Speaker
Description
Studying the cellular phenotype of plant cells is crucial for understanding how plants grow, develop, and respond to mutations, pathogens, or environmental stimuli. To this end, a new procedure is presented, based on the application of cryo–soft X-ray tomography (cryoSXT) on protoplasts, enabling the accurate determination of the tridimensional subcelluar structure of plant-derived cells at a spatial resolution of ~50 nm.
The described pipeline takes advantage of the technique's large depth of field and minimal requirements for sample manipulation, allowing it to be applied directly to frozen, hydrated, unfixed, and unstained whole cells. To overcome initial imaging limitations caused by high carbohydrate content in the cell walls, the protoplast preparation protocol was optimized using a cryoSXT-compatible buffer that maintains osmotic pressure. Additionally, data collection strategy was adjusted to increase the effective depth of field to match the protoplast thickness and raise the signal-to-noise ratio.
Using this optimized protocol at the BL09-MISTRAL beamline, several tomograms with improved signal-to-noise ratios were yielded, representing the first nanometer-resolution reconstructions of full cells from land plants. Reconstructed leaf protoplasts from uninfected Arabidopsis thaliana and tomato (Solanum lycopersicum) plants maintained their typical brick shape and continuous plasma membrane, clearly showing the internal morphology and distribution of organelles like the nucleus, chloroplasts, mitochondria, vacuoles, and endoplasmic reticulum. To highlight its potential, the pipeline was applied to tomato leaves infected with Potato spindle tuber viroid (PSTVd). Quantitative and qualitative comparison of infected and uninfected cellular volumes revealed new details on viroid-induced phenotype alterations. Specifically, a highly electrodense RNA accumulation was observed in the host nucleus, divided into a small round zone compatible with the nucleolus and a larger arrow-shaped volume packed against the nuclear membrane.
These examples demonstrate the vast potential of cryoSXT in plant biology for detailed in-depth qualitative and quantitative analyses of cellular substructures.