Sunlight is a major source of the energy used to drive life processes on earth. Photosynthetic prokaryotic and eukaryotic organisms have existed for 1-2 billion years and, throughout that time, have had an immeasurable influence on the planet and its atmosphere. Land plants and their products constitute the major food and energy sources for humans. Furthering our understanding of the ways in which plants sense and adapt to their light environment and how they respond to environmental light variation arising from competition, seasonality, and atmospheric change represent critical areas of scientific study. In plants, red (R) and far-red (FR) light directly regulate development, reproduction, and circadian rhythms. R and FR are sensed by the phytochromes, a family of five photoreceptor types that exhibit R/FR-reversible structural changes and biological activities. The objectives of this project are to determine the physical and genetic interactions between the different phytochrome forms in plant cells and between the phytochromes and the partner proteins that transmit light signals within cells. In addition, the roles of specific regions of phytochrome molecules in mediating their functions will be characterized and new systems for directing the formation of specific combinations of phytochromes in plants will be developed. It is already clear that phytochrome interactions are critical to light sensing/signaling in plants and the objectives of this project target a deeper and more comprehensive understanding these photoreceptors and their mechanisms of action. Challenges in science education facing universities in predominantly rural areas include geographical and cultural isolation of rural students, notably minority and women students, limited access to mentoring relationships, and lack of opportunities to participate in enrichment programs. This proposal will directly result in improved access for undergraduate and graduate students to individual research experiences and interaction with the broader national and international scientific community.
Plants require light for photosynthesis and have evolved to actively sense and respond to the ever-changing characteristics of their light environment including the wavelength, intensity, and direction of available light and the seasonally varying day length. In order to do this, plants utilize sensory photoreceptors, analogous to "visual" pigments, which are distinct from the light-absorbing components of the photosynthetic apparatus. Among these "visual" receptors, the phytochromes are proteins with an attached chemical group that absorbs red and far-red photons of light (R and FR). Following activation by R/FR photons, phytochromes signal through molecular pathways in plant cells, ultimately modulating the growth, gene expression, and development in those cells in ways that confer fitness under the prevailing light conditions. In addition to plants, phytochrome-like molecules are present and active as R/FR receptors in bacteria, photosynthetic bacteria, fungi, and algae but they have not been observed in animals. This award addressed several aspects of plant phytochrome structure and function. First, the diversity of phytochrome forms present in a plant and the specific functions of those forms have been investigated. Five genes encode five different phytochrome proteins and we have shown that these assemble into a large number of paired, or dimeric, combinations that are in fact the active receptors. Genetic experiments utilizing mutants lacking one of more of the five phytochromes were used to assess their individual roles in R/FR-controlled responses such as seed germination, seedling growth characteristics, gene expression patterns, and flowering time. Second, a molecular engineering system was developed in which protein domains from well-characterized yeast proteins that mediate protein-protein binding interactions were used to assemble defined phytochrome forms in transgenic plants and to test their functions. This novel "directed dimerization" approach may have applicability in other efforts to engineer biologically-active complexes for biotechnological uses. A third area of study focused on an important aspect of the phytochrome signaling mechanism which entails movement of the light-activated receptor itself from one plant cell compartment, the cytoplasm, into the nucleus. This cellular re-localization was studied via fluorescence microscopy through a collaboration with the University of Freiburg, Germany. Fourth, in an effort to better understand the functions of two highly-conserved regions, or domains, found in all plant phytochrome proteins, a large number of mutations in these regions were constructed and their effects on photo-sensing were tested in transgenic plants. Outcomes of these experiments comprise a more detailed understanding of some of the most critical general components of phytochrome protein structure and a first assessment of the importance of phytochrome diversity in R/FR signaling. The goals of the project and the outcomes are also related to each other in that they target development of new tools and strategies for modifying critical plant responses to their changing light environment. With regard to initiating and improving educational opportunities and exposure to scientific research, this award provided funding for many undergraduate research projects and presentation of these projects by the students at scientific conferences. It funded the complete training of one PhD graduate student and for the development and presentation of a major portion of a summer science education program for Native American high school/tribal college students at Montana State University.
