Collaborative Research: An F-box protein targeting PIF1 and PIF3

Seeds comprise 70% of our food. They are fodder, a method of bulk food transport, storage, and germ plasm preservation, a vehicle for technology delivery and molecular farming, and vital to establishing the next crop. Seed germination, its completion, and subsequent seedling establishment are exquisitely attuned to environmental stimuli, but these early developmental events in the plant's life cycle remain poorly understood. This project's focus is the characterization of the existence and consequences of protein-protein interaction between an FBOX protein (COLD TEMPERATURE GERMINATING10, CTG10), and basic-Helix-Loop-Helix (bHLH) transcription factors (PHYTOCHROME INTERACTING FACTORs, PIF1 and PIF3). This interaction is a step in the phytochrome siqnalinq cascade following light perception. The interaction of CTG10 with PIF1/PIF3 has a profound influence on photomorphogenesis, leading to the completion of seed germination (PIF1) and the degree to which the seedling alters its morphology to allow it to achieve an autotrophic existence (PIF1, PIF3). Genetic, physiological, and molecular evidence demonstrating an interaction between CTG10 and PIF1 have been accrued but more work is required to convince ourselves of the association. Some evidence implicates CTG10 in the polyubiquitination of PIF3. Based on preliminary data, 3 objectives will be pursued. 1: Establish the veracity of CTG10:PIF1/PIF3 interaction by assessing single- and double-mutant seed germination- and seedling photomorphogenic-phenotypes, as well as western blot results. 2. Employ fluorescently labeled YFP-CTG10 with either PIF1-CFP, or PIF3- CFP in stably transformed WT and various mutant plants to explore the co-localization of the two proteins in nuclear speckles upon illumination. To the same end, use bimolecular fluorescence complementation (BiFC) with a split YFP cDNA cloned in-frame with CTG10 and the complementing portion with PIF1 or PIF3. 3. Demonstrate protein-protein interaction in vitro and in vivo using reciprocal co-immunoprecipitation-, pull down- and TAPa-tag-analyses. Particularly edifying will be plants harboring a LUC-PIF1 triple point mutation LUC-PIF1 (3M) that has poor affinity for phyA and B and greatly enhanced stability. Results from these experiments will contribute to our understanding of light-induced degradation of PIF1 and PIF3, a poorly understood mechanism used by the phytochrome (phy) signal transduction cascade to promote photomorphogenesis.