Tissue-specific changes in polyamine levels of neural tissue and fat body in adult crickets

The three major polyamines—putrescine, spermidine, and spermine—were studied and changes of their levels were examined in extracts of cerebral ganglia and fat body from adult Acheta domesticus. In nervous tissue, only spermidine and spermine were present and spermine was two- to three-fold more abundant than spermidine. The polyamine levels were high up to day 3, decreased on day 4, and then remained relatively unchanged up to day 10. The spermidine/spermine ratios decreased during the imaginal life. Higher spermidine titres were observed in the neural tissue of egg-laying females compared to virgin females. In the fat body, putrescine was detected together with spermidine and spermine. Spermidine and spermine levels were two-fold higher than putrescine. Fat body of virgin females contained two times more polyamines than male fat body. Low at emergence, spermidine and spermine concentrations peaked on days 2–3 only in females, and egg-laying was characterized by an increase of putrescine and spermidine titres. Starvation did not change polyamine contents, implying homeostatic regulation of the intracellular polyamine metabolism. These data showing tissue specific changes in polyamine levels during the imaginal life of Acheta domesticus point to the physiological importance of polyamines as possible intracellular regulators during adult insect development. © 1993 Wiley-Liss, Inc.     

Polyamine content of the Plodia interpunctella granulosis virus and its larval host

The presence of polyamines in the granulosis virus and its larval host, Plodia interpunctella, was examined using thin-layer chromatography of the dansylated compounds. The uninfected and infected larvae contain the three polyamines, putrescine, spermidine, and spermine. Although purified granulosis virus infecting this host does not contain any typical polyamines, an acidsoluble dansylated compound with properties indicative of a polyamine was observed.     

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.     

Unique polyamines produced by an extreme thermophile, <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Summary. Recent research progress on polyamines in extreme thermophiles is reviewed. Extreme thermophiles produce two types of unique polyamines; one is longer polyamines such as caldopentamine and caldohexamine, and the other is branched polyamines such as tetrakis(3-aminopropyl)ammonium. The protein synthesis catalyzed by a cell-free extract of Thermus thermophilus, an extreme thermophile, required the presence of a polyamine and the highest activity was found in the presence of tetrakis(3-aminopropyl)ammonium. In vitro experiments, longer polyamines efficiently stabilized double stranded nucleic acids and a branched polyamine, tetrakis(3-aminropyl)ammonium, stabilized stem-and-loop structures. In T. thermophilus, polyamines are synthesized from arginine by a new metabolic pathway; arginine is converted to agmatine and then agmatine is aminopropylated to N¹-aminopropylagmatine which is converted to spermidine by an enzyme coded by a gene homologous to speB (a gene for agmatinase). In this new pathway spermidine is not synthesized from putrescine. Reverse genetic studies indicated that the unique polyamines are synthesized from spermidine.     

Seasonal changes of polyamines in habitat adaptation of different ecotypes of reed plants

The leaves of four reed ecotypes (Phragmites communis Trinius) growing in the desert regions of northwest China were investigated for levels of polyamines and activity of arginine decarboxylase (ADC; EC 4.1.1.19) during the growing season of 5 months. The polyamines in the leaves of all reed ecotypes consisted of putrescine, spermidine and spermine. The polyamine levels of the leaves were lower in the swamp reed than in the terrestrial reed ecotypes. Leaf polyamine levels decreased in all ecotypes over the course of the season. Compared to the swamp reed, the terrestrial reed ecotypes maintained higher ADC activity and a predominance of spermine, resulting in a lower ratio of putrescine to spermidine and spermine. It seems that the adaptation of reed plants to drought and saline habitats may be correlated with putrescine synthesis via the ADC pathway, and with a successful conversion of putrescine to spermidine and spermine.     

Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. 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 [¹⁴C]methionine. Putrescine and spermidine 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.     

Cytoplasmic polyamines block the fast-activating vacuolar cation channel

The fast-activating vacuolar (FV) channel dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Since polyamines are known to increase in plant cells in response to stress, the regulation of FV channels by polyamines was investigated. Patch-clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. The trivalent polyamine spermidine and the tetravalent polyamine spermine blocked FV channels with K_d≈ 100 μM and K_d≈ 5 μM, respectively. Increasing cytosolic and vacuolar Ca²⁺ had no effect on putrescine and spermidine binding to FV channels but slightly decreased the affinity for spermine. The inhibition of FV channels by all three polyamines was not voltage-dependent. This points to a different mode of binding compared to inward rectifier K⁺ channels and Ca²⁺-permeable glutamate receptor channels from animal cells, which show rectification due to a voltage-dependent block by polyamines. In plant cells, the common polyamines (putrescine, spermidine and spermine) are likely to mediate a salt stress-induced decrease of ion flux across the vacuolar membrane by blocking FV channels.     

Changes in free polyamines and gene expression during peach flower development

Free polyamine contents and expressions of five genes encoding for polyamine biosynthetic enzymes were investigated at four different stages during peach (Prunus persica L. Batsch cv. Akatsuki) flower development. Fresh mass of peach flowers increased, accompanied by reduction in contents of total polyamines and putrescine/spermidine ratio due to decrease in putrescine content. Spermidine, the largest fraction, and spermine, the least part, underwent minor change. Expressions of the five key genes involved in polyamine biosynthesis during flower development did not parallel the changes in free polyamines.     

Comparison of soybean cultivars for enhancement of the polyamine contents in the fermented soybean natto using Bacillus subtilis (natto)

Polyamines have beneficial properties to prevent aging-associated diseases. Raw soybean has relatively high polyamine contents; and the fermented soybean natto is a good source of polyamines. However, detailed information of diversity of polyamine content in raw soybean is lacking. The objectives of this study were to evaluate differences of polyamines among raw soybeans and select the high polyamine-containing cultivar for natto production. Polyamine contents were measured chromatographically in 16 samples of soybean, which showed high variation among soybeans as follows: 93–861 nmol/g putrescine, 1055–2306 nmol/g spermidine, and 177–578 nmol/g spermine. We then confirmed the high correlations of polyamine contents between raw soybean and natto (r = 0.96, 0.95, and 0.94 for putrescine, spermidine, and spermine, respectively). Furthermore, comparison of the polyamine contents among 9 Japanese cultivars showed that ‘Nakasen-nari’ has the highest polyamine contents, suggesting its suitability for enhancement of polyamine contents of natto.     

Polyamines and environmental challenges: recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.     

Role of polyamines in the cytokinin-dependent physiological processes. I. Effect of benzyladenine on polyamine levels during chloroplast differentiation in the tissue culture of Dianthus caryophyllus

Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N⁶-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry. Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin. Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus. Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process. Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.     

Recombinant polyamine-binding protein of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 specifically binds to and is induced by polyamines

His-tagged Synechocystis sp. PCC 6803 PotD protein (rPotD) involved in polyamine transport was overexpressed in Escherichia coli. The purified rPotD showed saturable binding kinetics with radioactively labeled polyamines. The rPotD exhibited a similar binding characteristic for three polyamines, with putrescine having less preference. The K _d values for putrescine, spermine, and spermidine were 13.2, 8.3, and 7.8 μM, respectively. Binding of rPotD with polyamines was maximal at pH 8.0. Docking of these polyamines into the homology model of Synechocystis PotD showed that all three polyamines are able to interact with Synechocystis PotD. The binding modes of the docked putrescine and spermidine in Synechocystis are similar to those of PotF and PotD in E. coli, respectively. Competition experiments showed specific binding of rPotD with polyamines. The presence of putrescine and spermidine in the growth medium could induce an increase in PotD contents, suggesting the role of PotD in mediating the transport of polyamine in Synechocystis sp. PCC 6803.     

Separation and quantitation of polyamines in plant tissue by high performance liquid chromatography of their dansyl derivatives

High performance liquid chromatography in combination with fluorescence spectrophotometry can be used to separate and quantitate polyamines (putrescine, cadaverine, spermidine, spermine), prepared as their dansyl derivatives, from plant tissue. The procedure gives sensitive and consistent results for polyamine determinations in plant tissue. In a standard mixture, the minimal detection level was less than 1 picomole of polyamines.     

Metabolism of polyamines in NaCl-resistant cell lines from Nicotiana sylvestris

The polyamine titers in three cell lines of Nicotiana sylvestris were compared: Type 1, rapidly adapting to NaCl; Type 2, constantly resistant to NaCl; Type 3, a saltsensitive wild strain. During short-term cultivation in MS medium in the presence of 170 mM NaCl (1 passage, 14 d) the changes in polyamine titer in cell suspensions of type 1 (in a slightly adapted state) and non-adapted wild strain (type 3) showed a considerable increase in spermidine and spermine and a decrease in putrescine. After prolonged adaptation to NaCl (20 passages) the putrescine content in the cells of type 1 and of type 2 was increased at the expense of the polyamines. This suggests that the pattern of polyamine titer varies under short- and long-term adaptation to NaCl. The inverse ratio between growth processes and changes in polyamine and proline level indicates that polyamines fulfil primarily a protective and osmorepulatory function in plant cells under NaCl stress.     

Participation of polyamines in the flowering of the short-day plant Pharbitis nil

The effect of the exogenous application of polyamines on the flowering induction of the short-day plant Pharbtis nil was investigated. Putrescine, spermidine and spermine applied on the cotyledons of 4-day seedlings had no significant effect on the flowering of this plant under conditions of full induction caused by a 16-hour-long inductive night. Under the conditions of partial induction caused by a 13-hour-long subinductive night, polyamines inhibit or stimulate flowering, depending on the time of application. Also, inhibitors of the biosynthesis of polyamines influenced the flowering process. Analysis of endogenous polyamines revealed significant fluctuations in their content in cotyledons during an inductive night, as well as under continuous light conditions. Particularly large changes occurred in spermidine and spermine levels. The putrescine level in induced seedlings was lower than in non-induced ones. However, induced seedlings contained a higher level of spermine and spermidine. The highest spermidine and spermine levels were observed at the 8th h of the night, although the total concentration of spermine during photoinduction was always 2–3 times lower than that of spermidine. A break in the inductive night, leading to a complete inhibition of flowering, had caused significant changes in the polyamine level by the end of the night. The results suggest that the flowering induction of Pharbitis nil took place at a low putrescine level and increased spermidine and spermine levels.     

Acetylation of spermidine and spermine by rat liver and kidney chromatin

The in vitro enzymatic acetylation of the polyamines, spermidine and spermine, is described. The reaction is catalyzed by chromatin preparations from rat liver and kidney and is dependent on acetyl-CoA. Spermidine, spermine, and putrescine are each converted to the corresponding monoacetyl derivatives. s_0.5 values of 0.5 ± 0.1, 1.0 ± 0.1, and 2.6 ± 0.7 mm (mean ± standard deviation) were obtained for spermidine, spermine, and putrescine, respectively. These values for s_0.5 are similar to the concentrations of polyamines reported for tissues, and therefore, suggest the occurrence of polyamine acetylation in vivo. Evidence is also presented for the metabolism of acetylated polyamines by the 100,000g supernatant fraction of rat liver. The physiological function of polyamine acetylation is unknown, but the possibility of an effect on the association of polyamines with nucleic acids is discussed.     

Polyamines in the photosynthetic apparatus

The three main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were characterized by HPLC in intact spinach leaf cells, intact chloroplasts, thylakoid membranes, Photosystem II membranes, the light-harvesting complex and the PS II complex. All contain the three polyamines in various ratios; the HPLC polyamine profiles of highly resolved PS II species (a Photosystem II core and the rection center) suggest an enrichment in the polyamine Spm.Abbreviations Chl chlorophyll - HPLC high performance liquid chromatography - LHC light-harvesting complex - PS II Photosystem II - PS II-RC Photosystem II reaction center - Put putrescine - Spd spermidine - Spm spermine - 10%S-core D₁-D₂-Cyt b559-47 kD-43 kD complex     

Sensitive fluorimetric method for the determination of putrescine,spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles.The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 ± 3.1, and spermine 8.4 ± 2.8 nmoles/ml packed erythrocytes. Putrescine is not present in normal human erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml.The polyamine content of the erythrocytes from patients with malignant neoplasm was significantly elevated.