Analyzing the In Vivo Status of Exogenously Applied Auxins: A HPLC-Based Method to Characterize the Intracellularly Localized Auxin Transporters
Exogenous application of biologically important molecules for plant growth promotion and/or regulation is very common both in plant research and horticulture. Plant hormones such as auxins and cytokinins are classes of compounds which are often applied exogenously. Nevertheless, plants possess a well-established machinery to regulate the active pool of exogenously applied compounds by converting them to metabolites and conjugates. Consequently, it is often very useful to know the in vivo status of applied compounds to connect them with some of the regulatory events in plant developmental processes. The in vivo status of applied compounds can be measured by incubating plants with radiolabeled compounds, followed by extraction, purification, and HPLC metabolic profiling of plant extracts. Recently we have used this method to characterize the intracellularly localized PIN protein, PIN5. Here we explain the method in detail, with a focus on general application.
Key wordsAuxin Auxin metabolism BY-2 tobacco cells PIN proteins HPLC Plant cell cultures
This work was supported by grants from the Czech Ministry of Education (MSM0021622415), the Grant Agency of the Academy of Sciences of the Czech Republic (IAA601630703), and the Odysseus program of Research Foundation-Flanders (JF), Grant Agency of the Czech Republic, projects n. P305/11/0797 (JP), and P305/11/0797 (EZ). We thank Annick Bleys for critical reading of this chapter.
- 3.Mravec J, Skůpa P, Bailly A, Hoyerová K, Křeček P, Bielach A, Petrášek J, Zhang J, Gaykova V, Stierhof Y-D, Dobrev PI, Schwarzerová K, Rolčík J, Seifertová D, Luschnig C, Benková E, Zažímalová E, Geisler M, Friml J (2009) Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature 459:1136–1140PubMedCrossRefGoogle Scholar
- 4.Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int Rev Cytol 132:1–30Google Scholar