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1.  Global Identification of Multiple OsGH9 Family Members and Their Involvement in Cellulose Crystallinity Modification in Rice 
PLoS ONE  2013;8(1):e50171.
Plant glycoside hydrolase family 9 (GH9) comprises typical endo-β-1,4-glucanase (EGases, EC3.2.1.4). Although GH9A (KORRIGAN) family genes have been reported to be involved in cellulose biosynthesis in plants, much remains unknown about other GH9 subclasses. In this study, we observed a global gene co-expression profiling and conducted a correlation analysis between OsGH9 and OsCESA among 66 tissues covering most periods of life cycles in 2 rice varieties. Our results showed that OsGH9A3 and B5 possessed an extremely high co-expression with OsCESA1, 3, and 8 typical for cellulose biosynthesis in rice. Using two distinct rice non-GH9 mutants and wild type, we performed integrative analysis of gene expression level by qRT-PCR, cellulase activities in situ and in vitro, and lignocellulose crystallinity index (CrI) in four internodes of stem tissues. For the first time, OsGH9B1, 3, and 16 were characterized with the potential role in lignocellulose crystallinity alteration in rice, whereas OsGH9A3 and B5 were suggested for cellulose biosynthesis. In addition, phylogenetic analysis and gene co-expression comparison revealed GH9 function similarity in Arabidopsis and rice. Hence, the data can provide insights into GH9 function in plants and offer the potential strategy for genetic manipulation of plant cell wall using the five aforementioned novel OsGH9 genes.
doi:10.1371/journal.pone.0050171
PMCID: PMC3537678  PMID: 23308094
2.  Perturbation of Brachypodium distachyon CELLULOSE SYNTHASE A4 or 7 results in abnormal cell walls 
BMC Plant Biology  2013;13:131.
Background
Cellulose is an integral component of the plant cell wall and accounts for approximately forty percent of total plant biomass but understanding its mechanism of synthesis remains elusive. CELLULOSE SYNTHASE A (CESA) proteins function as catalytic subunits of a rosette-shaped complex that synthesizes cellulose at the plasma membrane. Arabidopsis thaliana and rice (Oryza sativa) secondary wall CESA loss-of-function mutants have weak stems and irregular or thin cell walls.
Results
Here, we identify candidates for secondary wall CESAs in Brachypodium distachyon as having similar amino acid sequence and expression to those characterized in A. thaliana, namely CESA4/7/8. To functionally characterize BdCESA4 and BdCESA7, we generated loss-of-function mutants using artificial microRNA constructs, specifically targeting each gene driven by a maize (Zea mays) ubiquitin promoter. Presence of the transgenes reduced BdCESA4 and BdCESA7 transcript abundance, as well as stem area, cell wall thickness of xylem and fibers, and the amount of crystalline cellulose in the cell wall.
Conclusion
These results suggest BdCESA4 and BdCESA7 play a key role in B. distachyon secondary cell wall biosynthesis.
doi:10.1186/1471-2229-13-131
PMCID: PMC3847494  PMID: 24024469
Artificial microRNA; Brachypodium distachyon; Crystallinity; Secondary cell wall; Sum-frequency-generation vibration spectroscopy; Xylem
3.  The cellulose synthase superfamily in fully sequenced plants and algae 
BMC Plant Biology  2009;9:99.
Background
The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl) families and one cellulose synthase (CesA) family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses.
Results
A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ), providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome.
Conclusion
Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.
doi:10.1186/1471-2229-9-99
PMCID: PMC3091534  PMID: 19646250
4.  Functional analysis of complexes with mixed primary and secondary cellulose synthases 
Plant Signaling & Behavior  2013;8(3):e23179.
In higher plants, cellulose is synthesized by cellulose synthase complexes, which contain multiple isoforms of cellulose synthases (CESAs). Among the total 10 CESA genes in Arabidopsis, recessive mutations at three of them cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis (irx1cesa8, irx3cesa7 and irx5cesa4). These CESA genes are considered secondary cell wall CESAs. The others (the function CESA10 is still unknown) are thought to be specialized for cellulose synthesis in the primary cell wall. A split-ubiquitin membrane yeast two-hybrid system was used to assess interactions among four primary CESAs (CESA1, CESA2, CESA3, CESA6) and three secondary CESAs (CESA4, CESA7, CESA8). Our results showed that primary CESAs could physically interact with secondary CESAs in a limited fashion. Analysis of transgenic lines showed that CESA1 could partially rescue irx1cesa8 null mutants, resulting in complementation of the plant growth defect, collapsed xylem and cellulose content deficiency. These results suggest that mixed primary and secondary CESA complexes are functional using experimental set-ups.
doi:10.4161/psb.23179
PMCID: PMC3676487  PMID: 23299322
cellulose; cellulose synthase complex (CSC); primary cell wall; secondary cell wall; promoter swap
5.  The CELLULOSE SYNTHASE-LIKE A and CELLULOSE SYNTHASE-LIKE C families: recent advances and future perspectives 
The CELLULOSE SYNTHASE (CESA) superfamily of proteins contains several sub-families of closely related CELLULOSE SYNTHASE-LIKE (CSL) sequences. Among these, the CSLA and CSLC families are closely related to each other and are the most evolutionarily divergent from the CESA family. Significant progress has been made with the functional characterization of CSLA and CSLC genes, which have been shown to encode enzymes with 1,4-β-glycan synthase activities involved in the biosynthesis of mannan and possibly xyloglucan backbones, respectively. This review examines recent work on the CSLA and CSLC families from evolutionary, molecular, and biochemical perspectives. We pose a series of questions, whose answers likely will provide further insight about the specific functions of members of the CSLA and CSLC families and about plant polysaccharide biosynthesis is general.
doi:10.3389/fpls.2012.00109
PMCID: PMC3359485  PMID: 22654891
CELLULOSE SYNTHASE-LIKE; mannan; xyloglucan; CSLA; CSLC; plant cell wall
6.  Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis 
Journal of Experimental Botany  2011;62(13):4495-4506.
The phytohormones, brassinosteroids (BRs), play important roles in regulating cell elongation and cell size, and BR-related mutants in Arabidopsis display significant dwarf phenotypes. Cellulose is a biopolymer which has a major contribution to cell wall formation during cell expansion and elongation. However, whether BRs regulate cellulose synthesis, and if so, what the underlying mechanism of cell elongation induced by BRs is, is unknown. The content of cellulose and the expression levels of the cellulose synthase genes (CESAs) was measured in BR-related mutants and their wild-type counterpart. The chromatin immunoprecipitation (CHIP) experiments and genetic analysis were used to demonstrate that BRs regulate CESA genes. It was found here that the BR-deficient or BR-perceptional mutants contain less cellulose than the wild type. The expression of CESA genes, especially those related to primary cell wall synthesis, was reduced in det2-1 and bri1-301, and was only inducible by BRs in the BR-deficient mutant det2-1. CHIP experiments show that the BR-activated transcription factor BES1 can associate with upstream elements of most CESA genes particularly those related with the primary cell wall. Furthermore, over-expression of the BR receptor BRI1 in CESA1, 3, and 6 mutants can only partially rescue the dwarf phenotypes. Our findings provide potential insights into the mechanism that BRs regulate cellulose synthesis to accomplish the cell elongation process in plant development.
doi:10.1093/jxb/err164
PMCID: PMC3170551  PMID: 21617247
Arabidopsis; brassinosteroids; cell elongation; cellulose; cellulose synthase; transcription factor
7.  Large-Scale Co-Expression Approach to Dissect Secondary Cell Wall Formation Across Plant Species 
Plant cell walls are complex composites largely consisting of carbohydrate-based polymers, and are generally divided into primary and secondary walls based on content and characteristics. Cellulose microfibrils constitute a major component of both primary and secondary cell walls and are synthesized at the plasma membrane by cellulose synthase (CESA) complexes. Several studies in Arabidopsis have demonstrated the power of co-expression analyses to identify new genes associated with secondary wall cellulose biosynthesis. However, across-species comparative co-expression analyses remain largely unexplored. Here, we compared co-expressed gene vicinity networks of primary and secondary wall CESAsin Arabidopsis, barley, rice, poplar, soybean, Medicago, and wheat, and identified gene families that are consistently co-regulated with cellulose biosynthesis. In addition to the expected polysaccharide acting enzymes, we also found many gene families associated with cytoskeleton, signaling, transcriptional regulation, oxidation, and protein degradation. Based on these analyses, we selected and biochemically analyzed T-DNA insertion lines corresponding to approximately twenty genes from gene families that re-occur in the co-expressed gene vicinity networks of secondary wall CESAs across the seven species. We developed a statistical pipeline using principal component analysis and optimal clustering based on silhouette width to analyze sugar profiles. One of the mutants, corresponding to a pinoresinol reductase gene, displayed disturbed xylem morphology and held lower levels of lignin molecules. We propose that this type of large-scale co-expression approach, coupled with statistical analysis of the cell wall contents, will be useful to facilitate rapid knowledge transfer across plant species.
doi:10.3389/fpls.2011.00023
PMCID: PMC3355677  PMID: 22639584
secondary cell wall; comparative co-expression analysis; Arabidopsis; cellulose
8.  Understanding Plant Cellulose Synthases through a Comprehensive Investigation of the Cellulose Synthase Family Sequences 
The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA) family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized clades and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair CesA function, and compare these sites to those observed in the closest cellulose synthase-like (Csl) families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.
doi:10.3389/fpls.2011.00005
PMCID: PMC3355508  PMID: 22629257
cellulose; cellulose synthase; CesA; CslD; CslF; comparative phylogenetics; cellulose synthase superfamily; sequence-based analysis
9.  Cellulose Synthase (CesA) Genes in the Green Alga Mesotaenium caldariorum 
Eukaryotic Cell  2002;1(6):847-855.
Cellulose, a microfibrillar polysaccharide consisting of bundles of β-1,4-glucan chains, is a major component of plant and most algal cell walls and is also synthesized by some prokaryotes. Seed plants and bacteria differ in the structures of their membrane terminal complexes that make cellulose and, in turn, control the dimensions of the microfibrils produced. They also differ in the domain structures of their CesA gene products (the catalytic subunit of cellulose synthase), which have been localized to terminal complexes and appear to help maintain terminal complex structure. Terminal complex structures in algae range from rosettes (plant-like) to linear forms (bacterium-like). Thus, algal CesA genes may reveal domains that control terminal complex assembly and microfibril structure. The CesA genes from the alga Mesotaenium caldariorum, a member of the order Zygnematales, which have rosette terminal complexes, are remarkably similar to seed plant CesAs, with deduced amino acid sequence identities of up to 59%. In addition to the putative transmembrane helices and the D-D-D-QXXRW motif shared by all known CesA gene products, M. caldariorum and seed plant CesAs share a region conserved among plants, an N-terminal zinc-binding domain, and a variable or class-specific region. This indicates that the domains that characterize seed plant CesAs arose prior to the evolution of land plants and may play a role in maintaining the structures of rosette terminal complexes. The CesA genes identified in M. caldariorum are the first reported for any eukaryotic alga and will provide a basis for analyzing the CesA genes of algae with different types of terminal complexes.
doi:10.1128/EC.1.6.847-855.2002
PMCID: PMC138757  PMID: 12477785
10.  Cellular Localization and Biochemical Characterization of a Chimeric Fluorescent Protein Fusion of Arabidopsis Cellulose Synthase-Like A2 Inserted into Golgi Membrane 
The Scientific World Journal  2014;2014:792420.
Cellulose synthase-like (Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily of CslA is putatively involved in the biosynthesis of β-mannans. Here we report a study on the cellular localization and the enzyme activity of an Arabidopsis CslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out the in vitro synthesis of a 14C-mannan starting from GDP-d-[U-14C]mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen.
doi:10.1155/2014/792420
PMCID: PMC3914377  PMID: 24558328
11.  Rice SLENDER LEAF 1 gene encodes cellulose synthase-like D4 and is specifically expressed in M-phase cells to regulate cell proliferation 
Journal of Experimental Botany  2013;64(7):2049-2061.
Cellulose synthase-like (CSL) genes are predicted to catalyse the biosynthesis of non-cellulosic polysaccharides such as the β-d-glycan backbone of hemicelluloses and are classified into nine subfamilies (CSLA–CSLH and CSLJ). The CSLD subfamily is conserved in all land plants, and among the nine CSL subfamilies, it shows the highest sequence similarity to the cellulose synthase genes, suggesting that it plays fundamental roles in plant development. This study presents a detailed analysis of slender leaf 1 (sle1) mutants of rice that showed rolled and narrow leaf blades and a reduction in plant height. The narrow leaf blade of sle1 was caused by reduced cell proliferation beginning at the P3 primordial stage. In addition to the size reduction of organs, sle1 mutants exhibited serious developmental defects in pollen formation, anther dehiscence, stomata formation, and cell arrangement in various tissues. Map-based cloning revealed that SLE1 encodes the OsCSLD4 protein, which was identified previously from a narrow leaf and dwarf 1 mutant. In situ hybridization experiments showed that OsCSLD4 was expressed in a patchy pattern in developing organs. Double-target in situ hybridization and quantitative RT-PCR analyses revealed that SLE1 was expressed specifically during the M-phase of the cell cycle, and suggested that the cell-cycle regulation was altered in sle1 mutants. These results suggest that the OsCSLD4 protein plays a pivotal role in the M phase to regulate cell proliferation. Further study of OsCSLD4 is expected to yield new insight into the role of hemicelluloses in plant development.
doi:10.1093/jxb/ert060
PMCID: PMC3638827  PMID: 23519729
CSLD; cytokinesis; leaf blade; M-phase; rice; slender leaf 1.
12.  Four Novel Cellulose Synthase (CESA) Genes from Birch (Betula platyphylla Suk.) Involved in Primary and Secondary Cell Wall Biosynthesis 
Cellulose synthase (CESA), which is an essential catalyst for the generation of plant cell wall biomass, is mainly encoded by the CesA gene family that contains ten or more members. In this study; four full-length cDNAs encoding CESA were isolated from Betula platyphylla Suk., which is an important timber species, using RT-PCR combined with the RACE method and were named as BplCesA3, −4, −7 and −8. These deduced CESAs contained the same typical domains and regions as their Arabidopsis homologs. The cDNA lengths differed among these four genes, as did the locations of the various protein domains inferred from the deduced amino acid sequences, which shared amino acid sequence identities ranging from only 63.8% to 70.5%. Real-time RT-PCR showed that all four BplCesAs were expressed at different levels in diverse tissues. Results indicated that BplCESA8 might be involved in secondary cell wall biosynthesis and floral development. BplCESA3 appeared in a unique expression pattern and was possibly involved in primary cell wall biosynthesis and seed development; it might also be related to the homogalacturonan synthesis. BplCESA7 and BplCESA4 may be related to the formation of a cellulose synthase complex and participate mainly in secondary cell wall biosynthesis. The extremely low expression abundance of the four BplCESAs in mature pollen suggested very little involvement of them in mature pollen formation in Betula. The distinct expression pattern of the four BplCesAs suggested they might participate in developments of various tissues and that they are possibly controlled by distinct mechanisms in Betula.
doi:10.3390/ijms131012195
PMCID: PMC3497266  PMID: 23202892
birch; CESA; primary cell wall; secondary cell wall; transcript; wood; cellulose; gene expression
13.  Enhanced disease resistance to Botrytis cinerea in myb46 Arabidopsis plants is associated to an early downregulation of CesA genes 
Plant Signaling & Behavior  2011;6(6):911-913.
The cell wall is a protective barrier of paramount importance for the survival of plant cells. Monitoring the integrity of the cell wall allows plants to quickly activate defense pathways to minimize pathogen entry and reduce the spread of disease. Counterintuitively, however, pharmacological effects as well as genetic lesions that affect cellulose biosynthesis and content confer plants with enhanced resistance against necrotrophic fungi. These kind of pathogens target cellulose for degradation to facilitate penetration and to generate glucose units as a food source. Our results point towards the existence of a transcriptional reprogramming mechanism in genes encoding cellulose synthases (CesAs) that occurs very soon after Botrytis cinerea attack and that result in a temporary shut down of some CesA genes. Interestingly, the observed coordinated downregulation of CesA genes is more pronounced, and occurs earlier in myb46 mutant plants. In the resistant myb46 plants, pathogen infection induces transient downregulation of CesA genes that concur with a selective transcriptional reprogramming in a set of genes encoding structural cell wall proteins and extracellular remodeling enzymes. Together with previous indications, our results favor the hypothesis that CesAs are part of a surveillance system of the cell wall integrity that senses the presence of a pathogen and transduces that signal into a rapid transcriptional reprogramming of the affected cell.
doi:10.4161/psb.6.6.15354
PMCID: PMC3218503  PMID: 21617373
necrotrophs; cellulose; cell wall; peroxidase; B. cinerea
14.  The Barley Genome Sequence Assembly Reveals Three Additional Members of the CslF (1,3;1,4)-β-Glucan Synthase Gene Family 
PLoS ONE  2014;9(3):e90888.
An important component of barley cell walls, particularly in the endosperm, is (1,3;1,4)-β- glucan, a polymer that has proven health benefits in humans and that influences processability in the brewing industry. Genes of the cellulose synthase-like (Csl) F gene family have been shown to be involved in (1,3;1,4)-β-glucan synthesis but many aspects of the biosynthesis are still unclear. Examination of the sequence assembly of the barley genome has revealed the presence of an additional three HvCslF genes (HvCslF11, HvCslF12 and HvCslF13) which may be involved in (1,3;1,4)-β-glucan synthesis. Transcripts of HvCslF11 and HvCslF12 mRNA were found in roots and young leaves, respectively. Transient expression of these genes in Nicotiana benthamiana resulted in phenotypic changes in the infiltrated leaves, although no authentic (1,3;1,4)-β-glucan was detected. Comparisons of the CslF gene families in cereals revealed evidence of intergenic recombination, gene duplications and translocation events. This significant divergence within the gene family might be related to multiple functions of (1,3;1,4)-β-glucans in the Poaceae. Emerging genomic and global expression data for barley and other cereals is a powerful resource for characterising the evolution and dynamics of complete gene families. In the case of the CslF gene family, the results will contribute to a more thorough understanding of carbohydrate metabolism in grass cell walls.
doi:10.1371/journal.pone.0090888
PMCID: PMC3940952  PMID: 24595438
15.  A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces▿  
Journal of Bacteriology  2008;190(14):4971-4978.
Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing β-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslASc) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslASc replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslASc. cslASc was constitutively transcribed, and an enhanced green fluorescent protein-CslASc fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslASc interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslASc membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development.
doi:10.1128/JB.01849-07
PMCID: PMC2446991  PMID: 18487344
16.  Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls 
Journal of Experimental Botany  2011;62(6):2053-2062.
The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.
doi:10.1093/jxb/erq395
PMCID: PMC3060685  PMID: 21209026
Brittle culm; cellulose synthesis; CesA protein; dominant-negative form; hemicellulose; rice; secondary cell wall; transgenic plant
17.  Cellulose synthase interacting protein 
Plant Signaling & Behavior  2010;5(12):1571-1574.
Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities.
doi:10.4161/psb.5.12.13621
PMCID: PMC3115106  PMID: 21150290
cellulose; CESA; terminal complexes; primary cell walls; Armadillo repeat
18.  De novo assembly and characterization of transcriptome using Illumina paired-end sequencing and identification of CesA gene in ramie (Boehmeria nivea L. Gaud) 
BMC Genomics  2013;14:125.
Background
Ramie fiber, extracted from vegetative organ stem bast, is one of the most important natural fibers. Understanding the molecular mechanisms of the vegetative growth of the ramie and the formation and development of bast fiber is essential for improving the yield and quality of the ramie fiber. However, only 418 expressed tag sequences (ESTs) of ramie deposited in public databases are far from sufficient to understand the molecular mechanisms. Thus, high-throughput transcriptome sequencing is essential to generate enormous ramie transcript sequences for the purpose of gene discovery, especially genes such as the cellulose synthase (CesA) gene.
Results
Using Illumina paired-end sequencing, about 53 million sequencing reads were generated. De novo assembly yielded 43,990 unigenes with an average length of 824 bp. By sequence similarity searching for known proteins, a total of 34,192 (77.7%) genes were annotated for their function. Out of these annotated unigenes, 16,050 and 13,042 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 19,846 unigenes were mapped to 126 KEGG pathways, and 565 genes were assigned to http://starch and sucrose metabolic pathway which was related with cellulose biosynthesis. Additionally, 51 CesA genes involved in cellulose biosynthesis were identified. Analysis of tissue-specific expression pattern of the 51 CesA genes revealed that there were 36 genes with a relatively high expression levels in the stem bark, which suggests that they are most likely responsible for the biosynthesis of bast fiber.
Conclusion
To the best of our knowledge, this study is the first to characterize the ramie transcriptome and the substantial amount of transcripts obtained will accelerate the understanding of the ramie vegetative growth and development mechanism. Moreover, discovery of the 36 CesA genes with relatively high expression levels in the stem bark will present an opportunity to understand the ramie bast fiber formation and development mechanisms.
doi:10.1186/1471-2164-14-125
PMCID: PMC3610122  PMID: 23442184
19.  Higher plant cellulose synthases 
Genome Biology  2000;1(4):reviews3001.1-reviews3001.6.
The sole function of cellulose synthases, which are found in plants bacteria, fungi, and animals, is to produce the biopolymer cellulose. Although no crystal structure has yet been solved, a considerable amount is known about their structure, function and evolution.
Cellulose, an aggregate of unbranched polymers of β-1,4-linked glucose residues, is the major component of wood and thus paper, and is synthesized by plants, most algae, some bacteria and fungi, and even some animals. The genes that synthesize cellulose in higher plants differ greatly from the well-characterized genes found in Acetobacter and Agrobacterium sp. More correctly designated as 'cellulose synthase catalytic subunits', plant cellulose synthase (CesA) proteins are integral membrane proteins, approximately 1,000 amino acids in length. The sequences for more than 20 full-length CesA genes are available, and they show high similarity to one another across the entire length of the encoded protein, except for two small regions of variability. There are a number of highly conserved residues, including several motifs shown to be necessary for processive glycosyltransferase activity. No crystal structure is known for cellulose synthase proteins, and the exact enzymatic mechanism is unknown. There are a number of mutations in cellulose synthase genes in the model organism Arabidopsis thaliana. Some of these mutants show altered morphology due to the lack of a properly developed primary or secondary cell wall. Others show resistance to well-characterized cellulose biosynthesis inhibitors.
PMCID: PMC138876  PMID: 11178255
20.  Mannan synthase activity in the CSLD family 
Plant Signaling & Behavior  2011;6(10):1620-1623.
Cellulose Synthase Like (CSL) proteins are a group of plant glycosyltransferases that are predicted to synthesize β-1,4-linked polysaccharide backbones. CSLC, CSLF and CSLH families have been confirmed to synthesize xyloglucan and mixed linkage β-glucan, while CSLA family proteins have been shown to synthesize mannans. The polysaccharide products of the five remaining CSL families have not been determined. Five CSLD genes have been identified in Arabidopsis thaliana and a role in cell wall biosynthesis has been demonstrated by reverse genetics. We have extended past research by producing a series of double and triple Arabidopsis mutants and gathered evidence that CSLD2, CSLD3 and CSLD5 are involved in mannan synthesis and that their products are necessary for the transition between early developmental stages in Arabidopsis. Moreover, our data revealed a complex interaction between the three glycosyltransferases and brought new evidence regarding the formation of non-cellulosic polysaccharides through multimeric complexes.
doi:10.4161/psb.6.10.17989
PMCID: PMC3256401  PMID: 21904114
mannan; mannose; plant cell wall; glycosyltransferase; cellulose synthase like; CSL; biosynthesis; hemicellulose
21.  Cellulose synthase interactive protein 1 (CSI1) mediates the intimate relationship between cellulose microfibrils and cortical microtubules 
Plant Signaling & Behavior  2012;7(7):714-718.
Cellulose is synthesized at the plasma membrane by protein complexes known as cellulose synthase complexes (CSCs). The cellulose-microtubule alignment hypothesis states that there is a causal link between the orientation of cortical microtubules and orientation of nascent cellulose microfibrils. The mechanism behind the alignment hypothesis is largely unknown. CESA interactive protein 1 (CSI1) interacts with CSCs and potentially links CSCs to the cytoskeleton. CSI1 not only co-localizes with CSCs but also travels bi-directionally in a speed indistinguishable from CSCs. The linear trajectories of CSI1-RFP coincide with the underlying microtubules labeled by YFP-TUA5. In the absence of CSI1, both the distribution and the motility of CSCs are defective and the alignment of CSCs and microtubules is disrupted. These observations led to the hypothesis that CSI1 directly mediates the interaction between CSCs and microtubules. In support of this hypothesis, CSI1 binds to microtubules directly by an in vitro microtubule-binding assay. In addition to a role in serving as a messenger from microtubule to CSCs, CSI1 labels SmaCCs/MASCs, a compartment that has been proposed to be involved in CESA trafficking and/or delivery to the plasma membrane.
doi:10.4161/psb.20338
PMCID: PMC3583948  PMID: 22751327
cellulose microfibril; cellulose synthase complex; microtubule; plasma membrane
22.  Involvement of TBL/DUF231 proteins into cell wall biology 
Plant Signaling & Behavior  2010;5(8):1057-1059.
Through map-based cloning we determined TRICHOME BIREFRINGENCE (TBR) belongs to a plant-specific, yet anonymous gene family with 46 members in Arabidopsis thaliana. These genes all encode the domain of unknown function 231 (DUF231). TBR and its homolog TRICHOME BIREFRINGENCE-LIKE3 (TBL3) are transcriptionally coordinated with CELLULOSE SYNTHASE (CESA) genes, and loss of TBR or TBL3 results in decreased levels of crystalline secondary wall cellulose in trichomes and stems, respectively. Loss of TBR or TBL3 further results in increased pectin methylesterase (PME) activity and reduced pectin esterification in etiolated Arabidopsis hypocotyls. Together, the results suggest that DUF231 proteins might function in the maintenance of pectin- and probably homogalacturonan esterification, and that this is a requirement for normal secondary wall cellulose synthesis, at least in some tissues and organs. Here we expand the discussion about the role of TBL/DUF231 proteins in cell wall biology based on sequence and structure analyses. Our analysis revealed structural similarities of TBR with a rhamnogalacturonan acetylesterase (RGAE) of Aspergillus aculeatus and the protein LUSTRIN A-LIKE (Oryza sativa). The implications of these findings in regard to TBL functions are discussed.
doi:10.4161/psb.5.8.12414
PMCID: PMC3115197  PMID: 20657172
cellulose; cell wall; pectin; esterase; lustrin A; DUF231; TBL; Arabidopsis
23.  Functional characterization of barley betaglucanless mutants demonstrates a unique role for CslF6 in (1,3;1,4)-β-D-glucan biosynthesis 
Journal of Experimental Botany  2011;63(1):381-392.
(1,3;1,4)-β-D-glucans (mixed-linkage glucans) are found in tissues of members of the Poaceae (grasses), and are particularly high in barley (Hordeum vulgare) grains. The present study describes the isolation of three independent (1,3;1,4)-β-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-β-D-glucan in all the tissues tested. The bgl phenotype cosegregates with the cellulose synthase like HvCslF6 gene on chromosome arm 7HL. Each of the bgl mutants has a single nucleotide substitution in the coding region of the HvCslF6 gene resulting in a change of a highly conserved amino acid residue of the HvCslF6 protein. Microsomal membranes isolated from developing endosperm of the bgl mutants lack detectable (1,3;1,4)-β-D-glucan synthase activity indicating that the HvCslF6 protein is inactive. This was confirmed by transient expression of the HvCslF6 cDNAs in Nicotiana benthamiana leaves. The wild-type HvCslF6 gene directed the synthesis of high levels of (1,3;1,4)-β-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-β-D-glucans. The fine structure of the (1,3;1,4)-β-D-glucan produced in the tobacco leaf was also very different from that found in cereals having an extremely low DP3/DP4 ratio. These results demonstrate that, among the seven CslF and one CslH genes present in the barley genome, HvCslF6 has a unique role and is the key determinant controlling the biosynthesis of (1,3;1,4)-β-D-glucans. Natural allelic variation in the HvCslF6 gene was found predominantly within introns among 29 barley accessions studied. Genetic manipulation of the HvCslF6 gene could enable control of (1,3;1,4)-β-D-glucans in accordance with the purposes of use.
doi:10.1093/jxb/err285
PMCID: PMC3245474  PMID: 21940720
Cell wall; grasses; Hordeum vulgare; mixed-linkage glucans; polysaccharide
24.  Cellulose synthesis in two secondary cell wall processes in a single cell type 
Plant Signaling & Behavior  2011;6(11):1638-1643.
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 cell's 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.
doi:10.4161/psb.6.11.17709
PMCID: PMC3329324  PMID: 22057330
cell shape; cellulose biosynthesis; embryogenesis; mucilage; radial cell wall; secondary cell wall biosynthesis; seed coat; seed development
25.  Features of the primary wall CESA complex in wild type and cellulose-deficient mutants of Arabidopsis thaliana 
Journal of Experimental Botany  2008;59(10):2627-2637.
Evidence from genetics, co-precipitation and bimolecular fluorescence complementation suggest that three CESAs implicated in making primary wall cellulose in Arabidopsis thaliana form a complex. This study shows the complex has a Mr of approximately 840 kDa in detergent extracts and that it has undergone distinctive changes when extracts are prepared from some cellulose-deficient mutants. The mobility of CESAs 1, 3, and 6 in a Triton-soluble microsomal fraction subject to blue native polyacrylamide gel electrophoresis was consistent with a Mr of about 840 kDa. An antibody specific to any one CESA pulled down all three CESAs consistent with their occupying the same 840 kDa complex. In rsw1, a CESA1 missense mutant, extracts of seedlings grown at the permissive temperature have an apparently normal CESA complex that was missing from extracts of seedlings grown at the restrictive temperature where CESAs precipitated independently. In prc1-19, with no CESA6, CESAs 1 and 3 were part of a 420 kDa complex in extracts of light-grown seedlings that was absent from extracts of dark-grown seedlings where the CESAs precipitated independently. Two CESA3 missense mutants retained apparently normal CESA complexes as did four cellulose-deficient mutants defective in proteins other than CESAs. The 840 kDa complex could contain six CESA subunits and, since loss of plasma membrane rosettes accompanies its loss in rsw1, the complex could form one of the six particles which electron microscopy reveals in rosettes.
doi:10.1093/jxb/ern125
PMCID: PMC2486462  PMID: 18495638
Arabidopsis thaliana; cellulose synthase; CESA complex; membrane proteins; primary wall; rsw mutants

Results 1-25 (531011)