The decrease in polyamine levels is not associated with growth inhibition in suspension-cultured rice cells under nitrogen deficiency

The effects of nitrogen deficiency on the growth and polyamine levels of suspensioncultured rice cells were investigated. Nitrogen deficiency markedly inhibited cell growth and resulted in lower levels of putrescine, spermidine and spermine than in the control culture supplied with nitrogen. The growth inhibition of rice cells induced by nitrogen deficiency could be recovered by the addition of a mixture of amino acids (glycine, L-aspartic acid, L-glutamic acid and L-arginine), but not by polyamines (putrescine, spermidine or spermine). Our results indicate that the decline of polyamine levels is not a factor causing growth inhibition of suspension-cultured rice cells under nitrogen deficiency.     

Intracellular putrescine and spermidine deprivation induces increased uptake of the natural polyamines and methylglyoxal bis(guanylhydrazone).

Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1'-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous "priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect.     

Role of spermidine in the expression of late markers of adipose conversion. Effects of growth hormone.

Confluent Ob1771 cells treated with an inhibitor of spermidine and spermine synthesis, methylglyoxyal bis(guanylhydrazone), were dependent on putrescine addition for the expression of glycerol-3-phosphate dehydrogenase and acyl-CoA synthetase, which behaved as late markers of adipose conversion. A similar dependence was observed with drug-treated Ob17MT18 and 3T3-F442A preadipocyte cells, but not with non-differentiating 3T3-C2 cells. Studies in drug-treated Ob1771 cells at the mRNA level showed that the parallel expression of mRNAs encoding for glycerol-3-phosphate dehydrogenase and an homologue of serine proteinases of Mr 28,000 [Cook, Groves, Min & Spiegelman (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6480-6484] was also dependent on putrescine addition. Double-isotope experiments with [14C]putrescine and [3H]spermidine, as well as analysis of the polyamine content in drug-treated Ob1771 cells under various conditions, demonstrate after putrescine addition that the expression of late markers of adipose conversion was highly correlated with a 2-fold increase in the intracellular concentration of spermidine. No correlation was observed with changes in the intracellular concentrations of putrescine and spermine. Long-term exposure of untreated Ob1771 cells to growth hormone, which led to the expression of late markers of adipose conversion [Doglio, Dani, Grimaldi & Ailhaud (1986) Biochem. J. 238, 123-129] was also accompanied by the same increase in spermidine concentration, which attained values identical with those determined in drug-treated cells supplemented with putrescine. This observation suggests that the permissive effect of growth hormone on the terminal differentiation of adipose cells might e related to changes in the intracellular concentration of spermidine.     

Levels of endogenous polyamines and NaCl-inhibited growth of rice seedlings

The role of endogenous polyamines in the control of NaCl-inhibited growth of rice seedlings was investigated. Putrescine, spermidine and spermine were all present in shoots and roots of rice seedlings. NaCl treatment did not affect spermine levels in shoots and roots. Spermidine levels in shoots and roots were increased with increasing concentrations of applied NaCl. NaCl at a concentration of 50 mM, which caused only slight growth inhibition, drastically lowered the level of putrescine in shoots and roots. Addition of precursors of putrescine biosynthesis (L-arginine and L-ornithine) resulted in an increase in putrescine levels in NaCl-treated shoots and roots, but did not allow recovery of the growth inhibition of rice seedlings induced by NaCl. Pretreatment of rice seeds with putrescine caused an increase in putrescine level in shoots, but could not alleviate the inhibition effect of NaCl on seedling growth. The current results suggest that endogenous polyamines may not play a significant role in the control of NaCl-inhibited growth of rice seedlings.Abbreviations PUT putrescine - SPD spermidine - SPM spermine     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Treatment with phorbol esters leads to spermine depletion and inhibition of DNA synthesis in phytohemagglutinin-activated bovine lymphocytes

Phorbol 12-myristate-13-acetate (PMA) inhibited an increase in [3H]thymidine incorporation induced by phytohemagglutinin (PHA) in cultured bovine lymphocytes. Cellular levels of putrescine increased in the presence of PHA and PMA but the levels of spermidine and spermine had decreased to the control levels by 40 h. In cells treated with PHA and PMA, the activity of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme in polyamine biodegradation, was stimulated synergistically. Phorbol esters with tumor-promoting ability also stimulated the enzyme activity and a reciprocal correlation between the enzyme activity and DNA synthesis was observed. Addition of spermine reversed the PHA- and PMA-induced inhibition of DNA synthesis but putrescine and spermidine failed to restore it. These results suggest that the enhancement of spermidine/spermine N1-acetyltransferase activity results in the depletion of intracellular spermine and a concomitant decrease in DNA synthesis.     

Effect of 5''-deoxy-5''-isobutylthioadenosine on putrescine uptake and polyamine biosynthesis by chick embryo fibroblasts.

The effect of 5'-deoxy-5'-S-isobutylthioadenosine (SIBA) on polyamine biosynthesis has been studied by using cultured chick embryo fibroblasts. It has been shown that the drug inhibits the uptake of [14C]putrescine and its conversion into labelled spermidine or spermine. The inhibitory effect is reversed by removing the inhibitor after exposing the cells to the drug for 24 h. SIBA also caused a significant decrease in cellular spermine levels and an accumulation of putrescine. These changes are reversed by removing the inhibitor. SIBA had the same effect on chick embryo fibroblasts transformed by Rous sarcoma virus; a decrease in cellular spermine levels in SIBA-treated cells was observed. In all the experiments SIBA caused a reduction in the spermine/putrescine and spermidine/putrescine ratios. It is suggested that SIBA is not only an inhibitor of transmethylation but also interferes with polyamine biosynthesis, probably by blocking aminopropyltransferase.     

The induction of α-amylase activity by sucrose starvation in suspension-cultured rice cells is regulated by polyamines

When suspension-cultured rice ( Oryza sativa L. cv. Tainan 5) cells were deprived of sucrose, α-amylase (EC 3.2.1.1) activity in the cells and the culture medium increased markedly. The increase in activity of α-amylase caused by sucrose starvation in the cells and the medium was strongly reduced in the presence of exogenously added spermine. Putrescine and spermidine also inhibited, though only slightly, the increase in α-amylase activity caused by sucrose starvation. Preincubation of the enzyme extract or enzyme in the medium with polyamines had no effect on α-amylase activity. Sucrose starvation resulted in lower polyamine levels in rice suspension cells. D-Arginine and α-methylomithine, inhibitors of polyamine biosynthesis, caused reduced levels of polyamines and increased activity of α-amylase in rice suspension cells cultured in the presence of sucrose. Our results indicate that the induction of α-amylase activity by sucrose starvation in rice suspension cells is mediated, at least partly, through the internal level of polyamines.     

Inhibition of spermidine and spermine synthesis leads to growth arrest of rat embryo fibroblasts in G1

Following growth stimulation of rat embryo fibroblast (REF) cells previously arrested in G₁ by serum deprivation, there occurs a large increase in the synthesis of the polyamines putrescine, spermidine and spermine. Methylglyoxal bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase can block the accumulation of both spermidine and spermine over a period of several days. Under such conditions REF cells treated with MGBG will approximately double in number and then become growth-arrested again predominantly in the G₁ phase of the cell cycle. REF cells therefore appear to contain sufficient spermidine and spermine to progress through one cell cycle before the intracellular levels of these polyamines is reduced sufficiently to arrest growth in the absence of continued polyamine synthesis. Limitation of intracellular polyamine levels is therefore not the mechanism by which deprivation of serum growth factors arrests cell growth. While continued growth is nevertheless dependent on polyamine synthesis, this cell type is capable of limited proliferation in its absence. Addition of spermidine or spermine to MGBG-arrested REF cells results in a rapid resumption of proliferation demonstrating that either polyamine can fulfill the role played by these polyamines in the growth process. Low levels of spermidine and spermine therefore arrest this cell type at a resriction point in G₁ at which it is decided whether the intracellular level of these polyamines is sufficiently high to enable a cell to enter into and complete a new cell cycle. This polyamine-sensitive restriction point is considered to be analogous to the restriction point(s) in G₁ at which serum and nutrient limitation act.     

Metabolism of polyamines in mouse neuroblastoma cells in culture: formation of GABA and putreanine.

—Polyamine metabolism of mouse neuroblastoma cells grown in culture was studied with special reference to the synthesis of GABA from putrescine and putreanine from spermidine. This study shows that neuroblastoma cells in the presence of a complete culture medium containing calf serum readily metabolized [¹⁴C]putrescine to GABA; the rate of synthesis is similar to the rate of synthesis of spermidine from putrescine. In the absence of serum the conversion of putrescine to GABA is minimal. In the presence of serum GABA formation is completely inhibited by the diamine oxidase inhibitor aminoguanidine. GABA synthesis does not occur in the absence of cells. The GABA synthesized is not readily metabolized to succinate or homocarnosine. Mouse neuroblastoma cells metabolized [¹⁴C]ornithine to putrescine, GABA, and spermidine. Spermidine was metabolized to putrescine, putreanine and spermine.     

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

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 putrescine, 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.     

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.     

Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing <Emphasis Type="Italic">Datura stramonium</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine decarboxylase

Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.     

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.     

Ammonium accumulation in relation to growth of rice cell suspension under phosphate deprivation

Suspension-cultured rice cells growth was markedly inhibited and ammonium content increased when rice cells were deprived of phosphate. When rice cells were cultured at increasing concentrations of ammonium chloride, ammonium content increased, however, no significant inhibition of cell growth was observed. Addition of D-arginine, an inhibitor of putrescine biosynthesis, resulted in a complete recovery of growth in rice cells under phosphate deprivation, but did not decrease the content of ammonium. Our results indicate that the growth inhibition induced by phosphate deprivation is not associated with ammonium accumulation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

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

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

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Changes in endogenous polyamine levels are associated with differentiation in Dictyostelium discoideum.

This is the first report correlating levels of polyamines and its fractions with differentiation in Dictyostelium discoideum. Temporal changes in endogenous levels of free, conjugated and bound putrescine, spermidine and spermine were analysed at critical stages of morphogenesis in this organism. No spermine was found at any given stage and putrescine was the most abundant polyamine. There was a sharp increase in the levels of both free (and total) and conjugated forms of putrescine and spermidine at the slug stage as compared to the growth phase. The levels of putrescine and spermidine were found to be higher in isolated prespore cells as compared to the prestalk cells. Remarkably, the levels of polyamine decreased at the early culminant stage. Data suggest that a moderate level of polyamines is needed for growth but it is important to have high levels of polyamines at the time of differentiation.     

Polyamines as modulators of salt tolerance in rice cultivars

The effect of NaCl on the endogenous levels of diamine, putrescine and polyamines, spermidine and spermine, was studied in the shoot system of salt-tolerant and salt-sensitive lines of rice (Oryza sativa L.) cultivars during three growth stages. Salt stress increased the levels of diamine and polyamine in varying degrees among nine rice cultivars investigated. Salt tolerant AU1, Co43, and CSC1 were effective in maintaining high concentrations of spermidine and spermine, while the content of putrescine was not significantly altered in all the growth stages when plants were exposed to salinity. The salt sensitivity in rice was associated with excessive accumulation of putrescine and with low levels of spermidine and spermine in the shoot system of salt-sensitive cultivars Co36, CSC2, GR3, IR20, TKM4, and TKM9 under saline condition. One of the possible mechanisms of saline resistance was observed to be due to the highly increased polyamines against the low increase in diamines. Alternatively, the salt sensitivity could be due to high increase of diamines and an incapacity to maintain high levels of polyamines.