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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

Activation by VrCBF1 or VrCBF4 on CRT (M2) and mutated core CRT sequence. M2: TGCCGACAT, M7: TGAAGACAT, M8: TGCCGCCAT, M9: TGCCGAAAT. Error bars represent the standard deviation. Infiltrations without CBF effector were included as control. Statistical analysis was performed on the set of no CBF, VrCBF1 and VrCBF4 data separately and significantly different activation values (ANOVA p < 0.05) are indicated by different letters. Similar results were obtained for two other independent experiments.
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Fig3: Activation by VrCBF1 or VrCBF4 on CRT (M2) and mutated core CRT sequence. M2: TGCCGACAT, M7: TGAAGACAT, M8: TGCCGCCAT, M9: TGCCGAAAT. Error bars represent the standard deviation. Infiltrations without CBF effector were included as control. Statistical analysis was performed on the set of no CBF, VrCBF1 and VrCBF4 data separately and significantly different activation values (ANOVA p < 0.05) are indicated by different letters. Similar results were obtained for two other independent experiments.

Mentions: To determine whether the transactivation by VrCBF1 and VrCBF4 has strict requirements regarding the TACCGACAT sequence present in the RiLUC reporter gene promoter, various mutations were made in this sequence and tested for transactivation. Examination of the results with the regular CRT sequence (GCCGAC = M2) showed that both VrCBF1 and VrCBF4 gave higher RiLUC/FiLUC/GUS values, respectively about 6 and 14 times higher, than the values obtained in the absence of a CBF (Figure 3). Mutations in the CCGAC core DRE/CRT sequence significantly reduced the activation by VrCBF1 and VrCBF4 to values that were not statistically different from those obtained without CBF.Figure 3


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)

Activation by VrCBF1 or VrCBF4 on CRT (M2) and mutated core CRT sequence. M2: TGCCGACAT, M7: TGAAGACAT, M8: TGCCGCCAT, M9: TGCCGAAAT. Error bars represent the standard deviation. Infiltrations without CBF effector were included as control. Statistical analysis was performed on the set of no CBF, VrCBF1 and VrCBF4 data separately and significantly different activation values (ANOVA p < 0.05) are indicated by different letters. Similar results were obtained for two other independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Activation by VrCBF1 or VrCBF4 on CRT (M2) and mutated core CRT sequence. M2: TGCCGACAT, M7: TGAAGACAT, M8: TGCCGCCAT, M9: TGCCGAAAT. Error bars represent the standard deviation. Infiltrations without CBF effector were included as control. Statistical analysis was performed on the set of no CBF, VrCBF1 and VrCBF4 data separately and significantly different activation values (ANOVA p < 0.05) are indicated by different letters. Similar results were obtained for two other independent experiments.
Mentions: To determine whether the transactivation by VrCBF1 and VrCBF4 has strict requirements regarding the TACCGACAT sequence present in the RiLUC reporter gene promoter, various mutations were made in this sequence and tested for transactivation. Examination of the results with the regular CRT sequence (GCCGAC = M2) showed that both VrCBF1 and VrCBF4 gave higher RiLUC/FiLUC/GUS values, respectively about 6 and 14 times higher, than the values obtained in the absence of a CBF (Figure 3). Mutations in the CCGAC core DRE/CRT sequence significantly reduced the activation by VrCBF1 and VrCBF4 to values that were not statistically different from those obtained without CBF.Figure 3

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