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

PLoS One. 2012;7(9):e44908. doi: 10.1371/journal.pone.0044908. Epub 2012 Sep 13.

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 findings: We 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/significance: This 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biomarkers / metabolism
  • Cell Separation / methods*
  • Culture Media / chemistry
  • Gene Expression
  • Genes, Plant / genetics
  • Mesophyll Cells / cytology*
  • Mesophyll Cells / drug effects
  • Mesophyll Cells / metabolism
  • Organelles / drug effects
  • Organelles / metabolism
  • Plant Growth Regulators / pharmacology
  • Populus / cytology*
  • Populus / drug effects
  • Populus / genetics*
  • Protein Interaction Mapping
  • Protein Transport / drug effects
  • Protoplasts / cytology*
  • Protoplasts / drug effects
  • Protoplasts / metabolism
  • Signal Transduction / drug effects
  • Time Factors

Substances

  • Biomarkers
  • Culture Media
  • Plant Growth Regulators

Grants and funding

This work was supported by the Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science Program, United States Department of Energy, Office of Science, Biological and Environmental Research. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the United States Department of Energy under contract DE-AC05-00OR22725. JG was supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.