Journal: Frontiers in Plant Science

Article Title: Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2

doi: 10.3389/fpls.2023.1181311

Figure Lengend Snippet: Effect of two N-terminal regions of the OsPSY2 protein including the hydrophobic helix (HH) 1 on subcellular localization in rice protoplasts. (A) Individual and merged images of sGFP, chlorophyll, and visible protoplasts using confocal microscopy. sG and RTp-sG were used as a cytosolic (Cy) and stromal (St) localization markers, respectively. Scale bars indicate 5 μm. (B) Western blot analysis to compare the amount and size of sGFP. Rice protoplasts were cotransfected with each experimental vector and an OsPSY2:mC vector as a transfection control. This blot was performed with three antibodies simultaneously: Anti-GFP (blue circle), anti-mCherry (pink rectangle), and anti-rice actin (marked at 45 kDa, to be used as an internal reference of the total protein amount). (C) Prediction of cleavage probability in the N-terminal 80-amino-acid region of OsPSY2 as implemented by the TargetP 2.0 program ( https://services.healthtech.dtu.dk/service.php?TargetP ). Estimated cleavage scores of the 80 amino acids of PTp are shown in Supplementary Table S1 .

Article Snippet: Next, a Western blot was performed using an anti-rabbit polyclonal antibody against GFP (Abcam, Cambridge, UK), an antibody against mCherry (Novus Biologicals, Denver, USA), and an antibody against actin (Abcam, Cambridge, UK).

Techniques: Confocal Microscopy, Western Blot, Plasmid Preparation, Transfection, Control

Journal: Frontiers in Plant Science

Article Title: Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2

doi: 10.3389/fpls.2023.1181311

Figure Lengend Snippet: Effect of the hydrophobic region (HR) 2 on subcellular and suborganellar localization in rice protoplasts. (A) Individual and merged images of sGFP, mCherry, chlorophyll, and visible protoplasts using confocal microscopy. PTp-sG, OsPSY2:mC, and mC were used as markers for dual localization into plastoglobules (PGs) and stroma (St) as well as single localization into a PG and the cytosol (Cy), respectively. Scale bars indicate 5 μm. (B) Western blot analysis to confirm the cleavage in HR2 when placed right after a Tp signal such as RTp or HR1 within PTp. (C) Western blot analysis to confirm that no cleavage occurred in HR2 when placed at the C-terminus of sGFP. In both (B, C) , rice protoplasts were cotransfected with an experimental vector and an OsPSY2:mC vector used as a transfection control. In one exceptional case we used RTp-sG-HR2-mC with intrinsic mC. The blot shown in (B) was performed with three antibodies simultaneously: Anti-GFP (blue circle), anti-mCherry (red diamond for the experimental vector or pink rectangle for the control vector), and anti-rice actin (marked at 45 kDa and used as an internal reference indicating total protein amount). The blot shown in (C) was performed with two antibodies simultaneously: Anti-GFP and anti-rice actin in the left panel, and anti-mCherry and anti-rice actin in the right panel, respectively. The expected protein size is noted under the blot.

Article Snippet: Next, a Western blot was performed using an anti-rabbit polyclonal antibody against GFP (Abcam, Cambridge, UK), an antibody against mCherry (Novus Biologicals, Denver, USA), and an antibody against actin (Abcam, Cambridge, UK).

Techniques: Confocal Microscopy, Western Blot, Plasmid Preparation, Transfection, Control

Journal: Frontiers in Plant Science

Article Title: Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2

doi: 10.3389/fpls.2023.1181311

Figure Lengend Snippet: Effect of the hydrophobic region (HR) 3 on PG localization in rice protoplasts. (A) Individual and merged images of mCherry, chlorophyll, and visible protoplasts using confocal microscopy. OsPSY2:mC and OsPSY2(ΔPTp):mC were used as localization markers of plastoglobules (PGs) and the cytosol (Cy), respectively. Scale bars indicate 5 μm. (B) Western blot analysis to confirm the presence of a PG-targeting signal in an area other than PTp in OsPSY2. Rice protoplasts were individually transfected with three experimental vectors. This blot was conducted with anti-mCherry (red diamond) and a polyacrylamide gel electrophoresis (PAGE) image is shown to indicate total protein amounts. (C) Individual and merged images of sGFP, mCherry, chlorophyll, and visible protoplasts using confocal microscopy. RTp-sG and OsPSY2:mC were used as localization markers for the stroma (St) and plastoglobules (PGs), respectively. Scale bars indicate 5 μm. (D) Western blot analysis to reveal the precise location of HR3 that exhibits PG-targeting capacity. Rice protoplasts were cotransfected with an experimental vector and an OsPSY2:mC vector, used as a transfection control. The blot was performed with three antibodies simultaneously: Anti-GFP (blue circle), anti-mCherry (pink rectangle), and anti-rice actin (marked at 45 kDa and used as an internal reference of total protein amount). (E) Quantitative real-time-PCR to indicate relative transcript levels depending on different protein levels for the three vectors. The rice ubiquitin gene (AK061988) was used as a reference to normalize RNA amounts. qRT-PCR was performed using three technical replicates with three biological samples per vector.

Article Snippet: Next, a Western blot was performed using an anti-rabbit polyclonal antibody against GFP (Abcam, Cambridge, UK), an antibody against mCherry (Novus Biologicals, Denver, USA), and an antibody against actin (Abcam, Cambridge, UK).

Techniques: Confocal Microscopy, Western Blot, Transfection, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Quantitative RT-PCR

Journal: Frontiers in Plant Science

Article Title: Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2

doi: 10.3389/fpls.2023.1181311

Figure Lengend Snippet: Differential effects of the hydrophobic region (HR) 3 on the PG-localization depending on PTp in rice protoplasts. (A) Individual and merged images of sGFP, mCherry, chlorophyll, and visible protoplasts using confocal microscopy. OsPSY2:mC was used as a plastoglobule (PG) localization marker. Scale bars indicate 5 μm. (B) Western blot analysis to determine the factors influencing aggregation and loss of fluorescence. Rice protoplasts were cotransfected with an experimental vector and an OsPSY2:mC vector used as a transfection control. In one exceptional case PTp-sG-HR3-mC was used, which has intrinsic mC. The blot was performed with two antibodies simultaneously: Anti-GFP (blue circle) and anti-rice actin (marked at 45 kDa) on the left panel, and anti-mCherry (red diamond for experimental vector or pink rectangle for the control vector) and anti-rice actin on the right panel, respectively.

Article Snippet: Next, a Western blot was performed using an anti-rabbit polyclonal antibody against GFP (Abcam, Cambridge, UK), an antibody against mCherry (Novus Biologicals, Denver, USA), and an antibody against actin (Abcam, Cambridge, UK).

Techniques: Confocal Microscopy, Marker, Western Blot, Fluorescence, Plasmid Preparation, Transfection, Control