In vitro synthesis of cellulose microfibrils by a membrane protein from protoplasts of the non-vascular plant Physcomitrella patens.

Plant cellulose synthases (CesAs) form a family of membrane proteins that are associated with hexagonal structures in the plasma membrane called CesA complexes (CSCs). It has been difficult to purify plant CesA proteins for biochemical and structural studies. We describe CesA activity in a membrane protein preparation isolated from protoplasts of Physcomitrella patens overexpressing haemagglutinin (HA)-tagged PpCesA5. Incubating the membrane preparation with UDP-glucose predominantly produced cellulose. Negative-stain EM revealed microfibrils. Cellulase bound to and degraded these microfibrils. Vibrational sum frequency generation (SFG) spectroscopic analysis detected the presence of crystalline cellulose in the microfibrils. Putative CesA proteins were frequently observed attached to the microfibril ends. Combined cross-linking and gradient centrifugation showed bundles of cellulose microfibrils with larger particle aggregates, possibly CSCs. These results suggest that P. patens is a useful model system for biochemical and structural characterization of plant CSCs and their components.

Effects of plant cell wall matrix polysaccharides on bacterial cellulose structure studied with vibrational sum frequency generation spectroscopy and X-ray diffraction.

The crystallinity, allomorph content, and mesoscale ordering of cellulose produced by Gluconacetobacter xylinus cultured with different plant cell wall matrix polysaccharides were studied with vibrational sum frequency generation (SFG) spectroscopy and X-ray diffraction (XRD). Crystallinity and ordering were assessed as the intensity of SFG signals in the CH/CH2 stretch vibration region (and confirmed by XRD), while Iα content was assessed by the relative intensity of the OH stretch vibration at 3240 cm(-1). A key finding is that the presence of xyloglucan in the culture medium greatly reduced Iα allomorph content but with a relatively small effect on cellulose crystallinity, whereas xylan resulted in a larger decrease in crystallinity with a relatively small decrease in the Iα fraction. Arabinoxylan and various pectins had much weaker effects on cellulose structure as assessed by SFG and XRD. Homogalacturonan with calcium ion reduced the SFG signal, evidently by changing the ordering of cellulose microfibrils. We propose that the distinct effects of matrix polysaccharides on cellulose crystal structure result, at least in part, from selective interactions of the backbone and side chains of matrix polysaccharides with cellulose chains during the formation of the microfibril.

Probing crystal structure and mesoscale assembly of cellulose microfibrils in plant cell walls, tunicate tests, and bacterial films using vibrational sum frequency generation (SFG) spectroscopy.

This study reports that the noncentrosymmetry and phase synchronization requirements of the sum frequency generation (SFG) process can be used to distinguish the three-dimensional organization of crystalline cellulose distributed in amorphous matrices. Crystalline cellulose is produced as microfibrils with a few nanometer diameters by plants, tunicates, and bacteria. Crystalline cellulose microfibrils are embedded in wall matrix polymers and assembled into hierarchical structures that are precisely designed for specific biological and mechanical functions. The cellulose microfibril assemblies inside cell walls are extremely difficult to probe. The comparison of vibrational SFG spectra of uniaxially-aligned and disordered films of cellulose Iß nanocrystals revealed that the spectral features cannot be fully explained with the crystallographic unit structure of cellulose. The overall SFG intensity, the alkyl peak shape, and the alkyl/hydroxyl intensity ratio are sensitive to the lateral packing and net directionality of the cellulose microfibrils within the SFG coherence length scale. It was also found that the OH SFG stretch peaks could be deconvoluted to find the polymorphic crystal structures of cellulose (Iα and Iß). These findings were used to investigate the cellulose crystal structure and mesoscale cellulose microfibril packing in intact plant cell walls, tunicate tests, and bacterial films.

Perturbation of Brachypodium distachyon CELLULOSE SYNTHASE A4 or 7 results in abnormal cell walls.

BACKGROUND: Cellulose is an integral component of the plant cell wall and accounts for approximately forty percent of total plant biomass but understanding its mechanism of synthesis remains elusive. CELLULOSE SYNTHASE A (CESA) proteins function as catalytic subunits of a rosette-shaped complex that synthesizes cellulose at the plasma membrane. Arabidopsis thaliana and rice (Oryza sativa) secondary wall CESA loss-of-function mutants have weak stems and irregular or thin cell walls. RESULTS: Here, we identify candidates for secondary wall CESAs in Brachypodium distachyon as having similar amino acid sequence and expression to those characterized in A. thaliana, namely CESA4/7/8. To functionally characterize BdCESA4 and BdCESA7, we generated loss-of-function mutants using artificial microRNA constructs, specifically targeting each gene driven by a maize (Zea mays) ubiquitin promoter. Presence of the transgenes reduced BdCESA4 and BdCESA7 transcript abundance, as well as stem area, cell wall thickness of xylem and fibers, and the amount of crystalline cellulose in the cell wall. CONCLUSION: These results suggest BdCESA4 and BdCESA7 play a key role in B. distachyon secondary cell wall biosynthesis.

Involvement of CesA4, CesA7-A/B and CesA8-A/B in secondary wall formation in Populus trichocarpa wood.

Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.

Modification of the nanostructure of lignocellulose cell walls via a non-enzymatic lignocellulose deconstruction system in brown rot wood-decay fungi.

Wood decayed by brown rot fungi and wood treated with the chelator-mediated Fenton (CMF) reaction, either alone or together with a cellulose enzyme cocktail, was analyzed by small angle neutron scattering (SANS), sum frequency generation (SFG) spectroscopy, Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Results showed that the CMF mechanism mimicked brown rot fungal attack for both holocellulose and lignin components of the wood. Crystalline cellulose and lignin were both depolymerized by the CMF reaction. Porosity of the softwood cell wall did not increase during CMF treatment, enzymes secreted by the fungi did not penetrate the decayed wood. The enzymes in the cellulose cocktail also did not appear to alter the effects of the CMF-treated wood relative to enhancing cell wall deconstruction. This suggests a rethinking of current brown rot decay models and supports a model where monomeric sugars and oligosaccharides diffuse from the softwood cell walls during non-enzymatic action. In this regard, the CMF mechanism should not be thought of as a "pretreatment" used to permit enzymatic penetration into softwood cell walls, but instead it enhances polysaccharide components diffusing to fungal enzymes located in wood cell lumen environments during decay. SANS and other data are consistent with a model for repolymerization and aggregation of at least some portion of the lignin within the cell wall, and this is supported by AFM and TEM data. The data suggest that new approaches for conversion of wood substrates to platform chemicals in biorefineries could be achieved using the CMF mechanism with >75% solubilization of lignocellulose, but that a more selective suite of enzymes and other downstream treatments may be required to work when using CMF deconstruction technology. Strategies to enhance polysaccharide release from lignocellulose substrates for enhanced enzymatic action and fermentation of the released fraction would also aid in the efficient recovery of the more uniform modified lignin fraction that the CMF reaction generates to enhance biorefinery profitability.

Multimodal Broadband Vibrational Sum Frequency Generation (MM-BB-V-SFG) Spectrometer and Microscope.

A broadband sum frequency generation (BB-SFG) spectrometer with multimodal (MM) capabilities was constructed, which could be routinely reconfigured for tabletop experiments in reflection, transmission, and total internal reflection (TIR) geometries, as well as microscopic imaging. The system was constructed using a Ti:sapphire amplifier (800 nm, pulse width = 85 fs, repetition rate = 2 kHz), an optical parameter amplification (OPA) system for production of broadband IR pulses tunable between 1000 and 4000 cm(-1), and two Fabry-Pérot etalons arranged in series for production of narrowband 800 nm pulses. The key feature allowing the MM operation was the nearly collinear alignment of the visible (fixed, 800 nm) and infrared (tunable, 1000-4000 cm(-1)) pulses which were spatially separated. Physical insights discussed in this paper include the comparison of spectral bandwidth produced with 40 and 85 fs pump beams, the improvement of spectral resolution using etalons, the SFG probe volume in bulk analysis, the normalization of SFG signals, the stitching of multiple spectral segments, and the operation in different modes for air/liquid and adsorbate/solid interfaces, bulk samples, as well as spectral imaging combined with principle component analysis (PCA). The SFG spectral features obtained with the MM-BB-SFG system were compared with those obtained with picosecond-scanning-SFG system and high-resolution BB-SFG system (HR-BB-SFG) for dimethyl sulfoxide, α-pinene, and various samples containing cellulose (purified commercial products, Cladophora cell wall, cotton and flax fibers, and onion epidermis cell wall).

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis.

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples--Avicel, bleached softwood, and bacterial cellulose--to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlate with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iß component.

Cellulose produced by Gluconacetobacter xylinus strains ATCC 53524 and ATCC 23768: Pellicle formation, post-synthesis aggregation and fiber density.

The pellicle formation, crystallinity, and bundling of cellulose microfibrils produced by bacterium Gluconacetobacter xylinus were studied. Cellulose pellicles were produced by two strains (ATCC 53524 and ATCC 23769) for 1 and 7 days; pellicles were analyzed with scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrational sum-frequency-generation (SFG) spectroscopy, and attenuated total reflectance infrared (ATR-IR) spectroscopy. The bacterial cell population was higher at the surface exposed to air, indicating that the newly synthesized cellulose is deposited at the top of the pellicle. XRD, ATR-IR, and SFG analyses found no significant changes in the cellulose crystallinity, crystal size or polymorphic distribution with the culture time. However, SEM and SFG analyses revealed cellulose macrofibrils produced for 7 days had a higher packing density at the top of the pellicle, compared to the bottom. These findings suggest that the physical properties of cellulose microfibrils are different locally within the bacterial pellicles.

Effect of mechanical disruption on the effectiveness of three reactors used for dilute acid pretreatment of corn stover Part 1: chemical and physical substrate analysis.

BACKGROUND: There is considerable interest in the conversion of lignocellulosic biomass to liquid fuels to provide substitutes for fossil fuels. Pretreatments, conducted to reduce biomass recalcitrance, usually remove at least some of the hemicellulose and/or lignin in cell walls. The hypothesis that led to this research was that reactor type could have a profound effect on the properties of pretreated materials and impact subsequent cellulose hydrolysis. RESULTS: Corn stover was dilute-acid pretreated using commercially relevant reactor types (ZipperClave(®) (ZC), Steam Gun (SG) and Horizontal Screw (HS)) under the same nominal conditions. Samples produced in the SG and HS achieved much higher cellulose digestibilities (88% and 95%, respectively), compared to the ZC sample (68%). Characterization, by chemical, physical, spectroscopic and electron microscopy methods, was used to gain an understanding of the effects causing the digestibility differences. Chemical differences were small; however, particle size differences appeared significant. Sum-frequency generation vibrational spectra indicated larger inter-fibrillar spacing or randomization of cellulose microfibrils in the HS sample. Simons' staining indicated increased cellulose accessibility for the SG and HS samples. Electron microscopy showed that the SG and HS samples were more porous and fibrillated because of mechanical grinding and explosive depressurization occurring with these two reactors. These structural changes most likely permitted increased cellulose accessibility to enzymes, enhancing saccharification. CONCLUSIONS: Dilute-acid pretreatment of corn stover using three different reactors under the same nominal conditions gave samples with very different digestibilities, although chemical differences in the pretreated substrates were small. The results of the physical and chemical analyses of the samples indicate that the explosive depressurization and mechanical grinding with these reactors increased enzyme accessibility. Pretreatment reactors using physical force to disrupt cell walls increase the effectiveness of the pretreatment process.