Cellular and Genetic Regulation of Coniferaldehyde Incorporation in Lignin of Herbaceous and Woody Plants by Quantitative Wiesner Staining

Abstract

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.

Importance of Lignin Coniferaldehyde Residues for Plant Properties and Sustainable Uses

“…The use of Arabidopsis represents an ideal model to quickly investigate multiple genetic engineering strategies and validate analytical method-ologies, both transposable to agronomically relevant lignocellulosic feedstock species. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In contrast to total G CHOH and S CHOH residue levels which could only be reduced or annulled compared to WT plants, total G CHO residue content varied by roughly threefold changes in either direction in Arabidopsis with specific genetic changes, namely increasing in the cad4xcad5 mutant and decreasing in the 4cl1x4cl2 mutant ( Figure 2A). Arabidopsis natural ecotype variant Wassilewskija (WS) presented~60 % more G CHO residues than the Columbia-0 (Col-0) ecotype, although their G CHOH and S CHOH residue amounts did not differ ( Figure S2).…”

“…In the herbaceous plant model Arabidopsis thaliana, the combined insertional mutagenesis of two CAD paralogs (cad4/c and cad5/d) exhibited large increases in lignin aldehyde levels ranging from 35 to 65 % of total measured lignin residues, without altering stem width, biomass weight or fruit yield per plant ( Figure 1). [6][7][8][9] Saccharification and catalytic fractionation yields of cad4xcad5 stem biomass were increased approximately two-and threefold respectively, compared to wild-type (WT) plants. [6,8] Similar increases of aldehyde residues in lignin by reducing CAD expression, using mutagenesis and transgenic approaches in poplar, tobacco, flax, brachypodium, switchgrass, rice and sorghum, have all showed either increased pulping, saccharification and/or biogas yields without affecting plant productivity.…”

“…Incorporated G CHO , but not sinapaldehyde, within the lignin polymer can moreover specifically cross-react in acid conditions and covalently bind other free phenolic compounds, such as phloroglucinol. [7] These G CHO residues can also react with NaHSO 3 /Na 2 SO 3 to form sulfonic acid derivatives. [26] This suggests that the lateral functionalization of lignin polymers, using internal G CHO residues as anchors, could be used similarly to the lateral functionalization of cellulose using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidative treatment.…”

“…[16,[18][19][20]28] Pyrolysis coupled to gas chromatography and mass spectroscopy (PyÀ GC/MS), which enables the quantitative measurement of G CHO , G CHOH and sinapyl alcohol (S CHOH ) residues, [29][30][31][32] showed that total G CHO content in lignin represented~9 % in spruce,~5 % in Arabidopsis,~2 % in eucalyptus and~0.2 % in poplar. [7,33,34] The content variability in lignin of G CHO , G CHOH and S CHOH residues was further examined using pyrolysis/GCÀ MS on a set of Arabidopsis thaliana mutants affected in one or several genes encoding for enzymes responsible of changing the C 6 and/or C 3 parts of lignin monomers ( Figure S1). The use of Arabidopsis represents an ideal model to quickly investigate multiple genetic engineering strategies and validate analytical method-ologies, both transposable to agronomically relevant lignocellulosic feedstock species.…”

“…One essential characteristic of lignin generally overlooked in the context of biomass optimization is its heterogeneity at the cellular and sub-cellular levels. [1,7,48] By neglecting this crucial aspect, opportunities are missed to design plant biomass with homogeneous lignin composition to improve its valorization potential. To characterize this cellular heterogeneity of lignin composition, the cell type specific accumulation of G CHO , G CHOH and S residues were measured using two in situ quantitative methods.…”