Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.

Jin-Gui Chen,Jessy L. Labbé,Wellington Muchero,Jennifer L. Morrell-Falvey,Jianjun Guo,Carl Ng,Gerald A. Tuskan,Udaya C. Kalluri

doi:10.1371/journal.pone.0044908

Abstract

Background Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.Methodology/Principal FindingsWe optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.Conclusions/SignificanceThis study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

Highlights

The establishment of renewable energy sources is essential for energy security and reduction of CO2 emissions
When we followed the same protocol for isolating protoplasts from leaf mesophyll cells of Populus plants grown in a greenhouse or a Magenta box as that used in Arabidopsis, we found that the yields and quality of protoplasts were very poor
In order to determine whether the Populus protoplasts respond to exogenous hormone treatment in a similar manner as that in intact leaves, we further examined the induction of these genes by plant hormones in intact Populus leaves

Summary

Introduction

The establishment of renewable energy sources is essential for energy security and reduction of CO2 emissions. One of the rate-limiting steps in studying the function of genes in Populus is its long life cycle (e.g., it takes a minimum 6 to 12 years to reach reproductive maturity [7]). The capacity of conventional genetic manipulation in Populus, one of the most commonly used approaches for studying gene function, is largely limited by the lengthy time required to obtain suitable genetic materials. This limitation is more pronounced when dealing with cellular signaling events that often involve multiple gene products. Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. Its lengthy life cycle impedes rapid characterization of gene function

Methods

Results

Conclusion