A correlative light electron microscopy approach reveals plasmodesmata ultrastructure at the graft interface

Abstract

Despite recent progress in our understanding of graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy (CLEM) approach to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing YFP or mRFP in the endoplasmic reticulum allowed efficient targeting of the grafting interface for examination under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that 4 classes of PD were formed at the interface and that PD introgression into the cell wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these incomplete PD gives us insights into the process of secondary PD biogenesis. We found that the establishment of successful symplastic connections between the scion and rootstock occurred predominantly in the presence of thin cell walls and endoplasmic reticulum–plasma membrane tethering. The resolution reached in this work shows that our CLEM method advances the study of biological processes requiring the combination of light and electron microscopy.

Keep in contact: multiple roles of endoplasmic reticulum–membrane contact sites and the organelle interaction network in plants

“…During virus infection, SYT1 is recruited to PDs by viral movement proteins to alter viral cell‐to‐cell movement (Levy et al ., 2015; Ishikawa et al ., 2020). Another great example of EPCS and PD interaction is from a study of grafting using correlative light electron microscopy (Chambaud et al ., 2022). Here, it was found that the formation of PD between the scion and rootstock requires ER–PM tethering at the place where cell walls are thinner.…”

Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants

“…The CLEM approach was performed similarly to previously described with minor modifications (Chambaud et al, 2022). Hypocotyls of 4-day old A. thaliana seedlings stably expressing 35S:AtEH1-GFP were cut and frozen in a Leica EM-PACT high-pressure freezer using 20% BSA as a cryoprotectant.…”

Bioimaging tools move plant physiology studies forward

“…Combining electron microscopy and light microscopy, correlative light and electron microscopy (CLEM) can be used to visualize the ultrastructure for studying intracellular function ( Begemann and Galic, 2016 ; Figure 1C ). Lysiane Brocard and her colleagues developed a CLEM approach to study the graft interface of Arabidopsis grafting hypocotyls ( Chambaud et al., 2022 ). With in-resin fluorescence CLEM combined with electron tomography, this method can provide the fine 3D ultrastructural details with up to the resolution of the bilayer of the plasma membrane ( Chambaud et al., 2022 ).…”

“…Lysiane Brocard and her colleagues developed a CLEM approach to study the graft interface of Arabidopsis grafting hypocotyls ( Chambaud et al., 2022 ). With in-resin fluorescence CLEM combined with electron tomography, this method can provide the fine 3D ultrastructural details with up to the resolution of the bilayer of the plasma membrane ( Chambaud et al., 2022 ). Classical CLEM usually results in low-resolution correlations because it is limited to the resolution of the light microscope, but this method can detect fluorescence signals and more accurately determine the ultrastructure position within electron tomograms.…”