While traditional genetics aims to understand the relationship between genotype and phenotype, the goal of chemical genetics is the targeted use of cell-permeable small molecules as modulating ligands for a particular phenotype. A plant's sessile lifestyle requires that it respond quickly to its environment. A cell wall is one of the most conserved evolutionary features of the plant cell and its dynamic structure regulates the passage of biomolecules, creates physical strength and acts as the first line of defense against biotic and abiotic stress. The project uses a chemical ligand that blocks cell wall sensing by targeting an integral protein kinase. The investigators will use chemical genetics, fluorescence biomarkers and molecular biochemistry to identify other genes in the signaling pathway, identify the substrate for the kinase and observe the system in real time. Ultimately, the project aims to discover the dynamics of cell wall signal transduction and transform our view of how plants perceive their environment and initiate downstream response. Synthesis of the project will provide training to a Ph.D student, several undergraduates and foster an educational outreach program with the science museum for children in Lexington Kentucky in order to engage the community with the exciting world of plant and agricultural sciences.
Intellectual merit Cellulose is the most abundant biopolymer on Earth and therefore researchers are keenly interested in how it is made, with hopes of improving the range of renewable products. Despite its importance, the mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved, which could be transformative in a bio-based economy. The project titles "From small molecule to gene: using chemical genetics to understand cell wall sensing and advance molecular resources: National Science Foundation NSF-IOS- 0922947" sought to overcome these challenges. The project developed a toolbox of pharmacological inhibitors and corresponding resistance-conferring mutations in the membrane spanning region of CESA1A903V and CESA3T942I in Arabidopsis thaliana. Using 13C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed structurally altered cellulose (lower crystallinity). Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated plants (CESA1A903V and CESA3T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggests crystallization of cellulose biophysically limits polymerization in plants. In terms of application, strategies identified herein provide insights about how to modify cellulose synthesis in planta for improved bioconversion. Broader impacts To improve science retention on a local stage, the project also created the Science Readiness Program. This program aimed to bridge the gap between high school and college focused on the sciences, particularly targeting at risk students interested in the STEM disciplines. A series of objectives targeting the K12 teacher, students themselves and the PI were instilled over 3 years impacting well over 100 K12 students, undergraduates interning in the laboratory as well as doctoral and post doctoral participants.
