PIN2 Turnover in Arabidopsis Root Epidermal Cells Explored by the Photoconvertible Protein Dendra2

• DOI: 10.1371/journal.pone.0061403
• Publication Date: 2013-04-18T21:18:03Z
• Authors: Ján Jásik; Barbara Boggetti; František Baluška; Dieter Volkmann; Thomas Gensch; Twan Rutten; Thomas Altmann; Elmon Schmelzer
• Keywords: 0301 basic medicine; Science; Recombinant Fusion Proteins; Arabidopsis; Cyclopentanes; Plant Roots; 03 medical and health sciences; Dimethyl Sulfoxide; Anaerobiosis; Oxylipins; Cycloheximide; Microscopy, Confocal; Arabidopsis Proteins; Q; Cell Membrane; R; Darkness; Plants, Genetically Modified; Cold Temperature; Luminescent Proteins; Dactinomycin; Medicine; info:eu-repo/classification/ddc/500; Research Article; Abscisic Acid

The steady state level of integral membrane proteins is dependent on a strictly controlled delivery and removal. Here we show that Dendra2, a green-to-red photoconvertible fluorescent protein, is a suitable tool to study protein turnover in plants. We characterized the fluorescence properties of Dendra2 expressed either as a free protein or as a tag in Arabidopsis thaliana roots and optimized photoconversion settings to study protein turnover. Dendra2 was fused to the PIN2 protein, an auxin transporter in the root tip, and by time-lapse imaging and assessment of red and green signal intensities in the membrane after photoconversion we quantified directly and simultaneously the rate of PIN2 delivery of the newly synthesized protein into the plasma membrane as well as the disappearance of the protein from the plasma membrane due to degradation. Additionally we have verified several factors which are expected to affect PIN2 protein turnover and therefore potentially regulate root growth.