Soluble Polyamines Research Articles

Polyelectrolytes with Main‐Chain Perylene Units

A new soluble polyamine with main chain perylene units is synthesized and subsequently converted into cationic or zwitterionic polyelectrolytes. The optical properties of the monomer 1,6,7,12-tetrakis(4-tert-butylphenoxy)-3,4,9,10-tetra(bromomethyl)perylene and the resulting polymers have been studied with special attention to ongoing de/aggregation processes. UV-Vis and PL spectra of the polymers show a distinct concentration and solvent dependency of the optical properties. In contrast to the corresponding monomer complete deaggregation did not occur for the polymers. The environment-sensitive optical properties should allow applications as sensor materials. The perylene-containing polyelectrolytes may be applied in orthogonal processing schemes toward multilayer electronic devices.

Arsenic accumulation and distribution in relation to young seedling growth in Atriplex atacamensis Phil.

Even at trace levels, arsenic is of environmental and health concern due to its high toxicity. The xerohalophyte plant species Atriplex atacamensis grows on an arsenic-contaminated mining area in North Chile. Young seedlings that were grown from seeds collected from these plants were grown in a nutrient solution under controlled environmental conditions and were exposed for 14 and 28 days to 0, 100 or 1000 μM arsenate. More than 75% of the plants that were exposed to the highest As dose survived until the end of the treatment. The seedling growth was reduced (100 μM As) or inhibited (1000 μM As) in the stress conditions, but the plants were able to efficiently close their stomata and perform osmotic adjustments to avoid secondary water stress. Arsenic accumulated up to 400 μg g −1 DW in the shoots and 3500 μg g −1 DW in the roots. Arsenate drastically impaired the P content and increased glycinebetaine content, although no arsenobetaine was found in the tissues. With the exception of arsenite and arsenate, no As-containing organic compound was detected. Arsenic was not excreted by the trichomes that were present at the leaf surface. Although an increase in the total level of non-protein thiols suggested that arsenite fixation on the sulfhydryl groups could occur in the stressed tissues, the majority of the soluble arsenic remained in its oxidized state As(V). Arsenate induced an increase in the free soluble polyamine concentrations in all of the organs, and it increased the proportion of spermidine and spermine and decreased the proportion of putrescine in the polyamine pool. Therefore, it is likely that these polycationic molecules may assist in arsenate sequestration in the stressed tissues, and A. atacamensis may represent a promising plant species that can be tested in field trials for its phytomanagement of As-contaminated sites in desert areas.

Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broad-spectrum tolerance to abiotic stresses in transgenic tobacco plants

Polyamines (PAs), such as putrescine, spermidine, and spermine, are present in all living organism and implicate in a wide range of cellular physiological processes. We have used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Sense construct of full-length cDNA for S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in PA biosynthesis, from carnation (Dianthus caryophyllus L.) flower was introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium tumefaciens-mediated transformation. Several transgenic lines overexpressing SAMDC gene under the control of cauliflower mosaic virus 35S promoter accumulated soluble total PAs by 2.2 (S16-S-4) to 3.1 (S16-S-1) times than wild-type plants. The transgenic tobacco did not show any difference in organ phenotype compared to the wild-type. The number and weight of seeds increased, and net photosynthetic rate also increased in transgenic plants. Stress-induced damage was attenuated in these transgenic plants, in the symptom of visible yellowing and chlorophyll degradation after all experienced stresses such as salt stress, cold stress, acidic stress, and abscisic acid treatment. H2O2-induced damage was attenuated by spermidine treatment. Transcripts for antioxidant enzymes (ascorbate peroxidase, manganase superoxide dismutase, and glutathione S-transferase) in transgenic plants and GUS activity transformed with SAMDC promoter::GUS fusion were induced more significantly by stress treatment, compared to control. These results that the transgenic plants with sense SAMDC cDNA are more tolerant to abiotic stresses than wild-type plants suggest that PAs may play an important role in contributing stress tolerance in plants.

Soluble polyamines in Salix myrsinifolia and S. myrsinites × S. myrsinifolia plantlets exposed to increased UV-B irradiation and decreased watering

Hybridisation between certain willow species is a common feature leading to novel genotypes varying in growth rate and stress tolerance. The objective of this 4-week study was to investigate the effects of decreased watering, enhanced ultraviolet-B irradiation (UV-BBE, 280–315 nm, 7.2 kJ m−2 day−1) and combined decreased watering and enhanced UV-B irradiation on di- and polyamines in the leaves of Salix myrsinifolia and its hybrid with S. myrsinites. Control plantlets were well-watered and exposed to ambient UV-B irradiation (UV-BBE, 3.6 kJ m−2 day−1). HPLC analyses showed that the constitutive concentrations of soluble di- and polyamines varied markedly between S. myrsinifolia and its hybrids. The degree of responses to treatments also varied: in S. myrsinifolia, concentrations of free putrescine were clearly increased by reduced watering, while in the hybrid willow, change in putrescine was less pronounced and not significant. Results also showed that the increase in putrescine in S. myrsinifolia by reduced watering was mitigated by concurrent enhancement of UV-B irradiation. There were no direct UV-B effects on the soluble polyamines.

Biosynthesis profile and endogenous titers of polyamines differ in totipotent and recalcitrant plant protoplasts

The expression of totipotency in plant protoplasts is a complex developmental phenomenon and is affected by genetic and physiological factors. Polyamines (PAs) are known to be involved in a variety of growth and developmental processes in higher plants, as well as in adaptation to stresses. In this study, we present the homeostatic characteristics of the endogenous PA putrescine (Put), spermidine (Spd), and spermine (Spm) in totipotent (T) and non‐totipotent (NT) tobacco protoplasts and in recalcitrant (R) grapevine protoplasts. T‐tobacco protoplasts, with high division rates, have the highest level of endogenous PAs. In these protoplasts, the soluble‐hydrolyzed fraction predominates and increases, and the insoluble‐hydrolyzed fraction also increases, whereas soluble (S) PAs decrease rapidly during culture. The isolation process contributes to the increased Put levels, which are higher in freshly isolated NT‐tobacco protoplasts than in T‐protoplasts. During culture, total Put predominates over Spd and Spm, and the highest accumulation is found in T‐protoplasts. Ornithine decarboxylase and arginase activities both increase in T‐protoplasts, whereas arginine decarboxylase activity causes Put accumulation in NT‐tobacco protoplasts. R‐grapevine protoplasts show a different PA profile, mostly due to the lower PA content, the higher S‐fraction, and the higher ratio of Spm to total PAs. The data suggest that the levels and metabolism of the intracellular PAs could be related to the expression of totipotency of plant protoplasts.

Downregulation of ethylene production and biosynthetic gene expression is associated to changes in putrescine metabolism in shoot-forming tobacco thin layers

The effect of aminoethoxyvinylglycine (AVG), an inhibitor of 1-aminocyclopropane-1-carboxylate synthase (ACS) activity, on ethylene emission and biosynthetic gene expression, on gene expression and/or activity of polyamine (putrescine, spermidine and spermine) biosynthetic enzymes, and on diamine oxidase (DAO, EC 1.4.3.6) activity was evaluated in tobacco ( Nicotiana tabacum L. cv. Samsun) thin layers cultured on a shoot-forming medium (1 μM indol-3-acetic acid (IAA) plus 10 μM benzyladenine (BA)). Northern analyses showed that ACS and 1-aminocyclopropane-1-carboxylate oxidase (ACO) transcripts were present throughout culture with a maximum accumulation on day 7. Besides ethylene emission, AVG (0.5 μM) increasingly reduced ACS and ACO messages. The time course of labelled methionine incorporation into spermidine and spermine, which share with ethylene the common precursor S-adenosylmethionine (SAM), as well as SAM decarboxylase (SAMDC, EC 4.1.1.21) activity and gene expression, were not affected by AVG treatment. On the contrary, labelled putrescine incorporation into the higher polyamines (spermidine and spermine) and into trichloroacetic acid (TCA)-soluble polyamine conjugates was enhanced early in culture (day 2) by the drug. Putrescine biosynthetic enzyme activities, arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17), were also increased in AVG-treated explants. Moreover, inhibition of ethylene synthesis by AVG led to a strong reduction in diamine oxidising activity, especially the one associated with a cell wall-enriched fraction. Changes in putrescine biosynthesis, oxidation and flux into higher polyamines are discussed in the light of the rejuvenating effect of AVG.

Differential responses of Brassica napus and Petunia hybrida to leaf protoplast isolation stress.

Changes in the response to abiotic stress during the isolation of leaf protoplasts were compared between a recalcitrant species of Brassica napus and regenerating species of Petunia hybrida. Initially, levels of soluble free putrescine (put), spermidine (spd) and spermine (spm) in leaves and protoplasts were determined. The sum of these three polyamines increased in petunia and B. napus leaf protoplasts by 1.6-fold and 1.1-fold, respectively. The soluble free fraction of spd and spm decreased in B. napus but not in petunia protoplasts. During the isolation of leaf protoplasts from B. napus, the ratio of soluble free put to the total PAs almost doubled, but that of spd and spm declined significantly. Petunia leaf protoplasts treated with cyclohexylamine (CHA), an inhibitor of spermidine synthase, accumulated ammonia and soluble putrescine, but lost the soluble spermidine. The soluble polyamine levels of CHA-treated petunia leaf protoplasts corresponded with those in B. napus. Leaves were subjected to abiotic stress during the isolation of protoplasts, namely wounding and osmotic stress which changed soluble free polyamine levels in B. napus and petunia, respectively. Both B. napus and petunia leaf protoplasts showed an increase in ammonia, but total free amino acid content and activation of proteases were only enhanced in B. napus leaf protoplasts. These results suggest that in B. napus wounding initiated senescence of leaf protoplasts during their isolation, leading to a constant production of ethylene early in the culture.

Are polyamines involved in the synthesis of heat-shock proteins in cell suspension cultures of tobacco and alfalfa in response to high-temperature stress?

Exposure of plants to sublethal doses of heat stress induces a complex set of responsive mechanisms in order to protect cells and organisms from severe damage and enable them to survive. In the present study, the contents of polyamines (putrescine, spermidine and spermine) and the patterns of heat-shock protein (HSP) production were determined in two differently thermosensitive suspension-cultured cell lines ( Nicotiana tabacum L. cv. Bright Yellow 2 (BY2) and Medicago varia cv. RAMBLER) subjected to various elevated temperatures for 3 h after gradual temperature increase. The aim of our investigations was the evaluation of a potential role of polyamines in regulating the synthesis of HSPs in plant cells under heat stress. The more heat-tolerant tobacco BY2 cells exhibited moderate ratios of free putrescine to free spermidine and contained relatively high titres of trichloroacetic acid- (TCA) soluble conjugated polyamines. HSP synthesis in tobacco cells could be detected up to 46 °C. In contrast, excessive accumulation of free putrescine and relatively low levels of TCA-soluble conjugated polyamines compared with those of the corresponding free forms characterised the more heat-susceptible alfalfa cell line. Under high-temperature conditions, the synthesis of HSPs ceased at 40 °C in alfalfa cells. Leakage of soluble polyamines from the cells was low in tobacco over the whole range of temperature investigated, but massive in alfalfa between 40 and 46 °C. The inhibition of putrescine biosynthesis in alfalfa cells by D-arginine decreased markedly the endogenous free putrescine level, reduced polyamine leakage between 40 and 42 °C and improved HSP synthesis. The results indicate that the polyamine metabolic status of cells might influence the extent of HSP synthesis under heat stress by affecting the integrity and properties of cell membranes.

In vitro morphogenic potential of differently aged Pinus radiata trees correlates with polyamines and DNA methylation levels

DNA methylation and polyamine content have been analysed in juvenile, mature vegetative and mature reproductive Pinus radiata trees. Juvenile individuals were characterised by a DNA methylation degree of 30% and a high ratio of free versus PCA (perchloric acid) soluble polyamine conjugates, while mature trees showed 60% methylcytosine and low ratio of free versus PCA-soluble polyamine conjugates. We propose that both indicators are related with the lost of morphogenic ability during pine ageing and so with the inability of mature trees to in vitro establishment through specific interactions. These interactions are discussed.

Cloning and expression of an arginine decarboxylase cDNA from Vitis vinifera L. cell-suspension cultures.

Arginine decarboxylase (ADC; EC 4.1.1.19) is a key enzyme in one of the two pathways to putrescine. We present the first ADC cDNA from a woody perennial plant species, the grapevine (Vitis vinifera L.), which exhibits 70-80% homology with other dicot ADCs. The effects of ammonium, nitrate, and putrescine on ADC specific activity, soluble polyamine levels, ADC-mRNA, endogeneous arginine and ornithine, and arginase specific activity were investigated in suspension cultures of grapevine cells. The addition of NH4+ to cells cultured in NH4(+)-free medium, resulted in a 4-fold increase in ADC activity and concomitantly in a 4-fold increase in putrescine and a 3-fold decrease in arginine. During this period ornithine increased and arginase activity followed a reverse pattern of changes compared with ADC. In contrast, the addition of NO3- did not markedly affect ADC activity, putrescine, arginine and ornithine, but transiently increased arginase activity. The addition of putrescine caused a 4-fold decrease in ADC activity and increased arginine, ornithine and arginase activity. The changes in ADC specific activity were not accompanied by analogous changes in the ADC transcript levels. These results further support the view that ADC regulation is not exhibited, at least for the factors considered in this work, at the transcriptional level.

Quantitation and determination of molecular weight distribution of residual water soluble extractable polyamines in DMP 504, a poly-alkylamine bile acid sequestrant, by aqueous high performance size exclusion chromatography

A high performance size exclusion chromatography (HPSEC) method with refractive index detection for quantitation and molecular weight determination of extractable water soluble polyamines (SPA) in a novel proprietary polymeric pharmaceutical compound (DMP 504) is described. The extracted polyamines are synthetic impurities as well as potential degradation products of the polymer. Data for calibration, precision, solution stability, and limits of detection and quantitation are presented to validate the suitability of this method for its intended purpose. Response linearity for the poly(2-vinylpyridine) standard is demonstrated between 10 and 1000 μg ml −1, equivalent to 0.005% and 0.5% (w/w) in DMP 504. Molecular weight distributions of SPA extracted from DMP 504 are presented. No increase in SPA is detected in DMP 504 over 6 months storage at room temperature.

Polyamine translocation following photoperiodic flowering induction in soybean

Radioactivity translocation after [14C]‐spermidine application over the third trifoliate leaf of soybean plants (Glycine max. [L.] Merr, cv. Williams) was checked during the first 72 h of short day (SD) treatment to study the involvement of polyamines (PAs) in photoperiodic flowering induction. PAs and/or their metabolites were translocated from the supplied leaf to all parts of the plant. Radioactivity reached its highest concentration in the upper portion of the stem, i.e. the apical bud and the youngest leaf. After the beginning of the first inductive night, the detected radioactivity showed two peaks of maximal concentration. The first arose after the first inductive night, coinciding with the proper flowering induction process; the second one arose after the third inductive night, coinciding with the first morphological symptoms of the transition of vegetative meristems to the reproductive condition. Soluble free PAs showed a different balance in the apical bud of SD‐induced plants compared with LD‐non induced control plants. Soluble conjugated PAs were detected as traces. It is suggested that under flowering inductive conditions, PAs play a different role according to the stage of the flowering process. Thus, their translocation from the leaves to the axillary and apical buds might be related, in a first step, to the fact that they were part of the complex mechanism of the flowering signal, and in a second step, to the flower transition of vegetative buds.

Changes in Polyamine Levels During Organ Induction in Cultured Melon Cotyledons

Summary A tissue culture system derived from cotyledons of melon was used to study changes in the titer of the soluble polyamines putrescine, spermidine and spermine during organogenesis. Spermidine was the predominant polyamine at all stages of development. Titers of all polyamines per unit protein increased with time in both the basal (organogenetic) and central (non-organogenetic) areas of the cotyledon. This indicates that the observed changes in polyamine titer are not caused by or dependent on root and/or shoot regeneration. Rather, they may be indicators of or causes of conditions leading to organogenesis.

Polyamine Metabolism in Ripening Tomato Fruit

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele a/c) and contain three times as much putrescine as the standard Rutgers variety (A/c) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-a/c-a near isogenic line possessing the allele a/c, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in a/c pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and a/c fruit showed a decrease in the metabolism of [1,4-(14)C]putrescine and [terminal labeled-(3)H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-a/c fruit, and as a result it was significantly higher in a/c fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in a/c fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase.

Application of Water Soluble Polyamine in the Pulp and Paper Industry

A study on the application of aminoethyl derivatives of starch and cellulose (AES and AEC) prepared through the Hofmann degradation was undertaken for the following purposes:a) Removal of color from the pulping waste liquor, b) Recovery of pulp fines and fillers in machine "white water", c) Acceleration of drainage, d) Retention of emulsified resins. As a result, it has been found that both AEC and AES have excellent properties as the coagulant for negative colloidal substances, and that the emulsified resins are retained uniformly on the surface of the pulp treated with AEC or AES contrary to the case of the alum-treament system where the resins are coagulated into large blocks.