| Low temperature, drought, and salinity, which eventually result in dehydration, are the most important environmental stresses limiting the planting area and causing devasting yield losses of many crop species worldwide. Over the past century, traditional breeding gained limited success in enhancing crop stress tolerance. Thus, understanding the molecular mechanisms of plant stress tolerance is required to provide an effective strategy to improve crop stress tolerance.; To elucidate the molecular mechanisms of plant cold acclimation, I focused on the understanding of Arabidopsis CBF1 (C-repeat binding factor 1, CRT) mediated plant cold-regulated (COR) gene trans-activation, using a combination of bioinformatic, biochemical, genetic, and molecular biology methods. The work was initiated (1) to mutationally analyze the CBF1 activation domain (AD), (2) and then to determine the epigenetic regulation on COR gene that is played by the putative associated chromatin-modifying factors of CBF1AD.; Mutagenesis strategies including alanine-scanning mutagenesis, truncation mutation, and leucine substitution were used to determine the functional roles of amino acids within the CBF1 COOH-terminus. I created all these mutants as fusions to the heterologous GAL4 DNA binding domain. Mutants were then transformed into yeast reporter strain harboring a GAL4 responsive reporter construct, consisting of a beta-galactosidase gene driven by a promoter with four GAL4 binding sites, or a GAL4-responsive GUS gene Arabidopsis reporter line. The trans-activity of mutants was then determined by the assay of reporter gene. Truncation mutation and alanine substitution analyses suggested there were multiple regions contributing to trans-activation. Using the computer program Hydrophobic Cluster Analysis (HCA), I identified several hydrophobic clusters (HC) composed of bulky hydrophobic or aromatic amino acids. The disruption of more than two HCs was required to significantly reduce the transactivation activity, suggesting a functional redundancy between HCs. Alignments of Arabidopsis CBFs with homologs from different species reveal that the hydrophobic patterns in Arabidopsis CBF1 are conserved across plant taxa. Further leucine substitution of the conserved alanine residues suggested that the residue size contributed to the CBF1 maximal trans-activity. Alanine substitution of the most conserved WSY motif revealed an inhibitory effect on the trans-activity. Furthermore, my mutagenesis also suggests that the growth retardation resulted from the overexpression of a trans-activator in Arabidopsis is related to the trans-activity of that trans-activator. Thus, the C-terminus of CBF1 contains conserved amino acid stretches with special functional roles, including alanine stretches, hydrophobic cluster, and WSY motif. My extensive analyses provide an example that how a plant transcription factor adopts a highly ordered structure to fine-tune gene expression.; The second part of this dissertation is to determine the role of epigenetic control of transcription in the regulation of COR gene expression. Several methylation mutant lines were subjected to low temperature in a time course treatment. RNA gel blot analyses revealed that a single T-DNA integration locus with multiple copies of rearranged transgene in these methylation mutants resulted in the reduction of CBF1, COR15a, and ABF1 gene expression, suggesting a link between the integrated repeats and transcriptional regulation of genes involved in plant cold acclimation. Plant freezing assays confirmed that the transgene locus affected the plant freezing tolerance.; Next, I determined the putative direct target of CBF1 activation domain, the homologs of yeast Tra1 (transcription domain-associated protein 1), and its role in regulation of COR gene expression. Tra1 is the only common and critical component of histone acetyltransferase complexes involved in chromatin-modifying that facilitate transcriptional activa... |
