Proteina desacopladora de plantas : efeito do estresse oxidativo na sua expressão e atividade e reconstituição funcional da isoforma de Arabidopsis thaliana

AUTOR(ES)
DATA DE PUBLICAÇÃO

2003

RESUMO

The present work is divided in two parts: the functional reconstitution of the Arabidopsis thaZiana uncoupling protein 1 (AtPUMPI) and the expression and activity of the uncoupling protein under several abiotic stresses. AtPUMPI expression in Saccharomyces cerevisae and Escherichia coZi allowed us to confirm that this gene codes for a true uncoupling protein, able to divert energy of the proton electrochemical gradient. In a thermoneutral environment (28°C), AtPUMPI expressing cells grew slower than control cells, while under low temperatures (14 °C) growth rates were similar. When expressed in a prokaryotic system and reconstituted in lipid vesicles, AtPUMPI showed similar biochemical patterns to those already described for other uncoupling proteins. Ir transport was induced by severa! fatty acids and was dependent on the length and insaturations of the alkyl chain of the substrates. Pmine nucleotides were able to inhibit this proton transport, while changes in external pH alter AtPUMPl affinity for the nucleotides. Reconstitution experiments helped us to determine the Km and the Vmax for AtPUMPl. The values are within the physiologicallevels and very similar to those already obtained for other uncoupling proteins. We also studied the expression and activity of the uncoupling protein under situations of abiotic stresses, such as salinity, low temperature (chilling) and hyperoxygenated atmosphere. These situations are known to generate an oxidative stress. In maize coleoptiles, ZmPUMP mRNA expression increases under salt stress and does not change under chilling. However, protein expression and availability of substrates increased significantly on both situations, suggesting a post - transcriptional regulation for this gene. Moreover, mitochondria isolated ftom salt and cold stressed maize coleoptiles showed smaller ADP/O ratio, which returns to controllevels in the presence of BSA or GTP. In tomatoes submmited to a hyperoxygenated atmosphere, PUMP concentration and AOX activity patterns were similar to those already described for post-harvest ripening. However, preliminary experiments show increase in lipid peroxidation, resulting in a release of ftee fàtty acids, and consequent AOX inhibition and PUMP activation, even with a decrease in PUMP protein contento Under situations ofhigh intracellular Ca2+, we are able to observe also that uncoupling protein activators inhibit the mitochondrial generation of Reactive Oxygen Species (ROS) and that its inhibitors increased this generation. Considering all the above results, we ean conelude that AtPUMPI is a true uneoupling protein able to influenee eell growth through an uncoupling mechanism mediated by fatty aeids and inhibited by purine nucleotides. It is also possible to suggest that plant uneoupling proteins have a signifieant role under abiotic stress situations, decreasing mitoehondrial ROS generation and thus proteeting the tissue ttom the resulting oxidative stress

ASSUNTO(S)

bioquimica vegetal calor - efeito fisiologico biologia molecular

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