Light is one of the most important environmental factors influencing plant development, controlling seed germination, growth, greening, and many other plant developmental processes in addition to driving photosynthesis. A central question in plant science concerns how, at the cellular level, the plant responds to light? Certain proteins are known to act as light receptors while others act as inhibitors of light responses. In darkness these proteins are kept separate within the cell but, upon illumination, arrive at the same destination, the nucleus. The receptor proximity somehow destroys the inhibitor through the action of a third, previously unidentified, mediator protein. This research seeks to clarify the identity of the mediator and investigate how it influences the stability of the inhibitor. It is anticipated that isolating the inhibitor protein from plant cells engineered to possess greater than usual amounts of the inhibitor protein, and exposed to light, will simultaneously isolate the mediator protein due to their affinity for one another while the converse will also be true. These in vivo protein-protein interactions will be demonstrated using antibodies against both proteins. This interaction will also be visualized using fluorescently tagged inhibitor and mediator proteins that are visible only when the inhibitor and mediator proteins are in physical contact. Visible signal should be generated only following exposure of the plant cells to light and this signal should then attenuate over time as the inhibitor is destroyed. A post-doctoral collaborator will be supported by this award as they spearhead efforts to elucidate this facet of the plant response to light. A greater understanding of light perception at the cellular level should provide opportunities to engineer crop plants for faster germination, greater stand establishment in the field, and potentiate the alteration of plant stature and structure.
Upon budget reductions to the NSF, the original scope of the project was reduced from three to two years funding which necessitated the removal of PIF3 from the project. All objectives should be read with this alteration in mind. Our specific objectives were to: 1: Examine seed germination and seedling photomorphogenic phenotypes in CTG10 overexpression (CTG10-OE), and CTG10 RNAi (CTG10-RNAi) lines in comparison with PIF1 or PIF3 overexpression (PIF1-OE; PIF3-OE) or knockout (pif1; pif3) lines. For several years, using RNA interference strategies (RNAi), we were unable to demonstrate a germination phenotype in darkness for the ctg10 knock down or CTG10 RNAi lines, despite evidence that CTG10 transcript and protein amounts were reduced in these seeds. However, upon switching germination media from half strength MS to 0.7% w/v agarose in water, a dramatic and statistically significant dark germination phenotype was demonstrated (Image 1). Seedling photomorphogenic phenotypes for ctg10 and CTG10 over expressing lines has been demonstrated by both our lab and that of our collaborator, Enamul Huq. 2. Co-localize CTG10 and the PIF proteins by employing live-cell imaging with fluorescently labeled YFP-CTG10 with either PIF1-CFP, or PIF3-CFP in stably transformed plants to explore the co-localization of the two proteins in nuclear speckles. 3. Explore protein-protein interaction between CTG10 and the PIFs using our CTG10 and PIF1 antibodies and PIF3 antibody (available from P. Quail, UC Berkeley, a collaborator, see letter), to perform reciprocal co-immunoprecipitation (Co-IP) assays. Despite many attempts we were unable to demonstrate a protein-protein interaction between CTG10 and PIF1 using co-immunoprecipitation. When examining plant protein extracts, we came to the conclusion that the nucleus, a sub-cellular compartment housing the genomic DNA and in which transcription factors such as PIF1 are known to reside, was resistant to lysis by mild detergent. Hence, when we used a mild detergent to try and break open isolated nuclei, most of the CTG10 and PIF1 signal remained in the insoluble pellet (unlysed nuclei). When more stringent detergent conditions were used, the protein protein interaction between CTG10 and PIF1 was disrupted. Mild cross linking of the proteins prior to nuclear lysis resulted in smeared Western signals, presumably due to irreversible epitope alterations. Because we were using fluorescence microscopy for objective 2, we altered our approach and used a technique called Bimolecular fluorescence complementation (BiFC) using a split-Yellow Fluorescence Protein (YFP) to satisfy both objectives 2 and 3. In this instance, the coding sequence of a natively fluorescent protein (YFP) is divided into two parts and one part is fused, in frame, to the coding region of either CTG10 or PIF1. The idea is that, the split YFP (:Y and :FP in two halves) does not fluoresce (Image 3, CTG10:Y and PIF1:FP, either construct alone). However, if the two different YFP halves are on two proteins that interact, the two YFP halves attached to these proteins are now brought into close proximity and reform a functionally fluorescent protein (Image 3, Viral proteins SYNV-P:Y and SYNV-N:FP). This can be visualized using fluorescence microscopy. Reconstitution of a functional fluorescent YFP from the split halves did indeed occur for the PIF1:FP and CTG10:Y proteins. Moreover, the fluorescence was localized to the nucleus and does appear to be in speckles (Image 4). Criteria II: this proposal is ideally suited for training the next generation of scientists, including undergraduate, graduate and postdoctoral students. We will preferentially recruit students from under-represented groups and minorities, and actively involve them in our research. Mentored Ms. Taylor Lloyd (undergraduate student) for 2 years. Goldman Scholar, NSF-REU recipient, Astronaut Scholar, ASPB-SURF award Recipient, co-author on 2 papers during this time. Chosen with Ms. Lloyd to provide highlights of undergraduate research experience and support at the University of Kentucky at the ASPB annual conference in Austin, Texas, 2012. Mentored Dr. Rekha Kushwaha (female in science) in the use of phage display and recombinant protein production to acquire PIF1 interacting proteins. Mentored Dr. Santosh Kumar for two years in protein-protein interaction techniques including tandem affinity purification, Bimolecular fluorescence complementation, and phage display.
