Collaborative Research: Investigations of the Plant and Bacterial LOV-domain Photoreceptor Flavo-proteins

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Intellectual Merit: Plant photoreceptors recognize light signals that detect light direction, light intensity, light color, and light duration. They use these signals to regulate almost every phase of plant development from seed germination through flowering, fruit development, and senescence. There are four known groups of plant photoreceptors: the phytochromes that can sense red and near-infrared light, and the cryptochromes, phototropins, and a family of three photoreceptor relatives of the phototropins, all of which can sense both blue and near-ultraviolet light. The phototropins are the photoreceptors that direct plant growth toward a light source (phototropism); adjust the leaf angle to maximize light capture for photosynthesis; adjust the positions of the small organelles that carry out photosynthesis (chloroplasts) to spread them out and minimize self-shading in order to maximize light capture in dim light (or to move into positions maximizing self-shading to avoid damage caused by too much light); and to induce the opening of small pores in the leaf surface (called stomata) to facilitate the uptake of carbon dioxide needed for photosynthesis. The phototropins contain two sequential segments of amino acids that form a pocket surrounding a molecule of a riboflavin derivative, a yellow pigment that is responsible for absorbing blue light. The segments are designated LOV domains for their similarity to domains that sense light, oxygen, or voltage. On absorption of blue light, the light-activated pigment alters the shape of the protein to initiate the steps leading to the eventual biological response - be it phototropism, leaf positioning, chloroplast movement, or stomatal opening. This light-absorbing protein pocket (the LOV domain) is also found in important proteins in lower plants, many different kinds of fungi, and a large number of bacteria. In the bacterium Brucella, a virulent animal pathogen, light absorption has been shown to induce a ten-fold increase in bacterial virulence. This response is mediated by a LOV-domain-containing protein. The present project has two foci: First, to continue to investigate how the change in shape of the LOV domain is transmitted into a signal that induces one of the several biological responses, using biochemical techniques to identify other proteins which interact with the LOV-domain-containing proteins on light excitation. The objective is to determine the complete chain of events from light absorption to response. Second, it will investigate, using biophysical and biochemical methods, the light responses of the bacterium Rhizobium, essential together with legumes for nitrogen fixation. Like the brucellosis pathogen, Rhizobium has a LOV-domain-containing signaling protein that affects its behavior on exposure to light. The research will investigate the previously un-described role of light in this agriculturally important bacterium using biophysical and biochemical methods to trace the steps from light absorption to alteration in the bacterial behavior. The project aims to elucidate the common mechanism by which a single small pigment-containing protein pocket can elevate pathogenesis in a bacterium on the one hand, and the opening of stomatal pores on a leaf on the other when activated by blue light. Broader Impact: Integration of research and education in the training of post-doctoral fellows and graduate students and mentoring of undergraduate students (a research thesis is required at UCSC) and summer interns, both undergraduate and high school is an essential component of this project. Many of the students are sponsored by minority programs - MARC (Minority Access to Research Careers), MBRS (NIH Minority Biomedical Research Support), Summer Community College Students working under the ACCESS program (ACCESS: Baccalaureate Bridges to the future, part of NIH Bridges program), and CAMP (California Alliance for Minority Participation). Winslow Briggs gives interpretive talks to the public at a local state park based partially on his research. Outreach to high-school students, teachers and college undergraduates will continue in the form of summer internships. Finally, it is anticipated that the research itself will ultimately impact both agriculture and health issues.