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An improved high-throughput screening assay for tunicamycin sensitivity in Arabidopsis seedlings.

McCormack ME, Liu X, Jordan MR, Pajerowska-Mukhtar KM - Front Plant Sci (2015)

Bottom Line: This study shows an improved method of testing Tm sensitivity in Arabidopsis using liquid Murashige and Skoog medium versus the traditional solid agar plates.Seedlings were subjected to comparative treatments with various concentrations of Tm on both solid and liquid media and allowed to recover.Determination of fresh weight, chlorophyll contents analysis and qRT-PCR results confirm the efficacy of using liquid medium to perform quantitative Tm stress assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Alabama at Birmingham, Birmingham, AL USA.

ABSTRACT
Tunicamycin (Tm) sensitivity assays are a useful method for studies of endoplasmic reticulum stress and the unfolded protein response in eukaryotic cells. While Tm sensitivity and Tm recovery assays have been previously described, these existing methods are time-consuming, labor intensive, and subjected to mechanical wounding. This study shows an improved method of testing Tm sensitivity in Arabidopsis using liquid Murashige and Skoog medium versus the traditional solid agar plates. Liquid medium bypasses the physical manipulation of seedlings, thereby eliminating the risk of potential mechanical damage and additional unwanted stress to seedlings. Seedlings were subjected to comparative treatments with various concentrations of Tm on both solid and liquid media and allowed to recover. Determination of fresh weight, chlorophyll contents analysis and qRT-PCR results confirm the efficacy of using liquid medium to perform quantitative Tm stress assays.

No MeSH data available.


Related in: MedlinePlus

Flowchart of the procedures to quantify tunicamycin (Tm) sensitivity of Arabidopsis seedlings using liquid media followed by chlorophyll measurement.
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Figure 1: Flowchart of the procedures to quantify tunicamycin (Tm) sensitivity of Arabidopsis seedlings using liquid media followed by chlorophyll measurement.

Mentions: Seeds were washed with a solution of 0.05% Triton and 70% Ethanol then stratified for 4 days at 4°C. For the assay on solid plates, seeds were placed in sterile 0.1% agar solution and pipetted onto solid Murashige Skoog (MS) growth medium (Phytotechnology Lab, Overland Park, KS, USA) with 1.2% sucrose (Fisher) and 50 mg/L ampicillin (Fisher) and allowed to grow for 5 days at 22°C under a 16 h light/8 h dark (for the initial dose-response assay) or 12 h light/12 h dark (remaining experiments) photoperiod. 15 seeds were transferred to solid MS medium containing either 0.15 or 0.3 μg/mL Tm, (Sigma-Aldrich T7765) and allowed to grow for 3 days at 22°C. The treated seeds were then transferred back to solid MS medium plates and allowed to grow for 3 days before collection for chlorophyll analysis. For the assay in liquid media, seeds were placed in sterile 0.1% agar solution and 15–20 seeds were pipetted into each well of a 12-well polystyrene plate (Fisher) containing 4 mL 0.5 × MS medium with 0.6% sucrose and ampicillin and grown for 5 days at 22°C. The MS medium was removed and replaced with fresh MS media or media containing either 0.15 or 0.3 μg/mL Tm and allowed to grow for 3 days at 22°C. The medium was again replaced with fresh MS medium and plants were allowed to grow for 3 days before collection for chlorophyll analysis. The experimental design is outlined in Figure 1.


An improved high-throughput screening assay for tunicamycin sensitivity in Arabidopsis seedlings.

McCormack ME, Liu X, Jordan MR, Pajerowska-Mukhtar KM - Front Plant Sci (2015)

Flowchart of the procedures to quantify tunicamycin (Tm) sensitivity of Arabidopsis seedlings using liquid media followed by chlorophyll measurement.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562274&req=5

Figure 1: Flowchart of the procedures to quantify tunicamycin (Tm) sensitivity of Arabidopsis seedlings using liquid media followed by chlorophyll measurement.
Mentions: Seeds were washed with a solution of 0.05% Triton and 70% Ethanol then stratified for 4 days at 4°C. For the assay on solid plates, seeds were placed in sterile 0.1% agar solution and pipetted onto solid Murashige Skoog (MS) growth medium (Phytotechnology Lab, Overland Park, KS, USA) with 1.2% sucrose (Fisher) and 50 mg/L ampicillin (Fisher) and allowed to grow for 5 days at 22°C under a 16 h light/8 h dark (for the initial dose-response assay) or 12 h light/12 h dark (remaining experiments) photoperiod. 15 seeds were transferred to solid MS medium containing either 0.15 or 0.3 μg/mL Tm, (Sigma-Aldrich T7765) and allowed to grow for 3 days at 22°C. The treated seeds were then transferred back to solid MS medium plates and allowed to grow for 3 days before collection for chlorophyll analysis. For the assay in liquid media, seeds were placed in sterile 0.1% agar solution and 15–20 seeds were pipetted into each well of a 12-well polystyrene plate (Fisher) containing 4 mL 0.5 × MS medium with 0.6% sucrose and ampicillin and grown for 5 days at 22°C. The MS medium was removed and replaced with fresh MS media or media containing either 0.15 or 0.3 μg/mL Tm and allowed to grow for 3 days at 22°C. The medium was again replaced with fresh MS medium and plants were allowed to grow for 3 days before collection for chlorophyll analysis. The experimental design is outlined in Figure 1.

Bottom Line: This study shows an improved method of testing Tm sensitivity in Arabidopsis using liquid Murashige and Skoog medium versus the traditional solid agar plates.Seedlings were subjected to comparative treatments with various concentrations of Tm on both solid and liquid media and allowed to recover.Determination of fresh weight, chlorophyll contents analysis and qRT-PCR results confirm the efficacy of using liquid medium to perform quantitative Tm stress assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Alabama at Birmingham, Birmingham, AL USA.

ABSTRACT
Tunicamycin (Tm) sensitivity assays are a useful method for studies of endoplasmic reticulum stress and the unfolded protein response in eukaryotic cells. While Tm sensitivity and Tm recovery assays have been previously described, these existing methods are time-consuming, labor intensive, and subjected to mechanical wounding. This study shows an improved method of testing Tm sensitivity in Arabidopsis using liquid Murashige and Skoog medium versus the traditional solid agar plates. Liquid medium bypasses the physical manipulation of seedlings, thereby eliminating the risk of potential mechanical damage and additional unwanted stress to seedlings. Seedlings were subjected to comparative treatments with various concentrations of Tm on both solid and liquid media and allowed to recover. Determination of fresh weight, chlorophyll contents analysis and qRT-PCR results confirm the efficacy of using liquid medium to perform quantitative Tm stress assays.

No MeSH data available.


Related in: MedlinePlus