Five amino acid mismatches in the zinc-finger domains of Cellulose Synthase 5 and Cellulose Synthase 6 cooperatively modulate their functional properties by controlling homodimerization in Arabidopsis.

Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain (ZN) of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. We found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5G43R + S49T+S54P+S80A+Y88F. Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5. Taken together, these five AA mismatches in the ZNs of CESA5 and CESA6 cooperatively modulate the functional properties of these CESAs by controlling their homodimerization capacity, which in turn imposes proportional changes on the incorporation of these CESAs into CSCs.

The N-terminal zinc finger of CELLULOSE SYNTHASE6 is critical in defining its functional properties by determining the level of homodimerization in Arabidopsis.

Primary cell wall cellulose is synthesized by the cellulose synthase complex (CSC) containing CELLULOSE SYNTHASE1 (CESA1), CESA3 and one of four CESA6-like proteins in Arabidopsis. It has been proposed that the CESA6-like proteins occupy the same position in the CSC, but their underlying selection mechanism remains unclear. We produced a chimeric CESA5 by replacing its N-terminal zinc finger with its CESA6 counterpart to investigate the consequences for its homodimerization, a crucial step in forming higher-order structures during assembly of the CSC. We found that the mutant phenotypes of prc1-1, a cesa6 null mutant, were rescued by the chimeric CESA5, and became comparable to the wild type (WT) and prc1-1 complemented by WT CESA6 in regard to plant growth, cellulose content, cellulose microfibril organization, CSC dynamics and subcellular localization. Bimolecular fluorescence complementation assays were employed to evaluate pairwise interactions between the N-terminal regions of CESA1, CESA3, CESA5, CESA6 and the chimeric CESA5. We verified that the chimeric CESA5 explicitly interacted with all the other CESA partners, comparable to CESA6, whereas interaction between CESA5 with itself was significantly weaker than that of all other CESA pairs. Our findings suggest that the homodimerization of CESA6 through its N-terminal zinc finger is critical in defining its functional properties, and possibly determines its intrinsic roles in facilitating higher-order structures in CSCs.

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.

Three AtCesA6-like members enhance biomass production by distinctively promoting cell growth in Arabidopsis.

Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plant's biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6-like genes (AtCesA2, AtCesA5, AtCesA6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (cellulose synthase complexes, CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild-type. The overexpression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, overexpression of the three AtCesA6-like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that overexpression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.

Cellobiohydrolase hydrolyzes crystalline cellulose on hydrophobic faces.

Biodegradation of plant biomass is a slow process in nature, and hydrolysis of cellulose is also widely considered to be a rate-limiting step in the proposed industrial process of converting lignocellulosic materials to biofuels. It is generally known that a team of enzymes including endo- and exocellulases as well as cellobiases are required to act synergistically to hydrolyze cellulose to glucose. The detailed molecular mechanisms of these enzymes have yet to be convincingly elucidated. In this report, atomic force microscopy (AFM) is used to image in real-time the structural changes in Valonia cellulose crystals acted upon by the exocellulase cellobiohydrolase I (CBH I) from Trichoderma reesei. Under AFM, single enzyme molecules could be observed binding only to one face of the cellulose crystal, apparently the hydrophobic face. The surface roughness of cellulose began increasing after adding CBH I, and the overall size of cellulose crystals decreased during an 11-h period. Interestingly, this size reduction apparently occurred only in the width of the crystal, whereas the height remained relatively constant. In addition, the measured cross-section shape of cellulose crystal changed from asymmetric to nearly symmetric. These observed changes brought about by CBH I action may constitute the first direct visualization supporting the idea that the exocellulase selectively hydrolyzes the hydrophobic faces of cellulose. The limited accessibility of the hydrophobic faces in native cellulose may contribute significantly to the rate-limiting slowness of cellulose hydrolysis.

Structure and function of the Clostridium thermocellum cellobiohydrolase A X1-module repeat: enhancement through stabilization of the CbhA complex.

The efficient deconstruction of lignocellulosic biomass remains a significant barrier to the commercialization of biofuels. Whereas most commercial plant cell-wall-degrading enzyme preparations used today are derived from fungi, the cellulosomal enzyme system from Clostridium thermocellum is an equally effective catalyst, yet of considerably different structure. A key difference between fungal enzyme systems and cellulosomal enzyme systems is that cellulosomal enzyme systems utilize self-assembled scaffolded multimodule enzymes to deconstruct biomass. Here, the possible function of the X1 modules in the complex multimodular enzyme system cellobiohydrolase A (CbhA) from C. thermocellum is explored. The crystal structures of the two X1 modules from C. thermocellum CbhA have been solved individually and together as one construct. The role that calcium may play in the stability of the X1 modules has also been investigated, as well as the possibility that they interact with each other. Furthermore, the results show that whereas the X1 modules do not seem to act as cellulose disruptors, they do aid in the thermostability of the CbhA complex, effectively allowing it to deconstruct cellulose at a higher temperature.

Differential Multiphoton Laser Scanning Microscopy.

Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot.

An NAC transcription factor orchestrates multiple features of cell wall development in Medicago truncatula.

To identify genes controlling secondary cell wall biosynthesis in the model legume Medicago truncatula, we screened a Tnt1 retrotransposon insertion mutant population for plants with altered patterns of lignin autofluorescence. From more than 9000 R1 plants screened, four independent lines were identified with a total lack of lignin in the interfascicular region. The mutants also showed loss of lignin in phloem fibers, reduced lignin in vascular elements, failure in anther dehiscence and absence of phenolic autofluorescence in stomatal guard cell walls. Microarray and PCR analyses confirmed that the mutations were caused by the insertion of Tnt1 in a gene annotated as encoding a NAM (no apical meristem)-like protein (here designated Medicago truncatula NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1, MtNST1). MtNST1 is the only family member in Medicago, but has three homologs (AtNST1-AtNST3) in Arabidopsis thaliana, which function in different combinations to control cell wall composition in stems and anthers. Loss of MtNST1 function resulted in reduced lignin content, associated with reduced expression of most lignin biosynthetic genes, and a smaller reduction in cell wall polysaccharide content, associated with reduced expression of putative cellulose and hemicellulose biosynthetic genes. Acid pre-treatment and cellulase digestion released significantly more sugars from cell walls of nst1 mutants compared with the wild type. We discuss the implications of these findings for the development of alfalfa (Medicago sativa) as a dedicated bioenergy crop.

Imaging cell wall architecture in single Zinnia elegans tracheary elements.

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production.

Structure of CBM4 from Clostridium thermocellum cellulase K.

Here, a 2.0 Å resolution X-ray structure of Clostridium thermocellum cellulase K family 4 carbohydrate-binding module (CelK CBM4) is reported. The resulting structure was refined to an R factor of 0.212 and an R(free) of 0.274. Structural analysis shows that this new structure is very similar to the previously solved structure of C. thermocellum CbhA CBM4. Most importantly, these data support the previously proposed notion of an extended binding pocket using a novel tryptophan-containing loop that may be highly conserved in clostridial CBM4 proteins.

Xylan Is Critical for Proper Bundling and Alignment of Cellulose Microfibrils in Plant Secondary Cell Walls.

Plant biomass represents an abundant and increasingly important natural resource and it mainly consists of a number of cell types that have undergone extensive secondary cell wall (SCW) formation. These cell types are abundant in the stems of Arabidopsis, a well-studied model system for hardwood, the wood of eudicot plants. The main constituents of hardwood include cellulose, lignin, and xylan, the latter in the form of glucuronoxylan (GX). The binding of GX to cellulose in the eudicot SCW represents one of the best-understood molecular interactions within plant cell walls. The evenly spaced acetylation and 4-O-methyl glucuronic acid (MeGlcA) substitutions of the xylan polymer backbone facilitates binding in a linear two-fold screw conformation to the hydrophilic side of cellulose and signifies a high level of molecular specificity. However, the wider implications of GX-cellulose interactions for cellulose network formation and SCW architecture have remained less explored. In this study, we seek to expand our knowledge on this by characterizing the cellulose microfibril organization in three well-characterized GX mutants. The selected mutants display a range of GX deficiency from mild to severe, with findings indicating even the weakest mutant having significant perturbations of the cellulose network, as visualized by both scanning electron microscopy (SEM) and atomic force microscopy (AFM). We show by image analysis that microfibril width is increased by as much as three times in the severe mutants compared to the wild type and that the degree of directional dispersion of the fibrils is approximately doubled in all the three mutants. Further, we find that these changes correlate with both altered nanomechanical properties of the SCW, as observed by AFM, and with increases in enzymatic hydrolysis. Results from this study indicate the critical role that normal GX composition has on cellulose bundle formation and cellulose organization as a whole within the SCWs.

Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production.

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.

Structure of a fibronectin type III-like module from Clostridium thermocellum.

The 1.6 A resolution structure of a fibronectin type III-like module from Clostridium thermocellum (PDB code 3mpc) with two molecules in the asymmetric unit is reported. The crystals used for data collection belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=35.43, b=45.73, c=107.72 A, and the structure was refined to an R factor of 0.166. Structural comparisons found over 800 similar structures in the Protein Data Bank. The broad range of different proteins or protein domains with high structural similarity makes it especially demanding to classify these proteins. Previous studies of fibronectin type III-like modules have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules or as proteins that help large enzyme complexes remain soluble.

Direct Measurement of Plant Cellulose Microfibril and Bundles in Native Cell Walls.

Plants use rigid cellulose together with non-cellulosic matrix polymers to build cell walls. Cellulose microfibrils comprise linear ß(1,4)-glucan chains packed through inter- and intra-chain hydrogen-bonding networks and van der Waals forces. Due to its small size, the number of glucan chains and their arrangement in a microfibril remains elusive. Here we used atomic force microscopy (AFM) to directly image primary cell walls (PCWs) and secondary cell walls (SCWs) from fresh tissues of maize (Zea mays) under near-native conditions. By analyzing cellulose structure in different types of cell walls, we were able to measure the individual microfibrils in elongated PCWs at the sub-nanometer scale. The dimension of the microfibril was measured at 3.68 ± 0.13 nm in width and 2.25 ± 0.10 nm in height. By superimposing multiple AFM height profiles of these microfibrils, the overlay area representing the cross-section was estimated at 5.6 ± 0.4 nm2, which fitted well to an 18-chain model packed as six sheets with 234432 conformation. Interestingly we found in PCW, all these individual microfibrils could be traced back to a bundle in larger imaging area, suggesting cellulose are synthesized as large bundles in PCWs, and then split during cell expansion or elongation. In SCWs where cell growth has ceased we observed nearly-parallel twined or individual microfibrils that appeared to be embedded separately in the matrix polymers without the splitting effect, indicating different mechanisms of cellulose biosynthesis in PCW and SCW. The sub-nanometer structure of the microfibril presented here was measured exclusively from elongated PCWs, further study is required to verify if it represents the inherent structure synthesized by the cellulose synthase complex in PCWs and SCWs.

Engineered carbohydrate-binding module (CBM) protein-suspended single-walled carbon nanotubes in water.

Engineered protein, CtCBM4, the first carbohydrate-binding module (CBM) protein is successfully used to debundle and suspend single-walled carbon nanotubes (SWNTs) effectively in aqueous solution, which opens up a new avenue in further functionalizing and potential selectively fractionating SWNTs for diverse biology- and/or energy-related applications.

A biophysical perspective on the cellulosome: new opportunities for biomass conversion.

The cellulosome is a multiprotein complex, produced primarily by anaerobic microorganisms, which functions to degrade lignocellulosic materials. An important topic of current debate is whether cellulosomal systems display greater ability to deconstruct complex biomass materials (e.g. plant cell walls) than nonaggregated enzymes, and in so doing would be appropriate for improved, commercial bioconversion processes. To sufficiently understand the complex macromolecular processes between plant cell wall polymers, cellulolytic microbes, and their secreted enzymes, a highly concerted research approach is required. Adaptation of existing biophysical techniques and development of new science tools must be applied to this system. This review focuses on strategies likely to permit improved understanding of the bacterial cellulosome using biophysical approaches, with emphasis on advanced imaging and computational techniques.

High-resolution mosaic imaging with multifocal, multiphoton photon-counting microscopy.

High-resolution mosaic imaging is performed for the first time to our knowledge with a multifocal, multiphoton, photon-counting imaging system. We present a novel design consisting of a home-built femtosecond Yb-doped KGdWO(4) laser with an optical multiplexer, which is coupled with a commercial Olympus IX-71 microscope frame. Photon counting is performed using single-element detectors and an inexpensive electronic demultiplexer and counters.

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately -7°C) and cold-acclimated (CA; leaf LT50 approximately -50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Heterologous expression of glycosyl hydrolases in planta: a new departure for biofuels.

The concept of expressing non-plant glycosyl hydrolase genes in plant tissue is nearly two decades old, yet relatively little work in this field has been reported. However, resurgent interest in technologies aimed at enabling processes that convert biomass to sugars and fuels has turned attention toward this intuitive solution. There are several challenges facing researchers in this field, including the development of better and more specifically targeted delivery systems for hydrolytic genes, the successful folding and post-translational modification of heterologous proteins and the development of cost-effective process strategies utilizing these transformed plants. The integration of these concepts, from the improvement of biomass production and conversion characteristics to the heterologous production of glycosyl hydrolases in a high yielding bioenergy crop, holds considerable promise for improving the lignocellulosic conversion of biomass to ethanol and subsequently to fuels.

Real-time imaging reveals that lytic polysaccharide monooxygenase promotes cellulase activity by increasing cellulose accessibility.

BACKGROUND: The high cost of enzymes is one of the key technical barriers that must be overcome to realize the economical production of biofuels and biomaterials from biomass. Supplementation of enzyme cocktails with lytic polysaccharide monooxygenase (LPMO) can increase the efficiency of these cellulase mixtures for biomass conversion. The previous studies have revealed that LPMOs cleave polysaccharide chains by oxidization of the C1 and/or C4 carbons of the monomeric units. However, how LPMOs enhance enzymatic degradation of lignocellulose is still poorly understood. RESULTS: In this study, we combined enzymatic assays and real-time imaging using atomic force microscopy (AFM) to study the molecular interactions of an LPMO [TrAA9A, formerly known as TrCel61A) from Trichoderma reesei] and a cellobiohydrolase I (TlCel7A from T. longibrachiatum) with bacterial microcrystalline cellulose (BMCC) as a substrate. Cellulose conversion by TlCel7A alone was enhanced from 46 to 54% by the addition of TrAA9A. Conversion by a mixture of TlCel7A, endoglucanase, and ß-glucosidase was increased from 79 to 87% using pretreated BMCC with TrAA9A for 72 h. AFM imaging demonstrated that individual TrAA9A molecules exhibited intermittent random movement along, across, and penetrating into the ribbon-like microfibril structure of BMCC, which was concomitant with the release of a small amount of oxidized sugars and the splitting of large cellulose ribbons into fibrils with smaller diameters. The dividing effect of the cellulose microfibril occurred more rapidly when TrAA9A and TlCel7A were added together compared to TrAA9A alone; TlCel7A alone caused no separation. CONCLUSIONS: TrAA9A increases the accessible surface area of BMCC by separating large cellulose ribbons, and thereby enhances cellulose hydrolysis yield. By providing the first direct observation of LPMO action on a cellulosic substrate, this study sheds new light on the mechanisms by which LPMO enhances biomass conversion.