Downregulation of ethylene production and biosynthetic gene expression is associated to changes in putrescine metabolism in shoot-forming tobacco thin layers.

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 mM indol-3-acetic acid (IAA) plus 10 mM 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 mM) 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

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers

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 muM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 muM) 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 muM 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

De Novo Root Formation in Tobacco Thin Layers is Affected by Inhibition of Polyamine Biosynthesis

The effect of various inhibitors of polyamine biosynthesis on free and bound polyamine accumulation and on rhizogenesis was followed in thin layers excised from the stem of Nicotiana tabacum L. cv. Samsun. Trichloroacetic acid-soluble (TCA-soluble) free, TCA-soluble bound, and TCA-insoluble bound putrescine and spermidine accumulated throughout the culture period in control explants, reaching a peak on days 15-18. All the inhibitors tested depressed the rhizogenic process, though to a different extent, while leaving most of the explants viable. DL-alpha-difluoromethylornithine plus DL-alpha-difluoromethylarginine (DFMO + DFMA), specific irreversible inhibitors of ornithine- and arginine decarboxylases, respectively, almost totally inhibited rhizogenesis and polyamine accumulation. DFMO alone allowed a certain concentration of putrescine and spermidine to accumulate and rhizogenesis to occur in 20% of the explants. Cyclohexylamine (CHA), a competitive inhibitor of spermidine synthesis, did not have a strong inhibitory effect on root formation but depleted free and bound spermidine almost totally. Methylglyoxal-bis(guanyl)hydrazone (MGBG), an inhibitor of spermidine synthesis, caused a strong reduction in rhizogenesis but an accumulation of polyamines at the end of the culture period. No noticeable reversion of the rooting inhibition was observed by supplying the drug together with the polyamine whose synthesis was inhibited. The uptake and accumulation of exogenously supplied polyamines throughout the culture period was followed. The involvement of the three classes of polyamines in rhizogenic response is discussed in relation to the effect of the inhibitors of their biosynthesis