| Abstract |
Polyamines spermidine and spermine and their obligatory precursor, putrescine, are small aliphatic amines, ubiquitous in living organisms. Even though their exact role and function is not yet understood, many studies relate them with various physiological procedures, including cellular development and differentiation to stress-induced responses. The preliminary and determinant stage of polyamine biosynthesis is the decarboxylation of ornithine and arginine from the homonymous decarboxylases, to putrescine and agmatine, respectively. In the present study, the regulation of polyamine biosynthesis from arginine decarboxylase (ADC) was examined. This was done in a woody perennial plant species, the grapevine (Vitis vinifera L.), which does not respond to in vitro morphogenetic manipulations. The optimum pH and temperature for grapevine enzyme were 8.0 and 37 oC, respectively. Grapevine ADC demanded the presence of soylfhydryl-groups and pyridoxal phosphate in order to exhibit maximum activity but there was no dependence on any metal factor. Agmatine and putrescine inhibited enzyme activity in relatively high concentrations. ADC activity was high in tissues with high metabolic activity and low in roots. In grapevine seedlings, ADC activity was high at the early stages of germination and was reduced later, similar to suspension cultures and calluses immediately after subculture. The deduced amino acid sequence of an isolated ADC cDNA clone (EMBL, accession no X96791) consists of 630 amino acid residues (about 100 amino acids are missing from the N-terminal) and includes the characteristic patterns of group IV of ornithine/ Dap/ arginine decarboxylases. The clone exhibits 70-75 % identity with other dicot ADCs, 40 % identity with the monocot oat ADC and 30 % identity with bacterial ADCs. Southern blot analysis supports the presence of a single ADC gene in grapevine genome. Northern analysis of total RNA from different grapevine tissues indicates that grapevine ADC gene is expressed as a single transcript of approximately 3,1 kb with a tissue specific expression pattern. The addition of ammonium ion and glutamine to grapevine cell suspension cultures increased the in vitro ADC activity to a larger extent than the addition of arginine and glutamate. On the contrary, ADC activity was not affected by the addition of nitrates whereas it was reduced by the addition of putrescine. The changes in ADC activity were not accompanied by analogous changes in the ADC transcript levels. The endogenous free putrescine and arginine titers followed the changes in ADC activity. Antibody prepared against ADC detected a protein band of about 80 kDa in size, in total protein extracts from different grapevine tissues. Protein extracts from leaves and shoots in different developmental stages, presented differences in the quantity of the 80-kDa band, but these differences did not follow the changes in ADC specific activity. Furthermore, protein extracts from cell suspension cultures treated with ammonium or nitrate ions or putrescine, with pronounced differences in ADC specific activity, did not show equivalent quantitative differences in the 80-kDa protein band. From the above results it is concluded that changes in ADC activity are not due to induction or suppression of its gene. Therefore, ADC regulation in grapevine is post-transcriptional and possibly post-translational as for other enzymes of polyamine biosynthesis in plants and other kingdoms.
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