Polyamine levels and tomato fruit development: possible interaction with ethylene

Fruits of tomato, Lycopersicon esculentum Mill. cv Liberty, ripen slowly and have a prolonged keeping quality. Ethylene production and the levels of polyamines in pericarp of cv Liberty, Pik Red, and Rutgers were measured in relation to fruit development. Depending on the stage of fruit development, Liberty produced between 16 and 38% of the ethylene produced by Pik Red and Rutgers. The polyamines putrescine, spermidine, and spermine were present in all cultivars. Cadaverine was detected only in Rutgers. Levels of putrescine and spermidine declined between the immature and mature green stages of development and prior to the onset of climacteric ethylene production. In Pik Red and Rutgers, the decline persisted, whereas in Liberty, the putrescine level increased during ripening. Ripe pericarp of Liberty contained about three and six times more free (unconjugated) polyamines than Pik Red and Rutgers, respectively. No pronounced changes in spermidine or cadaverine occurred during ripening. The increase in the free polyamine level in ripe pericarp of Liberty may account for the reduction of climacteric ethylene production, and prolonged storage life.     

Polyamine Metabolism in Ripening Tomato Fruit : II. Polyamine Metabolism and Synthesis in Relation to Enhanced Putrescine Content and Storage Life of a/c 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 [1,4-¹⁴C]putrescine and [terminal labeled-³H]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.     

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.     

Levels of Polyamines and Kinetic Characterization of Their Uptake in the Soybean Pathogen Phytophthora sojae

Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-¹⁴C]putrescine and [1,4-¹⁴C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection.     

Polyamine metabolism in ripening tomato fruit : I. Identification of metabolites of putrescine and spermidine

The metabolism of [1,4-¹⁴C]putrescine and [terminal methylene-³H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The ¹⁴C label from putrescine was incorporated into spermidine, γ-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [³H]Spermidine was catabolized into putrescine and β-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed.     

Changes in ripening-related processes in tomato conditioned by the alc mutant

Summary The alc mutation affects the ripening and storability of tomato fruit. The alteration of fruit color in alc lines is due to a reduction in total pigment and a reduction in lycopene relative to total carotinoids. Polygalacturonase (PG) activity is reduced to less than 5% of normal, and the isozymes PG2a and PG2b are absent in alc fruit. The level of anti-PG precipitable proteins is also reduced to less than 5% of normal. Total polyA + mRNA is not significantly reduced in ripening alc fruit, but hybridization of polyA + mRNA to different ripening-related cDNA clones showed that specific mRNAs are present at reduced levels in the mutant. Specific mRNA levels were reduced to 10%–80% of normal levels, depending on the cDNA clone used as the probe. PG mRNA was present at 5%–10% of the normal level.All effects of alc on fruit ripening are relived in the line Alcobaca-red, which arose spontaneously from the original alc line, Alcobaca. The Alcobaca-red trait segregates as a single dominant trait at or very near the alc locus, and it is probably the result of a reverse mutation at the alc locus.The chromosomal locations of regions homologous to 5 ripening-related cDNA probes were determined. Regions homologous to 4 of these probes map to chromosomes other than chromosome 10, indicating that the effects of alc are transactive. A cDNA clone for PG was homologous to only one chromosomal region. This region is located on chromosome 10, which is also the chromosome on which alc and nor are located.     

Conversion of exogenous spermidine into putrescine after administration to rats

Administration of large, but non-toxic doses of spermidine (0.4–1.25 mmol/kg) led to a substantial increase in putrescine in liver, kidney and a number of other tissues including muscle. The increase in putriscine peaked at 6 h after treatment and was completely prevented by administration of cycloheximide 3 h after the spermidine suggesting that the induction of a new protein was required. This protein is likely to be spermidine N¹-acetyltransferase which was induced by the treatment with spermidine and increased 3–4-fold in liver and kidney within 6 h. N¹-Acetylspermidine was detected in tissues at this time after spermidine treatment and experiments in which labeled spermidine was given indicated that a substantial fraction of the administered spermidine was converted into N¹-acetylspermidine and into putrescine. These results suggest that the rise in putrescine after spermidine treatment is brought about by the production of N¹-acetylspermidine which is converted into putrescine by the action of polyamine oxidase. The limiting step in this conversion is the activity of the acetylase which is induced in response to the rise in spermidine content. The acetylase/oxidase pathway, therefore, provides a means by which polyamine levels can be regulated and excess polyamine disposed of.     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

Endogenous levels of polyamines in the organs of cucumber plant (Cucumis sativus) and factors affecting leaf polyamine contents

Polyamine compositions of various organs from hydroponically cultivated cucumber plants ( Cucumis sativus L. cv. Sharp-1) and factors affecting the leaf polyamine content were examined. Diamine putrescine was found most abundantly in the root, while a relatively large amount of spermine was detected in the reproductive organs such as the immature fruit and the calyx (+stamen). Spermidine was present at the highest level in rapidly growing tissues such as newly expanded leaf and fruit at an early developing stage, implying the possible involvement of spermidine in the growth and development of these young tissues. Polyamine content of cucumber leaves changed during the day. Especially, the putrescine content of upper leaves showed a striking decrease from the morning to the night. Alterations of leaf Ca or Mg content did not significantly affect leaf polyamine composition. On the other hand, abnormal cucumber leaves showed altered polyamine composition. Yellowing of the leaf intervein resulted in a striking decrease in spermidine content without a significant change in putrescine and spermine content. By contrast, the leaves infected with the phytopathogen, powdery mildew, showed decreased putrescine and increased spermine content in response to the degree of fungi infection. The possible usefulness of polyamines as a diagnostic marker of plant development and physiological disorder is discussed.     

Acetylation of spermidine in polyamine catabolism

Treatment with thioacetamide (150 mg/kg)_ was used to enhance polyamine metabolism in rat liver. The increased uptake and catabolism of [¹⁴C]spermine and the changes of putrescine, spermidine and spermine concentrations indicated enhanced polyamine turnover rates. The increase of hepatic putrescine concentration was accompanied by an increase of monoacetylputrescine and N¹-monoacetylspermidine concentration. In control animals, the latter compound was below detection levels. Thioacetamide treatment also enhanced putrescine excretion, which again was concomitant with an increased excretion of N¹-acetylspermidine.The close time-dependent correlation between induced putrescine formation and enhanced formation of N¹-acetylsperimidine at a time when liver spermidine and spermine concentrations are not changed, favors the notion that acetylation is an essential step in polyamine degradation and elimination. The increase of polyamine oxidase and decrease of acetylpolyamine deacetylase activities in the liver of thioacetamide-treated rats is in line with an increased polyamine turnover, but these enzymes. although essential, are not rate-limiting in the catabolic reactions.     

Changes in gene expression during strawberry fruit ripening and their regulation by auxin

Changes in messenger RNA during the development of the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, were analysed by extracting total RNA and separating the in-vitro translated products by two-dimensional polyacrylamide gel electrophoresis. Alterations in numerous messenger RNAs accompanied fruit development between the immature green stage and the overripe stage, with prominent changes detected at or before the onset of ripening. A number of messenger RNAs undetectable in immature green fruit increased as the fruit matured and ripened. Others showed a marked decrease in advance of the ripening phase. A further group of messenger RNAs was prominent in immature and ripe fruit but absent just prior to the turning stage. Removing the achenes from a segment of the fruit accelerated anthocyanin accumulation in the de-achened portion and produced a pattern of translated polypeptides similar to normal ripe fruit. Application of the synthetic auxin 1-naphthaleneacetic acid to the de-achened receptacle produced a translation pattern similar to that in mature green fruit. These findings indicate that ripening in strawberry is associated with the expression of specific genes.     

Leishmania spp.: cellular levels and synthesis of polyamines during growth cycles

Polyamines were extracted from five different leishmanial strains (Leishmania sp., L. tropica major, L. mexicana, and two L. donovani isolates) and identified as pulrescine, spermidine, and spermine by thin-layer chromatography and mass spectrometry. These sensitive methods were also used to demonstrate the conversion of radioactive putrescine into spermidine and spermine. As in other types of cells, polyamine levels fluctuated during the growth cycle, maximal levels being attained during the logarithmic growth phase. In the five leishmanial strains, which were members of four different serotypes, the spermidine—putrescine ratios also varied, and in two strains of the same serotype, polyamine ratios were practically identical, suggesting that polyamine characteristics might serve as a further criterion for strain identification and classification.     

Changes in endogenous polyamine levels are associated with differentiation in Dictyostelium discoideum.

This is the first report correlating levels of polyamines and its fractions with differentiation in Dictyostelium discoideum. Temporal changes in endogenous levels of free, conjugated and bound putrescine, spermidine and spermine were analysed at critical stages of morphogenesis in this organism. No spermine was found at any given stage and putrescine was the most abundant polyamine. There was a sharp increase in the levels of both free (and total) and conjugated forms of putrescine and spermidine at the slug stage as compared to the growth phase. The levels of putrescine and spermidine were found to be higher in isolated prespore cells as compared to the prestalk cells. Remarkably, the levels of polyamine decreased at the early culminant stage. Data suggest that a moderate level of polyamines is needed for growth but it is important to have high levels of polyamines at the time of differentiation.     

Polyamine levels in pollinated and auxin-induced fruit of tomato (Lycopersicon esculentum) during development

The changes taking place during fruit development in the concentration of the 3 polyamine fractions, i.e. free, perchloric acid-soluble conjugates and perchloric acid-insoluble bound polyamines, were analyzed in tomato fruits ( Lycopersicon esculentum Mill, cv. F121) induced to set by either pollination or auxin application. Before the onset of cell division, total polyamines were 50% higher in auxin-treated fruits than in pollinated ones, most of the polyamines being found as perchloric acid-soluble conjugates in both fruit set treatments. At the onset the level of polyamines in both fruit types was 100 times higher than during cell expansion or ripening. The highest polyamine found during cell division was perchloric acid-soluble conjugated spermidine in both fruit set treatments. After cell division, polyamine concentration was similar in both fruit set treatments. The concentration of polyamines in the jelly was similar in pollinated and auxin-induced fruits during cell expansion but not during ripening. At the onset of ripening there was an increase of one order of magnitude in the concentration of perchloric acid-insoluble bound putrescine in the jelly of pollinated fruits. Polyamines were more than 5-fold higher in unpollinated ovaries than in fruits induced to set by either pollination or auxins. It is suggested that pollinated and parthenocarpic fruits differ in their polyamine metabolism during the initial stages of development, but not after cell division. It is also suggested that polyamines undergo rapid turnover during cell division. Perchloric acid-insoluble bound putrescine might play a role in seed formation in tomatoes.     

Developmental Regulation of the (1,3)-beta-Glucan (Callose) Synthase from Tomato : Possible Role of Endogenous Phospholipases

Activity levels of UDP-glucose: (1,3)-β-glucan (callose) synthase in microsomal membranes of pericarp tissue from tomato fruit (Lycoperisicon esculentum Mill, cv Rutgers) were determined during development and ripening. Addition of the phospholipase inhibitors O-phosphorylcholine and glycerol-1-phosphate to homogenization buffers was necessary to preserve enzyme activity during homogenization and membrane isolation. Enzyme activity declined 90% from the immature green to the red ripe stage. The polypeptide composition of the membranes did not change significantly during ripening. The enzyme from immature fruit was inactivated by exogenously added phospholipases A₂, C, and D. These results suggest that the decline in callose synthase activity during ontogeny may be a secondary effect of endogenous lipase action.     

Modulation of polyamine levels in ginseng hairy root cultures subjected to salt stress

Modulation of differential gene expression and change of polyamine content by salt stress are analyzed for the first time in a well-known medicinal plant, Panax ginseng C.A. Meyer. Three ginseng genes (PgSPD, PgSAMDC, and PgADC) involved in polyamine biosynthesis showed differential up-regulation patterns after 1 and 7 days of salt treatments. The modulation of gene expression resulted in the elevation of total polyamine content with relatively high levels of spermidine and spermine, while putrescine level diminished depending on the salt concentration. Conversely, salt stress led to a significant increase in diamine oxidase and subsequent decline in polyamine oxidase. The proline content caused by salinity follows a similar pattern as the total polyamine content and exogenous spermidine also resulted in the alleviation of proline content under salinity. Further, polyamine biosynthesis inhibitors, such as cyclohexylamine and methylglyoxal bis-(guanylhydrazone) mediated down-regulation of PgSPD and PgSAMDC, and affected cellular polyamine levels. Thus, polyamines may enhance the ginseng plant tolerance in response to the salt stress by increasing the levels of endogenous polyamines.     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.