CBFomics and the Molecular Genetics of Low Temperature and Freezing Tolerance in Plants

Background

Plants vary widely in their ability to survive low temperatures. Some plants are able to withstand prolonged periods at subzero temperatures, whereas others such as tropical & warm season plants have their growth and development severely and permanently impaired by temperatures of 10‑15oC. However, the maximal freezing tolerance level of even the most extremely freezing tolerant plants is not constitutive. Rather, it is an induced phenomenon. These plants cold acclimate, a process in which plants increase in freezing tolerance in response to a period of low, non-freezing temperatures.

During cold acclimation a subset of a plant's genes are activated, or turned on. Most of the proteins encoded by these newly activated genes fulfill a structural role in protecting the plant cell from the physical effects imposed during a freeze thaw. A much smaller subset of the newly activated genes play a regulatory role in controlling the expression of these structural protein-encoding genes. One particular set of regulatory genes, the CBFs (for C-Repeat Binding Factors), were identified in Arabidopsis thaliana and encode proteins that function as a molecular switch to exert regulatory control over the majority of the low temperature induced structural protein-encoding genes.



 Our research currently revolves around two central themes. One of these is to determine whether components of the Arabidopsis thaliana CBF regulatory system are conserved across plant taxa and to determine the role that these homologous systems play in mediating low temperature responsiveness, cold acclimation and increasing freezing tolerance. Another important research area we are pursuing is to understand the mechanisms by which the CBFs activate expression of their target gene.
 

To address the question of conservation across plant taxa we are taking a comparative genomics approach. We are presently focused on a number of key plants within the Solanaceae (tomato and potato), the Fabaceae (alfalfa, barrel medic and soybean) and the Poaceae (wheat, barley and rye). Each offers a unique set of questions that we can pose into understanding the molecular genetic nature of low temperature and freezing tolerance.  In Arabidopsis there are 3 linked genes on chromosome IV that encode CBF transcriptional activators. All three play a role in controlling pathways leading to increased freezing tolerance.

Tomato, a chilling-sensitive plant suffers irreversible damage when exposed to temperatures below 10oC.

 

 Tomato also harbors 3 linked genes that encode CBFs.  However, only one of these genes appears to function in response to cold temperatures. Moreover downstream target genes orthologous to the Arabidopsis thaliana targets are not induced by CBFs in tomato. Thus there is a reduced response to cold temperatures at multiple points in the CBF pathway in tomato.

 In the cereal crops, the CBFs may also play a key role in winter survival. In general, the cereal crops: wheat, barley and rye; are generally one of either two types: winter or spring. Winter types require fall planting and must survive the winter. In contrast spring types are planted in the spring and are unable to survive colder winters.



Molecular genetic analyses have revealed that a large cluster of CBF genes in one region of the genome is correlated with low temperature tolerance. This has led us to hypothesize that the genetic basis underlying the difference in cold hardiness between winter and spring cereal types may be due to structural and functional differences in a cluster of cereal CBF genes. To investigate this we are molecularly cloning and sequencing this region of the genome from a select group of winter and spring barley genotypes and concomitantly determining individual CBF gene expression patterns.


To address the question of how the CBFs activate expression of their target genes we have taken a mutational analysis strategy. This approach revealed that the CBF activation domain is comprised of numerous motifs that impart substantial functional redundancy in trans-activation. We are presently pursuing the identification of the activation domain targets in order to have a more complete picture as to how these multiple activating motifs effect trans-activation.

Development of Bacteriophage l Plant Genomic Libraries

Since one of our objectives was to determine the structure of the genes encoding the CBF proteins from a wide range of plant taxa, my lab has constructed, screened, isolated, and deduced the DNA sequence of the genomic regions encompassing the CBF genes from more than 15 different genotypes and species of plants. Aliquots of these amplified Bacteriophage λ Genomic Libraries are available for scientific research. Part of this endeavor also necessitated the de novo development of many of the techniques, including Bacteriophage l Plant Genomic Library Construction, and Shotgun Library Construction. Together these two procedures (downloadable in pdf format) describe in substantial detail, essential technical steps that will allow one to rapidly create, screen, subclone and sequence bacteriophage l genomic inserts. Additionally, we have a collection of standard laboratory protocols that may also be helpful and of interest.