Highly Efficient Isolation of <em>Populus</em> Mesophyll Protoplasts and Its Application in Transient Expression Assays

<div><h3>Background</h3><p><em>Populus</em> is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.</p> <h3>Methodology/Principal Findings</h3><p>We optimized a <em>Populus</em> leaf mesophyll protoplast isolation protocol and established a <em>Populus</em> protoplast transient expression system. We demonstrated that <em>Populus</em> protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the <em>Populus</em> protoplasts. Furthermore, we showed that the <em>Populus</em> protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.</p> <h3>Conclusions/Significance</h3><p>This study established a method for efficient isolation of protoplasts from <em>Populus</em> leaf and demonstrated the efficacy of using <em>Populus</em> protoplast transient expression assays as an <em>in vivo</em> system to characterize genes and pathways.</p> </div

Subcellular localization of various organelle markers in <i>Populus</i> protoplasts.

<p>(<b>A</b>) Plasma membrane; (<b>B</b>) Golgi apparatus; (<b>C</b>) Nucleus; (<b>G</b>) Peroxisome; (<b>H</b>) endoplasmic reticulum (ER); (<b>I</b>) An ubiquitously-localized protein (RACK1, Receptor for Activated C-protein Kinase 1). Shown in (<b>D</b>), (<b>E</b>), (<b>F</b>), (<b>J</b>), (<b>K</b>) and (<b>L</b>) are bright field images for fluorescent images of (<b>A</b>), (<b>B</b>), (<b>C</b>), (<b>G</b>), (<b>H</b>) and (<b>I</b>), 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.</p

<i>Populus</i> leaf mesophyll protoplasts.

<p>(<b>A</b>) Optimal yield and quality of protoplasts can be isolated from one month-old <i>Populus</i> plants grown on MS medium in a magenta box. (<b>B</b>) High transfection efficiency is indicated with GFP signal.</p

Energy sensing signaling in <i>Populus</i> protoplasts.

<p>(<b>A</b>) Semi-quantitative RT-PCR analysis of <i>PtrDIN6</i> transcripts in response to dark and hypoxia treatments. L-Light; D-Dark; H-Hypoxia. The <i>PtrUBQ10</i> gene was used as a control. (<b>B</b>) The change of <i>PtrDIN6</i> transcripts in response to overexpression of AthKIN10 protein. After transfection, protoplasts were incubated overnight to allow the expression of AthKIN10 before samples were harvested for qRT-PCR and western blot analysis. Western blot was used to detect the presence of the introduced HA-tagged AthKIN10 protein. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are presented in the graph. * indicates a significant difference (at P≤0.01, student’s t-test) between protoplasts expressing AthKIN10 and the control (ctrl). (<b>C</b>) The expression of three <i>Populus KIN10</i> homologues in transfected protoplasts examined by semi-quantitative RT-PCR. The expression of <i>PtrUBQ10</i> was used as an internal control. (<b>D</b>) The response of <i>PtrDIN6</i> transcript to the overexpression of three <i>Populus KIN10</i> homologues. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are shown. Protoplasts transfected with an empty vector was used as control (ctrl) for each comparison. (<b>E</b>) The activation of <i>PtrDIN6</i> by PtrKIN10 in a GUS reporter assay. For each co-transfection, a 35S::LUC (Luciferase) was included and the LUC activity was used to normalize GUS activity to account for the potential variations in the transfection efficiency. The averages of three technical replicates ± standard errors are shown. * indicates a significant difference (at P≤0.01, student’s t-test) between each treatment and the control (ctrl).</p

The response of <i>Populus</i> protoplasts to various plant hormone treatments.

<p>Shown are the change <i>POPTR_01s30560</i> transcript in response to different concentrations of NAA in protoplasts (A) and intact leaves (E), the change of <i>POPTR_14s08030</i> transcript in response to different concentrations of GA₃ in protoplasts (B) and intact leaves (F), the change of <i>POPTR_10s00320</i> transcript in response to different concentrations of BAP in protoplasts (C) and intact leaves (G), and the change of <i>POPTR_10s08300</i> transcript in response to different concentrations of ACC in protoplasts (D) and intact leaves (H). The protoplasts or intact leaves were incubated with various concentrations of plant hormones for 3h before being harvested for qRT-PCR analysis. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are shown. * indicates a significant difference (at P≤0.01, student’s t-test) between each treatment and the untreated control.</p