Immunoanalysis of dehydrins in Emphasis Type="Ita

Authors: Francine Lunardi FariasSoares Hernán Pablo Burrieza Neusa Steiner Sara Maldonado Miguel Pedro Guerra

Publish Date: 2012/12/21

Volume: 250, Issue: 4, Pages: 911-918

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Abstract

The aim of this study was to describe the dehydrin content of mature Araucaria angustifolia embryos a species of endangered and economically important conifers native to southern Brazil northeastern Argentina and eastern Paraguay The A angustifolia seeds have been categorized as recalcitrant Dehydrins were studied by western blot analysis and in situ immunolocalization microscopy using antibodies raised against the K segment a highly conserved lysinerich 15amino acid sequence extensively used to recognize proteins immunologically related to the dehydrin family Western blot analysis of the heatstable protein fraction as estimated by 15  SDSPAGE revealed three main bands of approximately 20 26 and 29kDa when 175  SDSPAGE was used each band resolved into two other bands Two thermosensitive dehydrin bands of around 16 and 35 kDa were common to the axis and cotyledons and another thermosensitive band with molecular mass of approximately 10 kDa was present in the cotyledons only Following alkaline phosphatase AP treatment a gel mobility shift was detected for each one of the four main bands that can be due to phosphorylation Dehydrins were detected in all axis and cotyledon tissues using in situ immunolocalization microscopy At the subcellular level dehydrins were immunolocalized in the nuclei protein bodies and microbodies In the nucleus dehydrins were found to be associated with chromatin We concluded that the gel mobility shift for the four main bands probably due to phosphorylation the presence of thermosensitive bands and the specific localizations in nuclei and protein bodies provide key starting points to understand the function of dehydrins in the embryo cells of this speciesWe would like to thank Timothy Close for supplying the antidehydrin antiserum and peptide salt Funding for this work was provided by the Universidad de Buenos Aires X125 to SM Ministerio de Ciencia y Tecnología/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior MINCyT/CAPES 08/10 to SM and MPG and Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina FAPESC to MPG For their fellowships FLF thanks the CNPq and FAPESC and HPB the Universidad de Buenos Aires

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