Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture

Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.Abbreviations ADC arginine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DCHAS dicyclohexylammonium sulfate - SAMDC S-adenosylmethionine decarboxylase     

Influence of copper ions on growth,lipid peroxidation,and proline and polyamines content in carrot rosettes obtained from anther culture

Effect of Cu (0.1, 1, 10, and 100 μM) on the regeneration of carrot (Daucus carota L.) androgenic embryos of var. Feria and 1014 breeding line as well as on polyamines (PAs), proline contents, lipid peroxidation and Cu accumulation after 16 and 24 weeks was studied. Generally, growth of Feria rosettes was better than that of the 1014 line. Significant increase in Cu content in tissues was observed in both cultures grown at the highest Cu concentration (100 μM). The dose-dependent increase in proline in the 16-week-old culture of Feria was observed, while in 1014 its level increased only at the highest applied Cu concentration. On the contrary, in the 24-week-old culture, significant increase in the proline content were observed at 100 and 10 μM Cu in Feria and in 1014 breeding lines, respectively. The decline in proline content and decrease in embryogenic ability in the line 1014 grown in the presence of the highest Cu concentration for 24 weeks may indicate that a certain threshold of intracellular Cu was crossed. Both in Feria and 1014 line, putrescine and spermidine were the most abundant free PAs. The increased content of proline and higher contents of the constitutive free putrescine and spermidine in Feria cultivated for 24 weeks at the highest Cu concentration point to better protection of this cultivar. Thus, it seems that the higher tolerance of Feria to oxidative stress (characterized by lower thiobarbituric acid reactive substances value) may result from higher constitutive level of PAs. These data confirm the suggestion that variations in PA levels depend not only on the concentrations of metals tested, but also on plant species and cultivars. The role of PAs and proline in the carrot cultures treated with Cu is discussed.     

Polyamines in relation to growth in carrot cell cultures

Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed.     

Endogenous polyamine content and metabolism in the ectomycorrhizal fungus Paxillus involutus

Endogenous polyamine content of the ectomycorrhizal fungus Paxillus involutus , as well as the activity of its biosynthetic enzymes in relation to mycelia ageing were investigated in this work. Polyamines in free, PCA-soluble and insoluble conjugated forms, are present in Paxillus involutus mycelia in relatively high amounts and the ratio of putrescine to spermidine is age-dependent. Both arginine- and ornithine-decarboxylases are present, but putrescine biosynthesis proceeds mostly via ornithine decarboxylase and decreases with the age of mycelia. There was a large release of free polyamines from mycelia which showed age-dependent features. Clear polyamine uptake was observed in 2-wk-old mycelia and no competition between putrescine and cadaverine was detected. Putrescine uptake seems to reduce ornithine decarboxylase activity, but does not affect arginine decarboxylase.     

Polyamine levels as related to growth,differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa

We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events.In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors -difluoromethyl-arginine (specific inhibitor of arginine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.     

Light quality influences the polyamine content of lettuce (Lactuca sativa L.) cotyledon explants during shoot production <Emphasis Type="Italic">in vitro</Emphasis>

Light quality has previously been shown to influence morphogenesis in lettuce cotyledon explants, with white or red light promoting adventitious shoot production, and blue light inhibiting it. Endogenous polyamine (PA) concentrations were compared between explants cultured under different light qualities. Explants cultured under white or red light accumulated PAs during shoot primordia production, with a 5.6-fold increase compared to initial concentrations under white light, and 6.7-fold increase under red light. These results suggest polyamines are involved in the formation of shoot primordia. After 18 days in culture PA concentrations decreased under white light, and to a lesser extent under red light, signaling a shift in polyamine metabolism that correlates with shoot expansion, which occurs more readily under white light. Explants cultured under blue light accumulated polyamines for the first 7 days, to a level 1.3 times greater than initial values, followed by a gradual decline during the remainder of the culture period. Explants cultured under blue light also contained a greater proportion of PCA-insoluble conjugated PAs, compared to explants under white or red light, which contained greater proportions of free or PCA-soluble conjugated polyamines. The ratio of putrescine to spermidine was also different with a lower Put:Spd ratio being associated with shoot production under white or red light, and higher Put:Spd ratio being associated with culture under blue light.     

The effects of spermidine synthesis inhibitors on in-vitro plant development

The spermidine synthesis inhibitors methylglyoxal bis-(guanylhydrazone) (MGBG) and dicyclohexylammonium sulfate (DCHA) were found to reduce growth and embryogenesis in wild carrot cultures. Cellular polyamine levels were also affected by the inhibitors, with spermidine levels being especially reduced by DCHA. Similarly, MGBG reduced organogenetic development of shoots on excised aspen hypocotyls. These data suggest that the polyamines, especially spermidine, play an important role in the growth and development of plants.     

Study on the role of endogenous polyamines in glucagon, isoproterenol or serum-mediated induction of tyrosine aminotransferase in cultured heart cells

In confluent and serum-starved embryonic heart cell cultures, the addition of serum (10%), glucagon (GLU, 0.1 microM) or isoproterenol (ISO, 10 microM), causes the onset of ornithine decarboxylase (ODC) activity, with a maximum after 5-6 hr. This is paralleled by polyamine accumulation and by the induction of TAT, which, in the case of GLU and ISO, exhibits maximal activity at 4-3 hr respectively, followed by a net decline. Cyclic AMP (cAMP) also accumulates after exposure to GLU or ISO. However, under different conditions of ODC inhibition, serum fails to induce TAT, thus supporting a relevant role of cellular polyamines in serum action. Conversely, cAMP and TAT responses to GLU or ISO are markedly improved under prevention of polyamine accumulation, which also leads to a longer lasting TAT inducibility. The suggestion is made that polyamines are not required in the cAMP-dependent mechanism of TAT induction, but rather in the restoration of the basal activity of the enzyme.     

Floral morphogenesis in Rudbeckia hirta in relation to polyamine concentration

Changes in polyamine concentration in the long day (LD) plant Rudbeckia hirta were examined over the course of floral initiation and development. Plants of R. hirta were grown to maturity under 9h, non-floral-inductive photoperiods. At maturity, half the plants were placed in ambient day length plus a 4- h night interruption. Plants were sampled at 0, 4, 8, 12 or 16 days for polyamine content and floral initiation. Polyamines were extracted from fully expanded leaves and the meristems were examined histologically. In another experiment, polyamines were extracted from the meristems under paired LD and short day (SD) conditions every 2 days from 0 to 22 days. A rise in free polyamines was linked to important cytological events during floral initiation. Free putrescine and spermidine levels increased after 4 LD and continued until 14 to 16 LD when the levels in the meristem began to decline. Events of floral initiation began between 4 and 8 LD with cell proliferation and the start of stem elongation. Initiation was irreversible after 14 to 16 LD, the period when putrescine and spermidine began to decline. After 4 LD, the polyamine level was consistently higher in the photoinduced plants. Our results from this study, using direct histological comparisons of meristematic development and polyamine concentration, clearly demonstrate a correlation of polyamines and flowering.     

Metabolism of l[U-14C]-arginine and l[U-14C]-ornithine in maturing and vernalised embryos and megagametophytes of Picea abies

The metabolism of the polyamine precursors arginine and ornithine was studied in maturing and vernalised seeds of Picea abies (L.) Karst. (Norway spruce) in feeding experiments. Incorporation of radioactivity from these 14 C-labelled amino acids into liberated CO₂, amino acids, polyamines, proteins and cell wall fractions, as well as polyamine levels were determined in embryos and megagametophytes. Ornithine and especially arginine decarboxylation was more active in the embryo than in the megagametophytic cells, and vernalisation increased arginine metabolism more than it increased ornithine metabolism. Both precursors were metabolised to each other, to other amino acids, and to polyamines. The only polyamine in which radioactivity incorporated was free putrescine, showing either a slow synthesis or a high degradation rate of spermidine and spermine in maturing spruce seeds. The putrescine level was approximately 10 times higher in the embryo than in the megagametophytic tissues, whereas spermidine and spermine levels were almost the same in both tissues. The label from arginine and ornithine was also incorporated into proteins as amino acids and post-translationally as polyamines. Higher radioactivity was seen in the small ≤14-kDa polypeptides. Protein hydrolysates of the embryo and the megagametophytic tissues contained spermidine and spermine and their degradation product 1,3-diaminopropane (DAP), suggesting that polyamines may play a role in the accumulation of seed storage protein and in the maturation of spruce seeds.     

Free and protein-conjugated polyamines in mouse epidermal cells. Effect of high calcium and retinoic acid

We have investigated polyamine metabolism in primary cultures of mouse epidermal cells. These cells, which grow at low Ca2+ levels as a monolayer with characteristics of basal cells, terminally differentiate when the extracellular Ca2+ level is raised above 1 mM. The cellular levels of free polyamines were measured, and, after incubation of cell cultures with [3H]putrescine, the distribution of label in both acid-soluble and acid-insoluble cellular components was examined. Free polyamine levels were reduced in cells induced to differentiate. Treatment with retinoic acid, which prevents differentiation and causes increased proliferation, resulted in an increase in free putrescine. Upon adjustment of the calcium concentration to a level that induces differentiation, the enzyme transglutaminase was activated, and a concomitant increase in the level of both protein-bound mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine was observed. Isolation of a material of apparent molecular weight about 6000 which contains only mono-gamma-glutamylpolyamines and the finding of both mono- and bis-gamma-glutamylpolyamines in the protein fraction containing cornified cell envelopes provided the basis for speculation on polyamines in envelope formation. Our data suggest that polyamines play a role during epidermal cell differentiation through transglutaminase-mediated post-translational modification.     

Regulation of ornithine aminotransferase gene expression and activity by all-transretinoic acid in Caco-2 intestinal epithelial cells

Ornithine aminotransferase (OAT) is a crucial enzyme in the synthesis of citrulline and arginine from glutamine/glutamate and proline by enterocytes of the small intestine. However, a role for OAT in intestinal polyamine synthesis and cell growth is not known. All-transretinoic acid (RA), an active metabolite of vitamin A, regulates the activity of several metabolic enzymes related to OAT, including ornithine decarboxylase and arginase, which may influence the function of OAT through effects on substrate (ornithine) availability. The objective of the present study was to test the hypothesis that RA regulates OAT mRNA expression and enzymatic activity in intestinal epithelial cells. Caco-2 cells were cultured for 12-72 h in the presence of 0, 0.01 and 1 microM RA and then used for measurements of OAT mRNA levels and enzyme activity as well as ornithine and polyamines. Treatment with RA induced increases in OAT gene expression and enzymatic activity, which resulted in decreased intracellular concentrations of ornithine and polyamines (putrescine, spermidine and spermine) in a dose-dependent manner. These changes occurred concomitantly with a decrease in the total number of cells, and the increase in OAT activity was due to increased OAT mRNA expression. In cells treated with 1 microM RA, addition of 10 microM putrescine to culture medium restored both cellular levels of polyamines and cell numbers to the values for the control group (without addition of RA). We conclude that exposure of Caco-2 cells to RA induces OAT expression for increasing ornithine catabolism. This leads to a reduced availability of intracellular ornithine for polyamine synthesis, thereby decreasing cell proliferation. These novel findings indicate a functional role for OAT in regulating intestinal polyamine synthesis and growth.     

Polyamine analogues bind human serum albumin

Polyamine analogues show antitumor activity in experimental models, and their ability to alter activity of cytotoxic chemotherapeutic agents in breast cancer is well documented. Association of polyamines with nucleic acids and protein is included in their mechanism of action. The aim of this study was to examine the interaction of human serum albumin (HSA) with several polyamine analogues, such as 1,11-diamino-4,8-diazaundecane (333), 3,7,11,15-tetrazaheptadecane.4HCl (BE-333), and 3,7,11,15,19-pentazahenicosane.5HCl (BE-3333), in aqueous solution at physiological conditions using a constant protein concentration and various polyamine contents (microM to mM). FTIR, UV-visible, and CD spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind nonspecifically (H-bonding) via polypeptide polar groups with binding constants of K333 = 9.30 x 10(3) M(-1), KBE-333 = 5.63 x 10(2) M(-1), and KBE-3333 = 3.66 x 10(2) M(-1). The protein secondary structure showed major alterations with a reduction of alpha-helix from 55% (free protein) to 43-50% and an increase of beta-sheet from 17% (free protein) to 29-36% in the 333, BE-333, and BE-3333 complexes, indicating partial protein unfolding upon polyamine interaction. HSA structure was less perturbed by polyamine analogues compared to those of the biogenic polyamines.     

Uptake and excretion of polyamines from baby hamster kidney cells (BHK-21/C13) the effect of serum on confluent cell cultures

In confluent cultures of BHK-21/C13 cells there was little uptake oxogenous polyamines and only a low level of polyamine biosynthesis. These cultures continously excreted polyamines into the extracellular medium. Spermidine, in both the free and bound form, was the predominant excretion product, whereas the major intracellular polyamine was spermine implying that excretion of polyamines was specific. Reinitiation of growth by the addition of fresh serum immediately increased the uptake of exogenous putrescine, increased the biosynthesis of polyamines and decreased the excretion of polyamines. Thus, polyamine transport into and out of the cell appears to be regulated by the growth status of that cell.     

Polyamine metabolism during the growth cycle of tobacco BY-2 cells.

We studied polyamine (PA) biosynthesis, oxidation and conjugation in asynchronously dividing cells of tobacco BY-2 cell suspension culture (Nicotiana tabacum L.) during 7-day growth cycle. We analyzed the levels of free and conjugated PAs and the activities of biosynthetic and catabolic enzymes during the subculture interval. The contents of free spermidine and spermine started to increase after the inoculation into the fresh medium, positively correlated with the mitotic activity of BY-2 cells and reached their maxima at the beginning of exponential phase on day 3. On the contrary, the endogenous level of free Put showed a transient decline in the lag-phase, and then increased till the end of exponential phase (day 5). The time-course of the content of PCA-soluble conjugates showed a trend similar to that of the free PAs. The inoculation of BY-2 cells into the fresh medium resulted in a sharp increase in the activities of ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50). Arginine decarboxylase (ADC; EC 4.1.1.19) activity remained low during the whole subculture interval. The rise of diamine oxidase (DAO; EC 1.4.3.6) in the first day after subculture coincided with the decrease in free Put level. De novo synthesis of PAs in BY-2 cells after inoculation into the fresh medium and the participation of both PA conjugation with hydroxycinnamic acids and Put oxidative degradation in maintaining of free PA levels during the growth cycle are discussed.     

Polyamines and somatic embryogenesis in two Vitis vinifera cultivars

Polyamine content and activities of enzymes of polyamine biosynthesis were assayed during somatic embryogenesis in Vitis vinifera callus cultures of Chardonnay and Brachetto 'a grappolo lungo' (Brachetto g.l.) cultivars. The analyses were carried out on embryogenic callus samples, embryos at different stages and developing plants. Polyamine content, both in the free and PCA-soluble conjugated form, was higher in Brachetto g.l. than in Chardonnay, and putrescine was present at higher concentrations than the other polyamines. In all samples of both cultivars, ornithine decarboxylase activity (ODC, EC 4.1.1.17) was higher than arginine decarboxylase (ADC, EC 4.1.1.19), with a maximum in developing plant roots. S -Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity displayed a similar trend. The activities of all three enzymes were detected both in the supernatant and pellet fractions, indicating for the first time the presence of SAMDC activity in the particulate fraction. Particularly in the Chardonnay cultivar, an increase in the mRNAs expression patterns of ODC and SAMDC during morphogenesis from small embryos to plantlets was detected by northern blot, suggesting a direct correlation with enzymatic activities.     

Peroxynitrite inhibits the activity of ornithine decarboxylase

Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO-), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO- resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 microM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO-, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis.