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.

Vascular plants reinforce the cell walls of the different xylem cell types with lignin phenolic polymers. Distinct lignin chemistries differ between each cell wall layer and each cell type to support their specific functions. Yet the mechanisms controlling the tight spatial localization of specific lignin chemistries remain unclear. Current hypotheses focus on control by monomer biosynthesis and/or export, while cell wall polymerization is viewed as random and nonlimiting. Here, we show that combinations of multiple individual laccases (LACs) are nonredundantly and specifically required to set the lignin chemistry in different cell types and their distinct cell wall layers. We dissected the roles of Arabidopsis thaliana LAC4, 5, 10, 12, and 17 by generating quadruple and quintuple loss-of-function mutants. Loss of these LACs in different combinations led to specific changes in lignin chemistry affecting both residue ring structures and/or aliphatic tails in specific cell types and cell wall layers. Moreover, we showed that LAC-mediated lignification has distinct functions in specific cell types, waterproofing fibers, and strengthening vessels. Altogether, we propose that the spatial control of lignin chemistry depends on different combinations of LACs with nonredundant activities immobilized in specific cell types and cell wall layers.

Lignin is indispensable for vascular plants. It allows their cells to coalesce into gravity-defying giants, hardens them to withstand pressures and predators, and waterproofs them to allow the flow of water only where it is advantageous. Lignin fulfils these different functions as a structural component of specialised cell walls in a wide range of different tissues and cell types. Between them, lignin shows great heterogeneity in its concentration and composition. The biosynthesis of lignin proceeds via monomer biosynthesis in the cell, export of the monomers into the apoplast and oxidative polymerisation by laccases (LACs) and class III peroxidases (PRXs) in the cell wall. In this thesis, I investigated how these processes are regulated to allow distinct lignification programs in different cell types and even adjacent cell wall layers (I–III) and what physiological advantages these differences in lignin amount and composition confer to the plant (IV). In paper I and II, we optimised and validated the histochemical Wiesner test and Raman microspectroscopy for the in situ quantitative analysis of lignin. We then used those techniques to map the cell autonomous and cell–cell cooperative genetic programs that regulate lignin monomer biosynthesis in the vasculature of Arabidopsis thaliana and Populus. Because lignin monomers are mobile in the cell wall prior to polymerisation, the sophisticated, cell type-specific genetic regulation of lignin monomer biosynthesis alone cannot explain the lignin differences observed between adjacent cell wall layers. In paper III, we therefore characterised five LACs paralogs involved in lignification, showing that they fine-tuned lignification at the nanoscale through distinct patterns of activity and substrate specificity. But what is the advantage of such a complex, layered control of lignification? In paper IV we began to answer this question by showing that different cell types – and even the same cell type in different developmental contexts – relied on distinct lignin amounts and compositions to withstand the unique stresses they were exposed to. Altogether, the work presented herein highlights how finely lignification is controlled the on cellular and sub-cellular scale, and how this regulation allows plants to fully exploit the versatile functions of lignin.

The biopolymer lignin is deposited in the cell walls of vascular cells and is essential for long-distance water conduction and structural support in plants. Different vascular cell types contain distinct and conserved lignin chemistries, each with specific aromatic and aliphatic substitutions. Yet, the biological role of this conserved and specific lignin chemistry in each cell type remains unclear. Here, we investigated the roles of this lignin biochemical specificity for cellular functions by producing single cell analyses for three cell morphotypes of tracheary elements, which all allow sap conduction but differ in their morphology. We determined that specific lignin chemistries accumulate in each cell type. Moreover, lignin accumulated dynamically, increasing in quantity and changing in composition, to alter the cell wall biomechanics during cell maturation. For similar aromatic substitutions, residues with alcohol aliphatic functions increased stiffness whereas aldehydes increased flexibility of the cell wall. Modifying this lignin biochemical specificity and the sequence of its formation impaired the cell wall biomechanics of each morphotype and consequently hindered sap conduction and drought recovery. Together, our results demonstrate that each sap-conducting vascular cell type distinctly controls their lignin biochemistry to adjust their biomechanics and hydraulic properties to face developmental and environmental constraints. 

The metabolism of polyphenolic polymers is essential to the development and response to environmental changes of organisms from all kingdoms of life, but shows particular diversity in plants. In contrast to other biopolymers, whose polymerisation is catalysed by homologous gene families, polyphenolic metabolism depends on phenoloxidases, a group of heterogeneous oxidases that share little beyond the eponymous common substrate. In this review, we provide an overview of the differences and similarities between phenoloxidases in their protein structure, reaction mechanism, substrate specificity, and functional roles. Using the example of laccases (LACs), we also performed a meta-analysis of enzyme kinetics, a comprehensive phylogenetic analysis and machine-learning based protein structure modelling to link functions, evolution, and structures in this group of phenoloxidases. With these approaches, we generated a framework to explain the reported functional differences between paralogs, while also hinting at the likely diversity of yet undescribed LAC functions. Altogether, this review provides a basis to better understand the functional overlaps and specificities between and within the three major families of phenoloxidases, their evolutionary trajectories, and their importance for plant primary and secondary metabolism.

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.

The diverse morphological shapes of plants are made possible by the structural rigidity provided by cell walls. In order to support vertical growth and long distance water transport, cell walls need to resist a variety of biological and physical stresses. Lignin, a cell wall polyphenolic unique to vascular plants, has long been considered to structurally support the cell walls of xylem vessels and other specialised cell types against these forces. Lignin is a complex polymer whose monomeric composition and biochemical properties vary widely between different species, tissues and cell types. However, the precise characterisation of this micro-scale variation poses considerable methodological hurdles. As a result, it has yet to be understood how differences in lignin composition contribute to the cell-type specific functions of the cell wall. In the works presented herein, we optimise and validate the Wiesner test and Raman microspectroscopy for the quantitative characterisation of lignin in situ and use these techniques to show how cell-type specific genetic regulation of lignification is crucial for cell wall function. Using synthetic lignin monomers and polymers, as well as genetically altered Arabidopsis and Populus plants in conjunction with biochemical lignin composition analyses, we establish the Wiesner test as a specific high-resolution method to quantify coniferaldehyde (I), and show that Raman microspectroscopy allows the relative quantification of total lignin, guaiacyl lignin subunits (G-units), coniferyl alcohol and syringyl lignin subunits (S-units) (II). We then use these methods to characterise cell-autonomous and cell-cell cooperative lignification patterns and show that cell walls of different vessel types depend on distinct amounts of lignin and specific G-units for structural reinforcement (III). S-unit incorporation into vessel lignin and increased adjacency to neighbouring vessels on the other hand compromise their resistance to collapse (III). Altogether, we provide evidence for a lignification process consisting of a fine scale, cell-type specific regulatory network of lignin biosynthesis, cell-to-cell cooperative monomer supply, and cell wall layer specific monomer incorporation. Crucially, it is this dynamic small-scale regulation that allows lignified plant cell walls to fulfil their cell-type specific functions.

Lignin is a phenolic polymer accumulatig in the cell walls of specific plant cell types to confer unique properties such as hydrophobicity, mechanical strengthening, and resistance to degradation. Different cell types accumulate lignin with specific concentration and composition to support their specific roles in the different plant tissues. Yet the genetic mechanisms controlling lignin quantity and composition differently between the different lignified cell types and tissues still remain poorly understood. To investigate this tissue-specific genetic regulation, we validated both the target molecular structures as well as the linear semi-quantitative capacity of Raman microspectroscopy to characterize the total lignin amount, S/G ratio, and coniferyl alcohol content in situ directly in plant biopsies. Using the optimized method on stems of multiple lignin biosynthesis loss-of-function mutants revealed that the genetic regulation of lignin is tissue specific, with distinct genes establishing nonredundant check-points to trigger specific compensatory adjustments affecting either lignin composition and/or cell wall polymer concentrations.

Increases in coniferaldehyde content, a minor lignin residue, significantly improves the sustainable use of plant biomass for feed, pulping, and biorefinery without affecting plant growth and yields. Herein, different analytical methods are compared and validated to distinguish coniferaldehyde from other lignin residues. It is shown that specific genetic pathways regulate amount, linkage, and position of coniferaldehyde within the lignin polymer for each cell type. This specific cellular regulation offers new possibilities for designing plant lignin for novel and targeted industrial uses.