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.
Figure 3.
Using Populus protoplast system to test protein-protein interaction.
Arabidopsis RACK1A (AtRACK1A) and eIF6A (AteIF6A) were fused with the N-terminal and C-terminal half of YFP, respectively. GUS-nYFP/cYFP fusions were used as negative controls. YFP, yellow fluorescent protein. BF, Bright field.
Figure 4.
The response of Populus protoplasts to various plant hormone treatments.
Shown are the change POPTR_01s30560 transcript in response to different concentrations of NAA in protoplasts (A) and intact leaves (E), the change of POPTR_14s08030 transcript in response to different concentrations of GA3 in protoplasts (B) and intact leaves (F), the change of POPTR_10s00320 transcript in response to different concentrations of BAP in protoplasts (C) and intact leaves (G), and the change of POPTR_10s08300 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.
Figure 5.
Energy sensing signaling in Populus protoplasts.
(A) Semi-quantitative RT-PCR analysis of PtrDIN6 transcripts in response to dark and hypoxia treatments. L-Light; D-Dark; H-Hypoxia. The PtrUBQ10 gene was used as a control. (B) The change of PtrDIN6 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). (C) The expression of three Populus KIN10 homologues in transfected protoplasts examined by semi-quantitative RT-PCR. The expression of PtrUBQ10 was used as an internal control. (D) The response of PtrDIN6 transcript to the overexpression of three Populus KIN10 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. (E) The activation of PtrDIN6 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).