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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.

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.

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

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.

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.