Yaowu Yuan - US grants

Carotenoids are yellow, orange, and red pigments that contribute to the beautiful colors and nutritive value of many flowers (e.g., daffodils, daylilies, sunflowers) and fruits (e.g., oranges, tomatoes, mangos). They serve an important function in the ecology and evolution of plants as visual pigments to attract animals for pollination and seed dispersal. The tremendous diversity of carotenoid pigmentation patterns found in flowers and fruits is primarily determined by regulatory genes that control when and where the carotenoid pigments are produced. However, little is known about the identity and function of these regulatory genes. This study employs a new plant genetic model system, monkeyflowers, to identify carotenoid-regulating genes and to characterize their function. The discovery and characterization of these regulatory genes are crucial to understanding how carotenoid production is controlled during flower development and how carotenoid-based flower color variation is generated during evolution. From a practical perspective, because carotenoids are also essential components of human diets as precursors for vitamin A biosynthesis, the regulatory genes discovered in this study have the potential for high translational impact in plant breeding to enhance carotenoid production in crop plants. The plant materials will be incorporated into a live plant exhibit at the University of Connecticut to demonstrate concepts of genetics and evolution for student and public education. Furthermore, the flower images, pollination videos, and interactive, multimedia illustrations resulting from this project will be developed into a new Plant Biology section on the nationally and internationally recognized Learn.Genetics website as online educational resources.

This study leverages recently developed genomic resources, stable transgenic tools, and chemically induced mutants of monkeyflowers (Mimulus) to identify and characterize transcription factors that regulate flower carotenoid pigmentation. Results from preliminary work in the PIs laboratory allows the formulation of the central hypothesis: a transcriptional regulatory complex, containing at least an R2R3-MYB and a tetratricopeptide repeat (TPR) protein, directly activates carotenoid biosynthetic genes during flower development in Mimulus, and differential expression of the regulatory genes contributes to pigment pattern variation between species. In this proposed project, additional components of the transcriptional regulatory complex will be identified by bulk segregant analysis of another non-allelic mutant and by yeast two-hybrid screening using the MYB and TPR proteins as baits. The functional mechanisms of the MYB and TPR genes in the regulation of carotenoid pigmentation will be determined by protein-protein and protein-DNA interaction assays. The role of these regulatory genes in producing flower color variation will be examined by gene expression analysis and transgenic experiments in Mimulus species that display distinct carotenoid pigmentation patterns.

A fundamental goal in biology is to understand the genes and developmental processes that affect ecological success and adaptation of organisms in natural environments. Historically, a mechanistic understanding of ecologically important phenotypes has been limited to a small number of genes in very few model organisms. The research will improve genetic tools in the wildflower genus Mimulus (monkeyflowers), a system that exhibits rich diversity in form, physiology, and life history. By connecting this diversity with functional genetic analysis and field experiments, the research will enable molecular genetic investigations of previously unexplored of phenotypic variation. With the tools proposed here, the growing Mimulus scientific community will be in an excellent position to identify the causal genetic mechanisms underlying ecologically important traits. The research will provide hands-on learning opportunities for two different undergraduate laboratory courses taught at University of Georgia and University of California, Riverside. In addition, the research will offer training experiences for local high school teachers in Georgia to develop and refine lab activities for dissemination in the high school classroom. Furthermore, the plant materials generated in this research will be incorporated into live plant outreach activities at the University of Connecticut for student and public education. Finally, the actives will be broadly disseminated via the Plant Biology section on the "Learn.Genetics" website as online educational resources.

Identifying the genetic and developmental determinants of natural phenotypic variation is fundamentally important for understanding organismal biology and the processes that generate biodiversity. The genus Mimulus is an exceptionally diverse group of approximately 200 wildflower species with abundant natural populations that is ideally suited for linking naturally occurring genotypes to their ecologically important phenotypes. The research will enable Mimulus as a powerful new model for ecological functional genomics and plant developmental genetics. The specific scientific objectives are as follows: 1) Optimize genetic transformation protocols in seven Mimulus species that are actively studied by many researchers and represent a wide range of phylogenetic diversity. 2) Generate large-scale chemical and transposon insertion mutant libraries for three of the most actively studied species. Additionally, a major goal of the research is to enable Mimulus researchers to take full advantage of the newly developed functional genomics protocols and resources in their own labs. This project includes an annual, weeklong summer lab short course that will rotate between the University of Connecticut and the University of Georgia. The project will also include a website to disseminate detailed instructions and video tutorials of protocols, as well as a searchable database with information on plant lines, seed stocks, and other important tools generated from this work.

This award was co-funded by funds from Enabling Discovery through GEnomic Tools (EDGE) Program and the Evolutionary Process Program within the Division of Environmental Biology.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary The emergence of qualitatively distinct new structures or patterns (e.g., turtle shells, beetle horns, butterfly color patterns, plant flowers) is one of the most fascinating aspects of organismal evolution. How such novel, complex traits have originated and evolved remains one of the most important yet challenging problems of evolutionary biology. It is widely assumed that novel structures or patterns arise by co-option or rewiring of pre-existing developmental programs, but we know very little about the specific genetic changes that trigger such co-option or developmental rewiring, how the genetic changes translate to novel phenotypes, what kind of developmental pre-settings or cryptic pre-patterns are required for the the trigger gene(s) to produce the novel phenotype and how these cryptic pre-patterns come about, and how the novel phenotypes rise in frequency in natural habitats (i.e., the evolutionary process). This project is to address these fundamental questions by studying a novel, qualitatively distinct, pigmentation pattern that evolved very recently in a wild population of Mimulus verbenaceus (crimson monkeyflower), a species amenable to genetic and developmental manipulations as well as evolutionary analyses or/and ecological interrogations. We propose to: (i) Identify the causal gene and mutation(s) underlying the novel pigmentation pattern by genetic mapping and transgenic experiments; (ii) Characterize the developmental pre-settings (cryptic pre- patterns) required for the formation of the novel pigmentation pattern by identifying multiple upstream activators and repressors, through analysis of chemically induced mutants with altered pigmentation patterns; (iii) Elucidate the evolutionary process through which the novel phenotype rose in frequency in the natural habitat by population genomics analyses and field experiments. We anticipate that these studies will, for the first time, provide a detailed view of the genetic and developmental mechanisms as well as the evolutionary process driving the emergence of a novel phenotype in nature. The successful completion of this project is also expected to help move the field forward by shifting the focus from correlational studies and just-so stories of morphological innovations that happened in the distant past, to rigorous genetic, developmental, and evolutionary analyses of novel phenotypes that are still in the initial stage of emergence.

Project Summary The emergence of complex tissue patterns from seemingly uniform, undifferentiated cells during development is an essential feature of all multicellular organisms. One of the most prominent theoretical mechanisms often invoked to explain biological pattern formation is the reaction-diffusion (RD) model, which postulates that local activation of pattern differentiation factors combined with long-range inhibition of the activity of those factors can produce dynamic, self-organizing spatial patterns. Numerous empirical and simulation studies have suggested that the RD mechanism underlies a wide range of pattern formation processes. However, we still know very little about the actual genes encoding the hypothetical activation and inhibition factors in most empirical systems, even less about the biophysical properties of these factors where candidate genes have been identified, and virtually nothing about how modulation of the properties of these activators and inhibitors affects pattern evolution in nature. The overall objective of this project is to address these fundamental questions by elucidating the detailed genetic and developmental mechanisms of pigment pattern formation and evolution in the wildflower genus Mimulus (monkeyflowers), a system amenable to rigorous genetic analysis, developmental interrogation, and phenotypic perturbation. The work proposed here will build on our prior efforts that identi?ed a pair of MYB proteins underlying the formation of dispersed anthocyanin pigment spots in Mimulus flowers. This MYB pair forms a local autocatalytic feedback loop and a long-range inhibitory feedback loop, fulfilling the tenets of a classical activator-inhibitor RD model. Our goals in the coming years are to: (i) experimentally determine the biophysical properties of the activator-inhibitor pair, including their diffusion coefficients, degradation (clearance) rates, and relative activation and inhibition constants; (ii) characterize the genetic and developmental bases of pattern evolution from dispersed spots to longitudinal stripes between closely related species; and (iii) identify the key cis-regulatory elements that constitute the activator-inhibitor interacting network and test the function of this two-component, activator-inhibitor module in other tissue types and heterologous systems. Towards these ends we will use a suite of approaches, including fluorescence imaging, genetic mapping, transgenic manipulation, and mathematical modeling. Together our efforts will provide an in-depth view of how the RD mechanism generates self-organizing spatial patterns and modulates pattern evolution in a real biological system, lending empirical support to the mathematically elegant but somewhat controversial RD model.

Whole genome sequencing across the tree of life has shown that new genes arise frequently in evolution. However, relatively little is known about the function or evolutionary importance of these genes in the derivation of novel phenotypes. This project will recruit postbaccalaureate trainees to engage in a cohort project to describe the genomes of species that are not well studied and to identify potentially new genes in these genomes. In addition, trainees will engage in individual projects in a network of labs to characterize the function of novel genomic elements and their potential links to new traits in diverse taxa. These research projects will promote integrated research opportunities in molecular and computational biology. The program will support the training of a diverse set of 30 postbaccalaureate mentees, enriched for members from groups underrepresented in biology, for future success in STEM. Integral to the program are the broader impacts that include mentor training for PIs and co-mentors; training, mentoring, and networking opportunities; public outreach about genomes; and connections across departments, institutions, and industry. <br/><br/>A major challenge in biology is determining how information encoded in the genome generates individual phenotypes and thus how differences in that information lead to unique phenotypic outcomes. Comparative genomic studies identified a highly conserved ‘toolkit’ of developmental genes that predates the diversification of major clades. One way that morphological novelty originates is through regulatory tinkering with these ancient toolkit genes. More recently, comparative studies have identified a profusion of new ‘taxon-restricted genes,’ which may also be important in the origin of phenotypic novelty. The functional characterization of genomic novelty in diverse taxa and diverse subfields of biology will be leveraged as the intellectual focal point for a new postbaccalaureate research training program. Individual research projects will address specific questions about the function of new genes in diverse taxa. Collectively, these projects address broader question about the role of new genes in evolution and development. Sequencing, assembly, and annotation of genomes will be done in collaboration with researchers at regional partner institutions. This project will help build a cross-institutional research network, provide trainees with bioinformatics training, and disseminate training materials for genomics. The training program will deploy individualized mentoring networks, cohort training workshops, professional development and networking opportunities, and interactions with PIs, mentors, peers and near-peers to help trainees identify and meet their professional goals.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.