Studies on low temperature induced gene regulation and freezing stress tolerance in Arabidopsis

Low temperature is a significant environmental stress factor limiting growth and productivity of agronomically important plants in many parts of the world. Plant responses to low temperature are complex, involving coordinated regulation of multiple biochemical pathways leading to expression of a number of genes encoding proteins that contribute to cold acclimation. Genes whose expression is increased during low temperature stress include those associated with freezing stress as well as the cellular protective enzymes, proteins and other compounds involved in osmotic adaptation to and tolerance of the cellular dehydration caused by freezing stress. Other up regulated genes include the several signaling proteins such as protein kinases and transcription factors that play roles in adapting plants to tolerate freezing temperature. Some of the regulatory DNA sequences that confer responsiveness to low temperature and associated stresses have been identified and transcription factors that interact with such cis-elements have been partially characterized. Work to identify additional sequences and factors involved in the sensation of low temperature and the transduction of the earliest gene response signals is continuing through the use of reverse genetics and mutagenesis studies. In particular, the following describes efforts to characterize some of the functions of the CBF genes as transcriptional activators in the low temperature signal transduction pathway and also to identify sequences responsible for regulation of their own activation. Through the use of promoter deletion analyses, a 155bp fragment of the CBF2 promoter was found to be responsible for low temperature inducible expression of the gene as well as responsiveness to mechanical agitation, ABA and the translation inhibitor cycloheximide. Some transcript accumulation at warm temperatures in the presence of cycloheximide also led to the idea that an inhibitor of CBF as well as an inducer of CBF expression (ICE) may be working to precisely regulate CBF expression.; Work to identify additional genes associated with the low temperature signal transduction pathway upstream of the COR genes using a COR gene promoter and marker gene fusion system and chemical mutagenesis was performed. Markers included the BAR gene, codA, and the R gene. Several mutants affected in their expression of both the R gene marker and one of the COR genes were isolated but the change in expression was likely due to gene silencing and not interruption of the cold signaling pathway. Plants exhibiting loss of the endogenous COR78 expression were examined with an ion leakage assay to assess the significance of the loss of this one COR gene on freezing tolerance. No significant change was seen in these plants, however, a wealth of information was obtained concerning the use of different reporter genes and possible methods to improve the search for genes.