对精胺（Ｓｐｍ ）无明显影响。在稀脉浮萍中,精氨酸脱羧酶（ＡＤＣ）活性占优势。ＫＴ 可轻微促进ＡＤＣ 活性,而对鸟氨酸脱羧酶（ＯＤＣ）和Ｓ－腺苷甲硫氨酸脱羧酶（ＳＡＭＤＣ）活性无显著影响。讨论了多胺与细胞分裂素在抑制植物叶片衰老过程中作用途径的可能关系     

Involvement of polyamines in root development at low temperature in the subantarctic cruciferous species Pringlea antiscorbutica

Polyamine involvement in root development at low temperature was studied in seedlings of Pringlea antiscorbutica R. Br. This unique endemic cruciferous species from the subantarctic zone is subjected to strong environmental constraints and shows high polyamine contents. In the present study, free polyamine levels were modified by inhibitors of polyamine biosynthesis (D-arginine, difluoromethylornithine, cyclohexylammonium, and methylglyoxal-bis-guanylhydrazone) and variations of the endogenous pools were compared to changes in root growth. The arginine decarboxylase pathway, rather than that of ornithine decarboxylase, seemed to play a major role in polyamine synthesis in Pringlea antiscorbutica seedlings. Root, but not shoot, phenotypes were greatly affected by these treatments, which modified polyamine endogenous levels according to their expected effects. A positive correlation was found between agmatine level and growth rate of the primary root. Spermidine and spermine contents also showed positive correlations with primary root growth whereas the putrescine level showed neutral or negative effects on this trait. Free polyamines were therefore found to be differentially involved in the phenotypic plasticity of root architecture. A comparison of developmental effects and physiological concentrations suggested that agmatine and spermine in particular may play a significant role in the control of root development.     

Ornithine and arginine decarboxylases and polyamine involvement during in vivo differentiation and in vitro dedifferentiation of Datura innoxia leaf explants

The effects of exogenous ornithine, arginine and polyamines added to media leading to root, callus or bud initiation of Datura innoxia Mill. leaf explants growing in vitro were examined. Ornithine and arginine decarboxylase activities (ODC, EC 4.1.1.17; ADC, EC 4.1.1.19) as well as endogenous polyamine levels were also determined during the course of in vivo differentiation of the leaves and their subsequent in vitro dedifferentiation under rooting, callusing, or budding conditions. Decarboxylase activities were determined by measuring the ¹⁴CO₂ released and the polyamines were quantified after dansylation by thin-layer chromatography. In vivo, ODC and ADC activities decreased from shoots to young to old leaves. In vitro, synergistic effects between ornithine and indole-3-acetic acid on rhizogenesis were detected, while arginine was not effective. Exogenous putrescine also acted synergistically with auxin by promoting root growth. A close relationship was found between rhizogenesis, ODC activity and increase in endogenous putrescine and spermidine levels. ODC increased during the induction and time course of cell dedifferentiation and seemed to support these events, while ADC seemed to support only the later events involving redifferentiation.     

The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves

In the present study we determined the effects of methionine, intermediates of polyamine catabolic pathways and inhibitors of either ethylene biosynthetic or polyamine catabolic pathways on polyamine accumulation in soybean leaves. Inhibitors to SAM decarboxylase and spermidine synthase, methylglyloxal-bis-(guanylhy-drazone) and cyclohexylamine, respectively, suggest that methionine may provide aminopropyl groups for the synthesis of polyamine via S-adenosylmethionine (SAM). Results from experiments that utilized a combination of compounds which altered either ethylene or polyamine biosynthesis, namely, aminoethoxyvinyl glycine, CoSO₄, 2,5-norbornadiene, and CuSO₄, suggest the two pathways compete for a common precursor. However, exogenous addition of ethylene (via ethephon treatments) had little or no effect on polyamine biosynthesis. Likewise, polyamine treatments had little or no effect on ethylene biosynthesis. These data suggest that there are few or no inhibitory effects from the end products of one pathway on the synthesis of the other. Data from leaves treated with metabolic intermediates in the catabolic pathway of polyamines and inhibitors of enzymes in the catabolic pathway, i.e. aminoguanidine, hydroxyethyldrazine and gabaculine, suggest that the observed increases in polyamine titers were not due to decreased catabolism of the polyamines. One catabolic intermediate, γ-aminobutyric acid (GABA), elevated putrescine, spermidine and spermine by 12-, 1.4-, and 2-fold, respectively, Ethylene levels decreased (25%) in GABA-treated leaves. This small decrease in ethylene could not account for such large increase in putrescine titers. Further analysis demonstrated that the GABA-mediated polyamine accumulation was inhibited by difluoromethylarginine, an inhibitor of arginine decarboxylase, but not by difluoromethylornithine, an inhibitor of ornithine decarboxylase. These data suggest that GABA directly or indirectly affects the biosynthesis of polyamines via arginine decarboxylase.     

Arginase,Arginine Decarboxylase,Ornithine Decarboxylase,and Polyamines in Tomato Ovaries (Changes in Unpollinated Ovaries and Parthenocarpic Fruits Induced by Auxin or Gibberellin)

Arginase (EC 3.5.3.1) activity has been found in the ovaries and Young fruits of tomato (Lycopersicon esculentum Mill. cv Rutgers).Changes in arginase, arginine decarboxylase (EC 4.1.1.19), and ornithine decarboxylase activity (EC 4.1.1.17) and levels of free and conjugated putrescine, spermidine, and spermine were determined in unpollinated ovaries and in parthenocarpic fruits during the early stages of development induced by 2,4-dichlorophenoxyacetic acid (2,4-D) or gibberellic acid (GA3). Levels of arginase, free spermine, and conjugates of the three polyamines were constant in unpollinated ovaries and characteristic of a presenescent step. A marked decrease in arginase activity, free spermine, and polyamine conjugates was associated with the initiation of fruit growth due to cell division, and when cell expansion was initiated, the absence of arginase indicated a redirection of nitrogen metabolism to the synthesis of arginine. A transient increase in arginine decarboxylase and ornithine decarboxylase was also observed in 2,4-D-induced fruits. In general, 2,4-D treatments produced faster changes than GA3, and without treatment, unpollinated ovaries developed only slightly and senescence was hardly visible. Sensitivity to 2,4-D and GA3 treatment remained for at least 2 weeks postanthesis.     

Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Both the polyamine content and the route of acquisition of polyamines by Rickettsia prowazekii, an obligate intracellular parasitic bacterium, were determined. The rickettsiae grew normally in an ornithine decarboxylase mutant of the Chinese hamster ovary (C55.7) cell line whether or not putrescine, which this host cell required in order to grow, was present. The rickettsiae contained approximately 6 mM putrescine, 5 mM spermidine, and 3 mM spermine when cultured in the presence or absence of putrescine. Neither the transport of putrescine and spermidine by the rickettsiae nor a measurable rickettsial ornithine decarboxylase activity could be demonstrated. However, we demonstrated the de novo synthesis of polyamines from arginine by the rickettsiae. Arginine decarboxylase activity (29 pmol of 14CO2 released per h per 10(8) rickettsiae) was measured in the rickettsiae growing within their host cell. A markedly lower level of this enzymatic activity was observed in cell extracts of R. prowazekii and could be completely inhibited with 1 mM difluoromethylarginine, an irreversible inhibitor of the enzyme. R. prowazekii failed to grow in C55.7 cells that had been cultured in the presence of 1 mM difluoromethylarginine. After rickettsiae were grown in C55.7 in the presence of labeled arginine, the specific activities of arginine in the host cell cytoplasm and polyamines in the rickettsiae were measured; these measurements indicated that 100% of the total polyamine content of R. prowazekii was derived from arginine.     

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.     

The possible involvement of polyamines in the development of tomato fruits in vitro

The apparent involvement of ornithine decarboxylase (ODC) and putrescine in the early stages of fruit growth in tomato (Lycopersicon esculentum Mill.) has been previously described. Further evidence presented here supports the direct involvement of ODC and putrescine in the cell division process in tomato fruits. In tomato fruits grown in vitro, in which basic growth processes are inhibited, the activity of ODC and arginine decarboxylase (ADC) and the level of free polyamines were reduced. While ODC and ADC activity was correlated with the period of cell division in the tomato fruit, the free polyamine content was correlated with the DNA content, cell size, and fruit fresh weight. The addition of exogenous putrescine, however, did not restore the basic growth processes in the fruits grown in vitro.     

Polyamines in plant physiology

The diamine putrescine, the triamine spermidine, and the tetramine spermine are ubiquitous in plant cells, while other polyamines are of more limited occurrence. Their chemistry and pathways of biosynthesis and metabolism are well characterized. They occur in the free form as cations, but are often conjugated to small molecules like phenolic acids and also to various macromolecules. Their titer varies from approximately micromolar to more than millimolar, and depends greatly on environmental conditions, especially stress. In cereals, the activity of one of the major polyamine biosynthetic enzymes, arginine decarboxylase, is rapidly and dramatically increased by almost every studied external stress, leading to 50-fold or greater increases in putrescine titer within a few hours. The physiological significance of this increase is not yet clear, although most recent work suggests an adaptive, protective role. Polyamines produced through the action of ornithine decarboxylase, by contrast, seem essential for DNA replication and cell division. The application of exogenous polyamines produces effects on patterns of senescence and morphogenesis, suggesting but not proving a regulatory role for polyamines in these processes. The evidence for such a regulatory role is growing.     

Polyamine metabolism in Acanthamoeba: polyamine content and synthesis of ornithine, putrescine, and diaminopropane

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

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     

A probable new pathway for the biosynthesis of putrescine in Escherichia coli.

Some cultures of Escherichia coli BGA8, a mutant unable to synthesize putrescine, showed a change of behaviour and could grow almost equally well in either the absence or the presence of polyamines after repeated periods of polyamine starvation. Experiments in vivo with radioactive precursors showed that the bacteria which evaded the polyamine requirement had recovered their ability to synthesize putrescine from glucose or glutamic acid, but not from ornithine or arginine. These results are in agreement with the fact that the polyamine-independent cells were still deficient in the enzymes ornithine decarboxylase and agmatinase. Our findings seem to indicate the existence of a new pathway synthesize putrescine which does not involve ornithine or arginine as intermediates.     

Mutants of Escherichia coli that do not contain 1,4-diaminobutane (putrescine) or spermidine.

Strains of Escherichia coli K12 have been constructed which do not contain any of the polyamines normally present in a wild type strain, namely, 1,4-diaminobutane (putrescine) and spermidine. This phenotype arises as a consequence of the assembly into these strains of deletion mutations in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). The polyamine-deficient strains grow indefinitely in the absence of polyamines but with a growth rate one-third of that found in the presence of polyamines. These strains can act as hosts for bacteriophages T4, T7, and f2, although the latter phage is poorly adsorbed; they can also maintain F' factors, ColE1 and P1 plasmids, and lysogeny by bacteriophage lambda. In contrast, the production of bacteriophage lambda in the absence of polyamines is strikingly decreased (greater than 99%) either after infection of a nonlysogen or after induction of a lysogen. A polyamine-deficient Hfr strain can transfer its chromosome to a recipient at a normal rate, but the number of recombinants observed in a cross is decreased approximately 300-fold. No such effect is observed when only the F- recipient strain in a cross is polyamine deficient.