Differential responses of Brassica napus and Petunia hybrida to leaf protoplast isolation stress

Changes in the response to abiotic stress during the isolation of leaf protoplasts were compared between a recalcitrant species of Brassica napus and regenerating species of Petunia hybrida . Initially, levels of soluble free putrescine (put), spermidine (spd) and spermine (spm) in leaves and protoplasts were determined. The sum of these three polyamines increased in petunia and B. napus leaf protoplasts by 1.6-fold and 1.1-fold, respectively. The soluble free fraction of spd and spm decreased in B. napus but not in petunia protoplasts. During the isolation of leaf protoplasts from B. napus , the ratio of soluble free put to the total PAs almost doubled, but that of spd and spm declined significantly. Petunia leaf protoplasts treated with cyclohexylamine (CHA), an inhibitor of spermidine synthase, accumulated ammonia and soluble putrescine, but lost the soluble spermidine. The soluble polyamine levels of CHA-treated petunia leaf protoplasts corresponded with those in B. napus . Leaves were subjected to abiotic stress during the isolation of protoplasts, namely wounding and osmotic stress which changed soluble free polyamine levels in B. napus and petunia, respectively. Both B. napus and petunia leaf protoplasts showed an increase in ammonia, but total free amino acid content and activation of proteases were only enhanced in B. napus leaf protoplasts. These results suggest that in B. napus wounding initiated senescence of leaf protoplasts during their isolation, leading to a constant production of ethylene early in the culture.     

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.     

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.     

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.     

Polyamine content as a marker of radiation injury in the rat spleen

The modifications of the polyamines putrescine (put), spermidine (spd) and spermine (spm) in rat spleen after 3 Gy whole-body irradiation were studied. Rats were irradiated at four different times of the day (00.00, 06.00, 12.00 and 18.00) and sacrificed between 12 h and 62 days after irradiation. Control animals, sacrificed at the same times of the day, showed higher levels of the spd/spm ratio during the hours of light. After irradiation the polyamine content was rapidly and significantly reduced over a period of 20 days. The modification of the amount of spm lasted for a longer period of time. Normal values of polyamine content were reached at later times when the mitotic activity was restored. The results show a close correlation between polyamine concentration and [3H]thymidine uptake.     

Enzymatic Synthesis of a Branched Trisaccharide, 2,4-di-α-Glucosyl-glucose

New procedures for determining putrescine, spermidine and spermine were first established here by the end point assay method using polyamine oxidase from Penicillium chrysogenum or Aspergillus terreus and putrescine oxidase from Micrococcus rubens. Method 1: Spermidine and spermine were first oxidized with polyamine oxidase (step A). To the reaction mixture, putrescine oxidase was added to oxidize putrescine (step B). Putrescine and spermidine in another reaction mixture were oxidized with putrescine oxidase (step C). Method 2 : Putrescine and spermidine were first oxidized with putrescine oxidase (step A). To the reaction mixture, polyamine oxidase was added to oxidize spermine (step B). Spermidine and spermine in another reaction mixture were oxidized with polyamine oxidase (step C). The amounts of putrescine, spermidine and spermine were determined from the absorbance values at each steps A, B and C.     

Effects of external osmolality on polyamine metabolism in HeLa cells

The polyamine content of Escherichia coli is inversely related to the osmolality of the growth medium. The experiments described here demonstrate that a similar phenomenon occurs in mammalian cells. When grown in media of low NaCl concentration, HeLa cells and human fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels of enzyme activity appear to result solely from variations in the rate of enzyme degradation.A sudden increase in NaCl concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaCl concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.     

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.     

Biochemical changes during seed development in <Emphasis Type="Italic">Pinus taeda</Emphasis> L.

Amino acids, polyamines, 3-indoleacetic acid (IAA), abscisic acid (ABA), buffer-soluble protein and starch contents and dry matter accumulation were analyzed in megagametophytes containing developing embryos during seed development in Pinus taeda. The highest total amino acids and polyamine contents occurred at the cotyledonary stage, followed by a significant decrease in the mature seed. Free polyamines exhibited higher levels than conjugated ones, with putrescine being the predominant type until the cotyledonary stage, and spermidine at the mature seed stage. IAA content increased continually from the globular stage reaching the maximum at the cotyledonary stage, followed by a decrease in the mature seed. The highest ABA level occurred at the globular stage, followed by a continuous reduction until stabilization at the pre-cotyledonary stage. Buffer-soluble protein and starch contents, and dry matter increased progressively during development, reaching their maximum values at the mature stage.     

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     

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.     

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.     

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.     

Effect of organic and inorganic nitrogen sources on endogenous polyamines and growth of ectomycorrhizal fungi in pure culture

 The abilities of three ectomycorrhizal fungi, Paxillus involutus, Suillus variegatus and Lactarius rufus, to utilize organic and inorganic nitrogen sources were determined by measuring the growth and endogenous free polyamines (putrescine, spermidine and spermine) of pure culture mycelium. Differences were found in the utilization of the nitrogen sources and in the polyamine concentrations between the fungal species and between isolates of L. rufus. All the fungi grew well on ammonium and on several amino acids. Endogenous polyamine levels varied with the nitrogen source. Spermidine was commonly the most abundant polyamine; however, more putrescine than spermidine was found in P. involutus growing on inorganic nitrogen or arginine. Low amounts of spermine were found in S. variegatus and some samples of L. rufus. None or only a trace of spermine was found in P. involutus mycelium. In all fungi, putrescine concentrations were higher with ammonium than with the nitrate treatment. The total nitrogen content of peat did not determine the ability of L. rufus strains isolated from peatland forest sites to utilize organic nitrogen. Accepted: 27 November 1998     

Cadaverine: a common polyamine in zygotic embryos and somatic embryos of the species Capsicum chinense Jacq.

The behavior of endogenous polyamines was studied in somatic embryos and zygotic embryos of Habanero pepper (Capsicum chinense). In the first part of the work, the polyamine content was evaluated in both types of embryos (somatic and zygotic). As a result, in addition to the common polyamines (putrescine, spermidine and spermine), it was also possible to detect cadaverine, a polyamine rarely found in plants. In general, all the polyamines were found to be more abundant in somatic embryos than in zygotic embryos, with significantly higher contents of putrescine and cadaverine. Subsequently, the content of putrescine, spermidine, spermine and cadaverine, in their different forms (free, bound and conjugated) was determined in somatic embryos which were cultured in non-ventilated and ventilated containers. Detection of polyamines was carried out at 28 and 42 days of culture by the HPLC method. The ethylene content was monitored during the process in both culture conditions (ventilated and non-ventilated). As a result of the analysis, cadaverine was always found present, indicating that it is a common polyamine in the species. Ethylene was detected in containers without ventilation throughout the culture, except during replenishment of the culture medium (R1, R2 and R3). The behavior pattern of each polyamine, analyzed under different culture conditions (ventilated and non-ventilated) and at different moments of culture (28 and 42 days of culture), show that the polyamines are not only involved in morphogenic processes in plants; polyamines are also significantly affected by the surrounding environment. However, the most novel result, presented for the first time in this paper, is that cadaverine is found to be a common polyamine in C. chinense since it is present in both zygotic embryos and somatic embryos.

     

Polyamines and ethylene in relation to adventitious root formation in Prunus avium shoot cultures

An attempt was made to identify some of the hormonal factors that control adventitious root formation in our Prunus avium micropropagation system in order to improve rooting in difficult-to-root genotypes. Changes in endogenous contents of free polyamines were determined at intervals during auxin-induced rooting of shoot cultures. Accumulation of putrescine and spermidine peaked between days 9 and 11. Spermine was only present in traces, Exogenously supplied putrescine or spermine (50-500 μM), in the presence of optimal or suboptimal levels of indolebutyric acid (IBA), had no effect on rooting percentage or root density, except for spermine at 500 μM. At this external concentration spermine caused a substantial accumulation in both free spermine and putrescine. The use of several inhibitors of polyamine biosynthesis, namely α-difluoromethylornithine (DFMO), α-difluoromethylarginine (DFMA), dicyclohexylammonium sulphate (DCHA) and methylglyoxal-bis-guanyl-hydrazone (MGBG) alone or in combination in the 0.1 to 5 μM range, resulted in an inhibition of rooting that was partially reversed by the addition of the corresponding polyamine. Cellular polyamine levels were significantly reduced by DFMO and DFMA but not by DCHA and MGBG, Labeled putrescine incorporation into spermidine increased somewhat in the presence of the ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG). A system based on [3,4-¹⁴C]methionine incorporation was used to measure ethylene synthesis by the in vitro cultured shoots. Label incorporation was drastically reduced by 10 μM AVG and increased 3.5-fold in the presence of 50 μM IBA with respect to controls (no IBA). Labeled methionine incorporation into spermidine increased to some extent when ethylene synthesis was inhibited by AVG. Adding the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) to the rooting medium significantly inhibited rooting percentage; AVG caused the formation of a greater number of roots per shoot but delayed their growth. Supplying the shoots with both compounds resulted in an intermediate rooting response, in which both rooting percentage and root density were affected. These results indicate that polyamines may play a significant role at least in some stages of root formation. The polyamine and ethylene biosynthetic pathways seem to be competitive but under our conditions, the enhancement of one pathway when the other was inhibited, was not dramatic. Although IBA promoted ethylene synthesis, AVG, which drastically reduced it, also promoted root formation. Thus, the auxin effect on root induction cannot be directly related to its ability to enhance ethylene synthesis.