Visualizing polymeric components that define distinct root barriers across plant lineages

Hydrophobic cell wall depositions in roots play a key role in plant development and interaction with the soil environment, as they generate barriers that regulate bidirectional nutrient flux. Techniques to label the respective polymers are emerging, but are efficient only in thin roots or sections. Moreover, simultaneous imaging of the barrier constituents lignin and suberin remains problematic owing to their similar chemical compositions. Here, we describe a staining method compatible with single- and multiphoton confocal microscopy that allows for concurrent visualization of primary cell walls and distinct secondary depositions in one workflow. This protocol permits efficient separation of suberin- and lignin-specific signals with high resolution, enabling precise dissection of barrier constituents. Our approach is compatible with imaging of fluorescent proteins, and can thus complement genetic markers or aid the dissection of barriers in biotic root interactions. We further demonstrate applicability in deep root tissues of plant models and crops across phylogenetic lineages. Our optimized toolset will significantly advance our understanding of root barrier dynamics and function, and of their role in plant interactions with the rhizospheric environment.