Profile of polyamines during sprouting and growth of saffron (Crocus sativus L.) Corms

The profile of free and conjugated polyamines putrescine, spermidine, and spermine was studied at the onset of sprouting and during various stages of vegetative growth in saffron (Crocus sativus L.) corms. Polyamines were extracted from the shoot meristems and estimated by high performance liquid chromatography. Free putrescine was not detected at the onset of sprouting, whereas free spermidine and spermine levels increased rapidly on sprouting and decreased during further stages of corm development. The levels of conjugated polyamines were several times higher than the free forms indicating their possible role in the developmental processes. A comparison of polyamine levels of vegetative and floral corms showed higher titers of free polyamines in vegetative and conjugated polyamines in floral corms.     

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.     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

Changes in endogenous polyamines during flower development in two diverse species of rose

Changes in the concentrations of endogenous free, conjugated and bound polyamine were determined in petals of two different species of rose, viz. Rosa damascena and Rosa bourboniana, from small bud (stage 1) till full bloom (stage 8). High free putrescine and spermidine concentrations were associated with early stages of flower development and then decreased in R. damascena. At full bloom, the concentration of free putrescine was higher than rest of the polyamines measured. A steady increase in conjugated putrescine, spermidine and spermine was observed during entire period of flower development with predominance of conjugated putrescine at full bloom. In R. damascena the bound spermine was higher than rest of the polyamines during full bloom. In R. bourboniana, during the early stages of flower development, similar situation was observed, however, at full bloom, free spermidine concentration was higher than rest of the polyamines. In this species, the concentration of conjugated and bound spermine was higher than rest of the polyamines during full bloom. Polyamine concentrations were generally lower in the petals of R. bourboniana than R. damascena which may be due to genotypic differences. The possible roles of the observed polyamines are discussed in relation to flower development.IHBT Communication no, 0345.     

Polyamines and Morphogenesis - Effects of Methylglyoxal-bis(guanylhydrazone)

The effects of methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis were studied on tuberization and cellular polyamine content using in vitro Solanum tuberosum (cv Binjte) plants. When MGBG was added to the culture medium, it produced a partial inhibition of the growth of stems and leaves; it totally blocked rhizogenesis and strongly stimulated tuber formation. Morphogenetic effects of MGBG were correlated to a 40 % decrease in free putrescine, spermidine, spermine content of the leaves and to a 28 % decrease in spermidine titer of the stems. In the tubers, this inhibitor did not change the free polyamine titer but increased by up to 85 % the titer of conjugated putrescine, spermidine, spermine. When the plants were grown in the dark, MGBG produced, like benzyladenine, a stimulation of the rate of tuberization and enhanced the content of conjugated polyamines in the tuber. These results support the hypothesis that polyamines play an important role in the morphogenesis of potato plants. The role of polyamine conjugation in tuber development is discussed.     

Polyamine metabolism in McCoy cells: I. Comparative studies of extracellular polyamine conjugated proteins of human fibroblast and McCoy cultures

polyamine conjugated proteins were identified in culture medium from both human skin fibroblasts and transformed mouse cells (McCoy cells). Sephadex G-100 column chromatography of medium allowed identification of two polyamine conjugated proteins from both types of cell cultures; one with Mr greater than 100,000 (MP1) and one with Mr = 60,000-70,000 (MP2). Human skin fibroblast MP1 contained putrescine and spermidine while MP1 from McCoy cultures contained putrescine, spermidine and spermine. MP2 isolated from both cultures contained all three polyamines. The relative concentration of polyamines in MP1 and MP2 for human fibroblasts and McCoy cells were different. The spermidine and spermine associated with MP1 and MP2 of McCoy cultures was covalently bound while for putrescine only 70.5% in MP1 and MP2 of McCoy cultures was covalently bound while for putrescine only 70.5% in MP1 and 74.5% in MP2 was covalently bound. The covalent nature of the polyamine protein conjugation was confirmed by autoradiography following isogel agarose isoelectric focusing. MP2 was resolved into three radiolabeled proteins with pI's between 5.25 and 5.20. Both MP1 and MP2 of McCoy cultures were heterogeneous. MP1 consisted of at least five proteins with Mr's of 180,000, 38,000, 76,000 and 68,000. The major protein (or proteins) had a pI of 5.25. MP2 consisted of at least three proteins with Mr's 72,000, 68,000 and 62,000; their pI's were between 5.20 and 5.25.     

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.     

Formation of Polyamines in the Rumen of Goats During Growth

Ornithine decarboxylase (E.G. 4.1.1.17) and S-adenosylmethionine decarboxylase (E.G. 4.1.1.50) and their products putrescine, spermidine and spermine were estimated in the rumen liquid from 3 groups of growing kids and 23 adult goats. Polyamines were also estimated in the feedstuff used. Marked differences in polyamine synthesis in rumen liquid were observed between the different groups of kids. Two groups of kids growing up together with adult goats had at an age of 2–4 months a peak of a few days duration in enzyme activity as well as in polyamine concentration. In these groups ornithine decarboxylase activity reached maximal values of 158±79 s (n = 4) and 100 (66–117) (n = 3) nmol[14CO2]/ml rumen liquid/h at an age of 120 and 77 days, respectively. The corresponding activity in rumen liquid from kids who were isolated from other animals was only about 1/10 of this value. By comparison ornithine decarboxylase activity in adult goats was 30.7±20 (n = 43) nmol[14CO]/ml/h. In rumen liquid from kids grown up together with adults, concentrations of the polyamines reached maximum at about the same time as ornithine decarboxylase activity. The mean maximal concentration of putrescine in the 2 groups was about 350 and 500 nmol/ml, while the corresponding value for spermidine was about 200 nmol/ml in both groups. Relatively constant and high concentration of polyamines were present in the feedstuff used. However, in growing kids the ruminai putrescine and spermidine concentration at times far exceeded those that could be accounted for by the estimated intake of polyamines by the food. The results therefore strongly indicate that polyamines are formed in considerable amounts in rumen content of kids during the phase of rapid growth. Results from a few experiments with calves also indicate that this may be true for cattle. polyamines; putrescine; spermidine; spermine; ornithine-decarboxylase; rumen liquid.     

Polyamines,floral induction and floral development of strawberry (Fragaria ananassa Duch.)

In the short-day plant, strawberry (Fragaria ananassa Duch.), polyamines (putrescine, spermidine and spermine), conjugated spermidine (water-insoluble compounds) and bound amines (putrescine, spermidine, phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine) accumulated in the shoot tips during floral induction and before floral emergence. Different associations of free amines and conjugated amines were observed during floral induction, as compared with the reproductive phase. During the whole period of floral development, phenylethylamine (an aromatic amine) was the predominant amine, representing 80 to 90% of the total free amine pool. Phenylethylamine conjugates (water-insoluble compounds) were the predominant amides observed prior to fertilization. These substances decreased drastically after fertilization. In vegetative shoot tips from plants grown continously under long days, free polyamines (putrescine, spermidine) and bound polyamines (putrescine, spermidine) were low and no change was observed. Free amines (spermine and phenylethylamine), bound aromatic amines (phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine), conjugated spermidine and phenylethylamine did not appear. Male-sterile flowers were distinguished by their lack of conjugated spermidine and phenylethyalamine and by a decrease in free phenylethylamine. In normal and sterile strawberry plants -DL-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), caused inhibition of flowering and free and polyamine conjugates. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with -DL-difluoromethylarginine (DFMA), a specific, irreversible inhibitor of arginine decarboxylase (ADC), did not affect flowering and polyamine titers. These results suggest that ornithine decarboxylase (ODC) and polyamines are involved in regulating floral initiation in strawberry. The relationship between polyamines, aromatic amines, conjugates, floral initiation and male sterility is discussed.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - Put putrescine - Spd spermidine - Spm spermine - Phen phenylethylamine - 3H4M Phen 3-hydroxy, 4-methoxyphenylethylamine     

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.     

Occurrence of uncommon polyamines in cultured tissues of maize

Summary The uncommon polyamines, norspermidine and norspermine, were detected in maizein vitro cultures of three different genotypes. The common polyamines, spermidine and spermine, along with the diamine, putrescine, were also observed. The total amounts of the uncommon polyamines, norspermidine and norspermine, were comparable to the total amounts of the common polyamines, spermidine and spermine, in the maize tissues. The titer for norspermidine was 6- to 15-fold greater than that of its common counterpart (spermidine) in the three genotypes. Norspermidine was the predominant polyamine among all triamines and tetramines detected in cell cultures of two of the three genotypes of maize examined and was predominant along with spermine in the third genotype. Enzyme assays performed with extracts from callus of one of the genotypes suggested a likely mechanism to account for the biosynthesis of the uncommon polyamines in cultured maize cells, through the actions of putrescine aminopropyltransferase, polyamine oxidase, and Schiff-base reductase/decarboxylase enzyme activities. This is the first report of the detection of uncommon polyamines in maize tissues, as well as the first report of these uncommon polyamines in a monocotyledonous plant.     

Changes in Polyamine Contents Development of the Frog, Microhyla ornata

Putrescine, spermidine and spermine levels were measured during development, metamorphosis and adult life of the frog, Microhyla ornata . Development of Microhyla was accompanied by high fluctuating levels of putrescine and spermidine with low and steady levels of spermine. Putrescine was the major polyamine during development from egg to mature tadpole. During metamorphosis both putrescine and spermidine decreased significantly; but the decrease in putrescine content was more rapid than that of spermidine. Thus, in the freshly metamorphosed frog, the concentration of spermidine exceeded that of putrescine. In most of the adult tissues also spermidine concentration was higher than putrescine and spermine. While the free form of putrescine and spermidine increased during early development of the fertilized egg to tadpole, the levels of protein conjugated polyamines decreased. In the free form, putrescine was the major polyamine while in the protein conjugated form spermidine concentration was higher than putrescine and spermine. Thus polyamine pattern is different in early development, during metamorphosis and in differentiated adult tissues of this frog. ∞-Difluoromethylornithine treatment at early blastula stage did not interfere with the normal development of Microhyla embryos.     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Ratio of free to bound polyamines during maturation in mung-bean hypocotyl cells

Free- and bound-polyamine levels were estimated in successive segments of the mung-bean hypocotyl. Three aliphatic polyamines (putrescine, spermidine and spermine) were found in proportions which depended on the state of maturation. In young cells, most of the polyamines were located in the protoplasm whereas in older cells they were mostly bound to the cell walls. Spermidine was always the main bound polyamine, and putrescine, the main free polyamine.Abbreviation EDTA ethylenediaminetetraacetic acid     

Levels of Polyamines and Kinetic Characterization of Their Uptake in the Soybean Pathogen Phytophthora sojae

Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-¹⁴C]putrescine and [1,4-¹⁴C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection.     

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.     

Polyamines of Anacystis nidulans and metabolism of exogenous spermidine and spermine.

Several biochemical parameters, including that of polyamine content, accompanying the growth of the cyanobacterium Anacystis nidulans were studied. At all stages of growth under autotrophic conditions, the organisms were found to be rich in spermidine and lacking in spermine, as is typical of procaryotic organisms. The cells were quite low in putrescine, and no unusual polyamine was observed to be present as a major component. Conjugated polyamines were not detected in the cultures. At maximal culture density, the levels of spermidine, DNA, RNA, protein, and chlorophyll were also maximal. Shortly after the inception of the stationary phase, the spermidine content of the cells was the first parameter observed to decrease in cultures which were shortly to become yellow. Spermidine lost from the cells was not recovered in the medium in a free or conjugated form. This indication of degradation of spermidine was studied by the addition of polyamines to growing cultures. Exogenous spermidine and spermine were found to be metabolized rapidly by the organisms, of which diaminopropane was one product. Putrescine was found to be markedly toxic, whereas spermidine, some other triamines, and spermine were much less toxic.     

Oxidized polyamines and the growth of human vascular endothelial cells. Prevention of cytotoxic effects by selective acetylation.

The responses of human umbilical-vein vascular endothelial cells in culture to the naturally occurring polyamines spermine, spermidine and putrescine, their acetyl derivatives and oxidation products were examined. In the absence of human polyamine oxidase, exposure of cells to polyamines (up to 160 microM) had no adverse effects. In the presence of polyamine oxidase, spermine and spermidine were cytotoxic, but putrescine was not. Acetylation of the aminopropyl group of spermidine or both aminopropyl groups of spermine prevented this cytotoxicity. The amino acids corresponding to the polyamines, representing a further stage of oxidation, were also without effect. The cytotoxic effects were irreversible. Use of bovine serum amine oxidase in place of the human enzyme gave qualitatively similar results.