Cellulose Synthase

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles an...

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles an...

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles an...

Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a nontargeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity.

Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a nontargeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity.

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles an...

SummaryGenetic improvement of cellulose production in commercially important trees is one of the formidable goals of current forest biotechnology research. To achieve this goal, we must first decipher the enigmatic and complex process of cellulose biosynthesis in trees. The recent availability of rich genomic resources in poplars make Populus the first tree genus for which genetic augmentation of cellulose may soon become possible. Fortunately, because of the structural conservation of key cellulose biosynthesis genes between Arabidopsis and poplar genomes, the lessons learned from exploring the functions of Arabidopsis genes may be applied directly to poplars. However, regulation of these genes will most likely be distinct in these two‐model systems because of their inherent biological differences. This research review covers the current state of knowledge about the three major cellulose biosynthesis‐related gene families from poplar genomes: cellulose synthases, sucrose synthases ...

Diploid wheat, Triticum monococcum L. (einkorn) is an ideal plant material for wheat functional genomics. Brittle culm mutant was identified by screening of the ethyl methane sulphonate-treated M 2 progenies of a T. monococcum accession pau14087 by banding the plant parts manually. The brittle culm mutant with drooping leaves, early flowering, reduced tiller numbers and susceptible to lodging had also exhibited brittleness in all plant parts than the wild-type parents. Comprehensive mechanical strength, histological, biochemical, scanning electron microscopy, and Fourier transform infrared spectroscopy analyses of brittle culm mutant supplemented and complemented the findings that the mutant had defective cellulose biosynthesis pathway and deposition of cell wall materials on secondary cell wall of mechanical tissues. Microscopic studies demonstrated that the decrease in cellulose contents resulted in the irregular cell wall organization in xylem vessels of leaf vascular bundles. To map the brc5 mutant, mapping populations were developed by crossing the brittle culm mutant with wild Triticum boeoticum acc. pau5088, having non-brittle characters. The brittle culm mutation was mapped between SSR markers, Xcfd39 and Xgwm126 on 5A m L chromosome of T. monococcum, with genetic distances of 2.6 and 4.8 cM, respectively. The brc5 mutant mapped on 5A m L, being distinct from a previously mapped brittle culm mutant in wheat, has been designated as brc5. The work on fine mapping and map-based cloning of brc5 gene regulating synthesis and deposition of cellulose on the secondary cell wall is in progress. Keywords brc5 mutant Á Cellulose Á Gene mapping Á Irregular xylem vessels Á SSR marker Á Triticum monococcum (L.) Abbreviations BSA Bulk segregant analysis brc5 Brittle culm mutant 5 EMS Ethyl methane sulfonate IIT Indian Institute of Technology M 2 Second generation after mutagenesis F 2 Second filial generation PCR Polymerase chain reaction RILs Recombinant inbred lines SEM Scanning electron microscopy FTIR Fourier transform infrared spectroscopy VBs Vascular bundles Communicated by P. Sowinski.

Diploid wheat, Triticum monococcum L. (einkorn) is an ideal plant material for wheat functional genomics. Brittle culm mutant was identified by screening of the ethyl methane sulphonate-treated M 2 progenies of a T. monococcum accession pau14087 by banding the plant parts manually. The brittle culm mutant with drooping leaves, early flowering, reduced tiller numbers and susceptible to lodging had also exhibited brittleness in all plant parts than the wild-type parents. Comprehensive mechanical strength, histological, biochemical, scanning electron microscopy, and Fourier transform infrared spectroscopy analyses of brittle culm mutant supplemented and complemented the findings that the mutant had defective cellulose biosynthesis pathway and deposition of cell wall materials on secondary cell wall of mechanical tissues. Microscopic studies demonstrated that the decrease in cellulose contents resulted in the irregular cell wall organization in xylem vessels of leaf vascular bundles. To map the brc5 mutant, mapping populations were developed by crossing the brittle culm mutant with wild Triticum boeoticum acc. pau5088, having non-brittle characters. The brittle culm mutation was mapped between SSR markers, Xcfd39 and Xgwm126 on 5A m L chromosome of T. monococcum, with genetic distances of 2.6 and 4.8 cM, respectively. The brc5 mutant mapped on 5A m L, being distinct from a previously mapped brittle culm mutant in wheat, has been designated as brc5. The work on fine mapping and map-based cloning of brc5 gene regulating synthesis and deposition of cellulose on the secondary cell wall is in progress. Keywords brc5 mutant Á Cellulose Á Gene mapping Á Irregular xylem vessels Á SSR marker Á Triticum monococcum (L.) Abbreviations BSA Bulk segregant analysis brc5 Brittle culm mutant 5 EMS Ethyl methane sulfonate IIT Indian Institute of Technology M 2 Second generation after mutagenesis F 2 Second filial generation PCR Polymerase chain reaction RILs Recombinant inbred lines SEM Scanning electron microscopy FTIR Fourier transform infrared spectroscopy VBs Vascular bundles Communicated by P. Sowinski.

Diploid wheat, Triticum monococcum L. (einkorn) is an ideal plant material for wheat functional genomics. Brittle culm mutant was identified by screening of the ethyl methane sulphonate-treated M 2 progenies of a T. monococcum accession pau14087 by banding the plant parts manually. The brittle culm mutant with drooping leaves, early flowering, reduced tiller numbers and susceptible to lodging had also exhibited brittleness in all plant parts than the wild-type parents. Comprehensive mechanical strength, histological, biochemical, scanning electron microscopy, and Fourier transform infrared spectroscopy analyses of brittle culm mutant supplemented and complemented the findings that the mutant had defective cellulose biosynthesis pathway and deposition of cell wall materials on secondary cell wall of mechanical tissues. Microscopic studies demonstrated that the decrease in cellulose contents resulted in the irregular cell wall organization in xylem vessels of leaf vascular bundles. To map the brc5 mutant, mapping populations were developed by crossing the brittle culm mutant with wild Triticum boeoticum acc. pau5088, having non-brittle characters. The brittle culm mutation was mapped between SSR markers, Xcfd39 and Xgwm126 on 5A m L chromosome of T. monococcum, with genetic distances of 2.6 and 4.8 cM, respectively. The brc5 mutant mapped on 5A m L, being distinct from a previously mapped brittle culm mutant in wheat, has been designated as brc5. The work on fine mapping and map-based cloning of brc5 gene regulating synthesis and deposition of cellulose on the secondary cell wall is in progress. Keywords brc5 mutant Á Cellulose Á Gene mapping Á Irregular xylem vessels Á SSR marker Á Triticum monococcum (L.) Abbreviations BSA Bulk segregant analysis brc5 Brittle culm mutant 5 EMS Ethyl methane sulfonate IIT Indian Institute of Technology M 2 Second generation after mutagenesis F 2 Second filial generation PCR Polymerase chain reaction RILs Recombinant inbred lines SEM Scanning electron microscopy FTIR Fourier transform infrared spectroscopy VBs Vascular bundles Communicated by P. Sowinski.

The cause of low cellulose content in brittle mutants of barley was studied. No differences were found in the dimension of crystalline cellulose microfibrils among mutant and normal strains by X-ray diffraction analysis. By contrast, the number of cellulose synthesizing terminal complexes (TCs) in a selected brittle mutant, Kobinkatagi 4, decreased to one fifth of that in the normal strain. These findings suggest that the low cellulose content in brittle mutants of barley is caused by the decrease in the number of TCs on its plasma membrane.

The physicochemical nature of the cell wall was determined in the fourth internode of three isogenic brittle mutants of barley (Hordeum vulgare L.) and corresponding nonbrittle strains. Cellulose contents of the brittle culms were 17.5 to 20.3% of those of corresponding nonbrittle strains. No major difference was found in lignin and noncellulose components (except glucose) between brittle and nonbnttle strains. Maximum bending stresses of brittle culms were 38.0 to 54.2% of those of corresponding nonbrittle strains. The degree of polymerization of cellulose, measured by viscometry, was similar between the brittle and the nonbrittle strains. Mole number of cellulose molecules in a unit length of brittle culms, calculated by dividing cellulose mass by molecular weight, was 7.7 to 17.3% of those of the nonbrittle strains. These results indicate that brittleness of mutant culms is due to fewer numbers of cellulose molecules in the cell walls. ' Supported by a Grant-in-Aid for Science Research (No. 02640525) from the Ministry of Education, Science, and Culture of Japan.

Understanding how cellulose synthesis occurs is key to understanding the formation of the plant cell wall. This understanding could also be key to modifying cellulose production to permit more efficient extraction of glucose from cellulose for the production of biobased materials. Cellulose biosynthesis is carried out by cellulose synthases; transmembrane multimeric processive glycosyltransferases responsible for polymerizing UDP-glucose into glucan chains. Thirty-six glucan chains bind together in parallel to form elementary cellulose microfibrils. Due to the essential nature of cellulose synthases for plant survival and the recalcitrant nature of the cell wall to chemical and enzymatic digestion, the cellulose synthases can be very difficult to analyze by traditional approaches. In an attempt to circumvent some of the issues of studying cellulose synthases, the cellulose synthase genes CESA1 and CESA3, along with the cell wall associated genes COBRA, DET3 and POM1 were recombined into an engineered Autographa californica nucleopolyhedron virus and expressed in Spodoptera fruigiperda ovarian cells. Although recombinant protein could be detected for CESA1 and CESA3, C 14-glucose incorporation on baculovirus infected cell lines have given inconclusive results to the cellulose synthase activity of the CESA1 and CESA3 proteins. With further optimization of the protein expression of CESA1 and optimization of the variability in the C 14-glucose incorporation assays, the baculovirus system may prove a useful tool for studying the cellulose synthases. I would also like to thank the members of my committee, Dr. Ayush Kumar and Dr. Janice Strap for their expertise, support and advice to guide me in the right direction. I would like to thank my colleagues from the Applied Bioscience Program at the University Of Ontario Institute Of Technology for their help and perspective. Finally, I would like to thank my Fiancée Jessica and my family for their constant support during my studies.

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles an...

Plants have evolved to live in environments where they are often exposed to different stress factors. Being sessile, they have developed specific mechanisms that allow them to detect precisely environmental changes and respond to complex conditions, minimizing damage while conserving valuable resources for growth and reproduction. The cell wall polysaccharide β-D-glucan observed in some species of Poales can determine responses to various environmental factors in specific plant developmental stages. It is located in the outer epidermal layer, at the place of stress attack and therefore its metabolism could relate to response of plant to environmental factors within moderate, physiological range. Putative protective role of β-D-glucan during heat stress was indicated through naked oats with higher content of β-D-glucan. It appeared that oats with higher β-D-glucan content are better adapted to stress conditions. The presented article discusses the β-D-glucan as a possible protective ...

Quantitative expression analysis of three cellulose synthases/subunits encoding genes (cesA1, cesA1b, and cesA2) during three fibre development stages [3, 15 and 35 days post anthesis (dpa)] in Gossypium arboreum, G. anomalum, and G. hirsutum was undertaken. Exceptionally high expression was observed in G. hirsutum at 15 dpa stage for cesA1 and cesA2 (6.5 and 7.7 magnitude increase over β tubulin) genes, which might be responsible for longer fibre in G. hirsutum. Moderately high expression of cesA1 gene was recorded in G. arboreum (at 15 dpa), and in G. anomalum (at 3 and 15 dpa). However cesA1 gene expression was negligible in G. arboreum, and G. hirsutum at 3 dpa and in all three species at 35 dpa. Strong increase in expression levels of cesA1b gene (6.4 magnitude increase over β tubulin) was observed in G. arboreum at 15 dpa stage. Expression levels of cesA1b gene were moderate in G. hirsutum (at 3 and 15 dpa) and in G. anomalum (at 15 dpa); while its expression was low to neglig...

Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a nontargeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity.

© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-β-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-β-glucan content. (1,3;1,4)-β-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-β-glucan content to wild-type (WT). Howeve...

Polycyclic aromatic compounds (PAHs) are toxic compounds that are released in the environment as a consequence of industrial activities. The restoration of PAH-polluted sites considers the use of bacteria capable of degrading aromatic compounds to carbon dioxide and water. Here we characterize a new Xanthobacteraceae strain, Starkeya sp. strain N1B, previously isolated during enrichment under microaerophilic conditions, which is capable of using naphthalene crystals as the sole carbon source. The strain produced a structured biofilm when grown on naphthalene crystals, which had the shape of a half-sphere organized over the crystal. Scanning electron microscopy (SEM) and GC-MS analysis indicated that the biofilm was essentially made of cellulose, composed of several micron-long nanofibrils of 60 nm diameter. A cellulosic biofilm was also formed when the cells grew with glucose as the carbon source. Fourier transformed infrared spectroscopy (FTIR) confirmed that the polymer was type I cellulose in both cases, although the crystallinity of the material greatly depended on the carbon source used for growth. Using genome mining and mutant analysis, we identified the genetic complements required for the transformation of naphthalene into cellulose, which seemed to have been successively acquired through horizontal gene transfer. The capacity to develop the biofilm around the crystal was found to be dispensable for growth when naphthalene was used as the carbon source, suggesting that the function of this structure is more intricate than initially thought. This is the first example of the use of toxic aromatic hydrocarbons as the carbon source for bacterial cellulose production. Application of this capacity would allow the remediation of a PAH into such a value-added polymer with multiple biotechnological usages.

The desmid Micrasterias denticulata Bréb. is useful for the study of streptophyte cell wall biology and morphology. However, no tools to analyze cell biological processes in vivo in this species are available. In the present study, transient gene expression under the control of the chl a ⁄ b-binding protein gene of the Closterium peracerosum-strigosum-littorale complex (CpCAB1) promotor was achieved for M. denticulata and illustrated by the intracellular localization of an endogenous cellulose synthase (MdCesA1). A transformation efficiency of 1 ⁄ 5,000 cells was achieved following microparticle bombardment. The free green fluorescent protein (GFP) signal was detected both in the nucleus and in the cytoplasm. The MdCesA1-GFP fusion protein, on the other hand, occurred at the plasma membrane in particles concentrated at the lobe indentations, the lobe tips, and, to a lesser extent, along the lobe sides. Hence, the multipolar growth mechanism of the cell is reflected. In addition, the margins of cytoplasmic compartments, most likely dictyosomes, were labeled, in accordance with the known secretory pathway of cellulose synthase complexes. Besides intracellular localization studies, the utility of the system for overexpression phenotyping is discussed.

Background Streptophyte green algae share several characteristics of cell growth and cell wall formation with their relatives, the embryophytic land plants. The multilobed cell wall of Micrasterias denticulata that rebuilds symmetrically after cell division and consists of pectin and cellulose, makes this unicellular streptophyte alga an interesting model system to study the molecular controls on cell shape and cell wall formation in green plants. Results Genome-wide transcript expression profiling of synchronously growing cells identified 107 genes of which the expression correlated with the growth phase. Four transcripts showed high similarity to expansins that had not been examined previously in green algae. Phylogenetic analysis suggests that these genes are most closely related to the plant EXPANSIN A family, although their domain organization is very divergent. A GFP-tagged version of the expansin-resembling protein MdEXP2 localized to the cell wall and in Golgi-derived vesicl...

Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM image...

Background: Setaria viridis has emerged as a model species for the larger C 4 grasses. Here the cellulose synthase (CesA) superfamily has been defined, with an emphasis on the amounts and distribution of (1,3;1,4)-β-glucan, a cell wall polysaccharide that is characteristic of the grasses and is of considerable value for human health. Methods: Orthologous relationship of the CesA and Poales-specific cellulose synthase-like (Csl) genes among Setaria italica (Si), Sorghum bicolor (Sb), Oryza sativa (Os), Brachypodium distachyon (Bradi) and Hordeum vulgare (Hv) were compared using bioinformatics analysis. Transcription profiling of Csl gene families, which are involved in (1,3;1,4)-β-glucan synthesis, was performed using real-time quantitative PCR (Q-PCR). The amount of (1,3;1,4)-β-glucan was measured using a modified Megazyme assay. The fine structures of the (1,3;1,4)-β-glucan, as denoted by the ratio of cellotriosyl to cellotetraosyl residues (DP3:DP4 ratio) was assessed by chromatography (HPLC and HPAEC-PAD). The distribution and deposition of the MLG was examined using the specific antibody BG-1 and captured using fluorescence and transmission electron microscopy (TEM). Results: The cellulose synthase gene superfamily contains 13 CesA and 35 Csl genes in Setaria. Transcript profiling of CslF, CslH and CslJ gene families across a vegetative tissue series indicated that SvCslF6 transcripts were the most abundant relative to all other Csl transcripts. The amounts of (1,3;1,4)-β-glucan in Setaria vegetative tissues ranged from 0.2% to 2.9% w/w with much smaller amounts in developing grain (0.003% to 0.013% w/w). In general, the amount of (1,3;1,4)-β-glucan was greater in younger than in older tissues. The DP3:DP4 ratios varied between tissue types and across developmental stages, and ranged from 2.4 to 3.0:1. The DP3:DP4 ratios in developing grain ranged from 2.5 to 2.8:1. Micrographs revealing the distribution of (1,3;1,4)-β-glucan in walls of different cell types and the data were consistent with the quantitative (1,3;1,4)-β-glucan assays. Conclusion: The characteristics of the cellulose synthase gene superfamily and the accumulation and distribution of (1,3;1,4)-β-glucans in Setaria are similar to those in other C 4 grasses, including sorghum. This suggests that Setaria is a suitable model plant for cell wall polysaccharide biology in C 4 grasses.

Sensors deployed in natural environments, such as rivers, beaches and glaciers, experience large forces and damaging environmental conditions. Sensors need to be robust, securely operate for extended time periods and be readily relocated and serviced. The sensors must be housed in materials that mimic natural conditions of size, density, shape and roughness. We have developed an encasement system for sensors required to measure large forces experienced by mobile river sediment grains. Sensors are housed within two discrete cases that are rigidly conjoined. The inner case exactly fits the sensor, radio components and power source. This case can be mounted within outer cases of any larger size and can be precisely moulded to match the shapes of natural sediment. Total grain mass can be controlled by packing the outer case with dense material. Case design uses Solid-Works TM software, and shape-matching involved 3D laser scanning of natural pebbles. The cases were printed using a HP Designjet TM 3D printer that generates high precision parts that lock rigidly in place. The casings are watertight and robust. Laboratory testing produces accurate results over a wider range of accelerations than previously reported.

Oriented cellulose deposition is critical to plant patterning and models suggest microtubules constrain cellulose synthase movements through the plasma membrane. Though widespread in plants, urochordates are the only animals that synthesize cellulose. We characterized the distinctive cellulose microfibril scaffold of the larvacean house and its interaction with house structural proteins (oikosins). Targeted disruption of cytoskeletal elements, secretory pathways, and plasma membrane organization, suggested a working model for templating extracellular cellulose microfibrils from animal cells that shows both convergence and differences to plant models. Specialized cortical F-actin arrays template microfibril orientation and glycosylphosphatidylinositol-anchored proteins in lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules deliver and maintain cellulose synthase complexes to specific cell membrane sites rather than orienting their movement through the membrane. Oikosins are incorporated into house compartments directly above their corresponding cellular field of expression and interact with the cellulose scaffold to a variable extent. Keywords F-actin Á Microtubules Á GPI-anchored proteins Á Cellulose synthase Á Larvacean Electronic supplementary material The online version of this article (

The Committee Firenze 2016, on the occasion of the 50th anniversary of the tragic 1966 flood, invited six engineers and scientists to form an International Technical Scientific Committee (ITSC) to assess the current status of flood protection for the city of Florence and identify steps to reduce the risk of flooding facing the city. In this final Report, ITSC concludes that Florence remains at risk to significant flooding and this risk grows each day. It is not a question of whether a flood of the magnitude of 1966 or greater will occur, but when. In fact, the level of protection that exists in Florence now is not on a level appropriate to the citizens and treasures that rest within the city. If, under current conditions, a 1966-like flood occurred, the consequences to human lives, treasures, properties and community infrastructure could be much more catastrophic than they were in 1966.

Quantitative expression analysis of three cellulose synthases/subunits encoding genes (cesA1, cesA1b, and cesA2) during three fibre development stages [3, 15 and 35 days post anthesis (dpa)] in Gossypium arboreum, G. anomalum, and G. hirsutum was undertaken. Exceptionally high expression was observed in G. hirsutum at 15 dpa stage for cesA1 and cesA2 (6.5 and 7.7 magnitude increase over β tubulin) genes, which might be responsible for longer fibre in G. hirsutum. Moderately high expression of cesA1 gene was recorded in G. arboreum (at 15 dpa), and in G. anomalum (at 3 and 15 dpa). However cesA1 gene expression was negligible in G. arboreum, and G. hirsutum at 3 dpa and in all three species at 35 dpa. Strong increase in expression levels of cesA1b gene (6.4 magnitude increase over β tubulin) was observed in G. arboreum at 15 dpa stage. Expression levels of cesA1b gene were moderate in G. hirsutum (at 3 and 15 dpa) and in G. anomalum (at 15 dpa); while its expression was low to neglig...

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles an...

Background Streptophyte green algae share several characteristics of cell growth and cell wall formation with their relatives, the embryophytic land plants. The multilobed cell wall of Micrasterias denticulata that rebuilds symmetrically after cell division and consists of pectin and cellulose, makes this unicellular streptophyte alga an interesting model system to study the molecular controls on cell shape and cell wall formation in green plants. Results Genome-wide transcript expression profiling of synchronously growing cells identified 107 genes of which the expression correlated with the growth phase. Four transcripts showed high similarity to expansins that had not been examined previously in green algae. Phylogenetic analysis suggests that these genes are most closely related to the plant EXPANSIN A family, although their domain organization is very divergent. A GFP-tagged version of the expansin-resembling protein MdEXP2 localized to the cell wall and in Golgi-derived vesicl...

A key aspect of plant growth is the synthesis and deposition of cell walls. In specific tissues and cell types including xylem and fibre, a thick secondary wall comprised of cellulose, hemicellulose and lignin is deposited. Secondary cell walls provide a physical barrier that protects plants from pathogens, promotes tolerance to abiotic stresses and fortifies cells to withstand the forces associated with water transport and the physical weight of plant structures. Grasses have numerous cell wall features that are distinct from eudicots and other plants. Study of the model species Brachypodium distachyon as well as other grasses has revealed numerous features of the grass cell wall. These include the characterisation of xylosyl and arabinosyltransferases, a mixedlinkage glucan synthase and hydroxycinnamate acyltransferases. Perhaps the most fertile area for discovery has been the formation of lignins, including the identification of novel substrates and enzyme activities towards the synthesis of monolignols. Other enzymes function as polymerising agents or transferases that modify lignins and facilitate interactions with polysaccharides. The regulatory aspects of cell wall biosynthesis are largely overlapping with those of eudicots, but salient differences among species have been resolved that begin to identify the determinants that define grass cell walls.

Mixed-linkage (1 fi 3),(1 fi 4)-b-D-glucan (MLG) is widely considered to be a defining feature of the cell walls of plants in the Poales order. However, we conducted an extensive survey of cell-wall composition in diverse land plants and discovered that MLG is also abundant in the walls of the horsetail Equisetum arvense. MALDI-TOF MS and monosaccharide linkage analysis revealed that MLG in E. arvense is an unbranched homopolymer that consists of short blocks of contiguous 1,4-blinked glucose residues joined by 1,3-b linkages. However, in contrast to Poaceae species, MLG in E. arvense consists mostly of cellotetraose rather than cellotetriose, and lacks long 1,4-blinked glucan blocks. Monosaccharide linkage analyses and immunochemical profiling indicated that, in E. arvense, MLG is a component of cell walls that have a novel architecture that differs significantly from that of the generally recognized type I and II cell walls. Unlike in type II walls, MLG in E. arvense does not appear to be co-extensive with glucuroarabinoxylans but occurs in walls that are rich in pectin. Immunofluorescence and immunogold localization showed that MLG occurs in both young and old regions of E. arvense stems, and is present in most cell types apart from cells in the vascular tissues. These findings have important implications for our understanding of cell-wall evolution, and also demonstrate that plant cell walls can be constructed in a way not previously envisaged.

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.

Testing in real life conditions is not as easy as it first seems, because often it is costly and difficult to reproduce Internet behavior in controlled environment. WAN Emulator (WANE) proposed herein is a software tool that helps controlling the IP traffic parameters of a network, like delay, packet loss or packet duplication. The advantage of this software is that is using free tools implemented in Linux kernels but offers a friendly graphical interface, so that the researcher does not have to bother with time consuming operations like creating the tc tree, marking the specific traffic and filtering it.

We tested Wilhelm Hofmeister's hypothesis that the outer layers of herbaceous stem tissues are held in a preferential state of longitudinal tension by more internal stem tissues that are held in a reciprocal state of compression. We measured (1) the biaxial stiffness of dandelion peduncles that were barometrically inflated with a Scholander pressure bomb, and (2) the stiffness and mechanical behavior of different layers of tissues that were surgically manipulated as longitudinal strips placed in uniaxial tension. Hofmeister's hypothesis predicts that stems will shorten and expand in girth as their volume transiently increases (due to barometric or hydrostatic inflation), that they will longitudinally rupture when excessively inflated, and that the principal stiffening agents in their outer tissues will be aligned in the longitudinal direction with respect to stem length. Our experiments confirmed these predictions: (1) the longitudinal strains observed for inflated peduncles were negative and smaller than the circumferential strains such that stems contracted in length and expanded in girth, (2) peduncles longitudinally ruptured when excessively inflated, (3) surgical experiments indicated that the epidermis was stiffer in longitudinal tension than any other immature peduncle tissue and was as stiff as any other tissue region in mature stems, and (4) microscopic analyses showed that the net orientation of cellulose microfibrils in the cell walls of the outer region of stem tissues was parallel to stem length. A strong positive correlation existed between the tensile stiffness of tissues and the net orientation of cell wall microfibrils. A biomechanical model for the dandelion peduncle wall was constructed based on the thermal behavior of a cross-ply unidirectional composite material whose adjacent plies contain orthogonally oriented stiffening agents. This model accurately predicted the magnitudes of the tensile stiffness of different tissue regions, the proportional thickness of these regions, and the formation of a bulge in the epidermis and a crease on the inner surface of the stem wall when the innermost region of stem tissues was transversely cut. For each of its variants possessing an outer ply with longitudinally aligned stiffening agents (i.e., a 0Њ ply) and containing at least one internal ply with transversely aligned stiffening agents (i.e., a 90Њ ply), the model predicted a state of preferential longitudinal tension in the 0Њ ply (or plies) and of preferential longitudinal compression in the 90Њ ply as posited by Hofmeister's hypothesis.

Rod-shaped high-purity samples of CrO2 have been synthesized by an inexpensive and simplified procedure. Here, we have prepared pure CrO2 without applying any external pressure or control it during synthesis. The sample prepared exhibited an improvement in saturation magnetization values, 68 emu/g at 300 K, 136 emu/g at 80 K, and uniform grained microstructure. The complex permittivity, permeability, and microwave absorption properties of high-purity CrO2 sample were investigated in the 8.2–12.2 GHz (X-band) microwave frequency range. Microwave measurements have shown the high shielding effectiveness due to absorption (SEA) of 20.3 dB. The high value of SEA suggests that CrO2 can be used as a promising electromagnetic shielding, EMI, material in 8.2–12.2 GHz (X-band) microwave frequency range.

Accurate, reliable, and computationally inexpensive models of the dynamic state of combustion engines are a fundamental tool to investigate new engine designs, develop optimal control strategies, and monitor their performance. The use of those models would allow to improve the engine cost-efficiency trade-off, operational robustness, and environmental impact. To address this challenge, two state-of-the-art alternatives in literature exist. The first one is to develop high fidelity physical models (e.g., mean value engine, zero-dimensional, and one-dimensional models) exploiting the physical principles that regulate engine behaviour. The second one is to exploit historical data produced by the modern engine control and automation systems or by high-fidelity simulators to feed data-driven models (e.g., shallow and deep machine learning models) able to learn an accurate digital twin of the system without any prior knowledge. The main issues of the former approach are its complexity and the high (in some case prohibitive) computational require

Background The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses. Results We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature st...

Plants have evolved to live in environments where they are often exposed to different stress factors. Being sessile, they have developed specific mechanisms that allow them to detect precisely environmental changes and respond to complex conditions, minimizing damage while conserving valuable resources for growth and reproduction. The cell wall polysaccharide β-D-glucan observed in some species of

La evaluación del perfil lipídico debe realizarse de forma sistemática en varones adultos de más de 40 años, y en mujeres de más de 50 años, especialmente en presencia de otros factores de riesgo. Se considera en alto riesgo a todas las personas con clínica de aterosclerosis y con diabetes mellitus tipo 2, fumadores, hipertensos, obesidad abdominal e insuficiencia renal. Finalmente, sujetos con historia familiar de enfermedad cardiovascular prematura y/o dislipidemia son firmes candidatos al estudio del perfil lipídico. Una vez obtenido el perfil lipídico se valorará el riesgo cardiovascular (SCORE), y a partir del mismo se plantearán los objetivos terapéuticos. En prevención secundaria o en presencia de equivalentes de riesgo, los objetivos de tratamiento están plenamente establecidos.

Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the β-1,4 glucan backbone of XyG, we exploited a property of nasturtium ( Tropaeolum majus ) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like ( CSL ) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a β-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL ...

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.