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Analysis of the Vitis C-Repeat Binding Factor (CBF) genes and their potential roles in both the CBF and stomatal development pathways

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Title: Analysis of the Vitis C-Repeat Binding Factor (CBF) genes and their potential roles in both the CBF and stomatal development pathways
Author: Carlow, Chevonne
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Nassuth, Annette
Abstract: Vitis vinifera species are adapted for growth in moderate climates, so freezing temperature damage during Ontario winters results in significant revenue loss for producers. Highly conserved Drought Response Element Binding 1/C-Repeat Binding factor (DREB1/CBF) genes have been shown to be crucial in the acquisition of frost tolerance, while Drought Response Element Binding factor 2 (DREB2) genes were thought to function in drought tolerance. Seven CBF genes and two DREB2 genes were cloned from both Vitis vinifera and the more cold hardy Vitis riparia. Vitis CBFs showed differing expression patterns under ambient and cold conditions, with specific subsets expressed in leaf and bud tissues. Transcripts for the two DREB2 genes were not observed in conditions where CBFs had been detected. Transient transactivation assays showed that all Vitis CBFs but one and both DREB2 proteins could induce transcription via the core C-Repeat (CRT)/Drought Response Element (DRE) promoter sequence element. Investigation revealed that the C-terminal hydrophobic domains present in CBF6 but absent from CBF5 contributed to activation. Predicted PEST motifs in both CBF4 and DREB2-3 affect activation by CBF4 only. Further investigation found that CBF and DREB2 proteins had a preference for the sequence surrounding the DRE core based on their classification; CBF proteins preferred an AT-rich sequence, while DREB2 proteins preferred a GC-rich sequence. From the evidence presented in this thesis, we predict that Vitis CBFs and DREB2s have roles in different abiotic stress tolerance pathways, and that the individual members of each of these families have different functions within their respective regulons. Evidence from transient transactivation and overexpression studies suggests that Vitis DREB2-5 and CBF8 may also have a role in the stomatal development pathway. Their overexpression induced a higher stomatal lineage plus pavement cell density, similar to phenotypes previously found for Vitis SPEECHLESS and ICE4. We hypothesize that these four proteins are involved in the first step of the Vitis stomatal development pathway, an increase in stomatal lineage cells. How they do that remains unclear, but the work presented here provides a basis of understanding to help direct forthcoming investigations.
URI: http://hdl.handle.net/10214/9645
Date: 2016-05


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