Study of C-Repeat Binding Factors (CBF) structural elements conferring stability under adverse conditions
Winter wheat is a crop with a lot of potential for the Canadian Prairies. It has many agronomic, environmental and economic advantages over spring wheat, such as a 20-30 % higher yield potential and a lower rate of Fusarium infections. However, wheat production on the Prairies is dominated by spring varieties because the current winter wheat cultivars do not accumulate sufficient frost resistance during cold acclimation in the autumn to survive the winters on a consistent basis. Efforts to improve frost tolerance genes in winter wheat via conventional breeding practices has been attempted but proven to be difficult as the cold tolerance in the available varieties cannot be exceeded. A possible approach to increase frost hardiness in plants is through the optimization of CRT (C-repeat)-Binding Factor (CBF) expression, activities and stability under cold conditions. These master transcription factors upregulate a wide range of cold stress responses and represent about 40% of the difference in freezing tolerance observed between Canadian winter wheat cultivars with low and very high winter-hardiness. Cold-hardy winter wheat produces many CBF variants, but little information is available about the role of the individual proteins. From initial studies of the CBF family encoded by cold-hardy cultivar Norstar, several members display high resilience to extreme temperatures and denaturing conditions, which may be important for winter survival. The high CBF stability is also associated with anomalous slow migration on SDS-PAGE gels. To further investigate the abnormal gel behavior of CBF12, various modifications to the protein were made and analyzed by SDS-PAGE. It was concluded that the conserved CMIII3, CMIII-1 motifs flanking the DNA-binding AP2 domain or the AP2 domain itself do not slow down CBF12 migration during SDS-PAGE. Rather, removal of the secondary structures within the AP2 region increased the degree of anomalous migration. Instead, it was found that changes in the ratio and distribution of charged residues and the degree of disorder predicted for CBF12 correlated with increased anomalous migration during SDS-PAGE. Similar features are also present for other stress response proteins and transcription factors. Whether the specific amino acid residues contributing to gel shifting play a role in enhancing cold tolerance in planta remains to be verified.
SDS-PAGE, cold tolerance, charge distribution, flexibility, anomalous migration, winter wheat, CBF
Master of Science (M.Sc.)