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.
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.
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.
Figure 1:Populus leaf mesophyll protoplasts.
(A) Optimal yield and quality of protoplasts can be isolated from one month-old Populus plants grown on MS medium in a magenta box. (B) High transfection efficiency is indicated with GFP signal.
Figure 2:Subcellular localization of various organelle markers in Populus protoplasts.
(A) Plasma membrane; (B) Golgi apparatus; (C) Nucleus; (G) Peroxisome; (H) endoplasmic reticulum (ER); (I) An ubiquitously-localized protein (RACK1, Receptor for Activated C-protein Kinase 1). Shown in (D), (E), (F), (J), (K) and (L) are bright field images for fluorescent images of (A), (B), (C), (G), (H) and (I),
respectively. The organelle markers were fused with mCherry fluorescent
protein, and RACK1 was fused with YFP fluorescent protein. The mCherry
signal was separated from chloroplast autofluorescence using spectral
imaging and linear unmixing. The mCherry and YFP signals are
false-colored green and the chloroplast autofluorescence is shown in
red. Scale bar, 1 µm.
Guo J, Morrell-Falvey JL, Labbé JL, Muchero W, Kalluri UC, et al. (2012) Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays. PLoS ONE 7(9): e44908. doi:10.1371/journal.pone.0044908