Putative occurrence of lysine decarboxylase isoforms in soybean (Glycine max) seedlings.

The activity of lysine decarboxylase was studied in 3-day-old soybean (Glycine max (L.) Meer cv. Sakai) seedlings also in relation to light conditions. Lysine decarboxylase activity was mainly localized in the roots and to a lesser extent in the hypocotyls and was detectable in both the soluble and particulate fractions. The enzyme activity levels were similar during germination under light and dark conditions. With respect to lysine concentration, the initial decarboxylation rate of the soluble fraction showed a saturating curve. Conversely, the initial decarboxylation rate of the particulate fraction showed a sigmoidal curve. These results could suggest that at least two isoforms of lysine decarboxylase are present in different organs of soybean seedlings. In the root soluble fraction, the suicide inhibitor alpha-difluoromethyl-lysine suppressed the activity of lysine decarboxylase and of ornithine decarboxylase to the same extent, but had no effect on arginine decarboxylase activity.

Bis(guanylhydrazones) negatively affect in vitro germination of kiwifruit pollen and alter the endogenous polyamine pool.

Bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines (PAs). Among them, the methylglyoxal derivative (MGBG) has been studied most thoroughly. Because PAs and their biosynthetic enzymes are strongly involved in pollen tube organization, emergence and elongation, a number of these inhibitors have been studied in the present work for their effects on the in vitro performance of kiwifruit (Actinidia deliciosa) pollen. Increasing concentrations of several bis(guanylhydrazones) in the range 0.05-1 mM were checked for their effect on pollen germination. Most of the compounds tested showed a dose-dependent inhibitory effect on tube emergence, which was established very early during incubation. At 0.5 mM, the methylpropylglyoxal derivative (MPGBG) had a stronger inhibitory effect than MGBG. To verify whether the inhibitors reached their metabolic target, PA levels and S-adenosylmethionine decarboxylase (SAMDC) activity were determined in pollen germinated in the presence or absence (controls) of 0.5 mM bis(guanylhydrazones). Spermidine (Spd) content was significantly reduced in the treated pollen, and this effect was more pronounced after treatment with MGBG than with MPGBG. An early and strong reduction in SAMDC activity was observed after exposure to either inhibitor. Inhibition of pollen germination by MGBG or MPGBG could not be reversed by the addition of exogenous Spd, which per se was inhibitory. Taken together, our results suggest that bis(guanylhydrazones) alter PA metabolism and negatively affect kiwifruit pollen germination, even though a strict cause-effect relationship could not be established, and other mechanisms, unrelated to PA activity, must be involved.

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.

Polyamine Binding to Plasma Membrane Vesicles Isolated from Zucchini Hypocotyls.

The general features of [14C]spermidine binding to plasmalemma vesicles isolated from zucchini (Cucurbita pepo L.) etiolated hypocotyls are reported in the present paper. The specific interaction of the polyamine with the plasma membranes was reversible and thermolabile, since it decreased by about 50% in the assay performed at 40[deg]C compared to that carried out on ice. On the contrary, nonspecific binding was unaffected by temperature. Specific spermidine binding showed a pH dependence with a maximum at pH 8.0 and it reached saturation between 0.75 and 1 mM external spermidine concentration. The value of the dissociation constant calculated from Scatchard analysis was 4.4 x 10-5 M. Specific spermidine interaction appeared to be sensitive to detergents and was markedly reduced by the presence of divalent cations, such as Mg2+ and Ca2+, whereas it was stimulated by monovalent cations. Polyamine binding sites were highly sensitive to pronase treatment. Competition experiments, performed using a series of compounds structurally related to spermidine, may provide some indication of the characteristics of spermidine binding sites. The results presented here suggest that specific spermidine binding occurs mainly with the protein component of the plasma membrane.

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.

Effect of polyamine biosynthesis inhibitors on mycelial growth and concentrations of polyamines in Ophiostoma ulmi (Buism.) Nannf.

Several inhibitors of polyamine biosynthesis reduced mycelial growth of the fungus Ophiostoma ulmi (Buism.) Nannf. cultured on malt extract-agar medium. Growth inhibition was particularly evident when expressed in terms of fresh weight rather than colony diameter. Of the different drugs tested, the most effective was difluoromethylarginine (DFMA), while difluoromethylornithine (DFMO) and methylglyoxal(bis)guanyl-hydrazone (MGBG) plus cyclohexylamine (CHA) reduced growth only by 75 and 62% respectively. The growth inhibition by DFMO + DFMA, measured as colony diameter, was apparently reversed by putrescine (PUT), but not when expressed in terms of fresh weight. Spermidine (SPD) was the most abundant polyamine in control cultures, followed by PUT and spermine (SPM). PUT was no longer detectable 8 d after inoculation. On day 10, DFMO and DFMA, alone and in combination, caused a significant reduction in cellular SPD concentrations, while exogenously supplied PUT restored the levels of this polyamine to control values. MGBG + CHA caused a conspicuous accumulation of PUT and an approximately 50% reduction in SPD titres. DFMA, alone or in combination with DFMO and with or without PUT, led to increased cellular levels of SPM. The latter polyamine, but not PUT or SPD, strongly retarded growth when added to the growth medium. As suggested by the effectiveness of DFMA in inhibiting growth, arginine decarboxylase activity was shown to be prevalent over ornithine decarboxylase activity in this fungus.

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.

Jasmonates induce over-accumulation of methylputrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures.

Jasmonic acid (JA) and its methyl ester (MeJA) at concentrations ranging from 0.001 to 10 µM provoked large increases in methylputrescine levels in normal and hairy roots of Hyoscyamus muticus L.; generally, levels of free putrescine and perchloric acid-soluble conjugated putrescine, spermidine and spermine also increased dramatically. More 14C-putrescine was formed when hairy roots were incubated with labelled ornithine than with arginine; conjugated 14C-putrescine was also rapidly formed. In accord with these results, ornithine decarboxylase (EC 4.1.1.17) activity was higher than that of arginine decarboxylase (EC 4.1.1.19), and MeJA enhanced these activities about two- and fourfold, respectively. Although treatment of root cultures with jasmonates enhanced precursor (putrescine, methylputrescine) levels and accumulation of secondary metabolites such as acid-soluble conjugated di-/polyamines, it provoked only modest increases in tropane alkaloid tissue levels.

Polyamine biosynthesis and control of the development of functional pollen in kiwifruit.

The role of polyamines (PAs) in plant reproduction, especially pollen development and germination has been demonstrated in several higher plants. The aim of the present research was to investigate PA involvement in pollen development and germination in dioecious kiwifruit (Actinidia deliciosa). Differences in PA content, level and gene expression for PA biosynthetic enzymes, and the effect of PA biosynthetic inhibitors were found during pollen development (or abortion in female flowers). Whereas PAs, especially spermidine (Spd), remained high throughout the development of functional pollen, the levels collapsed by the last stage of development of sterile pollen. Mature and functional pollen from male-fertile anthers showed S-adenosyl methionine decarboxylase activity (SAMDC; involved in Spd biosynthesis) throughout microgametogenesis, with high levels of soluble SAMDC found starting from the late uninucleate microspore stage. Soluble SAMDC was absent in male-sterile anthers. Arginine decarboxylase [ADC; for putrescine (Put) biosynthesis] showed little difference in functional vs sterile pollen; ornithine decarboxylase [ODC; also for putrescine (Put) biosynthesis] was present only in sterile pollen. Ultrastructural studies of aborted pollen grains in male-sterile flowers showed that cytoplasmic residues near the intine contain vesicles, extruding towards the pollen wall. Very high SAMDC activity was found in the wall residues of the aborted pollen. The combined application in planta of competitive inhibitors of S-adenosylmethionine decarboxylase (MGBG) and of spermidine synthase (CHA), or of D-arginine (inhibitor of Put synthesis), to male-fertile plants led to abnormal pollen grains with reduced viability. The importance of PAs during male-fertile pollen germination was also found. In fact, PA biosynthetic enzymes (ADC and, mainly, SAMDC) were active early during pollen hydration and germination in vitro. Two different SAMDC gene transcripts were expressed in germinating pollen together with a lower level of ADC transcript. Gene expression preceded PA enzyme activity. The application of PA inhibitors in planta drastically reduced pollen germination. Thus, low free Spd can lead either to degeneration or loss of functionality of kiwifruit pollen grains.

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.

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.

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.

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.

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.

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.

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.

Kinetics and calcium-specificity of polyamine uptake in carrot protoplasts.

The kinetics of putrescine and spermidine uptake and the influence of calcium on the kinetic parameters of the transport process were investigated in protoplasts isolated from carrot phloem parenchyma. Spermidine uptake dependence on external concentration was biphasic, both in the absence and in the presence of 1 mM CaCl2. In the first case, saturation was reached at 0.1 to 0.25 mM and the Km value was 43µM. When calcium was added, the Km and Vmax increased. A similar pattern was found with regard to putrescine uptake. Moreover, in order to clarify the mode of action of calcium on polyamine uptake, lanthanides (lanthanum and gadolinium) were utilised as Ca(+2)-channel antagonists. When protoplasts were preincubated with these lanthanides, the stimulatory effect exerted by Ca(+2) on polyamine uptake was almost totally abolished. On the other hand, if lanthanum was supplied instead of calcium, it gave rise to a small enhancement of polyamine transport. These results induce us to suggest that calcium acts on polyamine uptake both by binding to external sites on the plasmalemma and by penetrating into the cell.