Navigating Guaiacyl and Syringyl Lignin Structural Complexity in Intact Plant Cell Walls Through 13 C Chemical Shift Mapping by Two‐Dimensional Solid‐State NMR

Lignin is a structurally complex and heterogeneous biopolymer whose in situ architecture remains difficult to resolve because of extensive spectral overlap and structural perturbations introduced during isolation and dissolution. These challenges have limited direct interrogation of native lignin within intact plant cell walls and underscore the need for analytical approaches capable of resolving lignin structure in its native context. Here, we present a framework that assists systematic 13 C solid‐state NMR identification of guaiacyl‐ and syringyl‐based lignin motifs, including their associated monolignol units and interunit linkages with reduced level of ambiguity directly in native lignocellulosic materials. This approach is applicable to solid‐state NMR analysis of 13 C‐enriched plant systems or dynamic nuclear polarization (DNP) analysis of unlabeled plant samples. By compiling and organizing diagnostic chemical shift patterns for syringyl and guaiacyl units across selected major linkage types, we delineate regions of uniqueness and overlap in 2D 13 C correlation spectra. The scope is restricted to G/S‐based lignin mapping, with applications to Arabidopsis, hardwoods, and softwoods. Application to Arabidopsis wild‐type and lignin mutants, as well as to the hardwood species poplar and eucalyptus and the softwood spruce, reveals species‐, genotype‐, and developmental stage‐dependent structural variations. The analysis further highlights the uncertainty arising from the structural complexity of the lignin network and demonstrates the need for higher dimensional correlation approaches for assignment of lignin structure. This work establishes a reference for the continued development of solid‐state NMR‐based G/S lignin analysis and advances the ability to interrogate native lignin architecture in intact plant cell walls.