Low temperature is a major environmental factor that affects the yield and quality of food and bioenergy crop plants. To cope with low temperature stress, many plants can acquire freezing tolerance through cold acclimation, in which remodeling of the metabolome and biological pathways leads to physiological adaptation to low temperatures. Many of these adjustments are brought about by intensively reconfiguring the expression of cold-regulated (COR) genes during cold acclimation. Some of the COR genes are regulated by the well-studied CBF pathway and its parallel ZAT12 pathway. However, the regulation and functions of other CBF-independent pathways that are involved in COR gene regulation are not well understood. Freezing tolerance in plants is affected by external signals as well as the growth conditions of plants themselves, but cross talk between cold and other signals has not been fully elucidated. The goal of this dissertation is to understand how freezing tolerance is established during cold acclimation in plants by addressing two key questions: first, how do plants perceive and integrate photoperiod as seasonal signals to modulate the expression of COR genes and subsequent freezing tolerance; second, what are the CBF-independent pathways that have major roles in COR gene expression and their roles in tolerance to low temperatures?In the first part of the dissertation, the photoperiodic regulation of the cold-responsive CBF pathway and freezing tolerance was demonstrated in Arabidopsis. The CBF transcript levels in short-day (SD) plants were higher than in long-day (LD) plants. Genetic analysis indicated that phytochrome B (PHYB) functions with two phytochrome interacting factors, PIF4 and PIF7, to down-regulate the CBF pathway and freezing tolerance under LD conditions. Down-regulation of the CBF pathway in LD plants correlated with higher PIF4 and PIF7 transcript levels and greater stability of the PIF4 and PIF7 proteins under LD conditions. These findings provide a mechanism of how plants sense and integrate photoperiod as seasonal signals to regulate cold responsive pathways and freezing tolerance. To address the second question, fifteen early cold-induced transcription factors (TF) sharing similar cold-induction kinetics with CBFs and ZAT12 were identified as regulators of CBF-independent pathways in parallel with the CBF and ZAT12 pathways. These TFs regulate about 29% of the COR genes; they regulate different sets of COR genes but with substantial overlap, which indicates that highly co-regulation of COR genes exists. Functional enrichment analysis of the COR genes regulated by each TF indicated that they are involved in diverse but overlapping biological pathways during cold acclimation. My results shed light on the regulation of COR genes by CBF-independent pathways and also on interactions between different biological pathways in cold acclimation. In summary, this dissertation provides substantial contributions to the understanding of gene regulatory networks in cold acclimation by integrating photoperiod signals into cold responsive pathways, and by dissecting the cold-regulatory pathways. In addition, the knowledge can be potentially applied for crop engineering and improvements.
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