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Transcriptomic and transgenic approaches to understanding abiotic stress response in wine grape (Vitis vinifera).
AuthorTillett, Richard Leonard
AdvisorCushman, John C.
Biochemistry and Molecular Biology
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As a woody perennial, cultivated grapevine (Vitis vinifera L.) and related wild species must adapt to repeated environmental challenges in a lifetime. Abiotic stresses such as water deficits, soil salt accumulation, and freezing temperatures provoke complex molecular responses in plants that ultimately determine a plant's ability to tolerate or survive the stress conditions. In these studies, we investigate abiotic stress in grapevine employing transcriptomic and transgenic approaches to characterize stress response and improve abiotic stress tolerance in Vitis vinifera.The vast majority of transcript sequence information in grapevine has been obtained by the generation of libraries of expressed sequence tags (ESTs), partial transcript fragments, though this method is currently being displaced by "Next- Generation Sequencing" or RNA-Seq deep-sequencing technologies. We first review the progress of research in grapevine molecular biology that has been powered partly by ESTs as well as the most recent studies making use of the revolutionary RNA-Seq platforms (Chapter I).In initial analyses, we generated EST libraries from the abiotically stressed leaves, berries, and roots of grapevines and examined changes in gene expression under stress in these tissues, as well as identify genes with root-specific or root-enriched gene expression, using EST frequency. Additionally, we compared performance of this method vs. closed-platform microarray-based gene expression analysis. (Chapter II).Among the tens of thousand of sequences in our stressed tissue EST libraries, a transcript of a grape gene similar to the CBF family of stress-responsive transcription factors, VvCBF4, was found. The function of VvCBF4 was investigated and we characterized transgenic grapevines in which the VvCBF4 gene was constitutively overexpressed. VvCBF4 overexpression enhanced freezing stress survival of grapevine. Global changes in transcript and protein expression were investigated with microarray expression profiling and DIGE proteomics to observe the molecular effects of VvCBF4 over-expression which might contribute to freezing survival improvement. (Chapter III).Technological advances in the last decade have allowed researchers around the world to construct two drafts of the grapevine genome and generate great volumes of transcriptomic and marker sequence for Vitis vinifera L. and related Vitis species. This growth is likely to continue to accelerate with the adoption of RNA-Seq technologies. We conclude with proposals for future experiments to leverage these and other techniques to expand our knowledge of the stress biology of grapevine (Chapter IV).