Deposition Of Cellulose Microfibrils Research Articles

Enhancing the flexural properties of CFRP with vacuum-assisted deposition of cellulose microfibrils to create a multiscale reinforcement network

ABSTRACT Carbon fiber (CF) was coated with micro-fibrillated cellulose (MFC) using vacuum-assisted deposition in aqueous suspensions at concentrations of 0.1%, 0.2%, and 0.4%. The impact of distribution mesh configurations (DM arrangements) and fabric layer configuration on cellulose deposition was examined. Multiscale CF-MFC-Epoxy composites were created using resin infusion under flexible tooling, forming a 3D network of MFC between CFs that transferred stress and acted as a node at the intersection of 0–90 bundles. The addition of 0.1% MFC suspensions improved flexural properties, using no distribution media enhanced deposition and penetration efficiency, and drying stacked layers of CF fabrics improved load transference between them. This combination of parameters resulted in composites with a 16% increase in flexural modulus and 45% increase in flexural strength.

Forces on and in the cell walls of living plants.

Environmental influences and differential growth subject plants to mechanical forces. Forces on the whole plant resolve into tensile forces on its primary cell walls and both tensile and compression forces on the secondary cell wall layers of woody tissues. Forces on cell walls are further resolved into forces on cellulose microfibrils and the noncellulosic polymers between them. Many external forces on plants oscillate, with time constants that vary from seconds to milliseconds. Sound waves are a high-frequency example. Forces on the cell wall lead to responses that direct the oriented deposition of cellulose microfibrils and the patterned expansion of the cell wall, leading to complex cell and tissue morphology. Recent experiments have established many of the details of which cell wall polymers associate with one another in both primary and secondary cell walls, but questions remain about which of the interconnections are load bearing, especially in primary cell walls. Direct cellulose-cellulose interactions appear to have a more important mechanical role than was previously thought, and some of the noncellulosic polymers may have a role in keeping microfibrils apart rather than cross-linking them as formerly envisaged.

Lockhart with a twist: Modelling cellulose microfibril deposition and reorientation reveals twisting plant cell growth mechanisms

Plant morphology emerges from cellular growth and structure. The turgor-driven diffuse growth of a cell can be highly anisotropic: significant longitudinally and negligible radially. Such anisotropy is ensured by cellulose microfibrils (CMF) reinforcing the cell wall in the hoop direction. To maintain the cell’s integrity during growth, new wall material including CMF must be continually deposited. We develop a mathematical model representing the cell as a cylindrical pressure vessel and the cell wall as a fibre-reinforced viscous sheet, explicitly including the mechano-sensitive angle of CMF deposition. The model incorporates interactions between turgor, external forces, CMF reorientation during wall extension, and matrix stiffening. Using the model, we reinterpret some recent experimental findings, and reexamine the popular hypothesis of CMF/microtubule alignment. We explore how the handedness of twisting cell growth depends on external torque and intrinsic wall properties, and find that cells twist left-handedly ‘by default’ in some suitable sense. Overall, this study provides a unified mechanical framework for understanding left- and right-handed twist-growth as seen in many plants.

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported to play a key role in this process. Here, we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers, and cellulose microfibrils as components of the core pathway behind hook formation in the presence or absence of external mechanical cues.

Shear-induced unidirectional deposition of bacterial cellulose microfibrils using rising bubble stream cultivation

In crystalline cellulose I, all glucan chains are ordered from reducing ends to non-reducing ends. Thus, the polarity of individual chains is added forming a large dipole within the crystal. If one can engineer unidirectional alignment (parallel packing) of cellulose crystals, then it might be possible to utilize the material properties originating from polar crystalline structures. However, most post-synthesis manipulation methods reported so far can only achieve the uniaxial alignment with bi-directionality (antiparallel packing). Here, we report a method to induce the parallel packing of bacterial cellulose microfibrils by applying unidirectional shear stress during the synthesis and deposition through the rising bubble stream in a culture medium. Driving force for the alignment is explained with mathematical estimation of the shear stress. Evidences of the parallel alignment of crystalline cellulose Iα domains were obtained using nonlinear optical spectroscopy techniques.

KATANIN-dependent mechanical properties of the stigmatic cell wall mediate the pollen tube path in Arabidopsis

Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules. Pollen tubes first grow within the cell wall of the papilla cells, applying pressure to the cell. Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules (CMTs), which in turn mediate deposition of cellulose microfibrils (CMFs). Here, by combining imaging, genetic and chemical approaches, we show that isotropic reorientation of CMTs and CMFs in aged Col-0 and katanin1-5 (ktn1-5) papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae. We show that this coiled phenotype is associated with specific mechanical properties of the cell walls that provide less resistance to pollen tube growth. Our results reveal an unexpected role for KTN1 in pollen tube guidance on the stigma by ensuring mechanical anisotropy of the papilla cell wall.

The Rice BZ1 Locus Is Required for Glycosylation of Arabinogalactan Proteins and Galactolipid and Plays a Role in both Mechanical Strength and Leaf Color

BackgroundThe cell wall and chloroplast are two fundamental structures determining plant mechanical strength and grain yield. Therefore, understanding mechanisms that improve plants’ ability to develop a robust cell wall and well-developed chloroplast is of utmost importance for agricultural activities.ResultsIn this study, we report the functional characterization of a novel rice mutant, brittle stem and zebra leaf (bz1), which displays altered cell wall composition and collapsed chloroplast membrane. Molecular and biochemical analysis revealed that BZ1 encodes a functional UDP-galactose/glucose epimerase (UGE) and is ubiquitously expressed with higher expression in stem and leaf tissues. Multiple techniques analyses, including immunoblots, immuno-gold, and cryogenic scanning electron microscopy, demonstrated a significantly impaired glycosylation of arabinogalactan proteins (AGPs) and disordered cellulose microfibril deposition in bz1. Lipid profiling assay showed that the amount of monogalactosyldiacylglycerols (MGDG), a major chloroplast membrane glycolipid, was significantly decreased in bz1. Taken together, these results strongly demonstrate that BZ1 participates in UDP-galactose supply for the sugar chains biosynthesis of AGPs and MGDG, which thereby, respectively, results in altered cell wall and abnormal chloroplast development. Due to inferior mechanical strength and reduced photosynthesis, bz1 plants displayed detrimental agronomic traits, whereas BZ1 overexpressing lines showed enhanced plant growth. Transcriptome analysis of stems and leaves further showed that numerous key genes involved in AGPs biosynthesis and photosynthesis metabolism were substantially suppressed in bz1.ConclusionsOur finding identifies BZ1 as a dual-targeting UGE protein for glycosylation of AGPs and MGDG and suggests a strategy for breeding robust elite crops.

Microtubule self-organisation during seed germination in Arabidopsis

BackgroundUpon water uptake and release of seed dormancy, embryonic plant cells expand, while being mechanically constrained by the seed coat. Cortical microtubules (CMTs) are key players of cell elongation in plants: their anisotropic orientation channels the axis of cell elongation through the guidance of oriented deposition of load-bearing cellulose microfibrils in the cell wall. Interestingly, CMTs align with tensile stress, and consistently, they reorient upon compressive stress in growing hypocotyls. How CMTs first organise in germinating embryos is unknown, and their relation with mechanical stress has not been investigated at such an early developing stage.ResultsHere, we analysed CMT dynamics in dormant and non-dormant Arabidopsis seeds by microscopy of fluorescently tagged microtubule markers at different developmental time points and in response to abscisic acid and gibberellins. We found that CMTs first appear as very few thick bundles in dormant seeds. Consistently, analysis of available transcriptome and translatome datasets show that limiting amounts of tubulin and microtubule regulators initially hinder microtubule self-organisation. Seeds imbibed in the presence of gibberellic acid or abscisic acid displayed altered microtubule organisation and transcriptional regulation. Upon the release of dormancy, CMTs then self-organise into multiple parallel transverse arrays. Such behaviour matches the tensile stress patterns in such mechanically constrained embryos. This suggests that, as CMTs first self-organise, they also align with shape-derived tensile stress patterns.ConclusionsOur results provide a scenario in which dormancy release in the embryo triggers microtubule self-organisation and alignment with tensile stress prior to germination and anisotropic growth.

Interaction between Autonomous and Microtubule Guidance Systems Controls Cellulose Synthase Trajectories.

The organization of cellulose microfibrils is critical for the strength and growth of plant cell walls. Microtubules have been shown to play a key role in controlling microfibril organization by guiding cellulose synthase complexes [1-4]. However, cellulose synthase trajectories can be maintained when microtubules are removed by drugs, suggesting a separate guidance mechanism is also at play [1, 5, 6]. By slowing down microtubule dynamics, we reveal such a mechanism by showing that cellulose synthase complexes can interact with the trails left by other complexes, causing them to follow the trails or disappear. The stability of the trails, together with the sensitivity of their directions to cellulase treatment, indicates they most likely reflect nascent cellulose microfibrils. Over many hours, this autonomous mechanism alone can lead to a change in the dominant orientation of cellulose synthase trajectories. However, the mechanism can be overridden by the microtubule guidance system. Our findings suggest a dual guidance model, in which an autonomous system, involving interaction between cellulose synthases and microfibrils, can maintain aligned cellulose synthase trajectories, while a microtubule guidance system allows alignments to be steered by environmental and developmental cues.

Role of cytoskeleton in plant cell wall formation

Cell wall, which is one important and characteristic structure of plant cells, represents the most abundant renewable resource on the earth. It exists as part of a continuum with the plasma membrane and cytoskeleton. The cell wall provides external support and the cytoskeletons including the microtubules and the microfilaments constitute an internal framework. In recent years, great progress has been made in the understanding of the roles of cytoskeleton involved in regulating plant cell wall formation. In this review, we summarized the role of cytoskeleton in trafficking of vesicles containing cell wall precursors and in the oriented deposition of cellulose microfibrils, the regulation of secondary cell wall thickening by the cytoskeleton, and the involvement of cytoskeleton in cell wall signaling. In the end, we also give some prospects for the future studies.

Cortical Microtubule Organization during Petal Morphogenesis in Arabidopsis.

Cortical microtubules guide the direction and deposition of cellulose microfibrils to build the cell wall, which in turn influences cell expansion and plant morphogenesis. In the model plant Arabidopsis thaliana (Arabidopsis), petal is a relatively simple organ that contains distinct epidermal cells, such as specialized conical cells in the adaxial epidermis and relatively flat cells with several lobes in the abaxial epidermis. In the past two decades, the Arabidopsis petal has become a model experimental system for studying cell expansion and organ morphogenesis, because petals are dispensable for plant growth and reproduction. Recent advances have expanded the role of microtubule organization in modulating petal anisotropic shape formation and conical cell shaping during petal morphogenesis. Here, we summarize recent studies showing that in Arabidopsis, several genes, such as SPIKE1, Rho of plant (ROP) GTPases, and IPGA1, play critical roles in microtubule organization and cell expansion in the abaxial epidermis during petal morphogenesis. Moreover, we summarize the live-confocal imaging studies of Arabidopsis conical cells in the adaxial epidermis, which have emerged as a new cellular model. We discuss the microtubule organization pattern during conical cell shaping. Finally, we propose future directions regarding the study of petal morphogenesis and conical cell shaping.

Changes in the orientations of cellulose microfibrils during the development of collenchyma cell walls of celery (Apium graveolens L.).

During development, cellulose microfibrils in collenchyma walls become increasingly longitudinal, as determined by small-angle X-ray scattering, despite the walls maintaining a fine structure indicative of a crossed-polylamellate structure. Collenchyma cells have thickened primary cell walls and provide mechanical support during plant growth. During their development, these cells elongate and their walls thicken considerably. We used microscopy and synchrotron small-angle X-ray scattering to study changes in the orientations of cellulose microfibrils that occur during development in the walls of collenchyma cells present in peripheral strands in celery (Apium graveolens) petioles. Transmission electron microscopy showed that the walls consisted of many lamellae (polylamellate), with lamellae containing longitudinally oriented cellulose microfibrils alternating with microfibrils oriented at higher angles. The lamellae containing longitudinally oriented microfibrils predominated at later stages of development. Nevertheless, transmission electron microscopy of specially stained, oblique sections provided evidence that the cellulose microfibrils were ordered throughout development as crossed-polylamellate structures. These results are consistent with our synchrotron small-angle X-ray scattering results that showed the cellulose microfibrils become oriented increasingly longitudinally during development. Some passive reorientation of cellulose microfibrils may occur during development, but extensive reorientation throughout the wall would destroy ordered structures. Atomic force microscopy and field emission scanning electron microscopy were used to determine the orientations of newly deposited cellulose microfibrils. These were found to vary widely among different cells, which could be consistent with the formation of crossed-polylamellate structures. These newly deposited cellulose microfibrils are deposited in a layer of pectic polysaccharides that lies immediately outside the plasma membrane. Overall, our results show that during development of collenchyma walls, the cellulose microfibrils become increasingly longitudinal in orientation, yet organized, crossed-polylamellate structures are maintained.

Pectin Chemistry and Cellulose Crystallinity Govern Pavement Cell Morphogenesis in a Multi-Step Mechanism.

Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (Arabidopsis thaliana) cotyledons. We show that nonuniform distribution of cellulose microfibrils and demethylated pectin coincides with spatial differences in cell wall stiffness but may intervene at different developmental stages. We also show that lobe period can be reduced when demethyl-esterification of pectins increases under conditions of reduced cellulose crystallinity. Our data suggest that lobe initiation involves a modulation of cell wall stiffness through local enrichment in demethylated pectin, whereas subsequent increase in lobe amplitude is mediated by the stress-induced deposition of aligned cellulose microfibrils. Our results reveal a key role of noncellulosic polymers in the biomechanical regulation of cell morphogenesis.

Whole-Genome Sequencing Identifies a Rice Grain Shape Mutant, gs9\u20131

BackgroundBreeding for genes controlling key agronomic traits is an important goal of rice genetic improvement. To gain insight into genes controlling grain morphology, we screened M3 plants derived from 1,000 whole-genome sequenced (WGS) M2 Kitaake mutants to identify lines with altered grain size.ResultsIn this study, we isolated a mutant, named fast-neutron (FN) 60–4, which exhibits a significant reduction in grain size. We crossed FN60–4 with the parental line Kitaake and analyzed the resulting backcross population. Segregation analysis of 113 lines from the BC2F2 population revealed that the mutant phenotype is controlled by a single semi-dominant locus. Mutant FN60–4 is reduced 20% in plant height and 8.8% in 1000-grain weight compared with Kitaake. FN60–4 also exhibits an 8% reduction in cell number and a 9% reduction in cell length along the vertical axis of the glume. We carried out whole-genome sequencing of DNA pools extracted from segregants with long grains or short grains, and revealed that one gene, LOC_Os09g02650, cosegregated with the grain size phenotype in the BC1F2 and BC2F2 populations. This mutant allele was named grain shape 9–1 (gs9–1). gs9–1 carries a 3-bp deletion that affects two amino acids. This locus is a new allele of the BC12/GDD1/MTD1 gene that encodes a kinesin-like protein involved in cell-cycle progression, cellulose microfibril deposition and gibberellic acid (GA) biosynthesis. The GA biosynthesis-related gene KO2 is down-regulated in gs9–1. The dwarf phenotype of gs9–1 can be rescued by adding exogenous GA3. In contrast to the phenotypes for the other alleles, the gs9–1 is less severe, consistent with the nature of the mutation, which does not disrupt the open reading frame as observed for the other alleles.ConclusionsIn this study, we isolated a mutant, which exhibits altered grain shape and identified the mutated gene, gs9–1. Our study reveals that gs9–1 is a semi-dominant gene that carries a two-amino acid mutation. gs9–1 is allelic to the BC12/GDD1/MTD1 gene involved in GA biosynthesis. These results demonstrate the efficiency and convenience of cloning genes from the whole-genome sequenced Kitaake mutant population to advance investigations into genes controlling key agronomic traits in rice.

Mechanical Conflicts in Twisting Growth Revealed by Cell-Cell Adhesion Defects.

Many plants grow organs and tissues with twisted shapes. Arabidopsis mutants with impaired microtubule dynamics exhibit such a phenotype constitutively. Although the activity of the corresponding microtubule regulators is better understood at the molecular level, how large-scale twisting can emerge in the mutants remains largely unknown. Classically, oblique cortical microtubules would constrain the deposition of cellulose microfibrils in cells, and such conflicts at the cell level would be relaxed at the tissue scale by supracellular torsion. This model implicitly assumes that cell-cell adhesion is a key step to transpose local mechanical conflicts into a macroscopic twisting phenotype. Here we tested this prediction using the quasimodo1 mutant, which displays cell-cell adhesion defects. Using the spriral2/tortifolia1 mutant with hypocotyl helical growth, we found that qua1-induced cell-cell adhesion defects restore straight growth in qua1-1 spr2-2. Detached cells in qua1-1 spr2-2 displayed helical growth, confirming that straight growth results from the lack of mechanical coupling between cells rather than a restoration of SPR2 activity in the qua1 mutant. Because adhesion defects in qua1 depend on tension in the outer wall, we also showed that hypocotyl twisting in qua1-1 spr2-2 could be restored when decreasing the matrix potential of the growth medium, i.e., by reducing the magnitude of the pulling force between adjacent cells, in the double mutant. Interestingly, the induction of straight growth in qua1-1 spr2-2 could be achieved beyond hypocotyls, as leaves also displayed a flat phenotype in the double mutant. Altogether, these results provide formal experimental support for a scenario in which twisted growth in spr2 mutant would result from the relaxation of local mechanical conflicts between adjacent cells via global organ torsion.

Effects of exogenous 24-epibrassinolide and brassinazole on negative gravitropism and tension wood formation in hybrid poplar (Populus deltoids × Populus nigra).

Exogenous 24-epibrassinolide (BL) and brassinazole (BRZ) have regulatory roles in G-fiber cell wall development and secondary xylem cell wall carbohydrate biosynthesis during tension wood formation in hybrid poplar. Brassinosteroids (BRs) play important roles in regulating gravitropism and vasculature development. Here, we report the effect of brassinosteroids on negative gravitropism and G-fiber cell wall development of the stem in woody angiosperms. We applied exogenous 24-epibrassinolide (BL) or its biosynthesis inhibitor brassinazole (BRZ) to slanted hybrid poplar trees (Populus deltoids × Populus nigra) and measured the morphology of gravitropic stems, anatomy and chemistry of secondary cell wall. We furthermore analyzed the expression levels of auxin transport and cellulose biosynthetic genes after 24-epibrassinolide (BL) or brassinazole (BRZ) application. The BL-treated seedlings showed no negative gravitropism bending, whereas application of BRZ dramatically enhanced negative gravitropic bending. BL treatment stimulated secondary xylem fiber elongation and G-fiber formation on the upper side of stems but delayed G-fiber maturation. BRZ inhibited xylem fiber elongation but induced the production of more mature G-fibers on the upper side of stems. Wood chemistry analyses and immunolocalization demonstrated that BL and BRZ treatments increased the cellulose content and modified the deposition of cell wall carbohydrates including arabinose, galactose and rhamnose in the secondary xylem. The expression of cellulose biosynthetic genes, especially those related to cellulose microfibril deposition (PtFLA12 and PtCOBL4) was significantly upregulated in BL- and BRZ-treated TW stems compared with control stems. The significant differences of G-fibers development and negative gravitropism bending between 24-epibrassinolide (BL) and brassinazole (BRZ) application suggest that brassinosteroids are important for secondary xylem development during tension wood formation. Our findings provide potential insights into the mechanism by which BRs regulate G-fiber cell wall development to accomplish negative gravitropism in TW formation.

Rab‐H1b is essential for trafficking of cellulose synthase and for hypocotyl growth in Arabidopsis thaliana

Cell-wall deposition of cellulose microfibrils is essential for plant growth and development. In plant cells, cellulose synthesis is accomplished by cellulose synthase complexes located in the plasma membrane. Trafficking of the complex between endomembrane compartments and the plasma membrane is vital for cellulose biosynthesis; however, the mechanism for this process is not well understood. We here report that, in Arabidopsis thaliana, Rab-H1b, a Golgi-localized small GTPase, participates in the trafficking of CELLULOSE SYNTHASE 6 (CESA6) to the plasma membrane. Loss of Rab-H1b function resulted in altered distribution and motility of CESA6 in the plasma membrane and reduced cellulose content. Seedlings with this defect exhibited short, fragile etiolated hypocotyls. Exocytosis of CESA6 was impaired in rab-h1b cells, and endocytosis in mutant cells was significantly reduced as well. We further observed accumulation of vesicles around an abnormal Golgi apparatus having an increased number of cisternae in rab-h1b cells, suggesting a defect in cisternal homeostasis caused by Rab-H1b loss function. Our findings link Rab GTPases to cellulose biosynthesis, during hypocotyl growth, and suggest Rab-H1b is crucial for modulating the trafficking of cellulose synthase complexes between endomembrane compartments and the plasma membrane and for maintaining Golgi organization and morphology.

The self-organization of plant microtubules inside the cell volume yields their cortical localization, stable alignment, and sensitivity to external cues.

Many cell functions rely on the ability of microtubules to self-organize as complex networks. In plants, cortical microtubules are essential to determine cell shape as they guide the deposition of cellulose microfibrils, and thus control mechanical anisotropy of the cell wall. Here we analyze how, in turn, cell shape may influence microtubule behavior. Building upon previous models that confined microtubules to the cell surface, we introduce an agent model of microtubules enclosed in a three-dimensional volume. We show that the microtubule network has spontaneous aligned configurations that could explain many experimental observations without resorting to specific regulation. In particular, we find that the preferred cortical localization of microtubules emerges from directional persistence of the microtubules, and their interactions with each other and with the stiff wall. We also identify microtubule parameters that seem relatively insensitive to cell shape, such as length or number. In contrast, microtubule array anisotropy depends on local curvature of the cell surface and global orientation follows robustly the longest axis of the cell. Lastly, we find that geometric cues may be overcome, as the network is capable of reorienting toward weak external directional cues. Altogether our simulations show that the microtubule network is a good transducer of weak external polarity, while at the same time, easily reaching stable global configurations.

Update: Plant Cortical Microtubule Arrays

Cortical microtubules play a critical role in plant morphogenesis by creating array patterns that template the deposition of cellulose microfibrils.

Emerging roles of cortical microtubule-membrane interactions.

Plant cortical microtubules have crucial roles in cell wall development. Cortical microtubules are tightly anchored to the plasma membrane in a highly ordered array, which directs the deposition of cellulose microfibrils by guiding the movement of the cellulose synthase complex. Cortical microtubules also interact with several endomembrane systems to regulate cell wall development and other cellular events. Recent studies have identified new factors that mediate interactions between cortical microtubules and endomembrane systems including the plasma membrane, endosome, exocytic vesicles, and endoplasmic reticulum. These studies revealed that cortical microtubule-membrane interactions are highly dynamic, with specialized roles in developmental and environmental signaling pathways. A recent reconstructive study identified a novel function of the cortical microtubule-plasma membrane interaction, which acts as a lateral fence that defines plasma membrane domains. This review summarizes recent advances in our understanding of the mechanisms and functions of cortical microtubule-membrane interactions.