Darby Harris

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1A903V and CESA3T942I in Arabidopsis thaliana. Using 13C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1A903V and CESA3T942I displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1A903V and CESA3T942I have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

Synthesis, regulation and utilization of lignocellulosic biomass

Increasing the range of fuels and bioproducts that are derived from lignocellulosic biomass and the efficiency at which they are produced hinges on a detailed understanding of the cell wall biosynthetic process. Herein, we review the structure and biosynthesis of lignocellulosic biomass and also highlight recent breakthroughs that demonstrate a complex regulatory system of transcription factors, small interfering RNAs and phosphorylation that ultimately dictate the development of the polyalaminate cell wall. Finally, we provide an update on cases where plant biotechnology has been used to improve lignocellulosic biomass utilization as a second-generation biofuel source.

CELLULOSE SYNTHASE9 serves a nonredundant role in secondary cell wall synthesis in Arabidopsis epidermal testa cells.

Herein, we sought to explore the contribution of cellulose biosynthesis to the shape and morphogenesis of hexagonal seed coat cells in Arabidopsis (Arabidopsis thaliana). Consistent with seed preferential expression of CELLULOSE SYNTHASE9 (CESA9), null mutations in CESA9 caused no change in cellulose content in leaves or stems, but caused a 25% reduction in seeds. Compositional studies of cesa9 seeds uncovered substantial proportional increases in cell wall neutral sugars and in several monomers of cell wall-associated polyesters. Despite these metabolic compensations, cesa9 seeds were permeable to tetrazolium salt, implying that cellulose biosynthesis, via CESA9, is required for correct barrier function of the seed coat. A syndrome of depleted radial wall, altered seed coat cell size, shape, and internal angle uniformity was quantified using scanning electron micrographs in cesa9 epidermal cells. By contrast, morphological defects were absent in cesa9 embryos, visually inspected from torpedo to bent cotyledon, consistent with no reduction in postgermination radical or hypocotyl elongation. These data implied that CESA9 was seed coat specific or functionally redundant in other tissues. Assessment of sections from glutaraldehyde fixed wild-type and cesa9 mature seeds supported results of scanning electron micrographs and quantitatively showed depletion of secondary cell wall synthesis in the radial cell wall. Herein, we show a nonredundant role for CESA9 in secondary cell wall biosynthesis in radial cell walls of epidermal seed coats and document its importance for cell morphogenesis and barrier function of the seed coat.

Genetic modification in cellulose-synthase reduces crystallinity and improves biochemical conversion to fermentable sugar

The cellulose synthase (CESA) membrane complex synthesizes microfibrils of cellulose that surround all plant cells. Cellulose is made of sugar (β,1‐4 glucan) and accessing the sugar in cellulose for biofuels is of critical importance to stem the use of fossil fuels and avoid competition with food crops and pristine lands associated with starch‐based biofuel production. The recalcitrance of cellulose to enzymatic conversion to a fermentable form of sugar is related to the degree of hydrogen bonding or crystallization of the glucan chain. Herein, we isolate the first viable low biomass‐crystallinity mutant by screening for altered cell wall structure using X‐ray scattering as well as screening for enzymatic conversion efficiency on a range of cell wall mutants in the model plant Arabidopsis thaliana (L.) Heynh. Through detailed analysis of the kinetics of bioconversion we identified a mutant that met both selection criteria. This mutant is ixr1‐2, which contains a mutation in a highly conserved consensus sequence among the C‐terminal transmembrane regions within CESA3. A 34% lower biomass crystallization index and 151% improvement in the efficiency of conversion from raw biomass to fermentable sugars was measured relative to that of wild type (Col‐0). Recognizing the inherent ambiguities with an insoluble complex substrate like cellulose and how little is still understood regarding the regulation of CESA we propose a general model for how to manipulate CESA enzymes to improve the recalcitrance of cellulose to enzymatic hydrolysis. This study also raises intriguing possibilities as to the functional importance of transmembrane anchoring in CESA complex and microfibril formation.

Tools for cellulose analysis in plant cell walls.

Cellulose is intimately associated with multiple facets of human civilization: central to clothing, shelter, heat, medicine, and food, there are few moments in the average human life that are not spent in direct contact with cellulose or a by-product of its composition. It should come as no surprise

Relative Crystallinity of Plant Biomass: Studies on Assembly, Adaptation and Acclimation

Plant biomechanical design is central to cell shape, morphogenesis, reproductive performance and protection against environmental and mechanical stress. The cell wall forms the central load bearing support structure for plant design, yet a mechanistic understanding of its synthesis is incomplete. A key tool for studying the structure of cellulose polymorphs has been x-ray diffraction and fourier transform infrared spectroscopy (FTIR). Relative crystallinity index (RCI) is based on the x-ray diffraction characteristics of two signature peaks and we used this technique to probe plant assembly, adaptation and acclimation. Confocal microscopy was used to visualize the dynamics of cellulose synthase in transgenic Arabidopsis plants expressing a homozygous YFP::CESA6. Assembly: RCI values for stems and roots were indistinguishable but leaves had 23.4 and 21.6% lower RCI than stems and roots respectively. Adaptation: over 3-fold variability in RCI was apparent in leaves from 35 plant species spanning Ordovician to Cretaceous periods. Within this study, RCI correlated positively with leaf geometric constraints and with mass per unit area, suggestive of allometry. Acclimation: biomass crystallinity was found to decrease under conditions of thigmomorphogenesis in Arabidopsis. Further, in etiolated pea hypocotyls, RCI values also decreased compared to plants that were grown in light, consistent with alterations in FTIR cellulose fingerprint peaks and live cell imaging experiments revealing rapid orientation of the YFP::cellulose synthase-6 array in response to light. Herein, results and technical challenges associated with the structure of the cell wall that gives rise to sample crystallinity are presented and examined with respect to adaptation, acclimation and assembly in ecosystem-level processes.

Cellulose synthesis in two secondary cell wall processes in a single cell type.

Plant cells have a rigid cell wall that constrains internal turgor pressure yet extends in a regulated and organized manner to allow the cell to acquire shape. The primary load-bearing macromolecule of a plant cell wall is cellulose, which forms crystalline microfibrils that are organized with respect to a cells function and shape requirements. A primary cell wall is deposited during expansion whereas secondary cell wall is synthesized post expansion during differentiation. A complex form of asymmetrical cellular differentiation occurs in Arabidopsis seed coat epidermal cells, where we have recently shown that two secondary cell wall processes occur that utilize different cellulose synthase (CESA) proteins. One process is to produce pectinaceous mucilage that expands upon hydration and the other is a radial wall thickening that reinforced the epidermal cell structure. Our data illustrate polarized specialization of CESA5 in facilitating mucilage attachment to the parent seed and CESA2, CESA5 and CESA9 in radial cell wall thickening and formation of the columella. Herein, we present a model for the complexity of cellulose biosynthesis in this highly differentiated cell type with further evidence supporting each cellulosic secondary cell wall process.

The Synthesis, Regulation and Modification of Lignocellulosic Biomass as a Resource for Biofuels and Bioproducts

Most of the plant biomass is cell wall and therefore represents a renewable carbon source that could be exploited by humans for bioenergy and bioproducts. A thorough understanding of the type of cell wall being harvested and the molecules available will be crucial in developing the most efficient conversion processes. Herein, we review the structure, function, and biosynthesis of lignocellulosic biomass, paying particular attention to the most important bioresources present in the plant cell wall: cellulose, hemicellulose, and lignin. We also provide an update on key improvements being made to lignocellulosic biomass with respect to utilization as a second-generation biofuel and as a resource for bioproducts.