Metabolomics and Genome Wide Association Mapping for the Elucidation of Triterpene Saponin Molecular Biochemistry in Medicago

Noble Research Institute, Llc, Ardmore OK

Investigators

Abstract

The production and role of plant compounds, known as secondary compounds, is a key scientific problem that has many agricultural and ecological dimensions. The fundamental goal of this project is to identify and characterize novel genes responsible for triterpene saponin biosynthesis, in important class of plant secondary compounds, in Medicago truncatula using revolutionary technologies. The plant species Medicago truncatula has emerged as an important experimental model for leguminous crop species such as alfalfa. Triterpene saponins are a class of structurally diverse plant natural products with a wide range of demonstrated bioactivities including allelopathic, antifungal, antibacterial, antiinsect, anticancer, antinutritive activities. The antinutritive properties of triterpene saponins in legume forages such as alfalfa and soybean are of particular and substantial economic importance. However, the biosynthesis of triterpene saponins is poorly characterized and this pathway is absent from most textbooks. This project will use cutting-edge metabolomics, genome wide association mapping, correlated gene expression profiling, and traditional molecular validation approaches for gene discovery and characterization related to triterpene saponin biosynthesis in M. truncatula. The goal of this project will be accomplished through the following specific aims: 1) Metabolome analyses of a large and diverse M. truncatula germplasm collection to identify hyper- (high) and hypo- (low) saponin accumulating lines and to enable genome-wide association mapping between specific loci with saponin content 2) Comparative gene expression analyses of hyper and hypo saponin accumulating lines to identify putative genes involved in triterpene saponin biosynthesis and regulation, and 3) Molecular and biochemical confirmation of select targeted saponin biosynthetic genes prioritized based upon multiple levels of evidence. Preliminary data document substantial metabolic diversity in saponin accumulation which is a necessary prerequisite for successful association mapping and comparative microarray analyses. Broader Impacts: This project will identify and characterize specific genes involved in legume triterpenoid saponin biosynthesis. Specific genes identified in this project have the potential for future metabolic-engineering of crop varieties with: 1) improved fitness and defense, 2) decreased bloat and antinutritive properties resulting in optimized livestock weight gain performance, and 3) potential plant neutraceuticals and adjuvants related to human and animal health. High resolution biochemical phenotype data generated for a large number of M. truncatula accessions core collection will be generated and will be made publicly available via an anonymous FTP server (ftp://sumnerftp.noble.org/pub). This data will highlight specific germplasm critical for further molecular and biochemical dissection of saponin biosynthesis. More importantly, the copious metabolomics data generated will provide substantial community value in that it will enable association mapping with a wealth of other metabolites (both known and unknown) and related fitness traits including, but not limited, to symbiosis (flavonoids, isoflavonoids, dicarboxylic acids), drought/salinity tolerance (proline, manitol, and other osmoprotectants), and/or aluminum tolerance (malate). Funding of this project provides multidisciplinary training for two postdoctoral researchers and one undergraduate summer intern/hourly worker in advanced technologies, plant molecular biology, large-scale integrated systems biology, and largescale analytical biochemistry. A postdoctoral researcher is being mentored in scientific writing, scientific presentations, publishing, grant applications, constructive peer review, project management, time management, and personnel management to further mold a great scientific mind more towards the realistic and professional expectations of modern academic and commercial research. A series of enrichment workshops focused upon local high school students is being provided. Participating groups include Oklahoma Upward Bound Science/Math Program, Ardmore High School AP and Science Club, and Southern Oklahoma Technology Center Biotechnology Program. These annual enrichment opportunities stimulate student interests in science, plant biology, and natural product biochemistry. An international symposium is being be hosted in year three of the project. This will provide a forum for the latest advances in saponin research and serve to highlight the outcomes of this project. Postdocs funded by this proposal are serving key leadership roles in organizing and executing the workshops.