Application of Cadaverine to Inhibit Biotin Biosynthesis in Plants

Abstract

Biotin is an essential vitamin in plants. However, characterization of biotin deficiency has been limited by embryo lethality in mutants, which can only be rescued by supplementation of biotin. Here, we describe a protocol to characterize biotin deficiency in Arabidopsis thaliana through application of the polyamine cadaverine. Cadaverine induces changes in primary root growth. Protein biotinylation in Arabidopsis seedlings can be quantified through an assay similar to a western blot, in which protein biotinylation is detected by a streptavidin probe. This technique provides a chemical means of inhibiting biotin synthesis, allowing for further characterization of biotin deficiency on a physiological and molecular level.

Keywords: Arabidopsis thaliana; Biotin; Cadaverine; Primary root growth; Streptavidin.

Figure 1.. Experimental scheme for detection of biotinylated proteins in Arabidopsis.

To analyze the biotinylation of Arabidopsis proteins, seedlings are homogenized in protein extraction buffer and separated by SDS Page. Following transfer to a membrane, the membrane is probed with a streptavidin dye, which allows for visualization of the biotinylated proteins on the membrane. The signal intensity of each band on the membrane can be quantified, to determine the level of biotinylated protein in the corresponding sample.