High-pressure freezing/freeze substitution-CLEM: Visualization of ER bodies involved in the massive transport of β-glucosidase to vacuole in the root tip

Abstract

Many small endoplasmic reticulum (ER) bodies are constantly present in the lateral root cap region of Arabidopsis thaliana. We analyzed the morphology and function of these structures by a combined method of high-pressure freezing-freeze substitution (HPF-FS) and correlative light and electron microscopy (CLEM). Analysis of transformed root tips labeled with red fluorescent protein for PYK10, the major enzyme β-glucosidase of ER bodies, revealed that PYK10 is present in small ER bodies in the lateral root cap, and that PYK10 is frequently in contact with ER bodies and vacuoles from the second layer of the lateral root cap to the outer layers, and is influxed into and localized within vacuoles. Since PYK10 is not localized in the Golgi apparatus, it was transported directly from the ER to the vacuole by the ER body of the lateral root cap, without passing through the Golgi apparatus. To clarify this process, it was important to improve HPF-FS method to optimize for CLEM that can retain ultrastructure and fluorescence and can image a wide area. This paper describes the details of the combined method of HPF-FS and CLEM, its combination with immunogold staining and serial section techniques, and the new findings obtained from the method.