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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

Dose-response assay to determine Tm concentrations resulting in appearance of retarded growth and chlorosis in Arabidopsis seedlings. Long-day grown 5-day-old seedlings were subjected to Tm treatment for 3 days and photographed. Wells corresponding to Tm concentrations of 0.15 and 0.3 μg/mL are circled in red.
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Figure 2: Dose-response assay to determine Tm concentrations resulting in appearance of retarded growth and chlorosis in Arabidopsis seedlings. Long-day grown 5-day-old seedlings were subjected to Tm treatment for 3 days and photographed. Wells corresponding to Tm concentrations of 0.15 and 0.3 μg/mL are circled in red.

Mentions: It is well established that Tm treatment can be conducted on solid MS media and leads to a stress response that is equivalent to UPR (Iwata and Koizumi, 2005; Oslowski and Urano, 2011). The most frequently used concentrations of Tm on solid plates range between 0.1 and 0.5 μg/mL (Iwata and Koizumi, 2005; Pajerowska-Mukhtar et al., 2012). However, liquid MS culture of Arabidopsis seedlings offers a more uniform delivery of the chemical compound to the plant and is a widespread method to test responses to chemicals (Schreiber et al., 2008). We set out to adapt the liquid assay to quantify Tm sensitivity through a recovery assay. First, we subjected 5-day-old wild type Col-0 seedlings to increasing doses of Tm in liquid culture (Figure 2) for 3 days followed by replacing the MS medium containing Tm with fresh aliquot of regular MS medium. We observed progressively smaller and more chlorotic seedlings as the Tm concentrations increased, with a nearly complete arrest in growth and development and profound bleaching at concentrations of 0.4 μg/ml and above. Based on this dose-response experiment, we determined that under liquid MS conditions the Tm concentrations of 0.15 and 0.3 μg/ml can serve as appropriate conditions to test for severe and mild defects in the UPR tolerance, respectively.


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)

Dose-response assay to determine Tm concentrations resulting in appearance of retarded growth and chlorosis in Arabidopsis seedlings. Long-day grown 5-day-old seedlings were subjected to Tm treatment for 3 days and photographed. Wells corresponding to Tm concentrations of 0.15 and 0.3 μg/mL are circled in red.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Dose-response assay to determine Tm concentrations resulting in appearance of retarded growth and chlorosis in Arabidopsis seedlings. Long-day grown 5-day-old seedlings were subjected to Tm treatment for 3 days and photographed. Wells corresponding to Tm concentrations of 0.15 and 0.3 μg/mL are circled in red.
Mentions: It is well established that Tm treatment can be conducted on solid MS media and leads to a stress response that is equivalent to UPR (Iwata and Koizumi, 2005; Oslowski and Urano, 2011). The most frequently used concentrations of Tm on solid plates range between 0.1 and 0.5 μg/mL (Iwata and Koizumi, 2005; Pajerowska-Mukhtar et al., 2012). However, liquid MS culture of Arabidopsis seedlings offers a more uniform delivery of the chemical compound to the plant and is a widespread method to test responses to chemicals (Schreiber et al., 2008). We set out to adapt the liquid assay to quantify Tm sensitivity through a recovery assay. First, we subjected 5-day-old wild type Col-0 seedlings to increasing doses of Tm in liquid culture (Figure 2) for 3 days followed by replacing the MS medium containing Tm with fresh aliquot of regular MS medium. We observed progressively smaller and more chlorotic seedlings as the Tm concentrations increased, with a nearly complete arrest in growth and development and profound bleaching at concentrations of 0.4 μg/ml and above. Based on this dose-response experiment, we determined that under liquid MS conditions the Tm concentrations of 0.15 and 0.3 μg/ml can serve as appropriate conditions to test for severe and mild defects in the UPR tolerance, respectively.

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