Limits...
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

Unfolded protein response (UPR) target genes are upregulated during Tm-induced ER stress. Five-day-old Arabidopsis seedlings were pre-grown in liquid MS medium, then subjected to stress by replacing the media with liquid MS supplemented with designated concentrations of Tm for 3 days. The recovery was performed by incubating stressed seedlings in liquid MS medium for another 3 days. The transcript accumulation of (A)CRT1, (B)CRT3, (C)CNX1, and (D)bZIP60 splicing were measured by real-time PCR and normalized to a housekeeping gene UBQ5. Statistical analysis was performed with Student’s t-test comparing values from Tm-exposed seedlings to regular MS-grown seedlings, *p < 0.05, **p < 0.01, ***p < 0.001. Experiments were performed in at least three independent biological replications. Error bars represent SE between these replications.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4562274&req=5

Figure 3: Unfolded protein response (UPR) target genes are upregulated during Tm-induced ER stress. Five-day-old Arabidopsis seedlings were pre-grown in liquid MS medium, then subjected to stress by replacing the media with liquid MS supplemented with designated concentrations of Tm for 3 days. The recovery was performed by incubating stressed seedlings in liquid MS medium for another 3 days. The transcript accumulation of (A)CRT1, (B)CRT3, (C)CNX1, and (D)bZIP60 splicing were measured by real-time PCR and normalized to a housekeeping gene UBQ5. Statistical analysis was performed with Student’s t-test comparing values from Tm-exposed seedlings to regular MS-grown seedlings, *p < 0.05, **p < 0.01, ***p < 0.001. Experiments were performed in at least three independent biological replications. Error bars represent SE between these replications.

Mentions: To firmly establish that the phenotypic responses of seedlings grown on Tm are indeed caused by the UPR stress, we next tested expression levels of several canonical ER stress marker genes. We treated liquid MS-grown 5-day-old wild type Col-0 seedlings with 0.15 μg/mL and 0.3 μg/mL Tm for 3 days, then replaced the media with plain MS and continued to culture the seedlings for another 3 days. We sampled the seedlings at 3-days post Tm treatment (Tm stress) and after the 3-days recovery period (Tm recovery). We used qRT-PCR (quantitative Reverse Transcription PCR) to determine levels of transcriptional induction of several UPR stress markers: Calreticulin 1 (CRT1), CRT3, and Calnexin 1 (CNX1Figures 3A–C). Calreticulins (CRTs) are ER-localized proteins that bind Ca2++ and function in the proper folding of proteins through oligosaccharide modification. Arabidopsis contains three isoforms of CRT: CRT1, CRT2, and CRT3. All three family members are essential for the ER stress response (Wang et al., 2005; Christensen et al., 2008; Saijo et al., 2009; Pajerowska-Mukhtar et al., 2012) and function in many other signaling pathways, including defense against pathogen infection (Christensen et al., 2010). Calnexin (CNX) is also an ER-localized chaperone that monitors protein folding activity within the ER. Specifically, the function of CNX is to retain misfolded N-linked glycoproteins. Arabidopsis CNX was demonstrated to be implicated in pathogen defense (Wang et al., 2005). The seedlings exhibited a marked increase in UPR marker genes at 3-days post exposure to both concentrations of Tm (Figure 3). The amplitude of induction ranged from 2.5 to 5-fold for 0.15 μg/ml Tm and 7–20-fold for the higher 0.3 μg/ml Tm dose. Thus, we confirmed that the liquid assay can reliably induce UPR marker genes in Arabidopsis seedlings. Next, we asked whether the recovery in plain MS media will translate into a deactivation of the UPR response. Following a 3-days period of incubation under stress-free conditions, the ER stress markers expression decreased dramatically compared to samples collected directly after Tm exposure, and reverted to nearly basal levels. This observation confirms that the wild type Col-0 seedlings can effectively recover from the ER stress under the conditions tested (Figures 3A–C). To further corroborate these data, we also measured the activity of bZIP60 splicing, which reflects activation of the IRE1 signaling branch of UPR, using our previously established qRT-PCR assay (Moreno et al., 2012). We observed a 2.5 and 12-fold increase in bZIP60 splicing in the samples treated with Tm doses of 0.15 and 0.30 μg/ml, respectively. This bZIP60 splicing activity is specific to Tm-induced ER stress as we did not observe an induction of bZIP60 splicing in the Tm recovery samples (Figure 3D). Taken together, we conclude that Tm can specifically induce UPR stress in Arabidopsis grown in liquid MS medium.


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)

Unfolded protein response (UPR) target genes are upregulated during Tm-induced ER stress. Five-day-old Arabidopsis seedlings were pre-grown in liquid MS medium, then subjected to stress by replacing the media with liquid MS supplemented with designated concentrations of Tm for 3 days. The recovery was performed by incubating stressed seedlings in liquid MS medium for another 3 days. The transcript accumulation of (A)CRT1, (B)CRT3, (C)CNX1, and (D)bZIP60 splicing were measured by real-time PCR and normalized to a housekeeping gene UBQ5. Statistical analysis was performed with Student’s t-test comparing values from Tm-exposed seedlings to regular MS-grown seedlings, *p < 0.05, **p < 0.01, ***p < 0.001. Experiments were performed in at least three independent biological replications. Error bars represent SE between these replications.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Unfolded protein response (UPR) target genes are upregulated during Tm-induced ER stress. Five-day-old Arabidopsis seedlings were pre-grown in liquid MS medium, then subjected to stress by replacing the media with liquid MS supplemented with designated concentrations of Tm for 3 days. The recovery was performed by incubating stressed seedlings in liquid MS medium for another 3 days. The transcript accumulation of (A)CRT1, (B)CRT3, (C)CNX1, and (D)bZIP60 splicing were measured by real-time PCR and normalized to a housekeeping gene UBQ5. Statistical analysis was performed with Student’s t-test comparing values from Tm-exposed seedlings to regular MS-grown seedlings, *p < 0.05, **p < 0.01, ***p < 0.001. Experiments were performed in at least three independent biological replications. Error bars represent SE between these replications.
Mentions: To firmly establish that the phenotypic responses of seedlings grown on Tm are indeed caused by the UPR stress, we next tested expression levels of several canonical ER stress marker genes. We treated liquid MS-grown 5-day-old wild type Col-0 seedlings with 0.15 μg/mL and 0.3 μg/mL Tm for 3 days, then replaced the media with plain MS and continued to culture the seedlings for another 3 days. We sampled the seedlings at 3-days post Tm treatment (Tm stress) and after the 3-days recovery period (Tm recovery). We used qRT-PCR (quantitative Reverse Transcription PCR) to determine levels of transcriptional induction of several UPR stress markers: Calreticulin 1 (CRT1), CRT3, and Calnexin 1 (CNX1Figures 3A–C). Calreticulins (CRTs) are ER-localized proteins that bind Ca2++ and function in the proper folding of proteins through oligosaccharide modification. Arabidopsis contains three isoforms of CRT: CRT1, CRT2, and CRT3. All three family members are essential for the ER stress response (Wang et al., 2005; Christensen et al., 2008; Saijo et al., 2009; Pajerowska-Mukhtar et al., 2012) and function in many other signaling pathways, including defense against pathogen infection (Christensen et al., 2010). Calnexin (CNX) is also an ER-localized chaperone that monitors protein folding activity within the ER. Specifically, the function of CNX is to retain misfolded N-linked glycoproteins. Arabidopsis CNX was demonstrated to be implicated in pathogen defense (Wang et al., 2005). The seedlings exhibited a marked increase in UPR marker genes at 3-days post exposure to both concentrations of Tm (Figure 3). The amplitude of induction ranged from 2.5 to 5-fold for 0.15 μg/ml Tm and 7–20-fold for the higher 0.3 μg/ml Tm dose. Thus, we confirmed that the liquid assay can reliably induce UPR marker genes in Arabidopsis seedlings. Next, we asked whether the recovery in plain MS media will translate into a deactivation of the UPR response. Following a 3-days period of incubation under stress-free conditions, the ER stress markers expression decreased dramatically compared to samples collected directly after Tm exposure, and reverted to nearly basal levels. This observation confirms that the wild type Col-0 seedlings can effectively recover from the ER stress under the conditions tested (Figures 3A–C). To further corroborate these data, we also measured the activity of bZIP60 splicing, which reflects activation of the IRE1 signaling branch of UPR, using our previously established qRT-PCR assay (Moreno et al., 2012). We observed a 2.5 and 12-fold increase in bZIP60 splicing in the samples treated with Tm doses of 0.15 and 0.30 μg/ml, respectively. This bZIP60 splicing activity is specific to Tm-induced ER stress as we did not observe an induction of bZIP60 splicing in the Tm recovery samples (Figure 3D). Taken together, we conclude that Tm can specifically induce UPR stress in Arabidopsis grown in liquid MS medium.

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