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N Bagni

Publications and source records attributed to N Bagni.

At least 19 recordsLinked to original sources
PubMed2006

Polyamine metabolism and biosynthetic gene expression in Arabidopsis thaliana under salt stress.

In the present study we analysed polyamine metabolism in Arabidopsis thaliana (ecotype Columbia) rosette leaves collected at vegetative and reproductive stages from plants germinated and grown under increasing salt stress (0-75 mM NaCl) conditions. The expression level of the different isoforms of polyamine biosynthetic enzymes was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and the polyamine biosynthetic enzyme activities were determined both in supernatant and pellet fractions. Free and perchloric acid (PCA)-conjugated (soluble and insoluble) polyamines, were measured. At vegetative stage, plants were able to adapt up to 50 mM NaCl, showing a significant growth inhibition only at 75 mM NaCl. At this growth stage and NaCl concentration there was an up-regulation of spermine biosynthesis. At reproductive stage, plants were able to flower up to 50 mM NaCl, even if with a delay of 7 days. On the contrary, at 75 mM NaCl two different phenotypes were isolated: 75/01 (salt sensitive) and 75/02 (salt tolerant). The sensitive plants (75/01) showed a severely stressed phenotype, compared to the tolerant ones (75/02), and the polyamine metabolism was up-regulated, with the increase of free putrescine and spermine.

Arabidopsis↗
PubMed2001

Characterization of monocot and dicot plant S-adenosyl-l-methionine decarboxylase gene families including identification in the mRNA of a highly conserved pair of upstream overlapping open reading frames.

S-Adenosyl-L-methionine decarboxylase (AdoMetDC; EC 4.1.1.50) is one of the key regulatory enzymes in the biosynthesis of polyamines. Isolation of genomic and cDNA sequences from rice and Arabidopsis had indicated that this enzyme is encoded by a small multigene family in monocot and dicot plants. Analysis of rice, maize and Arabidopsis AdoMetDC cDNA species revealed that the monocot enzyme possesses an extended C-terminus relative to dicot and human enzymes. Interestingly, we discovered that all expressed plant AdoMetDC mRNA 5' leader sequences contain a highly conserved pair of overlapping upstream open reading frames (uORFs) that overlap by one base. The 5' tiny uORF consists of two or three codons and the 3' small uORF encodes 50-54 residues. Sequences of the small uORFs are highly conserved between monocot, dicot and gymnosperm AdoMetDC mRNA species and the C-terminus of the plant small uORFs is conserved with the C-terminus of nematode AdoMetDC uORFs; such a conserved arrangement is strongly suggestive of a translational regulatory mechanism. No introns were found in the main AdoMetDC proenzyme ORF from any of the plant genes encoding AdoMetDC, whereas introns were found in conserved positions flanking the overlapping uORFs. The absence of the furthest 3' intron from the Arabidopsis gene encoding AdoMetDC2 suggests that this intron was lost recently. Reverse-transcriptase-mediated PCR analysis of the two Arabidopsis genes for AdoMetDC indicated that AdoMetDC1 is abundant and ubiquitous, whereas the gene for AdoMetDC2 is expressed preferentially in leaves and inflorescences. Investigation of recently released Arabidopsis genome sequences has revealed that in addition to the two genes encoding AdoMetDC isolated as part of the present work, four additional genes are present in Arabidopsis but they are probably not expressed.

Adenosylmethionine Decarboxylase↗
PubMed2001

Biosynthesis, oxidation and conjugation of aliphatic polyamines in higher plants.

This chapter will focus on polyamine biosynthesis, oxidation, conjugation processes, mainly to hydroxycinnamic acids, and compartmentation of enzymes, substrates and products, giving an overview about recent results especially in higher plants. New research advances regarding the cloning of the main cDNA encoding for polyamine biosynthetic and oxidative enzymes, will be taken into consideration.

Cloning, Molecular↗
PubMed1999

Effect of Bis(guanylhydrazones) on Growth and Polyamine Uptake in Plant Cells.

In the present work the effect of several bis(guanylhydrazones) on the growth of Helianthus tuberosus tuber explants was studied. Different aliphatic congeners of glyoxal bis(guanylhydrazone) were tested. Most of the compounds displayed an inhibitory effect on growth, and a correlation between the structure of the molecule and the inhibitory activity was observed. Experiments carried out with glyoxal bis(guanylhydrazone) and its congeners methyl-, ethylmethyl-, and methylpropylglyoxal bis(guanylhydrazones) show that as the total number of side chain carbon atoms in the molecule increases, the inhibitory potency also increases. A depletion of spermidine levels was also found in the explants treated with ethylmethylglyoxal bis(guanylhydrazone), which turned out to be one of the most potent growth inhibitors. The addition of spermidine caused a significant reversion of the antiproliferative action of glyoxal bis(guanylhydrazone). The effect of these compounds on spermidine uptake in protoplasts isolated from carrot phloem parenchyma was also investigated. Only a slight competition was found when antagonists were present at concentrations 20 times higher than the polyamine, thus suggesting that bis(guanylhydrazones) do not share, at least at low concentrations, the polyamine transport system in plant cells.Key Words. Bis(guanylhydrazones)-Carrot protoplasts-Growth-Helianthus tuberosus-Polyamines-Uptakehttp://link.springer-ny.com/link/service/journals/00344/bibs/18n1p39.html

Journal Article↗
PubMed1998

Characterization of spermidine binding to solubilized plasma membrane proteins from zucchini hypocotyls

In this work [14C]spermidine binding to total proteins solubilized from plasma membrane purified from zucchini (Cucurbita pepo L.) hypocotyls was investigated. Proteins were solubilized using octyl glucoside as a detergent. Specific polyamine binding was thermolabile, reversible, pH dependent with an optimum at pH 8.0, and had a Kd value of 5 &mgr;M, as determined by glass-fiber-filter assays. Sephadex G-25 M gel-filtration assays confirmed the presence of a spermidine-protein(s) complex with a specific binding activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis of collected fractions having the highest specific spermidine-binding activity, several protein bands (113, 75, 66, and 44 kD) were identified. The specificity of spermidine binding was examined by gel-filtration competition experiments performed using other polyamines and compounds structurally related to spermidine. Partial purification on Sephadex G-200 led to the identification of 66- and 44-kD protein bands, which may represent the putative spermidine-binding protein(s) on the plasmalemma.

Journal Article↗
PubMed1995

Improved method for polyamine determination in TMV, a rod-shaped virus.

Polyamines were measured in viruses using different techniques. An improved method of polyamine analysis is reported for tobacco mosaic virus (TMV), a rod-shaped virus (95% protein and 5% RNA), based on HPLC of sonicated PCA-treated highly purified virus suspensions. This method allowed higher and more reliable recovery of TMV polyamines (putrescine, spermidine and spermine) when compared to the HPLC of non-sonicated samples and to thin layer chromatography. It is suggested that sonication acts on PCA-precipitated protein aggregates causing the release of trapped polyamine molecules.

Chromatography, High Pressure Liquid↗
PubMed1992

Evidence for polyamine channels in protoplasts and vacuoles of Arabidopsis thaliana cells.

The presence of ion channels permeable to polyamines in the plasma membrane and tonoplast of Arabidopsis thaliana cultured cells was investigated by means of the patch-clamp technique in the whole-cell configuration. Evidence is shown for channels, activated by depolarizations in protoplasts and by hyperpolarizations in vacuoles, with slow time course of activation, permeable to putrescine, spermidine and spermine.

Cell Membrane↗
PubMed1990

Spermidine Uptake by Mitochondria of Helianthus tuberosus.

In the present work evidence is provided that spermidine, a polyamine largely present in plant tissues, may be transported, at physiological concentrations, into the matrix space of mitochondria isolated from tubers of Helianthus tuberosus L. cv OB1 (Jerusalem artichoke). It is concluded that the movement of spermidine strictly depends on membrane potential, since it is drastically blocked by valinomycin and only slightly sensitive to nigericin. Mg(2+) and K(+) inhibit the transport of spermidine in line with the general concept that these cations compete for the same binding sites on the mitochondrial membrane. In contrast to previous data on mammalian mitochondria, spermidine uptake by plant mitochondria does not depend on the presence of inorganic phosphate. This latter result, along with evidence that Ca(2+) does not affect accumulation of spermidine, indicates that the control of the polyamine uptake mechanism in plant mitochondria is distinct from that of mammalian systems.

Journal Article↗
PubMed1988

Transport and subcellular localization of polyamines in carrot protoplasts and vacuoles.

Putrescine and spermidine uptake in carrot (Daucus carota L., cv "Tip top") protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K(m) = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca(2+) affected only surface binding but not transport into the cells. Nonpermeant polycations such as La(3+) and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast.

Journal Article↗
PubMed1987

In vitro interactions between polyamines and pectic substances.

Putrescine, spermidine and spermine induce a decrease in the pH value of 1 mM polygalacturonic acid or pectin solutions; spermidine and spermine also cause the precipitation of the polymers. The association constants between polyamines and polygalacturonic acid were in the order of 10(5) for putrescine and spermidine, and 10(6) for spermine. The number of galacturonic units per binding sites are proportional to the number of positive charges on the polyamine molecule. Low affinity binding sites appear at high polyamine concentrations. Calcium ions seem to compete weakly with spermine by lowering the association constant 4- to 6-fold. Two natural pectins tested, showed that methylation of the carboxylic groups influences only the number of galacturonic units per site but not the association constant.

Hydrogen-Ion Concentration↗
PubMed1987

Polyamine Biosynthesis and Effect of Dicyclohexylamine during the Cell Cycle of Helianthus tuberosus Tuber.

Polyamine content and the activities of their main biosynthetic enzymes, ornithine decarboxylase (ODC, EC 4.1.1.17), arginine decarboxylase (ADC, EC 4.1.1.19), S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), and arginase (EC 3.5.3.1.), were examined in crude extracts of Helianthus tuberosus tuber slices during the first synchronous cell cycle, induced by synthetic auxin, with or without the addition of 1 or 5 millimolar dicyclohexylamine (DCHA), an inhibitor of spermidine synthase. In the DCHA-treated slices a peak of accumulation of the drug was observed at 12 hours. Bound DCHA was also found. Free polyamine content generally increased, reaching a maximum at 12 to 18 hours in the S phase of the cycle; while spermidine content was decreased slightly with DCHA after 12 hours, putrescine almost doubled at 18 hours. Bound polyamines were also present. ODC and ADC showed a maximum activity at 15 and 18 to 21 hours, respectively, i.e. in the S phase; both activities increased slightly in the presence of 5 millimolar DCHA at or near the time of maximum activity. Arginase was initially very high and then rapidly decreased although a small peak of activity occurred at 15 hours. SAMDC, which had two peaks of activity, was initially inhibited by DCHA, and then stimulated, especially at 12 hours and in coincidence with the main peak, at 21 hours. Thus ODC, ADC, and SAMDC activities as well as polyamine titer increased before and during the S phase of the cell cycle and all declined during cell division. The slight inhibitory effect of DCHA was possibly due to its degradation in the tissue and to the fact that putrescine could substitute for the function(s) of spermidine.

Journal Article↗
PubMed1987

Polyamine uptake in carrot cell cultures.

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca(2+). The effects of Ca(2+) were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca(2+) and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca(2+) concentration range between 10 micromolar and 1 millimolar. La(3+) nullified the stimulatory effect of 10 micromolar Ca(2+) on putrescine uptake and that of 1 millimolar Ca(2+) on spermidine uptake. La(3+) at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule.

Journal Article↗
PubMed1986

Polyamine Uptake, Kinetics, and Competition among Polyamines and between Polyamines and Inorganic Cations.

Polyamine uptake, the kinetics of this uptake, and the competition among polyamines and between polyamines and inorganic cations were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments using (14)C-labeled polyamines were carried out on single petals, at room temperaure (20 degrees C) and in the light. The results show that putrescine, spermidine, and spermine uptake was dependent on the external pH and occurred up to high external polyamine concentrations with K(m) values of 8.6, 1.2, and 2.1 millimolar, respectively, with spermidine being the most absorbed at low concentration (17 micromolar). Putrescine and spermidine did not seem to compete for the same site of absorption. Furthermore, putrescine and spermidine uptake was not inhibited by Ca(2+), Mg(2+), and K(+) at the same concentrations (17 micromolar), whereas 1.7 millimolar Ca(2+) inhibited and K(+) enhanced spermidine uptake. The intracellular localization of the absorbed putrescine was determined using two different methods. Very little label was found in the apoplast, while most of it was localized in the 98,500g supernatant. According to our data the vacuole, which represents a substantial part of Saintpaulia parenchyma cells, could be a site of putrescine accumulation. 2,4-Dinitrophenol and diethylstilbestrol did not inhibit uptake; however, at 0 degrees C there was a 35% inhibition of spermidine uptake, compared with the controls kept at 20 degrees C as well as a 68% inhibition with 20 millimolar NaSCN.

Journal Article↗
PubMed1985

Putrescine uptake in saintpaulia petals.

Putrescine uptake and the kinetics of this uptake were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments of [(3)H] or [(14)C] putrescine were done on single petals at room temperature at various pH values. The results show that putrescine uptake occurs against a concentration gradient at low external putrescine concentration (0.5-100 micromolar) and follows a concentration gradient at higher external putrescine concentrations (100 micromolar to 100 millimolar). 2,4-Dinitrophenol and carbonylcyanide-m-chlorophenylhydrazone, two uncouplers, had no effect on putrescine uptake. Uptake rates were constant for 2 hours, reaching a maximum after 3 to 4 hours. Putrescine uptake depended markedly on the external pH and two maxima were observed: at low external concentrations of putrescine, the optimum was at pH 5 to 5.5; at higher concentrations the optimum was at pH 8.

Journal Article↗
PubMed1981

RNA, proteins and polyamines during tube growth in germinating apple pollen.

Variations of RNA, protein, and free- and trichloroacetic acid-soluble bound polyamine levels were determined during tube growth in germinating Malus domestica Borkh. cv. Starkrimson pollen.During rehydration of pollen no marked differences were observed, whereas, during germination, RNA, proteins, and polyamines showed parallel decreases. At the same time, there was synthesis of RNA and polyamines as indicated by use of labeled precursors. The data indicate that during germination: (a) the genes for rRNA, tRNA, and probably mRNA are active; (b) the enzymes involved in polyamine biosynthesis are very active. High levels of free arginine during the first 15 minutes were observed, probably in response to a demand for this precursor in polyamine biosynthesis. Moreover, profiles of the variations in the specific activities of RNA and polyamines showed similar patterns. The results indicate that biosynthesis of RNA and polyamines precedes tube emergence. The possible role of these compounds, which are known to be released into the medium in the progamic phase of the fertilization processes, is considered.

Journal Article↗
PubMed1981

Effect of various inhibitors of polyamine synthesis on the growth of Helianthus tuberosus.

The growth effect of various inhibitors of polyamine synthesis was studied on explants obtained from Helianthus tuberosus tubers during dormancy and dormancy break and cultured for 20 days on a sterile agarized medium. Explant growth was strongly inhibited by canavanine and canaline, natural non-protein amino acid analogues of arginine and ornithine, respectively, as well as by canavanine in combination with putrescine. Methylglyoxal-bis-guanylhydrazone, an inhibitor of S-adenosylmethionine decarboxylase, did not increase growth inhibition caused by canavanine. Methylglyoxal-bis-guanylhydrazone alone, as well as alpha-methylornithine, 1,3-diaminopropane and 1,3-diaminopropan-2-o1 - which are inhibitors of ornithine decarboxylase - showed no growth inhibition with respect to the control treated with 2,4-dichlorophenoxyacetic acid. 1,3-Diaminopropane caused a paradoxic enhancement of callus growth and a much greater polyamine accumulation than in the control with 2,4-dichlorophenoxyacetic acid alone. During the first cell cycle inactivated tuber slices, 40 microM methylglyoxal-bis-guanylhydrazone inhibited spermidine and spermine synthesis up to 6 h, and their accumulation up to 1 h; RNA synthesis and accumulation and DNA accumulation were reduced at 1 h, and later enhanced. During the same period, 1 mM canavanine inhibited putrescine synthesis in the S and M phases, and spermidine and spermine synthesis only at 24 h; accumulation of putrescine and spermidine was reduced only at 12 h. Canavanine also reduced RNA synthesis throughout the S and M phases, while RNA accumulation was reduced at 18 and 24 h. Two different hypotheses are put forward concerning the induction of new pathways of polyamine synthesis.

Adenosylmethionine Decarboxylase↗