Courtney J. Murren, Ph.D. - US grants

What is the effect of a mutation that "knocks out" a single gene in an organism's genome? Unsatisfyingly, today's plant geneticists can only say: "It depends." Only a small minority of mutations are lethal or have a significant impact. For the vast majority, at most only subtle effects have been detected. Plant genetics is poised to progress rapidly in remedying this situation, using a collection of "knockout" mutant strains of the model species Arabidopsis thaliana. Knockouts, carrying a tiny tagged segment of disruptive DNA in one targeted gene, are already available for ~67% of the 27,000+ unique genes in this plant's genome. Inspired by progress in yeast genetics, the project will grow large subsets of these knockouts in benign and challenging environments, quantifying ecological performance attributes (e.g., population growth rate, fruit yield). We predict that, as in yeast, such an approach will reveal important phenotypic differences between mutant and control lines and possibly reveal undiscovered genome-wide patterns. The project is a collaboration of primarily undergraduate institutions and will train young scientists in how major genomic questions are addressed by networks of researchers.

The project will involve screening 7000 available T-DNA lines to identify those that have mutations at only one gene, providing these results to the biological community. Traits relevant to plant performance will be measured in over 3000 single-mutant lines, across multiple environments. Cyberinfrastructure will be developed to disseminate common protocols and to archive and index data. Over 50 undergraduate researchers will be involved in collecting and analyzing data and communicating results. The project also includes a study of how participating in a distributed research network influences undergraduates' access to social capital within the scientific community. Results from this aspect of the work will lay the foundation for a larger scale, empirical study of the outcomes undergraduates realize by participating in research networks.

Complex plant phenotypes are influenced by the environment, genetics and their interactions. The root system has been largely overlooked in natural or ecologically relevant settings in the plant species Arabidopsis thaliana. An overarching goal of this project is to examine in Arabidopsis thaliana how traits and their relationships within the root, within the shoot, and between the root and shoot change across habitats in nature. In this project, natural variation of root traits and soil nutrient features in field populations will be examined and plants will be grown in varying natural ecological settings. Genetic tools will be employed to examine the effects of the disruption of single genes on root traits and their relationships with shoot traits. These data will form the largest ecological dataset for Arabidopsis roots in the field. Roots are critical to successful agriculture and may have positive or negative relationships with other agriculturally important traits. These data will therefore inform agricultural scientists on how root traits, shoot traits, and trait relationships change across environments and with genetic perturbations. This project will engage a diverse group of undergraduate students in the study of whole plant phenotypes as research apprentices, in classroom inquiry based learning, and through service learning experiences to the urban kindergarten community.

Overview:
Biological introductions, defined as the establishment of species in geographic regions outside the reach of their natural dispersal mechanisms, have dramatically increased in frequency during the 20th century and are now altering community structure and ecosystem function of virtually all marine habitats. To date, studies on marine invasions focus principally on demographic and ecological processes, and the importance of evolutionary processes has been rarely tested. This knowledge gap has implications for management policies, which attempt to prevent biological introductions and mitigate their impacts. The Asian seaweed Gracilaria vermiculophylla has been introduced to every continental margin in the Northern Hemisphere, and preliminary data indicate that non-native populations are both more resistant to heat stress and resistant to snail herbivory. The project will integrate population genetics, field survey and common-garden laboratory experiments to comprehensively address the role of rapid evolutionary adaptation in the invasion success of this seaweed. Specifically, the PIs will answer the following. What is the consequence of introductions on seaweed demography and mating systems? How many successful introductions have occurred in North America and Europe? Where did introduced propagules originate? Do native, native-source and non-native locations differ in environmental conditions? Do native, native-source and non-native populations differ in phenotype?

Intellectual Merit:
The intellectual merit of this project is based on three gaps in the literature. First, while biological invasions are widely recognized as a major component of global change, there are surprisingly few studies that compare native and non-native populations in their biology or ecology. Native and non-native populations will be surveyed in a similar manner, allowing assessment of differences in population dynamics, mating system, epifaunal and epiphytic communities, and the surrounding abiotic and biotic environment. Second, G. vermiculophylla exhibits a life cycle typical of other invasive species (including some benthic invertebrates), yet we still lack data on the effects of decoupling the haploid and diploid stages on genetic structure, and in turn, on the evolvability of their populations. Finally, this project will provide unequivocal evidence of an adaptive shift in a marine invasive. To our knowledge, such evolutionary change has been described previously for only a complex of marine copepod species. G. vermiculophylla will serve as a model for understanding evolution in other nuisance invasions, and perhaps lead to novel methods to counter future invasions or their spread.

Broader Impacts:
There are several broader impacts afforded by this project. The postdoctoral researcher will receive extensive training in population and ecological genetics, develop skills necessary to further development as an independent researcher, and develop career-enhancing interpersonal skills by leading the field trips and mentoring undergraduates. At least four undergraduates will be recruited, all of whom will generate data and will be encouraged to pursue independent projects. The results will be published in peer-reviewed journals and be presented at national and international meetings of both applied and basic scientists, during lectures and seminars at our home universities and at other universities. Finally, a co-PI has successfully implemented after-school hands on plant-ecology programs at the urban public library in their after-school programming for K-5 students (http://www.cclpl.org) with undergraduate research apprentices and collaboration with undergraduate senior education majors. Offerings will be expanded in this after-school program by developing hands on quantitative inquiry based modules on marine invasive species, marine algae and other topics. Undergraduate students on this project will have the opportunity to interact with undergraduate students in other ongoing projects in our group (see http://arabidopsisunpak.org). Thus research students across different areas of ecological-genetics will have the opportunity to share experiences and expertise.

The unPAK (undergraduates Phenotyping Arabidopsis Knockouts) project uses the plant Arabidopsis thaliana to examine how and when differences in DNA sequences lead to differences in traits related to survival, growth, and reproduction. It will detect effects, if any, of mutation in a comprehensive collection of mutants generated by the Salk Institute, and will then compile results in a comprehensive database. To examine the interaction of genetic and environmental effects, unPAK's experiments are replicated within and across multiple growth facilities and labs, and a subset of experiments deliberately manipulate soil factors, moisture, and temperature to see how these important ecological factors interact with genetics.

unPAK will survey enough mutant lines to reach coverage of a third of the A. thaliana genome (approximately 9,000 genes). All data will be uploaded into a public database at arabidopsisunpak.org, and this database of observed traits is linked to existing genomic databases. Evidence produced by unPAK will enable testing central hypotheses about the relationship between genomic features and important plant characteristics. For example, the data can be used to test whether the effects of mutation are predicted by gene attributes such as molecular signatures of selection in the past, gene function, gene family size, or by patterns of variability in natural populations.

unPAK is centered around participation of undergraduate researchers in the lab and the classroom, educating and training undergraduates integratively in genetics, ecology, evolution, and bioinformatics. The network extends across diverse post-secondary institutions, with over 100 undergraduate apprentices participating across 13 laboratories in 3 years. Greater than 20 course-based undergraduate research experiences (CUREs) will be supported by the program, reaching over 600 students. Any discoveries about the influence of particular genes on fruit production, survival, or life history will have significant broader impacts in potential application to crop species.

As a final part of the project the unPAK research network itself is being studied. Using mixed-methods approaches from the social sciences, researchers are documenting interactions among network participants to analyze changes in network structure over time, and to investigate factors that influence student participation and outcomes, including students' accrual of human, cultural, and social capital.

This project aims to serve the national interest by expanding student opportunities for learning through research by developing, implementing and evaluating a digital course-based undergraduate research experience module in plant biology using quantitative methods. This project will increase research experiences in foundational and upper division courses in experimental plant biology by expanding materials for use in a digital environment. Substantial evidence indicates that participation in undergraduate research in a course where students contribute to new knowledge discovery increases student learning, science self-efficacy, and persistence in STEM. Digital course-based undergraduate research experience (CURE) has been successfully utilized for genomic sequence data, and this new project will introduce these methods to other biology sub-disciplines. This project will build on an existing live plant research and education program that addresses a grand challenge in biology – the link between genes and traits. In the course-based research module, students will study variation among plants in their response to genetic changes and environmental treatments by measuring plant traits from photographs using digital tools. This project aims to advance our understanding of student learning gains in quantitative and plant biology, and identity as scientists from this new digital curricular approach. Significant student learning gains, and plant biology discoveries benefits are expected through this project, as well as the expansion of access to quantitative biology research experience by more students in a broad range of institutional types.

The goals of this project are to: 1) design, modify, and curate plant biology and data science materials for quantitative biology literacy and learning via a CURE using digital images of plants in an ecological genetic experiment by adapting an in-person CURE for the digital environment, 2) provide faculty development and professional networking opportunities associated with student learning modules through yearly workshops and Slack communication, and 3) evaluate teaching and learning for a digital CURE using qualitative and quantitative methods. The project will adapt photography and plant science techniques in novel ways such that students can make observations to facilitate discoveries by collecting trait data using digital approaches. The novel CURE instructional materials will include videos, web materials, digital images of experimental plants, and tools for data analysis and data communication. The project will involve faculty instructors and students from public, private colleges, including minority-serving institutions, in foundational and upper division courses. Faculty and students will collaborate across campuses and contribute novel discoveries through their findings. To evaluate the goals of the project, the planned evaluation will use student focus group (qualitative) and survey methodologies (quantitative) prior to and after each course offering for formative and summative evaluation. Evaluation will also include faculty focus groups which will inform and expand knowledge on the course-based approaches. Yearly faculty development workshops will contribute to support for adoption of this new curriculum, networking and support among instructors committed to innovative pedagogy. This project will add to our knowledge of faculty instructional gains following workshops and iterative course development. Results of this project will expand student access to experimental research in courses in institutional settings with limited laboratory space, and develop transferable STEM research skills in data management, data display and data communication. The NSF IUSE: EHR Program supports research and development projects to improve the effectiveness of STEM education for all students. Through the Engaged Student Learning track, the program supports the creation, exploration, and implementation of promising practices and tools.

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.