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.

S-Adenosyl-L-methionine decarboxylase (AdoMetDC; EC 4.1.1.50) is one of the key regulatory enzymes in the biosynthesis of polyamines. Isolation of genomic and cDNA sequences from rice and Arabidopsis had indicated that this enzyme is encoded by a small multigene family in monocot and dicot plants. Analysis of rice, maize and Arabidopsis AdoMetDC cDNA species revealed that the monocot enzyme possesses an extended C-terminus relative to dicot and human enzymes. Interestingly, we discovered that all expressed plant AdoMetDC mRNA 5' leader sequences contain a highly conserved pair of overlapping upstream open reading frames (uORFs) that overlap by one base. The 5' tiny uORF consists of two or three codons and the 3' small uORF encodes 50-54 residues. Sequences of the small uORFs are highly conserved between monocot, dicot and gymnosperm AdoMetDC mRNA species and the C-terminus of the plant small uORFs is conserved with the C-terminus of nematode AdoMetDC uORFs; such a conserved arrangement is strongly suggestive of a translational regulatory mechanism. No introns were found in the main AdoMetDC proenzyme ORF from any of the plant genes encoding AdoMetDC, whereas introns were found in conserved positions flanking the overlapping uORFs. The absence of the furthest 3' intron from the Arabidopsis gene encoding AdoMetDC2 suggests that this intron was lost recently. Reverse-transcriptase-mediated PCR analysis of the two Arabidopsis genes for AdoMetDC indicated that AdoMetDC1 is abundant and ubiquitous, whereas the gene for AdoMetDC2 is expressed preferentially in leaves and inflorescences. Investigation of recently released Arabidopsis genome sequences has revealed that in addition to the two genes encoding AdoMetDC isolated as part of the present work, four additional genes are present in Arabidopsis but they are probably not expressed.

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.

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Summary• 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). [ 3 H]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.

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.

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.