Plants, unlike animals, are unable to run away from adversity (e.g., herbivores and drought) or toward their next meal (e.g., sunlight and water). Instead plants have evolved strategies that involve organ bending in order to respond to changes in their environment. Dramatic, rapid, and reversible changes in morphology can result from differential growth - unequal cellular elongation in one position of an organ relative to an opposing position. One such response is phototropism, or organ bending in response to directional blue light (BL). Based on preliminary results and recent paradigms developed in fungal and animal systems the investigators hypothesize that NPH3, a protein required for normal phototropism that interacts with the primary phototropic receptor phototropin 1 (phot1), functions as a core component of a CULLIN3-based E3 ubiquitin ligase. They further hypothesize that ubiquitination of phot1 by this complex leads to relocalization of the receptor from the plasma membrane to endomembranes in response to BL, and that this is necessary for the furthered progression of phototropic signals. This project is aimed at addressing this hypothesis by: 1) characterizing phot1 ubiquitylation in response to BL, as well as the role of NPH3 and a CUL3-based E3-ubiquitin ligase in this response; and 2) characterize the influence of ubiquitylation on phot1 localization and phototropic function. Results from these studies will not only provide important insights into the mechanisms by which phototropic signaling occurs, but will also contribute to the blossoming field of CULLIN3-based E3 ligase study, which to this point has been largely dominated by fungal and animal models. This project will provide theoretical and practical training for one technician (female), at least one PhD student (female), one undergraduate student, and two high school students. Genetic resources from the project will be utilized in the PREP (Partnership for Research and Education in Plants) program that the PI participates in to provide hands-on research experience to high school students in Columbia and Jefferson City, MO. Insights into the regulation of plant growth and cellular signaling resulting from these studies, which will published in publicly-accessible and high-profile scientific journals, will hopefully be integrated into current crop improvement programs aimed at increasing economic and ecological aspects of US agriculture.
The main goal of this project was to assess the role of protein ubiquitination in the phototropic response of a plant, or how this protein modification process allows a plant to bend toward directional light cues. In nature and in agricultural settings phototropism is an important adaptive response to helps to orient plant organs for maximal photosynthetic light capture - thus optimizing production and bio-yield. We have found that the receptor protein, phototropin 1 (phot1), that is necessary for phototropism in plants, is ubiquitinated in response to light perception and that this occurs through the cooperative function of a number of proteins forming an ubiquitin ligase enzyme complex, including the previously identified by uncharacterized protein NONPHOTOTROPIC HYPOCOTYL 3 (NPH3). Surprisingly, but interestingly from an adaptive point of view, we found that phot1 is modified by ubiquitin in different ways depending upon how much light the plant 'sees'. Under low light conditions (e.g., shade conditions that would promote the greatest adaptive phototropic response) phot1 is ubiquitinated on very unique sites with single ubiquitin moieties thus promoting its intracellular relocalization and functional activation. This allows the phototropic response to develop. Under high light conditions (e.g., condition under which the plant does not need to seek light) phot1 is ubiquitinated in multiple places with chains of ubiquitin, thus targeting the receptor for degradation by cellular machinery. This is an adaptive response as well but one which saves energy of making and maintaining a receptor protein that is not essential because the plant is already growing in an optimal condition. A majority of the experiments done were performed by graduate students and undergraduates at the University of Missouri as part of their training toward PhD and BS degrees. The Principle Investigator and PhD student also presented this work (within a larger context of 'how plants move') in local Columbia, MO public elementary schools. Through these educational venues we are reaching both K-12 and post high school populations of students in Missouri, an important outreach activity to promote STEM education. Training of the undergraduate and graduate students has helped to prepare them to enter the scientific workforce in the US.
