Lignin is a group of cell wall localised heterophenolic polymers varying in the chemistry of the aromatic and aliphatic parts of its units. The lignin residues common to all vascular plants have an aromatic ring with one para hydroxy group and one meta methoxy group, also called guaiacyl (G). The terminal function of the aliphatic part of these G units, however, varies from alcohols, which are generally abundant, to aldehydes, which represent a smaller proportion of lignin monomers. The proportions of aldehyde to alcohol G units in lignin are, nevertheless, precisely controlled to respond to environmental and development cues. These G aldehyde to alcohol unit proportions differ between each cell wall layer of each cell type to fine-tune the cell wall biomechanical and physico-chemical properties. To precisely determine changes in lignin composition, we, herein, describe the various methods to detect and quantify the levels and positions of G aldehyde units, also called coniferaldehyde residues, of lignin polymers in ground plant samples as well as in situ in histological cross-sections.

Lignin accumulates in the cell walls of specialized cell types to enable plants to stand upright and conduct water and minerals, withstand abiotic stresses, and defend themselves against pathogens. These functions depend on specific lignin concentrations and subunit composition in different cell types and cell wall layers. However, the mechanisms controlling the accumulation of specific lignin subunits, such as coniferaldehyde, during the development of these different cell types are still poorly understood. We herein validated the Wiesner test (phloroglucinol/HCl) for the restrictive quantitative in situ analysis of coniferaldehyde incorporation in lignin. Using this optimized tool, we investigated the genetic control of coniferaldehyde incorporation in the different cell types of genetically-engineered herbaceous and woody plants with modified lignin content and/or composition. Our results demonstrate that the incorporation of coniferaldehyde in lignified cells is controlled by (a) autonomous biosynthetic routes for each cell type, combined with (b) distinct cell-to-cell cooperation between specific cell types, and (c) cell wall layer-specific accumulation capacity. This process tightly regulates coniferaldehyde residue accumulation in specific cell types to adapt their property and/or function to developmental and/or environmental changes.