Summary. Overall Objectives. This project, entitled “Molecular Mechanisms of Phytochrome Signaling,” (PI J. Clark Lagarias, UC Davis), focuses on gaining fundamental knowledge about the phytochrome family of protein light sensors. Phytochromes utilize linear tetrapyrroles (bilins) as chromophores to sense light quality, quantity and duration. Photochemical light sensing by phytochromes triggers conformational changes that modulate the behavior of living systems via downstream transcriptional cascades. The proposed investigations address the hypothesis that eukaryotic phytochromes share a common signaling mechanism (light-mediated nuclear translocation) despite diverse spectral responses and signaling architectures generated over billions of years of evolution. There are three specific aims focused on 1) conserved protein- chromophore interactions and the interplay between environment and photocycle in plant and algal phytochromes, 2) photoperception and signaling by phytochromes and phytochrome eukaryotic kinases in cryptophyte algae, and 3) intramolecular signal propagation and intermolecular signal transduction by phytochrome in land plants. By examining phytochromes from evolutionarily distant species, our studies seek to elucidate the basis of light sensing and the intramolecular structural changes that are used to control gene expression. To test these hypotheses, we leverage computational analyses to guide experimental design, protein biochemistry and molecular biology to express and purify photoreceptors, enzymology and spectroscopy to understand light-induced changes in photoreceptor structure, forward and reverse genetics for in vivo assessment of nuclear translocation and function in the model land plant Arabidopsis thaliana. Significance. Studies on phytochromes provide fundamental knowledge about how living systems regulate their behavior in response to the external environment. Phytochromes are key regulators for seed germination, seedling establishment, vegetative development and flowering (sexual development). Because of their role in shade sensing, phytochromes are an important limiting factor for yield at high crop densities in modern agriculture. Application of the insights from our studies can improve nutrition, enhance health, lengthen life, and reduce the burdens of illness and disability. Moreover, photosensory proteins are valuable tools for studying function and localization of mammalian proteins (optogenetics), and this work yields new tools for fundamental research on cellular processes of biomedical relevance.