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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

Chlorophyll a and b content after Tm treatment. Chlorophyll a was extracted with methanol from seedlings 3 days after exposure to various concentrations of Tm in liquid (A) and solid (B) media. Chlorophyll b was extracted following the same procedure in liquid (C) and solid (D) media. Chlorophyll a and b content was read using a microplate reader at 652 and 665 nm wavelengths. Error bars represent standard error of the means. Statistical analysis was performed with Tukey’s HSD test. Bars within a class connected by the same letter did not differ from each other at p < 0.05. Experiments were performed in at least three independent biological replications.
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Figure 6: Chlorophyll a and b content after Tm treatment. Chlorophyll a was extracted with methanol from seedlings 3 days after exposure to various concentrations of Tm in liquid (A) and solid (B) media. Chlorophyll b was extracted following the same procedure in liquid (C) and solid (D) media. Chlorophyll a and b content was read using a microplate reader at 652 and 665 nm wavelengths. Error bars represent standard error of the means. Statistical analysis was performed with Tukey’s HSD test. Bars within a class connected by the same letter did not differ from each other at p < 0.05. Experiments were performed in at least three independent biological replications.

Mentions: To substantiate the fresh weight data, we next quantified seedling chlorosis through a quantitative measurement of chlorophylls a and b contents per unit of fresh weight resulting from Tm exposure on both liquid MS medium and on solid MS plates. Seedlings were collected after 3 days of recovery from Tm treatment and analyzed for chlorophyll accumulation at 652 and 665 nm wavelengths using a microplate reader. Col-0 showed 30 times higher chlorophyll a and b contents than the ire1a-2 ire1b-4 double mutant in liquid culture (Figures 6A,C). The lowered chlorophyll contents per unit of fresh weight in Col-0 seedlings treated with 0.3 μg/mL Tm on solid medium might be attributed to chlorosis without a substantial decrease in fresh weight (Figures 5B and 6B,D). It is also plausible that this decrease in chlorophyll contents was caused by mechanical damage to the fragile seedlings during transfer between plates. The ire1a-2 ire1b-4 showed severe reduction of chlorophyll a and b content on both liquid and solid plates. Chlorophyll a content was 11–13 times lower in ire1a-2 ire1b-4 mutants compared to ire1a-2 ire1b-4 controls and 5–11 times lower compared to ire1b-4 single mutants treated with 0.3 μg/mL Tm, indicating the sensitivity of the ire1a-2 ire1b-4 double knock-out as well as the severity of chlorosis at higher Tm doses. The reduction of chlorophyll b content showed a similar although less severe pattern, which is consistent with chlorophyll a being the dominant chlorophyll pigment accumulated by leaf tissue. The bip2-1 bip3-1 double mutants showed a similar trend in chlorophyll reduction comparable to that of ire1a-2 ire1b-4, although less dramatic (Figures 6A–D). The redundancy of BiP1 and BiP2 may contribute to the slightly higher levels of chlorophyll in the bip2-1 bip3-1 plants compared to ire1a-2 ire1b-4 double mutants. Altogether, these results indicate the effectiveness of using liquid medium to induce UPR through Tm treatment and highlight the numerous advantages of using this assay for large scale screens.


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)

Chlorophyll a and b content after Tm treatment. Chlorophyll a was extracted with methanol from seedlings 3 days after exposure to various concentrations of Tm in liquid (A) and solid (B) media. Chlorophyll b was extracted following the same procedure in liquid (C) and solid (D) media. Chlorophyll a and b content was read using a microplate reader at 652 and 665 nm wavelengths. Error bars represent standard error of the means. Statistical analysis was performed with Tukey’s HSD test. Bars within a class connected by the same letter did not differ from each other at p < 0.05. Experiments were performed in at least three independent biological replications.
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Related In: Results  -  Collection

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Figure 6: Chlorophyll a and b content after Tm treatment. Chlorophyll a was extracted with methanol from seedlings 3 days after exposure to various concentrations of Tm in liquid (A) and solid (B) media. Chlorophyll b was extracted following the same procedure in liquid (C) and solid (D) media. Chlorophyll a and b content was read using a microplate reader at 652 and 665 nm wavelengths. Error bars represent standard error of the means. Statistical analysis was performed with Tukey’s HSD test. Bars within a class connected by the same letter did not differ from each other at p < 0.05. Experiments were performed in at least three independent biological replications.
Mentions: To substantiate the fresh weight data, we next quantified seedling chlorosis through a quantitative measurement of chlorophylls a and b contents per unit of fresh weight resulting from Tm exposure on both liquid MS medium and on solid MS plates. Seedlings were collected after 3 days of recovery from Tm treatment and analyzed for chlorophyll accumulation at 652 and 665 nm wavelengths using a microplate reader. Col-0 showed 30 times higher chlorophyll a and b contents than the ire1a-2 ire1b-4 double mutant in liquid culture (Figures 6A,C). The lowered chlorophyll contents per unit of fresh weight in Col-0 seedlings treated with 0.3 μg/mL Tm on solid medium might be attributed to chlorosis without a substantial decrease in fresh weight (Figures 5B and 6B,D). It is also plausible that this decrease in chlorophyll contents was caused by mechanical damage to the fragile seedlings during transfer between plates. The ire1a-2 ire1b-4 showed severe reduction of chlorophyll a and b content on both liquid and solid plates. Chlorophyll a content was 11–13 times lower in ire1a-2 ire1b-4 mutants compared to ire1a-2 ire1b-4 controls and 5–11 times lower compared to ire1b-4 single mutants treated with 0.3 μg/mL Tm, indicating the sensitivity of the ire1a-2 ire1b-4 double knock-out as well as the severity of chlorosis at higher Tm doses. The reduction of chlorophyll b content showed a similar although less severe pattern, which is consistent with chlorophyll a being the dominant chlorophyll pigment accumulated by leaf tissue. The bip2-1 bip3-1 double mutants showed a similar trend in chlorophyll reduction comparable to that of ire1a-2 ire1b-4, although less dramatic (Figures 6A–D). The redundancy of BiP1 and BiP2 may contribute to the slightly higher levels of chlorophyll in the bip2-1 bip3-1 plants compared to ire1a-2 ire1b-4 double mutants. Altogether, these results indicate the effectiveness of using liquid medium to induce UPR through Tm treatment and highlight the numerous advantages of using this assay for large scale screens.

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