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

Ján Jásik,František Baluška,Thomas Altmann,Dieter Volkmann,Barbara Boggetti,Elmon Schmelzer,Twan Rutten,Thomas Gensch,Luis Herrera-Estrella

doi:10.1371/journal.pone.0061403

Abstract

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.

Highlights

The plasma membrane is a highly dynamic structure whose components undergo continuous modification and renewal
Protein abundance can be evaluated by classical approaches such as western blotting and, at the single cell level, by GFP tagging technology and immunocytochemistry, these approaches are not able to explain which process contributes to the fluctuations in the steady state levels of the proteins
48 from 721 F2 progenies of self-pollinated double hemizygous F1 plants showed gravitropic defects. This means a segregation ratio (SR) of 14.1:1 of gravitropic to agravitropic seedlings instead of a SR of 3:1 expected providing that the mutation is not complemented by PIN2-Dendra2

Summary

Introduction

The plasma membrane is a highly dynamic structure whose components undergo continuous modification and renewal. We supposed that timelapse imaging in photoconverting experiments combined with measurement of red and green signal intensities emitted by the PIN2-Dendra2 recombinant protein by laser confocal microscopy may allow to quantify protein internalization and delivery to the plasma membrane.

Results

Conclusion