Search

Lignin is a complex polymer deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrients homeostasis and plant growth.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity., (© 2020 John Wiley & Sons Ltd.)

Lignin engineering is a promising tool to reduce the energy input and the need of chemical pre-treatments for the efficient conversion of plant biomass into fermentable sugars for downstream applications. At the same time, lignin engineering can offer new insight into the structure-function relationships of plant cell walls by combined mechanical, structural and chemical analyses. Here, this comprehensive approach was applied to poplar trees (Populus tremula × Populus alba) downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD) in order to gain insight into the impact of lignin reduction on mechanical properties. The downregulation of CAD resulted in a significant decrease in both elastic modulus and yield stress. As wood density and cellulose microfibril angle (MFA) did not show any significant differences between the wild type and the transgenic lines, these structural features could be excluded as influencing factors. Fourier transform infrared spectroscopy (FTIR) and Raman imaging were performed to elucidate changes in the chemical composition directly on the mechanically tested tissue sections. Lignin content was identified as a mechanically relevant factor, as a correlation with a coefficient of determination (r²) of 0.65 between lignin absorbance (as an indicator of lignin content) and tensile stiffness was found. A comparison of the present results with those of previous investigations shows that the mechanical impact of lignin alteration under tensile stress depends on certain structural conditions, such as a high cellulose MFA, which emphasizes the complex relationship between the chemistry and mechanical properties in plant cell walls., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply., (© 2014 American Society of Plant Biologists. All rights reserved.)

Bioorthogonal click chemistry was commissioned to visualize the plant cell wall lignification process in vivo. This approach uses chemical reporter-tagged monolignol mimics that can be metabolically incorporated into lignins and subsequently derivatized via copper-assisted or copper-free click reactions.

To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of plants, N-ACETYLGLUCOSAMINYLTRANSFERASE (NodC) of Azorhizobium caulinodans was expressed in Arabidopsis (Arabidopsis thaliana). The corresponding enzyme catalyzes the polymerization of GlcNAc and, accordingly, β-1,4-GlcNAc oligomers accumulated in the plant. A phenotype characterized by difficulties in developing an inflorescence stem was visible when plants were grown for several weeks under short-day conditions before transfer to long-day conditions. In addition, a positive correlation between the oligomer concentration and the penetrance of the phenotype was demonstrated. Although NodC overexpression lines produced less cell wall compared with wild-type plants under nonpermissive conditions, no indications were found for changes in the amount of the major cell wall polymers. The effect on the cell wall was reflected at the transcriptome level. In addition to genes encoding cell wall-modifying enzymes, a whole set of genes encoding membrane-coupled receptor-like kinases were differentially expressed upon GlcNAc accumulation, many of which encoded proteins with an extracellular Domain of Unknown Function26. Although stress-related genes were also differentially expressed, the observed response differed from that of a classical chitin response. This is in line with the fact that the produced chitin oligomers were too small to activate the chitin receptor-mediated signal cascade. Based on our observations, we propose a model in which the oligosaccharides modify the architecture of the cell wall by acting as competitors in carbohydrate-carbohydrate or carbohydrate-protein interactions, thereby affecting noncovalent interactions in the cell wall or at the interface between the cell wall and the plasma membrane.

Lignin is an abundant phenylpropanoid polymer produced by the oxidative polymerization of p-hydroxycinnamyl alcohols (monolignols). Lignification, i.e., deposition of lignin, is a defining feature of secondary cell wall formation in vascular plants, and provides an important mechanism for their disease resistance; however, many aspects of the cell wall lignification process remain unclear partly because of a lack of suitable imaging methods to monitor the process in vivo. In this study, a set of monolignol analogs γ-linked to fluorogenic aminocoumarin and nitrobenzofuran dyes were synthesized and tested as imaging probes to visualize the cell wall lignification process in Arabidopsis thaliana and Pinus radiata under various feeding regimens. In particular, we demonstrate that the fluorescence-tagged monolignol analogs can penetrate into live plant tissues and cells, and appear to be metabolically incorporated into lignifying cell walls in a highly specific manner. The localization of the fluorogenic lignins synthesized during the feeding period can be readily visualized by fluorescence microscopy and is distinguishable from the other wall components such as polysaccharides as well as the pre-existing lignin that was deposited earlier in development., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

The plant hormone auxin (indole-3-acetic acid, IAA) has a crucial role in plant development. Its spatiotemporal distribution is controlled by a combination of biosynthetic, metabolic and transport mechanisms. Four families of auxin transporters have been identified that mediate transport across the plasma or endoplasmic reticulum membrane. Here we report the discovery and the functional characterization of the first vacuolar auxin transporter. We demonstrate that WALLS ARE THIN1 (WAT1), a plant-specific protein that dictates secondary cell wall thickness of wood fibres, facilitates auxin export from isolated Arabidopsis vacuoles in yeast and in Xenopus oocytes. We unambiguously identify IAA and related metabolites in isolated Arabidopsis vacuoles, suggesting a key role for the vacuole in intracellular auxin homoeostasis. Moreover, local auxin application onto wat1 mutant stems restores fibre cell wall thickness. Our study provides new insight into the complexity of auxin transport in plants and a means to dissect auxin function during fibre differentiation.

By combining Zinnia elegans in vitro tracheary element genomics with reverse genetics in Arabidopsis, we have identified a new upstream component of secondary wall formation in xylary and interfascicular fibers. Walls are thin 1 (WAT1), an Arabidopsis thaliana homolog of Medicago truncatula NODULIN 21 (MtN21), encodes a plant-specific, predicted integral membrane protein, and is a member of the plant drug/metabolite exporter (P-DME) family (transporter classification number: TC 2.A.7.3). Although WAT1 is ubiquitously expressed throughout the plant, its expression is preferentially associated with vascular tissues, including developing xylem vessels and fibers. WAT1:GFP fusion protein analysis demonstrated that WAT1 is localized to the tonoplast. Analysis of wat1 mutants revealed two cell wall-related phenotypes in stems: a defect in cell elongation, resulting in a dwarfed habit and little to no secondary cell walls in fibers. Secondary walls of vessel elements were unaffected by the mutation. The secondary wall phenotype was supported by comparative transcriptomic and metabolomic analyses of wat1 and wild-type stems, as many transcripts and metabolites involved in secondary wall formation were reduced in abundance. Unexpectedly, these experiments also revealed a modification in tryptophan (Trp) and auxin metabolism that might contribute to the wat1 phenotype. Together, our data demonstrate an essential role for the WAT1 tonoplast protein in the control of secondary cell wall formation in fibers., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula x Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

Lignin is an important component of secondarily thickened cell walls. Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are two key enzymes that catalyse the penultimate and last steps in the biosynthesis of the monolignols. Downregulation of CCR in tobacco (Nicotiana tabacum) has been shown to reduce lignin content, whereas lignin in tobacco downregulated for CAD incorporates more aldehydes. We show that altering the expression of either or both genes in tobacco has far-reaching consequences on the transcriptome and metabolome. cDNA-amplified fragment length polymorphism-based transcript profiling, combined with HPLC and GC-MS-based metabolite profiling, revealed differential transcripts and metabolites within monolignol biosynthesis, as well as a substantial network of interactions between monolignol and other metabolic pathways. In general, in all transgenic lines, the phenylpropanoid biosynthetic pathway was downregulated, whereas starch mobilization was upregulated. CCR-downregulated lines were characterized by changes at the level of detoxification and carbohydrate metabolism, whereas the molecular phenotype of CAD-downregulated tobacco was enriched in transcript of light- and cell-wall-related genes. In addition, the transcript and metabolite data suggested photo-oxidative stress and increased photorespiration, mainly in the CCR-downregulated lines. These predicted effects on the photosynthetic apparatus were subsequently confirmed physiologically by fluorescence and gas-exchange measurements. Our data provide a molecular picture of a plant's response to altered monolignol biosynthesis.

Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.