SUMMARY
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.
Significance Statement
We found that the N‐terminal zinc‐finger domain (ZN) plays a critical role in defining the functional properties of CESA6 by determining the level of homodimerization. Furthermore, using bimolecular fluorescence complementation assays we verified that CESA6 and CESA5 form homodimers at different rates. Based on our findings, we speculate that homodimerization through its ZN is critical in defining the functional properties of CESA6, and possibly determines its intrinsic roles in facilitating higher‐order structures in CSCs.
Higher plant cell walls are the major source of the cellulose used in a variety of industries. Cellulose in plant forms nanoscale fibrils that are embedded in non-cellulosic matrix polymers in the ...cell walls. The morphological features of plant cellulose fibrils such as the size, shape, and arrangement, are still poorly understood due to its inhomogeneous nature and the limited resolution of the characterization techniques used. Here, we sketch out a proposed model of plant cellulose fibril and its arrangement that is based primarily on review of direct visualizations of different types of cell walls in maize using atomic force microscopy at sub-nanometer scale, and is also inspired by recent advances in understanding of cellulose biosynthesis and biodegradation. We propose that the principal unit of plant cellulose fibril is a 36-chain cellulose elementary fibril (CEF), which is hexagonally shaped and 3.2 × 5.3 nm in cross-section. Macrofibrils are ribbon-like bundles containing variable numbers of CEFs associated through their hydrophilic faces. As the cell expands and/or elongates, large macrofibril may split to become smaller bundles or individual CEFs, which are simultaneously coated with hemicelluloses to form microfibrils of variable sizes during biosynthesis. The microfibrils that contain one CEF are arranged nearly parallel, and the hydrophobic faces of the CEF are perpendicular to the cell wall surface. Structural disordering of the CEF may occur during plant development while cells expand, elongate, dehydrate, and die, as well as during the processing to prepare cellulose materials.
Summary
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.
Greater understanding of the mechanisms contributing to chemical and enzymatic solubilization of plant cell walls is critical for enabling cost-effective industrial conversion of cellulosic biomass ...to biofuels. Here, we report the use of correlative imaging in real time to assess the impact of pretreatment as well as the resulting nanometer-scale changes in cell wall structure, upon subsequent digestion by two commercially relevant cellulase systems. We demonstrate that the small, noncomplexed fungal cellulases deconstruct cell walls using mechanisms that differ considerably from those of the larger, multienzyme complexes (cellulosomes). Furthermore, high-resolution measurement of the microfibrillar architecture of cell walls suggests that digestion is primarily facilitated by enabling enzyme access to the hydrophobic cellulose face. The data support the conclusion that ideal pretreatments should maximize lignin removal and minimize polysaccharide modification, thereby retaining the essentially native microfibrillar structure.
Lignocellulosic biomass has long been recognized as a potential sustainable source of mixed sugars for fermentation to biofuels and other biomaterials. Several technologies have been developed during ...the past 80 years that allow this conversion process to occur, and the clear objective now is to make this process cost-competitive in today's markets. Here, we consider the natural resistance of plant cell walls to microbial and enzymatic deconstruction, collectively known as "biomass recalcitrance." It is this property of plants that is largely responsible for the high cost of lignocellulose conversion. To achieve sustainable energy production, it will be necessary to overcome the chemical and structural properties that have evolved in biomass to prevent its disassembly.
Plants use rigid cellulose together with non-cellulosic matrix polymers to build cell walls. Cellulose microfibrils comprise linear β(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 (
) 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 nm
, 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.
Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that ...converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components is not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance, to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan and lignin) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.
Understanding the molecular architecture of the plant cell wall is critical to reducing the biomass recalcitrance problem, which currently impedes economic bioconversion processing. The parenchyma ...cell walls from field senesced, maize stem pith have been directly visualized without extraction processes using high-resolution atomic force microscopy (AFM). By imaging the cell wall inner surfaces from different cells and different faces of the same cell, we were able to map the native primary cell wall ultrastructures. Depending on the thickness of non-cellulosic deposition, the parallel-microfibrils appear in various morphologies ranging from clearly defined to completely embedded in the wall matrixes forming cell wall lamella. Macrofibrils were found to exist only on the uppermost layer of the native primary cell wall and appeared to be bundles of elementary fibrils. This novel observation led us to a new hypothesis for the cell wall fibrillar network and biosynthesis processes. Put concisely, a number of elementary fibrils are synthesized at one locus, that of the cellulose synthase complex (CelS), and coalesce into much larger macrofibrils. These macrofibrils eventually split at the ends to form parallel microfibrils with deposition of other cell wall components (i.e. hemicelluloses, pectin, etc.) also evident. On the basis of these AFM surface measurements and current supportive evidence from cell wall biophysics, biosynthesis, and genomics, we propose a new molecular model consisting of a 36-glucan-chain elementary fibril, in which the 36-glucan chains form both crystalline and subcrystalline structures. We also propose a modified model of CelS based on recently reported experimental evidence from plant cell wall biosynthesis. Keywords: Microfibril; elementary fibril; maize, cellulose; plant cell wall; atomic force microscopy
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