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Immunoanalysis of dehydrins in Araucaria angustifolia embryos

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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 lysine-rich 15-amino acid sequence extensively used to recognize proteins immunologically related to the dehydrin family. Western blot analysis of the heat-stable protein fraction, as estimated by 15 % SDS-PAGE, revealed three main bands of approximately 20-, 26-, and 29-kDa; when 17.5 % SDS-PAGE 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 species.

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Acknowledgments

We 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 S.M.), 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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Authors and Affiliations

  1. Plant Developmental Physiology and Genetics Laboratory Graduate Program in Plant Genetic Resources, Federal University of Santa Catarina, 88034-000, Florianópolis, SC, Brazil

    Francine Lunardi Farias-Soares & Miguel Pedro Guerra

  2. Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA, Buenos Aires, Argentina

    Hernán Pablo Burrieza & Sara Maldonado

  3. Curitibanos Campus, Federal University of Santa Catarina, 89550-000, PO Box 101, Curitibanos, SC, Brazil

    Neusa Steiner

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  1. Francine Lunardi Farias-Soares
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  2. Hernán Pablo Burrieza
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  3. Neusa Steiner
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  4. Sara Maldonado
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  5. Miguel Pedro Guerra
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Correspondence to Sara Maldonado.

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Handling Editor: Friedrich W. Bentrup

Francine Lunardi Farias-Soares and Hernán Pablo Burrieza contributed equally.

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Farias-Soares, F.L., Burrieza, H.P., Steiner, N. et al. Immunoanalysis of dehydrins in Araucaria angustifolia embryos. Protoplasma 250, 911–918 (2013). https://doi.org/10.1007/s00709-012-0474-7

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