Essential amino acids in the Plant-Conserved and Class-Specific Regions of cellulose synthases.

The Plant-Conserved Region (P-CR) and the Class-Specific Region (CSR) are two plant-unique sequences in the catalytic core of cellulose synthases (CESAs) for which specific functions have not been established. Here, we used site-directed mutagenesis to replace amino acids and motifs within these sequences predicted to be essential for assembly and function of CESAs. We developed an in vivo method to determine the ability of mutated CesA1 transgenes to complement an Arabidopsis (Arabidopsis thaliana) temperature-sensitive root-swelling1 (rsw1) mutant. Replacement of a Cys residue in the CSR, which blocks dimerization in vitro, rendered the AtCesA1 transgene unable to complement the rsw1 mutation. Examination of the CSR sequences from 33 diverse angiosperm species showed domains of high-sequence conservation in a class-specific manner but with variation in the degrees of disorder, indicating a nonredundant role of the CSR structures in different CESA isoform classes. The Cys residue essential for dimerization was not always located in domains of intrinsic disorder. Expression of AtCesA1 transgene constructs, in which Pro417 and Arg453 were substituted for Ala or Lys in the coiled-coil of the P-CR, were also unable to complement the rsw1 mutation. Despite an expected role for Arg457 in trimerization of CESA proteins, AtCesA1 transgenes with Arg457Ala mutations were able to fully restore the wild-type phenotype in rsw1. Our data support that Cys662 within the CSR and Pro417 and Arg453 within the P-CR of Arabidopsis CESA1 are essential residues for functional synthase complex formation, but our data do not support a specific role for Arg457 in trimerization in native CESA complexes.

Redesigning plant cell walls for the biomass-based bioeconomy.

Lignocellulosic biomass-the lignin, cellulose, and hemicellulose that comprise major components of the plant cell well-is a sustainable resource that could be utilized in the United States to displace oil consumption from heavy vehicles, planes, and marine-going vessels and commodity chemicals. Biomass-derived sugars can also be supplied for microbial fermentative processing to fuels and chemicals or chemically deoxygenated to hydrocarbons. However, the economic value of biomass might be amplified by diversifying the range of target products that are synthesized in living plants. Genetic engineering of lignocellulosic biomass has previously focused on changing lignin content or composition to overcome recalcitrance, the intrinsic resistance of cell walls to deconstruction. New capabilities to remove lignin catalytically without denaturing the carbohydrate moiety have enabled the concept of the "lignin-first" biorefinery that includes high-value aromatic products. The structural complexity of plant cell-wall components also provides substrates for polymeric and functionalized target products, such as thermosets, thermoplastics, composites, cellulose nanocrystals, and nanofibers. With recent advances in the design of synthetic pathways, lignocellulosic biomass can be regarded as a substrate at various length scales for liquid hydrocarbon fuels, chemicals, and materials. In this review, we describe the architectures of plant cell walls and recent progress in overcoming recalcitrance and illustrate the potential for natural or engineered biomass to be used in the emerging bioeconomy.

Rhamnogalacturonan-I is a determinant of cell-cell adhesion in poplar wood.

The molecular basis of cell-cell adhesion in woody tissues is not known. Xylem cells in wood particles of hybrid poplar (Populus tremula × P. alba cv. INRA 717-1B4) were separated by oxidation of lignin with acidic sodium chlorite when combined with extraction of xylan and rhamnogalacturonan-I (RG-I) using either dilute alkali or a combination of xylanase and RG-lyase. Acidic chlorite followed by dilute alkali treatment enables cell-cell separation by removing material from the compound middle lamellae between the primary walls. Although lignin is known to contribute to adhesion between wood cells, we found that removing lignin is a necessary but not sufficient condition to effect complete cell-cell separation in poplar lines with various ratios of syringyl:guaiacyl lignin. Transgenic poplar lines expressing an Arabidopsis thaliana gene encoding an RG-lyase (AtRGIL6) showed enhanced cell-cell separation, increased accessibility of cellulose and xylan to hydrolytic enzyme activities, and increased fragmentation of intact wood particles into small cell clusters and single cells under mechanical stress. Our results indicate a novel function for RG-I, and also for xylan, as determinants of cell-cell adhesion in poplar wood cell walls. Genetic control of RG-I content provides a new strategy to increase catalyst accessibility and saccharification yields from woody biomass for biofuels and industrial chemicals.

Expression profiles of cell-wall related genes vary broadly between two common maize inbreds during stem development.

BACKGROUND: The cellular machinery for cell wall synthesis and metabolism is encoded by members of large multi-gene families. Maize is both a genetic model for grass species and a potential source of lignocellulosic biomass from crop residues. Genetic improvement of maize for its utility as a bioenergy feedstock depends on identification of the specific gene family members expressed during secondary wall development in stems. RESULTS: High-throughput sequencing of transcripts expressed in developing rind tissues of stem internodes provided a comprehensive inventory of cell wall-related genes in maize (Zea mays, cultivar B73). Of 1239 of these genes, 854 were expressed among the internodes at ≥95 reads per 20 M, and 693 of them at ≥500 reads per 20 M. Grasses have cell wall compositions distinct from non-commelinid species; only one-quarter of maize cell wall-related genes expressed in stems were putatively orthologous with those of the eudicot Arabidopsis. Using a slope-metric algorithm, five distinct patterns for sub-sets of co-expressed genes were defined across a time course of stem development. For the subset of genes associated with secondary wall formation, fifteen sequence motifs were found in promoter regions. The same members of gene families were often expressed in two maize inbreds, B73 and Mo17, but levels of gene expression between them varied, with 30% of all genes exhibiting at least a 5-fold difference at any stage. Although presence-absence and copy-number variation might account for much of these differences, fold-changes of expression of a CADa and a FLA11 gene were attributed to polymorphisms in promoter response elements. CONCLUSIONS: Large genetic variation in maize as a species precludes the extrapolation of cell wall-related gene expression networks even from one common inbred line to another. Elucidation of genotype-specific expression patterns and their regulatory controls will be needed for association panels of inbreds and landraces to fully exploit genetic variation in maize and other bioenergy grass species.

Biomass recalcitrance: a multi-scale, multi-factor, and conversion-specific property.

Recalcitrance of plant biomass to enzymatic hydrolysis for biofuel production is thought to be a property conferred by lignin or lignin-carbohydrate complexes. However, chemical catalytic and thermochemical conversion pathways, either alone or in combination with biochemical and fermentative pathways, now provide avenues to utilize lignin and to expand the product range beyond ethanol or butanol. To capture all of the carbon in renewable biomass, both lignin-derived aromatics and polysaccharide-derived sugars need to be transformed by catalysts to liquid hydrocarbons and high-value co-products. We offer a new definition of recalcitrance as those features of biomass which disproportionately increase energy requirements in conversion processes, increase the cost and complexity of operations in the biorefinery, and/or reduce the recovery of biomass carbon into desired products. The application of novel processing technologies applied to biomass reveal new determinants of recalcitrance that comprise a broad range of molecular, nanoscale, and macroscale factors. Sampling natural genetic diversity within a species, transgenic approaches, and synthetic biology approaches are all strategies that can be used to select biomass for reduced recalcitrance in various pretreatments and conversion pathways.

The maize mixed-linkage (1->3),(1->4)-beta-D-glucan polysaccharide is synthesized at the golgi membrane.

With the exception of cellulose and callose, the cell wall polysaccharides are synthesized in Golgi membranes, packaged into vesicles, and exported to the plasma membrane where they are integrated into the microfibrillar structure. Consistent with this paradigm, several published reports have shown that the maize (Zea mays) mixed-linkage (1-->3),(1-->4)-beta-D-glucan, a polysaccharide that among angiosperms is unique to the grasses and related Poales species, is synthesized in vitro with isolated maize coleoptile Golgi membranes and the nucleotide-sugar substrate, UDP-glucose. However, a recent study reported the inability to detect the beta-glucan immunocytochemically at the Golgi, resulting in a hypothesis that the mixed-linkage beta-glucan oligomers may be initiated at the Golgi but are polymerized at the plasma membrane surface. Here, we demonstrate that (1-->3),(1-->4)-beta-D-glucans are detected immunocytochemically at the Golgi of the developing maize coleoptiles. Further, when maize seedlings at the third-leaf stage were pulse labeled with [(14)C]O(2) and Golgi membranes were isolated from elongating cells at the base of the developing leaves, (1-->3),(1-->4)-beta-D-glucans of an average molecular mass of 250 kD and higher were detected in isolated Golgi membranes. When the pulse was followed by a chase period, the labeled polysaccharides of the Golgi membrane diminished with subsequent transfer to the cell wall. (1-->3),(1-->4)-beta-D-Glucans of at least 250 kD were isolated from cell walls, but much larger aggregates were also detected, indicating a potential for intermolecular interactions with glucuronoarabinoxylans or intermolecular grafting in muro.

The thanatos mutation in Arabidopsis thaliana cellulose synthase 3 (AtCesA3) has a dominant-negative effect on cellulose synthesis and plant growth.

Genetic functional analyses of mutants in plant genes encoding cellulose synthases (CesAs) have suggested that cellulose deposition requires the activity of multiple CesA proteins. Here, a genetic screen has led to the identification of thanatos (than), a semi-dominant mutant of Arabidopsis thaliana with impaired growth of seedlings. Homozygous seedlings of than germinate and grow but do not survive. In contrast to other CesA mutants, heterozygous plants are dwarfed and display a radially swollen root phenotype. Cellulose content is reduced by approximately one-fifth in heterozygous and by two-fifths in homozygous plants, showing gene-dosage dependence. Map-based cloning revealed an amino acid substitution (P578S) in the catalytic domain of the AtCesA3 gene, indicating a critical role for this residue in the structure and function of the cellulose synthase complex. Ab initio analysis of the AtCesA3 subdomain flanking the conserved proline residue predicted that the amino acid substitution to serine alters protein secondary structure in the catalytic domain. Gene dosage-dependent expression of the AtCesA3 mutant gene in wild-type A. thaliana plants resulted in a than dominant-negative phenotype. We propose that the incorporation of a mis-folded CesA3 subunit into the cellulose synthase complex may stall or prevent the formation of functional rosette complexes.

Structure of cellulose-deficient secondary cell walls from the irx3 mutant of Arabidopsis thaliana.

In the Arabidopsis mutant irx3, truncation of the AtCesA7 gene encoding a xylem-specific cellulose synthase results in reduced cellulose synthesis in the affected xylem cells and collapse of mature xylem vessels. Here we describe spectroscopic experiments to determine whether any cellulose, normal or abnormal, remained in the walls of these cells and whether there were consequent effects on other cell-wall polysaccharides. Xylem cell walls from irx3 and its wild-type were prepared by anatomically specific isolation and were examined by solid-state NMR spectroscopy and FTIR microscopy. The affected cell walls of irx3 contained low levels of crystalline cellulose, probably associated with primary cell walls. There was no evidence that crystalline cellulose was replaced by less ordered glucans. From the molecular mobility of xylans and lignin it was deduced that these non-cellulosic polymers were cross-linked together in both irx3 and the wild-type. The disorder previously observed in the spatial pattern of non-cellulosic polymer deposition in the secondary walls of irx3 xylem could not be explained by any alteration in the structure or cross-linking of these polymers and may be attributed directly to the absence of cellulose microfibrils which, in the wild-type, scaffold the organisation of the other polymers into a coherent secondary cell wall.

Modulation of the cellulose content of tuber cell walls by antisense expression of different potato (Solanum tuberosum L.) CesA clones.

Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis.

Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity.

The maize (Zea mays) brittle stalk2 (bk2) is a recessive mutant, the aerial parts of which are easily broken. The bk2 phenotype is developmentally regulated and appears 4 weeks after planting, at about the fifth-leaf stage. Before this time, mutants are indistinguishable from wild-type siblings. Afterward, all organs of the bk2 mutants turn brittle, even the preexisting ones, and they remain brittle throughout the life of the plant. Leaf tension assays and bend tests of the internodes show that the brittle phenotype does not result from loss of tensile strength but from loss in flexibility that causes the tissues to snap instead of bend. The Bk2 gene was cloned by a combination of transposon tagging and a candidate gene approach and found to encode a COBRA-like protein similar to rice (Oryza sativa) BC1 and Arabidopsis (Arabidopsis thaliana) COBRA-LIKE4. The outer periphery of the stalk has fewer vascular bundles, and the sclerids underlying the epidermis possess thinner secondary walls. Relative cellulose content is not strictly correlated with the brittle phenotype. Cellulose content in mature zones of bk2 mature stems is lowered by 40% but is about the same as wild type in developing stems. Although relative cellulose content is lowered in leaves after the onset of the brittle phenotype, total wall mass as a proportion of dry mass is either unchanged or slightly increased, indicating a compensatory increase in noncellulosic carbohydrate mass. Fourier transform infrared spectra indicated an increase in phenolic ester content in the walls of bk2 leaves and stems. Total content of lignin is unaffected in bk2 juvenile leaves before or after appearance of the brittle phenotype, but bk2 mature and developing stems are markedly enriched in lignin compared to wild-type stems. Despite increased lignin in bk2 stems, loss of staining with phloroglucinol and ultraviolet autofluorescence is observed in vascular bundles and sclerid layers. Consistent with the infrared analyses, levels of saponifiable hydroxycinnamates are elevated in bk2 leaves and stems. As Bk2 is highly expressed during early development, well before the onset of the brittle phenotype, we propose that Bk2 functions in a patterning of lignin-cellulosic interactions that maintain organ flexibility rather than having a direct role in cellulose biosynthesis.

Cell-wall structure and anisotropy in procuste, a cellulose synthase mutant of Arabidopsis thaliana.

In dark-grown hypocotyls of the Arabidopsis procuste mutant, a mutation in the CesA6 gene encoding a cellulose synthase reduces cellulose synthesis and severely inhibits elongation growth. Previous studies had left it uncertain why growth was inhibited, because cellulose synthesis was affected before, not during, the main phase of elongation. We characterised the quantity, structure and orientation of the cellulose remaining in the walls of affected cells. Solid-state NMR spectroscopy and infrared microscopy showed that the residual cellulose did not differ in structure from that of the wild type, but the cellulose content of the prc-1 cell walls was reduced by 28%. The total mass of cell-wall polymers per hypocotyl was reduced in prc-1 by about 20%. Therefore, the fourfold inhibition of elongation growth in prc-1 does not result from aberrant cellulose structure, nor from uniform reduction in the dimensions of the cell-wall network due to reduced cellulose or cell-wall mass. Cellulose orientation was quantified by two quantitative methods. First, the orientation of newly synthesised microfibrils was measured in field-emission scanning electron micrographs of the cytoplasmic face of the inner epidermal cell wall. The ordered transverse orientation of microfibrils at the inner face of the cell wall was severely disrupted in prc-1 hypocotyls, particularly in the early growth phase. Second, cellulose orientation distributions across the whole cell-wall thickness, measured by polarised infrared microscopy, were much broader. Analysis of the microfibril orientations according to the theory of composite materials showed that during the initial growth phase, their anisotropy at the plasma membrane was sufficient to explain the anisotropy of subsequent growth.