Limits...
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

Analysis of reporter gene expression after agroinfiltration of tobacco leaves with VrCBF4 effector in combination with four different 4XCRTmin35S reporter constructs. [A] GUS/protein (blue bars) and FiLUC/protein (black bars), [B] FiLUC/GUS, [C] RiLUC/FiLUC, [D] RiLUC/FiLUC/GUS. Shown are the averages of three technical replicates and their standard deviation. Different letters indicate statistically significant differences (ANOVA p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Analysis of reporter gene expression after agroinfiltration of tobacco leaves with VrCBF4 effector in combination with four different 4XCRTmin35S reporter constructs. [A] GUS/protein (blue bars) and FiLUC/protein (black bars), [B] FiLUC/GUS, [C] RiLUC/FiLUC, [D] RiLUC/FiLUC/GUS. Shown are the averages of three technical replicates and their standard deviation. Different letters indicate statistically significant differences (ANOVA p < 0.05).

Mentions: Various assay conditions were tested for compatibility with an analysis of the activities of the beta-glucuronidase, renilla luciferase and firefly luciferase reporter enzymes in a single extract. It was determined that extracts prepared in CCLR and diluted in PLB buffer (both from Promega) could be used for either glucuronidase or luciferase assays. Dilutions between 75 and 100x gave values below the maximum value for the fluorescence reader and were in a linear range. This indicates that all reporter enzyme activity values could be determined from the same extract.The GUS/protein and FiLUC/protein values were expected to be the same for each effector/reporter infiltration treatment if the amount of DNA taken up and expressed in leaves infiltrated with VrCBF4 effector and reporter plasmid-containing agrobacteria (reporters containing the M1, M2, M4 and M5 variants of the DRE/CRT element, for details on these variants see a later section), was similar. The results show that both GUS/protein and FiLUC/protein values vary (Figure 2A), indicating that the amount of DNA transferred and expressed in the plant cells after the different infiltrations varied for both effector (GUS) and reporter (FiLUC) plasmid. Also the FiLUC/GUS values vary (Figure 2B), indicating that the ratio between effector DNA and reporter DNA uptake and expression varies. This means that the amounts of CBF protein and reporter gene in the cells might vary independently from each other, and this can cause variations in activation between separate infiltrations. The RiLUC/FiLUC ratio can therefore not be taken as a true measure of transactivation (Figure 2C). Instead it is better to normalize for the amount and expression of effector plasmid (represented by GUS) as well and therefore the RiLUC/FiLUC/GUS was taken as a measure of transactivation (Figure 2D).Figure 2


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)

Analysis of reporter gene expression after agroinfiltration of tobacco leaves with VrCBF4 effector in combination with four different 4XCRTmin35S reporter constructs. [A] GUS/protein (blue bars) and FiLUC/protein (black bars), [B] FiLUC/GUS, [C] RiLUC/FiLUC, [D] RiLUC/FiLUC/GUS. Shown are the averages of three technical replicates and their standard deviation. Different letters indicate statistically significant differences (ANOVA p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Analysis of reporter gene expression after agroinfiltration of tobacco leaves with VrCBF4 effector in combination with four different 4XCRTmin35S reporter constructs. [A] GUS/protein (blue bars) and FiLUC/protein (black bars), [B] FiLUC/GUS, [C] RiLUC/FiLUC, [D] RiLUC/FiLUC/GUS. Shown are the averages of three technical replicates and their standard deviation. Different letters indicate statistically significant differences (ANOVA p < 0.05).
Mentions: Various assay conditions were tested for compatibility with an analysis of the activities of the beta-glucuronidase, renilla luciferase and firefly luciferase reporter enzymes in a single extract. It was determined that extracts prepared in CCLR and diluted in PLB buffer (both from Promega) could be used for either glucuronidase or luciferase assays. Dilutions between 75 and 100x gave values below the maximum value for the fluorescence reader and were in a linear range. This indicates that all reporter enzyme activity values could be determined from the same extract.The GUS/protein and FiLUC/protein values were expected to be the same for each effector/reporter infiltration treatment if the amount of DNA taken up and expressed in leaves infiltrated with VrCBF4 effector and reporter plasmid-containing agrobacteria (reporters containing the M1, M2, M4 and M5 variants of the DRE/CRT element, for details on these variants see a later section), was similar. The results show that both GUS/protein and FiLUC/protein values vary (Figure 2A), indicating that the amount of DNA transferred and expressed in the plant cells after the different infiltrations varied for both effector (GUS) and reporter (FiLUC) plasmid. Also the FiLUC/GUS values vary (Figure 2B), indicating that the ratio between effector DNA and reporter DNA uptake and expression varies. This means that the amounts of CBF protein and reporter gene in the cells might vary independently from each other, and this can cause variations in activation between separate infiltrations. The RiLUC/FiLUC ratio can therefore not be taken as a true measure of transactivation (Figure 2C). Instead it is better to normalize for the amount and expression of effector plasmid (represented by GUS) as well and therefore the RiLUC/FiLUC/GUS was taken as a measure of transactivation (Figure 2D).Figure 2

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