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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

Phenotypic responses of Arabidopsis wild type Col-0 and ire1a-2 ire1b-4 seedlings to varied concentrations of Tm. (A) Seeds were grown in liquid MS media for 5 days, then the media was removed and replaced by fresh MS supplemented with designated concentrations of Tm for 3 days. Phenotypic responses, such as dwarfism and chlorosis, were documented. The ire1a-2 ire1b-4 plants display more profound stress symptoms compared to Col-0 under both Tm concentrations. (B) Seeds were grown on solid plates under the same conditions as seeds grown in (A). Seeds were transferred to plates containing various concentrations of Tm after 5 days of growth on MS and were transferred again to MS plates after 3 days of growth on plates supplemented with Tm. Seeds were allowed 3 days recovery before data analysis.
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Figure 4: Phenotypic responses of Arabidopsis wild type Col-0 and ire1a-2 ire1b-4 seedlings to varied concentrations of Tm. (A) Seeds were grown in liquid MS media for 5 days, then the media was removed and replaced by fresh MS supplemented with designated concentrations of Tm for 3 days. Phenotypic responses, such as dwarfism and chlorosis, were documented. The ire1a-2 ire1b-4 plants display more profound stress symptoms compared to Col-0 under both Tm concentrations. (B) Seeds were grown on solid plates under the same conditions as seeds grown in (A). Seeds were transferred to plates containing various concentrations of Tm after 5 days of growth on MS and were transferred again to MS plates after 3 days of growth on plates supplemented with Tm. Seeds were allowed 3 days recovery before data analysis.

Mentions: Two primary phenotypes of Tm-stressed seedlings are a decrease in their size and visible yellowing and chlorosis (Figures 2 and 4A,B). A combined quantitative assessment of these two plant responses offers detailed insights in the level of sensitivity to UPR stress. After 3 days of recovery from Tm exposure, 10 seedlings per genotype were collected and weighed from both liquid and solid plates (Figure 5). We observed progressively decreased weight of liquid-grown Col-0 following recovery after exposure to 0.15 and 0.3 μg/mL concentrations of Tm, indicating an induction of Tm-dependent UPR (Figure 5A). However, Col-0 seedlings weighed 2–5 times more than ire1a-2 ire1b-4 double mutant seedlings at 0.15 and 0.3 μg/mL concentrations of Tm, respectively. This indicates that the ire1a-2 ire1b-4 double mutant exhibits a decreased ability to cope with and recover from UPR, consistent with the previous findings (Chen and Brandizzi, 2012; Moreno et al., 2012). As expected, ire1a-2 ire1b-4 double mutants weighed nearly two times less than ire1b-4 mutants. Similarly, bip2-1 bip3-1 double mutants weighed two times less than ire1b-4 and 3 times less than Col-0 at the higher dose of Tm (Figure 5A). However, the reduction in the fresh weight observed on solid MS plates under the conditions tested, although statistically significant, was not as dramatic compared to liquid assay. This was true for all the genotypes included in our assay and in particular the bip2-1 bip3-1 double mutants (Figure 5B). This effect can be, at least in part, attributed to the non-uniform Tm exposure on solid plates, which is a known limitation of this assay, especially since these mutants have previously been shown to have severe phenotypic responses to Tm (Noh et al., 2003). In addition, it is worth noting that all genotypes grown on solid plated were smaller than their counterparts cultured in liquid MS medium (Figures 5A,B).


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)

Phenotypic responses of Arabidopsis wild type Col-0 and ire1a-2 ire1b-4 seedlings to varied concentrations of Tm. (A) Seeds were grown in liquid MS media for 5 days, then the media was removed and replaced by fresh MS supplemented with designated concentrations of Tm for 3 days. Phenotypic responses, such as dwarfism and chlorosis, were documented. The ire1a-2 ire1b-4 plants display more profound stress symptoms compared to Col-0 under both Tm concentrations. (B) Seeds were grown on solid plates under the same conditions as seeds grown in (A). Seeds were transferred to plates containing various concentrations of Tm after 5 days of growth on MS and were transferred again to MS plates after 3 days of growth on plates supplemented with Tm. Seeds were allowed 3 days recovery before data analysis.
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Figure 4: Phenotypic responses of Arabidopsis wild type Col-0 and ire1a-2 ire1b-4 seedlings to varied concentrations of Tm. (A) Seeds were grown in liquid MS media for 5 days, then the media was removed and replaced by fresh MS supplemented with designated concentrations of Tm for 3 days. Phenotypic responses, such as dwarfism and chlorosis, were documented. The ire1a-2 ire1b-4 plants display more profound stress symptoms compared to Col-0 under both Tm concentrations. (B) Seeds were grown on solid plates under the same conditions as seeds grown in (A). Seeds were transferred to plates containing various concentrations of Tm after 5 days of growth on MS and were transferred again to MS plates after 3 days of growth on plates supplemented with Tm. Seeds were allowed 3 days recovery before data analysis.
Mentions: Two primary phenotypes of Tm-stressed seedlings are a decrease in their size and visible yellowing and chlorosis (Figures 2 and 4A,B). A combined quantitative assessment of these two plant responses offers detailed insights in the level of sensitivity to UPR stress. After 3 days of recovery from Tm exposure, 10 seedlings per genotype were collected and weighed from both liquid and solid plates (Figure 5). We observed progressively decreased weight of liquid-grown Col-0 following recovery after exposure to 0.15 and 0.3 μg/mL concentrations of Tm, indicating an induction of Tm-dependent UPR (Figure 5A). However, Col-0 seedlings weighed 2–5 times more than ire1a-2 ire1b-4 double mutant seedlings at 0.15 and 0.3 μg/mL concentrations of Tm, respectively. This indicates that the ire1a-2 ire1b-4 double mutant exhibits a decreased ability to cope with and recover from UPR, consistent with the previous findings (Chen and Brandizzi, 2012; Moreno et al., 2012). As expected, ire1a-2 ire1b-4 double mutants weighed nearly two times less than ire1b-4 mutants. Similarly, bip2-1 bip3-1 double mutants weighed two times less than ire1b-4 and 3 times less than Col-0 at the higher dose of Tm (Figure 5A). However, the reduction in the fresh weight observed on solid MS plates under the conditions tested, although statistically significant, was not as dramatic compared to liquid assay. This was true for all the genotypes included in our assay and in particular the bip2-1 bip3-1 double mutants (Figure 5B). This effect can be, at least in part, attributed to the non-uniform Tm exposure on solid plates, which is a known limitation of this assay, especially since these mutants have previously been shown to have severe phenotypic responses to Tm (Noh et al., 2003). In addition, it is worth noting that all genotypes grown on solid plated were smaller than their counterparts cultured in liquid MS medium (Figures 5A,B).

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