Yeast One-Hybrid Techniques to Construct Cold-Responsive Regulatory Networks in Arabidopsis

As sessile organisms, plants are constantly challenged with abiotic and biotic stresses such as salt, drought, cold, heat, bacteria, fungi, and insects. To achieve proper growth and reproduction, plants have evolved mechanisms to overcome these stresses. We are studying cold acclimation, the process whereby certain plants increase in freezing tolerance when exposed to low, non-freezing temperatures. It has been established by us and others that the C-repeat/DRE binding factors of Arabidopsis – CBF1, CBF2, and CBF3 – are major transcription factors involved in cold acclimation. When Arabidopsis plants are exposed to low temperature, CBF1, -2, and -3 are rapidly induced (within 15 min) prior to the induction of cold-responsive CBF-targeted genes such as COR15 and COR78. Ectopic constitutive expression of CBF1, -2, and -3 results in constitutive expression of CBF-targeted COR genes and enhanced freezing tolerance without cold acclimation. The goal of this project is to identify the regulatory and structural proteins that comprise the “thermometer” that mediates cold induction of CBF1, -2, and -3 and other genes encoding transcription factors that are rapidly induced in response to low temperature. Toward this end, we developed a library of nearly 1000 yeast clones carrying individual Arabidopsis transcription factors that can be used in a mating-based yeast one-hybrid screening procedure to identify transcription factors that bind to any gene promoter of interest. We have used the procedure to screen for transcription factors that bind to the promoters of CBF1, CBF2, CBF3, ZAT10, and ZAT12 and have identified a number of factors that were previously not known to bind to these promoters. The roles of these factors in cold-regulated gene expression are now being explored.