Magda Zaoralova, SLAC/SSRL/CryoEM

Abstract

Nearly all aspects of plant growth are orchestrated by an interplay of complex sensing and signaling networks, including plant receptor-like kinases (RLKs). A central RLK is FERONIA, which has been implicated in numerous pathways including sexual reproduction, cellular morphogenesis, root hair growth, and stress responses. Due to the complexity of signaling, detailed structural and functional analysis is as important as the spatial-temporal distribution of individual players within a plant tissue. Cryogenic imaging of plant cells could offer an unprecedented level of detail; however, the preparation has been notoriously challenging due to various morphological and physiological features that are unique to terrestrial plants and dissimilar from both unicellular and multicellular organisms that are amenable for cryoEM study using standard robotic plunge-freezing – in order achieve vitrification, we optimize high-pressure freezing technique as an alternative to plunge-freezing methods.

To successfully target the proteins of interest within a specific environment, we combine cryoEM imaging with biosensor cryogenic correlative light microscopy. Fluorescent biosensors are fluorescent labels that have been engineered to have photophysical changes in response to specific aspects of the local environment, such as Ca2+, pH, membrane potential, etc. These changes manifest in excitation or emission spectra, lifetime, or brightness, allowing us to bridge tissue imaging with precise localization and physiology changes.

Here we present results from the development of a workflow for visualizing spatial and temporal responses of plant cells to the environment that has the potential to be transformative to the field of plant biology by demonstrating that plant tissues can be observed with molecular-scale resolution and physiological context provided by fluorescent biosensors.