Exploring Lignification Complexity in Plant Cell Walls with Airyscan Super-resolution Microscopy and Bioorthogonal Chemistry

Abstract

In this paper, we present the use of multiplex click/bioorthogonal chemistry combined with super-resolution Airyscan microscopy to track biomolecules in living systems with a focus on studying lignin formation in plant cell walls. While laser scanning confocal microscopy (LSCM) provided insights into the tissue-scale dynamics of lignin formation and distribution in our previous reports, its limited resolution precluded an in-depth analysis of lignin composition at the unique cell wall or substructure level. To overcome this limitation, we explored the use of Airyscan microscopy, which, among the super-resolution techniques available, offers an optimal balance between performance, cost, accessibility, and ease of implementation. Our study demonstrates that a triple labeling strategy using copper-catalyzed azide–alkyne cycloaddition (CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC), and inverse electronic-demand Diels–Alder cycloaddition (IEDDA) to label modified lignin metabolic precursors can be combined with Airyscan microscopy to reveal the zones of active lignification at the single cell level with improved sensitivity and resolution. This approach enables insights into the lignin composition in wall substructures, such as pits or in wall layers that are otherwise not distinguishable by classical LSCM. Our work emphasizes the importance of studying lignin formation in plant cell walls and demonstrates the potential of combining bioorthogonal chemistry and super-resolution microscopy techniques for studying biomolecules in living systems.

Deconvoluting Structures of Component Plant Biopolymers Using Deuterium Labeled Brassica oleracea Stems

“…This implies that the contrast match point of ∼48% D 2 O observed in the low- Q region is mostly associated with features that contain a partially deuterated lignin biopolymer. With lignin found to concentrate within 1–2 μm on the surface of the cell wall lumen, it implies that the low- Q surface scattering for the d-kale stem sample is due to the cell wall lumen and not aggregates of cellulose microfibrils.…”

Bioorthogonal Reactions in Bioimaging

“…Bioorthogonal labeling combined with superresolution imaging has advanced lipid research as well, revealing nanoscale lipid heterogeneity in living cells [ 127 ]. Furthermore, Lion et al applied this approach to plant cells to reveal the zones of active lignification using Airyscan microscopy [ 128 ].…”