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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.

• Change is reported in the biosynthetic and oxidative activity of hypersensitive (NN) and susceptible (nn) tobacco (Nicotiana tabacum) plants in response to tobacco mosaic virus (TMV). • Mature leaves of nn and NN tobacco were collected over 0–72 h as uninoculated controls or after inoculation with TMV or phosphate buffer (mock-inoculation). The polyamine response to inoculation was analysed by measuring activity and gene expression of S-adenosylmethionine decarboxylase (SAMDC), arginine-(ADC) and ornithine decarboxylases (ODC); incorporation of labelled putrescine; and activity of diamine oxidase (DAO). • In NN leaves SAMDC activity and transcript levels, and DAO activity increased in the TMV-inoculated plants but not in the other treatments; a two-fold increase in DAO activity was seen after 72 h. Both ADC and ODC activity increased in NN leaves at 72 h in TMV-inoculated plants; ADC mRNA increased with activity. The increase in SAMDC mRNA (24 h) preceded the rise in activity (72 h). [3H]putrescine added to NN leaves led to enhanced label recovery and incorporation into spermidine and spermine in TMV-inoculated plants. No significant changes in biosynthetic or oxidative activity occurred in nn plants. • After TMV inoculation, NN, unlike nn, tobacco plants upgrade polyamine synthesis and oxidation; this leads to changes in cellular components which might induce programmed cell death.

To further our understanding of the mechanisms governing salt stress responses and adaptation in halophytes, we explored morphological, metabolic, and gene expression responses to high salinity in quinoa (Chenopodium quinoa Willd). The main objective of this study was to analyze selected responsive genes in a time-course experiment to test for expression kinetics and to compare short-term salt-induced effects at the transcript level between two Chilean landraces belonging to different ecotypes. Quinoa genotypes exhibit a large variability in their responses to salinity, but it is not clear whether this is strictly related to the ecotype to which they belong. We tested this hypothesis by comparing the expression levels of genes involved in growth, ion homeostasis, abscisic acid (ABA) biosynthesis, perception, and conjugate cleavage, polyamine (PA) biosynthesis and oxidation, and proline biosynthesis as well as genes encoding ABA-dependent and −independent transcription factors. Landraces R49 (salares ecotype) and Villarrica (VR, coastal-lowlands ecotype) were analyzed from 0.5 to 120Âh after transfer to saline (300ÂmM NaCl) or non-saline (control) medium. All the genes, except CqSOS1 and CqNHX, were investigated here for the first time in quinoa under salt stress. Transcript levels were determined by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis. Germination, seedling growth, ABA, and PA contents were evaluated in parallel. Even though on saline medium germination was inhibited in VR but not in R49, seedling growth reduction at 120Âh was not substantially different in the two landraces. The ABA biosynthetic enzyme NCED was the most strongly salt-induced gene; ABA content was similarly enhanced (shoots) or unaffected (roots) in both R49 and VR. NaCl treatment also altered transcript levels of some PA metabolic enzymes and the PA profile leading to an enhanced ratio between the higher PAs and putrescine. All other genes also exhibited similar expression profiles in response to salinity in the two landraces especially in roots, while in shoots some differences were observed. Our results provide new information indicating that crucial salt adaptation strategies at the molecular level and in terms of ABA and PA contents are shared by the coastal-lowlands and salares landraces; however, the timing of the onset of transcriptional changes (e.g., NCED, ABF3, and RD22) may reflect genotype-dependent constitutive and/or inducible adaptive strategies.

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.

none 5 no This research was supported by funds from the University of Bologna (RFO2014) to S.B.and from the University of Urbino (RFO 2014) to V.S.. Quinoa (Chenopodium quinoa Willd), an ancient Andean seed crop, exhibits exceptional nutritional properties and resistance to abiotic stress. The species' tolerance to heavy metals has, however, not yet been investigated nor its ability to take up and translocate chromium (Cr). This study aimed to investigate the metabolic adjustments occurring upon exposure of quinoa to several concentrations (0.01–5mM) of CrCl3. Young hydroponically grown plants were used to evaluate Cr uptake, growth, oxidative stress, and other biochemical parameters three and/or seven days after treatment. Leaves accumulated the lowest amounts of Cr, while roots and stems accumulated the most at low and at high metal concentrations, respectively. Fresh weight and photosynthetic pigments were reduced only by the higher Cr(III) doses. Substantially increased lipid peroxidation, hydrogen peroxide, and proline levels were observed only with 5mM Cr(III). Except for a significant decrease at day 7 with 5mM Cr(III), total polyphenols and flavonoids maintained control levels in Cr(III)-treated plants, whereas antioxidant activity increased in a dose-dependent manner. Maximum polyamine accumulation was observed in 1mM CrCl3-treated plants. Even though α- and γ-tocopherols also showed enhanced levels only with the 1mM concentration, tyrosine aminotransferase (TAT, EC 2.6.1.5) activity increased under Cr(III) treatment in a dose- and time-dependent manner. Taken together, results suggest that polyamines, tocopherols, and TAT activity could contribute to tolerance to 1mM Cr(III), but not to the highest concentration that, instead, generated oxidative stress. open Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania

Neochloris oleoabundans (Chlorophyta) is widely considered one of the most promising microalgae for biotechnological applications. However, the large-scale production of microalgae requires large amounts of water. In this perspective, the possibility of using exhausted growth media for the re-cultivation of N. oleoabundans was investigated in order to simultaneously make the cultivation more economically feasible and environmentally sustainable. Experiments were performed by testing the following media: autotrophic exhausted medium (E+) and mixotrophic exhausted medium after cultivation with glucose (EG+) of N. oleoabundans cells grown in a 20-L photobioreactor (PBR). Both exhausted media were replenished with the same amounts of nitrate and phosphate as the control brackish medium (C). Growth kinetics, nitrate and phosphate consumption, photosynthetic pigments content, photosynthetic efficiency, cell morphology, and lipid production were evaluated. Moreover, the free fatty acid (FFA) composition of exhausted media and the polyamine (PA) concentrations of both algae and media were analyzed in order to test if some molecules, released into the medium, could influence algal growth and metabolism. Results showed that N. oleoabundans can efficiently grow in both exhausted media, if appropriately replenished with the main nutrients (E+ and EG+), especially in E+ and to the same extent as in C medium. Growth promotion of N. oleoabundans was attributed to PAs and alteration of the photosynthetic apparatus to FFAs. Taken together, results show that recycling growth medium is a suitable solution to obtain good N. oleoabundans biomass concentrations, while providing a more sustainable ecological impact on water resources.

S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine, namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on polyamine metabolism, ethylene production and plant morphology was analysed in primary transformants (R0) and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls, all of which were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analysis, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and polyamine titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with polyamines the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and polyamine titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms of polyamine conjugation was higher and differentially affected in transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are discussed in relation to plant generation, culture conditions and response to stress.

Putrescine (5, 10 and 20 mM), spermidine (0.5, 1 and 2 mM) and aminoethoxyvinylglycine (AVG; 0.32, 0.64 and 1.28 mM) were applied to peach trees (Prunus persica L. Batsch cv Stark Red Gold) under open field conditions. Treatments were performed 28 (polyamines; PAs) and 21 or 7 (AVG) days before harvest at 115 dAFB. Both PAs and AVG reduced ethylene production of fruit, delayed loss of firmness, retained titratable acidity, and prevented the increase in dry matter (DM) and soluble solids concentration (SSC). Fruit drop was consistently reduced by AVG at both application times, and by the highest spermidine concentration. Endogenous PA levels in treated fruit were transiently affected (7 days after treatment), but returned to control values at harvest in both mesocarp and epicarp tissues. Northern analysis of ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO), and of those involved in PA biosynthesis, arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), was performed in mesocarp tissue at harvest. AVG and putrescine reduced the accumulation of ACO and SAMDC message, putrescine also influenced ACS transcript levels, while spermidine did not affect any of these genes. Results are discussed on the basis of the reduction by PAs and AVG of ethylene production, and confirm the capacity of these naturally occurring substances to modulate fruit ripening.

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.

Previous results indicated that in shoot-forming tobacco thin layers the putative inhibitor of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) activity methylglyoxal(bis-guanylhydrazone) (MGBG) inhibited meristemoid/primordia formation while enhancing conjugated polyamine accumulation and ethylene biosynthesis, thus showing an inverse relationship between the two phenomena ( Scaramagli et al. 1999). In order to better understand how MGBG causes polyamine overproduction and by using the same model system, we further examined the effect of 0.5 mM MGBG on the incorporation of labelled methionine into spermidine and spermine, on SAMDC transcript levels and activity and on putrescine oxidising (DAO) activity at different times in culture. MGBG-induced ethylene accumulation was also evaluated by gas chromatography (GC). In shoot-forming controls (grown on a medium supplemented with IAA+BA) label was mainly incorporated into free spermidine (82–86% of the total amount incorporated into polyamines) while only 6–12% of radioactivity was recovered in free spermine. Some label was also found in putrescine. In the presence of MGBG, spermidine biosynthetic capacity was enhanced throughout the culture by 28–45% compared with controls. The same occurred for spermine and putrescine. MGBG-enhanced spermidine synthesis was supported by increased SAMDC activity (days 1–2) and higher levels of SAMDC message (days 1–7). On the contrary, the effect of the drug on putrescine oxidation was inhibitory starting from day 2. These results, together with the previous ones, suggest that MGBG, in fact, does not behave as a SAMDC inhibitor in shoot-forming tobacco thin layers and that its negative effect on organised growth is not due to SAMDC inhibition/polyamine depletion.

Summary Aliphatic polyamines, putrescine, spermidine and spermine are ubiquitous in all living organisms. They occur in the free form as cations, but are often conjugated to small molecule like phenolics acids and also to various macromolecules. Polyamines and their biosynthetic enzymes may play an important role in many aspects of plants development including growth, differentiation, senescence and response to differents types of stress. Recent work suggest, but has not proven, a regulatory role of polyamines in these processes. Putrescine accumulation in response to stress, suggests a protective role. By contrast, increased spermidine and spermine synthesis appears essential for DNA replication and cell division.

The phytopathogenic actinomycete Rhodococcus fascians drives its host to form a nutrient-rich niche by secreting a mixture of cytokinins that triggers plant cell division and shoot formation. The discrepancy between the relatively low amount of secreted cytokinins and the severe impact of R. fascians infection on plant development has puzzled researchers for a long time. Polyamine and transcript profiling of wild-type and cytokinin receptor mutant plants revealed that the bacterial cytokinins directly stimulated the biosynthesis of plant putrescine by activating arginine decarboxylase expression. Pharmacological experiments showed that the increased levels of putrescine contributed to the severity of the symptoms. Thus, putrescine functions as a secondary signal that impinges on the cytokinin-activated pathway, amplifying the hormone-induced changes that lead to the formation of a leafy gall. Exogenous putrescine and treatment with polyamine biosynthesis inhibitors combined with transcript and polyamine analyses of wild-type and mutant plants indicated that the direct target of both the bacterial cytokinins and plant putrescine was the expression of D3-type cyclins. Hence, the activated d-type cyclin/retinoblastoma/E2F transcription factor pathway integrates both external and internal hormonal signals, stimulating mitotic cell divisions and inducing pathological plant organogenesis.

The optimal assay conditions and the trend with time in culture (28 days) of arginine decarboxylase (ADE; EC 4.1.1.19), omithine decarboxylase (ODC; EC 4.1.1.17) and diamine oxidase (DAO; EC 1.4.3.6) activities in habituated (H) and normal (N) auxin- and cytokinin-requiring sugar beet callus were compared. Although the response to variations in buffer pH and EDTA and pyridoxal phosphate (PLP) concentrations varied for ADC and ODC activities between the two callus types, pH 8.3, 50 μM PLP and 5 mM EDTA were generally optimal or near-optimal for both H and N callus. In most cases the addition of ornithine or arginine in the ADC and ODC assays, respectively, given to block the interconversion between the two substrates, resulted in lower 14CO2 recovery. DAO activity was very differently affected in H and N callus by the presence of polyvinylpyrrolidone in the extration buffer. However, in both cases, this activity increased with time in culure. ADC activity was always predominant in both cell lines and always higher in N callus. In the latter, ADC activity rose sharply between days 14 and 21 and then leveled off while in H callus it incresed steadily from day 14 onwards. ODC activity was also higher in N callus and peaked sharply on day 21 while in H callus it was not detectable in the second half of the culture period. In both cell lines this activity was low or nil on day 28. 3,4-[14C]-methionine incorporation into ethylene and polyamines was also compared in N and H callus. In the latter, ethylene synthesis was lower and [14C]-spermidine formation higher than in N callus. This is in accord with the significantly higher spermidine titres found in H callus.

Three ex situ collections of poplar clones from natural populations of Populus alba and P. nigra growing in northern Italy were assessed for their genetic dissimilarity (GD) by means of amplified fragment length polymorphism (AFLP). The high GD evidenced within populations was exploited for screening 168 clones in a field trial on heavy metal-polluted soil. After one growth season, clonal differences in plant survival and growth were observed. On the basis of performance, six clones were singled out, and used to evaluate copper and zinc accumulation in different organs. Clonal differences in metal concentrations were most evident for leaves and stems; one clone of P. alba (AL35) had a distinctly higher concentration of both metals in the roots. Leaf polyamine (putrescine, spermidine, spermine) profiles correlated with tissue metal concentrations, depending on the clone, plant organ and metal. In particular, the high metal-accumulating clone AL35 exhibited a dramatically higher concentration of free and conjugated putrescine. Overall, the results indicate that, given the high GD of Populus even within populations, it is possible to identify genotypes best suited for soil clean-up, and useful also for investigating physiological markers associated with high metal accumulation/tolerance.

Long-sized oligogalacturonides (OGs) are plant cell wall fragments involved in defence responses and developmental processes. A hormone/OG interaction in the control of different organogenic processes is known. However, hormones also modulate polyamine (PA) effects on organogenesis. Furthermore, OGs are known to affect mitotic activity leading to specific morphogenic events, and PAs are known to affect mitotic activity leading to xylogenesis. Thus, it may be reasonable to assume that OGs and PAs affect mitotic activity in the same cell types, and in the same hormone-induced morphogenic processes, e.g., xylogenesis. To gain further insight into this aspect, the effects of OGs, and of putrescine (Put) and spermidine (Spd), on auxin (indoleacetic acid, IAA) plus benzyladenine (BA)-induced morphogenesis in tobacco leaf explants were investigated histologically. The effect of PA biosynthetic inhibitors in the culture medium was also monitored, as well as the combined application of the inhibitor with ...

Long-sized oligogalacturonides (OGs) are cell wall fragments that induce defence and developmental responses. The Ca(2+)-dependent "egg-box" conformation is required for their activity, and polyamines may prevent them from adopting this conformation. Although OGs are known to inhibit auxin-induced growth processes, their effect on cytokinin-induced ones requires investigation. In the present work OGs were shown to promote cytokinin (benzyladenine, BA)-induced vegetative shoot formation from tobacco leaf explants, independent of the presence of CaCl(2) in the medium and of auxin (indoleacetic acid, IAA) supply. The effect of polyamines, putrescine (PU) and spermidine (SD) supplied with/without their biosynthetic inhibitors (DFMO, CHA) was also investigated, and showed that spermidine enhanced adventitious vegetative shoot formation, but only on medium containing Ca(2+) and IAA. Treatments with inhibitors blocked this promotive effect. OGs did not alter free polyamine concentrations, but caused a moderate increase of conjugated ones, and exhibited an early inhibitory effect on polyamine biosynthetic gene expression. OGs, but not SD, caused long-term changes in calcium-associated epifluorescent signals in the cell walls, and, later, inside the cells of specific tissues. Electron microscopy analysis (ESI system) demonstrated that calcium accumulated in the cell walls and vacuoles of OG-cultured explants. The relationship between OGs, cytokinin, calcium, and polyamines in adventitious vegetative shoot formation is discussed.

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. Copyright © 1990, Wiley Blackwell. All rights reserved

Jasmonates (JAs), which derive from α -linolenic acid through allene oxide synthase (AOS), are signal molecules which elicit stress responses through JA-induced proteins (JIPs). They are also involved in growth processes such as cell division, tuber formation and fruit development. JAs induce various biological responses mostly correlated with defense responses; however, their reciprocal relationships with ethylene are not well established. In apple and cherries endogenous JAs peak during the early phase of fruit growth; but only in apple they rise again at climacteric. A JA derivative, methyl jasmonate (MJ), stimulates ethylene production in pre-climacteric apples but inhibits it in climacteric ones. However, MJ-induced apple degreening is partly independent of ethylene. A better established relation is be that between JAs and PAs. PAs are ubiquitous growth regulators involved in fruit set and development; when applied to developing fruit they inhibit ethylene production and delay ripening in peach and other climacteric fruit. To better understand the relationships JA-ethylene in a climacteric fruit, in the present study, MJ and its synthetic analogous propyl jasmonate (PJ) were applied to peach (Prunus persica L. Batsch, cv Stark Red Gold) branches during S2 phase (rapid growth). In treated fruit ethylene production and the evolution of the main fruit quality parameters were inhibited and transcript levels of PpACO1, PpERS1 and PpETR1 were altered in comparison to respective controls. Increases in polyamine titres were associated to JA-induced ripening delay probably contributing to the observed antisenescent effect. A fragment of PpAOS cDNA was isolated and its expression monitored during fruit ripening showing that the corresponding transcript was enhanced in JA-treated fruit. Finally, a microarray comparative trascriptome profiling of treated versus control fruit indicated that MJ down-regulate genes positively involved in ripening thus confirming that JAs slow down such process in peach fruit.

An in vitro model system of micropropagated shoots of the commercial clone ‘Villafranca’ of Populus alba (L.) was used to investigate polyamine (PA) biosynthesis and accumulation in response to high concentrations of zinc (Zn) and copper (Cu). Based on leaf symptoms, rate of adventitious root formation and ethylene production, 0.5–1 mM Zn was transiently toxic while 2–4 mM Zn concentrations were increasingly toxic. Free and conjugated putrescine and spermidine accumulated in a dose–response manner and proportionally to toxicity. The expression profiles of the PA biosynthetic genes were analysed by reverse transcriptase polymerase chain reaction in plants exposed to Zn. In both leaves and stems, PaADC and PaODC transcript levels were early enhanced by all Zn concentrations, while those of PaSAMDC were not. In adventitious roots, free and conjugated PA levels also rose, although their composition and reciprocal ratios were different from those of leaves and no changes in transcript levels of PA biosynthetic genes were detected. Several Cu concentrations (5–500 mM) were tested in shoots showing severe leaf toxicity, strongly impaired adventitious root formation and high ethylene production. No changes in PA levels were detected until end of culture. The different timing of the PA response to Zn and Cu, despite their rapid uptake and translocation already at 24 h, is discussed in relation to the extent of leaf toxicity.

The time course of ethylene biosynthesis and perception was investigated in ripening peach fruit (Prunus persica) following treatments with the polyamines putrescine (Pu) and spermidine (Sd), and with aminoethoxyvinylglycine (AVG). Fruit treatments were performed in planta. Ethylene production was measured by gas chromatography, and polyamine content by high-performance liquid chromatography; expression analyses were performed by Northern blot or real-time polymerase chain reaction. Differential increases in the endogenous polyamine pool in the epicarp and mesocarp were induced by treatments; in both cases, ethylene production, fruit softening and abscission were greatly inhibited. The rise in 1-aminocyclopropane-1-carboxylate oxidase (PpACO1) mRNA was counteracted and delayed in polyamine-treated fruit, whereas transcript abundance of ethylene receptors PpETR1 (ethylene receptor 1) and PpERS1 (ethylene sensor 1) was enhanced at harvest. Transcript abundance of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) was transiently reduced in both the epicarp and mesocarp. AVG, here taken as a positive control, exerted highly comparable effects to those of Pu and Sd. Thus, in peach fruit, increasing the endogenous polyamine pool in the epicarp or in the mesocarp strongly interfered, both at a biochemical and at a biomolecular level, with the temporal evolution of the ripening syndrome.

1-Methylcyclopropene (1-MCP) inhibits ethylene action and fruit quality achievement by binding to ethylene receptors. Developmental stage, timing, concentration and storage temperature may modulate 1-MCP effects. Ethylene and the antisenescent growth regulators polyamines (PAs) share a common precursor and may exert opposite effects. Nectarines (Prunus persica L. Batsch ‘Stark Red Gold’) were harvested and treated with 1-MCP for 12 h at 25°C, and then stored for 3 days at 25 or 4°C. At 25°C, 1-MCP strongly inhibited ethylene production and Pp-ACO gene expression, and development of quality parameters (softening, SSC, loss of acidity). At 4°C, ethylene production was not inhibited, but enhanced on the third day. A transient increase in endogenous PAs is associated with 1-MCP treatment, which at 4°C is accentuated, suggesting a combined negative effect of 1-MCP and cold stress. In fact, the 1-MCP-induced increase in PA levels seems to be more correlated with the cold stress than with preventing post-harvest ripening. Thus, in nectarines a single dose of 1-MCP is sufficient to improve post-harvest storage at 25°C, but has a less beneficial effect at 4°C.

“Stark Red Gold” nectarines were harvested at 30 N of flesh firmness (FF) and 1 nl g −1 h −1 of ethylene production, and treated in sealed plastic containers with 1 μl l −1 (1 ppm) 1-methylcyclopropene (1-MCP) for 12 h at 25 °C. Treated and control fruit were then transferred either to a growth chamber at 25 °C or to a cold room at 4 °C for 3 days. At the end of treatment with 1-MCP; ethylene production in control fruit had increased relative to production at harvest, and this rise was abolished by the presence of the chemical. Moreover, treated fruit showed lower soluble solids content (SSC) and higher FF and titratable acidity (TA) compared to control ones, and putrescine and spermine levels were moderately enhanced in the mesocarp at the end of treatment. In contrast with the inhibition of ethylene production, 1-aminocyclopropane-1-carboxylate synthase (ACS) and especially 1-aminocyclopropane-1-carboxylate oxidase (ACO1 and ACO2) transcript levels were enhanced relative to controls. During storage, 1-MCP affected ethylene production and biosynthetic gene expression, fruit softening and other quality parameters in a temperature-dependent manner: in fruit held at 25 °C a strong decrease in ethylene production, a delay in ripening and lower ACS and ACO1/ACO2 levels were recorded, while in fruit held at 4 °C an opposite trend was observed. Results suggest that 1-MCP application followed by storage at 25 °C appears effective in controlling postharvest ripening. The lack of efficacy of the chemical in cold-stored fruit is discussed in relation to changes in SAMDC gene expression and putrescine accumulation in treated fruit relative to controls, which may be part of a stress response.

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H2O2-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H2O2 only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H2O2 produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.

Transcript levels and activities of the polyamine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19), ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), as well as free polyamine titres, were analysed throughout the four growth stages S1-S4 leading up to ripening in the mesocarp from peach fruit (Prunus persica L. Batsch cv. Redhaven) grown under field conditions. SAMDC mRNA, which was northern analysed by using a PCR-generated homologous SAMDC probe, and ADC mRNA levels appeared quite stable during fruit development, while ODC transcript accumulation showed a discontinuous trend. The pattern of transcript levels during growth did not correlate with that of the relative enzyme activity, which instead correlated well with free polyamine levels. Both exhibited maximum levels in S1 and a smaller peak in S3. The behaviour of the polyamine biosynthetic machinery is discussed in relation to the different cell growth rates occurring during fruit development.

In several in vitro systems, treatment with methyl jasmonate (MJ) stimulates polyamine biosynthesis as well as accumulation of acid-soluble and -insoluble conjugated polyamines. This effect is attributed to changes in gene expression and enzyme activity of arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S -adenosylmethionine decarboxylase (SAMDC). In the present study, we used tobacco ( Nicotiana tabacum L. cv. Samsun) leaf discs to investigate the interaction between this MJ-induced response and those triggered by indole-3-acetic acid (IAA), N 6 -benzyladenine (BA) and ethylene. Our results indicate that MJ-induced accumulation of conjugated polyamines is further stimulated by auxin and counteracted by BA. The MJ-induced stimulation of ODC and SAMDC, but not ADC, mRNA levels was diminished by IAA±BA, whereas the corresponding enzyme activities were further enhanced in the presence of hormones. MJ enhanced ethylene production only when combined with hormones, and this trend was reflected in 1-aminocyclopropane-1-carboxylate oxidase transcript levels. Results suggest that the induction by MJ of conjugated polyamine levels, and of the polyamine biosynthetic genes and activities are differentially modulated by hormones, and that ethylene does not seem to be directly involved in this response.

The effect of various concentrations of aminoethoxyvinylglycine (AVG; 0.32 and 1.28 mM), an ethylene biosynthesis inhibitor, and of the polyamines putrescine (10 mM), spermidine (0.1, 1 and 5 mM) and spermine (2 mM) 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 methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 microM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 microM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 microM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S:-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants.

Eggplant (Solanum melongena L. cv. Violetta lunga 2) cotyledon expiants grown on hormone-free medium (controls) or on medium containing either naphthaleneacetic acid alone (root forming) or in combination with zeatin riboside (shoot forming) showed minor differences in free polyamine titres during culture. In contrast, conjugated polyamines (particularly those in the trichloroacetic acid-soluble fraction) accumulated only in hormonetreated explants, but not in controls. The extent and the temporal changes in soluble-conjugate levels differed between root-forming and shoot-forming expiants; in the former, accumulation began earlier (within 1 day of culture) and reached the highest levels. In both organogenic programmes, maximum conjugate accumulation occurred just before and during organ emergence. Adventitious roots and shoots were formed along the cut surfaces. The regions closest to these (“borders”) displayed a significantly higher ratio of conjugated to free spermidine and/or putrescine than the nonorganogenic regions (“centres”) of the explant. Ornithine decarboxylase activity was higher than arginine decarboxylase activity both in control and hormone-treated explants. However, both activities increased markedly on day 2 of culture in the presence of hormones. Thereafter ornithine decarboxylase activity remained high in shoot-forming explants, but not in root-forming ones. Putrescine oxidising activity was also enhanced by exogenously supplied hormones starting from day 4 of culture. This activity remained high up to day 12 in the presence of auxin plus cytokinin, whereas it peaked on day 6 in auxin-treated explants. Spermidine oxidising activity was the only enzyme activity which was consistently higher in controls than in hormone-treated tissue. Differences between the two organogenic programmes with respect to temporal changes in polyamine content, and putrescine biosynthetic and oxidative activities are discussed in relation to the timing of organ formation. The latter was monitored both histologically and macroscopically.

The effects of two inhibitors of polyamine (spermidine and spermine) biosynthesis, cyclohexylamine (CHA; 5 and 10 mM) and methylglyoxal(bis-guanylhydrazone) (MGBG; 0.1, 0.5 and 1 mM), on the organogenic response in vegetative bud-forming tobacco (Nicotiana tabacum L. cv. Samsun) thin layer explants were evaluated micro- and macroscopically at different times during culture. The final number of buds formed and the percentage of organogenic explants was significantly reduced by both inhibitors, but much more so by MGBG than CHA. This inhibitory effect was already evident in MGBG-treated explants on day 5, in terms of the number of meristemoids per explant. On the contrary, in the presence of CHA, the number of meristemoids on day 5 was higher than that in the controls. Between days 9 and 13, meristemoid formation slowed down considerably in inhibitor-treated explants compared with controls. On day 13, the number of bud primordia was similar in control and CHA-treated explants, but significantly lower in MGBG-treated explants. This inhibitor also induced peculiar cytohistological events, such as a reduced formation of oval-shaped cell aggregates on the explant surface and more frequent cases of nucleolar extrusion, while CHA led to the appearance of hypertrophic epidermal cells; callus formation at the basal end of the explant and xylogenesis were also affected by the inhibitors. Ethylene biosynthesis, measured as [ C]methionine incorporation, was stimulated 2- (day 2) to 3-fold (15 h) by 0.5 mM MGBG, whereas CHA (10 mM) had little effect and aminoethoxyvinylglycine (AVG; 0.1 μM), an ethylene synthesis inhibitor, was strongly inhibitory. In control explants, the incorporation of labelled methionine into ethylene and spermidine followed an inverse trend up to day 8. In these explants, free putrescine increased 32-fold and spermidine increased about 10-fold between days 0 and 8. Trichloroacetic acid (TCA)-soluble conjugated putrescine also accumulated dramatically during culture. While CHA provoked a decline in spermidine levels, MGBG caused an unexpected increase in free spermidine and spermine titres; however, its most conspicuous effect was on the further enhancement of putrescine conjugate accumulation, while CHA and AVG had the opposite effect. Results are discussed in view of establishing a putative link between MGBG-enhanced ethylene synthesis, increased conjugate titres and inhibition of meristemoid formation.

Biondi, S., Torrigiani, P., Sansovini, A. and Bagni, N. 1988. Inhibition of polyamine biosynthesis by dicyclohexylamine in cultured cotyledons of Pinus radiata. - Physiol. Plant. 72: 471–476. The effect of 1 mAf dicyclohexylamine (DCHA) on the synthesis of spermidine and spermine was examined in excised cotyledons of radiata pine (Pinus radiata D. Don) cultured under shoot-forming (with cytokinin) and non-shoot-forming (minus cytokinin) conditions by incubation with [14C]-putrescine. In control cotyledons incorporation into spermidine showed a peak at day 2 in the presence and at day 5 in the absence of N6-benzyldenine (BA). DCHA-treated cotyledons gave the same labeling pattern, both in the presence and absence of benzyladenine, with a much smaller peak at day 2. The incorporation into spermidine and spermine was insignificant at day 5 and later. The total radioactivity in the trichloroacetic acid supernatant indicated that precursor uptake was strongly reduced by the drug. In addition, the percentage label found in the benzene phase and combined in the 3 polyamines was lower in DCHA-treated cotyledons. Thus, treatment with DCHA not only inhibited the conversion from putrescine to spermidine and spermine, but also reduced its conversion to other benzene-extractable compounds. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity, which furnishes the propylamine group to spermidine and spermine synthases (EC 2.5.1.16 and EC 2.5.1.-), was not significantly affected by DCHA and appeared to be independent of the spermidine and spermine synthase reactions, suggesting that spermine synthesis decreased as a result of substrate depletion. The correlation between morphological development and polyamine biosynthesis is discussed.

Summary The biochemical characterization of a carrot cell line repeatedly subcultured on a medium containing 5 mM α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, is reported. The cells grown on DFMO had an identical growth rate to the controls but a larger surface area that decreased with increasing numbers of subcultures. During the exponential phase of growth the DFMO treated cells displayed an increase in polyamine and protein content and a decrease in arginine and ornithine decarboxylase activities. The uptake of the inhibitor was slower in treated cells, and occurred against a concentration gradient. After the 12th subculture the amount of 14C-DFMO-bound to the protein fraction of crude cell extracts was followed over a 50 h period after the 12th subculture. The ratio between free and bound labelled DFMO was initially the same in both control and treated cells but increased with time in the latter probably as a result of the high endogenous pool of DFMO accumulated.

Summary In the present work ornithine-, arginine- and S-adenosylmethionine decarboxylase activities were found in chloroplasts of Pinus radiata D. Don cotyledons and in isolated mitochondria from activated slices of Helianthus tuberosus L. tubers. Buffer molarity and pH optima were determined for each enzyme. In organelles different ratios between free polyamine contents were found with respect to the entire cell. Most of the S-adenosylmethionine decarboxylase activity was found in the 26,000 g supernatant of both plants where arginine and ornithine decarboxylase activities were markedly lower. Arginase activity was also detected in mitochondria. The different ratios between the enzyme activities in the various subcellular fractions of both systems may be an indication that these are effectively compartmentalized.

Excised cotyledons of radiata pine (Pinus radiata D. Don) were cultured in the presence or absence of benzyladenine and two concentrations of dicyclohexylamine, a potent inhibitor of spermidine synthesis in animals and bacteria. Cellular levels of the drug and of putrescine, spermidine and spermine were determined by dansylation followed by thin layer chromatography. The rate of uptake of the drug was rapid during the first 6 h of incubation and then diminished; nevertheless, its cellular content increased with time in culture and uptake was greater if more was present in the medium. Cotyledons cultured on benzyladenine-containing media accumulated more dicyclohexylamine than on benzyladenine-free media and this resulted in toxic side-effects. The drug had no significant effect on the elongation growth occurring in the absence of the hormone. In cotyledons treated with the drug, spermidine levels fell to zero after a 5- to 10-day exposure. Putrescine increased transiently at 24 h and then declined significantly. Spermine levels also declined to 11% of control values by day 5 and remained low throughout. Except for a marked decrease in all three polyamines during the first 24 h of culture, all control cotyledons maintained significantly higher polyamine levels than dicyclohexylamine-treated ones.