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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.

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.

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.