No abstract

The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

Stable expression of foreign genes was achieved in sweet potato (Zpomoea batatas (L.) Lam) plants using an Agrobacterium tumefaciens mediated system. Embryogenic calluses produced from apical meristems of the cultivar White Star were multiplied and cocultivated with A. tumefizciens strain EHAlOl harboring a binary vector containing the P-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes. The calluses were transferred to selective regeneration medium and kanamycin resistant embryos were recovered which developed into morphologically normal plants. Histochemical and fluorimetric GUS assays of plants developed from the kanamycin resistant embryos were positive. Amplified DNA fragments were produced in polymerase chain reactions using GUS-specific primers and DNA from these plants. Transformation was confirmed by Southern analysis of the GUS gene. With the developed method, transgenic sweet potato plants were obtained within 7 weeks. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.

Arginine decarboxylase (ADC; EC 4.1.1.19) is a key enzyme in one of the two possible ways to synthesize putrescine (Put) in plants. In previous work (Masgrau et al. 1997), we observed an altered phenotype (growth inhibition, leaf chlorosis and necrosis) in tobacco transgenic plants (Nicotiana tabacum L. var. Wisconsin-38) containing the oat ADC cDNA under the control of a tetracycline inducible promoter, the severity of which was correlated with Put content. Now we have analysed the T2 generation of a selected transgenic line (line 52), which in previous generations was characterized by presenting a moderate increase in ADC activity and polyamine levels, but no phenotype alterations. Studying two selected individuals, one with a high expression level of the transgene and the other with a moderate expression level, we demonstrate that only the one with increased polyamine content displays the altered (toxic) phenotype. The possible causes of toxicity have been analysed. The results suggest that either Put or its oxidation products, via diamine oxidase (DAO; EC 1.4.3.6), are the responsible factors for the deleterious effects observed in the transgenic plants.

Introduction of foreign genes into plant tissues via Agrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L.), five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors.