Free Spermidine Research Articles

Spatial and Temporal Changes in Endogenous Polyamine Levels Associated with Somatic Embryogenesis from Different Hypocotyl Segments of Eggplant ( Solanum melongena L.)

Summary In Solanum Melongena L. Cv. Pusa Purple Long, Significant Differences Were Observed For Embryogenic Potential Within A Single Explant (Hypocotyl System). Terminal Hypocotyl Segments (Apical and Basal) Yielded A Higher Number Of Somatic Embryos Than The Medial (Sub-Apical and Sub-Basal) Segments. High Levels Of Conjugated Spermidine Along With High Levels Of Total Polyamines (Primarily Free Fraction) Could Be Correlated With The Formation Of Somatic Embryos In Terminal Segments. Temporal Changes In Endogenous Levels Of Free, Conjugated and Bound Putrescine, Spermidine and Spermine Were Analyzed At Critical Stages Of Somatic Embryogenesis From Four Different Hypocotyl Segments. Within 3 Days Of Culture, There Was A Sharp Decline In Free (and Total) Putrescine and An Increase In Conjugated Spermidine Levels. All Hypocotyl Segments Attained Similar Levels Of Free Spermidine, Irrespective Of Their Subsequent Embryogenic Response. As The Tissues Become Committed For Embryogenesis, Free, Conjugated and Total Putrescine and Spermidine Reach Uniformly Minimal Levels For All Segments. Just Before The Onset Of Embryogenesis, There Was A Dramatic Increase In All Fractions Of Putrescine, and In Free and Conjugated (Along With Total) Spermidine Levels. Intermediate Levels Of Conjugated and Total Putrescine and Spermidine Were Reached In Tissues With Good Embryogenic Potential: tissues with poor embryogenic potential attained lower or higher levels. After the embryos were formed, their levels fell sharply, and continued to decline. Free putrescine, however, reached another peak when embryos had fully matured. The changes in bound putrescine and spermidine, and in spermine (all forms) could not be correlated with somatic embryogenesis.

Polyamines in Solanum tuberosum in vitro: Free and Conjugated Polyamines in Hormone-Induced Tuberization

Summary The putative role of polyamines (PAs) in in vitro tuberization in single leaved cuttings of Solanum tuberosum was investigated by comparing the pattern of distribution of acid-soluble free and bound PAs (putrescine (PUT), spermidine (SPD), spermine (SPM)) in shoots grown on non-tuber-inducing, hormone- free and tuber-inducing hormone-augmented medium. Exogenous free PAs (PUT, SPD, SPM each at 10 -4 M) and/or phenolic acids ( t -cinnamic, caffeic, chlorogenic, p -coumaric and ferulic acid, each at 10 -5 M) had no tuber-inducing effect when supplied in hormone-free medium, and growth, morphology and endogenous distribution of PAs were largely unaffected. Under non-tuberizing conditions, PUT was the main free PA followed more or less closely by free SPD and, by a wider margin, free SPM. The profile of conjugated PAs was dominated by conjugated SPD. Conjugated PUT was the least abundant. Distinct modifications of the profile of both free and conjugated endogenous PAs were found to be associated with the process of tuberization. On tuber induction by cytokinin (benzylaminopurine 2 mg/L) and auxin (indol-3-ylacetic acid 1 mg/L) free SPD became the main PA relegating free PUT to second place. The overall content of conjugated PAs increased. The distribution of the individual conjugated PAs shifted: conjugated SPD decreased and conjugated SPM increased with tuberization. In the system used, tuberizing conditions can be characterized by ratios between the individual PAs: free PUT/SPD below 1, conjugated PUT/SPD above 1 and conjugated SPD/SPM at least below 0.5.

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.

Differential recognition of free and covalently bound polyamines by the monoclonal anti-spermine antibody SPM8-2

The reactivity of an anti-spermine MAb (SPM8-2) toward polyamines either free or bound to a solid surface was investigated using equilibrium dialysis and ELISA methods. When polyamines were covalently linked to hydrophilized microtiter plates using carbodiimide, the MAb SPM8-2 reacted both with spermine and spermidine, with a higher affinity for the latter, but did not show any reactivity towards bound putrescine. In contrast, the MAb SPM8-2 reacted with all three polyamines bound to the microtiter plates with glutaraldehyde, with an affinity in the order: putrescine > spermidine > spermine. Equilibrium dialysis and competitive ELISA tests showed that the MAb SPM8-2 exhibited high affinity for free spermine and 50% and 5% cross-reactivity with free spermidine and putrescine respectively. The affinity of the MAb SPM8-2 for putrescine, spermidine and spermine appears to depend on whether the polyamine is free or bound. The antigenicity of the polyamines differs according to the nature of their link to the solid phase. These observations are discussed in the light of the structural modification produced by covalent binding of the polyamines. It is also concluded that when antibodies are used, due care has to be exercised in choosing the appropriate immunoassay for determining the specificity of antibodies directed against small haptens such as the polyamines.

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.

Changes in polyamine synthesis during anther development and pollen germination in tobacco (Nicotiana tabacum)

Changes in polyamine levels and in the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and omithine decarboxylase (ODC. EC 4.1.1.17) were studied during anther development in Nicotiana tabacum as well as during the first 120 min of dry pollen germination in vitro. Arginine decarboxylase activity was more abundant than that of ODC in the whole anther and isolated pollen: activities of both enzymes reached a maximum at the binucleate stage of pollen development and increased with elongation of the pollen tube. During anther development, no alteration in free putrescine (Put) or in free spermidine (Spd) was found from the microspore to the binucleate stage. However, during maturation, free Put decreased, probably because of its transformation into Spd. Bound polyammes were more abundant than the free polyamines during anther development. The presence of a‐amanitin. on inhibitor of transcription, did not affect ADC activity during the germination process, whereas cycloheximide. on inhibitor of protein synthesis, had a strong inhibitory effect. Compared to their levels in dry pollen, free Put. Spd and spermine (Spm), and bound Put were present at increased levels during pollen tube elongation. However, in the presence of cycloheximide. which had little effect on elongation of the pollen tube, free polyamine synthesis was inhibited. In light of the results obtained with cycloheximide and difluoromethvlargi‐nine. An inhibitor of arginine decarboxylase. which inhibited elongation but not emergence of the pollen tube, we propose that the endogenous polyamine liter in dry pollen was sufficient to contribute to the emergence of the pollen tube and. once emergence was established, that pre‐existent ADC and ODC were responsible for the synthesis of those polyaraines related to tube elongation, since the polyamine levels of the dry pollen appeared to be insufficient.

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.

Polyamine content and translocation in soybean plants

Summary Polyamine content was investigated in all parts of soybean plants (Glycine max (L.) Merr. cv. Williams) at the fifth or sixth leaf stage. Trichloroacetic acid-soluble free spermidine was the most abundant polyamine found. Apical buds and internodes had a free polyamine content greater than the other parts of the plant. Bound polyamines were also detected, even though their content was quite low. Both free and bound cadaverine and DAO activity, measured in the different organs, were found only in hypocotyls and roots. [1,4-14C]-spermidine was supplied to a leaf in order to study the uptake and translocation through the plant; in the same organ the metabolic fate of the labelled spermidine was followed and radioactive putrescine and traces of spermine were also found. Uptake experiments, carried out utilising detached leaves, showed a time-dependent process. In translocation experiments, performed on the whole plant, a gradient of radioactivity distribution along the stem was evident, while the amount of radioactivity present in the leaves was lower than that found in internodes. Label translocation was not polar and its rate in the two internodes under the treated leaf was higher than in the internodes above it.

Role of basal shoot darkening and exogenous putrescine treatments on in vitro rooting and on endogenous polyamine changes in difficult-to-root woody species

The effect of in vitro putrescine treatment on several difficult-to-root fuuit tree species was studied in order to improve rooting efficiency. Putrescine was added to a double liquid layer at the shoot proliferation phase and during the rooting phase. Shoots were rooted in either a 16-h photoperiod or a 16-h photoperiod with shoot bases in the dark (basal darkening). Putrescine, at 1 mM, in a double liquid layer, increased the number of pear shoots with expanded leaf lamina but did not increase the rooting efficiency of these shoots. Putrescine increased the rooting of apple (M9 rootstock, clone P3) and the basal explants of olive, rooted in 16-h photoperiod; decreased it in walnut but did not affect chestnut, almond, jojoba and apricot rooting. Basal shoot darkening was essential for root formation in chestnut, walnut and almond. The endogenous content of perchloric acid (PCA)-soluble free polyamines (PA) was determined at the end of the proliferation phase in olive, chestnut and walnut. The shoots proliferated under the 16-h photoperiod contained a lesser concentration of free spermidine and spermine compared with those kept in the dark for 10 days before being excised for rooting. High levels of spermine were detected in walnut shoots. Total free polyamine content was relatively low in olive, at an intermediate level in chestnut and high in walnut. The content seems to be inversely correlated with the response to exogenous putrescine treatments. In addition, PCA-soluble free and PCA-soluble bound polyamines in olive shoots, both basal and apical, either in a 16-h photoperiod or in basal darkening, showed an increase at Day 2 of rooting and then decreased in the following days. In 16-h photoperiods basal explants of olive, which under these conditions require applied exogenous putrescien for a strong rooting response, accumulated all three Pa as bound forms at Day 2. However, those which rooted with basal shoot darkening did not respond to exogenous putrescine or accumulate PA.

Estimation of Polyamine Distribution and Polyamine Stimulation of Protein Synthesis in Escherichia coli

To estimate the polyamine distribution in Escherichia coli, the binding constants (K) for DNA, RNA, phospholipids, and ATP were calculated under the condition of 10 mM Tris-HCl, pH 7.5, 150 mM K+, and 10 mM Mg2+. The binding constants of spermidine for E. coli DNA, E. coli 16S rRNA, phospholipids in E. coli membrane, and ATP were 0.015, 0.066, 0.028, and 0.081 mM−1, respectively. Similarly, those of putrescine were 0.0 10, 0.0 10, 0.007, and 0.037 mM−1, respectively. The concentrations of putrescine, spermidine, and ATP and phosphates in DNA, RNA, and phospholipids in E. coli harvested at A600 = 0.3 were 32.2, 6.88, and 2.66 and 96.4, 436, and 57.2 mM, respectively. Accordingly, the percentage of spermidine bound to DNA, RNA, phospholipids, and ATP and that of free spermidine were 5.1, 90, 0.7, 0.8, and 3.8%, respectively. The percentage of putrescine bound to DNA, RNA, phospholipids, and ATP and that of free putrescine were 9.3, 48, 1.4, 2.6, and 39%, respectively. The results indicate that most spermidine exists as a spermidine-RNA complex, and about 40% and 50% of putrescine exists as a free form and a putrescine-RNA complex in cells, respectively. Under the conditions that the synthesis of specific proteins such as RNA replicase is stimulated by polyamines in a cell-free system, the amount of spermidine and putrescine bound to RNA was close to the value estimated in cells. Experiments to demonstrate the polyamine stimulation of MS2 RNA-directed RNA replicase synthesis in vivo were thus performed, and the results were confirmed.

Inhibition of ethylene production in sunflower cell suspensions by the plant growth retardant BAS 111..W: Possible relations to changes in polyamine and cytokinin contents

At a concentration of 10−5 mol · L−1 the triazole-type growth retardant BAS 111..W completely inhibited the transiently elevated ethylene production in the exponential growth phase of heterotrophic sunflower cell suspensions. This effect, which could not be restored by adding gibberellin A3, was accompanied by transiently increased levels of 1-aminocyclopropanecarboxylic acid (ACC) in the cells, which was increasingly converted to N-malonyl-ACC. Thus, the reactions from ACC to ethylene catalyzed by the ethylene-forming enzyme appeared to be blocked by the retardant. Concomitantly, higher endogenous levels of free spermidine and particularly spermine were found over control, whereas free putrescine, the direct precursor of both polyamines, simultaneously decreased. We assume that the remaining S-adenosylmethionine from ethylene biosynthesis was increasingly incorporated into spermidine and finally spermine. A further relation appears to exist between the reduced ethylene production and enhanced levels of cytokinins in the cells. The application of both BAS 111..W and aminoethoxyvinylglycine depressed ethylene formation while immunoreactive cytokinins from isopentenyladenosine-, trans-zeatin ribo-side-, and dihydrozeatin riboside-type increased. By additional treatment with ACC, the effects could partially be reversed. On the other hand, stimulation of ethylene production by ACC alone or ethephon considerably lowered cytokinin levels.

Effect of phosphate ion on the activity of bovine plasma amine oxidase

The system bovine plasma amine oxidase-polyamine-phosphate ion was investigated by activity measurements and 31P NMR spectroscopy. Lineweaver-Burk plots showed that phosphate ion, under physiological conditions, is an apparent competitive inhibitor of bovine plasma amine oxidase. While NMR measurements of the T1 of 31P do not suggest the binding of phosphate to/or near the paramagnetic Cu(II) sites of bovine plasma amine oxidase, the chemical shift dependence of 31P on spermidine concentration indicates the formation of a spermidine-phosphate complex. The value of the dissociation constant of this complex was found 18.5 ± 1.4 mM, at pH 7.2, by NMR, in good agreement with the value 17.0 ± 0.8 mM calculated from activity measurements, assuming the enzyme activity is proportional to the free amine concentration, under second order conditions. Our data suggest that the decrease of the free spermidine, due to the binding of phosphate ion, is responsible of the observed inhibition of bovine plasma amine oxidase.

Time Course of Polyamine Levels during Starvation of Medicago Varia Cells and its Correlation with Cell Cycle Data

Summary Enhanced levels of polyamines have been shown to be associated with the proliferative status of cells (see Bagni and Biondi, 1987). We investigated free, conjugated and bound polyamines during the growth cycle of cell suspension cultures of Medicago varia . In addition to the commonly determined free polyamines, considerable amounts of conjugated polyamines as well as small amounts of bound putrescine and spermidine were detected. In order to investigate the correlation of polyamine levels with cell division activities we employed various starvation treatments, resulting in a cessation of cell division activities. Flow cytometric investigation of the cell cycle revealed that different starvation treatments led to an arretation of cells at specific phases of the cell cycle. In all cases free putrescine and spermidine levels declined markedly following the cessation of cell divisions.

Distribution and Seasonal Variation of Polyamines in Shootaxes of Spruce ( Picea abies (L.) KARST.)

The distribution of free putrescine and free spermidine within 2-year-old and 6- to 7-year-old shoot-axes of Picea abies (L.) Karst. was determined on several dates during the growing season. Putrescine was the major amine in all tissue fractions investigated. The highest putrescine contents occurred in the meristematic cell layer of the cambial region during pronounced cambial activity. At the onset of latewood production, cambial putrescine levels declined sharply and thereafter remained more or less constant until fall. In the cortex, putrescine concentrations were in general significantly lower than in the cambial zone and exhibited less marked seasonal variation. The polyamine amounts measured in the wood were very small and nearly constant throughout the vegetation period. The possible significance of the predominant occurrence of polyamines in the cambial region of spruce stems is discussed with regard to cambial growth and developmental processes.

Enhancement by Ouabain and Calcium Ionophore A23187 of Outward Transport of Polyamines from Lymphocytes

Human lymphocytes in culture loaded with radioactive polyamines slowly release radioactivity into the medium. N1-Acetylspermidine is mostly released from spermidine and spermine. Both ouabain and calcium ionophore A23187 increase the outward transport, but by different mechanisms. Ouabain inhibits the acetylation of spermidine, and free spermidine is released, whereas A23187 increases both acetylation of spermidine and the efflux of N1-acetylspermidine.

The polyamines of Xanthium strumarium and their response to photoperiod.

Flowering plants of Xanthium strumarium L., grown in 8 h photoperiods, were analysed for polyamines. Putrescine, spermidine and spermine were found throughout the plant in three forms: (a) as free polyamines; (b) conjugates soluble in 5% trichloracetic acid (TCA); and (c) bound to the TCA-insoluble precipitate. On a fresh weight basis, total polyamines are most abundant in young leaves and buds, especially flower buds. Spermidine predominates in the free polyamine fractions, while spermine is dominant in the conjugated fraction. Transfer of vegetative plants from 16 h photoperiods to 1, 2, 3, or 4 inductive cycles (8 h light + 16 h uninterrupted dark) caused rapid and marked changes in the polyamine titer of the leaves and ultimately, floral initiation. The titer of free putrescine per mg protein declined progressively with induction in all leaf sizes, while the titers of free spermidine and spermine rose during days 2 and 3 in small and expanding leaves. Conjugated putrescine, spermidine and spermine rose sharply after only 1 inductive cycle, especially in small and expanding leaves, and maintained the higher level for at least several cycles. In plants given 4 inductive cycles, buds harvested after 4 additional days had sharply elevated levels of conjugated polyamines, especially spermine, on a protein basis.

Effects of chlorsulfuron on polyamine content in maize seedlings

The effects of chlorsulfuron, a herbicide known to inhibit plant growth by inhibiting cell cycle progression in both G 1 and G 2 stages, were examined on free polyamine content and on growth in Zea mays seedlings. Chlorsulfuron decreased the spermidine content in active growing tissue, such as root tips, completely unaffecting the spermidine content in the basal part of the root where mitotic activity is lacking. In addition to the effects of specific spermidine inhibitors, methylglyoxal-bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase activity, cyclohexylamine, a competitive inhibitor of spermidine synthase activity, and chlorsulfuron were examined on both free and bound polyamines. Chlorsulfuron proved to be able to decrease the free and bound spermidine content more than methylglyoxal-bis(guanylhydrazone) and cyclohexylamine but probably through a different mechanism. In fact the inhibition of free spermidine content induced by methylglyoxal-bis(guanylhydrazone) and cyclohexylamine was reversed by the addition of exogenous spermidine, while no reversal effect was obtained for the inhibition of spermidine content induced by chlorsulfuron. It has been hypothesized that the depletion of spermidine content, induced by chlorsulfuron, could be responsible for the inhibition of cell cycle progression in G 1 and G 2 stages leading to growth inhibition.

The effects of nor-spermidine and dicyclohexylamine on in vitro potato development and free polyamines content

The in vitro micropropagation of potato (Solanum tuberosum L., var. Spunta) on media containing nor-spermidine (nor-SPD), a natural polaymine (PA) or dicyclohexlamine (DCHA), a spermidine synthesis inhibitor was studied to test their effects on plantlet growth and on the level of free cellular polyamines. The triamine nor-SPD, inhibited spermidine synthesis and substantially reduced root growth. DCHA strongly inhibited potato growth but surprisingly the free spermidine level seemed unaffected. These data suggest that dicyclohexylamine acts on the growth and on the development of plants by mechanisms unrelated to polyamine metabolism. Conversely, nor-spermidine was effective in reducing cellular spermidine content and seems to be a spermidine biosynthesis inhibitor in plants.

Levels of polyamines, glutathione and glutathione-spermidine conjugates during growth of the insect trypanosomatid Crithidia fasciculata.

Levels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12.1 to 17.4 nmol per 10(8) cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 10(8) cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 10(8) cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.

Elevated levels of free putrescine and N1-acetylspermidine in cyst fluids of malignant brain tumours.

The contents of free and conjugated polyamines in cyst fluids of brain tumours were analysed. The putrescine and N1-acetylspermidine levels in cyst fluids of malignant brain tumours were significantly higher than those of benign tumours. The free spermidine levels in malignant and benign tumours were about the same, while the total spermidine levels were higher in malignant than those in benign tumours. These results suggest that polyamine levels in cyst fluids reflect the altered polyamine metabolism in brain tumours.