Pickering Emulsions and Hydrophobized Films of Amphiphilic Cellulose Nanofibers Synthesized in Deep Eutectic Solvent.

Herein, a dual-functioning deep eutectic solvent system based on triethylmethylammonium chloride and imidazole was harnessed as a swelling agent and a reaction medium for the esterification of cellulose with n-octyl succinic anhydride (OSA). The modified or amphiphilic cellulose nanofibers (ACNFs), synthesized using three different OSA-to-anhydroglucose unit molar ratios (0.5:1, ACNF-1; 1:1, ACNF-2; and 1.5:1, ACNF-3), were further converted into nanofibers with degree of substitution (DS) values of 0.24-0.66. The ACNFs possessed a lateral dimension of 4.24-9.22 nm and displayed surface activity due to the balance of hydrophobic and hydrophilic characteristics. The ACNFs made stable aqueous dispersions; however, the instability index of ACNF-3 (0.51) was higher than those of ACNF-1 (0.29) and ACNF-2 (0.33), which was attributed to the high DS-induced hydrophobicity, causing the instability in water. The amphiphilic nature of ACNFs promoted their performance as stabilizers in oil-in-water Pickering emulsions with average droplet sizes of 4.85 µm (ACNF-1) and 5.48 µm (ACNF-2). Self-standing films of ACNFs showed high contact angles for all the tested DS variants (97.48-114.12°), while their tensile strength was inversely related to DS values (ACNF-1: 115 MPa and ACNF-3: 49.5 MPa). Aqueous dispersions of ACNFs were also tested for coating fruits to increase their shelf life. Coatings improved their shelf life by decreasing oxygen contact and moisture loss.

Fast Response and Visual Transparency Switching Hydrochromic Film Based on the Rational Structure of Cellulose/Poloxamer Copolymers Design for Smart Window.

The authors are motivated to develop a series of hydrochromic copolymers with fast response, reversibility, repeatability, and visual transparency transition. The hydrochromic block copolymers are based on the rational ratio of hydrophilic segments of poloxamer block and hydrophobic segments of ethyl cellulose according to the preparation method of polyurethane. By tuning the ratio of hydrophilic segments or adding hygroscopic salts, the hydrochromic polymer is endowed with the ability to visualize the transparency in response to the relative humidity. Especially, the response time of the polymer is extremely shortened, up to 1 s for the optimized sample. Within the moisture stimulation, the hygroscopic swelling increases the film thickness, leading to a reversible transparency switching from a highly transparent state (82%) to an opaque white state (20.5%).

Role of silica-based porous cellulose nanocrystals in improving water absorption and mechanical properties.

Epoxy resins are important thermosetting polymers. They are widely used in many applications i.e., adhesives, plastics, coatings and sealers. Epoxy molding compounds have attained dominance among common materials due to their excellent mechanical properties. The sol-gel simple method was applied to distinguish the impact on the colloidal time. The properties were obtained with silica-based fillers to enable their mechanical and thermal improvement. The work which we have done here on epoxy-based nanocomposites was successfully modified. The purpose of this research was to look into the effects of cellulose nanocrystals (CNCs) on various properties and applications. CNCs have recently attracted a lot of interest in a variety of industries due to their high aspect ratio, and low density which makes them perfect candidates. Adding different amounts of silica-based nanocomposites to the epoxy system. Analyzed with different techniques such as Fourier-transformed infrared spectroscope (FTIR), thermogravimetric analysis (TGA) and scanning electronic microscopic (SEM) to investigate the morphological properties of modified composites. The various %-age of silica composite was prepared in the epoxy system. The 20% of silica was shown greater enhancement and improvement. They show a better result than D-400 epoxy. Increasing the silica, the transparency of the films decreased, because clustering appears. This shows that the broad use of CNCs in environmental engineering applications is possible, particularly for surface modification, which was evaluated for qualities such as absorption and chemical resistant behavior.

SCOBY Cellulose Modified with Apple Powder-Biomaterial with Functional Characteristics.

The need for new non-animal and non-petroleum-based materials is strongly emphasized in the sustainable and green economy. Waste materials have proven a valuable resource in this regard. In fact, there have been quite a large number of goods obtained from wastes called "Vegan leather" that have gained the clothing market's attention in recent years. In practice, they are mostly composites of waste materials like cactus, pineapples, or, eventually, apples with polymers like polyurethane or polyvinyl chloride. The article presents the results of work aimed at obtaining a material based entirely on natural, biodegradable raw materials. Bacterial cellulose produced as a byproduct of the fermentation carried out by SCOBY was modified with glycerol and then altered by the entrapment of apple powder. The effect of introducing apple powder into the SCOBY culture media on the mechanical properties of the obtained bacterial cellulose was also evaluated The resulting material acquired new mechanical characteristics that are advantageous in terms of strength. Microscopic observation of the apple powder layer showed that the coverage was uniform. Different amounts of apple powder were used to cover the cellulose surface from 10 to 60%, and it was found that the variant with 40% of this powder was the most favorable in terms of mechanical strength. Also, the application of the created material as a card folder showed that it is durable in use and retains its functional characteristics for at least 1 month. The mechanical properties of modified bacterial cellulose were favorably affected by the entrapment of apple powder on its surface, and as a result, a novel material with functional characteristics was obtained.

Structural hierarchy confers error tolerance in biological materials.

Structural hierarchy, in which materials possess distinct features on multiple length scales, is ubiquitous in nature. Diverse biological materials, such as bone, cellulose, and muscle, have as many as 10 hierarchical levels. Structural hierarchy confers many mechanical advantages, including improved toughness and economy of material. However, it also presents a problem: Each hierarchical level adds a new source of assembly errors and substantially increases the information required for proper assembly. This seems to conflict with the prevalence of naturally occurring hierarchical structures, suggesting that a common mechanical source of hierarchical robustness may exist. However, our ability to identify such a unifying phenomenon is limited by the lack of a general mechanical framework for structures exhibiting organization on disparate length scales. Here, we use simulations to substantiate a generalized model for the tensile stiffness of hierarchical filamentous networks with a nested, dilute triangular lattice structure. Following seminal work by Maxwell and others on criteria for stiff frames, we extend the concept of connectivity in network mechanics and find a similar dependence of material stiffness upon each hierarchical level. Using this model, we find that stiffness becomes less sensitive to errors in assembly with additional levels of hierarchy; although surprising, we show that this result is analytically predictable from first principles and thus potentially model independent. More broadly, this work helps account for the success of hierarchical, filamentous materials in biology and materials design and offers a heuristic for ensuring that desired material properties are achieved within the required tolerance.

Surface Modification of Bacterial Cellulose for Biomedical Applications.

Bacterial cellulose is a naturally occurring polysaccharide with numerous biomedical applications that range from drug delivery platforms to tissue engineering strategies. BC possesses remarkable biocompatibility, microstructure, and mechanical properties that resemble native human tissues, making it suitable for the replacement of damaged or injured tissues. In this review, we will discuss the structure and mechanical properties of the BC and summarize the techniques used to characterize these properties. We will also discuss the functionalization of BC to yield nanocomposites and the surface modification of BC by plasma and irradiation-based methods to fabricate materials with improved functionalities such as bactericidal capabilities.

Synthesis and Characterization of Cellulose Triacetate Obtained from Date Palm (Phoenix dactylifera L.) Trunk Mesh-Derived Cellulose.

Cellulosic polysaccharides have increasingly been recognized as a viable substitute for the depleting petro-based feedstock due to numerous modification options for obtaining a plethora of bio-based materials. In this study, cellulose triacetate was synthesized from pure cellulose obtained from the waste lignocellulosic part of date palm (Phoenix dactylifera L.). To achieve a degree of substitution (DS) of the hydroxyl group of 2.9, a heterogeneous acetylation reaction was carried out with acetic anhydride as an acetyl donor. The obtained cellulose ester was compared with a commercially available derivative and characterized using various analytical methods. This cellulose triacetate contains approximately 43.9% acetyl and has a molecular weight of 205,102 g·mol-1. The maximum thermal decomposition temperature of acetate was found to be 380 °C, similar to that of a reference sample. Thus, the synthesized ester derivate can be suitable for fabricating biodegradable and "all cellulose" biocomposite systems.

Structure of the Bacterial Cellulose Ribbon and Its Assembly-Guiding Cytoskeleton by Electron Cryotomography.

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength, and stiffness prevent dehydration and mechanical disruption of the biofilm. Bacteria in the genus Gluconacetobacter secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused-ion-beam milling of native bacterial biofilms to image cellulose-synthesizing Gluconacetobacter hansenii and Gluconacetobacter xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several micrometers in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, which we have named the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We found that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line to form higher-order cellulose structures, such as sheets and ribbons.IMPORTANCE This work's relevance for the microbiology community is twofold. It delivers for the first time high-resolution near-native snapshots of Gluconacetobacter spp. (previously Komagataeibacter spp.) in the process of cellulose ribbon synthesis, in their native biofilm environment. It puts forward a noncharacterized cytoskeleton element associated with the side of the cell where the cellulose synthesis occurs. This represents a step forward in the understanding of the cell-guided process of crystalline cellulose synthesis, studied specifically in the Gluconacetobacter genus and still not fully understood. Additionally, our successful attempt to use cryo-focused-ion-beam milling through biofilms to image the cells in their native environment will drive the community to use this tool for the morphological characterization of other studied biofilms.

Fabrication of Paper Sheets Coatings Based on Chitosan/Bacterial Nanocellulose/ZnO with Enhanced Antibacterial and Mechanical Properties.

Here, we designed paper sheets coated with chitosan, bacterial cellulose (nanofibers), and ZnO with boosted antibacterial and mechanical activity. We investigated the compositions, with ZnO exhibiting two different sizes/shapes: (1) rods and (2) irregular sphere-like particles. The proposed processing of bacterial cellulose resulted in the formation of nanofibers. Antimicrobial behavior was tested using E. coli ATCC® 25922™ following the ASTM E2149-13a standard. The mechanical properties of the paper sheets were measured by comparing tearing resistance, tensile strength, and bursting strength according to the ISO 5270 standard. The results showed an increased antibacterial response (assigned to the combination of chitosan and ZnO, independent of its shape and size) and boosted mechanical properties. Therefore, the proposed composition is an interesting multifunctional mixture for coatings in food packaging applications.

Valorization of Byproducts of Hemp Multipurpose Crop: Short Non-Aligned Bast Fibers as a Source of Nanocellulose.

Nanocellulose was extracted from short bast fibers, from hemp (Cannabis sativa L.) plants harvested at seed maturity, non-retted, and mechanically decorticated in a defibering apparatus, giving non-aligned fibers. A chemical pretreatment with NaOH and HCl allowed the removal of most of the non-cellulosic components of the fibers. No bleaching was performed. The chemically pretreated fibers were then refined in a beater and treated with a cellulase enzyme, followed by mechanical defibrillation in an ultrafine friction grinder. The fibers were characterized by microscopy, infrared spectroscopy, thermogravimetric analysis and X-ray diffraction after each step of the process to understand the evolution of their morphology and composition. The obtained nanocellulose suspension was composed of short nanofibrils with widths of 5-12 nm, stacks of nanofibrils with widths of 20-200 nm, and some larger fibers. The crystallinity index was found to increase from 74% for the raw fibers to 80% for the nanocellulose. The nanocellulose retained a yellowish color, indicating the presence of some residual lignin. The properties of the nanopaper prepared with the hemp nanocellulose were similar to those of nanopapers prepared with wood pulp-derived rod-like nanofibrils.

Atomic force microscopy imaging of delignified secondary cell walls in liquid conditions facilitates interpretation of wood ultrastructure.

Deep understanding of the physicochemical and structural characteristics of wood at the nanoscale is essential for improving wood usage in biorefining and advancing new high performance materials design. Herein, we use in situ atomic force microscopy and a simple delignification treatment to elucidate the nanoscale architecture of individual secondary cell wall layers. Advantages of this approach are: (i) minimal sample preparation that reduces the introduction of potential artifacts; (ii) prevention of structural rearrangements due to dehydration; (iii) increased accessibility to structural details masked by the lignin matrix; and (iv) possibility to complement results with other analytical techniques without sample manipulation. The methodology permits the visualization of parallel and helicoidally arranged microfibril aggregates in the S1 layer and the determination of lignin contribution to microfibril aggregates forming S2 layers. Cellulose and hemicelluloses constitute the core of the aggregates with a mean diameter of approximately 19 nm, and lignin encloses the core forming single structural entities of about 30 nm diameter. Furthermore, we highlight the implications of sample preparation and imaging parameters on the characterization of microfibril aggregates by AFM.

Artificially lignified cell wall catalyzed by peroxidase selectively localized on a network of microfibrils from cultured cells.

MAIN CONCLUSION: An artificial lignified cell wall was synthesized in three steps: (1) isolation of microfibrillar network; (2) localization of peroxidase through immunoreaction; and (3) polymerization of DHP to lignify the cell wall. Artificial woody cell wall synthesis was performed following the three steps along with the actual formation in nature using cellulose microfibrils extracted from callus derived from Cryptomeria japonica. First, we constructed a polysaccharide network on a transmission electron microscopy (TEM) grid. The preparation method was optimized by chemical treatment, followed by mechanical fibrillation to create a microfibrillated network. Morphology was examined by TEM, and chemical characterization was by Fourier transform infrared (FTIR) spectroscopy. Second, we optimized the process to place peroxidase on the microfibrils via an immunoreaction technique. Using a xyloglucan antibody, we could ensure that gold particles attached to the secondary antibodies were widely and uniformly localized along with the microfibril network. Third, we applied the peroxidase attached to secondary antibodies and started to polymerize the lignin on the grid by simultaneously adding coniferyl alcohol and hydrogen peroxide. After 30 min of artificial lignification, TEM observation showed that lignin-like substances were deposited on the polysaccharide network. In addition, FTIR spectra revealed that the bands specific for lignin had increased, demonstrating the successful artificial formation of woody cell walls. This approach may be useful for studying woody cell wall formation and for producing made-to-order biomaterials.

Cladophora Cellulose: Unique Biopolymer Nanofibrils for Emerging Energy, Environmental, and Life Science Applications.

Because of its natural abundance, hierarchical fibrous structure, mechanical flexibility, potential for chemical modification, biocompatibility, renewability, and abundance, cellulose is one of the most promising green materials for a bio-based future and sustainable economy. Cellulose derived from wood or bacteria has dominated the industrial cellulose market and has been developed to produce a number of advanced materials for applications in energy storage, environmental, and biotechnology areas. However, Cladophora cellulose (CC) extracted from green algae has unprecedented advantages over those celluloses because of its high crystallinity (>95%), low moisture adsorption capacity, excellent solution processability, high porosity in the mesoporous range, and associated high specific surface area. The unique physical and chemical properties of CC can add new features to and enhance the performance of nanocellulose-based materials, and these attributes have attracted a great deal of research interest over the past decade. This Account summarizes our recent research on the preparation, characterization, functionalization, and versatile applications of CC. Our aim is to provide a comprehensive overview of the uniqueness of CC with respect to material structure, properties, and emerging applications. We discuss the potential of CC in energy storage, environmental science, and life science, with emphasis on applications in which its properties are superior to those of other nanocellulose forms. Specifically, we discuss the production of the first-ever paper battery based on CC. This battery has initiated a rising interest in the development of sustainable paper-based energy storage devices, where cellulose is used as a combined building block and binder for paper electrodes of various types in combination with carbon, conducting polymers, and other electroactive materials. High-active-mass and high-mass-loading paper electrodes can be made in which the CC acts as a high-surface-area and porous substrate while a thin layer of electroactive material is coated on individual nanofibrils. We have shown that CC membranes can be used directly as battery separators because of their low moisture content, high mesoporosity, high thermal stability, and good electrolyte wettability. The safety, stability, and capacity of lithium-ion batteries can be enhanced simply by using CC-based separators. Moreover, the high chemical modifiability and adjustable porosity of dried CC papers allow them to be used as advanced membranes for environmental science (water and air purification, pollutant adsorption) and life science (virus isolation, protein recovery, hemodialysis, DNA extraction, bioactive substrates). Finally, we outline some concluding perspectives on the challenges and future directions of CC research with the aim to open up yet unexplored fields of use for this interesting material.

Bacterial Nanocellulose in Dentistry: Perspectives and Challenges.

Bacterial cellulose (BC) is a natural polymer that has fascinating attributes, such as biocompatibility, low cost, and ease of processing, being considered a very interesting biomaterial due to its options for moldability and combination. Thus, BC-based compounds (for example, BC/collagen, BC/gelatin, BC/fibroin, BC/chitosan, etc.) have improved properties and/or functionality, allowing for various biomedical applications, such as artificial blood vessels and microvessels, artificial skin, and wounds dressing among others. Despite the wide applicability in biomedicine and tissue engineering, there is a lack of updated scientific reports on applications related to dentistry, since BC has great potential for this. It has been used mainly in the regeneration of periodontal tissue, surgical dressings, intraoral wounds, and also in the regeneration of pulp tissue. This review describes the properties and advantages of some BC studies focused on dental and oral applications, including the design of implants, scaffolds, and wound-dressing materials, as well as carriers for drug delivery in dentistry. Aligned to the current trends and biotechnology evolutions, BC-based nanocomposites offer a great field to be explored and other novel features can be expected in relation to oral and bone tissue repair in the near future.

Nanocellulose Hybrids with Metal Oxides Nanoparticles for Biomedical Applications.

Cellulose is one of the most affordable, sustainable and renewable resources, and has attracted much attention especially in the form of nanocellulose. Bacterial cellulose, cellulose nanocrystals or nanofibers may serve as a polymer support to enhance the effectiveness of metal nanoparticles. The resultant hybrids are valuable materials for biomedical applications due to the novel optical, electronic, magnetic and antibacterial properties. In the present review, the preparation methods, properties and application of nanocellulose hybrids with different metal oxides nanoparticles such as zinc oxide, titanium dioxide, copper oxide, magnesium oxide or magnetite are thoroughly discussed. Nanocellulose-metal oxides antibacterial formulations are preferred to antibiotics due to the lack of microbial resistance, which is the main cause for the antibiotics failure to cure infections. Metal oxide nanoparticles may be separately synthesized and added to nanocellulose (ex situ processes) or they can be synthesized using nanocellulose as a template (in situ processes). In the latter case, the precursor is trapped inside the nanocellulose network and then reduced to the metal oxide. The influence of the synthesis methods and conditions on the thermal and mechanical properties, along with the bactericidal and cytotoxicity responses of nanocellulose-metal oxides hybrids were mainly analyzed in this review. The current status of research in the field and future perspectives were also signaled.

Antibacterial Activity of Bacterial Cellulose Loaded with Bacitracin and Amoxicillin: In Vitro Studies.

The use of bacterial cellulose (BC) in skin wound treatment is very attractive due to its unique characteristics. These dressings' wet environment is an important feature that ensures efficient healing. In order to enhance the antimicrobial performances, bacterial-cellulose dressings were loaded with amoxicillin and bacitracin as antibacterial agents. Infrared characterization and thermal analysis confirmed bacterial-cellulose binding to the drug. Hydration capacity showed good hydrophilicity, an efficient dressing's property. The results confirmed the drugs' presence in the bacterial-cellulose dressing's structure as well as the antimicrobial efficiency against Staphylococcus aureus and Escherichia coli. The antimicrobial assessments were evaluated by contacting these dressings with the above-mentioned bacterial strains and evaluating the growth inhibition of these microorganisms.

Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags.

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.

The Crystallinity and Aspect Ratio of Cellulose Nanomaterials Determine Their Pro-Inflammatory and Immune Adjuvant Effects In Vitro and In Vivo.

Nanocellulose is increasingly considered for applications; however, the fibrillar nature, crystalline phase, and surface reactivity of these high aspect ratio nanomaterials need to be considered for safe biomedical use. Here a comprehensive analysis of the impact of cellulose nanofibrils (CNF) and nanocrystals (CNC) is performed using materials provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences. An intermediary length of nanocrystals is also derived by acid hydrolysis. While all CNFs and CNCs are devoid of cytotoxicity, 210 and 280 nm fluorescein isothiocyanate (FITC)-labeled CNCs show higher cellular uptake than longer and shorter CNCs or CNFs. Moreover, CNCs in the 200-300 nm length scale are more likely to induce lysosomal damage, NLRP3 inflammasome activation, and IL-1ß production than CNFs. The pro-inflammatory effects of CNCs are correlated with higher crystallinity index, surface hydroxyl density, and reactive oxygen species generation. In addition, CNFs and CNCs can induce maturation of bone marrow-derived dendritic cells and CNCs (and to a lesser extent CNFs) are found to exert adjuvant effects in ovalbumin (OVA)-injected mice, particularly for 210 and 280 nm CNCs. All considered, the data demonstrate the importance of length scale, crystallinity, and surface reactivity in shaping the innate immune response to nanocellulose.

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis.

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis (Arabidopsis thaliana) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis.

A mutation in the catalytic domain of cellulose synthase 6 halts its transport to the Golgi apparatus.

Cellulose microfibrils, which form the mechanical framework of the plant cell wall, are synthesized by the cellulose synthase complex in the plasma membrane. Here, we introduced point mutations into the catalytic domain of cellulose synthase 6 (CESA6) in Arabidopsis to produce enhanced yellow fluorescent protein (EYFP)-tagged CESA6D395N, CESA6Q823E, and CESA6D395N+Q823E, which were exogenously produced in a cesa6 null mutant, prc1-1. Comparison of these mutants in terms of plant phenotype, cellulose content, cellulose synthase complex dynamics, and organization of cellulose microfibrils showed that prc1-1 expressing EYFP:CESA6D395N or CESA6D395N+Q823E was nearly the same as prc1-1, whereas prc1-1 expressing EYFP:CESA6Q823E was almost identical to wild type and prc1-1 expressing EYFP:WT CESA6, indicating that CESA6D395N and CESA6D395N+Q823E do not function in cellulose synthesis, while CESA6Q823E is still functionally active. Total internal reflection fluorescence microscopy and confocal microscopy were used to monitor the subcellular localization of these proteins. We found that EYFP:CESA6D395N and EYFP:CESA6D395N+Q823E were absent from subcellular regions containing the Golgi and the plasma membrane, and they appeared to be retained in the endoplasmic reticulum. By contrast, EYFP:CESA6Q823E had a normal localization pattern, like that of wild-type EYFP:CESA6. Our results demonstrate that the D395N mutation in CESA6 interrupts its normal transport to the Golgi and its eventual participation in cellulose synthase complex assembly.