Ethylene-induced putrescine accumulation modulates K+ partitioning between roots and shoots in barley seedlings

We investigated the cause and effect relationships among ethylene, polyamines, and K⁺ in barley ( Hordeum vulgare L. cv. Amagi) seedlings. Application of 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, to the growth medium caused a decrease in K⁺ concentration in roots and an increase in shoots. Addition of ACC induced putrescine accumulation in roots, while spermidine and spermine levels remained unchanged. Exogenous supply of putrescine led to putrescine accumulation and reduced K⁺ concentration. Application of Co²⁺, an inhibitor of ethylene biosynthesis, together with ACC, inhibited putrescine accumulation with a decrease in K⁺ concentration in roots. ACC-treated roots showed K⁺ uptake capacity equivalent to that of control roots, implying that the majority of K⁺ is translocated to shoots. These results suggest that ethylene regulates K⁺ partitioning between roots and shoots through the level of accumulation of putrescine in barley seedlings.     

Effects of exogenous polyamines and difluoromethylornithine on seed germination and root growth of Arabidopsis thaliana

Effects of exogenous polyamines and difluoromethylornithine (DFMO) on seed germination and seedling root growth of Arabidopsis thaliana were investigated. Root growth was stimulated by low concentrations of putrescine but was increasingly inhibited by high concentrations of putrscine. DFMO inhibited root growth and this inhibition was reversed by applying putrescine. In contrast, both spermidine and spermine had no effect on root growth but inhibited seed germination. The results suggest a possible requirement of endogeneous putrescine for normal root growth of Arabidopsis seedlings.Abbreviations DFMO difluoromethylornithine - DFMA difluoromethylarginine - ODC ornithine decarboxylase - Put Putrescine - Spd Spermidine - Spm Spermine     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

Polyamines and ethylene in relation to adventitious root formation in Prunus avium shoot cultures

An attempt was made to identify some of the hormonal factors that control adventitious root formation in our Prunus avium micropropagation system in order to improve rooting in difficult-to-root genotypes. Changes in endogenous contents of free polyamines were determined at intervals during auxin-induced rooting of shoot cultures. Accumulation of putrescine and spermidine peaked between days 9 and 11. Spermine was only present in traces, Exogenously supplied putrescine or spermine (50-500 μM), in the presence of optimal or suboptimal levels of indolebutyric acid (IBA), had no effect on rooting percentage or root density, except for spermine at 500 μM. At this external concentration spermine caused a substantial accumulation in both free spermine and putrescine. The use of several inhibitors of polyamine biosynthesis, namely α-difluoromethylornithine (DFMO), α-difluoromethylarginine (DFMA), dicyclohexylammonium sulphate (DCHA) and methylglyoxal-bis-guanyl-hydrazone (MGBG) alone or in combination in the 0.1 to 5 μM range, resulted in an inhibition of rooting that was partially reversed by the addition of the corresponding polyamine. Cellular polyamine levels were significantly reduced by DFMO and DFMA but not by DCHA and MGBG, Labeled putrescine incorporation into spermidine increased somewhat in the presence of the ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG). A system based on [3,4-¹⁴C]methionine incorporation was used to measure ethylene synthesis by the in vitro cultured shoots. Label incorporation was drastically reduced by 10 μM AVG and increased 3.5-fold in the presence of 50 μM IBA with respect to controls (no IBA). Labeled methionine incorporation into spermidine increased to some extent when ethylene synthesis was inhibited by AVG. Adding the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) to the rooting medium significantly inhibited rooting percentage; AVG caused the formation of a greater number of roots per shoot but delayed their growth. Supplying the shoots with both compounds resulted in an intermediate rooting response, in which both rooting percentage and root density were affected. These results indicate that polyamines may play a significant role at least in some stages of root formation. The polyamine and ethylene biosynthetic pathways seem to be competitive but under our conditions, the enhancement of one pathway when the other was inhibited, was not dramatic. Although IBA promoted ethylene synthesis, AVG, which drastically reduced it, also promoted root formation. Thus, the auxin effect on root induction cannot be directly related to its ability to enhance ethylene synthesis.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores

The pathways for putrescine biosynthesis and the effects of polyamine biosynthesis inhibitors on the germination and hyphal development of Gigaspora rosea spores were investigated. Incubation of spores with different radioactive substrates demonstrated that both arginine and ornithine decarboxylase pathways participate in putrescine biosynthesis in G. rosea. Spermidine and spermine were the most abundant polyamines in this fungus. The putrescine biosynthesis inhibitors alpha-difluoromethylarginine and alpha-difluoromethylornithine, as well as the spermidine synthase inhibitor cyclohexylamine, slightly decreased polyamine levels. However, only the latter interfered with spore germination. The consequences of the use of putrescine biosynthesis inhibitors for the control of plant pathogenic fungi on the viability of G. rosea spores in soil are discussed.     

Peach (Prunus persica) fruit ripening: aminoethoxyvinylglycine (AVG) and exogenous polyamines affect ethylene emission and flesh firmness

The effect of various concentrations of aminoethoxyvinylglycine (AVG; 0.32 and 1.28 m M ), an ethylene biosynthesis inhibitor, and of the polyamines putrescine (10 m M ), spermidine (0.1, 1 and 5 m M ) and spermine (2 m M ) on peach ( Prunus persica L. Batsch cv. Redhaven) fruit ripening was evaluated under field conditions. Treatments were performed 19 (polyamines) and 8 (AVG) days before harvest. Fruit growth (diameter, fresh and dry weight), flesh firmness, soluble solids content and ethylene emission were determined on treated and untreated (controls) fruits. Moreover, endogenous polyamine content and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) activity were determined to check for a possible competition between polyamines and ethylene for their common precursor S-adenosylmethionine (SAM). Both treatments strongly inhibited ethylene emission and delayed flesh softening. On a biochemical level, AVG and exogenous polyamines both reduced the free-to-conjugate ratio of endogenous polyamines, and transiently altered SAMDC activity. The possible use of these compounds to control fruit ripening is discussed also in the light of their rejuvenating effect on peach fruits.     

The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves

In the present study we determined the effects of methionine, intermediates of polyamine catabolic pathways and inhibitors of either ethylene biosynthetic or polyamine catabolic pathways on polyamine accumulation in soybean leaves. Inhibitors to SAM decarboxylase and spermidine synthase, methylglyloxal-bis-(guanylhy-drazone) and cyclohexylamine, respectively, suggest that methionine may provide aminopropyl groups for the synthesis of polyamine via S-adenosylmethionine (SAM). Results from experiments that utilized a combination of compounds which altered either ethylene or polyamine biosynthesis, namely, aminoethoxyvinyl glycine, CoSO₄, 2,5-norbornadiene, and CuSO₄, suggest the two pathways compete for a common precursor. However, exogenous addition of ethylene (via ethephon treatments) had little or no effect on polyamine biosynthesis. Likewise, polyamine treatments had little or no effect on ethylene biosynthesis. These data suggest that there are few or no inhibitory effects from the end products of one pathway on the synthesis of the other. Data from leaves treated with metabolic intermediates in the catabolic pathway of polyamines and inhibitors of enzymes in the catabolic pathway, i.e. aminoguanidine, hydroxyethyldrazine and gabaculine, suggest that the observed increases in polyamine titers were not due to decreased catabolism of the polyamines. One catabolic intermediate, γ-aminobutyric acid (GABA), elevated putrescine, spermidine and spermine by 12-, 1.4-, and 2-fold, respectively, Ethylene levels decreased (25%) in GABA-treated leaves. This small decrease in ethylene could not account for such large increase in putrescine titers. Further analysis demonstrated that the GABA-mediated polyamine accumulation was inhibited by difluoromethylarginine, an inhibitor of arginine decarboxylase, but not by difluoromethylornithine, an inhibitor of ornithine decarboxylase. These data suggest that GABA directly or indirectly affects the biosynthesis of polyamines via arginine decarboxylase.     

Some morphological and anatomical observations during alleviation of salinity (NaCI) stress on seed germination and seedling growth of barley by polyamines

In this work, the effects of polyamine (cadaverine, putrescine, spermidine, spermine) pretreatments on the germination of barley seeds, seedling growth under saline (NaCI) conditions and on leaf anatomy of the seedlings grown for 20 days in pots with perlite containing different concentrations of NaCI prepared with Hoagland solutions following the germination period of 7 days were studied. The inhibitive effect of salt on seed germination and seedling growth was alleviated in varying degrees, and dramatically, by polyamine pretreatments at the levels of NaCI studied; particularly with lower levels of salt, the control seeds were able to germinate with great difficulty. Also, the successes of these pretreatments continued, even decreased, with higher levels of NaCI such that the control seeds showed no germination. On the other hand, on the various parameters of leaf anatomy of barley seedlings, interactions between pretreatments of polyamine and salinity, with some of these being statistically important, were observed.     

Regulation of cadaverine and putrescine levels in different organs of chick-pea seed and seedlings during germination

In chick-pea ( Cicer arietinum L.) seed germinated in the presence of ¹⁴C-lysine, the latter is taken up and partly metabolised to cadaverine and TCA-precipitable molecules. Labelled cadaverine is detectable in seedlings only after 3 days, on a labelled lysine-containing medium, as confirmed also by the presence of lysine decarboxylase (LDC) activity, measured in the embryo axis and cotyledons of the seed and in the epicotyl, cotyledons, hypocotyl and roots of the seedling on the basis of ¹⁴CO₂ evolution from the labelled precursor. Putrescine biosynthesis occurred only via arginine decarboxylase (ADC) activities in soaked seeds and via both ADC and ornithine decarboxylase (ODC) activities in seedlings. Both putrescine and cadaverine were present in soaked seed, and accumulated in very large amounts in the different portions of both 3- and 8-day-old seedlings, while spermidine and spermine titers were maintained at similar levels with respect to the seed. Diamine oxidase activity, measured by evaluating oxygen consumption in the presence of putrescine, was absent in ungerminated seed and appeared in 3- and 8-day-old seedlings. In order to clarify the metabolic relationships between cadaverine and the more common polyamines, gradients of biosynthesis, accumulation and degradation of putrescine and cadaverine along the seedling axis were compared, indicating that the two diamines behave similarly during seed germination and seedling development. Their conspicuous accumulation (up to 6 m M for putrescine) seems to be regulated mainly via oxidation rather than biosynthesis.     

Levels of endogenous polyamines and NaCl-inhibited growth of rice seedlings

The role of endogenous polyamines in the control of NaCl-inhibited growth of rice seedlings was investigated. Putrescine, spermidine and spermine were all present in shoots and roots of rice seedlings. NaCl treatment did not affect spermine levels in shoots and roots. Spermidine levels in shoots and roots were increased with increasing concentrations of applied NaCl. NaCl at a concentration of 50 mM, which caused only slight growth inhibition, drastically lowered the level of putrescine in shoots and roots. Addition of precursors of putrescine biosynthesis (L-arginine and L-ornithine) resulted in an increase in putrescine levels in NaCl-treated shoots and roots, but did not allow recovery of the growth inhibition of rice seedlings induced by NaCl. Pretreatment of rice seeds with putrescine caused an increase in putrescine level in shoots, but could not alleviate the inhibition effect of NaCl on seedling growth. The current results suggest that endogenous polyamines may not play a significant role in the control of NaCl-inhibited growth of rice seedlings.Abbreviations PUT putrescine - SPD spermidine - SPM spermine     

Polyamines influence morphogenesis and caffeine biosynthesis in in vitro cultures of <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

The influence of polyamines, polyamine inhibitors and ethylene inhibitors were tested in Coffea canephora for in vitro morphogenetic response and caffeine biosynthesis. Coffea canephora produced non-embryogenic and embryogenic calli. Somatic embryos were produced only from the embryogenic callus. Endogenous polyamine pools were estimated in these tissues. Somatic embryos were subjected to secondary embryogenesis under the influence of putrescine, silver nitrate and specific inhibitors of polyamine biosynthesis. Estimation of endogenous total polyamines revealed that embryogenic callus contained 11-fold more spermine and 3.3-fold higher spermidine when compared to non-embryogenic callus. Incorporation of polyamines resulted in 58% explant response for embryogenesis when compared to control with 42% response. Incorporation of silver nitrate resulted in 65% response for embryogenesis. Incorporation of polyamine biosynthetic pathway inhibitors DFMO and DFMA resulted in 83% reduction in embryogenic response with concomitant increase in caffeine levels by two-fold as compared to control. These results have clearly demonstrated that polyamines play a crucial role in embryogenesis and caffeine biosynthesis.     

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. -DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and -aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.     

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 paclobutrazol on water stress-induced ethylene biosynthesis and polyamine accumulation in apple seedling leaves

Ethylene biosynthesis and polyamine content were determined in [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol] (paclobutrazol) pre-treated and non-treated water-stressed apple seedling leaves. Paclobutrazol reduced water loss, and decreased endogenous putrescine spermidine content. Gibberellic acid (GA) counteracted the inhibitory effect of paclobutrazol on polyamine content. Paclobutrazol also prevented accumulation of water stress-induced 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), ethylene production and polyamines in apple leaves. α-Difluoromethylarginine (DFMA), but not α-difluoromethylornithine (DFMO), inhibited the rise of putrescine and spermidine in stressed leaves. S-Adenosylmethionine (SAM) was maintained at a steady state level even when ethylene and the polyamines were actively synthesized in stressed apple seedling leaves. The conversion of ACC to ethylene did not appear to be affected by paclobutrazol treatment.     

Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings

In this study, we examined the regulation by putrescine, spermidine and spermine of nitric oxide (NO) biosynthesis in Arabidopsis thaliana seedlings. Using a fluorimetric method employing the cell-impermeable NO-binding dye diaminorhodamine-4M (DAR-4M), we observed that the polyamines (PAs) spermidine and spermine greatly increased NO release in the seedlings, whereas arginine and putrescine had little or no effect. Spermine, the most active PA, stimulated NO release with no apparent lag phase. The response was quenched by addition of 2-aminoethyl-2-thiopseudourea (AET), an inhibitor of the animal nitric oxide synthase (NOS) and plant NO biosynthesis, and by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO), an NO scavenger. By fluorescence microscopy, using the cell-permeable NO-binding dye diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM), we observed that PAs induced NO biosynthesis in specific tissues in Arabidopsis seedlings. Spermine and spermidine increased NO biosynthesis in the elongation zone of the Arabidopsis root tip and in primary leaves, especially in the veins and trichomes, while in cotyledons little or no effect of PAs beyond the endogenous levels of NO-induced fluorescence was observed. We conclude that PAs induce NO biosynthesis in plants.     

Effects of indole amides on lettuce and onion germination and growth

Auxins, such as indole-3-acetic acid (IAA), are important in plant germination and growth, while physiological polyamines, such as putrescine, are involved in cell proliferation and differentiation, and their concentrations increase during germination. In this work, novel indole amides were synthesized in good yields by monoacylation of morpholine and unprotected symmetrical diamines with indole-3-carboxylic acid, a putative metabolite of IAA, possessing no auxin-like activity. These amides were tested for their effects on seed germination and growth of the radicles and shoots of Lactuca sativa (lettuce) and Allium cepa (onion) seedlings, at 100.0, 1.0, and 0.01 microM concentrations. Germination was generally stimulated, with the exception of amide 3, derived from morpholine, at 100 microM. On radicle and shoot growth, the effect of these compounds was predominantly inhibitory. Compound 3 was the best inhibitor of growth of lettuce and onion, at the highest concentration. Amides, such as propanil, among others, are described as having herbicidal activity.