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

Area of Science:

• Plant Biology
• Cell Biology
• Microscopy Techniques

Background:

• Graft union formation is crucial in plant science, but cellular events remain poorly understood.
• Studying the graft interface ultrastructure is challenging due to targeting difficulties in electron microscopy.

Purpose of the Study:

• To develop and apply a correlative light electron microscopy (CLEM) approach for high-resolution ultrastructural analysis of the graft interface.
• To investigate the formation and characteristics of secondary plasmodesmata (PD) during graft union establishment in Arabidopsis thaliana.

Main Methods:

• Developed a CLEM method using Arabidopsis lines expressing fluorescent proteins in the endoplasmic reticulum (ER) for precise interface targeting.
• Examined graft hypocotyls under both light and electron microscopy to analyze ultrastructure and PD formation.
• Characterized four classes of PD and their structural features at the graft interface.

Main Results:

• Successfully targeted the graft interface using fluorescent ER markers for CLEM analysis.
• Identified four classes of secondary PD at the graft interface, with initiation by both partners.
• Observed that approximately one-third of PD were incomplete, providing insights into PD biogenesis.
• Found that successful symplastic connections correlate with thin cell walls and ER-plasma membrane tethering.

Conclusions:

• The developed CLEM method enables high-resolution study of cellular events at the graft interface.
• Characterization of incomplete PD offers new insights into secondary PD biogenesis.
• Thin cell walls and ER-plasma membrane tethering are important for successful symplastic connections in grafted plants.