Intellectual Merit: This project investigates the roles and modes of action of the enzyme protein disulfide isomerase (PDI) in plants. PDI-regulated processes are important for seed formation and endosperm filling, which are central to agricultural yields and grain nutrition. New knowledge by which PDIs transfer electrons between proteins will advance the understanding of basic redox enzyme biochemistry. In Arabidopsis there are 12 genes for members of the PDI family. These 12 PDIs differ in the position and number of thioredoxin domains and in the presence of transmembrane domains. This project has demonstrated some PDIs are located in the endoplasmic reticulum (ER) where they interact with, and correctly fold, substrate polypeptides. In the protein folding process, PDIs use their thioredoxin domains to catalyze the reversible formation and rearrangement of disulfide bonds in substrates. Proper polypeptide folding is necessary for the activity, transport and assembly of the substrates. PDIs have also been shown to chaperone and regulate the activity of substrates outside the ER. PDI5 chaperones and inhibits cysteine proteases during trafficking from the ER to the Golgi apparatus and the vacuoles, where the proteases play essential roles in seed and embryo development. However, little is known about the roles of other members of the PDI family, their biochemical mechanisms of action, interacting partners, and subcellular locations. This research will define the functions and biochemical activities of PDIs, determine where they are located within cells, and identify substrate proteins with which they interact. A variety of molecular, cellular, biochemical, and genetic methods will be used to achieve these goals including yeast two hybrid assays, affinity-tag purification, fluorescence and immunoelectron microscopy, protein refolding activity assays and complementation of protein folding mutants in E. coli and yeast. The physiological, morphological, and cellular effects of over-expressing and eliminating PDIs will also be measured. This basic research forms the underpinnings for understanding fundamental and essential cellular processes common to all plants.
Broader Impacts: Methods to manipulate PDI activity as developed in this research will provide solutions for enzyme stability problems for environmental remediation, treating crop diseases and industrial uses. New biochemical knowledge will facilitate the design of novel catalysts and protein folding and stabilizing reagents for agricultural, environmental, food processing and bioenergy uses. Understanding and regulating protein folding is critical for over-expression of valuable proteins in transgenic plants. The graduate student exchange between Hawaii and Florida will promote interdisciplinary training in molecular genetics and cell biology. Central to the mission of this project is to integrate the resulting technologies with learning experiences that will inspire faculty and student development from small colleges lacking research programs. The project will partner with community colleges to continue a successful summer workshop (http://abe.leeward.hawaii.edu/). Faculty members obtain certificates of professional development, while students obtain mentoring, course credit and training in experimental problem-solving. Participants become aware of the fascinating array of activities and career choices in modern life sciences. Hands-on training in molecular biology, genomics, bioinformatics and cellular biological research coupled with synergistic interactions, partnerships and collaboratively developed teaching resources will have far-reaching educational impacts on faculty and students of Pacific Island and Asian decent.
This project studied the enzymes known as protein disulfide isomerases (PDI) in plants and defined their cellular functions and biological activities. PDI-regulated processes are important for cereal grain seed formation and growth, which are central to agricultural yields and grain nutrition. We discovered that one class of PDI can stimulate the formation of protein bodies essential for seed formation (Fig. 1). The project also developed methods to manipulate PDI activity, which can provide treatments to protein misfolding disorders, diseases and enzyme stability problems. For example, enzymes are used in detergents and in food processing but are inactivated by heat. This research can increase their resistance to heat inactivation so they operate for longer periods. This project embraces the transfer of new knowledge, technology, abilities and fascination to others, especially under-represented groups. Central to our mission has been providing learning experiences that will inspire faculty and student development from small colleges lacking research programs (Fig. 2 and Fig. 3, students doing research). Hands-on training in molecular biology, genomics, bioinformatics and cellular biological research coupled with synergistic interactions and collaboratively developed teaching resources have far-reaching educational impacts on faculty and students well beyond the project period. Students are better prepared for graduate-level research and the job market while teachers are given an opportunity to hone their professional skills and develop new educational resources. This boosts the level of instruction at the community colleges, the number of bioscience majors and their retention and graduation rates. Students and faculty at research institutions like the University of Hawaii at Manoa have greater access to sophisticated laboratory equipment and state-of-the-art methods than their colleagues at smaller community colleges. The community college students do not obtain real-life experience in biosciences to help their career and educational decisions. The NSF-sponsored research and education project bridges the technology and education gap by providing students and faculty from Leeward, Kapi‘olani, and Windward Community Colleges and Hawai‘i Pacific University the opportunity to learn a variety of molecular, cellular and genetic techniques throughout the year. After working in the summer project, the faculty members took small projects back with them to their schools to develop further. In addition, students are unsure about making the leap to a four-year degree program at the larger university. The two year program may be their final degree. Many bright university students from under-represented groups often face more personal challenges that affect their retention and graduation rates. Inspiring students by making a life-changing connection with them that can positively influence their desire to be bioscience majors and even go further into graduate school are also major goals of this NSF- project. The project has partnered with the Graduate Professional Access (GPA) program on the Manoa. GPA provides counseling services throughout the year that sustains good academic standing in STEM degree programs. GPA also provides guidance in character building, developing a code of ethics, social responsibility, CV/resume writing, and stress management.
