Análises moleculares das vias de sinalização de morte celular originadas no retículo endoplasmático

• Autor(a) principal: Reis, Pedro Augusto Braga dos
• Data de Publicação: 2010
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2419
• topic: Vias de sinalização; Retículo endoplasmático; Morte celular; Pathways signalling; Endoplasmic reticulum; Cell death; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

Abiotic and biotic stresses are responsible for major losses in agricultural productivity world wise. Therefore, the understanding of mechanisms of plant stress response and adaptation to such conditions should provide the foundation for engineering plant stress tolerance and hence maximizing the yield of crops growing under adverse environmental conditions. Recently, we identified a novel signaling pathway, mediated by the asparagine -rich proteins (NRPs), that transduces cell death signals emanated from ER and osmotic stresses. This novel signaling response was designated integrative pathway since it integrates the ER- and osmotic-stress signals into a common adaptive response. The goals of the present investigation were two-fold: (i) to identify components of the integrative pathway and (ii) to analyze the BiP function, a regulator of ER-stress response pathways, as a modulator of cell death in plants and its possible connection with the integrative pathway. Initially, we constructed a two-hibrid cDNA library of soybean mRNAs prepared from leaves stressed with tunicamycin, a potent inducer of ER-stress, and PEG that mimics osmotic stress. By screening the cDNA library with NRP-B as bait, we identified a soybean protein that belongs to the UspA (universal stress protein) family that was designated here as GmUspA. GmUspA-NRP-B interaction was mediated by the carboxyl-terminal of the protein and full-length GmUspA failed to Interact with NRP-B by the two-hybrid system in yeast. This result suggests that complex formation in vivo may be dependent on induced-conformational changes mediated by bound proteins. Like NRP-A and NRP-B, GmuspA was up-regulated by ER- and osmotic-stress although with a different induction kinetics.Furthermore, we demonstrated by confocal microscopy that GmUspA fused toGFP is located to the cytoplasm favoring the possibility of interactions with membrane-associated proteins, like NRP-B. Thepreviously identified membrane localization of NRP-B was further confirmed here by immunoblottings of microssomal fractions. Taken together, these results indicate that NRP-B -GmUspA interaction may occur in vivo and may be biologically relevant. In order to evaluate the role of BiP as modulator of programmed cell death (PCD) in plants, the BiP levels were manipulated in transgenic lines challenged with chemical inducers of cell death. We demonstrated that BiP overexpression attenuated cell death caused by ER stress, osmotic stress and cycloheximide induced stress. Ectopic expression of BiP in soybean transgenic lines reduced cotyledon leaf necrotic lesions promoted by the cell death inducers tunicamycin and cycloheximide, and maintained shoot turgidity under PEG-induced dehydration. The BiP-mediated attenuation of cell death was confirmed by the lack of induction and in some cases by delayed expression of senescenceassociated genes in BiP-overexpressing transgenic lines. This phenotype of attenuated cell death was coordinated with a decrease or delay in the induction of the cell-death genes NRP-A and NRP-B, which are involved in transducing a PCD signal emanated from ER stress and osmotic stress. The possible involvement of BiP in modulating NRPs-mediated cell death responses was directly examined through transient expression assays mediated by agroinoculation in tobacco leaves with enhanced (sense lines) or suppressed (antisense lines) expression of BiP. BiP overexprssion in sense lines prevented NRPs-induced chlorosis in agroinoculated leaf sectors, whereas silencing of BiP in antisense lines accelerated the onset of leaf senescence. These cell death phenotypes were confirmed by measuring chlorophyll loss and expression of senescence-associated genes in sense and antisense lines. Collectively, these results indicate a direct co-relation between the levels of BiP and NRPs-induced senescence and argue favorably for an involvement of BiP in modulating the NRPs-mediated PCD response.