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.
The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues. Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.
The maize abscisic acid (ABA)-responsive gene rab28 has been shown to be ABA-inducible in embryos and vegetative tissues, expression being mostly restricted to vascular elements during late embryogenesis. In the course of an expressed sequence tags (ESTs) programme, we have isolated an Arabidopsis thaliana gene, Atrab28, encoding the orthologue of maize rab28. The Atrab28 cDNA is 1090 bp long, including a poly(A)+ stretch, and encodes a polypeptide of 262 amino acids. Atrab28 antibody against the recombinant protein recognizes a polipeptide of about 30 kDa and pI 6, in close agreement with the predicted molecular mass and pI. As for maize rab28, expression studies with Atrab28 revealed high specificity for embryo tissues, transcription being stimulated by the transcriptional activator abi3. In contrast, Atrab28 was not induced in vegetative tissues by ABA, osmotic stress or dehydration. The expression of Atrab28 mRNA and the accumulation of Atrab28 protein was largely restricted to provascular tissues of mature embryos and in the seed coat outer tegument and embryo and silique epidermis, as revealed by in situ hybridization and immunocytochemistry with anti-Atrab28 antibodies.
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