Visualizing Spatial and Temporal Responses of Plant Cells to the Environment
Authors:
Peter Dalhberg1* ([email protected], PI), Magda Zaoralová1, Christopher A. Azaldegui2, Yue Rui3,
Anthony V. Sica2, Jose Dinneny3, Lydia-Marie Joubert1, Wah Chiu1
Institutions:
1SLAC National Accelerator Laboratory; 2University of Michigan; 3Stanford University
Abstract
Cryogenic electron tomography (cryo-ET) is a powerful approach to observe subcellular architecture and can even achieve near atomic resolution when specific complexes can be computationally identified, aligned, and averaged. Advances in this area have led to a situation where biological insight is often not limited by resolution, but instead by a lack of contextual information with which to interpret observed structures and by an inability to work with non-model systems, such as plant roots. This work aims to tackle both these issues through the development of biosensor cryogenic correlative light and electron microscopy (BioCryoCLEM) and advanced sample preparation techniques, including custom electron microscopy grids.
BioCryoCLEM correlates fluorescent biosensor data with electron tomography, providing essential physiological context alongside high-resolution structural information. This research group has calibrated biosensors for calcium, pH, and molecular crowding, demonstrating the workflow using the molecular crowding sensor. While broadly applicable, this project’s focus is on investigating the plant plasma membrane-cell wall interface and its response to biotic (microbes, pathogens) and abiotic (salinity, drought, nutrients) effectors.
Unfortunately, achieving high-quality cryogenic electron microscopy is challenging as soon as one deviates from model systems. Thick plant tissues pose specific difficulties due to their size and the presence of large vacuoles which both serve to slow freezing and makes sample preparation prone to crystalline ice formation. This presentation discusses the team’s work in employing the latest of sample preparation techniques, including high pressure freezing, cryogenic-lift-out, and custom grids to hold the roots and minimize sample volume as researchers work towards the goal of obtaining cryo-ET of the plasma membrane–cell wall interface.