Intellectual merit: Beets and roses are red for completely different reasons. Virtually all flowering plants contain one of two mutually exclusive red/purple pigment pathways, anthocyanins or betalains. The anthocyanin biosynthetic / regulatory network has been heavily studied and occurs in the vast majority of taxa including the major crop families: Poaceae (grasses), Brassicaceae (canola, mustards, cole crops), and Rosaceae (apples, pears, cherries). Betalains on the other hand are restricted to certain families of a single order, the Caryophyllales, where they assume the role of anthocyanin pigments in all biological contexts. These roles include attracting pollinators and seed dispersal agents, and as protective pigments during stress and aging. The betalain families contain crop species and ornamentals that are grown worldwide and form important staples in many agricultural economic systems, including Beets, Spinach, Amaranthus, Quinoa, and Prickly Pear cactus. This project is aimed at understanding the betalain biosynthetic and regulatory network at the molecular level and how pigment emergent properties are generated. While anthocyanins are based on the amino acid phenylalanine, while betalains derive from the animo acid tyrosine. Prior to the start of this project, the investigators identified a novel cytochrome P450 enzyme and a MYB-type transcription factor regulator that function in the betalain network. In particular, hypotheses will be tested regarding the identity of the last unknown betalain ring structure biosynthetic enzyme by silencing genes in beet to test their functions and beet gene expression in yeast to test function. One hypothesis is that betalains are regulated by the same set of interacting proteins that regulate the anthocyanin pigment pathway in other plants. The investigators will test this hypothesis using gene silencing and overexpression in beet. Anthocyanin pigments are regulated by well-characterized environmental signals. The hypothesis that betalains are regulated by these same signaling pathways will be tested. In order to ask the question about whether environmental signals work through increasing gene expression of the MYB regulator, plants will be treated with known elicitors and plant hormones, or nutrient deprivation (e.g. phosphate), and expression of all know betalain network genes will be measured. The hypothesis that the MYB regulator directly binds to cis regions of the betalain biosynthetic genes will be tested through protein-DNA binding assays.
Broader impacts: This research will enhance our basic understanding of the control of plant secondary metabolic networks. Betalains are important dietary antioxidants with high human availability and this project will lead to an ability to manipulate this network and to move the pathway into heterologous systems. Eventual impacts include the ability to create crops that are more nutritious for humans and livestock. The project will provide excellent training for all levels of students. Integral to this project is a new inquiry-based Freshman Research Initiative stream for 30+ students that emphasizes creative and critical thinking within science. Students receive lecture-based training in hypothesis building and testing, data collection, record-keeping, statistical analysis, and ethical research standards. In the spring, students enter the stream for two semesters of hands-on self-directed research. For the past ten years, all lab members, including undergrad and grad students, have participated in judging yearly science fairs at local elementary schools, including interactive interviews of the elementary students. For the past six years, the PI and his graduate students have conducted prefair outreach workshops at the schools. This past year, 3 graduate students, about 180 4th and 5th graders, and 6 teachers participated in the workshop on hypothesis driven scientific method, project conception, and project execution. This outreach activity will continue throughout the period of this project.