Storage domains of coniferin in cell wall forming tracheid cells in Pinus thunbergii stems evaluated by cryo microscopic chemical analyses

We investigated how lignin precursors are stored and used during secondary-wall formation in Pinus thunbergii stems, addressing the current lack of quantitative, spatially resolved information, especially on monolignols and their glucosides. Two complementary cryogenic workflows, microdissection (CryoMD) for µm-scale fractionation and secondary ion mass spectrometry with gas cluster ion beam (Cryo-GCIB-SIMS) for sub-µm imaging, were developed to quantify and visualize monolignols and their glucosides across defined cell-wall formation stages in compression and opposite woods of P. thunbergii. Coniferin (CF) and p-glucocoumaryl alcohol were the dominant storage forms, while free coniferyl alcohol was in trace amounts, and p-coumaryl alcohol was detected mainly in the mature xylem region; CF concentration peaked near the cambial zone and declined with cell wall thickening. GCIB-SIMS visualized CF in domains adjacent to the cell wall at the cell expansion-cell wall thickening boundary and revealed a continuous distribution over 10-20 μm along the cell axis direction. These findings indicate that monolignol glucosides reside in spatially restricted, possibly mobile storage domains that supply precursors precisely where and when lignification proceeds, refining current models of monolignol transport. The integrated cryo approach provides a framework for linking intracellular metabolism to cell and tissue-level cell wall architecture.