Precursor-Dependent Routing of Aromatic Amino Acids Determines Lignin Structure in Grasses by Sensitivity-Enhanced Solid-State NMR

Lignin biosynthesis in grasses exhibits unique metabolic flexibility, yet the precursor-specific routing of carbon into lignin polymers remains poorly resolved <i>in planta</i>. Here, we combine ¹³C-isotope labeling with solid-state NMR under sensitivity-enhancement by dynamic nuclear polarization (DNP), to directly track phenylalanine- and tyrosine-derived carbon incorporation into the lignin polymer in <i>Brachypodium distachyon</i>. Precursor-specific ¹³C labeling reveals that phenylalanine is the dominant contributor to canonical guaiacyl and syringyl lignins, whereas tyrosine preferentially enriches hydroxyphenyl lignin and hydroxycinnamates, including ferulates characteristic of grass cell walls. Two-dimensional ¹³C-¹³C correlation NMR resolves distinct lignin moieties arising from each precursor. Disruption of <i>p</i>-coumarate 3-hydroxylase (C3H) selectively impairs phenylalanine-derived lignification, while tyrosine-derived lignin remains comparatively unchanged, maintaining polymer assembly through alternative metabolic routes. These findings show precursor-dependent control of lignin composition and reveal tyrosine-mediated lignification as a compensatory pathway in grasses. This work also establishes precursor-resolved solid-state NMR and DNP as a powerful framework for dissecting lignin biosynthesis and metabolic plasticity in plant cell walls.