Downregulation of ethylene production and biosynthetic gene expression is associated to changes in putrescine metabolism in shoot- forming tobacco thin layers Patrizia Torrigiani a, *, Sonia Scaramagli a , Stefano Castiglione b , Maria Maddalena Altamura c , Stefania Biondi a a Dip. Biologia e.s., Universita ` di Bologna, Via Irnerio 42, 40126 Bologna, Italy b Dip. Biologia, Universita ` di Milano, Via Celoria 26, 20133 Milan, Italy c Dip. Biologia Vegetale, Universita ` La Sapienza, P.le A. Moro, 00185 Rome, Italy Received 29 October 2002; received in revised form 14 January 2003; accepted 26 February 2003 Abstract 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. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aminoethoxyvinylglycine; 1-Aminocyclopropane-1-carboxylic acid; Diamine oxidase; Ethylene biosynthesis; Polyamine biosynthesis; Tobacco 1. Introduction The interactions between the different classes of plant growth regulators are numerous and concomitant, making the dissection of the various components difficult. The case polyamines  /ethylene is intriguing but facilitated as they share a common precursor, S - adenosylmethionine (SAM). Only a small amount of the latter, which is the universal donor in transmethylation reactions, can indeed be decarboxylated to yield sper- midine and spermine or converted to 1-aminocyclopro- pane-1-carboxylic acid (ACC) by ACC synthase to produce ethylene via ACC oxidase [1]. Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ACO, 1-aminocyclopropane-1-carboxylate oxidase; ACS, 1- aminocyclopropane-1-carboxylate synthase; ADC, arginine decarboxylase; AVG, aminoethoxyvinylglycine; BA, benzyladenine; decaSAM, decarboxylated S -adenosylmethionine; DAO, diamine oxidase; DTT, dithiothreitol; IAA, indol-3-acetic acid; ODC, ornithine decarboxylase; ORF, open reading frame; PLP, pyridoxal phosphate; SAM, S -adenosylmethionine; SAMDC, S - adenosylmethionine decarboxylase; TCA, trichloroacetic acid. * Corresponding author. Tel.:  /39-051-209-1291; fax:  /39-051- 242-576. E-mail address: torrigia@alma.unibo.it (P. Torrigiani). Plant Science 164 (2003) 1087  /1094 www.elsevier.com/locate/plantsci 0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00115-8