Collaborative Research: TRTech-PGR: Development and Sharing of High-Resolution In Situ 3D Cryo Workflows to Transform Plant Cell Biology

Much can be learned about how an organism functions by understanding how its cells and tissues are organized. Cells are very small and so they can best be observed with specialized techniques like electron microscopy. Samples must be preserved carefully for study by electron microscopy and care must be taken not to introduce changes that could mislead interpretation of observations. The best method for preparing tissue for electron microscopy is to rapidly freeze samples and observe them under very low temperatures, a process called cryogenic preservation. Cryopreservation is challenging in plants, and the goal of the proposed research is to develop procedures that overcome these challenges. If successful, this proposal will allow plant cells and tissues to be observed after cryopreservation and give unprecedented information about how parts of plant cells and tissue work together. Understanding how plant cells work will help develop plants that are better able to meet human needs and ensure food security. The methods developed will be shared with others through public databases to allow other scientists to use the methods in their own studies. The research will include several early career scientists, and they will receive training in techniques and develop skills which are in high demand in science labs and in industry. Cryogenic preservation is the gold standard for determining cell ultrastructure in its “native state” by virtue of physical fixation (freezing) to immobilize cell constituents in milliseconds. Volume electron microscopy (vEM) has gained significant traction as a tool for discovering the structure and spatial organization of cell and tissue components. In combination with cryo-electron tomography (cryoET), cryo-vEM has the potential to yield a paradigm shift in multi-scale in situ structural biology. However, due to their physical and chemical properties, plant cells are uniquely recalcitrant to fixation. Thus, plant science is strikingly lagging other fields in the revolution of understanding of how macromolecular complexes and cell compartments coordinate to create life. Determining the structure of cells and organelles in fully frozen hydrated chemically untreated and unstained would allow, for the first time, the observation of these entities in their native states. This proposal will develop cryopreservation and nanoscale imaging procedures for vEM of plant tissues in a focused effort to address this challenge efficiently for the plant community. Whole cell structures will be elucidated in 3D using cryo-vEM, while nanoscale cellular structure and organelles will be determined using cryo-vEM and cryo-ET. The datasets, protocols, and analytical pipelines generated through this research will be made available to the scientific community and the public through public databases. The proposed work will also provide unique training opportunities for several students and postdocs, training them in state-of-the plant structural biology, much desired expertise in the scientific workforce. 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.