The role of the secondary cell wall in plant resistance to pathogens.

BackgroundBesides classical utilization of wood and paper, lignocellulosic biomass has become increasingly important with regard to biorefinery, biofuel production and novel biomaterials. For these new applications the macromolecular assembly of cell walls is of utmost importance and therefore further insights into the arrangement of the molecules on the nanolevel have to be gained. Cell wall recalcitrance against enzymatic degradation is one of the key issues, since an efficient degradation of lignocellulosic plant material is probably the most crucial step in plant conversion to energy. A limiting factor for in-depth analysis is that high resolution characterization techniques provide structural but hardly chemical information (e.g. Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM)), while chemical characterization leads to a disassembly of the cell wall components or does not reach the required nanoscale resolution (Fourier Tranform Infrared Spectroscopy (FT-IR), Raman Spectroscopy).ResultsHere we use for the first time Scanning Near-Field Optical Microscopy (SNOM in reflection mode) on secondary plant cell walls and reveal a segmented circumferential nanostructure. This pattern in the 100 nm range was found in the secondary cell walls of a softwood (spruce), a hardwood (beech) and a grass (bamboo) and is thus concluded to be consistent among various plant species. As the nanostructural pattern is not visible in classical AFM height and phase images it is proven that the contrast is not due to changes in surfaces topography, but due to differences in the molecular structure.ConclusionsComparative analysis of model substances of casted cellulose nanocrystals and spin coated lignin indicate, that the SNOM signal is clearly influenced by changes in lignin distribution or composition. Therefore and based on the known interaction of lignin and visible light (e.g. fluorescence and resonance effects), we assume the elucidated nanoscale structure to reflect variations in lignification within the secondary cell wall.

Cellulose microfibrils are the major structural component of plant secondary cell walls. Their arrangement in plant primary cell walls, and its consequent influence on cell expansion and cellular morphology, is directed by cortical microtubules; cylindrical protein filaments composed of heterodimers of alpha- and beta-tubulin. In secondary cell walls of woody plant stems the orientation of cellulose microfibrils influences the strength and flexibility of wood, providing the physical support that has been instrumental in vascular plant colonization of the troposphere. Here we show that a Eucalyptus grandisbeta-tubulin gene (EgrTUB1) is involved in determining the orientation of cellulose microfibrils in plant secondary fibre cell walls. This finding is based on RNA expression studies in mature trees, where we identified and isolated EgrTUB1 as a candidate for association with wood-fibre formation, and on the analysis of somatically derived transgenic wood sectors in Eucalyptus. We show that cellulose microfibril angle (MFA) is correlated with EgrTUB1 expression, and that MFA was significantly altered as a consequence of stable transformation with EgrTUB1. Our findings present an important step towards the production of fibres with altered tensile strength, stiffness and elastic properties, and shed light on one of the molecular mechanisms that has enabled trees to dominate terrestrial ecosystems.

The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.

The capacity of four xylan-directed probes (carbohydrate-binding modules CfCBM2b-1-2 and CjCBM15; monoclonal antibodies LM10 and LM11) to recognize xylan polysaccharides in primary and secondary cell walls of tobacco stem sections has been determined. Enzymatic removal of pectic homogalacturonan revealed differential recognition of xylans in restricted regions of cortical primary cell walls. Monoclonal antibody binding to these exposed xylans was more sensitive to xylanase action than carbohydrate-binding module (CBM) binding. In contrast, the recognition of xylans by CBMs in secondary cell walls of the same organ was more sensitive to xylanase action than the recognition of xylans by the monoclonal antibodies. A methodology was developed to quantify indirect immunofluorescence intensities, and to evaluate xylanase impacts. The four xylan probes were also used to detect xylan populations in chromatographic separations of solubilized cell wall materials from tobacco stems. Altogether, these observations reveal the heterogeneity of the xylans in plant cell walls. They indicate that although CBM and antibody probes can exhibit similar specificities against solubilized polymers, they can have differential capacities for xylan recognition in muro, and that the access of molecular probes and enzymes to xylan epitopes/ligands also varies between primary and secondary cell walls that are present in the same organ.

Lignin and carbohydrate rich secondary plant cell walls are abundantly available in the biosphere, and is a notable renewable feedstock for biofuels and biomaterials. Particularly for construction applications, wood that is resistant to fungal degradation is highly desirable. Chemical modifications, such as acetylation, have been successfully demonstrated to inhibit wood decay by microorganisms. It is well known that acetylation reduces wood moisture content, which slows down motions within the cell wall when measured by X-ray fluorescence microscopy experiments and molecular simulations. The open question is whether acetylation inhibits decay strictly by reducing moisture content, or if specific interactions with the acetyl group hinder motion within the cell wall and further inhibit decay. We investigate these hypotheses directly through molecular simulation, acetylating exposed hemicellulose and lignin hydroxyl groups in existing models for secondary plant cell wall structure to 5-18% weight-percent gain. By comparing diffusive behavior for cell wall polymers, water, and select ions (Na+ and Fe3+ ), we can track the dynamics within the cell wall and identify the causal mechanisms for reduced transport and uptake of these metal ions by acetylated cell walls. We find that the change from hydroxyl to acetyl group alone does not account for reduced transport, with only modest changes in diffusion when acetylated cell walls are expanded to provide constant moisture level. The most substantial changes in diffusion occur where the additional acetylation displaces water, reducing the moisture content for the cell wall. Utilizing these simulations, we further analyze the interactions between ions and cell wall polymers and the evolution of dynamic water pockets within the structure. Ions interact more frequently with the acetyl group than the hydroxyl groups they replace, yielding to increased ion interactions on aggregate upon acetylation. Collectively, these findings elucidate the molecular mechanism through which acetylation affects secondary plant cell walls at atomic resolution.

Confocal Raman microscopy and confocal fluorescence microscopy were used to understand the mechanism of ionic liquid (IL) pretreatment of corn stover stem using 1-ethyl-3-methylimidazolium acetate. Three different cell types including tracheids, sclerenchyma cells and parenchyma cells were analyzed during IL pretreatment. We have established a direct correlation between changes in both the morphology and chemical composition of these plant cell walls during IL pretreatment as well as specific cell type information. It was observed that cell wall swelling occurs primarily in the secondary plant cell walls and the IL had little effect on compound middle lamella in terms of swelling. Accordingly, more significant cell wall swelling and distortion was observed in sclerenchyma cells and tracheids than in parenchyma cells, which contain primary plant cell walls. Lignin dissolution was faster in the secondary cell walls, while there was no preferential cellulose dissolution. Surprisingly, with a much thicker cell wall and a much higher original lignin content than parenchyma cells, tracheids showed much faster lignin and cellulose dissolution and cell wall swelling. Sclerenchyma cells showed an intermediate rate of lignin dissolution, while the cellulose dissolution rate and degree of cell wall swelling was comparable to that observed for tracheids. These results suggest that there is a synergistic mechanism of lignocellulose dissolution regarding cellulose and lignin dissolution and cell wall swelling that occurs during IL pretreatment. This study provides valuable new insights towards the mechanism of ionic liquid pretreatment and can potentially assist researchers in cell wall engineering for efficient cell wall deconstruction using ILs, and the methods established can be easily extended to other systems.

Lignocellulosic biomass from the secondary cell walls of plants has a veritable potential to provide some of the most appropriate raw materials for producing second-generation biofuels. Therefore, we must first understand how plants synthesize these complex secondary cell walls that consist of cellulose, hemicellulose, and lignin in order to deconstruct them later on into simple sugars to produce bioethanol via fermentation. Knotted-like homeobox (KNOX) genes encode homeodomain-containing transcription factors (TFs) that modulate various important developmental processes in plants. While Class I KNOX TF genes are mainly expressed in the shoot apical meristems of both monocot and eudicot plants and are involved in meristem maintenance and/or formation, Class II KNOX TF genes exhibit diverse expression patterns and their precise functions have mostly remained unknown, until recently. The expression patterns of Class II KNOX TF genes in Arabidopsis, namely KNAT3, KNAT4, KNAT5, and KNAT7, suggest that TFs encoded by at least some of these genes, such as KNAT7 and KNAT3, may play a significant role in secondary cell wall formation. Specifically, the expression of the KNAT7 gene is regulated by upstream TFs, such as SND1 and MYB46, while KNAT7 interacts with other cell wall proteins, such as KNAT3, MYB75, OFPs, and BLHs, to regulate secondary cell wall formation. Moreover, KNAT7 directly regulates the expression of some xylan synthesis genes. In this review, we summarize the current mechanistic understanding of the roles of Class II KNOX TFs in secondary cell wall formation. Recent success with the genetic manipulation of Class II KNOX TFs suggests that this may be one of the biotechnological strategies to improve plant feedstocks for bioethanol production.

Cellulose, often touted as the most abundant biopolymer on Earth, is a critical component of the plant cell wall and is synthesized by plasma membrane-spanning cellulose synthase (CESA) enzymes, which in plants are organized into rosette-like CESA complexes (CSCs). Plants construct two types of cell walls, primary cell walls (PCWs) and secondary cell walls (SCWs), which differ in composition, structure, and purpose. Cellulose in PCWs and SCWs is chemically identical but has different physical characteristics. During PCW synthesis, multiple dispersed CSCs move along a shared linear track in opposing directions while synthesizing cellulose microfibrils with low aggregation. In contrast, during SCW synthesis, we observed swaths of densely arranged CSCs that moved in the same direction along tracks while synthesizing cellulose microfibrils that became highly aggregated. Our data support a model in which distinct spatiotemporal features of active CSCs during PCW and SCW synthesis contribute to the formation of cellulose with distinct structure and organization in PCWs and SCWs of Arabidopsis thaliana This study provides a foundation for understanding differences in the formation, structure, and organization of cellulose in PCWs and SCWs.

Our future bioeconomy depends on increased utilization of renewable lignocellulosic biomass. Controlling the diffusion of chemicals, such as inorganic ions, within secondary plant cell walls is central to many biomass applications. However, insufficient understanding of intra-cell-wall diffusion within secondary plant cell walls is hindering the advancement of many lignocellulosic biomass applications. In this work, X-ray fluorescence microscopy was used to measure diffusion constants of K+, Cu2+, and Cl− diffusing through loblolly pine (Pinus taeda) cell wall layers under 70%, 75%, or 80% relative humidity (RH). Results revealed that diffusion constants increased with RH, the larger Cu2+ diffused more slowly than the K+, and the Cl− diffusion constant was the same as that for the counter cation, indicating cations and anions diffused together to maintain charge neutrality. Comparison with electrical conductivity measurements showed that conductivity is being controlled by ion mobility over these RH. The results further support that intra-cell-wall diffusion of inorganic ions is a Fickian diffusion process occurring through rubbery amorphous polysaccharides, which contradicts previous assertions that intra-cell-wall diffusion is an aqueous process occurring through water pathways. Researchers can now utilize polymer science approaches to engineer the molecular architecture of lignocellulosic biomass to optimize properties for specific end uses.

The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. These walls are made principally from cellulose and hemicelluloses and are impregnated with lignin. Despite their importance as the main load bearing structure for plant growth, as well as their industrial importance as both a material and energy source, the precise arrangement of these constituents within the cell wall is not yet fully understood. We have adapted low temperature scanning electron microscopy (cryo-SEM) for imaging the nanoscale architecture of angiosperm and gymnosperm cell walls in their native hydrated state. Our work confirms that cell wall macrofibrils, cylindrical structures with a diameter exceeding 10 nm, are a common feature of the native hardwood and softwood samples. We have observed these same structures in Arabidopsis thaliana secondary cell walls, enabling macrofibrils to be compared between mutant lines that are perturbed in cellulose, hemicellulose, and lignin formation. Our analysis indicates that the macrofibrils in Arabidopsis cell walls are dependent upon the proper biosynthesis, or composed, of cellulose, xylan, and lignin. This study establishes that cryo-SEM is a useful additional approach for investigating the native nanoscale architecture and composition of hardwood and softwood secondary cell walls and demonstrates the applicability of Arabidopsis genetic resources to relate fibril structure with wall composition and biosynthesis.

Motivation and aim: Plant cell walls play a crucial role in growth and development by protecting plants from environmental stress and facilitating directed growth.They are generally categorized into two types: primary walls, which are dynamic structures that support the expansion of growing plant cells and are essential for plant morphogenesis, and secondary walls, which are formed after cell expansion and provide structural support and rigidity.Secondary walls make up the majority of a plant's biomass and are primarily composed of cellulose, hemicelluloses, and lignin.Plant fiber form quite remarkable cell walls, especially as many fiber crops, including flax, depositing an additional layer -a tertiary cell wall, also called the G-layer.The distinguishing features of such cell walls are high cellulose content, axial orientation of cellulose microfibrils, the virtual absence of xylan and lignin, and presence of rhamnogalacturonan I having a special structure and properties.The transcriptional network consisting of master transcriptional switches and their downstream transcription factors (TFs) that regulate secondary cell walls biosynthesis in plant fibers is well established, while TFs regulate tertiary cell wall deposition are not known.We performed transcriptome profiling of phloem flax fibers at different stage of development and revealed the upregulation of expression of a number of genes encoding TFs of different families, including the BZIP44 TF.To establish the possible role of BZIP44, we used the Arabidopsis nst1nst3 mutants, which lack secondary cell wall in fascicular fibers.These mutant plants are convenient models for investigation of phenotypes induced by expressing TFs of unknown function driven by the NST3 promoter, which is specific for xylary fibers.Materials and methods: Arabidopsis thaliana ecotype Columbia-0 and the nst1nst3 double mutants were transformed with constructs containing ATBZIP44 under the control of NST3 promoter and fused to strong transcriptional activation (VP16) or inhibition domains (SRDX).The phenotypes of the obtaining transgenic plants were analyzed using light microscopy.The expression of a set of genes encoding key TFs involved in secondary cell wall formation was analyzed using qPCR.Transcriptome analysis of the inflorescence stems of transgenic lines was also performed.Results: Transformation of the nst1nst3 mutants by NST3::ATBZIP44_SRDX induced recovery of cell wall deposition in interfascicular fibers.In the reconstructed thickened cell wall in interfascicular fibers of NST3::ATBZIP44_SRDX (nst1nst3 background), the distribution of cellulose and xylan was similar to that of the wild type, while in Arabidopsis plants Col-0 transformed by NST3::ATBZIP44_VP16, a reduction of secondary cell walls in interfascicular fibers was observed, and the phenotype of these transgenic plants resembles the nst1nst3 phenotype.The observed phenotype changes were confirmed by gene expression analysis using qPCR.Downregulation of genes involved in the biosynthesis of cellulose, lignin and xylan was revealed in wt_NST3::ATBZIP44_VP16.The expression pattern of the analyzed genes was very similar to that of the nst1nst3 mutants.Conversely, in nst1nst3 mutant plants transformed by NST3::ATBZIP44 with the repressor SRDX, upregulation of genes involved in secondary cell wall development occurred.TFs associated with secondary cell wall formation in interfascicular fibers were suppressed in wt_NST3::ATBZIP44 (NST1,2,3 and SDN2, 3), while the TFs involved in vessel elements formation (VND6 and VND7) did not change their expression.In nst1nst3_NST3::ATBZIP44_SRDX plants, the upregulation of the NST2 was revealed.The gene for WRKY12 known as a negative regulator of NST2 was not affected in the ATBZIP44 transgenic plants.Since VP16 chimeric constructs activate expression of target genes and SRDX constructs act as suppressor of target gene expression, the obtained data suggest that ATBZIP44 activates an unknown inhibitor (except WRKY12) that usually is not expressed in Arabidopsis inflorescence stem tissues; this inhibitor suppresses secondary cell wall formation in wt_NST3::ATBZIP44 transgenic plants.Suppression of the proposed inhibitor in nst1nst3 mutant plants transformed with NST3::ATBZIP44_SRDX stimulates secondary cell wall deposition, which is regulated rather by NST2 TF, but not NST1 and NST3.In the report data of transciptomic analysis for the analyzed transgenic plants will be presented as well to provide a deeper understanding of the observed phenomenon and to reveal new participants in cell wall regulation.Conclusions: In the course of the current research work, a novel transcription factor, BZIP44, involved in thickened cell wall formation was identified.This factor acts as a negative regulator of secondary cell wall formation, but could be associated with tertiary cell wall formation since the downregulation of the secondary cell wall is required to start the deposition of the tertiary cell wall layer.

The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi-dimensional solid-state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin-cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell-specific expression strategies to avoid negative effects on plant growth.

Secondary plant cell walls are composed of carbohydrate and lignin polymers, and collectively represent a significant renewable resource. Leveraging these resources depends in part on a mechanistic understanding for diffusive processes within plant cell walls. Common wood protection treatments and biomass conversion processes to create biorefinery feedstocks feature ion or solvent diffusion within the cell wall. X-ray fluorescence microscopy experiments have determined that ionic diffusion rates are dependent on cell wall hydration as well as the ionic species through non-linear relationships. In this work, we use classical molecular dynamics simulations to map the diffusion behavior of different plant cell wall components (cellulose, hemicellulose, lignin), ions (Na+, K+, Cu2+, Cl−) and water within a model for an intact plant cell wall at various hydration states (3–30 wt% water). From these simulations, we analyze the contacts between different plant cell wall components with each other and their interaction with the ions. Generally, diffusion increases with increasing hydration, with lignin and hemicellulose components increasing diffusion by an order of magnitude over the tested hydration range. Ion diffusion depends on charge. Positively charged cations preferentially interact with hemicellulose components, which include negatively charged carboxylates. As a result, positive ions diffuse more slowly than negatively charged ions. Measured diffusion coefficients are largely observed to best fit piecewise linear trends, with an inflection point between 10 and 15% hydration. These observations shed light onto the molecular mechanisms for diffusive processes within secondary plant cell walls at atomic resolution.

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays, and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.

Secondary cell walls are the major constituent of tracheary elements and fibres in wood, which is the most abundant biomass produced by plants. They provide strong mechanical strength to tracheary elements and fibres, and ultimately to plant organs. The principal components of secondary walls are cellulose, hemicellulose and lignin. Cellulose microfibrils together with hemicelluloses form the main load‐bearing network in secondary walls, in which lignin is impregnated to form another crosslinked network to provide hydrophobicity and more rigidity. The biosynthesis of secondary walls is a highly coordinated developmental process that involves a coordinated expression of secondary wall biosynthetic genes regulated by a cascade of transcription factors. Because secondary walls in the form of wood and fibres are the most abundant, renewable plant products, understanding how they are constructed will provide novel strategies for genetic improvement of wood and fibres to better suit our needs. Key concepts Cell walls are the plant ‘exoskeleton’ that dictates the cell shape and collectively the plant form. The ability for plant cells to make secondary cell walls is considered to be one of the most important evolutionary landmarks for vascular plants. Secondary cell walls are the main constituent of tracheary elements and fibres of wood, which is the most abundant biomass produced by land plants. Secondary cell walls in tracheary elements and fibres provide mechanical strength to plant organs. Secondary cell walls in tracheary elements are deposited in specific patterns, including helical, annular, scalariform, reticulated and pitted patterns. The principal components of secondary cell walls are cellulose, hemicelluloses and lignin. Cellulose is synthesized by the cellulose synthase complexes located at the plasma membrane. Hemicelluloses, including glucomannan and xylan, are synthesized at the Golgi and then secreted via vesicles into the cell walls. Lignin is synthesized through dehydrogenative polymerization of monolignols, p ‐coumaryl alcohol, coniferyl alcohol and syringyl alcohol, which is catalysed by oxidases including peroxidases and laccases. The biosynthesis of secondary cell walls is regulated by a transcriptional network comprising of a cascade of transcription factors.