title: Newly developed quantitative transactivation system shows difference in activation by Vitis CBF transcription factors on DRE/CRT elements.
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.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2 W1 Canada.
authors.forename: Annette
authors.initials: A
authors.lastname: Nassuth
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2 W1 Canada.
authors.forename: Mahbuba
authors.initials: M
authors.lastname: Siddiqua
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2 W1 Canada.
authors.forename: Huogen
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2 W1 Canada.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Moody
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2 W1 Canada.
authors.forename: Chevonne E
authors.initials: CE
authors.lastname: Carlow
keywords: Agroinfiltration normalization; CBF; DRE/CRT; Transactivation effector and reporter plasmids