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Analysis of the Role of Grape ICE Proteins in the CBF Pathway and Stomatal Development

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Title: Analysis of the Role of Grape ICE Proteins in the CBF Pathway and Stomatal Development
Author: Rahman, Mohammad
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Nassuth, Annette
Abstract: Arabidopsis ICE (Inducer of CBF expression) transcription factors play a role in freezing tolerance and stomatal development. This thesis examined the possible functions of ICE genes from grape. Four ICE genes were isolated and sequenced from both the freezing tolerant wild species Vitis riparia and the freezing sensitive cultivated species V. vinifera. All the encoded ICE proteins contain a bHLH domain with an ICE-specific sequence in their highly conserved C-terminus, and only a few amino acid differences between the ICE orthologs. RT-PCR analyses and sequencing showed that all genes produced spliced transcripts in leaves and buds at ambient and low temperature conditions. In addition, the alternative transcripts ICE1i1, ICE2i2 and ICE4i1 were detected in leaves but only ICE1i1 in mature buds. The functions of the various ICE variants in the CBF pathway were analyzed by Agrobacterium-mediated transactivation experiments. The results showed that all ICE proteins can activate the CBF4 promoter, albeit especially ICE2 and ICE3, via a MYC2g element. Cold increased activation, presumably because of cold-induced sumoylation that stabilizes these proteins. ICE1 and ICE4 induced the transcription better from CBF6 promoter, via different MYC elements. The truncated ICE proteins encoded by alternative transcripts were found to give a lower activation compared to their corresponding regular proteins. Homologs of the Arabidopsis stomatal genes SPCH, MUTE and FAMA were isolated and sequenced from wild and wine grape species. Each of these stomatal genes produced regular spliced transcripts, which for FAMA included 2 transcripts with different start sites, early FAMA(E) and late FAMA(L). The sequential presence of SPCH, MUTE and FAMA transcripts in different aged leaves and the effect of transient overexpression of these genes and of ICE genes on the formation of stomata and pavement cells supports their respective functions in three consecutive stages of stomatal development. The role of proteins encoded on alternatively spliced transcripts, SPCHi1, MUTEi1 FAMAi1(E) and FAMAi2(L), is as yet unclear. Transactivation results suggest that grape ICEs interact with grape FAMA(L) to activate VrCBF4, possibly to regulate both stomatal development and the freezing tolerance pathway. A model which suggests a role for all ICE and stomatal genes in either stomatal development or/and the acquisition of freezing tolerance is presented.
Date: 2015-01
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