PROJECT ABSTRACT

The goal of this project is to develop a novel optical technique, termed Bioluminescence Resonance Energy Transfer (BRET), as a resource for the plant functional genomics community, specifically in the model species Arabidopsis thaliana. BRET has shown promise as a tool to chart the physical contacts between specific cellular proteins, and their time-resolved interactions, in living tissue and in real time. While the importance of such interactions has long been recognized in the life sciences, the corresponding experimental tools have largely been limited to in vitro conditions or to heterologous, non-plant organisms. Therefore, the BRET technique will provide a unique contribution toward delineating the functions of the 25,000 genes thought to be contained within the Arabidopsis genome.

BRET is a form of radiation-free energy transfer that can occur when two compatible optical probes are brought into molecular proximity. In detail, to probe for a protein-protein interaction between two given partner proteins, the two proteins are genetically fused to a blue light emitting luciferase and to a blue light absorbing yellow fluorescent protein. If the two hybrid proteins interact, the excitation energy of the luciferase may be transferred to the fluorescent protein, resulting in an easily detected yellow-shift in the luminescence spectrum.

The first goal of this project is to construct molecular genetic tools, such as cloning vectors, for the expression of BRET hybrid proteins. Second, instrumentation and experimental protocols will be optimized for time-resolved BRET data acquisition. Moreover, as a prototype example of the efficacy of BRET for research in plants, BRET will be applied to illuminate the genetic networks that mediate the control of nuclear gene expression by light and the circadian clock. Resources developed in this project shall be widely disseminated to encourage their application within the entire plant science community.