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Newly developed quantitative transactivation system shows difference in activation by Vitis CBF transcription factors on DRE/CRT elements.

Nassuth A, Siddiqua M, Xiao H, Moody MA, Carlow CE - Plant Methods (2014)

Bottom Line: We developed new effector and reporter plasmids which carry additional reporter genes, as well as a procedure to assay all three reporter enzymes from a single extract.The system also detected a reduction in activation due to one amino acid change in Vitis CBF1.The newly developed effector and reporter plasmids improve the ability to quantitatively compare the activation on two different promoter elements by the same transcription factor, or between two different transcription factors on the same promoter element.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2 W1 Canada.

ABSTRACT

Background: Agroinfiltration-based transactivation systems can determine if a protein functions as a transcription factor, and via which promoter element. However, this activation is not always a yes or no proposition. Normalization for variation in plasmid delivery into plant cells, sample collection and protein extraction is desired to allow for a quantitative comparison between transcription factors or promoter elements.

Results: We developed new effector and reporter plasmids which carry additional reporter genes, as well as a procedure to assay all three reporter enzymes from a single extract. The applicability of these plasmids was demonstrated with the analysis of CBF transcription factors and their target promoter sequence, DRE/CRT. Changes in the core DRE/CRT sequence abolished activation by Vitis CBF1 or Vitis CBF4, whereas changes in the surrounding sequence lowered activation by Vitis CBF1 but much less so for Vitis CBF4. The system also detected a reduction in activation due to one amino acid change in Vitis CBF1.

Conclusions: The newly developed effector and reporter plasmids improve the ability to quantitatively compare the activation on two different promoter elements by the same transcription factor, or between two different transcription factors on the same promoter element. The quantitative difference in activation by VrCBF1 and VrCBF4 on various DRE/CRT elements support the hypothesis that these transcription factors have unique roles in the cold acclimation process.

No MeSH data available.


Related in: MedlinePlus

Comparison of activation by VrCBF1and VrCBF1E85K on the M2 and M5 CRT/DRE variants. M2 = CRT: TGCCGACAT, M5: GACCGACAA. The CRT variant M7 (TGAAGACAT) was included as a negative reporter control, whereas an empty effector was included as a negative effector control. Error bars represent the standard deviation. Statistical analysis was performed on all data together and significantly different activation values (ANOVA p < 0.05) are indicated by different letters. Similar results were obtained in two other independent experiments.
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Fig6: Comparison of activation by VrCBF1and VrCBF1E85K on the M2 and M5 CRT/DRE variants. M2 = CRT: TGCCGACAT, M5: GACCGACAA. The CRT variant M7 (TGAAGACAT) was included as a negative reporter control, whereas an empty effector was included as a negative effector control. Error bars represent the standard deviation. Statistical analysis was performed on all data together and significantly different activation values (ANOVA p < 0.05) are indicated by different letters. Similar results were obtained in two other independent experiments.

Mentions: Based on the higher transactivation by CBF1 from V. riparia compared to activation by CBF1 from V. vinifera in our previous transactivation system [30], we predicted that a change of the glutamic acid (E) in the AP2 DNA binding domain of VrCBF1 at position 85 to a lysine (K), as is present in VvCBF1, would decrease the transactivation. Therefore, transactivation by VrCBF1 was compared to that by the mutant VrCBF1-E85K in the newly developed dual luciferase transactivation system, on the two DRE/CRT variants that had given the highest values (M2 and M5, see Figure 5). An empty control effector plasmid was included to confirm that the activations observed with either CBF1 or CBF4 are higher than those observed in the presence of endogenous tobacco transcription factor only (control). The results showed that the mutant VrCBF1. E85K indeed had a lower activation of the reporter with either the M2 or M5 variants (4.0x or 1.1x), as compared to the wild type VrCBF1 (5.0x or 2.2x) (Figure 6). This difference in activation was observed in three independent experiments and was statistically significant in two of these.Figure 6


Newly developed quantitative transactivation system shows difference in activation by Vitis CBF transcription factors on DRE/CRT elements.

Nassuth A, Siddiqua M, Xiao H, Moody MA, Carlow CE - Plant Methods (2014)

Comparison of activation by VrCBF1and VrCBF1E85K on the M2 and M5 CRT/DRE variants. M2 = CRT: TGCCGACAT, M5: GACCGACAA. The CRT variant M7 (TGAAGACAT) was included as a negative reporter control, whereas an empty effector was included as a negative effector control. Error bars represent the standard deviation. Statistical analysis was performed on all data together and significantly different activation values (ANOVA p < 0.05) are indicated by different letters. Similar results were obtained in two other independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4196112&req=5

Fig6: Comparison of activation by VrCBF1and VrCBF1E85K on the M2 and M5 CRT/DRE variants. M2 = CRT: TGCCGACAT, M5: GACCGACAA. The CRT variant M7 (TGAAGACAT) was included as a negative reporter control, whereas an empty effector was included as a negative effector control. Error bars represent the standard deviation. Statistical analysis was performed on all data together and significantly different activation values (ANOVA p < 0.05) are indicated by different letters. Similar results were obtained in two other independent experiments.
Mentions: Based on the higher transactivation by CBF1 from V. riparia compared to activation by CBF1 from V. vinifera in our previous transactivation system [30], we predicted that a change of the glutamic acid (E) in the AP2 DNA binding domain of VrCBF1 at position 85 to a lysine (K), as is present in VvCBF1, would decrease the transactivation. Therefore, transactivation by VrCBF1 was compared to that by the mutant VrCBF1-E85K in the newly developed dual luciferase transactivation system, on the two DRE/CRT variants that had given the highest values (M2 and M5, see Figure 5). An empty control effector plasmid was included to confirm that the activations observed with either CBF1 or CBF4 are higher than those observed in the presence of endogenous tobacco transcription factor only (control). The results showed that the mutant VrCBF1. E85K indeed had a lower activation of the reporter with either the M2 or M5 variants (4.0x or 1.1x), as compared to the wild type VrCBF1 (5.0x or 2.2x) (Figure 6). This difference in activation was observed in three independent experiments and was statistically significant in two of these.Figure 6

Bottom Line: We developed new effector and reporter plasmids which carry additional reporter genes, as well as a procedure to assay all three reporter enzymes from a single extract.The system also detected a reduction in activation due to one amino acid change in Vitis CBF1.The newly developed effector and reporter plasmids improve the ability to quantitatively compare the activation on two different promoter elements by the same transcription factor, or between two different transcription factors on the same promoter element.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2 W1 Canada.

ABSTRACT

Background: Agroinfiltration-based transactivation systems can determine if a protein functions as a transcription factor, and via which promoter element. However, this activation is not always a yes or no proposition. Normalization for variation in plasmid delivery into plant cells, sample collection and protein extraction is desired to allow for a quantitative comparison between transcription factors or promoter elements.

Results: We developed new effector and reporter plasmids which carry additional reporter genes, as well as a procedure to assay all three reporter enzymes from a single extract. The applicability of these plasmids was demonstrated with the analysis of CBF transcription factors and their target promoter sequence, DRE/CRT. Changes in the core DRE/CRT sequence abolished activation by Vitis CBF1 or Vitis CBF4, whereas changes in the surrounding sequence lowered activation by Vitis CBF1 but much less so for Vitis CBF4. The system also detected a reduction in activation due to one amino acid change in Vitis CBF1.

Conclusions: The newly developed effector and reporter plasmids improve the ability to quantitatively compare the activation on two different promoter elements by the same transcription factor, or between two different transcription factors on the same promoter element. The quantitative difference in activation by VrCBF1 and VrCBF4 on various DRE/CRT elements support the hypothesis that these transcription factors have unique roles in the cold acclimation process.

No MeSH data available.


Related in: MedlinePlus