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.
Findings and Intellectual Merit: This collaborative research project between investigators from the Carnegie Institution for Science at Stanford University and the Department of Chemistry and Biochemistry at the University of California , Santa Cruz, focused on the biological role of a novel family of light sensor molecules first discovered in Plants by the Carnegie group and named the phototropins 1 and 2 because activation of phototropins by light cause a plant to grow toward a light source (Phototropism). We found that certain types of bacteria have sunlight-sensing molecules similar to those found in plants, and we discovered, and first reported, a bacterial species that needs light to maximize its virulence or infectivity. This bacterium, the Brucella, is responsible for causing Brucellosis, an infectious disease that results in abortion of the fetus in farm animals and severe fevers in humans (Brucellosis is also called undulating fever). Our work has revealed an entirely new model for bacterial virulence (infectivity) based on light sensitivity. The bacterial light sensors are closely related to the phototropins. They share protein components called LOV domains, so named because in association with appropriate receptor molecules locked inside their structures can detect Light, Oxygen, and/or Voltage. Over 100 bacterial species have been identified to carry these light-sensing substances and some of them are infectious in animals, plants or humans. We decided to study one infectious (Brucella) and on one non-infectious (Rhizobium) bacterium. The Rhizobium bacterium lives in small nodules present in the roots of legumes such as beans. Rhizobium synthesizes organic nitrogen compounds by trapping nitrogen gas from the air This process, called Nitrogen fixation is energy-intensive, the bacteria take carbon compounds from the plant to fuel it. The bacterium passes the nitrogen compounds to the plant to fulfill its nutritional need for nitrogen serving as the plant fertilizer. This bacteria/Plant association is called both a symbiosis and a mutualism, because both associated partners gain. When we disabled the LOV-domain protein gene in Brucella, its virulence—measured as the ability to reproduce efficiently inside the host cell enough to cause disease—dropped to less than 10% of normal, "wild-type" bacteria. In a simple experiment involving a layer of light-blocking aluminum foil, we obtained a similar drop in virulence, demonstrating that Brucella depends on sunlight to cause disease. Brucella has been studied for over 100 years because of its threat to livestock and the effect it has on our food supply and health. However its sensitivity to light was not discovered. We showed that light-enhanced infectivity in brucella relies on a light activation of the LOV domain protein and subsequent steps in which the activated LOV protein reacts with other cellular components that enhance the bacterium resistance against attack by the human or animal defenses to repel bacterial invasion, enhancing its virulence. When it is in the dark, a LOV domain uses weak chemical bonds to hold onto a light-sensing small molecular group known as a flavin chromophore. When it absorbs blue light, the LOV domain temporarily tightens its grip on the flavin chromophore by forming a more stable bond., and when the light source is removed, the LOV domain relaxes its grip on the chromophore once again and becomes ready for another cycle of light activation. Activated LOV domains can switch on yet another signaling molecule, known as a kinase enzyme, that initiates biochemical processes that ultimately activate genes that produce defensive compounds that protect the bacterium from the host cell, We found that the while initial activation mechanism by which Rhizobium uses light signals to promote its association with the plant is similar to the process by which Brucella uses light to infect the animals. The main differences between the two systems are that the genes that are stimulated in Rhizobium cause the enhancement of processes that favor the bacterial capabilities to enter the plant cell, while in Brucella the activated genes favor the survival of the bacterium once it has already entered the animal cell. Broader impact: On agricultural technology: Knowledge of these molecular mechanisms provides new strategies to chemically disturb the bacterial genetic machinery so that Brucella could not benefit from light in animal and human infection and Rhizobium could enhance the beneficial effect of light on promoting the symbiotic association with the plant. Inoculating the seeds (or spraying the planted fields) of economically important crops such as soybeans with liquid Rhizobium cultures is the current practice in the agriculture of this legume in many countries. Our findings could have major impact in agriculture because the use of pre-illumination of those cultures may considerably improve bacterial association with the plant and consequently crop yields. On education: We have sponsored 15 undergraduate and 6 graduate students researchers emphasizing the participation of underrepresented minorities during the duration of the project.
