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DNA methylation and proteome profiles of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures as affected by plant growth regulators supplementation

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Abstract

Araucaria angustifolia is a critically endangered conifer native to South America, and somatic embryogenesis (SE) is one of the most promising biotechnological tools for its conservation and mass propagation. In vitro tissue culture and chemical compounds supplemented to culture medium, especially plant growth regulators (PGRs), are known to affect DNA methylation and protein expression profiles, modulating the phenotype and/or the embryogenic potential. Here, we evaluated the global DNA methylation (GDM) levels of A. angustifolia embryogenic cultures (EC) during SE induction and multiplication steps for 1 year subcultures, and identified a wide range of differentially expressed proteins in PGR-free or -supplemented treatments. During long-term subcultures, PGR-supplementation proved to gradually increase the GDM, which may compromise genomic stability and evoke gene expression modifications. Label-free proteomics enabled a robust protein identification and quantification in A. angustifolia EC. Exclusively expression of PIN-like protein in PGR-supplemented treatment indicated a possible differential response of the EC to polar auxin transport, which can generate implications in its morphogenetic response to maturation step. Up-regulation of stress-related proteins in EC from PGR-supplemented treatment suggests its more stressful environment, triggering notable responses to hormonal, osmotic and oxidative stresses. Improved expression of proteins involved with protein folding and stabilization processes in PGR-free treatment could play a protective function in response to stress conditions caused by in vitro culture, and may provide an adaptive advantage to these EC. The expression of several proteins associated to terpenoid biosynthesis suggests that EC from both treatments and cell lines are possibly producing these compounds.

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Acknowledgments

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 478393/2013-0, and 306126/2013-3), and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC 14848/2011-2, 3770/2012, and 2780/2012-4). The authors are also grateful to the Unidade de Biologia Integrativa (BioInt) of Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF) for the support in the shotgun proteomics analysis.

Author contribution statement

Conceived and designed the experiments: HPFF, LNV and MPG; Performed the in vitro culture experiments and DNA methylation analysis: HPFF, LNV and CCP; Performed the label-free proteomics and data analysis: HPFF, ASH and VS; Contributed reagents/materials/analysis tools: VS and MPG; Wrote the paper: HPFF, LNV and MPG.

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  1. Laboratório de Fisiologia do Desenvolvimento e Genética Vegetal, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Florianópolis, SC, 88034-001, Brazil

    Hugo P. F. Fraga, Leila N. Vieira, Catarina C. Puttkammer & Miguel P. Guerra

  2. Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, 28013-602, Brazil

    Angelo S. Heringer & Vanildo Silveira

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  1. Hugo P. F. Fraga
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  2. Leila N. Vieira
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  3. Angelo S. Heringer
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  4. Catarina C. Puttkammer
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  6. Miguel P. Guerra
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Correspondence to Miguel P. Guerra.

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Fraga, H.P.F., Vieira, L.N., Heringer, A.S. et al. DNA methylation and proteome profiles of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures as affected by plant growth regulators supplementation. Plant Cell Tiss Organ Cult 125, 353–374 (2016). https://doi.org/10.1007/s11240-016-0956-y

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