Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata

Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don . During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no significant change in putrescine. This led to a several-fold increase in spermidine/putrescine ratios during development of both types of embryos. Cell cultures of plant-forming and non-plant-forming lines derived from the same clone and growing on proliferation (maintenance) medium differed significantly in their polyamine levels. Mature, cotyledonary stage somatic embryos capable of germination and formation of plants could be distinguished by their higher spermidine/putrescine ratios from abnormal cotyledonary stage somatic embryos which were incapable of forming plants.     

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     

New Metabolites of Streptomyces alboniger

A mutant unable to grow under acidic conditions was isolated from the acidophilic heterotrophic bacterium Acidiphilium facilis 24R. The growth of the mutant could be fully restored by the addition of spermidine or lysine at the concentration of 100 μm. The HPLC analysis of polyamine composition showed that spermidine and putrescine were major polyamine components in the parental strain. In the mutant strain, putrescine was replaced by cadaverine. It was found that some polyamines in the cells were conjugated with the other cell components. The growth of the bacterium in the medium below pH 4.5 was inhibited in the presence of α-methylornithine or methylglyoxal-bis(guanylhydrazone), which are inhibitors of rate-limiting enzymes involved in the biosynthesis of polyamines. The growth of the bacterium that had been inhibited in the presence of inhibitors could be fully restored by the addition of putrescine or spermidine. On the basis of these results, it was concluded that polyamines have a significant role in the growth of Acidiphilium facilis 24R under acidic conditions.     

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     

A specific role for spermidine in the initiation phase of somatic embryogenesis in Panax ginseng CA Meyer

Somatic embryogenesis of Panax ginseng CA Meyer was initiated from suspension aggregates of an embryogenic callus, in a liquid medium consisting of half strength Murashige and Skoog (1962) supplemented with the synthetic auxin benzoselenienyl-3 acetic acid. The addition of spermidine to this initiation medium significantly increased the production of somatic embryos. In this case, the total polyamine content of the embryogenic mass was higher than that of cultures without spermidine. At day 6 of the culture, a transient accumulation of free polyamines, mainly spermidine, was observed. After this peak, free and conjugated polyamines levels did not show significant variation nor did the polyamine oxidase activity. The results clearly demonstrated that spermidine supplied to the medium was oxidised by polyamine oxidase and partially metabolised into putrescine. The role of spermidine and its interaction with auxin in the initiation of the embryogenic process in Panax ginseng are discussed in relation to embryogenic potential.     

Do Polyamines Participate in the Long-Distance Translocation of Stress Signals in Plants?

Accumulation and ethylene-dependent translocation of free polyamines was studied in various organs, the phloem and xylem exudates of common ice plants (Mesembryanthemum crystallinum L.). Under normal conditions (23–25°C), spermidine predominated among polyamines. Cadaverine was found in old leaves, stems, and, in large quantities, in roots. The heat shock treatment (HS; 47°C, 2 h) of intact plant shoots induced intense evolution of ethylene from leaves but reduced the leaf content of polyamines. Under these conditions, the concentration of polyamines in roots, particularly of cadaverine, increased many times. The HS treatment of roots (40°C, 2 h) induced translocation of cadaverine to stems and putrescine to leaves. An enhanced polyamine content after HS treatment was also found in the xylem and phloem exudates. The exposure of detached leaves to ethylene led to a reduction in their putrescine and spermidine and accumulation of cadaverine, which implies the ethylene-dependent formation of cadaverine and a possible relation between the HS-induced translocation of this diamine to roots and the transient ethylene evolution by leaves. To validate this hypothesis, we compared the ethylene evolution rate and interorgan partitioning of cadaverine and other polyamines for two lines of Arabidopsis thaliana: the wild type (Col-0) and ein4 mutant with impaired ethylene reception. In plants grown in light at 20–21°C, the rate of ethylene evolution by rosetted leaves was higher in the mutant than in the wild type. The content of putrescine and spermidine was reduced in mutant leaves, whereas cadaverine concentration increased almost threefold compared with the wild type. In roots, cadaverine was found only in the wild type and not in the mutant line. Our data indicate the ethylene-dependent formation of cadaverine in leaves and possible involvement of cadaverine and ethylene in the long-distance translocation of stress (HS) signal in plants.     

Polyamines in photosynthetic eubacteria and extreme-halophilic archaebacteria

Qualitative and quantitative determinations of polyamines have been done in 4 photosynthetic eubacteria and 6 extreme-halophilic archaebacteria. For comparison, 5 moderate-halophilic eubacteria were also analyzed to determine their polyamine contents. Not only putrescine and spermidine but also homospermidine were found in the photosynthetic eubacteria, especially in the N2-fixing species, Rhodospirillum and Chromatium. Norspermidine, norspermine, and spermine were not detected in the phototrophic eubacteria. No appreciable amount of any polyamine was found in extreme-halophilic archaebacteria, Halobacterium and Halococcus, while moderate-halophilic eubacteria contained quite high concentrations of putrescine and spermidine and cadaverine. When arginine was incubated with cell lysates of these two archaebacteria, appreciable amounts of agmatine were produced; neither putrescine nor cadaverine was formed in the presence of ornithine or lysine. No detectable amount of spermidine was produced by the lysates on incubation with putrescine.     

Polyamine distribution patterns within the families Aeromonadaceae,Vibrionaceae, Pasteurellaceae,and Halomonadaceae,and related genera of the gamma subclass of the Proteobacteria

Polyamines of the four families and the five related genera within the gamma subclass of the class Proteobacteria were analyzed by HPLC with the objective of developing a chemotaxonomic system. The production of putrescine, diaminopropane, cadaverine, and agmatine are not exactly correlated to the phylogenetic genospecies within 36 strains of the genus Aeromonas (the family Aeromonadaceae) lacking in triamines. The occurrence of norspermidine was limited but not ubiquitous within the family Vibrionaceae, including 20 strains of Vibrio, Listonella, Photobacterium, and Salinivibrio. Spermidine was not substituted for the absence of norspermidine in the family. Agmatine was detected only in Photobacterium. Salinivibrio and some strains of Vibrio were devoid of polyamines. Vibrio ("Moritella") marinus contained cadaverine. Within the family Pasteurellaceae, Haemophilus contained cadaverine only and Actinobacillus contained no polyamine. Halomonas, Chromohalobacter, and Zymobacter, belonging to the family Halomonadaceae, ubiquitously contained spermidine and sporadically cadaverine and agmatine. Shewanella contained putrescine and cadaverine; Alteromonas macleodii, putrescine, 2-hydroxyputrescine, cadaverine, 2-hydroxyspermidine, and spermidine; Pseudoalteromonas, putrescine, cadaverine, and spermidine; Marinobacter, spermidine; and Marinomonas, putrescine and spermidine. Their polyamine profiles serve as a chemotaxonomic marker within the gamma subclass.     

Polyamines and their biosynthetic enzymes during somatic embryo development in red spruce (<Emphasis Type="Italic">Picea rubens</Emphasis> Sarg.)

Summary The major objective of this study was to determine if the observed changes in polyamines and their biosynthetic enzymes during somatic embryo development were specifically related to either the stage of the embryo development or to the duration of time spent on the maturation medium. Somatic embryos of red spruce (Picea rubens) at different developmental stages, grown in the embryo development and maturation media for various lengths of time, were separated from the associated subtending tissue (embryogenic and the suspensor cell masses) and analyzed for their polyamine content as well as for polyamine biosynthetic enzyme activities. Polyamine content was also analyzed in embryos representing different stages of developmentthat were collected from the sam culture plate at the same time and the subtending tissue surrouding them. Putrescine was the predominant polyamine in the pro-embryogenic tissue, while spermidine was predominant during embryo development. Significant changes in spermidine/putrescine and spermine/putrescine ratios were observed at all stages of embryo development as compared to the pro-embryogenic cell mass. Changes in the ratios of various polyamines were clearly correlated with the developmental stage of the embryo rather than the period of growth in the maturation medium. Whereas the activities of both ornithine decarboxylase and arginine decarboxylase increased by week 3 or 4 and stayed high during the subsequent 6 wk of growth, the activity of S-adenosylmethionine decarboxylase steadily declined during embryo development.     

Synthesis of novel polyamines in Paracoccus, Rhodobacter and Micrococcus

Abstract The Gram-negative facultative chemolithotroph, Paracoccus denitrificans contains putrescine, cadaverine, agmatine, spermidine, aminopropylcadaverine, spermine, thermospermine and aminopentylnorspermidine. This bacterium has the ability to produce norspermidine from supplemented diaminopropane. The halophile, Paracoccus halodenitrificans is devoid of any polyamines. Neither decarboxylation of ornithine, lysine or arginine, nor triamine synthetic activity from diamines was detected in this halophile. Two Gram-negative facultative photoautotrophs, Rhodobacter sphaeroides and Rhodobacter capsulatus contain putrescine, cadaverine, agmatine and spermidine and can produce norspermidine from supplemented diaminopropane. A Gram-negative eubacterium, Micrococcus cryophilus , contains histamine and homospermidine in addition to putrescine, cadaverine and spermidine. Hence, polyamine distribution patterns and polyamine biosynthetic activities were very different among the four groups of Gram-negative eubacteria examined.     

Polyamines and cytokinins in celery embryogenic cell cultures

The effect of inhibitors of polyamine biosynthesis on the development of embryogenic cell cultures of celery (Apium graveolus L.) was studied. Several developmental stages of somatic embryos were compared for differences in the content and biosynthesis of free polyamines and for cytokinin content. Cyclohexylamine and particularly methylglyoxal bis(guanylhydrazone), inhibited both cell division and the organization of polar embryos from globular embryos. Difluoromethylornithine slightly promoted embryo development, especially cell division.The free putrescine content of globular embryos was 6-fold that of fully differentiated plantlets, and that of spermidine 2-fold. Only a slight increase in the spermine content was found with embryo development. These differences were confirmed by data from polyamine biosynthesis. Incorporation of ¹⁴C-arginine into polyamines was slightly higher than that of ¹⁴C-ornithine. Over 96% of this incorporation was detected in the putrescine fraction. Incorporation of ¹⁴C into putrescine in globular embryos was 3 to 4-fold that in fully-differentiated plantlets. Incorporation into spermidine and spermine was, however, higher in plantlets than in globular embryos.Cytokinin analysis revealed considerable differences in the biological activity between the developmental stages of embryogenesis. This could be due to endogenous cytokinins and/or BA taken up from the maintenance medium. Cytokinin levels decreased with increased embryo development. Most of the detected cytokinin-like activity co-chromatographed with BA and its metabolites. Some as yet unidentified peaks of activity were recorded in the globular embryos.The results are considered with respect to the possible participation of polyamines and cytokinins in the development of embryogenic cell cultures of celery. It is suggested that the onset of embryogenesis is characterized by a high content of putrescine and cytokinins, while a decrease in putrescine synthesis and cytokinin content, and an increase in spermidine and spermine content, accompany further embryo development and plantlet formation.Abbreviation ADC arginine decarboxylase - ODC ornithine decarboxylase - 2,4-D dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DFMO difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone) - CHA cyclohexylamine - BA benzyladenine - BAR benzyladenine riboside     

A possible role of cadaverine in the biosynthesis of polyamines in the Japanese newt testis

Polyamine content in testes of various vertebrates was studied extensively. Putrescine, spermidine and spermine were detected in all the animals examined, although the distribution pattern varied greatly from animal to animal. Cadaverine was detected only in amphibian testes; sym-homospermidine was found not only in testes but also in various other tissues of amphibians and of some reptiles. In the newt testis the concentration of cadaverine was lower than that of any other polyamines in summer, but there was a great increase in cadaverine content from autumn to winter. The testicular content of cadaverine was greater than that of other polyamines in winter. There was a gradual decrease in the cadaverine content in spring. The spermidine and spermine levels, which were rather low in winter, increased in spring and reached a peak in summer when spermatogenesis was active. The testicular concentration of putrescine that was much higher than that of spermidine or spermine throughout the year, increased only a little in summer. There was a significant negative correlation between the cadaverine levels and four other polyamine levels. Exogenous cadaverine decreased the testicular levels of putrescine. Mammalian gonadotropins decreased the cadaverine levels and increased the levels of other polyamines. A partially purified LH fraction from pituitaries of bullfrog, Rana catesbeiana, was also potent in depleting cadaverine of the testes of newts kept at 8 degrees C. These results suggest that testicular cadaverine suppresses the biosynthesis of polyamines, especially spermidine and spermine which are closely associated with spermatogenesis.     

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.     

Endogenous polyamine profiles in different tissues of <Emphasis Type="Italic">Coffea</Emphasis> sp., and their levels during the ontogeny of fruits

Polyamines are essential compounds for growth and development in plants. An attempt has been made to find out the endogenous polyamine profiles in various parts and during the ontogeny of fruit formation of two commercially important Coffea species viz., arabica and canephora. Putrescine (Put), spermine (Spm) and spermidine (Spd) are the predominant polyamines during the ontogeny of fruit and their level increased with the advancement of fruit development. However, in the initial stages of flower and fruit development Spm levels were found to be decreased. Elevated levels of major polyamines Put, Spd, and Spm were observed in zygotic embryos than in somatic embryos. Along with this cadavarine (Cad) and other biogenic amines viz., tyramine (Tyr) and tryptamine (Try) were also found during the ontogeny of fruit in C. canephora. In this study the enodogenous polyamine profiles in coffee tissues and beans have been addressed.     

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-(14)C]putrescine and [1,4-(14)C]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.     

Spatial distribution of free and conjugated polyamines in leaves of Solanum melongena L. associated with differential morphogenetic capacity: efficient somatic embryogenesis with putrescine

In eggplant (Solanum melongena L., cv. Pusa Purple Long), explantsfrom different regions of the leaf showed significant differencesfor embryogenic potential. Discs from the apical region of leavesyielded more somatic embryos than those from the basal region.Apical discs showed consistently higher polya-mine titres thanthe basal discs. Putrescine treatment promoted somatic embryogenesisand at 0.5 mM it caused a remarkable increase (c. 6-fold) ina number of somatic embryos, accompanied by an increased putrescinetitre. On the other hand, spermidine and spermine had no stimulatoryeffect on embryogenesis; rather they were inhibitory at higherconcentrations. All tested inhibitors of polyamine biosynthesissuch as difluoromethylarginine, difluoromethylomithine, methylglyoxalbis (guanylhydrazone) and bis (cyclohex-ylammonium) sulphatesignificantly inhibited somatic embryogenesis. Difluoromethylarginineblocked somatic embryogenesis by lowering endogenous polyaminecontents (particularly putrescine) and such inhibitory effectswere totally restored by exogenous putrescine (0.5 mM), concomitantwith the revival of endogenous PA concentrations. These resultsdemonstrate (i) a positive correlation between the spatial distributionof free and conjugated polyamines and the embryogenic capacityof an explant and (ii) putrescine caused the promotion of somaticembryogenesis, suggesting the intricate regulatory role of polyamines,specifically putrescine, in somatic embryogenesis in eggplant. Key words: Solanum melongena, somatic embryogenesis, position effect, polyamines, putrescine, polyamine biosynthesis inhibitors, difluoromethylarginine     

Effect of polyamines on in vitro anther culture of Citrus clementina Hort. ex Tan

The improvement of the induction rate in Citrus anther culture is important for taking practical advantage of the haploid potential in breeding. The influence of polyamines on anther culture of Citrus clementina, cv Nules, with particular attention to the free, soluble and insoluble-conjugated polyamine levels, has been investigated. Putrescine, spermidine and putrescine plus spermidine, were added to the standard induction medium. Before culture, spermidine was the most abundant among the free polyamines detected in anthers. The exogenous supply of either putrescine or spermidine, either independently or combined, effected greater uptake and accumulation of polyamines. The addition of 2 mM spermidine to the medium stimulated gametic embryogenesis in clementine Nules, whereas putrescine did not influence embryo production. Regenerants were mostly tri-haploids; a few doubled-haploids and no haploid plants were obtained.     

Stable Ribonucleic Acid Synthesis in Stringent (rel+) and Relaxed (rel−) Polyamine Auxotrophs of Escherichia coli K-12

The relationship of polyamines to stable ribonucleic acid (RNA) synthesis under conditions of amino acid withdrawal or chloramphenicol treatment was examined with the use of a closely related rel(+), rel(-) pair conditionally incapable of synthesizing putrescine. Under conditions of polyamine starvation, the cellular sperimidine level fell to one-third to one-half of the value observed in putrescine-supplemented cultures and putrescine became undetectable; cadaverine was synthesized by both strains, but the relaxed strain, MA 252, accumulated less cadaverine per cell than its stringent twin, MA 254. Upon amino acid withdrawal, the stringent strain remained stringent whether starved of or supplemented with polyamines. Similarly, the relaxed strain was capable of making RNA either with or without polyamine starvation. On the addition of chloramphenicol or upon amino acid withdrawal in the relaxed strain, supplementation with spermidine had no effect on the initial rate of RNA synthesis, although RNA accumulation was greater in the presence of added spermidine. Spermidine added at the conclusion of RNA synthesis prompted additional synthesis, although preincubation with spermidine again had no effect on the initial rate. All forms of stable RNA species were made with polyamine supplementation. The present data appear to rule out the possibility that polyamines are primary causative agents in stimulating RNA synthesis, but rather suggest an indirect or secondary role for spermidine in which the polyamines "stimulate" stable RNA synthesis probably by relieving RNA product inhibition of RNA synthesis.     

Polyamines promote the development of embryonal-suspensor masses and the formation of somatic embryos in <Emphasis Type="Italic">Picea glehnii</Emphasis>

Effects of polyamines on the development of embryonal-suspensor masses (ESMs) and the formation of somatic embryos were examined in an effort to improve the frequency of somatic embryogenesis in Picea glehnii. Exogenous spermidine at 100 and 300 μM promoted the elongation of suspensors and the formation of somatic embryos. Exogenous putrescine at 100 μM increased the number of somatic embryos and decreased the time required for formation of somatic embryos. Thus, exogenous polyamines improved the maturation capacity of ESMs and promoted the formation of somatic embryos in P. glehnii. Light microscopy revealed that ESMs with high maturation capacity consisted of the three following cells: round or oval cells, elongated cells, and protean cells. These components might be related to the capacity for formation of somatic embryos.