Effect of repeated severe pruning on endogenous polyamine content in hazelnut trees

Endogenous polyamine content was determined in leaves and buds of adult and repeatedly severe pruned hazelnut trees ( Corvlus avellana L.). Polyamine content in leaves from shoots obtained by forced outgrowth of branches taken from adult and pruned trees was also determined. Variations of polyamine levels in relation to pruning treatments were observed in all the analysed, tissues. Free polyamines increased in response to pruning treatments, mostly due to an increase in free putrescine. Free spermidine and spermine seemed to decrease with pruning intensity, whereas bound polyamines did not seem to correlate with treatments. Significantly, in all the analysed tissues the putrescine to spermidine plus spermine ratio increased in the free polyamine fraction. The results indicate that polyamine metabolism could play a role as a physiological marker for juvenility and rejuvenation in relation to cloning of woody plants. The possible role of polyamines in mediating and/or regulating phase change and reinvigoration is discussed.     

Polyamine oxidase activity and polyamine content in maize during seed germination

Polyamine oxidase (PAO, EC 1.5.3.3) activity and polyamine content in the cell wall and soluble fractions obtained from embryos, endosperms and shoots and roots of etiolated or green seedlings of maize ( Zea mays L. cv. WF9) during the first 7 days of germination were investigated. Polyamine content was also determined in the trichloroacetic acid-soluble (free polyamines) and trichloroacetic acid insoluble (bound polyamines) fraction obtained from the same tissues. PAO activity, determined by the radiometric method based on the recovery of the labelled reaction product 1-pyrroline, was mostly localized in the cell wall fraction. The activity was very low in embryos and endosperms and present in traces in roots. In etiolated shoots PAO activity increased sharply, while in green shoots it was low and increased slowly. No polyamines were found in the cell wall fraction and only putrescine was detected in the soluble fraction, with the exception of the embryo, where spermidine and spermine were also present. In the TCA-soluble fraction of embryos, putrescine increased during imbibition, while spermidine and spermine decreased; in the endosperm no relevant changes in polyamines occurred. In the same fraction of green and etiolated seedlings, putrescine increased, giving a peak at days 3–5, while spermidine decreased to very low levels. The amount of bound polyamines was 1–4% of the free ones. The pattern of PAO activity seems to be unrelated to endogenous free polyamine content, which is the same in shoots and roots of etiolated and green seedlings. Enzyme activity, very low in ungerminated seeds, increased continuously during the progression of germination, especially in etiolated shoots, indicating a possible involvement in cell wall formation.     

Amelioration of NaCl Stress by Arginine in Rice Seedlings: Changes in Endogenous Polyamines

Effects of NaCl (0.1 – 0.2 M) alone or in combination with 1 mM arginine on growth and endogenous polyamine (PA) content have been observed in two cultivars of rice differing in NaCl stress tolerance. The germination, seedlings fresh mass and water content decreased with increase in salinity in both the cultivars. This inhibition was partially alleviated by application of arginine. Cv. CSR-27 exhibited relatively better germination than cv. Bas-370 at different salinities. Total PA content increased in both the cultivars under NaCl stress alone and in combination with arginine. Putrescine to spermidine and spermine ratio was higher in NaCl-treated seedlings being more in cv. Bas-370 as compared to cv. CSR-27 and the ratio reversed to almost control level when arginine was applied along with NaCl.     

Polyamines in normal and auxin-induced strawberry fruit development

The possible involvement of polyamines during strawberry ( Fragaria × ananassa Duch.) fruit development was investigated. Putrescine, spermidine, and spermine were identified in strawberry receptacles and achenes at all stages of development. Total (free) polyamine levels decreased from a maximum of 485 nmol g⁻¹ fresh weight at pollination to a minimum of 55 nmol g⁻¹ fresh weight in ripe receptacles. Total polyamine concentrations during corresponding stages of development were consistently higher in achenes than in receptacles, and ranged from 891 to 203 nmol g⁻¹ fresh weight. Removal of achenes from the surface of developing receptacles 10 days after pollination reduced receptacle growth, and re-initiation of growth by application of 1 m M α-naphtaleneacetic acid (α-NAA) was accompanied by a rapid increase in polyamine concentrations 24 h after treatment. Polyamine content per receptacle increased >3-fold in normally developing receptacles and in de-achened, auxin-treated receptacles 10 days after removal of achenes, but did not increase during this period in de-achened receptacles not treated with exogenous auxin. α-NAA increased growth and polyamine levels to a greater extent than the structurally related, but less effective auxin, β-NAA. Polyamine concentrations in receptacles with intact achenes remained similar to those of auxin depleted (de-achened) receptacles, implying that the concentration of these compounds may not be limiting following achene removal.     

Polyamines in normal and auxin-induced strawberry fruit development

The possible involvement of polyamines during strawberry ( Fragaria x ananassa Duch.) fruit development was investigated. Putrescine, spermidine, and spermine were identified in strawberry receptacles and achenes at all stages of development. Total (free) polyamine levels decreased from a maximum of 485 nmol g⁻¹ fresh weight at pollination to a minimum of 55 nmol g⁻¹ fresh weight in ripe receptacles. Total polyamine concentrations during corresponding stages of development were consistently higher in achenes than in receptacles, and ranged from 891 to 203 nmol g⁻¹ fresh weight. Removal of achenes from the surface of developing receptacles 10 days after pollination reduced receptacle growth, and re-initiation of growth by application of 1 m M α-naphtaleneacetic acid (α-NAA) was accompanied by a rapid increase in polyamine concentrations 24 h after treatment. Polyamine content per receptacle increased >3-fold in normally developing receptacles and in de-achened, auxin-treated receptacles 10 days after removal of achenes, but did not increase during this period in de-achened receptacles not treated with exogenous auxin. α-NAA increased growth and polyamine levels to a greater extent than the structurally related, but less effective auxin, β-NAA. Polyamine concentrations in receptacles with intact achenes remained similar to those of auxin depleted (de-achened) receptacles, implying that the concentration of these compounds may not be limiting following achene removal.     

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.     

Polyamine transport,accumulation, and release in brain

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.     

Methylated analogs of spermine and spermidine as tools to investigate cellular functions of polyamines and enzymes of their metabolism

Biogenic amines spermine (Spm) and spermidine (Spd) are essential for cell growth. Polyamine analogs are widely used to investigate the enzymes of polyamine metabolism and the functions of spermine and spermidine in vitro and in vivo. It was demonstrated recently that α-methylated derivatives of Spm and Spd are able to fulfill the key cellular functions of polyamines, moreover, in some cases, the effects of (R) and (S) isomers were actually different. Using these α-methylated analogs of Spm and Spd, it turned possible to prevent the development of acute pancreatitis in SSAT-transgenic rats with controllable expression of the Spm/Spd N¹-acetyltransferase gene. The analogs made it possible to reveal dormant stereospecificity of polyamine oxidase, Spm oxidase, and deoxyhypusine synthase. An original approach was suggested to regulate the stereospecificity of polyamine oxidase. Depletion of the intracellular polyamine pool was found to have both hypusine-related consequences and consequences unrelated to posttranslational modification of the eukaryotic translation initiation factor eIF5A. Possible applications of a new family of C-methylated polyamine analogs for the investigation and regulation of polyamine metabolism in vitro and in vivo are discussed.     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

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

Polyamine Levels During Growth, Sporulation, and Spore Germination of Bacillus megaterium

Spermidine was the major (>95%) polyamine of Bacillus megaterium in all stages of growth, although it could be replaced completely by spermine. Log-phase cells had 40 to 50% as much spermidine, based on ribonucleic acid (RNA) content, as did either stationary-phase cells or dormant spores; similar results were obtained in three other bacilli including an asporogenous mutant. Polyamine levels were essentially the same in B. megaterium grown in rich or poor media, or in media of high or low ionic strength. Polyamine levels were elevated three- to sixfold by exogenous spermidine without a major effect on growth, sporulation, or subsequent spore germination. During germination, the absolute amount of spermidine remained constant for almost 2 h until net RNA synthesis had lowered the polyamine/RNA ratio to a value close to that in log-phase cells. At this time, the spermidine level began to rise, and thereafter spermidine and RNA increased in parallel. This parallel relationship between the spermidine and RNA levels was abolished by actinomycin D, but not by chloramphenicol.     

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.     

The role of free polyamines in the alternate-bearing of pistachio (<Emphasis Type="Italic">Pistacia vera</Emphasis> cv. Pontikis)

The present study was conducted in order to examine the physiological role of free polyamines in flower bud abscission. For this reason five 15-year old pistachio trees cv. "Pontikis" were selected and half of the main branches were manually defruited in early May. Polyamines were analyzed in three different organs (shoots, leaves and flower buds) from both fruiting and non-fruiting branches, during the period of kernel growing. Five samplings took place and the polyamines putrescine, spermidine and spermine were assayed. The flower bud abscission percentage was recorded every 5–10 days during kernel formation. Polyamine concentration declined during the period that coincides with that of kernel development, in both fruiting and non-fruiting branches, while significant bud abscission occurred from mid-July till late September in fruiting branches. Polyamine concentration in organs from fruiting branches was in most cases lower than that of non-fruiting ones. Most of the individual polyamines exhibited a high and significant negative correlation with bud abscission. By measuring the spermidine content of leaves and the spermine content of buds, it was possible to estimate the forthcoming bud abscission with significant accuracy (approximately 93%). On the other hand, the total polyamine content of the buds exhibited a significant strong negative relationship with bud abscission. Consequently, polyamines could have an important physiological function in the development of flower bud abscission of pistachio.     

Manipulation of the polyamine content and senescence of apical buds of G2 peas

The effect of various treatments on the apical senescence and polyamine content of apical buds of G2 peas was analysed. Defruiting prevented senescence and increased bud size and polyamine content. Exogenous applications of GA₂₀ enhanced bud size and spermidine concentration. Applied spermidine had a slight effect on spermidine level but did not delay senescence. ACC strongly induced adecrease in bud size and, at 10 mM, apical senescence. This was accompanied by a steady decline in the level of all polyamines though their concentration remained constant until 10 mM ACC, where a drop was noted. Spermidine in the presence of ACC modulated the effect of ACC on the bud size while returning the internal polyamine content to control levels. AVG, an inhibitor of ACC synthesis produced pronounced increases in putrescine though no apparent effect on apical bud growth. Polyamine synthesis inhibitors were without effect on growth or internal polyamine content. The internal polyamine content appeared to correlate with apical bud size and vigor but did not show any consistent relationship to apical bud senescence.     

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.     

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 insect development: A comparison between the housefly Musca domestica and the plant bug Dysdercus koenigii

Polyamine levels were measured during the entire life cycle of a dipteran insect, Musca domestica and a hemipteran insect, Dysdercus koenigii. Putrescine, spermidine and spermine were found to be present at all stages of development in both the insects. A comparison of the polyamine changes in the two insects showed that spermidine is an important polyamine in insect development. Spermidine levels were very high in developing embryos of M. domestica before hatching of the egg. These spermidine levels decreased rapidly from first instar. On the contrary, in D. koenigii, spermidine levels were low in early development, started increasing in first instar and high levels were maintained from second to fifth instar nymphal stages. High levels of spermidine is probably indicative of rapid cell division in the above two groups of insects.     

Polyamines and Protein Metabolism in Maize Inbreds Differing in Seed Protein Content

Polyamine metabolism was evaluated in the embryo and the endosperm,during the early stages of seed germination, of two maize inbreds(Lo5 and B73) differing in the protein nitrogen content of thecaryopscs. On germination, the concentration of buffer-extractableproteins and of polyamines increases more quickly and to greatervalues in Lo5 than in B73. In the caryopses, the embryos havea higher polyamine content than the endosperms and in the seedlings,after three days of growth, the shoots show a higher polyaminecontent than in the case of the scutellum and the roots. Duringseed germination, spermidine is the main polyamine and its contentvaries while the spermine remains virtually constant. The polyaminesand protein pattern in the embryo and the endosperm of the twoinbreds are discussed in relation to the differences in theirgermination energy and early seedling growth.     

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.     

Polyamine Metabolism in the Roots of Phaseolus vulgaris. Interaction of the Inhibitors of Polyamine Biosynthesis with Putrescine in Growth and Polyamine Biosynthesis

The effects of the inhibitors of polyamine biosynthesis, canavanineand -methyl ornithine on growth, the activities of argininedecarboxylase (EC 4.1.1.19 [EC] ) and ornithine decarboxylase (EC4.1.1.17 [EC] ) and on polyamine content were examined in two differentgrowth regions of Phaseolus vulgaris L. cv. Taylor's Horticulturalroots. Separately, in the same manner, in the same bean rootsystem exogenous putrescine effect and the interaction of canavaninewith putrescine were determined. The arginine and ornithine decarboxylase activities found inroot apex were high where cell division activity was highest.Polyamine (putrescine and spermine) content did not correlatewith these activities, but polyamine level was high in the rootbase where cell elongation is the main process. The arginineanalogue, canavanine, inhibited arginine decayboxylase activityand polymine liters. Putrescine partially reversed the canavanineinhibition of root growth as well as arginine decarboxylaseactivity and polyamine content. Similarly -methyl ornithineslightly inhibited the root length and ornithine decarboxylaseactivity in the root apex. Besides, exogenous putrescine didnot effect significantly the endogenous polyamine titers. Theseresults reinforce the growing connection between polyaminesand the rates of cell devision in the roots of bean plants.Separately, arginine decarboxylase is the main enzyme in thebean roots. (Received November 10, 1986; Accepted March 3, 1987)     

Endogenous levels of polyamines in the organs of cucumber plant (Cucumis sativus) and factors affecting leaf polyamine contents

Polyamine compositions of various organs from hydroponically cultivated cucumber plants ( Cucumis sativus L. cv. Sharp-1) and factors affecting the leaf polyamine content were examined. Diamine putrescine was found most abundantly in the root, while a relatively large amount of spermine was detected in the reproductive organs such as the immature fruit and the calyx (+stamen). Spermidine was present at the highest level in rapidly growing tissues such as newly expanded leaf and fruit at an early developing stage, implying the possible involvement of spermidine in the growth and development of these young tissues. Polyamine content of cucumber leaves changed during the day. Especially, the putrescine content of upper leaves showed a striking decrease from the morning to the night. Alterations of leaf Ca or Mg content did not significantly affect leaf polyamine composition. On the other hand, abnormal cucumber leaves showed altered polyamine composition. Yellowing of the leaf intervein resulted in a striking decrease in spermidine content without a significant change in putrescine and spermine content. By contrast, the leaves infected with the phytopathogen, powdery mildew, showed decreased putrescine and increased spermine content in response to the degree of fungi infection. The possible usefulness of polyamines as a diagnostic marker of plant development and physiological disorder is discussed.