2. Academic Validation
  3. Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants

Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants

  • Methods Mol Biol. 2016;1405:121-35. doi: 10.1007/978-1-4939-3393-8_12.
Inês Carqueijeiro 1 2 3,  Viviana Martins 1 2 4 5,  Henrique Noronha 4 5,  Hernâni Gerós 4 5,  Mariana Sottomayor 6 7 8
Affiliations

Affiliations

  • 1 Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
  • 2 IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180, Porto, Portugal.
  • 3 Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007, Porto, Portugal.
  • 4 Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas (CITAB-UM), Braga, Portugal.
  • 5 Grupo de Investigação em Biologia Vegetal Aplicada e Inovação Agroalimentar, Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
  • 6 Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal. [email protected].
  • 7 IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180, Porto, Portugal. [email protected].
  • 8 Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007, Porto, Portugal. [email protected].
PMID: 26843171 DOI: 10.1007/978-1-4939-3393-8_12
Abstract

The characterization of membrane transport of specialized metabolites is essential to understand their metabolic fluxes and to implement metabolic engineering strategies towards the production of increased levels of these valuable metabolites. Here, we describe a set of procedures to isolate tonoplast membranes, to check their purity and functionality, and to characterize their transport properties. Transport is assayed directly by HPLC analysis and quantification of the metabolites actively accumulated in the vesicles, and indirectly using the pH sensitive fluorescent probe ACMA (9-amino-6- chloro-2-methoxyacridine), when a proton antiport is involved.

Keywords

ABC transporters; ACMA; Alkaloids; BY-2 cells; Catharanthus roseus; H+-antiport; MATE transporters; Tobacco; Tonoplast vesicles; Transmembrane pH gradient; Transmembrane transport; Uptake assay.

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